GB2357767A

GB2357767A - Methods of assaying human Aspartyl protease 1 alpha-secretase activity

Info

Publication number: GB2357767A
Application number: GB0023315A
Authority: GB
Inventors: Michael Jerome Bienkowkski; Mark Gurney
Original assignee: Pharmacia and Upjohn Co; Upjohn Co
Current assignee: Pharmacia and Upjohn Co
Priority date: 1998-09-24
Filing date: 2000-09-22
Publication date: 2001-07-04
Anticipated expiration: 2020-09-22
Also published as: GB0023315D0; GB2357767B

Abstract

A method for assaying hu-Asp1 . a. -secretase activity is claimed, comprising contacting hu-Asp1 protein with an amyloid precursor protein (APP) substrate containing an . a. -secretase cleavage site and measuring the cleavage of said substrate at said site. The hu-Asp1 amino acid sequence may comprise the sequence of SEQ ID NO:2 or preferably modified sequences are claimed lacking the amino terminal domain, transmembrane domain or cytoplasmic domain. Methods of identifying modulators of hu-Asp1 . a. -secretase activity as candidate Alzheimer's disease modulators and their use in therapeutic compositions are also claimed. Preferably the APP substrate contains the . a. -secretase cleavage site LVFFAEDF or KLVFFAED. Alternatively claimed is a hu-Asp1 substrate peptide comprising 50 or fewer amino acids including the cleavage site GLALALEP and methods of assaying hu-Asp1 proteolytic cleavage. The Hu-Asp2 . b. -secretase gene, polypeptide and assay for . b. -secretase activity is also described.

Description

2357767 Alzheimer's Disease Secretase, APP Substrates Therefor, and Uses

Therefor

The present application is a continuation-in-part of United States Patent Application 09/416,901, filed October 13, 1999 which claims priority benefit of United States Provisional Patent Application No. 601155,493, filed September 23, 1999 and United States Provisional Patent Application 60/169232, filed December 6, 1999. The present application also claims priority benefit as a continuation-in-part of United States Patent Application Serial No. 09/404,133 and PCT/US99/2088 1, both filed September 23, 1999, both of which in turn claim priority benefit of United States Provisional Patent Application No. 60/101,594, filed September 24, 1998. All of these priority applications are hereby incorporated by reference in their entirety. FIELD OF THE INVENTION

The present invention relates to Alzlieimer's Disease, arnyloid protein precursor, amyloid beta peptide, and human aspartyl proteases, as well as a method for the identification of agents that modulate the activity of these polypeptides and thereby are candidates to modulate the progression of Alzheimei's disease. BACKGROUND OF THE INVENTION

Alzheirner's disease (AD) causes progressive dementia with consequent formation of arnyloid plaques, neurofibrillary tangles, gliosis and neuronal loss. The disease occurs in both genetic and sporadic forms whose clinical course and pathological features are quite similar. Three genes have been discovered to date which, when mutated, cause an autosomal dominant form of Alzheimer's disease. These encode the amyloid protein precursor (APP) and two related proteins, presenilin- 1 (PSI) and presenilin-2 (PS2), which, as their names suggest, are structurally and functionally related. Mutations in any of the three proteins have been observed to enhance proteolytic processing of APP via an intracellular pathway that produces amyloid beta peptide (A0 peptide, or sometimes here as Abeta), a 40-42 aniino acid long peptide that is the primary component of amyloid plaque in AD.

Dysregulation of intracellular pathways for proteolytic processing may be central to the pathophysiology of AD. In the case of plaque formation, mutations in APP, PSI or PS2 consistently alter the proteolytic processing of APP so as to enhance -I- formation of Ap 1-42, a form of the AP peptide which seems to be particularly amyloidogenic, and thus very important in AD. Different forms of APP range in size from 695-770 amino acids, localize to the cell surface, and have a single C-terminal transmembrane domain. Examples of specific isotypes of APP which are currently known to exist in humans are the 695-amino acid polypeptide described by Kang et. al. (1987), Nature 325: 733-736 which is designated as the "normal" APP; the 751 amino acid polypeptide described by Ponte et al. (1988), Nature 331: 525-527 (1988) and Tanzi et al. (1988), Nature 331: 528-530; and the 770 amino acid polypeptide described by Kitaguchi et. al. (1988), Nature 331: 530-531 The Abeta peptide is derived from a region of APP adjacent to and containing a portion of the transmembrane domain. Normally, processing of APP at the et-secretase site cleaves the midregion of the AP sequence adjacent to the membrane and releases the soluble, extracellular domain of APP from the cell surface. This a-secretase APP processing creates soluble APP- a, (sAPPa) which is normal and not thought to contribute to AD.

Pathological processing of APP at the P- and V-secretase sites, which are located N-terminal and C-terminal to the a-secretase site, respectively, produces a very different result than processing at the a site. Sequential processing at the P- and,y-secretase sites releases the Ap peptide, a peptide possibly very important in AD pathogenesis. Processing at the P- and -f-secretase sites can occur in both the endoplasmic reticulum (in neurons) and in the endosomafflysosomal pathway after reinternalization of cell surface APP (in all cells). Despite intense efforts, for 10 years or more, to identify the enzymes responsible for processing APP at the P andy sites, to produce the Ap peptide, those proteases remained unknown until this disclosure.

SUMMARY OF THE INVENTION

Here, for the first time, we report the identification and characterization of the secretase enzyme, termed Aspartyl Protease 2 (Asp2). We disclose some known and some novel human aspartic proteases that can act as P-secretase proteases and, for the first time, we explain the role these proteases have in AD. We describe regions in the proteases critical for their unique function and for the first time characterize their substrate. This is the first description of expressed isolated purified active protein of this type, assays that use the protein, in addition to the identification and creation of useful cell lines and inhibitors. We also identify and characterize both a-secretase and P-secretase activities of a protease, designated as Asp I.

Here we disclose a number of variants of the Asp2 gene and peptide.

In one aspect, the invention provides any isolated or purified nucleic acid polynucleotide that codes for a protease capable of cleaving the beta (P) secretase cleavage site of APP that contains two or more sets of special nucleic acids, where the special nucleic acids are separated by nucleic acids that code for about 100 to 300 amino acid positions, where the amino acids in those positions may be any amino acids, where the first set of special nucleic acids consists of the nucleic acids that code for the peptide DTG, where the first nucleic acid of the first special set of nucleic acids is the first special nucleic acid, and where the second set of nucleic acids code for either the peptide DSG or DTG, where the last nucleic acid of the second set of nucleic acids is the last special nucleic acid, with the proviso that the nucleic acids disclosed in SEQ ID NO. 1 and SEQ ID NO. 3 are not included. In a preferred embodiment, the two sets of special nucleic acids are separated by nucleic acids that code for about 125 to 222 amino acid positions, which may be any amino acids. In a highly preferred embodiment, the two sets of special nucleic acids are separated by nucleic acids that code for about 150 to 196, or 150-190, or 150 to 172 amino acid positions, which may be any aniino acids. In a particular preferred embodiment, the two sets are separated by nucleic acids that code for about 172 amino acid positions, which may be any amino acids. An exemplary nucleic acid polynucleotide comprises the acid nucleotide sequence in SEQ ID NO. 5. In another particular preferred embodiment, the two sets are separated by nucleic acids that code for about 196 an-dno acids. An exemplary polynucleotide comprises the nucleotide sequence in SEQ ID NO. 5. In another particular embodiment, the two sets of nucleotides are separated by nucleic acids that code for about 190 amino acids. An exemplary polynucleotide comprises the nucleotide sequence in SEQ ID NO. 1. Preferably, the first nucleic acid of the first special set of amino acids, that is, the first special nucleic acid, is operably linked to any codon where the nucleic acids of that codon codes for any peptide comprising from 1 to 10,000 amino acid (positions). In one variation, the first special nucleic acid is operably linked to nucleic acid polymers that code for any peptide selected from the group consisting of: any reporter proteins or proteins which facilitate purification. For example, the first special nucleic acid is operably linked to nucleic acid polymers that code for any peptide selected from the group consisting of. immunoglobin-heavy chain, maltose binding protein, glutathione S transferase, Green Fluorescent protein, and ubiquitin. In another variation, the last nucleic acid of the second set of special amino acids, that is, the last special nucleic acid, is operably linked to nucleic acid polymers that code for any peptide comprising any amino acids from 1 to 10,000 amino acids. In still another variation, the last special nucleic acid is operably linked to nucleic acid polymers that code for any peptide selected from the group consisting of. any reporter proteins or proteins which facilitate purification. For example, the last special nucleic acid is operably linked to nucleic acid polymers that code for any peptide selected from the group consisting of. immunoglobin-heavy chain, maltose binding protein, glutathione S transferase, Green Fluorescent protein, and ubiquitin.

In a related aspect, the invention provides any isolated or purified nucleic acid polynucleotide that codes for a protease capable of cleaving the beta secretase cleavage site of APP that contains two or more sets of special nucleic acids, where the special nucleic acids are separated by nucleic acids that code for about 100 to 300 amino acid positions, where the arnine, acids in those positions may be any amino acids, where the first set of special nucleic acids consists of the nucleic acids that code for DTG, where the first nucleic acid of the first special set of nucleic acids is the first special nucleic acid, and where the second set of nucleic acids code for either DSG or DTG, where the last nucleic acid of the second set of special nucleic acids is the last special nucleic acid, where the first special nucleic acid is operably linked to nucleic acids that code for any number of amino acids from zero to 81 amino acids and where each of those codons may code for any an-iino acid. In a preferred embodiment, the first special nucleic acid is operably linked to nucleic acids that code for any number of from 64 to 77 amino acids where each codon may code for any amino acid. In a particular embodiment, the first special nucleic acid is operably linked to nucleic acids that code for 71 amino acids. For example, the first special nucleic acid is operably linked to 71 amino acids and where the first of those 71 amino acids is the amino acid T. In a preferred embodiment, the polynticleotide comprises a sequence that is at least 95 % identical to a human Asp 1 or Asp2 sequence as taught herein. In another preferred embodiment, the first special nucleic acid is operably linked to nucleic acids that code for any number of from 30 to 54 amino acids, or 35 to 47 amino acids, or 40 to 54 amino acids where each codon may code for any amino acid. In a particular embodiment, the first special nucleic acid is operably linked to nucleic acids that code for 47 amino acids. For example, the first special nucleic acid is operably linked to 47 codons where the first those 47 amino acids is the amino acid E.

In another related aspect, the invention provides for any isolated or purified nucleic acid polymicleotide that codes for a protease capable of cleaving the beta (P) secretase cleavage site of APP and that contains two or more sets of special nucleic acids, where the special nucleic acids are separated by nucleic acids that code for about 100 to 300 arnino acid positions, where the amino acids in those positions may.be any amino acids, where the first set of special nucleic acids consists of the nucleic acids that code for the peptide DTG, where the first nucleic acid of the first special set of amino acids is, the first special nucleic acid, and where the second set of special nucleic acids code for either the peptide DSG or DTG, where the last nucleic acid of the second set of special nucleic acids, the last special nucleic acid, is operably linked to nucleic acids that code for any number of codons from 50 to 170 codons. In a preferred embodiment, the last special nucleic acid is operably linked to nucleic acids comprising from 100 to 170 codons. In a highly preferred embodiment, the last special nucleic acid is operably linked to nucleic acids comprising from 142 to 163 codons. In a particular embodiment, the last special nucleic acid is operably linked to nucleic acids comprising about 142 codons, or about 163 codons, or about 170 codons. In a highly preferred embodiment, the polynucleotide comprises a sequence that is at least 95% identical to aspartyl-protease encoding sequences taught herein. In one variation, the second set of special nucleic acids code for the peptide DSG. In another variation, the first set of nucleic acid polynucleotide is operably linked to a peptide purification tag. For example, the nucleic acid polynucleotide is operably linked to a peptide purification tag which is six histidine. In still another variation, the first set of special nucleic acids are on one polynucleotide and the second set of special nucleic acids are on a second polynucleotide, where both first and second polynucleotides have at lease 50 codons. In one embodiment of this type, both of the polynucleotides are in the same solution. In a related aspect, the invention provides a vector which contains a polymicleotide as described above, or a cell or cell line which is transformed or transfected with a polynucleotide as described above or with a vector containing such a polynucleotide.

In still another aspect, the invention provides an isolated or purified peptide or protein comprising an amino acid polymer that is a protease capable of cleaving the beta (p) secretase cleavage site of APP that contains two or more sets of special amino acids, where the special amino acids are separated by about 100 to 300 amino acid positions, where each amino acid position can be any amino acid, where the first set of special amino acids consists of the peptide DTG, where the first amino acid of the first special set of an-tino acids is, the first special amino acid, where the second set of amino acids is selected from the peptide comprising either DSG or DTG, where the last amino acid of the second set of special amino acids is the last special amino acid, with the proviso that the proteases disclosed in SEQ ID NO. 2 and SEQ ID NO. 4 are not included. In preferred embodiments, the two sets of amino acids are separated by about 125 to 222 amino acid positions or about 150 to 196 anlino acids, or about 150190 amino acids, or about 150 to 172 amino acids, where in each position it may be any amino acid. In a particular embodiment, the two sets of amino acids are separated by about 172 amino acids. For example, the protease has the an-lino acid sequence described in SEQ ID NO 6. In another particular embodiment, the two sets of amino acids are separated by about 196 amino acids. For example, the two sets of amino acids are separated by the same an-lino acid sequences that separate the same set of special amino acids in SEQ ID NO 4. In another particular embodiment, the two sets of nucleotides are separated by about 190 amino acids. For example, the two sets of nueleotides are separated by the same amino acid sequences that separate the same set of special amino acids in SEQ ID NO 2. In one embodiment, the first amino acid of the first special set of amino acids, that is, the first special amino acid, is operably linked to any peptide comprising from 1 to 10,000 an-lino acids. In another embodiment, the first special amino acid is operably linked to any peptide selected from the group consisting of. any reporter proteins or proteins which facilitate purification. In particular embodiments, the first special amino acid is operably linked to any peptide selected from the group consisting of. immunoglobin-heavy chain, maltose binding protein, glutathione S transferase, Green Fluorescent protein, and ubiquitin. In still another variation, the last amino acid of the second set of special amino acids, that is, the last special amino acid, is operably linked to any peptide comprising any amino acids from 1 to 10, 000 amino acids. By way of nonlimiting example, the last special amino acid is operably linked any peptide selected from the group consisting of any reporter proteins or proteins which facilitate purification. In particular embodiments, the last special amino acid is operably linked to any peptide selected from the group consisting of. immunoglobin-heavy chain, maltose binding protein, glutathione S transferase, Green Fluorescent protein, and ubiquitin.

In a related aspect, the invention provides any isolated or purified peptide or protein comprising an amino acid polypeptide that codes for a protease capable of cleaving the beta secretase cleavage site of APP that contains two or more sets of special amino acids, where the special amino acids are separated by about 100 to 300 amino acid positions, where each amino acid in each position can be any amino acid, where the first set of special amino acids consists of the amino acids DTG, where the first amino acid of the first special set of amino acids is, the first special amino acid, D, and where the second set of amino acids is either DSG or DTG, where the last amino acid of the second set of special amino acids is the last special amino acid, G, where the first special amino acid is operably linked to amino acids that code for any number of amino acids from zero to 81 amino acid positions where in each position it may be any amino acid. In a preferred embodiment, the first special amino acid is operably linked to a peptide from about 30-77 or about 64 to 77 amino acids positions where each an-Lino acid position may be any amino acid. In a particular embodiment, the first special amino acid is operably linked to a peptide 3 5, 47, 7 1, or 77 an-lino acids. In a very particular embodiment, the first special amino acid is operably linked to 71 amino acids and the first of those 71 amino acids is the arnino acid T. For example, the polypeptide comprises a sequence that is at least 95% identical to an aspartyl protease sequence as described herein. In another embodiment, the first special amino acid is operably linked to any number of from 40 to 54 amino acids (positions) where each amino acid position may be any amino acid. In a particular embodiment, the first special amino acid is operably linked to amino acids that code for a peptide of 47 amino acids. In a very particular embodiment, the first special amino acid is operably linked to a 47 amino acid peptide where the first those 47 amino acids is the amino acid E. In another particular embodiment, the first special amino acid is operably linked to the same corresponding peptides from SEQ ID NO. 3 that are 3 5, 47, 7 1, or 77 peptides in length, beginning counting with the an-lino acids on the first special sequence, DTG, towards the N-terminal of SEQ ID NO. 3. In another particular embodiment, the polypeptide comprises a sequence that is at least 95% identical to the same corresponding amino acids in SEQ ID NO. 4, that is, identical to that portion of the sequences in SEQ ID NO. 4, including all the sequences from both the first and or the second special nucleic acids, toward the N terminal, through and including 71, 47, 35 amino acids before the first special amino acids. For example, the complete polypeptide comprises the peptide of 71 amino acids, where the first of the amino acid is T and the second is Q.

In still another related aspect, the invention provides any isolated or purified amino acid polypeptide that is a protease capable of cleaving the beta (p) secretase cleavage site of APP that contains two or more sets of special amino acids, where the special anlino acids are separated by about 100 to 300 arnino acid positions, where each amino acid in each position can be any amino acid, where the first set of special amino acids consists of the amino acids that code for DTG, where the first amino acid of the first special set of amino acids is, the first special amino acid, D, and where the second set of an-iino acids are either DSG or DTG, where the last an-iino acid of the second set of special amino acids is the last special amino acid, G, which is operably linked to any number of amino acids from 50 to 170 amino acids, which may be any amino acids. In preferred embodiments, the last special amino acid is operably linked to a peptide of about 100 to 170 amino acids or about 142-163 amino acids. In particular embodiments, the last special amino acid is operably linked to a peptide of about 142 amino acids, or about 163 amino acids, or about 170 arnino acids. For example, the polypeptide comprises a sequence that is at least 95% identical (and preferably 100% identical) to an aspartyl protease sequence as described herein. In one particular embodiment, the second set of special amino acids is comprised of the peptide with the amino acid sequence DSG. Optionally, the amino acid polypeptide is operably linked to a peptide purification tag, such as purification tag which is six histidine. In one variation, the first set of special amino acids are on one polypeptide and the second set of special amino acids are on a second polypeptide, where both first and second polypeptide have at lease 50 amino acids, which may be any amino acids. In one embodiment of this type, both of the polypeptides are in the same vessel. The invention further includes a process of making any of the polynucleotides, vectors, or cells described herein; and a process of making any of the polypeptides described herein.

In yet another related aspect, the invention provides a purified polynucleotide comprising a nucleotide sequence that encodes a polypeptide having aspartyl protease activity, wherein the polypeptide has an amino acid sequence characterized by: (a) a first tripeptide sequence DTG; (b) a second tripeptide sequence selected from the group consisting of DSG and DTG; and (c) about 100 to 300 amino acids separating the first and second tripeptide sequences, wherein the polypeptide cleaves the beta secretase cleavage site of amyloid protein precursor. In one embodiment, the polypeptide comprises an arnino acid sequence depicted in SEQ ID NO: 2 or 4, whereas in another embodiment, the polypeptide comprises an amino acid sequence other than the amino acid sequences set forth in SEQ ID NOs: 2 and 4. Similarly, the invention provides a purified polynucleotide comprising a nucleotide sequence that encodes a polypeptide that cleaves the beta secretase cleavage site of amyloid protein precursor; wherein the polynucleotide includes a strand that hybridizes to one or more of SEQ ID NOs: 3, 5, and 7 under the following hybridization conditions: hybridization overnight at 42'C for 2.5 hours in 6 X SSCIO. 1 % SDS, followed by washing in 1.0 X SSC at 650C, 0. 1 % SDS. In one embodiment, the polypeptide comprises an amino acid sequence depicted in SEQ ID NO: 2 or 4, whereas in another embodiment, the polypeptide comprises an amino acid sequence other than the amino acid sequences set forth in SEQ ID NOs: 2 and 4. Likewise, the invention provides a purified polypeptide having aspartyl protease activity, wherein the polypeptide is encoded by polynucleotides as described in the preceding sentences. The invention also provides a vector or host cell comprising such polynucleotides, and a method of making the polypeptides using the vectors or host cells to recombinantly express the polypeptide.

In yet another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide, said polynucleotide encoding a HuAsp polypeptide and having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of.

(a) a nucleotide sequence encoding a Hu-Asp polypeptide selected from the group consisting of Hu-Asp I, Flu-Asp2(a), and Hti-Asp2(b), wherein said Hu-Asp 1, Hu-Asp2(a) and Hti-Asp2(b) polypeptides have the complete amino acid sequence of SEQ ID NO. 2, SEQ ID NO. 4, and SEQ ID NO. 6, respectively; and (b) a nucleotide sequence complementary to the nucleotide sequence of (a).

Several species are particularly contemplated. For example, the invention provides a nucleic acid and molecule wherein said Hu-Asp polypeptide is Hu-Aspl, and said polynucleotide molecule of 1 (a) comprises the nucleotide sequence of SEQ ID NO. 1; and a nucleic acid molecule wherein said Hu-Asp polypeptide is Hu-Asp2(a), and said polynucleotide molecule of 1 (a) comprises the nucleotide sequence of SEQ ID NO. 4; and a nucleic acid molecule wherein said Hu-Asp polypeptide is Hu-Asp2(b), and said polynucleotide molecule of 1 (a) comprises the nucleotide sequence of SEQ ID NO. 5. In addition to the foregoing, the invention provides an isolated nucleic acid molecule comprising polynucleotide which hybridizes under stringent conditions to a polynucleotide having the nucleotide sequence in (a) or (b) as described above.

Additionally, the invention provides a vector comprising a nucleic acid molecule as described in the preceding paragraph. In a preferred embodiment, the nucleic acid molecule is operably linked to a promoter for the expression of a Hu-Asp polypeptide. Individual vectors which encode Hu-Aspl, and Hu-Asp2(a), and Hu-Asp2(b) are all contemplated. Likewise, the invention contemplates a host cell comprising any of the foregoing vectors, as well as a method of obtaining a Hu-Asp polypeptide comprising culturing such a host cell and isolating the Hu-Asp polypeptide. Host cells of the invention include bacterial cells, such as E. coli, and eukaryotic cells. Among the eukaryotic cells that are contemplated are insect cells, such as sf9 or High 5 cells; and mammalian cells, such as human, rodent, lagomorph, and primate. Preferred human cells include HEK293, and IMR-32 cells. Other preferred mammalian cells include COS-7, CHO-KI, Neuro-2A, and 3T3 cells. Also among the eukaryotic cells that are contemplated are a yeast cell and an avian cell.

In a related aspect, the invention provides an isolated Hu-Asp I polypeptide comprising an amino acid sequence at least 95% identical to a sequence comprising the amino acid sequence of SEQ ID NO. 2. The invention also provides an isolated Hu-Asp2(a) polypeptide comprising an amino acid sequence at least 95% identical to a sequence comprising the amino acid sequence of SEQ ID NO. 4. The invention also provides an isolated Hu-Asp2(a) polypeptide comprising an amino acid sequence at least 95% identical to a sequence comprising the amino acid sequence of SEQ ED NO. 8.

In still another aspect, the invention provides an isolated antibody that binds specifically to any Hu-Asp polypeptide described herein, especially the polypeptide described in the preceding paragraphs.

The invention also provides several assays involving aspartyl protease enzymes of the invention. For example, the invention provides a method to identify a cell that can be used to screen for inhibitors of secretase activity comprising:

(a) identifying a cell that expresses a protease capable of cleaving APP at the P secretase site, comprising:

i) collect the cells or the supernatant from the cells to be identified ii) measure the production of a critical peptide, where the critical peptide is selected from the group consisting of either the APP Cterminal peptide or soluble APP, iii) select the cells which produce the critical peptide.

In one variation, the cells are collected and the critical peptide is the APP C-terininal peptide created as a result of the P secretase cleavage. In another variation, the supernatant is collected and the critical peptide is soluble APP, where the soluble APP has a C-terminus created by P secretase cleavage. In preferred embodiments, the cells contain any of the nucleic acids or polypeptides described above and the cells are shown to cleave the P-secretase site of any peptide having the following peptide structure, P2, P 1, P V, P2', where P2 is K or N, where P 1 is M or L, where P 1' is D, where P2' is A. The method where P2 is K and P l is M. The method where P2 is N and PI is L.

In still another aspect, the invention provides novel isoforms of ainyloid protein precursor (APP) where the last two carboxy terminus amino acids of that isoform are both lysine residues. In this context, the term "isoform" is defined as any APP polypeptide, including APP variants (including mutations), and APP fragments that exists in humans, such as those described in US 5,766,846, col 7, lines 45-67, incorporated into this document by reference, modified as described herein by the inclusion of two C-terminal lysine residues. For example, the invention provides a polypeptide comprising the isoform known as APP695, modified to includetwo lysine residues as its last two carboxy terminus amino acids. An exemplary polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 16. The invention further includes APP isoform variants as set forth in SEQ ID N0s, 18 and 20. The invention further includes all polynucleotides that encode an APP protein that has been modified to include two C-terminal lysines; as well has any eukaryotic: cell line comprising such nucleic acids or polypeptides. Preferred cell lines include a mammalian cell line (e.g., HEK293, Neuro2a).

Thus, in one embodiment, the invention provides a polypeptide comprising the amino acid sequence of a mammalian arnyloid protein precursor (APP) or fragment thereof containing an APP cleavage site recognizable by a mammalian P-secretase, and further comprising two lysine residues at the carboxyl terminus of the amino acid sequence of the mammalian APP or APP fragment. As taught herein in detail, the addition of two additional lysine residues to APP sequences has been found to greatly increase AP processing of the APP in APP processing assays. Thus, the di-lysine modified APP reagents of the invention are particularly useful in assays to identify modulators of AP production, for use in designing therapeutics for the treatment or prevention of Alzheimer's disease. In one embodiment, the polypeptide comprises the complete amino acid sequence of a mammalian arnyloid protein precursor (APP), and further comprises the two lysine residues at the carboxyl terminus of the amino acid sequence of the mammalian amyloid protein precursor. In an alternative embodiment, the polypeptide comprises only a fragment of the APP, the fragment containing at least that portion of APP that is cleaved by a mammalian P-secretase (or et-secretase or V-secretase) in the formation of AP peptides.

The practice of assays that monitor cleavage of APP can be facilitated by attaching a marker to a portion of the APP. Measunnent of retained or liberated marker can be used to quantitate the amount of APP cleavage that occurs in the assay, e.g., in the presence or absence of a putative modulator of cleavage activity. Thus, in one preferred embodiment, the polypeptide of the invention further includes a marker. For example, the marker comprises a reporter protein amino acid sequence attached to the APP amino acid sequence. Exemplary reporter proteins include a fluorescing protein (e.g., green fluorescing proteins, luciferase) or an enzyme that is used to cleave a substrate to produce a colorimetric cleavage product. Also contemplated are tag sequences which are commonly used as epitopes for quantitative immunoassays.

In a prefer-red embodiment, the di-lysine-modified APP of the invention is a human APP. For example, human APP isoforms such as APP695, APP75 1, and APP770, modified to include the two lysines, are contemplated. In a preferred embodiment, the APP isoform comprises at least one variation selected from the group consisting of a Swedish KM --- >NL mutation and a London V717---W mutation, or any other mutation that has been observed in a subpopulation that is particularly prone to development of Alzheimer's disease. These mutations are recognized as mutations that influence APP processing into Ap. In a highly preferred embodiment, the APP protein or fragment thereof comprises the APP-Sw 0-secretase peptide sequence NLDA, which is associated with increased levels of AP processing and therefore is particularly useful in assays relating to Alzheimer's research. More particularly, the APP protein or fragment thereof preferably comprises the APP-Sw secretase peptide sequence SEVNLDAEFR (SEQ ID NO: 63).

In one preferred embodiment, the APP protein or fragment thereof further includes an APP transmembrane domain carboxy-terminal to the APP-Sw 0secretase peptide sequence. Polypeptides that include the TM domain are particularly useful in cell-based APP processing assays. In contrast, embodiments lacking the TM domain are useful in cell-free assays of APP processing.

In addition to working with APP from humans and various animal models, researchers in the field of Alzheimer's also have construct chimeric APP polypeptides which include stretches of amino acids from APP of one species (e.g., humans) fused to streches of APP from one or more other species (e.g., rodent). Thus, in another embodiment of the polypeptide of the invention, the APP protein or fragment thereof comprises a chimeric APP, the chimeric APP including partial APP amino acid sequences from at least two species. A chimeric APP that includes amino acid sequence of a human APP and a rodent APP is particularly contemplated.

In a related aspect, the invention provides a polynucleotide comprising a nucleotide sequence that encodes a polypeptide as described in the preceding paragraphs. Such a polynucleotide is useful for recominant expression of the polypeptide of the invention for use in APP processing assays. In addition, the polynucleotide is useful for transforming into cells to produce recombinant cells that express the polypeptide of the invention, which cells are useful in cell-based assays to identify modulators of APP processing. Thus, in addition to polynucleotides, the invention provides a vector comprising such polynucleotides, especially expression vectors where the polynucleotide is operably linked to a promoter to promote expression of the polypeptide encoded by the polynucleotide in a host cell. The invention further provides a host cell transformed or transfected with such a polynucleotide or a vector. Among the preferred host cells are mammalian cells, especially human cells.

In another, related embodiment, the invention provides a polypeptide useful for assaying for modulators of 0-secretase activity, said polypeptide comprising an amino acid sequence of the formula M2-X-Y-Z-KKCOOH; wherein X, Y, and Z each comprise an amino acid sequence of at least one amino acid; wherein-NH2-X comprises an arnino-terminal amino acid sequence having at least one amino acid residue; wherein Y comprises an amino acid sequence of a P-secretase recognition site of a mammalian amyloid protein precursor (APP); and wherein Z-KK-COOH comprises a carboxy-terminal amino acid sequence ending in two lysine (K) residues. In one preferred variation, the carboxyl-terminal arnino acid sequence Z includes a hyrdrophobic domain that is a transmembrane domain in host cells that express the polypeptide. Host cells that express such a polypeptide are particularly useful in assays described herein for identifying modulators of APP processing. In another preferred variation, the amino-terminal amino acid sequence X includes an amino acid sequence of a reporter or marker protein, as described above. In still another preferred variation, the P-secretase recognition site Y comprises the human APP-Sw P-secretase peptide sequence NWA. It will be apparent that these preferred variations are not mutually exclusive of each other -they may be combined in a single polypeptide. The invention further provides a polynucleotide comprising a nucleotide sequence that encodes such polypeptides, vectors which comprise such polynucleotides, and host cells which comprises such vectors, polynucleotides, andlor polypeptides.

In yet another aspect, the invention provides a method for identifying inhibitors of an enzyme that cleaves the beta secretase cleavable site of APP comprising:

a) culturing cells in a culture medium under conditions in which the enzyme causes processing of APP and release of amyloid beta-peptide into the medium and causes the accumulation of CTF99 fragments of APP in cell lysates, b) exposing the cultured cells to a test compound; and specifically determining whether the test compound inhibits the function of the enzyme by measuring the amount of amyloid beta-peptide released into the medium andlor the amount of CTF99 fragments of APP in cell lysates; c) identifying test compounds diminishing the amount of soluble amyloid beta peptide present in the culture medium and diminution of CTF99 fragments of APP in cell lysates as Asp2 inhibitors. In preferred embodiments, the cultured cells are a human, rodent or insect cell line. It is also preferred that the human or rodent cell line exhibits P secretase activity in which processing of APP occurs with release of ainyloid beta-peptide into the culture medium and accumulation of CTF99 in cell lysates. Among the contemplated test compounds are antisense oligomers directed against the enzyme that exhibits P secretase activity, which oligomers reduce release of soluble amyloid beta-peptide into the culture medium and accumulation of CTF99 in cell lysates.

In yet another aspect, the invention provides a method for the identification of an agent that decreases the activity of a Hu-Asp polypeptide selected from the group consisting of Hu-Asp 1, Hu-Asp2(a), and Hti-Asp2(b), the method comprising:

a) determining the activity of said Hu-Asp polypeptide in the presence of a test agent and in the absence of a test agent; and b) comparing the activity of said Hu-Asp polypeptide determined in the presence of said test agent to the activity of said Hu-Asp polypeptide detern-fined in the absence of said test agent; whereby a lower level of activity in the presence of said test agent than in the absence of said test agent indicates that said test agent has decreased the activity of said Hu-Asp polypeptide.

In a related aspect, the invention provides a method for assaying for modulators of P-secretase activity, comprising the steps of:

(a) contacting a first composition with a second composition both in the presence and in the absence of a putative modulator compound, wherein the first composition comprises a mammalian P-secretase polypeptide or biologically active fragment thereof, and wherein the second composition comprises a substrate polypeptide having an amino acid sequence comprising a P-secretase cleavage site; (b) measuring cleavage of the substrate polypeptide in the presence and in the absence of the putative modulator compound; and (c) identifying modulators of P-secretase activity from a difference in cleavage in the presence versus in the absence of the putative modulator compound. A modulator that is a P- secretase antagonist (inhibitor) reduces such cleavage, whereas a modulator that is a P-secretase agonist increases such cleavage. Since such assays are relevant to development of Alzheirner's disease therapeutics for humans, it will be readily apparent that, in one preferred embodiment, the first composition comprises a purified human Asp2 polypeptide. In one variation, the first composition comprises a soluble fragment of a human Asp2 polypeptide that retains Asp2 P- secretase activity. Several such fragments (including ATM fragments) are described herein in detail. Thus, in a particular embodiment, the soluble fragment is a fragment lacking an Asp2 transmembrane domain. Assaying to identify inhibitors of Aspl P-secretase activity also is contemplated.

The P-secretase cleavage site in APP is known, and it will be appreciated that the assays of the invention can be performed with either intact APP or fragments or analogs of APP that retain the P-secretase recognition and cleavage site. Thus, in one variation, the substrate polypeptide of the second composition comprises the amino acid sequence SEVNLDAEM, which includes the P-secretase recognition site of human APP that contains the "Swiss" mutation. In another variation, the substrate polypeptide of the second composition comprises the amino acid sequence EDAER In another variation, the second composition comprises a polypeptide having an amino acid sequence of a human amyloid precursor protein (APP). For example, the human arnyloid precursor protein is selected from the group consisting of: APP695, APP75 1, and APP770. Preferably, the human arnyloid precursor protein (irrespective of isoform selected) includes at least on mutation selected from a KM--->NL Swiss mutation and a V->F London mutation. As explained elsewhere, one preferred embodiment involves a variation wherein the polypeptide having an amino acid sequence of a human APP further comprises an amino acid sequence comprising a marker sequence attached amino-terminal to the amino acid sequence of the human arnyloid precursor protein. Preferably, the polypeptide having an amino acid sequence of a human APP further comprises two lysine residues attached to the carboxyl terminus of the amino acid sequence of the human APP. The assays can be performed in a cell free setting, using cell-free enzyme and cell-free substrate, or can be performed in a cell-based assay wherein the second composition comprises a eukaryotic cell that expresses amyloid precursor protein (APP) or a fragment thereof containing a P-secretase cleavage site. Preferably, the APP expressed by the host cell is an APP variant that includes two carboxyl-terminal lysine residues. It will also be appreciated that the 0-secretase enzyme can be an enzyme that is expressed on the surface of the same cells.

The present invention provides isolated nucleic acid molecules comprising a polynucleotide that codes for a polypeptide selected from the group consisting of human aspartyl proteases. In particular, human aspartyl protease 1 (Hu-Aspl) and two alternative splice variants of human aspartyl protease-2 (Hu-Asp2), a Iong" (L) form designated herein as Hu- Asp2(a) and a "short" (S) form designated Ilu-Asp2(b). As used herein, all references to "Hu-Asp" should be understood to refer to all of Hu-Asp I, Hu-Asp2(a), and Hu-Asp2(b). In addition, as used herein, all references to "Hu-Asp2" should be understood to refer to both Hu-Asp2(a) and Hu-Asp2(b). Hu-Asp I is expressed most abundantly in pancreas and prostate tissues, while Hu-Asp2(a) and Hu-Asp2(b) are expressed most abundantly in pancreas and brain tissues. The invention also provides isolated Hu-Aspi 1, Hu-Asp2(a), and Hu-Asp2(b) polypeptides, as well as fragments thereof which exhibit aspartyl protease activity.

In a preferred embodiment, the nucleic acid molecules comprise a polynucleotide having a nucleotide sequence selected from the group consisting of residues 1-1554 of SEQ H) NO. 1, encoding Hu-Aspl, residues 1-1503 of SEQ H) NO. 3, encoding Hu-Asp2(a), and residues 1-1428 of SEQ ID NO.5, encoding Hu-Asp2(b). In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent conditions to a polynucleotide encoding Hu-Asp 1, Hu-Asp2(a), Hu-Asp-2(b), or fragments thereof European patent application EP 0 848 062 discloses a polypeptide referred to as "Asp 1," that bears substantial homology to Hu-Aspl, while international application WO 98122597 discloses a polypeptide referred to as "Asp 2," that bears substantial homology to Hu-Asp2(a).

The present invention also provides vectors comprising the isolated nucleic acid molecules of the invention, host cells into which such vectors have been introduced, and recombinant methods of obtaining a HuAsp I, Hu-Asp2(a), or Hu-Asp2(b) polypeptide comprising culturing the above-described host cell and isolating the relevant polypeptide.

In another aspect, the invention provides isolated Hu-Aspl, Hu-Asp2(a), and Hu-Asp2(b) polypeptides, as well as fragments thereof. In a preferred embodiment, the Hu-Asp I, Hu-Asp2(a), and Hti-Asp2(b) polypeptides have the amino acid sequence given in SEQ H) NO. 2, SEQ ID NO. 4, or SEQ ID NO. 6, respectively. The present invention also describes active forms of HiiAsp2, methods for preparing such active forms, methods for preparing soluble forms, methods for measuring Hu-Asp2 activity, and substrates for Hu-Asp2 cleavage. The invention also describes antisense oligomers targeting the Hu-Asp 1, Hu-Asp2(a) and Hu-Asp2(b) niRNA transcripts and the use of such antisense reagents to decrease such mRNA and consequently the production of the corresponding polypeptide. Isolated antibodies, both polyclonal and monoclonal, that binds specifically to any of the Hu- Asp I, Hu-Asp2(a), and Hu-Asp2(b) polypeptides of the invention are also provided.

The invention also provides a method for the identification of an agent that modulates the activity of any of Hu-Asp-1, Hu-Asp2(a), and HuAsp2(b). The inventions describes methods to test such agents in cellfree assays to which Hu-Asp2 polypeptide is added, as well as methods to test such agents in human or other mammalian cells in which Hu-Asp2 is present.

The invention provides for methods for assaying for human Aspl(hu-Aspl) CLsecretase activity comprising contacting the hu-Asp 1 protein with an amyloid precursor protein (A.PP) substrate, wherein the substrate contains an a-sectetase cleavage site; and measuring cleavage of the APP substrate at the a-secretase cleavage site, thereby assaying hu-Aspl a- secretase activity. An example of a-secretase activity is APP processing wherein the APP substrate is cleaved at a site adjacent to the cell membrane (at residues Phe201A1a2' in relation to the A0 peptide). This cleavage results in the release of a soluble, extracellular domain of APP, known as amyloid alpha peptide (sAPPa), from the cell surface into the cytoplasm. The sAPPa within the cytoplasm can be detected and quantitated thereby measuring a-secretase activity.

The hu-Asp 1 enzyme used in the methods of the invention can be purified and isolated from a cell which is transfected or transformed with a polynucleotide that encodes hu-Asp I, such as SEQ ID NO: 1, or a polynucleotide sequence that encodes the the an-iino acid sequence of SEQ ID NO: 2. Further, the hu-Aspl protein used in the methods may be a fragment of the amino acid sequence of SEQ ID NO: 2 which retains asecretase activity. Possible fragments that may be of use for the methods include those lacking the transmembrane domain amino acids 469-492 of SEQ ID NO: 2, those fragments that lack the cytoplasmic amino acids 493-492 of SEQ ID NO: 2, those fragments that lack the amino terminal amino acids 1-62 of SEQ ID NO: 2 or combinations thereof.

The invention also encompasses methods of assaying for a-secretase activity where hu-Aspl protein and its substrate are brought into contact by a growing cell transfected or transformed with a polynucleotide encoding the hu-Aspl protein or a fragment thereof that retains asecretase activity under conditions where the cell expresses hu-Asp I protein in the presence of the APP substrate. The APP substrate in such circumstances can be exogenously introduced, or more preferably, is expressed by the cell that expresses Asp 1. These methods also encompass contacting hu-Asp 1 protein with a cell that expresses a polynucleotide that encodes an APP substrate containing an a-secretase cleavage site. For example, the cell may express a polynucleotide that encodes a polypeptide having an a-secretase cleavage site comprising the amino acid sequence LWFAEDF or KLVIFFAED. In addition, the APP substrate may comprise any human isoform of APP, such as "normal" APP (APP695), APP 75 1, or APP770. These APP substrates can be further modified to comprise a carboxy-terminal di-lysine motif.

To measure the cleavage of the substrates for the methods of assaying for asecretase activity of the invention, the substrates of the method can be further modified to comprise detectable labels such as radioactive, enzymatic, chemilumenescent or flourescent labels. In particular, shorter peptide substrates preferably comprise internally quenched labels that result in increased detectability after cleavage of the peptide substrates. The peptide substrates may be modified to have attached a paired flurophore and quencher including but not limited to 7-amino-4methyl coumarin and dinitrophenol, respectively, such that cleavage of the peptide by the hu-Asp 1 results in increased fluorescence due to physical separation of the flurophore and quencher. Other paired flurophores and quenchers include bodipy-tetramethylrhodamine and QSY-5 (Molecular Probes, Inc.) In a variant of this assay, biotin or another suitable tag may be placed on one end of the peptide to anchor the peptide to a substrate assay plate and a flurophore may be placed at the other end of the peptide. Useful flurophores include those listed above as well as Europium labels such as W8044 (EG&G Wallac, Inc.). A preferred label is oregon green that may be attached to a Cys residue. Cleavage of the peptide by Asp I will release the flurophore or other tag from the plate, allowing compounds to be assayed for inhibition of Asp I proteolytic cleavage as shown by an increase in retained fluorescence. Preferred colorimetric assays of hu-Asp I proteolytic activity utilize other suitable substrates that include the P2 and P, amino acids comprising the recognition site for cleavage linked to onitrophenol through an amide linkage, such that cleavage by the hu-Aspl results in an increase in optical density after altering the assay buffer to alkaline pH.

The prevent invention also provides for methods of assaying for asecretase activity comprising contacting hu-Asp 1 protein with an APP substrate, determining the level of hu-Asp I a-secretase activity in the presence and absence of a modulator of hu- Asp 1 a-secretase activity and comparing the hu-Asp 1 secretase activity in the presence and absence of the modulator. The modulators detennined to increase hu-Aspl asecretase activity will be identified as candidate Akheimer's disease therapeutics. The invention also encompasses methods which comprise a step for treating Akheimer's disease with identified candidate Akheimer disease therapeutics. The invention also provides for compositions comprising a candidate Akheimer's disease therpeutic identified by the a-secretase assaying methods of the invention. Aspl. modulators that reduce Aspl. 0-secretase activity and increase Aspl, a- secretase activity are highly preferred. Assays for Asp I P-secretase activity are preferred essentially as described in detail herein for Asp2.

The invention provides for Asp 1 protease substrate peptides or fragments thereof, wherein said peptides comprise an amino acid sequence consisting of fifty or fewer amino acids which comprise the Aspl cleavage site having the amino acid sequence GLALALEP. This peptide was derived from the Aspl amino acid sequence and the discovery of an apparent Aspl. autocatalytic cleavage in acidic conditions. The Aspl substrate of the invention may also comprise a detectable label, such as a radioactive label, chemiluminescent label, enzymatic label or a flourescent label. The flourescently labeled substrate can consist of internally quenched labels as described above.

The invention also encompasses methods comprising the steps of contacting huAsp 1 protein with an Asp 1 substrate under acidic conditions and determining the level of Asp 1 proteolytic activity. An example of Asp 1 proteolytic activity is the auto-catalytic processing hu-Asp undergoes in acidic environments, wherein cleavage occurs at an amino acid site surrounding AIP and cleaves the amino terminal arnino acids of the huAsp I pro-peptide. The hu-Asp I pro-peptide refers to a secreted form of Asp I that has completed intercellular processing which resulted in cleavage of its signal sequence.

For the methods of assaying Aspl proteolytic activity, the hu-Aspl may be produced in a cell transformed or transfected with a polynueleotide that encodes hu-Asp I. The hu-Asp 1 protein may be isolated and purified from these cells or the method may utilize a cell growing under conditions that it expresses hu-Asp I. The method may also be carried out with a fragment of hu-Aspl that retains its proteolytic activity. The fragments provided for by the invention include hu-Asp 1 polypeptide sequences which lack the arnino acids that encode a transmembrane domain such as amino acids 469-492 of SEQ ID NO: 2 or fragments that lacks the cytoplasmic domain such as amino acids 493-518 of SEQ ID NO: 2.

The invention provides for a purified polynucleotide comprising a nucleotide sequence encoding a polypeptide that comprises a fragment of a hu-Aspl protein, wherein said nucleotide sequence lacks the sequence that encodes amino acids 23-62 of SEQ ID NO: 2, and wherein the polypeptide has hu-Aspl a-secretase activity. This portion of the Aspl sequence corresponds to a punitive pro-peptide that is removed, apparently through autocatalysis, under acidic conditions. These polypeptide fragments also include those lacking the amino terminal amino acids 1-62 of SEQ ID NO: 2.

The invention encompasses a purified polynucleotide comprising a nucleotide sequence that hybridizes under stringent conditions to the noncoding strand complementary to SEQ ID NO: 1, wherein the nucleotide sequence encodes a polypeptide having Aspl, proteolytic activity and wherein the polynucleotide lacks nucleotides encoding a transmembrane domain. Further, the invention also provides for a purified polynxicleotide sequence comprising a nucleotide sequence that hybridizes under stringent conditions to the non-coding strand complementary to SEQ ID NO: 1,, wherein the nucleotide sequence encodes a polypeptide further lacking a pro-peptide domain corresponding to amino acids 23-62 of SEQ ID NO: 2.

The invention also provides for vectors comprising the hu-Asp 1 polynxicleotide of the invention and host cells transfected or transformed with these vectors. The invention also encompasses host cells transfected or transformed with the hu-Aspl polynucleotides of the invention.

Another embodiment of the invention is a purified polypeptide comprising a fragment of a hu-Aspl protein, wherein said polypeptide lacks the hu- Aspl transmembrane domain and retains hu-Aspl et-secretase activity. These polypeptides include fragments of hu-Asp I having the arnino acid sequence set forth in SEQ ID NO: 2, and wherein the polypeptide optionally lacks the transmembrane domain an-iino acids 469-492 of SEQ ID NO: 2, wherein the polypeptide lacks the cytoplasmic domain amino acids 493-518 of SEQ ID NO: 2 as well. In one variation, the invention provides a polypeptide that lacks amino terminal amino acids 1-62 of SEQ ID NO: 2 but retains Aspl proteolytic activity. Fragments lacking both the aforementioned arnino-terminal and carboxy terminal residues are contemplated.

The invention provides for a polypeptide comprising a fragment of hu-Aspl having the amino acid sequence set forth in SEQ ID NO: 2 and wherein said polypeptide lacks the amino terminal amino acids 1-62 and retains APP processing activity. For example, referring to the Aspl sequence in SEQ ID NO: 2, this pre-pro portion would correspond to residues 22 to 62. By performing conventional sequence analysis, the corresponding portions of the Aspl sequence can also be identified.

Another embodiment of the invention is a polypeptide comprising the andno acid sequence at least 95% identical to a fragment of hu-Aspl protein, wherein said polypeptide and said fragment lack a transmembrane domain and retain Ini-Aspl ctsecretase activity. In addition, the invention embodies a polypeptide comprising an amino acid sequence at least 95% identical to a fragment of hu-Aspl protein, wherein said polypeptide and said fragment lack the amino terminal amino acids corresponding to the pre-pro portion of hu-Asp I and retain APP processing activity.

Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, including the drawing and detailed description, and all such features are intended as aspects of the invention. Likewise, features of the invention described herein can be re-combined into additional embodiments that are also intended as aspects of the invention, irrespective of whether the combination of features is specifically mentioned above as an aspect or embodiment of the invention. Also, only such limitations which are described herein as critical to the invention should be viewed as such; variations of the invention lacking limitations which have not been described herein as critical are intended as aspects of the invention.

In addition to the foregoing, the invention includes, as an additional aspect, all ernbodiments of the invention narrower in scope in any way than the variations specifically mentioned above. Although theapplicant(s) invented the full scope of the claims appended hereto, the claims appended hereto are not intended to encompass within their scope the prior art work of others. Therefore, in the event that statutory prior art within the scope of a claim is brought to the attention of the applicants by a Patent Office or other entity or individual, the applicant(s) reserve the right to exercise amendment rights under applicable patent laws to redefine the subject matter of such a claim to specifically exclude such statutory prior art or obvious variations of statutory prior art from the scope of such a claim. Variations of the invention defined by such amended claims also are intended as aspects of the invention.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

Sequence ID No. 1: Human Asp- 1, nucleotide sequence. Sequence ID No. 2: Human Asp-1, predicted amino acid sequence. Sequence ID No. 3: Human Asp2(a), nucleotide sequence. Sequence ID No. 4: Human Asp-2(a), predicted amino acid sequence. The Asp2(a) amino acid sequence includes a putative signal peptide comprising residues 1 to 2 1; and a putative prepropeptide after the signal peptide that extends through residue 45 (as assessed by processing observed of recombinant Asp2(a) in CHO cells), and a putative propeptide that may extend to at least about residue 57, based on the observation of an observed GRRIGS sequence which has characteristics of a protease recognition sequence. The Asp2(a) further includes a transmembrane domain comprising residues 455-477, a cytoplasmic domain comprising residues 478-501, and a putative alpha- helical spacer region, comprising residues 420-454, believed to be unnecessary for proteolytic activity, between the protease catalytic domain and the transmembrane domain. Sequence ID No. 5: Human Asp-2(b), nucleotide sequence. Sequence ID No. 6: Human Asp-2(b), predicted amino acid sequence. The Asp2(b) amino acid sequence includes a putative signal peptide, pre-propeptide, and propeptide as described above for Asp2(a). The Asp2(b) further includes a transmembrane domain comprising residues 430-452, a cytoplasmic domain comprising residues 453-476, and a putative alphahelical spacer region, comprising residues 395-429, believed to be unnecessary for proteolytic activity, between the protease catalytic domain and the transmembrane domain.

Sequence ID No. 7: Murine Asp-2(a), nucleotide sequence.

Sequence ID No. 8: Murine Asp-2(a), predicted amino acid sequence. The proteolytic processing of murine Asp2(a) is believed to be analogous to the processing described above for human Asp2(a). In addition, a variant lacking amino acid residues 190-214 of SEQ ID NO: 8 is specifically contemplated as a murine Asp2(b) polypeptide.

Sequence ID No. 9: Human APP695, nucleotide sequence.

Sequence II) No.10: Human APP695, predicted amino acid sequence.

Sequence ID No. 11: Human APP695-Sw, nucleotide sequence.

Sequence ID No. 12: Human APP695-Sw. predicted amino acid sequence. In the APP695 isoform, the Sw mutation is characterized by a KM->NL alteration at positions 595-596 (compared to normal APP695).

Sequence ID No.13: Human APP695-W, nucleotide sequence.

Sequence IDNo.14: Human APP695-W, predicted an-fino acid sequence. In the APP 695 isoform, the V17 mutation is characterized by a V---F alteration at position 642 (compared to normal APP 695).

Sequence ID No. 15: Human APP695-KK, nucleotide sequence.

Sequence II) No. 16: Human APP695-KK, predicted amino acid sequence. (APP695 with two carboxy-terminal lysine residues.) Sequence ID No. 17: Human APP695-Sw-KK, nucleotide sequence.

Sequence ID No. 18: Human APP695-Sw-KIC, predicted amino acid sequence Sequence ID No. 19: Human APP695-W-KK, nucleotide sequence Sequence ID No. 20: Human APP695-VF-ICK, predicted amino acid sequence Sequence ID No.21: T7-Human-pro-Asp-2(a)ATM, nucleotide sequence Sequence ID No.22: T7-Humanpro-Asp-2fflATM, amino acid sequence Sequence ED No.23: T7-Caspase-Human-pro-Asp-2(a)ATM, nucleotide sequence Sequence ID No.24: T7-Caspase-Human-pro-Asp-2(a)ATM, amino acid sequence Sequence ID No.25: Human-pro-Asp-2(a)ATM (low GC), nucleotide sequence Sequence ID No.26: Human-pro-Asp-2(a)ATM, (low GC), amino acid sequence Sequence ID No.27: T7-Caspase-Caspase 8 cleavage-Human-pro-Asp-2(a)ATM, nucleotide sequence Sequence ID NO.28: T7-Caspase-Caspase 8 cleavage- Human-pro-Asp-2(a)ATM, amino acid sequence Sequence ID No.29: Human Asp- 2(a)ATM, nucleotide sequence Sequence ID No.30: Human Asp-2(a)ATM, amino acid sequence Sequence ID No.31: Human Asp-2(a)ATM(MS)6, nucleotide sequence Sequence 11) No. 32: Human Asp-2(a)ATM(HiS)6, amino acid sequence Sequence ID Nos. 3349 are short synthetic peptide and oligonucleotide sequences that are described below in the Detailed Description of the Invention.

Sequence ID No. 50: Human Asp2(b)ATM polynucleotide sequence.

Sequence ID No. 5 1: Human Asp2(b)AT'M polypeptide sequence (exemplary variant of Human Asp2(b) lacking transmembrane and intracellular domains of HuAsp2(b) set forth in SEQ ID NO: 6.

Sequence ID No. 52: Human Asp2(b)ATM(MS)6 polynucleotide sequence.

Sequence ID No. 53: Human Asp2(b)ATM(MS)6 polypeptide sequence (Human Asp2(b)ATM with six histidine tag attached to C-terminus).

Sequence ID No. 54: Human APP770-encoding polynucleotide sequence.

Sequence ID No. 55: Human APP770 polypeptide sequence. To introduce the KM---NL Swedish mutation, residues KM at positions 670-71 are changed to NL. To introduce the V->F London mutation, the V residue at position 717 is changed to F.

Sequence ID No. 56: Human APP751 encoding polynucleotide sequence.

Sequence ID No. 57: Human APP751 polypeptide sequence (Human APP751 isoform).

Sequence ID No. 58: Human APP770-KK encoding polynucleotide sequence.

Sequence ID No. 59: Human APP770-KK polypeptide sequence. (Human APP770 isoform to which two C-terminal lysines have been added).

Sequence 1D No. 60: Human APP75 1-KK encoding polynucleotide sequence.

Sequence ID No. 61: Human APP751-KK polypeptide sequence (Human APP751 isoform to which two C-terminal lysines have been added).

Sequence ID Nos. 62-65: Various short peptide sequences described in detail in detailed description.

Sequence 1D No. 66: Predicted amino acid sequence of human Asp1 ATM(MS)6 as described in Example 14.

Sequence 1D No. 67: Amino acid sequence of secreted recombinant Asp1 ATM(His)6 as described in Example 14. ' Sequence ID No. 68: Amino acid sequence of acid-processed form of AsplAfflis)6.

Sequence ID No. 69: Amino acid sequence of the self-activated acid processing site within Asp- 1 ATM.

Sequence ID No. 70: Amino acid sequence of a peptide that includes the secretase processing site within the Swedish mutant form of APP.

Sequence ID No. 7 1: Amino acid sequence of a peptide (residues 17-24) that includes the a-secretase processing site within the AP peptide (AP1228).

Sequence ID No. 72: Amino acid sequence of a peptide (residues 16-23) that includes the a-secretase processing site within the AP peptide (AP12- 28).

Sequence ID No. 73-74: PCR primers described in Example 14.

Sequence ID No.75: Amino acid sequence of ay-secretase substrate polypeptide described in Example 15.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Figure 1 shows the nucleotide (SEQ ID NOA) and predicted amino acid sequence (SEQ ID NO:2) of human Asp l.

Figure 2: Figure 2 shows the nucleotide (SEQ ID NO:3) and predicted an-iino acid sequence (SEQ ID NOA) of human Asp2(a).

Figure 3: Figure 3 shows the nucleotide (SEQ ID NO:5) and predicted amino acid sequence (SEQ ID NO:6) of human Asp2(b). The predicted transmembrane domain of Ilu-Asp2(b) is enclosed in brackets.

Figure 4: Figure 4 shows the nucleotide (SEQ ID No. 7) and predicted amino acid sequence (SEQ ID No. 8) of murine Asp2(a) Figure 5: Figure 5 shows the BestFit alignment of the predicted amino acid sequences of Hti-Asp2(a) and murine Asp2(a) Figure 6: Figure 6 shows the nucleotide (SEQ ID No. 21) and predicted amino acid sequence (SEQ ID No. 22) of T7-Hurnan-pro-Asp-2(a)ATM Figure 7: Figure 7 shows the nucleotide (SEQ ID No. 23) and predicted amino acid sequence (SEQ ID No. 24) of T7-caspase-Human-pro-Asp-2(a)ATM Figure 8: Figure 8 shows the nucleotide (SEQ ID No. 25) and predicted amino acid sequence (SEQ ID No. 26) of Hurnan-pro-Asp-2(a)ATM (low GC) Figure 9: Western blot showing reduction of CTF99 production by HEK125.3 cells transfected with antisense oligomers targeting the Hu-Asp2 mRNA.

Figure 10: Western blot showing increase in CTF99 production in mouse Neuro-2a cells cotransfected with APP-KK with and without Hti-Asp2 only in those cells cotransfected with Hu-Asp2. A further increase in CTF99 production is seen in cells cotransfected with APP-Sw-KK with and without Hti-Asp2 only in those cells cotransfected with Hti-Asp2 Figure 11: Figure 11 shows the predicted amino acid sequence (SEQ ID No. 30) of Hurnan-Asp2(a)ATM Figure 12: Figure 11 shows the predicted an-iino acid sequence (SEQ ID No. 30) of Hurnan-Asp2(a)ATM(M8)6 DETAILED DESCRIPTION OF THE INVENTION

A few definitions used in this invention follow, most definitions to be used are those that would be used by one ordinarily skilled in the art.

The term "P amyloid peptide" means any peptide resulting from beta secretase cleavage of APP. This includes peptides of 39, 40, 41, 42 and 43 amino acids, extending from the P-secretase cleavage site to 39, 40, 41, 42 and 43 amino acids C-ten-ninal to the P-secretase cleavage site. 0 arnyloid peptide also includes sequences 1-6, SEQ]ID N0s. 1-6 of US 5,750, 349, issued 12 May 1998 (incorporated into this document by reference).

A P-secretase cleavage fragment disclosed here is called CTF-99, which extends from P-secretase cleavage site to the carboxy terminus of APP.

When an isoform. of APP is discussed then what is meant is any APP polypeptide, including APP variants (including mutations), and APP fragments that exists in humans such as those described in US 5,766,846, col 7, lines 45-67, incorporated into this document by reference.

The term "0-amyloid precursor protein" (APP) as used herein is defined as a polypeptide that is encoded by a gene of the same name localized in humans on the long arm of chromosome 21 and that includes "PAP - here "pamyloid protein" see above, within its carboxyl third. APP is a glycosylated, single-membrane spanning protein expressed in a wide variety of cells in many mammalian tissues. Examples of specific isotypes of APP which are currently known to exist in humans are the 695 amino acid polypeptide described by Kang et. al. (1987) Nature 325:733-736 which is designated as the "normal" APP (SEQ]ID NOs: 9- 10); the 751 amino acid polypeptide described by Ponte et al. (1988) Nature 331:525- 527 (1988) and Tanzi et al. (1988) Nature 331:528-530 (SEQ I1D NOs: 56- 57); and the 770-amino acid polypeptide described by Kitaguchi et. al. (1988) Nature 331:530-532 (SEQ ID NOs: 54-55). Examples of specific variants of APP include point mutation which can differ in both position and phenotype (for review of known variant mutation see Hardy (1992) Nature Genet. 1:233-234). All references cited here incorporated by reference. The term "APP fragments" as used herein refers to fragments of APP other than those which consist solely of PAP or PAP fragments. That is, APP fragments will include unino acid sequences of APP in addition to those which form intact PAP or a fragment of PAP.

When the term "any amino aciC is used, the amino acids referred to are to be selected from the following, three letter and single letter abbreviations - which may also be used, are provided as follows:

Alanine, Ala, A; Arginine, Arg, R; Asparagine, Asn, N; Aspartic acid, Asp, D; Cysteine, Cys, C; Glutamine, G1n, Q; Glutan-fic Acid, Glu, E; Glycine, Gly, G; Histidine, His, H; Isoleucine, Be, I; Uucine, Leu, L; Lysine, Lys, K; Methionine, Met, M; Phenylalanine, Phe, F; Proline, Pro, P; Serine, Ser, S; Threonine, Thr, T; Tryptophan, Trp, W; Tyrosine, Tyr, Y; Valine, Val, V; Aspartic acid or Asparagine, Asx, B; Glutarnic acid or Glutarnine, Glx, Z; Any amino acid, Xaa, X.

The present invention describes a method to scan gene databases for the simple active site motif characteristic of aspartyl proteases. Eukaryotic aspartyl proteases such as pepsin and renin possess a two-domain structure which folds to bring two aspartyl residues into proximity within the active site. These are embedded in the short tripeptide motif DTG, or more rarely, DSG. Most aspartyl proteases occur as proenzyme whose N-tem-iinus must be cleaved for activation. The DTG or DSG active site motif appears at about residue 65-70 in the proenzyme (prorenin, pepsinogen), but at about residue 25-30 in the active enzyme after cleavage of the N-terminal prodomain. The limited length of the active site motif makes it difficult to search collections of short, expressed sequence tags (EST) for novel aspartyl proteases. EST sequences typically average 250 nucleotides or less, and so would encode 80-90 amino acid residues or less. That would be too short a sequence to span the two active site motifs. The preferred method is to scan databases of hypothetical or assembled protein coding sequences. The present invention describes a computer method to identify candidate aspartyl proteases in protein sequence databases. The method was used to identify seven candidate aspartyl protease sequences in the Caenorhabditis elegans genome. These sequences were then used to identify by homology search Hu- Asp 1 and two alternative splice variants of Hti-Asp2, designated herein as Hu-Asp2(a) and Hti-Asp2(b).

In a major aspect of the invention disclosed here we provide new information about APP processing. Pathogeneic processing of the amyloid precursor protein (APP) via the AP pathway requires the sequential action of two proteases referred to as P-secretase andy-secretase. Cleavage of APP by the P- secretase and V-secretase generates the N-terminus and C-terminus of the AP peptide, respectively. Because over production of the AP peptide, particularly the AP1-42, has been implicated in the initiation of Alzlieimer's disease, inhibitors of either the P-secretase and/or the V- secretase have potential in the treatment of Alzlieimer's disease. Despite the importance of the 0-secretase andy-secretase in the pathogenic processing of APP, molecular definition of these enzymes has not been accomplished to date. That is, it was not known what enzymes were required for cleavage at either the P-secretase or the y-secretase cleavage site. The sites themselves were known because APP was known and the API-42, peptide was known, see US 5,766,846 and US 5,837,672, (incorporated by reference, with the exception to reference to "soluble" peptides). But what enzyme was involved in producing the AP1-42, peptide was unknown.

Alignment of the amino acid sequences of HwAsp2 with other known aspartyl proteases reveals a similar domain organization. All of the sequences contain a signal sequence followed by a pro-segment and the catalytic domain containing 2 copies of the aspartyl protease active site motif (DTG/DSG) separated by approximately 180 an-lino acid residues. Comparison of the processing site for proteolytic removal of the prosegment in the mature forms of pepsin A, pepsin C, cathepsin D, cathepsin E and renin reveals that the mature forms of these enzymes contain between 31-35 amino acid residues upstream of the first DTG motif. Inspection of this region in the Hti-Asp-2 amino acid sequence indicates a preferred processing site within the sequence GRRIGS as proteolytic processing of pro-protein precursors commonly occurs at site following dibasic amino acid pairs (eg. RR). Also, processing at this site would yield a mature enzyme with 35 amino acid residues upstream of the first DTG, consistent with the processing sites for other aspartyl proteases. In the absence of self-activation of Hli-Asp2 or a knowledge of the endogenous protease that processes Hti-Asp2 at this site, a recombinant form was engineered by introducing a recognition site for the PreSission protease (LEVLFQIGP) into the expression plasmids for bacterial, insect cell, and mammalian cell expression of pro-Hu-Asp2. In each case, the Gly residue in Pl' position corresponds to the Gly residue 35 amino acids upstream of the first DW motif in Hu-Asp2.

The present invention involves the molecular definition of several novel human aspartyl proteases and one of these, referred to as Hu-Asp-2(a) and Hti-Asp2(b), has been characterized in detail. Previous forms of aspI and asp 2 have been disclosed, see EP 0848062 A2 and EP 0855444A2, inventors David Powel et al., assigned to Smith Kline Beecham Corp. (incorporated by reference). Herein are disclosed old and new forms of Hu-Asp 2. For the first time they are expressed in active form, their substrates are disclosed, and their specificity is disclosed. Prior to this disclosure cell or cell extracts were required to cleave the P-secretase site, now purified protein can be used in assays, also described here. Based on the results of (1) antisense knock out experiments, (2) transient transfection knock in experiments, and (3) biochemical experiments using purified recombinant Hti-Asp-2, we demonstrate that Hu-Asp-2 is the 0- secretase involved in the processing of APP. Although the nucleotide and predicted amino acid sequence of Hu-Asp-2(a) has been reported, see above, see EP 0848062 A2 and EP 0855444A2, no functional characterization of the enzyme was disclosed. Here the authors characterize the Hu-Asp-2 enzyme and are able to explain why it is a critical and essential enzyme required in the formation of API-42, peptide and possible a critical step in the development of AD.

In another embodiment the present invention also describes a novel splice variant of HuAsp2, referred to as Hu-Asp-2(b), that has never before been disclosed.

In another embodiment, the invention provides isolated nucleic acid molecules comprising a polynucleotide encoding a polypeptide selected from the group consisting of human aspartyl protease 1 (Hu-Aspl) and two alternative splice variants of human aspartyl protease-2 (Hu-Asp2), designated herein as Hu-Asp2(a) and Hti-Asp2(b). As used herein, all references to "Hu-Asp2" should be understood to refer to both Hti-Asp2(a) and Hti-Asp2(b). Hu-Asp 1 is expressed most abundantly in pancreas and prostate tissues, while Hti-Asp2(a) and Hu-Asp2(b) are expressed most abundantly in pancreas and brain tissues. The invention also provides isolated Hu-Asp 1, Hu-Asp2(a), and Hii-Asp2(b) polypeptides, as well as fragments thereof which exhibit aspartyl protease activity.

The predicted amino acid sequences of Hu-Asp I, Hti-Asp2(a) and HuAsp2(b) share significant homology with previously identified mammalian aspartyl proteases such as pepsinogen A, pepsinogen B, cathepsin D, cathepsin E, and renin. P.B.Szecs, Scand. J. Clin. Lab. Invest. 52:(Suppl. 210 5-22 (1992)). These enzymes are characterized by the presence of a duplicated DWIDSG sequence motif. The Hu-Asp I and HuAsp2 polypeptides disclosed herein also exhibit extremely high homology with the ProSite consensus motif for aspartyl proteases extracted from the SwissProt database.

The nucleotide sequence given as residues P 1554 of SEQ ID NO: 1 corresponds to the nucleotide sequence encoding Hu-Asp I, the nucleotide sequence given as residues 1- 1503 of SEQ H) NO: 3 corresponds to the nucleotide sequence encoding Hti-Asp2(a), and the nucleotide sequence given as residues 1-1428 of SEQ ID NO:5 corresponds to the nucleotide sequence encoding Hu-Asp2(b). The isolation and sequencing of DNA encoding Hu-Asp I, Hti-Asp2(a), and Hu-Asp2(b) is described below in Examples 1 and 2.

As is described in Examples 1 and 2, automated sequencing methods were used to obtain the nucleotide sequence of Hu-Asp I, Hu-Asp2(a), and HtiAsp-2(b). The Hu-Asp nucleotide sequences of the present invention were obtained for both DNA strands, and are believed to be 100% accurate. However, as is known in the art, nucleotide sequence obtained by such automated methods may contain some errors. Nucleotide sequences deterndned by automation are typically at least about 90%, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of a given nucleic acid molecule. The actual sequence may be more precisely determined using manual sequencing methods, which are well known in the art. An error in sequence which results in an insertion or deletion of one or more nueleotides may result in a frame shift in translation such that the predicted an-dno acid sequence will differ from that which would be predicted from the actual nucleotide sequence of the nucleic acid molecule, starting at the point of the mutation.

The Hu-Asp DNA of the present invention includes cDNA, chemically synthesized DNA, DNA isolated by PCR, genomic DNA, and combinations thereof. Genomic Hu-Asp DNA may be obtained by screening a genomic library with the Hti-Asp2 cDNA described herein, using methods that are well known in the art, or with oligonucleotides chosen from the Hti-Asp2 sequence that will prime the polymerase chain reaction (PCR). RNA transcribed from Hu-Asp DNA is also encompassed by the present invention.

Due to the degeneracy of the genetic code, two DNA sequences may differ and yet encode identical amino acid sequences. The present invention thus provides isolated nucleic acid molecules having a polynucleotide sequence encoding any of the Hu-Asp polypeptides of the invention, wherein said polynucleotide sequence encodes a Hu-Asp polypeptide having the complete amino acid sequence of SEQ ID NO:2, SEQ ID NOA, SEQ ID NO:6, or fragments thereof.

Also provided herein are purified Hu-Asp polypeptides, both recombinant and non-recombinant. Most importantly, methods to produce Hu-Asp2 polypeptides in active form are provided. These include production of HtiAsp2 polypeptides and variants thereof in bacterial cells, insect cells, and mammalian cells, also in forms that allow secretion of the Hu-Asp2 polypeptide from bacterial, insect or mammalian cells into the culture medium, also methods to produce variants of Hti-Asp2 polypeptide incorporating amino acid tags that facilitate subsequent purification. In a preferred embodiment of the invention the Hu-Asp2 polypeptide is converted to a proteolytically active form either in transformed cells or after purification and cleavage by a second protease in a cell-free system, such active forms of the Hti-Asp2 polypeptide beginning with the N-terminal sequence TQEIGIR or ETDEEP. The sequence TQHG1R represents the an-iino-tern-iinus of Asp2(a) or Asp2(b) beginning with residue 22 of SEQ ID NO: 4 or 6, after cleavage of a putative 21 residue signal peptide. Recombinant Asp2(a) expressed in and purified from insect cells was observed to have this arnino terminus, presumably as a result of cleavage by a signal peptidase. The sequence ETDEEP represents the amino-tem-iinus of Asp2(a) or Asp2(b) beginning with residue 46 of SEQ ID NO: 4 or 6, as observed when Asp2(a) has been recombinantly produced in CHO cells (presumably after cleavage by both a rodent signal peptidase and another rodent peptidase that removes a propeptide sequence). The Asp2(a) produced in the CHO cells possesses P-secretase activity, as described in greater detail in Examples 11 and 12. Variants and derivatives, including fragments, of Hu- Asp proteins having the native amino acid sequences given in SEQ ID Nos: 2, 4, and 6 that retain any of the biological activities of Hu-Asp are also within the scope of the present invention. Of course, one of ordinary skill in the art will readily be able to determine whether a variant, derivative, or fragment of a Hu-Asp protein displays Hu-Asp activity by subjecting the variant, derivative, or fragment to a standard aspartyl protease assay. Fragments of Hu-Asp within the scope of this invention include those that contain the active site domain containing the amino acid sequence DTG, fragments that contain the active site domain amino acid sequence DSG, fragments containing both the DW and DSG active site sequences, fragments in which the spacing of the DTG and DSG active site sequences has been lengthened, fragments in which the spacing has been shortened. Also within the scope of the invention are fragments of Hu-Asp in which the transmembrane domain has been removed to allow production of Hti-Asp2 in a soluble form. In another embodiment of the invention, the two halves of Hu-Asp2, each containing a single active site DW or DSG sequence can be produced independently as recombinant polypeptides, then combined in solution where they reconstitute an active protease.

Thus, the invention provides a purified polypeptide comprising a fragment of a mammalian Asp2 protein, wherein said fragment lacks the Asp2 transmembrane domain of said Asp2 protein, and wherein the polypeptide and the fragment retain the P-secretase activity of said mammalian Asp2 protein. In a preferred embodiment, the purified polypeptide comprises a fragment of a human Asp2 protein that retains the Psecretase activity of the human Asp2 protein from which it was derived. Examples include: a purified polypeptide that comprises a fragment of Asp2(a) having the amino acid sequence set forth in SEQ ID NO: 4, wherein the polypeptide lacks transmembrane domain an-iino acids 455 to 477 of SEQ ID NO: 4; a purified polypeptide as described in the preceding paragraph that further lacks cytoplasmic domain amino acids 478 to 501 of SEQ ID NO: 4; a purified polypeptide as described in either of the preceding paragraphs that further lacks arnino acids 420-454 of SEQ ID NO: 4, which constitute a putative alpha helical region between the catalytic domain and the transmembrane domain that is believed to be unnecessary for P-secretase activity; a purified polypeptide that comprises an amino acid sequence that includes amino acids 58 to 419 of SEQ ID NO: 4, and that lacks amino acids 22 to 57 of SEQ ID NO: 4; a purified polypeptide that comprises an amino acid sequence that includes amino acids 46 to 419 of SEQ ID NO: 4, and that lacks amino acids 22 to 45 of SEQ ID NO: 4; a purified polypeptide that comprises an amino acid sequence that includes amino acids 22 to 454 of SEQ ID NO: 4. a purified polypeptide that comprises a fragment of Asp2(b) having the amino acid sequence set forth in SEQ ID NO: 6, and wherein said polypeptide lacks transmembrane domain amino acids 430 to 452 of SEQ ID NO: 6; a purified polypeptide as described in the preceding paragraph that further lacks cytoplasmic domain an-fino acids 453 to 476 of SEQ ID NO: 6; a purified polypeptide as described in either of the preceding two paragraphs that further lacks amino acids 395- 429 of SEQ ID NO: 4, which constitute a putative alpha helical region between the catalytic domain and the transmembrane domain that is believed to be unnecessary for 0-secretase activity; a purified polypeptide comprising an amino acid sequence that includes amino acids 58 to 394 of SEQ ID NO: 4, and that lacks amino acids 22 to 57 of SEQ ID NO: 4; a purified polypeptide comprising an an-fino acid sequence that includes amino acids 46 to 394 of SEQ ID NO: 4, and that lacks amino acids 22 to 45 of SEQ ID NO: 4; and a purified polypeptide comprising an amino acid sequence that includes amino acids 22 to 429 of SEQ ID NO: 4. Also included as part of the invention is a purified polynucleotide comprising a nucleotide sequence that encodes such polypeptides; a vector comprising a polynucleotide that encodes such polypeptides; and a host cell transformed or transfected with such a polynucleotide or vector. Hu-Asp variants may be obtained by mutation of native Hu-Asp-encoding nucleotide sequences, for example. A Hu-Asp variant, as referred to herein, is a polypeptide substantially homologous to a native Hu-Asp polypeptide but which has an an-iino acid sequence different from that of native Hu-Asp because of one or more deletions, insertions, or substitutions in the amino acid sequence. The variant amino acid or nucleotide sequence is preferably at least about 80% identical, more preferably at least about 90% identical,and most preferably at least about 95% identical, to a native Hu-Asp sequence. Thus, a variant nucleotide sequence which contains, for example, 5 point mutations for every one hundred nucleotides, as compared to a native Hu-Asp gene, will be 95% identical to the native protein. The percentage of sequence identity, also termed homology, between a native and a variant Hu-Asp sequence may also be determined, for example, by comparing the two sequences using any of the computer programs commonly employed for this purpose, such as the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wisconsin), which uses the algorithm of Smith and Waterman (Adv. Appl. Math. 2: 482- 489 (198 1)). Alterations of the native amino acid sequence may be accomplished by any of a number of known techniques. For example, mutations may be introduced at particular locations by procedures well known to the skilled artisan, such as oligonucleotide-directed mutagenesis, which is described by Walder et al. (Gene 42:133 (1986)); Bauer et al. (Gene 37:73 (1985)); Craik (BioTechniques, January 1985, pp. 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press (1981)); and U.S. Patent Nos. 4,518,584 and 4,737, 462.

Hu-Asp variants within the scope of the invention may comprise conservatively substituted sequences, meaning that one or more amino acid residues of a Hu-Asp polypeptide are replaced by different residues that do not alter the secondary and/or tertiary structure of the Hu-Asp polypeptide. Such substitutions may include the replacement of an amino acid by a residue having similar physicochernical properties, such as substituting one aliphatic residue (I1e, Val, Leu or Ala) for another, or substitution between basic residues Lys and Arg, acidic residues Glu and Asp, amide residues G1n and Asn, hydroxyl residues Ser and Tyr, or aromatic residues Phe and Tyr. Further information regarding making phenotypically silent amino acid exchanges may be found in Bowie et al., Science 247:1306-1310 (1990). Other Hu-Asp variants which might retain substantially the biological activities of Hu-Asp are those where amino acid substitutions have been made in areas outside functional regions of the protein.

In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynueleotide which hybridizes under stringent conditions to a portion of the nucleic acid molecules described above, e. g., to at least about 15 nucleotides, preferably to at least about 20 nucleotides, more preferably to at least about 30 nucleotides, and still more preferably to at least about from 30 to at least about 100 nucleotides, of one of the previously described nucleic acid molecules. Such portions of nucleic acid molecules having the described lengths refer to, e.g., at least about 15 contiguous nucleotides of the reference nucleic acid molecule. By stringent hybridization conditions is intended overnight incubation at about 421'C for about 2.5 hours in 6 X SSC/0. 1 % SDS, followed by washing of the filters four times for 15 minutes in 1.0 X SSC at 650C, 0. 1 % SDS.

Fragments of the Hu-Asp encoding nucleic acid molecules described herein, as well as polynucleotides capable of hybridizing to such nucleic acid molecules may be used as a probe or as primers in a polymerase chain reaction (PCR). Such probes may be used, e.g., to detect the presence of Hu-Asp nucleic acids in in vitro assays, as well as in Southern and northern blots. Cell types expressing Hu-Asp may also be identified by the use of such probes. Such procedures are well known, and the skilled artisan will be able to choose a probe of a length suitable to the particular application. For PCR, 5'and Yprimers corresponding to the termini of a desired Hu-Asp nucleic acid molecule are employed to isolate and amplify that sequence using conventional techniques.

Other useful fragments of the Hu-Asp nucleic acid molecules are antisense or sense oligonucleotides comprising a single stranded nucleic acid sequence capable of binding to a target Hu-Asp mRNA (using a sense strand), or Hu-Asp DNA (using an antisense strand) sequence. In a preferred embodiment of the invention these Hu-Asp antisense oligonucleotides reduce Hu-Asp mRNA and consequent production of Hu-Asp polypeptides.

In another aspect, the invention includes Hu-Asp polypeptides with or without associated native pattern glycosylation. Both Hu-Asp I and HuAsp2 have canonical acceptor sites for Asn-linked sugars, with Hu-Asp l having two of such sites, and Hti-Asp2 having four. Hu-Asp expressed in yeast or mammalian expression systems (discussed below) may be similar to or significantly different from a native Hu-Asp polypeptide in molecular weight and glycosylation pattern. Expression of Hu-Asp in bacterial expression systems will provide non-glycosylated Hu-Asp.

The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. Hu-Asp polypeptides may be recovered and purified from tissues, cultured cells, or recombinant cell cultures by well-known methods, including ammonium sulfate or ethanol precipitation, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, and high performance liquid chromatography (HPLC). In a preferred embodiment, an amino acid tag is added to the Hu-Asp polypeptide using genetic engineering techniques that are well known to practitioners of the art which include addition of six histidine amino acid residues to allow purification by binding to nickel immobilized on a suitable support, epitopes for polyclonal or monoclonal antibodies including but not limited to the T7 epitope, the myc epitope, and the V5a epitope, and fusion of Hu-Asp2 to suitable protein partners including but not limited to glutathione-S- transferase or maltose binding protein. In a preferred embodiment these additional arnino acid sequences are added to the C-ten-ninus of Hu-Asp but may be added to the N-terminus or at intervening positions within the Hu-Asp2 polypeptide.

The present invention also relates to vectors comprising the polynucleotide molecules of the invention, as well as host cell transformed with such vectors. Any of the polynucleotide molecules of the invention may be joined to a vector, which generally includes a selectable marker and an origin of replication, for propagation in a host. Because the invention also provides Hu-Asp polypeptides expressed from the polynucleotide molecules described above, vectors for the expression of Hu-Asp are preferred. The vectors include DNA encoding any of the Hu- Asp polypeptides described above or below, operably linked to suitable transcriptional or translational regulatory sequences, such as those derived from a mammalian, microbial, viral, or insect gene. Examples of regulatory sequences include transcriptional promoters, operators. or enhancers, mRNA ribosornal binding sites, and appropriate sequences which control transcription and translation. Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA encoding Hu-Asp. Thus, a promoter nucleotide sequence is operably linked to a Hu-Asp DNA sequence if the promoter nucleotide sequence directs the transcription of the Hu-Asp sequence.

Selection of suitable vectors to be used for the cloning of polynucleotide molecules encoding Hu-Asp, or for the expression of Hu-Asp polypeptides, will of course depend upon the host cell in which the vector will be transformed, and, where applicable, the host cell from which the Hu-Asp polypeptide is to be expressed. Suitable host cells for expression of Hu-Asp polypeptides include prokaryotes, yeast, and higher eukaryotic cells, each of which is discussed below.

The Hu-Asp polypeptides to be expressed in such host cells may also be fusion proteins which include regions from heterologous proteins. Such regions may be included to allow, e.g., secretion, improved stability, or facilitated purification of the polypeptide. For example, a sequence encoding an appropriate signal peptide can be incorporated into expression vectors. A DNA sequence for a signal peptide (secretory leader) may be fused inframe to the Hu-Asp sequence so that Hu-Asp is translated as a fusion protein comprising the signal peptide. A signal peptide that is functional in the intended host cell promotes extracellular secretion of the Hu-Asp polypeptide. Preferably, the signal sequence will be cleaved from the Hu-Asp polypeptide upon secretion of Hu-Asp from the cell. Nonlimiting examples of signal sequences that can be used in practicing the invention include the yeast Ifactor and the honeybee melatin leader in sf9 insect cells.

In a preferred embodiment, the Hu-Asp polypeptide will be a fusion protein which includes a heterologous region used to facilitate purification of the polypeptide. Many of the available peptides used for such a function allow selective binding of the fusion protein to a binding partner. For example, the Hu-Asp polypeptide may be modified to comprise a peptide to form a fusion protein which specifically binds to a binding partner, or peptide tag. Nonlimiting examples of such peptide tags include the 6-His tag, thioredoxin tag, hemaglutinin tag, GST tag, and OmpA signal sequence tag. As will be understood by one of skill in the art, the binding partner which recognizes and binds to the peptide may be any molecule or compound including metal ions (e.g., metal affinity columns), antibodies, or fragments thereof, and any protein or peptide which binds the peptide, such as the FLAG tag.

Suitable host cells for expression of Hu-Asp polypeptides includes prokaryotes, yeast, and higher eukaryotic cells. Suitable prokaryotic hosts to be used for the expression of Hu-Asp include bacteria of the genera Escherichia, Bacillus, and Salmonella, as well as members of the genera Pseudomonas, Streptomyces, and Staphylococcus. For expression in, e.g., E. coli, a Hu-Asp polypeptide may include an N-tern-iinal methionine residue to facilitate expression of the recombinant polypeptide in a prokaryotic host. The N-terminal Met may optionally then be cleaved from the expressed Hu-Asp polypeptide. Other N-terminal amino acid residues, can be added to the Hu-Asp polypeptide to facilitate expression in Escherichia coli including but not limited to the T7 leader sequence, the T7-caspase 8 leader sequence, as well as others leaders including tags for purification such as the 6-His tag (Example 9). Hu-Asp polypeptides expressed in E. coli may be shortened by removal of the cytoplasmic tail, the transmembrane domain, or the membrane proximal region. Hu-Asp polypeptides expressed in E. coli may be obtained in either a soluble form or as an insoluble form which may or may not be present as an inclusion body. The insoluble polypeptide may be rendered soluble by guanidine HG, urea or other protein denaturants, then refolded into a soluble form before or after purification by dilution or dialysis into a suitable aqueous buffer. If the inactive proform of the Hu-Asp was produced using recombinant methods, it may be rendered active by cleaving off the prosegment with a second suitable protease such as human immunodeficiency virus protease.

Expression vectors for use in prokaryotic hosts generally comprises one or more phenotypic selectable marker genes. Such genes generally encode, e.g., a protein that confers antibiotic resistance or that supplies an auxotrophic requirement. A wide variety of such vectors are readily available from commercial sources. Examples include pSPORT vectors, pGEM vectors (Promega), pPROEX vectors (LTI, Bethesda, MD), Bluescript vectors (Stratagene), pET vectors (Novagen) and pQE vectors (Qiagen).

Hu-Asp may also be expressed in yeast host cells from genera including Saccharomyces, Pichia, and Kluveromyces. Preferred yeast hosts are S. cerevisiae and P. pastoris. Yeast vectors will often contain an origin of replication sequence from a 2T yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Vectors replicable in both yeast and E. coli (termed shuttle vectors) may also be used. In addition to the above-mentioned features of yeast vectors, a shuttle vector will also include sequences for replication and selection in E. coli. Direct secretion of Hu-Asp polypeptides expressed in yeast hosts may be accomplished by the inclusion of nucleotide sequence encoding the yeast I-factor leader sequence at the Yend of the Hu-Asp-encoding nucleotide sequence.

Insect host cell culture systems may also be used for the expression of Hu-Asp polypeptides. In a preferred embodiment, the Hu-Asp polypeptides of the invention are expressed using an insect cell expression system (see Example 10). Additionally, a baculovirus expression system can be used for expression in insect cells as reviewed by Luckow and Summers, BiolTechnology 6:47 (1988).

In another preferred embodiment, the Flu-Asp polypeptide is expressed in mammalian host cells. Nonlimiting examples of suitable mammalian cell lines include the COS7 line of monkey kidney cells (Gluzman et al., Cell 23:175 (198 1)), human embyonic kidney cell line 293, and Chinese hamster ovary (CHO) cells. Preferably, Chinese hamster ovary (CHO) cells are used for expression of Hu-Asp proteins (Example 11).

The choice of a suitable expression vector for expression of the Hu-Asp polypeptides of the invention will of course depend upon the specific mammalian host cell to be used, and is within the skill of the ordinary artisan. Examples of suitable expression vectors include pcDNA3 (Invitrogen) and pSVL (Pharmacia Biotech). A preferred vector for expression of Hu-Asp polypeptides is pcDNA3. 1 -Hygro (Invitrogen). Expression vectors for use in mammalian host cells may include transcriptional and translational control sequences derived from viral genomes. Commonly used promoter sequences and enhancer sequences which may be used in the present invention include, but are not limited to, those derived from human cytomegalovirus (CMV), Adenovirus 2, Polyoma virus, and Simian virus 40 (SV40). Methods for the construction of mammalian expression vectors are disclosed, for example, in Okayama and Berg (Mol. Cell. Biol. 3:280 (1983)); Cosman et al. (Mol. Immunol. 23:935 (1986)); Cosman et al. (Nature 312:768 (1984)); EP-A-0367566; and WO 91118982.

The polypeptides of the present invention may also be used to raise polyclonal and monoclonal antibodies, which are useful in diagnostic assays for detecting Hu-Asp polypeptide expression. Such antibodies may be prepared by conventional techniques. See, for example, Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988); Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980). Synthetic peptides comprising portions of Hu-Asp containing 5 to 20 amino acids may also be used for the production of polyclonal or monoclonal antibodies after linkage to a suitable carrier protein including but not limited to keyhole limpet hemacyanin (KIH), chicken ovalbumin, or bovine serum albumin using various cross-linking reagents including carbodin-lides, glutaraldehyde, or if the peptide contains a eysteine, N-methylmaleimide. A preferred peptide for immunization when conjugated to KLH contains the C-tenninus of Hu-Asp I or Hu-Asp2 comprising QRRPRDPEVVNDESSLVM (SEQ ID NO: 2, residues 497-518) or LRQQHDDFADDISLLK (SEQ ID NO:4, residues 486- 501), respectively. See SEQ ID Nos. 33-34.

The Hu-Asp nucleic acid molecules of the present invention are also valuable for chromosome identification, as they can hybridize with a specific location on a human chromosome. Hu-Aspl has been localized to chromosome 21, while Hti-Asp2 has been localized to chromosome 11 q23.324. 1. There is a current need for identifying particular sites on the chromosome, as few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location. Once a. sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. The relationship between genes and diseases that have been mapped to the same chromosomal region can then be identified through linkage analysis, wherein the coinheritance of physically adjacent genes is determined. Whether a gene appearing to be related to a particular disease is in fact the cause of the disease can then be determined by comparing the nucleic acid sequence between affected and unaffected individuals.

In another embodiment, the invention relates to a method of assaying HuAsp function, specifically Hti-Asp2 function which involves incubating in solution the Hu-Asp polypeptide with a suitable substrate including but not limited to a synthetic peptide containing the P-secretase cleavage site of APP, preferably one containing the mutation found in a Swedish kindred with inherited AD in which KM is changed to NL, such peptide comprising the sequence SEVNLDAEFR in an acidic buffering solution, preferably an acidic buffering solution of pH5. 5 (see Example 12) using cleavage of the peptide monitored by high performance liquid chromatography as a measure of Hu-Asp proteolytic activity. Preferred assays for proteolytic activity utilize internally quenched peptide assay substrates. Such suitable substrates include peptides which have attached a paired flurophore and quencher including but not limited to 7-amino-4-methyl cournarin and dinitrophenol, respectively, such that cleavage of the peptide by the Hu-Asp results in increased fluorescence due to physical separation of the flurophore and quencher. Other paired flurophores and quenchers include bodipy- tetramethylrhodamine and QSY-5 (Molecular Probes, Inc.). In a variant of this assay, biotin or another suitable tag may be placed on one end of the peptide to anchor the peptide to a substrate assay plate and a flurophore may be placed at the other end of the peptide. Useful flurophores include those listed above as well as Europium labels such as W8044 (EG&g Wallac, Inc.). Cleavage of the peptide by Asp2 will release the flurophore or other tag from the plate, allowing compounds to be assayed for inhibition of Asp2 proteolytic cleavage as shown by an increase in retained fluorescence. Preferred colorimetric assays of Hu- Asp proteolytic activity utilize other suitable substrates that include the P2 and P I amino acids comprising the recognition site for cleavage linked to o-nitrophenol through an amide linkage, such that cleavage by the Hu-Asp results in an increase in optical density after altering the assay buffer to alkaline pH.

In another embodiment, the invention relates to a method for the identification of an agent that increases the activity of a Hu-Asp polypeptide selected from the group consisting of Hu-Asp l, Hu-Asp2(a), and Hu-Asp2(b), the method comprising (a) determining the activity of said Hu-Asp, polypeptide in the presence of a test agent and in the absence of a test agent; and (b) comparing the activity of said Hu-Asp polypeptide determined in the presence of said test agent to the activity of said Hu-Asp polypeptide determined in the absence of said test agent; whereby a higher level of activity in the presence of said test agent than in the absence of said test agent indicates that said test agent has increased the activity of said Hu-Asp polypeptide. Such tests can be performed with Hu-Asp polypeptide in a cell free system and with cultured cells that express Hu-Asp as well as variants or isoforms thereof.

In another embodiment, the invention relates to a method for the identification of an agent that decreases the activity of a Hu-Asp polypeptide selected from the group consisting of Hu-Asp l, Hu-Asp2(a), and Hu-Asp2(b), the method comprising (a) determining the activity of said Hu-Asp polypeptide in the presence of a test agent and in the absence of a test agent; and (b) comparing the activity of said Hu-Asp polypeptide determined in the presence of said test agent to the activity of said Hu-Asp polypeptide determined in the absence of said test agent; whereby a lower level of activity in the presence of said test agent than in the absence of said test agent indicates that said test agent has decreased the activity of said Hu-Asp polypeptide. Such tests can be performed with Hu-Asp polypeptide in a cell free system and with cultured cells that express Hu- Asp as well as variants or isoforms thereof.

In another embodiment, the invention relates to a novel cell line (HEK125. 3 cells) for measuring processing of arnyloid P peptide (AP) from the arnyloid protein precursor (APP). The cells are stable transformants of human embryonic kidney 293 cells (HEK293) with a bicistronic vector derived from pWS-EGFP (Clontech) containing a modified human APP cDNA, an internal ribosorne entry site and an enhanced green fluorescent protein (EGFP) cDNA in the second cistron. The APP eDNA was modified by adding two lysine codons to the carboxyl terminus of the APP coding sequence. This increases processing of AP peptide from human APP by 2-4 fold. This level of AP peptide processing is 60 fold higher than is seen in nontransformed HEK293 cells. HEK125.3 cells will be useful for assays of compounds that inhibit AP peptide processing. This invention also includes addition of two lysine residues to the C-terminus of other APP isoforms including the 751 and 770 amino acid isoforms, to isoforms of APP having mutations found in human AD including the Swedish KM--->NL and V717---)Fmutations, to C-terminal fragments of APP, such as those beginning with the P-secretase cleavage site, to C-terminal fragments of APP containing the 0-secretase cleavage site which have been operably linked to an N-terminal signal peptide for membrane insertion and secretion, and to C-tenninal fragments of APP which have been operably linked to an N-terminal signal peptide for membrane insertion and secretion and a reporter sequence including but not limited to green fluorescent protein or alkaline phosphatase, such that 0-secretase cleavage releases the reporter protein from the surface of cells expressing the polypeptide.

Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.

Example 1

Development of a Search Algorithm Useful for the Identification of Aspartyl Proteases, and Identification of C. elegans Aspartyl Protease Genes in Wormpep 12 Materials and Methods: Classical aspartyl proteases such as pepsin and renin possess a two-domain structure which folds to bring two aspartyl residues into proximity within the active site. These are embedded in the short tripeptide motif DTG, or more rarely, DSG. The DW or DSG active site motif appears at about residue 25-30 in the enzyme, but at about 65-70 in the proenzyme (prorenin, pepsinogen). This motif appears again about 150- 200 residues downstream. The proenzyme is activated by cleavage of the N- terminal prodomain. This pattern exemplifies the double domain structure of the modem day aspartyl enzymes which apparently arose by gene duplication and divergence. Thus; NH2 ------------ X ------ D 2'TG --------------- Y ------- Dy+25TG --------------- C where X denotes the beginning of the enzyme, following the Nterminal prodomain, and Y denotes the center of the molecule where the gene repeat begins again.

In the case of the retroviral enzymes such as the HIV protease, they represent only a half of the two-domain structures of well-known enzymes like pepsin, cathepsin D, renin, etc. They have no prosegment, but are carved out of a polyprotein precursor containing the gag and pol proteins of the virus. They can be represented by:

N112 --------- D25TG --------------------- C100 This "monomer" only has about 100 aa, so is extremely parsimonious as compared to the other aspartyl protease "dimers" which have of the order of 330 or so aa, not counting the N-terniinal prodomain. The limited length of the eukaryotic aspartyl protease active site motif makes it difficult to search EST collections for novel sequences. EST sequences typically average 250 nucleotides, and so in this case would be unlikely to span both aspartyl protease active site motifs. Instead, we turned to the C. elegans genome. The C. elegans genome is estimated to contain around 13,000 genes. Of these, roughly 12,000 have been sequenced and the corresponding hypothetical open reading frame (ORF) has been placed in the database Wormpep12. We used this database as the basis for a whole genome scan of a higher eukaryote for novel aspartyl proteases, using an algorithm that we developed specifically for this purpose. The following AWK script for locating proteins containing two DTG or DSG motifs was used for the search, which was repeated four times to recover all pairwise combinations of the aspartyl motif.

BEGINIRS=">'} P defines ">" as record separator for FASTA format 1 1 pos = index($0,"DTG") /finds 'DTG' in record/ if (POSA) 1 rest = substr($0,pos+3) /get rest of record after first DTG1 pos2 = index(rest,"DTG") Pfind second DTG1 if (pos2>0) printf ("%s%s\n",">",$0)} /report hits/ The AWK script shown above was used to search Wormpep12, which was downloaded from ftp.sanger.ac.uk/pub/databases/wormpep, for sequence entries containing at least two DW or DSG motifs. Using AWK limited each record to 3000 characters or less. Thus, 35 or so larger records were eliminated manually from Wormpep12 as in any case these were unlikely to encode aspartyl proteases. Results and Discussion:

The Wormpep 12 database contains 12,178 entries, although some of these (< 10%) represent alternatively spliced transcripts from the same gene. Estimates of the number of genes encoded in the C. elegans genome is on the order of 13,000 genes, so Wormpep 12 may be estimated to cover greater than 90% of the C. elegans genome.

Eukaryotic aspartyl proteases contain a two-domain structure, probably arising from ancestral gene duplication. Each domain contains the active site motif D(SIT)G located from 20-25 amino acid residues into each domain. The retroviral (e.g., HIV protease) or retrotransposon proteases are homodimers of subunits which are homologous to a single eukaryotic aspartyl protease domain. An AWK script was used to search the Wormpep12 database for proteins in which the D(SIT)G motif occurred at least twice. This identified >60 proteins with two DTG or DSG motifs. Visual inspection was used to select proteins in which the position of the aspartyl domains was suggestive of a twodomain structure meeting the criteria described above.

In addition, the PROSITE eukaryotic and viral aspartyl protease active site pattern PS00141 was used to search Wormpep12 for candidate aspartyl proteases. (Bairoch A., Bucher P., Hofinann K., The PROSITE database: its status in 1997, Nucleic Acids Res. 24:217-221(1997)). This generated an overlapping set of Worinpep12 sequences. Of these, seven sequences contained two DTG or DSG motifs and the PROSITE aspartyl protease active site pattern. Of these seven, three were found in the same cosmid clone (F2 1 F8.3, F2 1 F8.4, and F2 1 F8.7) suggesting that they represent a family of proteins that arose by ancestral gene duplication. Two other ORFs with extensive homology to F2 1 F8.3, F2 1 F8.4 and F2 1 F8.7 are present in the same gene cluster (F2 1 F8.2 and F2 1 F8.6), however, these contain only a single DW motiL Exhaustive BLAST searches with these seven sequences against Wormpep12 failed to reveal additional candidate aspartyl proteases in the C. elegans genome containing two repeats of the DTG or WG motif.

BLASTX search with each C. elegans sequence against SWISS-PROT, GenPep and TREMBL revealed that R 121-17.2 was the closest worm homologue to the known mammalian aspartyl proteases, and that T 1 8H9.2 was somewhat moredistantly related, while CEASP 1, F2 1 F8.3, F2 1 F8.4, and F2 1 F8.7 formed a subcluster which had the least sequence homology to the mammalian sequences. Discussion:

APP, the presenilins, and p35, the activator of cdk5, all undergo intracellular proteolytic processing at sites which conform to the substrate specificity of the HIV protease. Dysregulation of a cellular aspartyl protease with the same substrate specificity, might therefore provide a unifying mechanism for causation of the plaque and tangle pathologies in AD. Therefore, we sought to identify novel human aspartyl proteases. A whole genome scan in C. elegans identified seven open reading frames that adhere to the aspartyl protease profile that we had identified. These seven aspartyl proteases probably comprise the complete complement of such proteases in a simple, multicellular eukaryote. These include four closely related aspartyl proteases unique to C. elegans which probably arose by duplication of an ancestral gene. The other three candidate aspartyl proteases (T 181-19.2, R 12117.2 and C 11 D2.2) were found to have homology to mammalian gene sequences. Example 2 Identification of Novel Human Aspartyl Proteases Using Database Mning by Genome Bridging Materials and Methods:

Computer-assisted analysis of EST databases, cDNA, and predicted polypeptide sequences:

Exhaustive homology searches of EST databases with the CEASP 1, F2 IF8.3, F21F8.4, and F21F8.7 sequences failed to reveal any novel mammalian homologues. TBLASTN searches with R 1 2H7.2 showed homology to cathepsin D, cathepsin E, pepsinogen A, pepsinogen C and renin, particularly around the DW motif within the active site, but also failed to identify any additional novel mammalian aspartyl proteases. This indicates that the C. elegans genome probably contains only a single lysosomal aspartyl protease which in mammals is represented by a gene family that arose through duplication and consequent modification of an ancestral gene.

TBLASTN searches with T 18119.2, the remaining C. elegans sequence, identified several ESTs which assembled into a contig encoding a novel human aspartyl protease (Hu-ASPI). As is described above in Example 1, BLASTX search with the Hti-ASP1 contig against SWISS-PROT revealed that the active site motifs in the sequence aligned with the active sites of other aspartyl p'roteases. Exhaustive, repetitive rounds of BLASTN searches against LifeSeq, LifeSeqFL, and the public EST collections identified 102 EST from multiple cDNA libraries that assembled into a single contig. The 51 sequences in this contig found in public EST collections also have been assembled into a single contig (THC213329) by The Institute for Genome Research (TIGR). The TIGR annotation indicates that they failed to find any hits in the database for the contig. Note that the TIGR contig is the reverse complement of the LifeSeq contig that we assembled. BLASTN search of Hu-ASP l against the rat and mouse EST sequences in ZooSeq revealed one homologous EST in each database (Incyte clone 700311523 and IMAGE clone 313341, GenBank accession number W10530, respectively).

TBLASTN searches with the assembled DNA sequence for Hu-ASP1 against both LifeSeqFL and the public EST databases identified a second, related human sequence (Hu-Asp2) represented by a single EST (2696295). Translation of this partial cDNA sequence reveals a single DW motif which has homology to the active site motif of a bovine aspartyl protease, NM 1.

BLAST searches, contig assemblies and multiple sequence alignments were performed using the bioinformatics tools provided with the LifeSeq, LifeSeqFL and LifeSeq Assembled databases from Incyte. Predicted protein motifs were identified using either the ProSite dictionary (Motifs in GCG 9) or the Pfarn database. Full-length cDNA cloning ofHu-Aspl The open reading frame of C. elegans gene T18H9.2CE was used to query Incyte LifeSeq and LifieSeq-FL databases and a single electronic assembly referred to as 1863920CE I was detected. The 5' most cDNA clone in this contig, 1863920, was obtained from Incyte and completely sequenced on both strands. Translation of the open reading frame contained within clone 1863920 revealed the presence of the duplicated aspartyl protease active site motif (DTWSG) but the 5' end was incomplete. The remainder of the Hu-Asp I coding sequence was determined by 5' Marathon RACE analysis using a human placenta Marathon ready cDNA template (Clontech). A Y- antisense oligonucleotide primer specific for the 5' end of clone 1863920 was paired with the Y-sense primer specific for the Marathon ready cDNA synthetic adaptor in the PCR. Specific PCR products were directly sequenced by cycle sequencing and the resulting sequence assembled with the sequence of clone 1863920 to yield the complete coding sequence of Hu- Asp-1 (SEQ ID No. 1).

Several interesting features are present in the primary amino acid sequence of Hu-Asp l (Figure 1, SEQ ID No. 2). The sequence contains a signal peptide (residues 1-20 in SEQ ID No. 2), a pro-segment, and a catalytic domain containing two copies of the aspartyl protease active site motif (DTGIDSG). The spacing between the first and second active site motifs is about 200 residues which should correspond to the expected size of a single, eukaryotic aspartyl protease domain. More interestingly, the sequence contains a predicted transmembrane domain (residues 469-492 in SEQ ID No.2) near its C-terminus which suggests that the protease is anchored in the membrane. This feature is not found in any other aspartyl protease.

Cloning of afull-length Hu-Asp-2 cDNAs:

As is described above in Example 1, genome wide scan of the Caenorhabditis elegans database WorniPep 12 for putative aspartyl proteases and subsequent mining of human EST databases revealed a human ortholog to the C. elegans gene T 1 8H9.2 referred to as Hu-Asp l. The assembled contig for Hu-Asp l was used to query for human paralogs using the BLAST search tool in human EST databases and a single significant match (2696295CE1) with approximately 60% shared identity was found in the LifeSeq FL database. Similar queries of either gb105PubEST or the family of human databases available from TIGR did not identify similar EST clones. eDNA clone 2696295, identified by single pass sequence analysis from a human uterus cDNA library, was obtained from Incyte and completely sequence on both strands. This clone contained an incomplete 1266 bp open-reading frame that encoded a 422 an-iino acid polypeptide but lacked an initiator ATG on the 5' end. Inspection of the predicted sequence revealed the presence of the duplicated aspartyl protease active site motif DTWSG, separated by 194 amino acid residues. Subsequent queries of later releases of the LifeSeq EST database identified an additional ESTs, sequenced from a human astrocyte cDNA library (4386993), that appeared to contain additional 5' sequence relative to clone 2696295. Clone 4386993 was obtained from Incyte and completely sequenced on both strands. Comparative analysis of clone 4386993 and clone 2696295 confirmed that clone 4386993 extended the open-reading frame by 31 amino acid residues including two in-frairne translation initiation codons. Despite the presence of the two in-frame ATGs, no in-frame stop codon was observed upstream of the ATG indicating that the 4386993 may not be full- length. Furthermore, alignment of the sequences of clones 2696295 and 4386993 revealed a 75 base pair insertion in clone 2696295 relative to clone 4386993 that results in the insertion of 25 additional amino acid residues in 2696295. The remainder of the Hti-Asp2 coding sequence was determined by 5' Marathon RACE analysis using a human hippocampus Marathon ready cDNA template (Clontech). A Y-antisense oligonucleotide primer specific for the shared 5'-region of clones 2696295 and 4386993 was paired with the Y-sense primer specific for the Marathon ready cDNA synthetic adaptor in the PCR. Specific PCR products were directly sequenced by cycle sequencing and the resulting sequence assembled with the sequence of clones 2696295 and 4386993 to yield the complete coding sequence of Hti-Asp2(a) (SEQ ID No. 3) and Hu-Asp2(b) (SEQ ID No. 5), respectively.

Several interesting features are present in the primary amino acid sequence of Hti-Asp2(a) (Figure 2 and SEQ ID No. 4) and Hti-Asp-2(b) (Figure 3, SEQ ID No. 6). Both sequences contain a signal peptide (residues 1-21 in SEQ ID No. 4 and SEQ ID No. 6), a pro-segment, and a catalytic domain containing two copies of the aspartyl protease active site motif (DTGIDSG). The spacing between the first and second active site motifs is variable due to the 25 amino acid residue deletion in Hu-Asp-2(b) and consists of 168-versus-194 amino acid residues, for Hu-Asp2(b) and Hu-Asp-2(a), respectively. More interestingly, both sequences contains a predicted transmembrane domain (residues 455-477 in SEQ 1D No.4 and 430-452 in SEQ ID No. 6) near their C-termini which indicates that the protease is anchored in the membrane. This feature is not found in any other aspartyl protease except Hu-Asp l. Example 3 Molecular cloning of mouse Asp2 cDNA and genomic DNA. Cloning and characterization of murine Asp2 cDNA.

The murine ortholog of Hti-Asp2 was cloned using a combination of cDNA library screening, PCR, and genomic cloning. Approximately 500,000 independent clones from a mouse brain cDNA library were screened using a 32P-labeled coding sequence probe prepared from Hu-Asp2. Replicate positives were subjected to DNA sequence analysis and the longest cDNA contained the entire 3' untranslated region and 47 amino acids in the coding region. PCR amplification of the same mouse brain cDNA library with an antisense oligonucleotide primer specific for the Y-most cDNA sequence detem-iined above and a sense primer specific for the 5' region of human Asp2 sequence followed by DNA sequence analysis gave an additional 980 bp of the coding sequence. The remainder of the 5' sequence of murine Asp-2 was derived from genomic sequence (see below). Isolation and sequence analysis of the murine Asp-2 gene.

* A murine EST sequence encoding a portion of the murine Asp2 cDNA was identified in the GenBank EST database using the BLAST search tool and the Hu-Asp2 coding sequence as the query. Clone g3160898 displayed 88% shared identity to the human sequence over 352 bp. Oligonucleotide primer pairs specific for this region of murine Asp2 were then synthesized and used to amplify regions of the murine gene. Murine genomic DNA, derived from strain 129/SvJ, was amplified in the PCR (25 cycles) using various primer sets specific for murine Asp2 and the products analyzed by agarose gel electrophoresis. The primer set Zoo-1 and Zoo-4 amplified a 750 bp fragment that contained approximately 600 bp of intron sequence based on comparison to the known cDNA sequence. This primer set was then used to screen a murine BAC library by PCR, a single genomic clone was isolated and this cloned was confirmed contain the murine Asp2 gene by DNA sequence analysis. Shotgun DNA sequencing of this Asp2 genomic clone and comparison to the cDNA sequences of both Hti-Asp2 and the partial murine cDNA sequences defined the full-length sequence of murine Asp2 (SEQ ID No. 7). The predicted amino acid sequence of murine Asp2 (SEQ ID No. 8) showed 96.4% shared identity (GCG BestFit algorithm) with 181501 amino acid residue substitutions compared to the human sequence (Figure 4). The proteolytic processing of murine Asp2(a) is believed to be analogous to the processing described above for human Asp2(a). In addition, a variant lacking an-iino acid residues 190-214 of SEQ ID NO: 8 is specifically contemplated as a murine Asp2(b) polypeptide. All forms of murine Asp2(b) gene and protein are intended as aspects of the invention. Example 4 Tissue Distribution of Expression of Hu-Asp2 Transcripts Materials and Methods:

The tissue distribution of expression of Hu-Asp-2 was determined using multiple tissue Northern blots obtained from Clontech (Palo Alto, CA). Incyte clone 2696295 in the vector pINCY was digested to completion with EcoRI1Notl and the 1.8 kb cDNA insert purified by preparative agarose gel electrophoresis. This fragment was radiolabeled to a specific activity > 1 X 109 dpmlgg by random priming in the presence of [a- 12 P-dATP1 (>3000 Ci/mmol, Amersham, Arlington Heights, lIL) and Klenow fragment of DNA polymerase 1. Nylon filters containing denatured, size fractionated poly A+ RNAs isolated from different human tissues were hybridized with 2 x 106 dpni/ml probe in ExpressHyb buffer (Clontech, Palo Alto, CA) for 1 hour at 68 'C and washed as recommended by the manufacture. Hybridization signals were visualized by autoradiography using BioMax XR film (Kodak, Rochester, NY) with intensifying screens at -80 'C.

Results and Discussion:

Limited information on the tissue distribution of expression of Hu-Asp-2 transcripts was obtained from database analysis due to the relatively small number of ESTs detected using the methods described above (< 5). In an effort to gain further information on the expression of the Hu-Asp2 gene, Northern analysis was employed to determine both the size(s) and abundance of Hu-Asp2 transcripts. PolyA' RNAs isolated from a series of peripheral tissues and brain regions were displayed on a solid support following separation under denaturing conditions and Hu-Asp2 transcripts were visualized by high stringency hybridization to radiolabeled insert from clone 2696295. The 2696295 cDNA probe visualized a constellation of transcripts that migrated with apparent sizes of 3.0kb, 4.4 kb and 8.0 kb with the latter two transcript being the most abundant.

Across the tissues surveyed, M-Asp2 transcripts were most abundant in pancreas and brain with lower but detectable levels observed in all other tissues examined except thymus and PBLs. Given the relative abundance of Hu-Asp2 transcripts in brain, the regional expression in brain regions was also established. A similar constellation of transcript sizes were detected in all brain regions examined [cerebellum, cerebral cortex, occipital pole, frontal lobe, temporal lobe and putamen] with the highest abundance in the medulla and spinal cord. Example 5 Northern Blot Detection of HuAsp-1 and HuAsp-2 Transcripts in Human Cell Lines A variety of human cell lines were tested for their ability to produce Hu- Asp 1 and Asp2 mRNA. Human embryonic kidney (HEK-293) cells, African green monkey (Cos-7) cells, Chinese hamster ovary (CHO) cells, HELA cells, and the neuroblastorna cell line M-32 were all obtained from the ATCC. Cells were cultured in DMEE containing 10% FCS except CHO cells which were maintained in a-MEMI 10% FCS at 37 'C in 5% C02 until they were near confluence. Washed monolayers of cells (3 X 107) were lysed on the dishes and poly A' RNA extracted using the Qiagen Oligotex Direct mRNA kit. Samples containing 2 pLg of poly A' RNA from each cell line were fractionated under denaturing conditions (glyoxal-treated), transferred to a solid nylon membrane support by capillary action, and transcripts visualized by hybridization with random-primed labeled (32p) coding sequence probes derived from either Hu-Asp I or Hu-Asp2. Radioactive signals were detected by exposure to X-ray film and by image analysis with a PhosphorImager.

The Hu-Asp 1 cDNA probe visualized a similar constellation of transcripts (2.6 kb and 3.5 kb) that were previously detected is human tissues. The relative abundance detern.lin ed by quantification of the radioactive signal was Cos-7 > BEK 292 = HELA > IMR32.

The Hti-Asp2 eDNA probe also visualized a similar constellation of transcripts compared to tissue (3.0 kb, 4.4 kb, and 8.0 kb) with the following relative abundance; HEK 293 > Cos 7 > IN4R32 > HELA.

Example 6

Modification of APP to increase AP processing for in vitro screening Human cell lines that process AP peptide ftom APP provide a means to screen in cellular assays for inhibitors of P- and y-secretase. Production and release of AP peptide into the culture supernatant is monitored by an enzyme-linked immunosorbent assay (EIA). Although expression of APP is widespread and both neural and non-neuronal cell lines process and release AP peptide, levels of endogenous APP processing are low and difficult to detect by EIA. AP processing can be increased by expressing in transformed cell lines mutations of APP that enhance AP processing. We made the serendipitous observation that addition of two lysine residues to the carboxyl terminus of APP695 increases AP processing still further. This allowed us to create a transformed cell line that releases AP peptide into the culture medium at the remarkable level of 20,000 pg/n-d. Materials And Methods Materials:

Human embryonic kidney cell line 293 (HEK293 cells) were obtained internally. The vector pIRES-EGFP was purchased from Clontech. Oligonucleotides for mutation using the polymerase chain reaction (PCR) were purchased from Genosys. A plasmid containing human APP695 (SEQ ID No. 9 [nucleotidel and SEQ ID No. 10 [arnino acid]) was obtained from Northwestern University Medical School. This was subcloned into pSK (Stratagene) at the Notl site creating the plasmid pAPP695. Mutagenesis protocol..

The Swedish mutation (K670N, M67 1 L) was introduced into pAPP695 using the Stratagene Quick Change Mutagenesis Kit to create the plasrnid pAPP695NL (SEQ ID No. 11 [nucleotide] and SEQ ID No. 12 [amino acid]). To introduce a di-lysine motif at the C-terminus of APP695, the forward primer #276 5' GACTGACCACWGACCAGGTTC (SEQ ID No. 47) was used with the "patch" primer #274 5' CGAATTAAATTCCAGCACACTGGCTACTTCTTGTTCTGCATCTCAAAGAAC (SEQ ID No. 48) and the flanking primer #275 CGAATTAAATTCCAGCACACTGGCTA (SEQ ID No. 49) to modify the 3' end of the APP695 cDNA (SEQ ID No. 15 [nucleotidel and SEQ H) No. 16 [amino aciffl). This also added a BstXl restriction site that will be compatible with the BstXl site in the multiple cloning site of pIRES-EWP. PCR amplification was performed with a Clontech HF Advantage cDNA PCR kit using the polymerase mix and buffers supplied by the manufacturer. For "patch" PCR, the patch primer was used at 1120th the molar concentration of the flanking primers. PCR amplification products were purified using a QlAquick PCR purification kit (Qiagen). After digestion with restriction enzymes, products were separated on 0.8% agarose gels and then excised DNA fragments were purified using a QlAquick gel extraction kit (Qiagen).

To reassemble a modified APP695-Sw cDNA, the 5' Not 1 -Bg12 fragment of the APP695-Sw cDNA and the 3' Bg12-BstX 1 APP695 cDNA fragment obtained by PCR were ligated into pIRES-EGFP plasmid DNA opened at the Notl and BstX1 sites. Ligations were performed for 5 minutes at room temperature using a Rapid DNA Ligation kit (Boehringer Mannheim) and transformed into Library Efficiency DH5a Competent Cells (GibcoBRL Life Technologies). Bacterial colonies were screened for inserts by PCR amplification using primers #276 and #275. Plasmid DNA was purified for mammalian cell transfection using a Q1Aprep Spin Miniprep kit (Qiagen). The construct obtained was designated pMG125.3 (APPSW-KK, SEQ ID No. 17 [nucleotide] and SEQ ID No. 18 [an-iino acid]). Mammalian Cell Transfection:

HEK293 cells for transfection were grown to 80% confluence in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum. Cotransfections were performed using LipofectArnine (Gibco-BRL) with 3 [tg pMG125.3 DNA and 9 gg pcDNA3.1 DNA per 10 X 106 cells. Three days posttransfection, cells were passaged into medium containing G418 at a concentration of 400 gg/ml. After three days growth in selective medium, cells were sorted by their fluorescence. Clonal Selection of 125.3 cells by FACS:

Cell samples were analyzed on an EPICS Elite ESP flow cytometer (Coulter, Hialeah, FL) equipped with a 488 nni excitation line supplied by an aircooled argon laser. EGFP emission was measured through a 525 nin bandpass filter and fluorescence intensity was displayed on a 4-decade log scale after gating on viable cells as determined by forward and right angle light scatter. Single green cells were separated into each well of one 96 well plate containing growth medium without G418. After a four day recovery period, G418 was added to the medium to a final concentration of 400 gg/ml. After selection, 32% of the wells contained expanding clones. Wells with clones were expanded from the 96 well plate to a 24 well plate and then a 6 well plate with the fastest growing colonies chosen for expansion at each passage. The final cell line selected was the fastest growing of the final six passaged. This clone, designated 125.3, has been maintained in G418 at 400 ug/M1 with passage every four days into fresh medium. No loss of AO production of EGFI fluorescence has been seen over 23 passages. APEL4Analysis (Double Antibody Sandwich ELISA for hA,6 140142 Cell culture supernatants harvested 48 hours after transfection were analyzed in a standard AP EIA as follows. Human AP 1-40 or 1-42 was measured using monoclonal antibody (mAb) 6E 10 (Senetek, St. Louis, MO) and biotinylated rabbit antiserum 162 or 164 (New York State Institute for Basic Research, Staten Island, NY) in a double antibody sandwich ELISA. The capture antibody 6E 10 is specific to an epitope present on the N-terminal amino acid residues 1- 16 of hAp. The conjugated detecting antibodies 162 and 164 are specific for hAP 1-40 and 1-42, respectively. Briefly, a Nunc Maxisorp 96 well immunoplate was coated with 100 gl/well of mAb 6E 10 (5 [tg/ml) diluted in 0. 1 M carbonate-bicarbonate buffer, pH 9.6 and incubated at 4'C overnight. After washing the plate 3x with 0.0 1 M DPBS (Modified Dulbecco's Phosphate Buffered Saline (0.008M sodium phosphate, 0.002M potassium phosphate, 0. 14M sodium chloride, 0.0 1 M potassium chloride, pH 7.4) from Pierce, RockfOrd, 11) containing 0.05% of Tween-20 (DPBST), the plate was blocked for 60 ininutes with 200 gI of 10% normal sheep serum (Sigina) in 0.01M DPBS to avoid non-specific binding. Human AO 1-40 or 1- 42 standards 100 gl/well (Bachem, Torrance, CA) diluted, from a lmglml stock solution in DMSO, in culture medium was added after washing the plate, as well as 100 gl/well of sample, e.g., conditioned medium of transfected cells.

The plate was incubated for 2 hours at room temperature and 4'C overnight. The next day, after washing the plate, 100 pd/well biotinylated rabbit antiserum. 162 1:400 or 164 1:50 diluted in DPBST + 0.5% BSA was added and incubated at room temperature for 1 hour, 15 minutes. Following washes, 100 gl/well neutravidin-horseradish peroxidase (Pierce, Rockford, E) diluted 1: 10,000 in DPBST was applied and incubated for 1 hour at room temperature. After the last washes 100 pd/well of o-phenylnediamine dihydrochloride (Sigma Chemicals, St. Louis, MO) in 50m.M citric acid/10OmM sodium phosphate buffer (Sigma Chemicals, St. Louis, MO), pH 5. 0, was added as substrate and the color development was monitored at 450nm, in a kinetic microplate reader for 20 minutes using Soft max Pro software. All standards and samples were run in triplicates. The samples with absorbance values falling within the standard curve were extrapolated from the standard curves using Soft max Pro software and expressed in pglml culture medium. Results:

Addition of two lysine residues to the carboxyl terminus of APP695 greatly increases AP processing in HEK293 cells as shown by transient expression (Table 1).

Addition of the di-lysine motif to APP695 increases AP processing to that seen with the APP695 containing the Swedish mutation. Combining the dilysine motif with the Swedish mutation further increases processing by an additional 2.8 fold.

Cotransformation of HEK293 cells with pMG125.3 and pcDNA3.1 allowed dual selection of transformed cells for G418 resistance and high level expression of EGM After clonal selection by FACS, the cell line obtained, produces a remarkable 20,000 pg AP peptide per mI of culture medium after growth for 36 hours in 24 well plates. Production of AP peptide under various growth conditions is summarized in Table 2.

TABLE 1

Release of AP peptide into the culture medium 48 hours after transient transfection of HEK293 cells with the indicated vectors containing wildtype or modified APP. Values tabulated are mean + SD and P-value for pairwise comparison using Student's Mest assuming unequal variances.

APP Construct AP 1-40 peptide Fold Increase P-value (pg/M1) pIRES-EGFP vector 147+28 1.0 wt APP695 (142.3) 194+15 1.3 0.051 wt APP695-KK (124. 1) 424+34 2.8 3 x 10-5 APP695-Sw (143.3) 457+65 3.1 2 x 10-3 APP695-SwKK (125.3) 1308+98 8.9 3 x 10-4 TABLE2 Release of AP peptide from HEK 125.3 cells under various growth conditions.

Type of Culture Volume of Duration of AP 1-40 AP 1-42 Plate Medium Culture (pg1M1) (P9/M1) 24 well plate 400 ul 36 hr 28,036 1,439 Example 7

Antisense oligomer inhibition of Abeta processing in HEK125.3 cells The sequences of Hu-Asp I and HwAsp2 were provided to Sequitur, Inc (Natick, MA) for selection of targeted sequences and design of 2nd generation chimeric antisense oligomers using prorietary technology (Sequitur Ver. D Pat pending #3002). Antisense oligomers Lot# S644, S645, S646 and S647 were targeted against Aspl. Antisense oligomers Lot# S648, S649, S650 and S651 were targeted against Asp2. Control antisense oligomers Lot# S652, S653, S655, and S674 were targeted against an irrelevant gene and antisense oligomers Lot #S656, S657, S658, and S659 were targeted against a second irrelevant gene.

For transfection with the antisense oligomers, HEK125.3 cells were grown to about 50% confluence in 6 well plates in Minimal Essential Medium (MEM) supplemented with 10% fetal calf serum. A stock solution of oligofectin G (Sequitur Inc., Natick, MA) at 2 mg/mI was diluted to 50 gg/ml in serum free MEM. Separately, the antisense oligomer stock solution at 100 gM was diluted to 800 nM in Opti-MEM (G11BCO-BRL, Grand Island, NY). The diluted stocks of oligofectin G and antisense oligomer were then mixed at a ratio of 1: 1 and incubated at room temperature. After 15 minutes incubation, the reagent was diluted 10 fold into MEM containing 10% fetal calf serum and 2 mI was added to each well of the 6 well plate after first removing the old medium. After transfection, cells were grown in the continual presence of the oligofectin G/antisense oligomer. To monitor AP peptide release, 400 pil of conditioned medium was removed periodically from the culture well and replaced with fresh medium beginning 24 hours after transfection. AP peptides in the conditioned medium were assayed via immunoprecipitation and Western blotting. Data reported are from culture supematants harvested 48 hours after transfection.

The 16 different antisense oligomers obtained from Sequitur Inc. were transfected separately into HEK 125.3 cells to determine their affect on AP peptide processing. Only antisense oligomers targeted against Asp2 significantly reduced Abeta processing by HEK125.3 cells. Both AP (P40) and AP (142) were inhibited by the same degree. In Table 3, percent inhibition is calculated with respect to untransfected cells. Antisense oligomer reagents giving greater than 50% inhibition are marked with an asterisk. Of the reagents tested, 3 or 4 antisense oligomers targeted against Aspl gave an average 52% inhibition of AP(P40) processing and 47% inhibition of AP(1 -42) processing. For Asp2, 4 of 4 antisense oligomers gave greater than 50% inhibition with an average inhibition of 62% of AP(1 -40) processing and 60% for AP(1 -42) processing.

TABLE3

Inhibition of AP peptide release from HEK125.3 cells treated with antisense oligomers.

Gene Targeted Antisense Oligomer Abeta (1 -40) Abeta (1 -42) Aspl-1 S644 62% 56% Aspl-2 S645 41% 38% Aspl-3 S646 52% 46% Aspl-4 S647 6% 25% Asp2-1 S648 71% 67% Asp2-2 S649 83% 76% Asp2-3 S650 46% 50% Asp24 S651 47% 46% Conl-1 S652 13% 18% Conl-2 S653 35% 30% Conl-3 S655 9% 18% Conl-4 S674 29% 18% Con2-1 S656 12% 18% Con2-2 S657 16% 19% Con2-3 S658 8% 35% Con24 S659 3% 18% Since HEK293 cells derive from kidney, the experiment was extended to human IMR-32 neuroblastoma cells which express all three APP isoforms and which release AP peptides into conditioned medium at measurablelevels. [See Neill et al., J. NeuroSci. Res., (1994) 39: 482-93; and Asami-Odaka et al., Biochem., (1995) 34:10272-8.1 Essentially identical results were obtained in the neuroblastoma cells as the HEK293 cells. As shown in Table 3B, the pair of Asp2 antisense oligomers reduced Asp2 mRNA by roughly one-half, while the pair of reverse control oligomers lacked this effect (Table 3B).

Table 3B

Reduction of AP40 and AP42 in human neuroblastoma IMR-32 cells and mouse neuroblastoma Neuro-2A cells treated with Asp2 antisense and control oligomers as indicated. Oligomers were transfected in quadruplicate cultures. Values tabulated are normalized against cultures treated with oligofectin-G TM only (mean + SD, p<0.001 compared to reverse control oligomer).

IN4R-32 cells Neuro-2A cells Asp2 AP40 AP42 A040 AP42 mRNA Asp2AA -75% -49 + 2% -42+ -70+ -67 + 2% 14% 7% Asp2AR 0.16 -0+3% 21.26 -9+15% 1.05 Asp2-2A -39% -43 + 3% -44 +18% -61 -61 +12% +12% Asp2-2R 0.47 12.2 19.22 6.15 -8+10% Together with the reduction in Asp2 mRNA there was a concomitant reduction in the release of AP40 and AP42 peptides into the conditioned medium. Thus, Asp2 functions directly or indirectly in a human kidney and a human neuroblastoma cell line to facilitate the processing of APP into AP peptides. Molecular cloning of the mouse Asp2 cDNA revealed a high degree of homology to human (>96% arnino acid identity, see Example 3), and indeed, complete nucleotide identity at the sites targeted by the Asp2- 1 A and Asp2-2A antisense oligorners. Similar results were obtained in mouse Neuro-2a cells engineered to express APP-Sw-KK. The Asp2 antisense oligomers reduced release of AP peptides into the medium while the reverse control oligorners did not (Table 3B). Thus, the three antisense experiments with HEK293, IMR-32 and Neuro-2a cells indicate that Asp2 acts directly or indirectly to facilitate AP processing in both somatic and neural cell lines. Example 8 Demonstration of Ilti-Asp2 P-Secretase Activity in Cultured Cells Several mutations in APP associated with early onset Alzheimer's disease have been shown to alter AP peptide processing. These flank the - and C- terminal cleavage sites that release AP from APP. These cleavage sites are referred to as the 0-secretase and y-secretase cleavage sites, respectively. Cleavage of APP at the 0-secretase site creates a C- terminal fragment of APP containing 99 an-iino acids of 11, 145 daltons molecular weight. The Swedish KM->NL mutation immediately upstream of the P-secretase cleavage site causes a general increase in production of both the 1-40 and 1-42 amino acid forms of AP peptide. The London W mutation (V717 ---- >F in the APP770 isoform) has little effect on total AP peptide production, but appears to preferentially increase the percentage of the longer 1-42 amino acid form of AP peptide by affecting the choice of P-secretase cleavage site used during APP processing. Thus, we sought to detem-fine if these mutations altered the amount and type of AP peptide produced by cultured cells cotransfected with a construct directing expression of Hu-Asp2.

Two experiments were performed which demonstrate Ilu-Asp2 P-secretase activity in cultured cells. In the first experiment, treatment of HEK125. 3 cells with antisense oligomers directed against Hu-Asp2 transcripts as described in Example 7 was found to decrease the amount of the Cterminal fragment of APP created by P-secretase cleavage (M99) (Figure 9). This shows that Hu-Asp2 acts directly or indirectly to facilitate 0-secretase cleavage. In the second experiment, increased expression of Hti-Asp2 in transfected mouse Neuro2A cells is shown to increase accumulation of the CTF99 P-secretase cleavage fragment (Figure 10). This increase is seen most easily when a mutant APP-KK clone containing a C-tern-linal dilysine motif is used for transfection. A further increase is seen when Hti-Asp2 is cotransfected with APP-Sw-KK containing the Swedish mutation KM --->NL. The Swedish mutation is known to increase cleavage of APP by the P-secretase.

A second set of experiments demonstrate Hu-Asp2 facilitates 7-secretase activity in cotransfection experiments with human embryonic kidney HEK293 cells. Cotransfection of Hu-Asp2 with an APP-KK clone greatly increases production and release of soluble API-40 and API-42 peptides from HEK293 cells. There is a proportionately greater increase in the release of AP 1 -42. A further increase in production of AP 1 -42 is seen when Hu-Asp2 is cotransfected with APP-VF (SEQ ID No. 13 [nucleotidel and SEQ ID No. 14 [arnino acid]) or APP-VF-KK SEQ ID No. 19 [nucleotide] and SEQ ID No. 20 [amino acid]) clones containing the London mutation V717--->F. The V717-->Fmutation is known to alter cleavage specificity of the APP -y-secretase such that the preference for cleavage at the AP42 site is increased. Thus, Asp2 acts directly or indirectly to facilitate -f-secretase processing of APP at the P42 cleavage site. Materials Antibodies 6E 10 and 4G8 were purchased from Senetek (St. Louis, MO). Antibody 369 was obtained from the laboratory of Paul Greengard at the Rockefeller University. Antibody C8 was obtained from the laboratory of Dennis Selkoe at the Harvard Medical School and Brigham and Women's Hospital. APP Constructs used The APP constructs used for transfection experiments comprised the following APP: wild-type APP695 (SEQ ID No. 9 and No. 10) APP-Sw: APP695 containing the Swedish KM---->NL mutation (SEQ ID No. 11 and No. 12, wherein the lysine (K) at residue 595 of APP695 is changed to asparagine (N) and the methionine (M) at residue 596 of APP695 is changed to leucine (L).), APP-VR APP695 containing the London V---F mutation (SEQ ID Nos. 13 & 14) (Affected residue 717 of the APP770 isoform. corresponds with residue 642 of the APP695 isoform. Thus, APP-VF as set in SEQ H) NO: 14 comprises the APP695 sequence, wherein the valine (V) at residue 642 is changed to phenylalanine (F).) APP-KK: APP695 containing a C-terminal KK motif (SEQ H) Nos. 15 & 16), APP-Sw-KK: APP695-Sw containing a C-terminal KK motif (SEQ ID No. 17 & 18), APP-W-KK: APP695-VF containing a C-terminal KK motif (SEQ ID Nos. 19 & 20).

These were inserted into the vector pIRES-EGFP (Clontech, Palo Alto CA) between the Notl and BstXl sites using appropriate linker sequences introduced by PCR. Transfection of antisense oligomers or plasmid DNA constructs in HEK293 cells, HEK125.3 cells and Neuro-2A cells, Human embryonic kidney HEK293 cells and mouse Neuro-2a cells were transfected with expression constructs using the Lipofectamine Plus reagent from Gibco/BRL. Cells were seeded in 24 well tissue culture plates to a density of 70-80% confluence. Four wells per plate were transfected with 2 [tg DNA (3: 1, APP:cotransfectant), 8 [tl Plus reagent, and 4 gI Lipofectarnine in OptiMEM. OptiMEM was added to a total volume of 1 rril, distributed 200 gI per well and incubated 3 hours. Care was taken to hold constant the ratios of the two plasmids used for cotransfection as well as the total amount of DNA used in the transfection. The transfection media was replaced with DMEM, 10%FBS, NaPyruvate, with antibiotic/antimycotic and the cells were incubated under normal conditions (37C, 5% C02) for 48 hours. The conditioned media were removed to polypropylene tubes and stored at -80'C until assayed for the content of AP 1 -40 and AP 1 -42 by EIA as described in the preceding examples. Transfection of antisense oligomers into HEK125.3 cells was as described in Example 7. Preparation of cell extracts, Western blot protocol Cells were harvested after being transfected with plasmid DNA for about 60 hours. First, cells were transferred to 15-mI conical tube from the plate and centrifuged at 1,500 rpm for 5 minutes to remove the medium. The cell pellets were washed once with PBS. We then lysed the cells with lysis buffer (10 mM HEPES, pH 7.9,150 mM NaCI, 10% glycerol, 1 niM EGTA, 1 mM EDTA, 0. 1 mM sodium vanadate and 1 % NP-40). The lysed cell mixtures were centrifuged at 5000 rpm and the supematant was stored at -20'C as the cell extracts. Equal amounts of extracts from HEK125.3 cells transfected with the Asp2 antisense oligomers and controls were precipitated with antibody 369 that recognizes the C-tern-iinus of APP and then CTF99 was detected in the immunoprecipitate with antibody 6E 10. The experiment was repeated using C8, a second precipitating antibody that also recognizes the C-terminus of APP. For Western blot of extracts from mouse Neuro-2a cells cotransfected with HwAsp2 and APP-KK, APP-Sw-KK, APP-W-KK or APP-W, equal amounts of cell extracts were electrophoresed through 4- 10% or 10-20% Tricine gradient gels (NOVEX, San Diego, CA). Full length APP and the CTF99 0-secretase product were detected with antibody 6E 10. Results Transfection of HEK125.3 cells with Asp2-1 or Asp2-2 antisense ofigorners reduces production of the CTF P-secretase product in comparison to cells similarly transfected with control oligomers having the reverse sequence (Asp2A reverse & Asp2-2 reverse), see Figure 9. Correspondingly, cotransfection of HwAsp2 into mouse Neuro-2a cells with the APP-KK construct increased the formation of M99. (See Fig. 10.) This was further increased if HwAsp2 was coexpressed with APP-Sw-KK, a mutant form of APP containing the Swedish KM->NI, mutation that increases P-secretase processing.

Effects of Asp2 on the production of Ab peptides from endogenously expressed APP isoforms were assessed in HEK293 cells transfected with a construct expressing Asp2 or with the empty vector after selection of transformants with the antibiotic G418. AP40 production was increased in cells transformed with the Asp2 construct in comparison to those transformed with the empty vector DNA. AP40 levels in conditioned medium collected from the Asp2 transformed and control cultures was 424 45 pg /mI and 113 5 8 pg/ml, respectively (p<0.00 1). AP42 release was below the limit of detection by the EIA, while the release of sAPPa was unaffected, 112 8 ngImI versus 111 40 ngIml. This further indicates that Asp2 acts directly or indirectly to facilitate the processing and release of AP from endogenously expressed APP.

Co-transfection of Hu-Asp2 with APP has little effect on A040 production but increases A042 production above background (Table 4). Addition of the di-lysine motif to the C-terminus of APP increases AP peptide processing about two fold, although AP40 and A042 production remain quite low (352 pglml and 21 pgln-d, respectively). Cotransfection of Asp2 with APP-KK further increases both AP40 and AP42 production.

The APP V717---W mutation has been shown to increase 'Y-secretase processing at the A042 cleavage site. Cotransfection of Hu-Asp2 with the APP-W or APP-W-KK constructs increased A042 production (a two fold increase with APP-W and a four-fold increase with APP-W-KK, Table 4), but had mixed effects on AP40 production (a slight decrease with APP-W, and a two fold increase with APP-W-KK in comparison to the pcDNA cotransfection control. Thus, the effect of Asp2 on A042 production was proportionately greater leading to an increase in the ratio of AP42/total Ab. Indeed, the ratio of AP42/total AP reaches a very high value of 42% in HEK293 cells cotransfected with Hu-Asp2 and APP-W-KK. Table 4 Results of cotransfecting Hu-Asp2 or pcDNA plasmid DNA with various APP constructs containing the V717---Wmutation that modifies -y-secretase processing. Cotransfection with Asp2 consistently increases the ratio of AP42/total Ap. Values tabulated are AP peptide pg/mL pcDNA Asp2 Cotransfection Cotransfection A040 A042 AP42/Tot AP40 AP42 AP42/Tot al al APP 192+1 <4 <2% 188+40 8+10 3.9% 8 APP-W 118+1 15+19 11.5% 85+7 24+12 22.4% APPKK 352+2 21+6 5.5% 1062+101 226+4 17.5% 4 9 APP-W-K 230+3 88+24 27.7% 491+35 355+3 42% K 1 6 Example 9

Bacterial expression of human Asp2(a) Expression of recombinant HuAsp2(a) in E. coli.

Hu-Asp2(a) can be expressed in E. coli after addition of N-terminal sequences such as a T7 tag (SEQ H) No. 21 and No. 22) or a T7 tag followed by a caspase 8 leader sequence (SEQ ID No. 23 and No. 24). Alternatively, reduction of the GC content of the 5' sequence by site directed mutagenesis can be used to increase the yield of Hu-Asp2 (SEQ ID No. 25 and No. 26). In addition, Asp2(a) can be engineered with a proteolytic cleavage site (SEQ ID No. 27 and No. 28). To produce a soluble protein after expression and refolding, deletion of the transmembrane domain and cytoplasmic tail, or deletion of the membrane proximal region, transmembrane domain, and cytoplasmic tail is preferred. Any materials (vectors, host cells, etc.) and methods described herein to express Hu-Asp2(a) should in principle be equally effective for expression of Hu-Asp2(b).

Methods PCR with primers containing appropriate linker sequences was used to assemble fusions of Asp2(a) coding sequence with N-terminal sequence modifications including a T7 tag (SEQ ID Nos. 21 and 22) or a T7-caspase 8 leader (SEQ ID Nos. 23 and 24). These constructs were cloned into the expression vector pet23 a(+) [Novagenj in which a T7 promoter directs expression of a T7 tag preceding a sequence of multiple cloning sites. To clone Hti-Asp2 sequences behind the T7 leader of pet23a+, the following oligonucleotides were used for amplification of the selected Hti-Asp2(a) sequence: #553=GTGGATCCACCCAGCACGGCATCCGGCTG (SEQ ID No. 35), #554=GAAAGCTTTCATGACTCATCTGTCTGTGGAATGTTG (SEQ ID No. 36) which placed BarnHI and HindM sites flanking the Yand Yends of the insert, respectively. The Asp2(a) sequence was amplified from the full length Asp2(a) cDNA cloned into pcDNA3.1 using the Advantage-GC eDNA PCR [Clontech] following the manufacturer's supplied protocol using annealing & extension at 680C in a two-step PCR cycle for 25 cycles. The insert and vector were cut with BarnHI and HindIII, purified by electrophoresis through an agarose gel, then ligated using the Rapid DNA Ligation kit [Boerhinger Mannheim]. The ligation reaction was used to transform the E. coli strain JM109 (Promega) and colonies were picked for the purification of plasmid (Qiagen,Qiaprep minispin) and DNA sequence analysis. For inducible expression using induction with isopropyl b-Dthiogalactopyranoside (IFTG), the expression vector was transferred into E. coli strain BL21 (Statagene). Bacterial cultures were grown in LB broth in the presence of ampicillin at 100 uglirll, and induced in log phase growth at an OD600 of 0.6-1.0 with 1 mM IPTG for 4 hour at 37'C. The cell pellet was harvested by centrifugation.

To clone Hti-Asp2 sequences behind the T7 tag and caspase leader (SEQ ID Nos. 23 and 24), the construct created above containing the T7-Hti-Asp2 sequence (SEQ ID Nos. 21 and 22) was opened at the BamHl site, and then the phosphorylated caspase 8 leader oligonucleotides #559=GATCGATGACTATCTCTGACTCTCCGCGTGAACAGGACG (SEQ ID No. 37), #560-GATCCGTCCTGTTCACGCGGAGAGTCAGAGATAGTCATC (SEQ ID No. 38) were annealed and ligated to the vector DNA. The Yoverhang for each set of oligonucleotides was designed such that it allowed ligation into the BamHI site but not subsequent digestion with BamHl. The ligation reaction was transformed into M 109 as above for analysis of protein expression after transfer to E. coli strain BL2 1.

In order to reduce the GC content of the Yterminus of asp2(a), a pair of antiparallel oligos, were designed to change degenerate codon bases in 15 amino acid positions from GIC to A/T (SEQ ID Nos. 25 and 26). The new nucleotide, sequence at the Yend of asp2 did not change the encoded amino acid and was chosen to optimize E. Coli expression. The sequence of the sense linker is 5' CGGCATCCGGCTGCCCCTGCGTAGCGGTCTGGGTGGTGCTCCACTGGGTCT GCGTCTGCCCCGGGAGACCGACGAA G Y(SEQ ID No. 39). The sequence of the antisense linker is: 5' CTTCGTCGGTCTCCCGGGGCAGACGCAGACCCAGTGGAGCACCACCCAGA CCGCTACGCAGGGGCAGCCGGATGCCG Y(SEQ ID No. 40). After annealing the phosphorylated linkers together in 0. 1 M NaCl- 10 mM Tris, pH 7.4 they were ligated into unique Cla 1 and Sma 1 sites in Hu-Asp2 in the vector pTAC. For inducible expression using induction with isopropyl b-Dthiogalactopyranoside (IIPTG), bacterial cultures were grown in LB broth in the presence of ampicillin at 100 ug/nil, and induced in log phase growth at an OD600 of 0.6-1.0 with 1 mM IPTG for 4 hour at 37'C. The cell pellet was harvested by centrifugation.

To create a vector in which the leader sequences can be removed by limited proteolysis with caspase 8 such that this liberates a Hu-Asp2 polypeptide beginning with the N-ten-ninal sequence GSFV (SEQ ID Nos. 27 and 28), the following procedure was followed. Two phosphorylated oligonueleotides containing the caspase, 8 cleavage site IETD, #57 1 =Y GATCGATGACTATCTCTGACTCTCCGCTGGACTCTGGTATCGAAACCGACG (SEQ ID No. 41) and #572= GATCCGTCGGTTTCGATACCAGAGTCCAGCGGAGAGTCAGAGATAGTCAT C (SEQ ID No. 42) were annealed and ligated into pET23a+ that had been opened with Bam.HI. After transformation into JMI09, the purified vector DNA was recovered and orientation of the insert was confirmed by DNA sequence analysis.

The following oligonucleotides were used for amplification of the selected Hti-Asp2(a) sequence: #573=5AAGGATCCMGTGGAGATGGTGGACAACCTG, (SEQ ID No. 43) #554=GAAAGCMCATGACTCATCTGTCTGTGGAATGTTG (SEQ ID No. 44) which placed BamHI and HindIH sites flanking the Yand Yends of the insert, respectively. The Hii-Asp2(a) sequence was amplified from the full length Hu-Asp2(a) cDNA cloned into pcDNA3.1 using the Advantage-GC cDNA PCR [Clontech] following the manufacturer's supplied protocol using annealing & extension at WC in a two-step PCR cycle for 25 cycles. The insert and vector were cut with BamHI and HindIH, purified by electrophoresis through an agarose gel, then ligated using the Rapid DNA Ligation kit [Boerhinger Mannheim]. The ligation reaction was used to transform the E. coli strain M 109 [Promegal and colonies were picked for the purification of plasmid Qiagen,Qiaprep rninispin) and DNA sequence analysis. For inducible expression using induction with isopropyl b-D- thiogalactopyranoside (IPTG), the expression vector was transferred into E. coli strain BL21 (Statagene). Bacterial cultures were grown in LB broth in the presence of ampicillin at 100 ugln-d, and induced in log phase growth at an OD600 of 0.6-1.0 with 1 mM IM for 4 hour at 37C. The cell pellet was harvested by centrifugation.

To assist purification, a 6-His tag can be introduced into any of the above constructs following the T7 leader by opening the construct at the BamHI site and then ligating in the annealed, phosphorylated oligonucleotides containing the six histidine sequence #565=GATCGCATCATCACCATCACCAM (SEQ ID No. 45), #566=GATCCATGGTGATGGTGATGATGC (SEQ ID No. 46). The Yoverhang for each set of oligonucleotides was designed such that it allowed ligation into the BarnHI site but not subsequent digestion with BamHI. Preparation of Bacterial Pellet:

36.34g of bacterial pellet representing 10.8L of growth was dispersed into a total volume of 20On-d using a 20mm tissue hornogenizer probe at 3000 to 5000 rpm in 2M KCl, 0. 1M Tris, 0.05M EDTA, 1 mM DTT. The conductivity adjusted to about 193m.Mhos with water. After the pellet was dispersed, an additional amount of the K0 solution was added, bringing the total volume to 500 n- il. This suspension was homogenized further for about 3 minutes at 5000 rpm using the same probe. The mixture was then passed through a Rannie high-pressure homogenizer at 10,000psi.

In all cases, the pellet material was carried forward, while the soluble fraction was discarded. The resultant solution was centrifuged in a GSA rotor for 1 hour at 12,500 rpm. The pellet was resuspended in the same solution (without the DTT) using the same tissue homogenizer probe at 2, 000 rpm. After homogenizing for 5 minutes at 3000 rpm, the volume was adjusted to 50OmI with the same solution, and spun for 1 hour at 12,500 rpm. The pellet was then resuspended as before, but this time the final volume was adjusted to 1.5L with the same solution prior to homogenizing for 5 minutes. After centrifuging at the same speed for 30 minutes, this procedure was repeated. The pellet was then resuspended into about 150mI of cold water, pooling the pellets from the six centrifuge tubes used in the GSA rotor. The pellet has homogenized for 5 ininutes at 3,000 rpm, volume adjusted to 250n-A with cold water, then spun for 30 minutes. Weight of the resultant pellet was 17.75g.

Summary: Lysis of bacterial pellet in KCl solution, followed by centrifugation in a GSA rotor was used to initially prepare the pellet. The same solution was then used an additional three times for resuspension/homogenization. A final water wash/homogenization was then performed to remove excess KG and EDTA. Solublization of Recombinant HuAsp2(a): A ratio of 9- 1 0m11gram of pellet was utilized for solubilizing the rHuAsp2L from the pellet previously described. 17.75g of pellet was thawed, and 150mI of 8M guanidine FICI, 5mM PME, 0. 1 % DEA, was added. 3M Tris was used to titrate the pH to 8.6. The pellet was initially resuspended into the guanidine solution using a 20 min. tissue homogenizer probe at 1000 rpm. The mixture was then stirred at 4'C for 1 hour prior to centrifugation at 12,500 rpm for 1 hour in GSA rotor. The resultant supernatant was then centrifuged for 30 ininutes at 40,000 x g in an SS-34 rotor. The final supematant was then stored at -20'C, except for 50 ml.

Immobilized Nickel Affinity Chromatography of Solubilized Recombinant HwAsp2(a):

The following solutions were utilized:

A) 6M Guanidine HCI, 0.1M NaP, pH 8.0, 0.01 M Tris, 5mM PME, 0.5mM Imidazole A') 6M Urea, 2OmM NaP, pH 6.80,50n1M NaCI B') 6M Urea, 20mM NaP, pH 6.20, 5OmM NaG, 12mM Imidazole C') 6M Urea, 20niM NaP, pH 6.80, 50mM NaCI, 30On- 1M L-nidazole Note: Buffers A' and C' were mixed at the appropriate ratios to give intermediate concentrations of Imidazole.

The 50rril of solubilized material was combined with 50M1 of buffer A prior to adding to 100- 125m]. Qiagen Ni-NTA SuperFlow (pre-equilibrated with buffer A) in a 5 X 10cm Bio-Rad econo column. This was shaken gently overnight at 4'C in the cold room.

Chromatography Steps: Drained the resultant flow through.

Washed with 5Onil buffer A (collecting into flow through fraction) Washed with 250rnI buffer A (wash 1) Washed with 250rnI buffer A (wash 2) Washed with 250rnI buffer A' Washed with 250mI buffer B' Washed with 25OmI buffer A' Eluted with 250mI 75mM lmidazole Eluted with 250mI 150mM Imidazole (150-1) Eluted with 250nil 150mM Imidazole (150-2) Eluted with 250rnI 30OmM hnidazole (300-1) Eluted with 250mI 30OmM Enidazole (300-2) Eluted with 250mI 30OmM hnidazole (300-3) Chromatography Results:

The Hu-Asp(a) eluted at 75niM Imidazole through 30OmM Imidazole. The 75MM fraction, as well as the first 150mM Iraidazole (150-1) fraction contained contaminating proteins as visualized on Coomassie Blue stained gels. Therefore, fractions 150-2 and 300-1 will be utilized for refolding experiments since they contained the greatest amount of protein as visualized on a Coomassie Blue stained gel.

Refolding Experiments of Recombinant Hu-Asp2(a):

Ex periment 1:

Forty m]. of 150-2 was spiked with 1 M DTT, 3M Tris, pH 7.4 and DEA to a final concentration of 6mM, 50mM, and 0. 1 % respectively. This was diluted suddenly (while stirring) with 20OmI of (4'C) cold 20mM NaP, pH 6. 8, 150mM NaCI. This dilution gave a final Urea concentration of I M. This solution remained clear, even if allowed to set open to the air at room temperature (RT) or at 4'C. After setting open to the air for 4-5 hours at 4'C, this solution was then dialyzed overnight against 20 niM NaP, pH 7.4,150 mM NaCI, 20% glycerol. This method effectively removes the urea in the solution without precipitation of the protein.

Ex periment 2:

Some of the 150-2 eluate was concentrated 2x on an Amicon Centriprep, 10, 000 MWCO, then treated as in Experiment 1. This material also stayed in solution, with no visible precipitation.

Experiment 3:

89mI of the 150-2 eluate was spiked with 1M DTT, 3M Tris, pH 7.4 and DEA to a final concentration of 6niM, 50mM, and 0. 1 % respectively. This was diluted suddenly (while stirring) with 445 mI of (4'C) cold 20 mM NaP, pH 6.8, 150 MM NaCI. This solution appeared clear, with no apparent precipitation. The solution was removed to RT and stirred for 10 minutes prior to adding MEA to a final concentration of 0. 1 mM. This was stirred slowly at RT for 1 hour. Cystarnine and CUS04 were then added to final concentrations of 1 mM and 10 PM respectively. The solution was stirred slowly at RT for 10 minutes prior to being moved to the 4'C cold room and shaken slowly overnight, open to the air.

The following day, the solution (still clear, with no apparent precipitation) was centrifuged at 100,000 x g for 1 hour. Supematants from multiple runs were pooled, and the bulk of the stabilized protein was dialyzed against 20mM NaP, pH 7.4, 150 mM NaCI, 20% glycerol. After dialysis, the material was stored at -20'C.

Some (about 10 nil) of the protein solution (still in 1M Urea) was saved back for biochemical analyses, and frozen at -20'C for storage. Example 10 Expression of Hu-Asp2 and Derivatives in Insect Cells Any materials (vectors, host cells, etc.) and methods that are useful to express Hu-Asp2(a) should in principle be equally effective for expression of HwAsp2(b). Expression by baculovirus infection.

The coding sequence of Hti-Asp2(a) and Hti-ASp2(b) and several derivatives were engineered for expression in insect cells using the PCR. For the full-length sequence, a Y-sense oligonucleotide primer that modified the translation initiation site to fit the Kozak consensus sequence was paired with a Y-antisense, primer that contains the natural translation terniination codon in the Hu-Asp2 sequence. PCR amplification of the pcDNA3. 1 (hygro)/Hu-Asp2(a) template was used to prepare two derivatives of Hti-Asp2(a) or Hu-Asp(b) that delete the C-terminal transmembrane domain (SEQ H) Nos. 29-30 and 50-51, respectively) or delete the transmembrane domain and introduce a hexa-histidine tag at the C-terminus (SEQ ID Nos. 31-32 and 52-53) respectively, were also engineered using PCR. The same Y-sense oligonucleotide primer described above was paired with either a Y-antisense primer that (1) introduced a translation termination codon after codon 453 (SEQ ID No. 3) or (2) incorporated a hexa-histidine tag followed by a translation termination codon in the PCR using peDNA3. 1 (hygro)/Hu-Asp-2(a) as the template. In all cases, the PCR reactions were performed amplified for 15 cycles using PwoI DNA polymerase (Boehringer-Mannheim) as outlined by the supplier. The reaction products were digested to completion with BamHI and Notl and ligated to BatnHI and NotI digested baculovirus transfer vector pVL1393 (Invitrogen). A portion of the ligations was used to transform competent E. coli DH5a cells followed by antibiotic selection on LB-Amp. Plasmid DNA was prepared by standard alkaline lysis and banding in CsCI to yield the baculovirus transfer vectors pVL 1 393/Asp2(a), pVL 13 93/Asp2(a)ATM and pVL1393/Asp2(a)ATM(HiS)6. Creation of recombinantbaculoviruses and infection of sf9 insect cells was performed using standard methods. Expression by transfection Transient and stable expression of Hu-Asp2(a)ATM and Hu-Asp2(a)ATM(HiS)6 in High 5 insect cells was performed using the insect expression vector pIZ/V5-His. The DNA inserts from the expression plasmids vectors pVL1393/Asp2(a),pVL1393/Asp2(a)ATM and pVL1393/Asp2(a)ATM(HiS)6 were excised by double digestion with BatnHI and Notl and subcloned into BamHI and NotI digested pEN5-His using standard methods. The resulting expression plasmids, referred to as p7Mu-Asp2ATM and pIZ/Hu-Asp2ATM(HiS)6, were prepared as described above.

For transfection, High 5 insect cells were cultured in High Five serum free medium supplemented with 10 gg/ml gentamycin at 27 'C in sealed flasks. Transfections were performed using High five cells, High five serum free media supplemented with 10 gg/ml gentamycin, and InsectinPlus liposomes (Invitrogen, CarIsbad, CA) using standard methods.

For large scale transient transfections, 1.2 x 107 high five cells were plated in a 150 mm tissue culture dish and allowed to attach at roomtemperature for 15-30 minutes. During the attachment time the DNA/ liposome mixture was prepared by mixing 6 mI of serum free media, 60 gg Hu-Asp2(a)ATM/plZ (+/- His) DNA and 120 gI of Insectin Plus and incubating at room temperature for 15 minutes. The plating media was removed from the dish of cells and replaced with the DNA/liposome mixture for 4 hours at room temperature with constant rocking at 2 rpm. An additional 6 n-d of media was added to the dish prior to incubation for 4 days at 27 'C in a humid incubator. Four days post transfection the media was harvested, clarified by centrifugation at 500 x g, assayed for Hu-Asp2(a) expression by Western blotting. For stable expression, the cells were treated with 50 [lgImI Zeocin and the surviving pool used to prepared clonal cells by limiting dilution followed by analysis of the expression level as noted above. Purification of Hu-Asp2(a)A TM and Bu-Asp2(a)A M(His)6 Removal of the transmembrane segment from Hu-Asp2(a) resulted in the secretion of the polypeptide into the culture medium. Following protein production by either baculovirus infection or transfection, the conditioned medium was harvested, clarified by centrifugation, and dialyzed against Tris-VIC1 (pH 8.0). This material was then purified by successive chromatography by anion exchange (Tris-HCI, pH 8.0) followed by cation exchange chromatography (Acetate buffer at pH 4.5) using NaCI gradients. The elution profile was monitored by (1) Western blot analysis and (2) by activity assay using the peptide substrate described in Example 12. For the Hu-Asp2(a)ATM(FliS)6, the conditioned medium was dialyzed against Tris buffer (pH 8.0) and purified by sequential chromatography on IN4AC resin followed by anion exchange chromatography.

Amino-terminal sequence analysis of the purified Hu-Asp2(a)ATM(HiS)6 protein revealed that the signal peptide had been cleaved [TQHG1RLPLR, corresponding to SEQ ID NO: 32, residues 22-31.

Example 11 Expression of Hu-Asp2(a) and Hu-Asp(b) in CHO cells The materials (vectors, host cells, etc.) and methods described herein for expression of Hu-Asp2(a) are intended to be equally applicable for expression of Hu-Asp2(b). Heterologous expression of Hu-Asp-2(a) in CHOKI cells The entire coding sequence of Hti-Asp2(a) was cloned into the mammalian expression vector pcDNA3. 1 (+)Hygro (Invitrogen, CarIsbad, CA) which contains the CMV immediate early promoter and bGH polyadenylation signal to drive over expression. The expression plasmid, pcDNA3. 1 (+)Hygro/Hu- Asp2(a), was prepared by alkaline lysis and banding in CsCI and completely sequenced on both strands to verify the integrity of the coding sequence.

Wild-type Chinese hamster ovary cells (CHO-KI) were obtained from the ATCC. The cells were maintained in monolayer cultures in a-MEM containing 10% FCS at 37'C in 5% C02. Two 100 mm dishes of CHO-K l cells (60% confluent) were transfected with pcDNA3. 1 (+)/Hygro alone (mock) or pcDNA3. 1 (+)Hygro/Hu-Asp2(a) or pcDNA3. 1 (+)Hygro/Hu-Asp2(b) using the cationic liposome DOTAP as recommended by the supplier (Roche, Indianapolis, IN). The cells were treated with the plasmid DNA/liposome mixtures for 15 hours and then the medium replaced with growth medium containing 500 UnitsIn-fl hygromycin B. In the case of pcDNA3.1(+ )Hygro/Hu-Asp2(a) or (b) transfected C110-KI.cells, individual hygromycin B-resistant cells were cloned by limiting dilution. Following clonal expansion of the individual cell lines, expression of Hu-Asp2(a) or HuAsp2(b) protein was assessed by Western blot analysis using a polyclonal rabbit antiserum raised against recombinant Hu-Asp2 prepared by expression in E. coli. Near confluent dishes of each cell line were harvested by scraping into PBS and the cells recovered by centrifugation. The cell pellets were resuspended in cold lysis buffer (25 mM Tris-11C1 (pH 8.0)/5 mM EDTA) containing protease inhibitors and the cells lysed by sonication. The soluble and membrane fractions were separated by centrifugation (105,000 x g, 60 min) and normalized amounts of protein from each fraction were then separated by SDS-PAGE. Following electrotransfer of the separated polypeptides to PVDF membranes, Hu-Asp- 2(a) or Hu-Asp2(b) protein was detected using rabbit anti-Hu-Asp2 antiserum (111000 dilution) and the antibody-antigen complexes were visualized using alkaline phosphatase conjugated goat anti-rabbit antibodies (l/2500). A specific immunoreactive protein with an apparent Mr value of 65 kDa was detected in pcDNA3. 1 (+)Hygro/Hu-Asp2 transfected cells and not mock-transfected cells. Also, the Hu-Asp2 polypeptide was only detected in the membrane fraction, consistent with the presence of a signal peptide and single transmembrane domain in the predicted sequence. Based on this analysis, clone #5 had the highest expression level of Hu-Asp2(a) protein and this production cell lines was scaled up to provide material for purification. Purification of recombinant Hu-Asp-2(a)from CHO-WI-M-Asp2 clone #5 In a typical purification, clone #5 cell pellets derived from 20 150 mm dishes of confluent cells, were used as the starting material. The cell pellets were resuspended in 50 mI cold lysis buffer as described above. The cells were lysed by polytron homogenization (2 x 20 sec) and the lysate centrifuged at 338,000 x g for 20 minutes. The membrane pellet was then resuspended in 20 M1 of cold lysis buffer containing 50 mM Poctylglucoside followed by rocking at 4 'C for 1 hour. The detergent extract was clarified by centrifugation at 338,000 x g for 20 minutes and the supernatant taken for further analysis.

The P-octy1glucoside extract was applied to a Mono Q anion exchange column that was previously equilibrated with 25 mM Tris-11C1 (pH 8.0)150 mM P-oetylglucoside. Following sample application, the column was eluted with a linear gradient of increasing NaCI concentration (0-1.0 M over 30 minutes) and individual fractions assayed by Westem blot analysis and for P-secretase activity (see below). Fractions containing both Hu-Asp-2(a) immunoreactivity and 0-secretase activity were pooled and dialyzed against 25 mM NaOAc (pH 4.5)50 mM P-oetylglucoside. Following dialysis, precipitated material was removed by centrifugation and the soluble material chromatographed on a MonoS cation exchange column that was previously equilibrated in 25 mM NaOAc (pH 4.5)l 50 mM P-octylglucoside. The column was eluted using a linear gradient of increasing NaCI concentration (0- 1.0 M over 30 minutes) and individual fractions assayed by Western blot analysis and for 0-secretase activity. Fractions containing both Hti-Asp2 immunoreactivity and P-secretase activity were combined and determined to be >95% pure by SDS-PAGE/Coomassie Blue staining.

The same methods were used to express and purify Hti-Asp2(b). Example 12 Assay of Hu-Asp2 P-secretase activity using peptide substrates flsecretase assay Recombinant human Asp2(a) prepared in CHO cells and purified as described in Example 11 was used to assay Asp2(a) proteolytic activity directly. Activity assays for Asp2(a) were performed using synthetic peptide substrates containing either the wildtype APP P-secretase site (SEVKM1DA. EFR), the Swedish KM--->NI, mutation (SEVNLIDAEFR), or the AP40 and 42 y- secretase sites (RRGGWIIA1WrVGER). Reactions were performed in 50 mM 2-[N- morpholinolethane-sulfonate ('Na-MES," pH 5.5) containing 1 % P- Octy1glucoside, 70 mM peptide substrate, and recombinant Asp2(a) (13 [tg protein) for various times at 37C. The reaction products were quantified by RP-HPLC using a linear gradient from 0-70 B over 30 minutes (A=O. 1 % TFA in water, B=0.1%TFA/10%water/90%AcCN). The elution profile was monitored by absorbance at 214 nm. In prelinfinary experiments, the two product peaks which eluted before the intact peptide substrate, were confirmed to have the sequence DAEFR and SEVNL using both Edman sequencing and MADLI-TOF mass spectrometry. Percent hydrolysis of the peptide substrate was calculated by comparing the integrated peak areas for the two product peptides and the starting material derived from the absorbance at 214 nm. The sequence of cleavagelhydrolysis products was confirmed using Edman sequencing and MADLI-TOF mass spectrometry.

The behavior of purified Asp2(a) in the proteolysis assays was consistent with the prior anti-sense studies which indicated that Asp2(a) possesses P-secretase activity. Maximal proteolysis was seen with the Swedish Psecretase peptide, which, after 6 hours, was about 10-fold higher than wild type APP.

The specificity of the protease cleavage reaction was determined by performing the 0-secretase assay in the presence of 8 [LM pepstatin A and the presence of a cocktail of protease inhibitors (10 gM leupeptin, 10 gM E64, and 5 mM EDTA). Proteolytic activity was insensitive to both the pepstatin and the cocktail, which areinhibitors of cathepsin D (and other aspartyl proteases), serine proteases, cysteinyl proteases, and metalloproteases, respectively.

Hu-Asp2(b) when similarly expressed in CHO cells and purified using identical conditions for extraction with 0-octy1glucoside and sequential chromatography over Mono Q and Mono, S also cleaves the Swedish Psecretase peptide in proteolysis assays using identical assay conditions.

Collectively, this data establishes that both forms of Asp2 (Hu-Asp2(a) and Hu-Asp2(b)) act directly in cell-free assays to cleave synthetic APP peptides at the secretase site, and that the rate of cleavage is greatly increased by the Swedish KM--->NI, mutation that is associated with Alzheimer's disease.

An alternative P-secretase assay utilizes internally quenched fluorescent substrates to monitor enzyme activity using fluorescence spectroscopy in a single sample or multiwell format. Each reaction contained 50 nim Na- MES (pH 5.5), peptide substrate MCA-EYKEF[K-DNP1 (BioSource International) (50 [tM) and purified Hu-Asp-2 enzyme. These components were equilibrated to 37 'C for various times and the reaction initiated by addition of substrate. Excitation was performed at 330 nm and the reaction kinetics were monitored by measuring the fluorescence emission at 390 nin. To detect compounds that modulate Hu-Asp-2 activity, the test compounds were added during the preincubation phase of the reaction and the kinetics of the reaction monitored as described above. Activators are scored as compounds that increase the rate of appearance of fluorescence while inhibitors decrease the rate of appearance of fluorescence.

Example 13 Demonstration that Aspl processes APP at the a-secretase site Increased expression of an oc-secretase candidate gene in human cells would be expected to increase basal release of sAPP(x and to decrease release of AP peptides. This the effect was observed when full length human Asp I is co-expressed with APP in HEK293 cells. The experiment utilized the APP 695 amino acid isoform which had been modified by the addition of a pair of lysine residues to the C-terminus (APP-KK). The Cterminal di-lysine motif increases the intracellular half-life of glycosylated APP and consequently the production of both sAPP(X and Ap. As shown in Table 5, cotransfection of HEK293 cells with APP-KK with Asp I increased the production of sAPPot by 3.5 fold (p<0.001) and decreased the production of AP40 by 2.8 fold. Thus, Asp l acts directly or indirectly to facilitate constitutive cc-secretase cleavage and this effect is competitive with the arnyloidogenic processing of APP to AP peptides. This implies that mutations or genetic polymorphisms in Aspi may affect AO production by affecting the balance between the competing pathways for constitutive (x-secretase cleavage and AP peptide production. Table 5.

Aspl stimulates basal release of sAPP(x from HEK293 cells after cotransfection with APP-KK.

Transfection sAPP(x Fold A040 Fold gg/rnl Increase pg/rn, Decrease Aspl 3.5+1.1 +3.5 113+7 -2.8 pcDNA 1.0+0.2 321+18 Specific methods used were as follows. The full length Asp l cDNA was cloned into the vector pcDNA3. 1/hygro+(lnvitrogen) for transfection studies as previously described (Yan etaL, (1999) Nature 402: 533-537). TheAPP-KKeDNA was cloned into the vector pIRES (Clontech) also as previously described. HEK293 cells were transfected with expression constructs using the Lipofectamine Plus reagent from Gibco/BRIL. Cells were seeded in 24 well tissue culture plates to a density of 70-80% confluence. Four wells per plate were transfected with 2 gg DNA (3: 1, APP:Asp 1 or empty pcDNA3. 1.1hygro+ vector), 8pl Plus reagent, and 4ptl Lipofectarnine in OptiMEM. OptiMEM was added to a total volume of 1 MI, distributed 200 gl per well and incubated 3 hours. Care was taken to hold constant the ratios of the two plasmids used for cotransfection as well as the total amount of DNA used in the transfection. The transfection media was replaced with DMEM supplemented with 10% FBS and NaPyruvate, with antibioticlantimycotic and the cells were incubated under normal conditions (37', 5% C02) for 48 hours. The conditioned media were removed to polypropylene tubes and stored at -80'C until assayed for the content of sAPP(X or A040/AP42 by enzyme-linked immunosorbent assay (EIA) as described above in Example 6. The AP EIA followed the protocol of Pirttila et aL (Neuro. Lett. (1999) 249: 21-4) using the 6E 10 monoclonal antibody (Senetek) as a capture antibody and biotinylated rabbit antiserurn 162 or 165 (New York State Institute for Basic Research, Staten Island, NY) for detection of AP40 and AP42, respectively. The 6E10 antibody recognizes residues 1- 16 of the AP peptide. The sAPPa EIA used LN27 antibody as a capture antibody and biotinylated 6E10 for detection as described previously (Yan et aL, (1999) supra.). The LN27 antibody recognized the first 20 amino acids of the human APP peptide.

Increased a-secretase activity and concomitant reduction of AP production in vivo represents an effect that may be desirable for the prevention, treatment (e.g., to show the progression of), or cure of Mheimer's disease. Thus, the activities demonstrated in this example provide an indication that modulators of Asp I activity, that achieve the same effects in vivo, will have utility for Alzheimer's disease therapy. Screening methods for such modulators are contemplated as an aspect of the invention.

Example 14 Expression of Pre-pro-11u-Aspl and Derivatives in Insect Cells Expression of hu-Asp-lTM(HisP by baculovirus infection.

The coding sequence of pre-pro-Hu-Asp 1 was engineered for production as a soluble, secreted form by insect cells. PCR primers were designed to (1) delete the predicted transmembrane domain and cytoplasmic tail of Asp I and (2) to introduce a Kozak consensus sequence for efficient translational initiation. The primers sequences were are follows: sense CGCMAAGCTTGCCACCATGGGCGCA CTGWCMGGW (SEQ ID NO: 74) and antisense CG=CWGAWTAA TGGTGATGGTGATGGTGCCACAAAATGGGCTCGCTCAAAGA (SEQ ID NO: 75) which replaced the deleted C-terminal transmembrane and cytoplasmic domains with a hexahistidine purification tag.

PCR reactions were carried out with 100 ng of full length Aspl pcDNA 3.1 hygro+ construct, 200 M NTPs, 300 riM of each primer, l x reaction buffer containing 2 mM MgSO4, and 5 units of Pwo I DNA polymerase (Roche Biochemicals). The reactions were cycled under the following conditions: WC for 5 minutes followed by 15 cycles of 94T for 30 seconds and 72T for 30 seconds, and then a final extension reaction at 72'C for 10 minutes. The predicted arnino acid sequence of this PCR generated derivative (denoted as Asp-lATM(HiSW is set out as SEQ ID NO: 66.

The reaction product was digested to completion with Hind111-XlioI and ligated into the expression vector p113 (Invitrogen) to yield the pIB/AspIATM(HiS)6 construct. Creation of recombinant baculovirus and infection of sf9 insect cells was performed using standard methods known in the art. Sf9 cells were transfected with either the p1B vector alone or the pIB/Asp- 1 ATM(MS)6 construct utilizing Insectin Plus reagent (Invitrogen) according to the manufacturer's instructions. After the transfection, the cells were cultured in High Five serum-fr6e media (Invitrogen) for 3 days. Subsequently, the conditioned medium was harvested and subjected to Western blot analysis. This analysis revealed specific expression and secretion of immunoreactive Asp-lATM(MS)6 polypeptide into the extracellular medium. The secreted proteins were detected on the Western blot with either the India probe (Pierce Chemicals) specific for the hexahistidine sequence tag or using a rabbit polyclonal antiserum. The polyclonal antisera (denoted as UP-199) was generated by injecting rabbits with recombinant Asp- 1 ATM(MS)6 (SEQ II) NO: 66). This recombinant peptide was prepared by heterologous expression in E. coli. The UP- 199 antibody recognizes the processed form of Asp- 1 ATM.

Direct analysis with the polyclonal antiscrum (UP-199) revealed an immunoreactive band of the expected molecular weight (50 kDa) only in pIB/Asp1 ATM(HiS)6 transfected cells. This signal was significantly enhanced in concentrated conditioned medium. A similar pattern was obtained using the India probe. No signal was detected in conditioned medium derived from mock-transfected cells using either UP- 199 antisera or the India probe.

Based on this result, transient and stable transfections of the pIB/AspI ATMO-ES)6 construct in sf9 insect cells were carried out as described above. Four days post transient transfection, the culture medium was collected to provide material for further characterization. In parallel, sf9 cells were stably transfected with the pIB/Asp-lATM(MS)6 construct and cultured in High Five serum-free medium (Invitrogen) supplemented with 50 gg/ml blasticidin for approximately 2 weeks.

After blasticidin selection, the resistant pool of cells was expanded to provide a stable source of conditioned medium for Asp- 1 ATM(HiS)6 purification. Purification ofrecombinantAsp-IATM(His)6 Conditioned media, from either transient or stably transfected A9 cells, were concentrated approximately 10-fold using a stirred cell concentrator equipped with a 30,000 MWCO membrane (Spectrum Medical Industries). This concentrate was then subjected to ammonium sulfate precipitation to further concentrate the sample and provide partial purification. Material precipitating between 0-40% saturation was discarded and the resulting supematant was brought to 80% saturation. Western blot analysis of the various ammonium sulfate precipitated fractions revealed that the majority of the immunoreactive material was contained within the 40-80% ammonium sulfate pellet. As a result, this material was subjected to further purification.

The 40-80% ammonium sulfate pellet was redisolved in approximately 1120 the original volume of Ni±NTA loading buffer (25 mM Tris-HG (pH 8.5)10.5 M NaCI/10 niM imidazole). Subsequently, the sample was applied to a Ni+NTA column previously equilibrated in Ni±NTA buffer. Following sample application, the column was washed with starting buffer (25 mM Tris-HO (ph 8.5)l 0.5 M NaCII 20 mM imidazole) until the A 280= of the column effluent returned to zero. After washing, the bound recombinant protein was eluted off the colunm with a linear gradient of Ni±NTA buffer containing increasing concentrations (10 mM, 50 MM, 100 mM, 250 mM, and 500 mM) of imidazole. The elution profile was monitored by Western blot analysis using the UP- 199 antiserum. Inununoreactive Asp- 1 ATM(HiS)6 was detected in the column load and eluted at 50 mM imidazole. NuPAGE gel analysis of the 50 mM imidazole fraction demonstrated a purity of Asp- 1 ATM(HiS)6 of approximately 50%, therefore further purification was required.

The positive fractions, eluted off the Ni±NTA column, were then pooled (denoted as post-INIAC pool), concentrated using a YM30 membrane (Amicron), and dialyzed with 25 mM Tris-HO (pH 8.0). The dialyzed postIN4AC pool was fractionated by MonoQ anion exchange chromatography (Amersham-Pharmacia Biotech) gradient elution containing increasing concentrations (0 -0.5 M) of NaCI (Buffer A: 25 mM Tris-11C1 (pH 8.0) and Buffer B: 25 n1M Tris-HG (pH 8. 0)10.5 M NaCI). The elution profile was determined by Western blot analysis which indicated immunoreactive fractions as those displaying immunoreactivity with the UP- 199 antisera. NuPAGE gel analysis with silver staining demonstrated that the material prepared in this manner was >90% pure. The immunoactive fractions eluted off the MonoQ anion exchange column were pooled, dialyzed with 25 rnM HEPES-Na+ (pH 8.0), and stored at 4C until further analysis. Acid-activation of recombinant Asp4 M(His)6 Recombinant Asp- 1 ATM(MS)6 migrated with an apparent molecular weight of 50 kD. Direct N-terminal sequence analysis carried out by automated Edman degradation for 20 cycles revealed a unique sequence beginning at Glu' (SEQ ID NO: 67), confirming the identity of the recombinant protein. Computer assisted prediction of the signal peptidase cleavage site indicated that the pro-form should initiate at Alal, suggesting either an unusual processing site by the signal peptidase during secretion or an additional processing step that removes an additional two amino acid residues.

To investigate the mechanism of pro-Asp- 1 ATM(MS)6 activation, aliquots of the purified protein were incubated in various acidic environments with pH values ranging from 3.0-8.0 at 37'C for 2 hours. Subsequently, the recombinant proteins were analyzed by Western blot. A faster migrating polypeptide species was detected after incubation at pH values of 4.0,43 and 5.0. The polypeptide migration was unaltered after incubation in environments which were either more acidic (pH 3.0 and 3.5) or more basic (pH 6.0, 7.0, and 8.0). Sequence analysis of this faster migrating 3 species revealed that it initiated exclusively at Ala ' consistent with removal of a 42 amino acid residue segment of the pro-peptide that was induced by treatment of the pro-enzyine at pH 4.5. The predicted amino acid sequence of the acid processed form of Asp- 1 ATM(MS)6 is set out as SEQ ID NO: 68.

To purify the acid-activated form of Asp- 1 ATM(His)6, the Asp- 1 ATM(HiS)6 post-C pool (generated as described above) was dialyzed to pH 4. 5 and then subjected to affinity chromatography on either pepstatin A agarose or sulfolink-PHA-292593E. Following sample application, the column was washed with n1M NaOAc (pH 4.5) and eluted with 50 mM Na-B03 (pH 9.5). The positive fractions eluted off the columns were dialyzed with 25 mM Hepes-Na (pH 7. 5) overnight at 0C which resulted in quantitative conversion of the pro- enzyme to the acid-processed form (SEQ ID NO: 68) described above. Western blot analysis of the elution profile revealed quantitative retention of immunoreactive Asp- 1 ATM(MS)6 on both affinity resins as evidenced by the lack of Asp- 1 ATM(HiS)6 in the unbound fraction as detected by UP-199 immunoreactivity on a Western blot. Step elution 50 mM NaB03 at pH 9.5 resulted in elution of immunoreactive Asp-1 ATM(MS)6, with variable recovery.

Comparison of the properties of the recombinant soluble catalytic domain of Asp I with the properties determined for Asp2 (see Example 10) revealed a number of significant differences. Processing of the pre-pro forms of either enzyme is distinct, with Aspl. undergoing efficient processing by the signal peptidase and additional processing to remove two additional amino acid residues from the N-teniiinus. Further processing of the pro-form of Asp I was not detected in neutral pH. In contrast, recombinant Asp2 produced, under similar conditions, yields an eqimolar mixture of the pro-form and a processed form that has 24 amino acid residues of the pro-segment removed.

Another distinction between the processing of these two enzymes involves processing initiated by acid-treatment. Systematic analysis of acidinduced processing of pro-Asp2 revealed that the purified polypeptide did not self-process. In contrast, acid dependent processing of pro-Asp I was readily demonstrated (as described above). Alignment of the selfprocessing site in Asp l with the processing site in Asp2 revealed that these two enzymes are processed at the same position, which is a different method of processing as compared with that of other known human aspartyl proteases.

In addition to providing valuable information about Asp l activity, the discovery of a site of apparent autocatalytic processing of Asp l provides an indication of a peptide sequences (surrounding AW3) that could be useful for performing screening assays to identify modulators of Aspl activity. This idea is explored in greater detail in Example 15.

Example 15 Development of an enzymatic assay for Asp-1ATM(MS)6 The relationship between Asp I and APP processing was explored by determining if APP a-secretase, APP P-secretase, or APP V-secretase peptide substrates were cleaved by recombinant Asp-lATM(HiS)6. These peptide substrates included the a-secretase specific substrates Apio-2o and AP12-28, the P-secretase specific substrates PHA-95812E (SEVICMDAEFR; SEQ ID NO: 64) and PHA-247574E (SEVNLDAEFR; SEQ 11) NO: 63), and V- secretase specific substrate PHA- 179111 E (RIZGGMATVIVGER; SEQ ID NO: 76). Each reaction consisted of incubating a peptide substrate (100 nM) with recombinant AspI ATM(MS)6 for 15 hours at pH 4.5 at 37T. Reaction products were quantified by RP-HPLC at A 211 n-. The elution profiles for Asp- 1 ATM(HiS)6 were compared to those obtained from parallel Asp 1 experiments. The identity of the cleavage products was detem-iined by MADLI-TOF mass spectrometry. Table 6 summarizes the Aspl substrates and indicates the cleavage site.

Table 6 Substrate Preferences of Asp-1ATM P4 P3 P2 P 1 P 1' P2' PY P4' SEQ ID NO:

G L A L A L E P Self Activation 69 E V K M D A E F P-Secretase, WT 70 E V N L D A E F P-Secretase, Sw 71 L V F F A E D V A012-28 (a-Secretase) 72 K L V F F A E D A012-28 (a-Secretase) 73 The peptides in Table 6 are described using the nomenclature by Schechter and Berger (Biochem. Biophys. Res. Commun. 27:157(1967) and Biochem. Biophys. Res. Commun. 32:898 (1968)), in which the amino acid residues in the peptide substrate that undergo the cleavage are defined as P1... P. toward the Nterminus and Pi'... PJtoward the C-terminus. Therefore, the scissile bond is between the Pi and the Pi'residue of the peptide subunits and is denoted herein throughout with a hyphen between the Pi and the Pi'.

Digestion of the a-secretase substrate (A12-28) revealed two Aspl cleavage sites. The major product was cleaved at Phe 211A1a2' and the minor product was cleaved at Phe191Phe20 (referring to the numbering convention in the APP AP) peptide. Analysis of the cleavage products obtained from the 0-secretase peptide substrates revealed that both the wild-type (PHA-95812E) and the Swedish mutation (PHA-247574E) substrates were hydrolyzed exclusively at the P-secretase site. Also, the relative rates of Asp-1 -dependent hydrolysis of the P-secretase peptide substrate containing the Swedish mutation was cleaved at least 10-times faster than the corresponding wild-type peptide. Conversion of the V-secretase peptide substrate was not detected under these reaction conditions.

Measurement of the cleavage of the a-secretase and P-secretase substrates can also be carried out with substrates comprising detectable labels such as radioactive, enzymati, chemiluminescent or flourescent labels. For example, the peptide substrates could comprise internally quenched labels that result in increased detectability after cleavage of the peptide substrates due to separation of the labels upon cleavage. The peptide substrates can be modified to have attached a paired fluorprobe and quencher such as 7-amino-4-methyl courarin and dinitrophenol, respectively.

This example illustrates the a-secretase and P-secretase activity exhibited by Asp-1, confirming the APP processing activity of Asp I indicating, e.g., in Examples 7 and 13. The substrates described herein may be used in combination with recombinant Asp 1 to measure Asp 1 proteolytic activity at the a-secretase and 0-secretase processing sites. These substrates are useful in screening assays for identification of modulators of Aspl. proteolytic activity.

In particular, production of AP species through the processing of APP atP-and.y-secretase sites may play a central role in Alzheimer's disease pathogenesis, and processing at the a-secretase site may have a protective role and may prevent AP production. Thus, a therapeutic andlor prophylactic indication exists for molecules that can increase Aspl asecretase activity and/or decrease Aspl P-secretase activity in vivo. The present invention includes screening assays for such modulators, and the foregoing substrate peptides are useful in such assays.

It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the invention. The entire disclosure of all publications cited herein are hereby incorporated by reference.

n:5 SEQUENCE LISTING <1lo> Pharmacia & Upjohn <l20> ALMEIMER'S DISEASE SECRETASE, APP SUBSTRATES THEREFOR, AND USES THEREFOR <l30> 28341/6280PCP <l40> <14l> <l50> 60/169,232 <15l> 1999-12-06 <l50> 09/416,901 <15l> 1999-10-13 <l50> 60/155,493 <15l> 1999-09-23 <15o> 09/404,133 <15l> 1999-09-23 <l50> PCT/US99/20881 <15l> 1999-09-23 <15o> 60/101,594 <15l> 1998-09-24 <l60> 76 <l70> PatentIn Ver. 2.0 <210> 1 <21l> 1804 <212> DNA <213> Homo sapiens <400> 1 atgggcgeac tggcccgggc gctgctgctg cctctgctgg cccagtggct cctgcgcgcc 60 gccccggagc tggcccccgc gcccttcacg ctgcccctcc gggtggccgc ggccacgaac 120 cgcgtagttg cgcccacccc gggacccggg acccctgccg agcgccacgc cgacggcttg 180 gcgctcgccc tggagcctgc cctggcgtcc cccgcgggcg ccgccaactt cttggccatg 240 gtagacaacc tgcaggggga ctctggccgc ggctactacc tggagatgct gatcgggacc 300 cccccgcaga agctacagat tctcgttgac actggaagca gtaactttgc cgtggcagga 360 accccgcact cctacataga cacgtacttt gacacagaga ggtctagcac ataccgctcc 420 aagggctttg acgtcacagt gaagtacaca caaggaagct ggacgggctt cgttggggaa 480 gacctcgtca ccatccccaa aggcttcaat acttcttttc ttgtcaacat tgccactatt 540 tttgaatcag agaatttctt tttgcctggg attaaatgga atggaatact tggcctagct 600 tatgccacac ttgccaagcc atcaagttct ctggagacct tcttcgactc cctggtgaca 660 caagcaaaca tccccaacgt tttctccatg cagatgtgtg gagccggctt gcccgttgct 720 ggatctggga ccaacggagg tagtcttgtc ttgggtggaa ttgaaccaag tttgtataaa 780 ggagacatct ggtatacccc tattaaggaa gagtggtact accagataga aattctgaaa 840 ttggaaattg gaggccaaag ccttaatctg gactgcagag agtataacgc agacaaggcc 900 atcgtggaca gtggcaccac gctgctgcgc ctgccccaga aggtgtttga tgcggtggtg 960 gaagctgtgg cccgcgcatc tctgattcca gaattctctg atggtttctg gactgggtcc 1020 cagctggcgt gctggacgaa ttcggaaaca ccttggtctt acttccctaa aatctccatc 1080 tacctgagag atgagaactc cagcaggtca ttccgtatca caatcctgcc tcagctttac 1140 attcagccca tgatg9999c cggcctgaat tatgaatgtt accgattcgg catttcccca 1200 tccacaaatg cgctggtgat cggtgccacg gtgatggagg gcttctacgt catcttcgac 1260 014w agagcccaga agagggtggg cttcgcagcg agcccctgtg cagaaattgc aggtgctgca 1320 gtgtctgaaa tttccgggcc tttctcaaca gaggatgtag ccagcaactg tgtccecgct 1380 cagtctttga gcgagcccat tttgtggatt gtgtcctatg cgctcatgag cgtctgtgga 1440 gccatcctcc ttgtcttaat cgtcctgctg ctgctgccgt tccggtgtca gcgtcgcccc 1500 cgtgaccctg aggtcgtcaa tgatgagtcc tctctggtca gacatcgctg gaaatgaata 1560 gccaggcctg acctcaagca accatgaact cagctattaa gaaaatcaca tttccagggc 1620 agcagccggg atcgatggtg gcgctttctc ctgtgcceac ccgtcttcaa tctctgttct 1680 getcccagat gccttctaga ttcactgtct tttgattett gattttcaag ctttcaaatc 1740 ctccctactt ccaagaaaaa taattaaaaa aaaaacttca ttctaaacca aaaaaaaaaa 1800 aaaa 1804 <210> 2 <21l> 518 <212> PRT <213> Homo sapiens <400> 2 Met Gly Ala Leu Ala Arg Ala Leu Leu Leu Pro Leu Leu Ala G1n Trp 1 5 10 15 Leu Leu Arg Ala Ala Pro Glu Leu Ala Pro Ala Pro Phe Thr Leu Pro 25 30 Leu Arg Val Ala Ala Ala Thr Asn Arg Val Val Ala Pro Thr Pro Gly 40 45 Pro Gly Thr Pro Ala Glu Arg His Ala Asp Gly Leu Ala Leu Ala Leu 55 60 Glu Pro Ala Leu Ala Ser Pro Ala Gly Ala Ala Asn Phe Leu Ala Met 70 75 80 Val Asp Asn Leu G1n Gly Asp Ser Gly Arg Gly Tyr Tyr Leu Glu Met 90 95 Leu Ile Gly Thr Pro Pro G1n Lys Leu G1n Ile Leu Val Asp Thr Gly 105 110 Ser Ser Asn Phe Ala Val Ala Gly Thr Pro His Ser Tyr Ile Asp Thr 120 125 Tyr Phe Asp Thr Glu Arg Ser Ser Thr Tyr Arg Ser Lys Gly Phe Asp 135 140 Val Thr Val Lys Tyr Thr G1n Gly Ser Trp Thr Gly Phe Val Gly Glu 150 155 160 Asp Leu Val Thr Ile Pro Lys Gly Phe Asn Thr Ser Phe Leu Val Asn 170 175 Ile Ala Thr lle Phe Glu Ser Glu Asn Phe Phe Leu Pro Gly Ile Lys 185 190 T.rp Asn Gly Ile Leu Gly Leu Ala Tyr Ala Thr Leu Ala Lys Pro Ser 200 205 Ser Ser Leu Glu Thr Phe Phe Asp Ser Leu Val Thr G1n Ala Asn Ile 210 215 220 Pro Asn Val Phe Ser Met G1n Met Cys Gly Ala Gly Leu Pro Val Ala 225 230 235 240 n_l Gly Ser Gly Thr Asn Gly Gly Ser Leu Val Leu Gly Gly Ile Glu Pro 245 250 255 Ser Leu Tyr Lys Gly Asp Ile Trp Tyr Thr Pro lle Lys Glu Glu Trp 260 265 270 Tyr Tyr G1n Ile Glu Ile Leu Lys Leu Glu Ile Gly Gly G1n Ser Leu 275 280 285 Asn Leu Asp Cys Arg Glu Tyr Asn Ala Asp Lys Ala lle Val Asp Ser 290 295 300 Gly Thr Thr Leu Leu Arg Leu Pro G1n Lys Val Phe Asp Ala Val Val 305 310 315 320 Glu Ala Val Ala Arg Ala Ser Leu Ile Pro Glu Phe Ser Asp Gly Phe 325 330 335 Trp Thr Gly Ser G1n Leu Ala Cys Trp Thr Asn Ser Glu Thr Pro Trp 340 345 350 Ser Tyr Phe Pro Lys Ile Ser Ile Tyr Leu Arg Asp Glu Asn Ser Ser 355 360 365 Arg Ser Phe Arg Ile Thr Ile Leu Pro G1n Leu Tyr Ile G1n Pro Met 370 375 380 Met Gly Ala Gly Leu Asn Tyr Glu Cys Tyr Arg Phe Gly Ile Ser Pro 385 390 395 400 Ser Thr Asn Ala Leu Val Ile Gly Ala Thr Val Met Glu Gly Phe Tyr 405 410 415 Val lle Phe Asp Arg Ala G1n Lys Arg Val Gly Phe Ala Ala Ser Pro 420 425 430 Cys Ala Glu Ile Ala Gly Ala Ala Val Ser Glu Ile Ser Gly Pro Phe 435 440 445 Ser Thr Glu Asp Val Ala Ser Asn Cys Val Pro Ala G1n Ser Leu Ser 450 455 460 Glu Pro Ile Leu Trp Ile Val Ser Tyr Ala Leu Met Ser Val Cys Gly 465 470 475 480 Ala Ile Leu Leu Val Leu Ile Val Leu Leu Leu Leu Pro Phe Arg Cys 485 490 495 G1n A-rg Arg Pro Arg Asp Pro Glu Val Val Asn Asp Glu Ser Ser Leu 500 505 510 Val Arg His Arg Trp Lys 515 <210> 3 <21l> 2070 <212> DNA <213> Homo sapiens <400> 3 atggcccaag ccctgccctg gctcctgctg tggatgggcg cgggagtgct gcctgcccac 60 ggcacccagc acggcatccg gctgcccctg cgcagcggcc tggggggcgc ccccctgggg 120 ctgcggctgc cccgggagac cgacgaagag cccgaggagc ccggccggag gggcagcttt 180 gtggagatgg tggacaacct gaggggcaag tcggggcagg gctactacgt ggagatgacc 240 gtgggcagcc ccccgcagac gctcaacatc ctggtggata caggcagcag taactttgca 300 gtgggtgctg ccccccaccc cttcctgcat cgctactacc agaggcagct gtccagcaca 360 taccgggacc tccggaaggg tgtgtatgtg ccctacaccc agggcaagtg ggaaggggag 420 ctgggcaccg acctggtaag catcccccat ggccccaacg tcactgtgcg tgccaacatt 480 gctgccatca ctgaatcaga caagttcttc atcaacggct ccaactggga aggcatcctg 540 gggctggcct atgctgagat tgccaggcct gacgactccc tggagccttt ctttgactct 600 ctggtaaagc agacccacgt tcccaacctc ttctccctgc acctttgtgg tgctggcttc 660 cccctcaacc agtctgaagt gctggcctct gtcggaggga gcatgatcat tggaggtatc 720 gaccactcgc tgtacacagg cagtctctgg tatacaccca tccggcggga gtggtattat 780 gaggtcatca ttgtgcgggt ggagatcaat ggacaggatc tgaaaatgga ctgcaaggag 840 tacaactatg acaagagcat tgtggacagt ggcaccacca accttcgttt gcccaagaaa 900 gtgtttgaag ctgcagtcaa atccatcaag gcagcctcct ccacggagaa gttccctgat 960 ggtttctggc taggagagca gctggtgtgc tggcaagcag gcaccacccc ttggaacatt 1020 ttcccagtca tctcactcta cctaatgggt gaggttacca accagtcctt ccgcatcacc 1080 atccttccgc agcaatacct gcggccagtg gaagatgtgg ccacgtccca agacgactgt 1140 tacaagtttg ccatctcaca gtcatccacg ggcactgtta tgggagctgt tatcatggag 1200 ggcttctacg ttgtctttga tcgggcccga aaacgaattg gctttgctgt cagcgcttgc 1260 catgtgcacg atgagttcag gacggcagcg gtggaaggcc cttttgtcac cttggacatg 1320 gaagactgtg gctacaacat tccacagaca gatgagtcaa ccctcatgac catagcctat 1380 gtcatggctg ccatctgcgc cctcttcatg ctgccactct gcctcatggt gtgtcagtgg 1440 cgctgcctcc gctgcetgcg ccagcagcat gatgactttg ctgatgacat ctccctgctg 1500 aagtgaggag gcecatgggc agaagataga gattcccctg gaccacacct ccgtggttca 1560 ctttggtcac aagtaggaga cacagatggc acctgtggcc agagcacctc aggaccctcc 1620 ccacccacca aatgcctctg ccttgatgga gaaggaaaag gctggcaagg tgggttccag 1680 ggactgtacc tgtaggaaac agaaaagaga agaaagaagc actctgctgg cgggaatact 1740 cttggtcacc tcaaatttaa gtcgggaaat tctgctgctt gaaacttcag ccctgaacct 1800 ttgtccacca ttCctttaaa ttctccaacc caaagtattc ttcttttctt agtttcagaa 1860 gtactggcat cacacgcagg ttaccttggc gtgtgtccct gtggtaccct ggcagagaag 1920 agaccaagct tgtttccctg ctggccaaag tcagtaggag aggatgcaca gtttgctatt 1980 tgctttagag acagggactg tataaacaag ectaacattg gtgcaaagat tgcctcttga 2040 attaaaaaaa aaaaaaaaaa aaaaaaaaaa 2070 <210> 4 <21l> 501 <212> PRT <213> Homo sapiens <400> 4 Met Ala Gin Ala Leu Pro Trp Leu Leu Leu Trp Met Gly Ala Gly Val 1 5 10 is Leu Pro Ala His Gly Thr G1n His Gly Ile Arg Leu Pro Leu Arg Ser 25 30 Gly Leu Gly Gly Ala Pro Leu Gly Leu Arg Leu Pro Arg Glu Thr Asp 40 45 Glu Glu Pro Glu Glu Pro Gly Arg Arg Gly Ser Phe Val Glu Met Val so 55 60 Asp Asn Leu Arg Gly Lys Ser Gly G1n Gly Tyr Tyr Val Glu Met Thr 70 75 80 Val Gly Ser Pro Pro G1n Thr Leu Asn Ile Leu Val Asp Thr Gly Ser 90 95 Ser Asn Phe A1A Val Gly Ala Ala Pro His Pro Phe Leu His Arg Tyr 105 110 Tyr G1n Arg G1n Leu Ser Ser Thr Tyr Arg Asp Leu Arg Lys Gly Val 120 125 Tyr Val Pro Tyr Thr G1n Gly Lys Trp Glu Gly Glu Leu Gly Thr Asp 135 140 Leu Val Ser Ile Pro His Gly Pro Asn Val Thr Val Arg Ala Asn Ile 150 155 160 Ala Ala Ile Thr Glu Ser Asp Lys Phe Phe Ile Asn Gly Ser Asn Trp 170 175 Glu Gly Ile Leu Gly Leu Ala Tyr Ala Glu Ile Ala Arg Pro Asp Asp 185 190 Ser Leu Glu Pro Phe Phe Asp Ser Leu Val Lys G1n Thr His Val Pro 200 205 Asn Leu Phe Ser Leu His Leu Cys Gly Ala Gly Phe Pro Leu Asn G1n 210 215 220 Ser Glu Val Leu Ala Ser Val Gly Gly Ser Met Ile Ile Gly Gly Ile 225 230 235 240 Asp His Ser Leu Tyr Thr Gly Ser Leu Trp Tyr Thr Pro Ile Arg A-rg 245 250 255 Glu Trp Tyr Tyr Glu Val Ile Ile Val Arg Val Glu Ile Asn Gly G1n 260 265 270 Asp Leu Lys Met Asp Cys Lys Glu Tyr Asn Tyr Asp Lys Ser Ile Val 275 280 285 Asp Ser Gly Thr Thr Asn Leu Arg Leu Pro Lys Lys Val Phe Glu Ala 290 295 300 Ala Val Lys Ser Ile Lys Ala Ala Ser Ser Thr Glu Lys Phe Pro Asp 305 310 315 320 Gly Phe Trp Leu Gly Glu G1n Leu Val Cys Trp G1n Ala Gly Thr Thr 325 330 335 Pro Trp Asn lle Phe Pro Val Ile Ser Leu Tyr Leu Met Gly Glu Val 340 345 350 Thr Asn G1n Ser Phe Arg Ile Thr Ile Leu Pro G1n G1n Tyr Leu Arg 355 360 365 Pro Val Glu Asp Val Ala Thr Ser G1n Asp Asp Cys Tyr Lys Phe Ala 370 375 380 Ile Ser G1n Ser Ser Thr Gly Thr Val Met Gly Ala Val Ile Met Glu 385 390 395 400 Gly Phe Tyr Val Val Phe Asp Arg Ala Arg Lys Arg Ile Gly Phe Ala 405 410 415 Val Ser Ala Cys His Val His Asp Glu Phe Arg Thr Ala Ala Val Glu 420 425 430 toe> Gly Pro Phe Val Thr Leu Asp Met Glu Asp Cys Gly Tyr Asn Ile Pro 435 440 445 G1n Thr Asp Glu Ser Thr Leu Met Thr Ile Ala Tyr Val Met Ala Ala 450 455 460 Ile Cys Ala Leu Phe Met Leu Pro Leu Cys Leu Met Val Cys G1n Trp 465 470 475 480 Arg Cys Leu Arg Cys Leu Arg G1n G1n His Asp Asp Phe Ala Asp Asp 485 490 495 Ile Ser Leu Leu Lys 500 <210> 5 <21l> 1977 <212> DNA <213> Homo sapiens <400> 5 atggcccaag ccctgcectg gctcctgctg tggatgggcg cgggagtgct gcctgcccac 60 ggcacccagc acggcatccg gctgcccctg cgcagcggcc t9999ggcgc ccccctgggg 120 ctgcggctgc cccgggagac cgacgaagag cccgaggagc ccggccggag gggcagcttt 180 gtggagatgg tggacaacct gaggggcaag tcggggcagg gctactacgt ggagatgacc 240 gtgggcagcc ccccgcagac gctcaacatc ctggtggata caggcagcag taactttgca 300 gtgggtgctg ccccccaccc cttcctgcat cgctactacc agaggcagct gtccagcaca 360 taccgggacc tccggaaggg tgtgtatgtg ccctacaccc agggcaagtg ggaaggggag 420 ctgggcaccg acctggtaag catcccccat ggccccaacg tcactgtgcg tgccaacatt 480 gctgccatca ctgaatcaga caagttcttc atcaacggct ccaactggga aggcatcctg 540 gggctggcct atgctgagat tgccaggctt tgtggtgctg gcttccccct caaccagtct 600 gaagtgctgg cctctgtcgg agggagcatg atcattggag gtatcgacca ctcgctgtac 660 acaggcagtc tct99tatac acccatccgg cgggagtggt attatgaggt gatcattgtg 720 cgggtggaga tcaatggaca ggatctgaaa atggactgca aggagtacaa ctatgacaag 780 agcattgtgg acagtggcac caccaacctt cgtttgccca agaaagtgtt tgaagctgca 840 gtcaaatcca tcaaggcagc ctcctccacg gagaagttcc ctgatggttt ctggctagga 900 gagcagctgg tgtgctggca agcaggcacc accccttgga acattttccc agtcatctca 960 ctctacctaa tgggtgaggt taccaaccag tccttccgca tcaccatcct tccgcagcaa 1020 tacctgcggc cagtggaaga tgtggccacg tcccaagacg actgttacaa gtttgccatc 1080 tcacagtcat ccacgggcac tgttatggga gctgttatca tggagggctt ctacgttgtc 1140 tttgatcggg cccgaaaacg aattggcttt gctgtcagcg cttgccatgt gcacgatgag 1200 ttcaggacgg cagcggtgga aggccctttt gtcaccttgg acatggaaga ctgtggctac 1260 aacattccac agacagatga gtcaaccctc atgaccatag cctatgtcat ggctgccatc 1320 tgcgccctct tcatgctgcc actctgcctc atggtgtgtc agtggcgctg cctccgctgc 1380 ctgcgccagc agcatgatga ctttgctgat gacatctccc tgctgaagtg aggaggccca 1440 tgggcagaag atagagattc ccctggacca cacctccgtg gttcactttg gtcacaagta 1500 ggagacacag atggcacctg tggccagagc acctcaggac cctccccacc caccaaatgc 1560 ctctgccttg atggagaagg aaaaggctgg caaggtgggt tccagggact gtacctgtag 1620 gaaacagaaa agagaagaaa gaagcactct gctggcggga atactcttgg tcacctcaaa 1680 tttaagtcgg gaaattctgc tgcttgaaac ttcagccctg aacctttgtc caccattcct 1740 ttaaattctc caacccaaag tattcttctt ttcttagttt cagaagtact ggcatcacac 1800 gcaggttacc ttggcgtgtg tccctgtggt accctggcag agaagagacc aagcttgttt 1860 ccctgctggc caaagtcagt aggagaggat gcacagtttg ctatttgctt tagagacagg 1920 gactgtataa acaa9cctaa cattggtgca aagattgcct cttgaaaaaa aaaaaaa 1977 <210> 6 <21l> 476 <212> PRT <213> Homo sapiens <400> 6 Met Ala G1n Ala Leu Pro Trp Leu Leu Leu Trp Met Gly Ala Gly Val 1 5 10 15 Leu Pro Ala His Gly Thr G1n His Gly Ile Arg Leu Pro Leu Arg Ser 25 30 Gly Leu Gly Gly Ala Pro Leu Gly Leu Arg Leu Pro Arg Glu Thr Asp 40 45 Glu Glu Pro Glu Glu Pro Gly Arg Arg Gly Ser Phe Val Glu Met Val 55 60 Asp Asn Leu Arg Gly Lys Ser Gly G1n Gly Tyr Tyr Val Glu Met Thr 70 75 80 Val Gly Ser Pro Pro G1n Thr Leu Asn Ile Leu Val Asp Thr Gly Ser 90 95 Ser Asn Phe Ala Val Gly Ala Ala Pro His Pro Phe Leu His Arg Tyr 105 110 Tyr G1n Arg G1n Leu Ser Ser Thr Tyr Arg Asp Leu Arg Lys Gly Val 120 125 Tyr Val Pro Tyr Thr G1n Gly Lys Trp Glu Gly Glu Leu Gly Thr Asp 135 140 Leu Val Ser Ile Pro His Gly Pro Asn Val Thr Val Arg Ala Asn lle 150 155 160 Ala Ala Ile Thr Glu Ser Asp Lys Phe Phe Ile Asn Gly Ser Asn Trp 170 175 Glu Gly Ile Leu Gly Leu Ala Tyr Ala Glu Ile Ala Arg Leu Cys Gly 185 190 Ala Gly Phe Pro Leu Asn G1n Ser Glu Val Leu Ala Ser Val Gly Gly 200 205 Ser Met Ile Ile Gly Gly Ile Asp His Ser Leu Tyr Thr Gly Ser Leu 210 215 220 Trp Tyr Thr Pro Ile Arg Arg Glu Trp Tyr Tyr Glu Val Ile Ile Val 225 230 235 240 Arg Val Glu Ile Asn Gly G1n Asp Leu Lys Met Asp Cys Lys Glu Tyr 245 250 255 Asn Tyr Asp Lys Ser Ile Val Asp Ser Gly Thr Thr Asn Leu Arg Leu 260 265 270 Pro Lys Lys Val Phe Glu Ala Ala Val Lys Ser Ile Lys Ala Ala Ser 275 280 285 Ser Thr Glu Lys Phe Pro Asp Gly Phe Trp Leu Gly Glu G1n Leu Val 290 295 300 Cys Trp G1n Ala Gly Thr Thr Pro Trp Asn Ile Phe Pro Val Ile Ser 305 310 315 320 Leu Tyr Leu Met Gly Glu Val Thr Asn G1P Ser Phe Arg Ile Thr Ile &c> 2- 325 330 335 Leu Pro G1n G1n Tyr Leu Arg Pro Val Glu Asp Val Ala Thr Ser G1n 340 345 350 Asp Asp Cys Tyr Lys Phe Ala Ile Ser G1n Ser Ser Thr Gly Thr Val 355 360 365 Met Gly Ala Val Ile Met Glu Gly Phe Tyr Val Val Phe Asp Arg Ala 370 375 380 Arg Lys Arg Ile Gly Phe Ala Val Ser Ala Cys His Val His Asp Glu 385 390 395 400 Phe Arg Thr Ala Ala Val Glu Gly Pro Phe Val Thr Leu Asp Met Glu 405 410 415 Asp Cys Gly Tyr Asn Ile Pro G1n Thr Asp Glu Ser Thr Leu Met Thr 420 425 430 Ile Ala Tyr Val Met Ala Ala Ile Cys Ala Leu Phe Met Leu Pro Leu 435 440 445 Cys Leu Met Val Cys G1n Trp Arg Cys Leu Arg Cys Leu Arg G1n G1n 450 455 460 His Asp Asp Phe Ala Asp Asp Ile Ser Leu Leu Lys 465 470 475 <210> 7 <21l> 2043 <212> DNA <213> Mus musculus <400> 7 atggccccag cgctgcactg gctcctgcta tgggtgggct cgggaatgct gcctgcccag 60 ggaacccatc tcggcatccg gctgcccctt cgcagcggcc tggcagggcc acccctgggc 120 ctgaggctgc cccgggagac tgacgaggaa tcggaggagc ctggccggag aggcagcttt 180 gtggagatgg tggacaacct gaggggaaag tccggccagg gctactatgt ggagatgacc 240 gtaggcagcc ccccacagac gctcaacatc ctggtggaca cgggcagtag taactttgca 300 gtgggggctg ccccacaccc tttcctgcat cgctactacc agaggcagct gtccagcaca 360 tatcgagacc tccgaaaggg tgtgtatgtg ccctacaccc agggcaagtg ggagggggaa 420 ctgggcaccg acctggtgag catccctcat ggccccaacg tcactgtgcg tgccaacatt 480 gctgccatca ctgaatcgga caagttcttc atcaatggtt ccaactggga gggcatccta 540 gggctggcct atgetgagat tgccaggccc gacgactctt tggagccctt ctttgactcc 600 ctggtgaagc agacccacat tcccaacatc ttttccctgc agctctgtgg cgctggcttc 660 cccctcaacc agaccgaggc actggcctcg gtgggaggga gcatgatcat tggtggtatc 720 gaccactcgc tatacacggg cagtctctgg tacacaccca tccggcggga gtggtattat 780 gaagtgatca ttgtacgtgt ggaaatcaat ggtcaagatc tcaagatgga ctgcaaggag 840 tacaactacg acaagagcat tgtggacagt gggaccacca accttcgctt gcccaagaaa 900 gtatttgaag ctgccgtcaa gtccatcaag gcagcctcct cgacggagaa gttcccggat 960 ggcttttggc taggggagca gctggtgtgc tggcaagcag gcacgacccc ttggaacatt 1020 ttcccagtca tttCacttta cctcatgggt gaagtcacca atcagtcctt ccgcatcacc 1080 atccttcctc agcaatacct acggccggtg gaggacgtgg ccacgtccca agacgactgt 1140 tacaagttcg ctgtctcaca gtcatccacg ggcactgtta tgggagccgt catcatggaa 1200 ggtttctatg tcgtcttcga tcgagcccga aagcgaattg gctttgctgt cagcgcttgc 1260 catgtgcacg atgagttcag gacggcggca gtggaaggtc cgtttgttac ggcagacatg 1320 gaagactgtg gctacaacat tccccagaca gatgagtcaa cacttatgac catagcctat 1380 gtcatggcgg ccatctgcgc cctcttcatg ttgccactct gcctcatggt atgtcagtgg 1440 cgctgcctgc gttgcctgcg ccaccagcac gatgactttg ctgatgacat ctccctgctc 1500 i02> aagtaaggag gctcgtgggc agatgatgga gacgcccctg gaccacatct gggtggttcc 1560 ctttggtcac atgagttgga gctatggatg gtacctgtgg ccagagcacc tcaggaccct 1620 caccaacctg ccaatgcttc tggcgtgaca gaacagagaa atcaggcaag ctggattaca 1680 gggcttgcac ctgtaggaca caggagaggg aaggaa9cag cgttctggtg gcaggaatat 1740 ccttaggcac cacaaacttg agttggaaat tttgctgctt gaagcttcag ccctgaccct 1800 ctgcccagca tcctttagag tctccaacct aaagtattct ttatgtcctt ccagaagtac 1860 tggcgtcata ctcaggetac ccggcatgtg tccctgtggt accctggcag agaaagggcc 1920 aatctcattc cctgctggcc aaagtcagca gaagaaggtg aagtttgeca gttgctttag 1980 tgatagggac tgcagaetca agectacact ggtacaaaga ctgcgtcttg agataaacaa 2040 gaa 2043 <210> 8 <21l> 501 <212> PRT <213> Mus musculus <400> 8 Met Ala Pro Ala Leu His Trp Leu Leu Leu Trp Val Gly Ser Gly Met 1 5 10 is Leu Pro Ala G1n Gly Thr His Leu Gly Ile Arg Leu Pro Leu Arg Ser 25 30 Gly Leu Ala Gly Pro Pro Leu Gly Leu Arg Leu Pro Arg Glu Thr Asp 40 45 Glu Glu Ser Glu Glu Pro Gly Arg Arg Gly Ser Phe Val Glu Met Val 55 60 Asp Asn Leu Arg Gly Lys Ser Gly Gin Gly Tyr Tyr Val Glu Met Thr 70 75 80 Val Gly Ser Pro Pro G1n Thr Leu Asn lle Leu Val Asp Thr Gly Ser 90 95 Ser Asn Phe Ala Val Gly Ala Ala Pro His Pro Phe Leu His Arg Tyr 105 110 Tyr G1n Arg Gin Leu Ser Ser Thr Tyr Arg Asp Leu Arg Lys Gly Val 120 125 Tyr Val Pro Tyr Thr G1n Gly Lys Trp Glu Gly Glu Leu Gly Thr Asp 135 140 Leu Val Ser Ile Pro His Gly Pro Asn Val Thr Val Arg Ala Asn Ile 150 155 160 Ala Ala Ile Thr Glu Ser Asp Lys Phe Phe Ile Asn Gly Ser Asn Trp 170 175 Glu Gly Ile Leu Gly Leu Ala Tyr Ala Glu lle Ala Arg Pro Asp Asp 185 190 Ser Leu Glu Pro Phe Phe Asp Ser Leu Val Lys G1n Thr His lle Pro 200 205 Asn Ile Phe Ser Leu G1n Leu Cys Gly Ala Gly Phe Pro Leu Asn G1n 210 215 220 Thr Glu Ala Leu Ala Ser Val Gly Gly Ser Met Ile Ile Gly Gly lle 225 230 235 240 )0(-k- Asp His Ser Leu Tyr Thr Gly Ser Leu Trp Tyr Thr Pro Ile Arg Arg 245 250 255 Glu Trp Tyr Tyr Glu Val Ile Ile Val Arg Val Glu Ile Asn Gly G1n 260 265 270 Asp Leu Lys Met Asp Cys Lys Glu Tyr Asn Tyr Asp Lys Ser Ile Val 275 280 285 Asp Ser Gly Thr Thr Asn Leu Arg Leu Pro Lys Lys Val Phe Glu Ala 290 295 300 Ala Val Lys Ser Ile Lys Ala Ala Ser Ser Thr Glu Lys Phe Pro Asp 305 310 315 320 Gly Phe Trp Leu Gly Glu G1n Leu Val Cys Trp G1n Ala Gly Thr Thr 325 330 335 Pro Trp Asn Ile Phe Pro Val lle Ser Leu Tyr Leu Met Gly Glu Val 340 345 350 Thr Asn G1n Ser Phe Arg Ile Thr Ile Leu Pro G1n G1n Tyr Leu Arg 355 360 365 Pro Val Glu Asp Val Ala Thr Ser G1n Asp Asp Cys Tyr Lys Phe Ala 370 375 380 Val Ser G1n Ser Ser Thr Gly Thr Val Met Gly Ala Val Ile Met Glu 385 390 395 400 Gly Phe Tyr Val Val Phe Asp Arg Ala Arg Lys Arg Ile Gly Phe Ala 405 410 415 Val Ser Ala Cys His Val His Asp Glu Phe Arg Thr Ala Ala Val Glu 420 425 430 Gly Pro Phe Val Thr Ala Asp Met Glu Asp Cys Gly Tyr Asn Ile Pro 435 440 445 G1n Thr Asp Glu Ser Thr Leu Met Thr Ile Ala Tyr Val Met Ala Ala 450 455 460 Ile Cys Ala Leu Phe Met Leu Pro Leu Cys Leu Met Val Cys G1n Trp 465 470 475 480 Arg Cys Leu Arg Cys Leu Arg His G1n His Asp Asp Phe Ala Asp Asp 485 490 495 Ile Ser Leu Leu Lys 500 <210> 9 <21l> 2088 <212> DNA <213> Homo sapiens <400> 9 atgctgcccg gtttggcact gctcctgctg gccgcctgga cggctcgggc gctggaggta 60 cccactgatg gtaatgctgg cctgctggct gaaccccaga ttgccatgtt ctgtggcaga 120 It> 5 ctgaacatgc acatgaatgt ccagaatggg aagtgggatt cagatccatc agggaccaaa 180 acctgcattg ataccaagga aggcatcctg cagtattgcc aagaagtcta ccctgaactg 240 cagatcacca atgtggtaga agccaaccaa ccagtgacca tccagaactg gtgcaagcgg 300 ggccgcaagc agtgcaagac ccatccccac tttgtgattc cctaccgctg cttagttggt 360 gagtttgtaa gtgatgccct tctcgttcct gacaagtgca aattcttaca ccaggagagg 420 atggatgttt gcgaaactca tcttcactgg cacaccgtcg ccaaagagac atgcagtgag 480 aagagtacca acttgcatga ctacggcatg ttgctgccct gcggaattga caagttccga 540 ggggtagagt ttgtgtgttg cccactggct gaagaaagtg acaatgtgga ttctgctgat 600 gcggaggagg atgactcgga tgtctggtgg ggcggagcag acacagacta tgcagatggg 660 agtgaagaca aagtagtaga agtagcagag gaggaagaag tggctgaggt ggaagaagaa 720 gaagccgatg atgacgagga cgatgaggat ggtgatgagg tagaggaaga ggctgaggaa 780 ccctacgaag aagccacaga gagaaccacc agcattgcca ccaccaccac caccaccaca 840 gagtctgtgg aagaggtggt tcgagttcct acaacagcag ccagtaeccc tgatgcegtt 900 gacaagtatc tcgagacacc tggggatgag aatgaacatg cccatttcca gaaagecaaa 960 gagaggcttg aggccaagca ccgagagaga atgtcecagg tcatgagaga atgggaagag 1020 gcagaacgtc aagcaaagaa cttgcctaaa gctgataaga aggcagttat ccagcatttc 1080 caggagaaag tggaatcttt ggaacaggaa gcagccaacg agagacagca gctggtggag 1140 acacacatgg ccagagtgga agccatgctc aatgaccgcc gccgcctggc cctggagaac 1200 tacatcaccg ctctgcaggc tgttcctcct cggcctcgtc acgtgttcaa tatgctaaag 1260 aagtatgtcc gcgcagaaca gaaggacaga cagcacaccc taaagcattt cgagcatgtg 1320 cgcatggtgg atcccaagaa agccgctcag atccggtccc aggttatgac acacctccgt 1380 gtgatttatg agcgeatgaa tcagtctctc tccctgctct acaacgtgcc tgcagtggcc 1440 gaggagattc aggatgaagt tgatgagctg cttcagaaag agcaaaacta ttcagatgac 1500 gtcttggcca acatgattag tgaaccaagg atcagttacg gaaacgatgc tctcatgcca 1560 tctttgaccg aaacgaaaac caccgtggag ctccttcccg tgaatggaga gttcagectg 1620 gacgatctce agccgtggca ttcttttggg gctgactctg tgccagccaa cacagaaaac 1680 gaagttgagc ctgttgatgc ccgccctgct gccgaccgag gactgaccac tcgaccaggt 1740 tctgggttga caaatatcaa gacggaggag atctctgaag tgaagatgga tgcagaattc 1800 cgacatgact caggatatga agttcatcat caaaaattgg tgttctttgc agaagatgtg 1860 ggttcaaaca aaggtgcaat cattggactc atggtgggcg gtgttgteat agcgacagtg 1920 atcgtcatca ccttggtgat gctgaagaag aaacagtaca catccattca tcatggtgtg 1980 gtggaggttg acgccgctgt caccccagag gagcgccacc tgtccaagat gcagcagaac 2040 ggctacgaaa atccaaccta caagttcttt gagcagatgc agaactag 2088 <210> 10 <21l> 695 <212> PRT <213- Homo sapiens <400> 10 Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15 Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 25 30 G1n Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val G1n 40 45 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp so 55 60 Thr Lys Glu Gly Ile Leu G1n Tyr Cys G1n Glu Val Tyr Pro Glu Leu 70 75 80 G1n Ile Thr Asn Val Val Glu Ala Asn G1n Pro Val Thr Ile G1n Asn 90 95 Trp Cys Lys Arg Gly Arg Lys G1n Cys Lys Thr His Pro His Phe Val 105 110 Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu lpG 120 125 Val Pro Asp Lys Cys Lys Phe Leu His G1n Glu Arg Met Asp Val Cys 135 140 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu 150 155 160 Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly lle 170 175 Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 185 190 Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 200 205 Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220 Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240 Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255 Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265 270 Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285 Val Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu 290 295 300 Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe G1n Lys Ala Lys 305 310 315 320 Glu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser G1n Val Met Arg 325 330 335 Glu Trp Glu Glu Ala Glu Arg G1n Ala Lys Asn Leu Pro Lys Ala Asp 340 345 350 Lys Lys Ala Val Ile G1n His Phe G1n Glu Lys Val Glu Ser Leu Glu 355 360 365 G1n Glu Ala Ala Asn Glu Arg G1n G1n Leu Val Glu Thr His Met Ala 370 375 380 Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn 385 390 395 400 Tyr Ile Thr Ala Leu G1n Ala Val Pro Pro Arg Pro Arg His Val Phe 405 410 415 Asn Met Leu Lys Lys Tyr Val Arg Ala Glu G1n Lys Asp Arg G1n His 420 425 430 Thr Leu Lys His Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala 435 440 445 i,c>-1 Ala G1n Ile Arg Ser G1n Val Met Thr His Leu Arg Val Ile Tyr Glu 450 455 460 Arg Met Asn G1n Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala 465 470 475 480 Glu Glu Ile G1n Asp Glu Val Asp Glu Leu Leu G1n Lys Glu G1n Asn 485 490 495 Tyr Ser Asp Asp Val Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser 500 505 510 Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr 515 520 525 Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu G1n 530 535 540 Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn 545 550 555 560 Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr 565 570 575 Thr Arg Pro Gly Ser Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser 580 585 590 Glu Val Lys Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val 595 600 605 His His G1n Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys 610 615 620 Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val 625 630 635 640 Ile Val Ile Thr Leu Val Met Leu Lys Lys Lys G1n Tyr Thr Ser Ile 645 650 655 His His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg 660 665 670 His Leu Ser Lys Met G1n G1n Asn Gly Tyr Glu Asn Pro Thr Tyr Lys 675 680 685 Phe Phe Glu G1n Met G1n Asn 690 695 <210> 11 <21l> 2088 <212> DNA <213> Homo sapiens <400> 11 atgctgcccg gtttggcact gctcctgctg gccgcctgga cggctcgggc gctggaggta 60 cccactgatg gtaatgctgg cctgctggct gaaccccaga ttgccatgtt ctgtggcaga 120 ctgaacatgc acatgaatgt ccagaatggg aagtgggatt cagatccatc agggaccaaa 180 acctgcattg ataccaagga aggcatcctg cagtattgcc aagaagtcta ccctgaactg 240 cagatcacca atgtggtaga agccaaccaa ccagtgacca tccagaactg gtgcaagcgg 300 (lc>2; ggccgcaagc agtgcaagac ccatccccac tttgtgattc cctaccgctg cttagttggt 360 gagtttgtaa gtgatgccct tctcgttcct gacaagtgca aattcttaca ccaggagagg 420 atggatgttt gcgaaactca tcttcactgg cacaccgtcg ccaaagagac atgcagtgag 480 aagagtacca acttgcatga ctacggcatg ttgctgccct gcggaattga caagttccga 540 ggggtagagt ttgtgtgttg cccactggct gaagaaagtg acaatgtgga ttctgctgat 600 gcggaggagg atgactcgga tgtctggtgg ggcggagcag acacagacta tgcagatggg 660 agtgaagaca aagtagtaga agtagcagag gaggaagaag tggctgaggt ggaagaagaa 720 gaagccgatg atgacgagga cgatgaggat ggtgatgagg tagaggaaga ggctgaggaa 780 ccctacgaag aagccacaga gagaaccacc agcattgcca ccaccaccac caccaccaca 840 gagtctgtgg aagaggtggt tcgagttcct acaacagcag ccagtacccc tgatgccgtt 900 gacaagtatc tcgagacacc tggggatgag aatgaacatg cccatttcca gaaagccaaa 960 gagaggcttg aggccaagca ccgagagaga atgtcccagg tcatgagaga atgggaagag 1020 gcagaacgtc aagCaaagaa cttgcctaaa gctgataaga aggcagttat ccagcatttc 1080 caggagaaag tggaatcttt ggaacaggaa gcagccaacg agagacagca gctggtggag 1140 acacacatgg ccagagtgga agccatgctc aatgaccgcc gccgcctggc cctggagaac 1200 tacatcaccg ctctgcaggc tgttcctcct cggcctcgtc acgtgttcaa tatgctaaag 1260 aagtatgtcc gcgcagaaca gaaggacaga cagcacaccc taaagcattt cgagcatgtg 1320 cgcatggtgg atcccaagaa agccgctcag atccggtccc aggttatgac acacctccgt 1380 gtgatttatg agcgcatgaa tcagtctctc tccctgctct acaacgtgcc tgcagtggcc 1440 gaggagattc aggatgaagt tgatgagctg cttcagaaag agcaaaacta ttcagatgac 1500 gtcttggcca acatgattag tgaaccaagg atcagttacg gaaacgatgc tctcatgcca 1560 tctttgaccg aaacgaaaac caccgtggag ctccttcccg tgaatggaga gttcagcctg 1620 gacgatctcc agccgtggca ttcttttggg gctgactctg tgccagccaa cacagaaaac 1680 gaagttgagc ctgttgatgc ccgccctgct gccgaccgag gactgaccac tcgaccaggt 1740 tctgggttga caaatatcaa gacggaggag atctctgaag tgaatctgga tgcagaattc 1800 cgacatgactcaggatatga agttcatcat caaaaattgg tgttctttgc agaagatgtg 1860 ggttcaaaca aaggtgcaat cattggactc atggtgggcg gtgttgtcat agcgacagtg 1920 atcgtcatca ccttggtgat gctgaagaag aaacagtaca catccattca tcatggtgtg 1980gtggaggttg acgccgctgt caccccagag gagcgccacc tgtccaagat gcagcagaac 2040 ggctacgaaa atccaaccta caagttcttt gagcagatgc agaactag 2088 <210> 12 <21l> 695 <212> PRT <213> Homo sapiens <400> 12 Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15 Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 25 30 G1n Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val G1n 40 45 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp 55 60 Thr Lys Glu Gly Ile Leu G1n Tyr Cys G1n Glu Val Tyr Pro Glu Leu 70 75 80 G1n Ile Thr Asn Val Val Glu Ala Asn G1n Pro Val Thr Ile G1n Asn 90 95 Trp Cys Lys Arg Gly Arg Lys G1n Cys Lys Thr His Pro His Phe Val 105 110 Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 120 125 Val Pro Asp Lys Cys Lys Phe Leu His G1n Glu Arg Met Asp Val Cys 135 140 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu 150 155 160 Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile 170 175 Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 185 190 Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 200 205 Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220 Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240 Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255 Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265 270 Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285 Val Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu 290 295 300 Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe G1n Lys Ala Lys 305 310 315 320 Glu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser G1n Val Met Arg 325 330 335 Glu Trp Glu Glu Ala Glu Arg G1n Ala Lys Asn Leu Pro Lys Ala Asp 340 345 350 Lys Lys Ala Val Ile G1n His Phe G1n Glu Lys Val Glu Ser Leu Glu 355 360 365 G1n Glu Ala Ala Asn Glu Arg G1n G1n Leu Val Glu Thr His Met Ala 370 375 380 Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn 385 390 395 400 Tyr Ile Thr Ala Leu G1n Ala Val Pro Pro Arg Pro Arg His Val Phe 405 410 415 Asn Met Leu Lys Lys Tyr Val Arg Ala Glu G1n Lys Asp Arg G1n His 420 425 430 Thr Leu Lys His Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala 435 440 445 Ala G1n Ile Arg Ser G1n Val Met Thr His Leu Arg Val Ile Tyr Glu 450 455 460 j I c Arg Met Asn G1n Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala 465 470 475 480 Glu Glu Ile G1n Asp Glu Val Asp Glu Leu Leu G1n Lys Glu G1n Asn 485 490 495 Tyr Ser Asp Asp Val Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser 500 505 510 Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr 515 520 525 Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu G1n 530 535 540 Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn 545 550 555 560 Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr 565 570 575 Thr Arg Pro Gly Ser Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser 580 585 590 Glu Val Asn Leu Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val 595 600 605 His His G1n Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys 610 615 620 Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val 625 630 635 640 Ile Val Ile Thr Leu Val Met Leu Lys Lys Lys G1n Tyr Thr Ser Ile 645 650 655 His His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg 660 665 670 His Leu Ser Lys Met G1n G1n Asn Gly Tyr Glu Asn Pro Thr Tyr Lys 675 680 685 Phe Phe Glu G1n Met G1n Asn 690 695 <210> 13 <21l> 2088 <212> DNA <213> Homo sapiens <400> 13 atgctgcccg gtttggcact gctcctgctg gccgcctgga cggctcgggc gctggaggta 60 cccactgatg gtaatgctgg cctgctggct gaaccccaga ttgccatgtt ctgtggcaga 120 ctgaacatgc acatgaatgt ccagaatggg aagtgggatt cagatccatc agggaccaaa 180 acctgcattg ataccaagga aggcatcctg cagtattgcc aagaagtcta ccctgaactg 240 cagatcacca atgtggtaga agccaaccaa ccagtgacca tccagaactg gtgcaagcgg 300 ggccgcaagc agtgcaagac ccatccccac tttgtgattc cctaccgctg cttagttggt 360 gagtttgtaa gtgatgccct tctcgttcct gacaagtgca aattcttaca ccaggagagg 420 atggatgttt gcgaaactca tcttcactgg cacaccgtcg ccaaagagac atgcagtgag 480 (11 aagagtacca acttgcatga ctacggcatg ttgctgccct gcggaattga caagttccga 540 ggggtagagt ttgtgtgttg cccactggct gaagaaagtg acaatgtgga ttctgctgat 600 gcggaggagg atgactcgga tgtctggtgg ggcggagcag acacagacta tgcagatggg 660 agtgaagaca aagtagtaga agtagcagag gaggaagaag tggctgaggt ggaagaagaa 720 gaagccgatg atgacgagga cgatgaggat ggtgatgagg tagaggaaga ggctgaggaa 780 ccctacgaag aagccacaga gagaaccacc agcattgcca ccaccaccac caccaccaca 840 gagtctgtgg aagaggtggt tcgagttcct acaacagcag ecagtaeccc tgatgccgtt 900 gacaagtatc tcgagacacc tggggatgag aatgaacatg cceatttcca gaaagccaaa 960 gagaggcttg aggccaagca ccgagagaga atgtcccagg tcatgagaga atgggaagag 1020 gcagaacgtc aagcaaagaa cttgcctaaa gctgataaga aggcagttat ccagcatttc 1080 caggagaaag tggaatcttt ggaacaggaa gcagccaacg agagacagca gctggtggag 1140 acacacatgg ccagagtgga agccatgetc aatgaccgcc gccgcctggc cctggagaac 1200 tacatcaccg ctctgcaggc tgttcctcct cggcctcgtc acgtgttcaa tatgctaaag 1260 aagtatgtcc gcgcagaaca gaaggacaga cagcacaccc taaagcattt cgagcatgtg 1320 cgcatggtgg atcccaagaa agccgctcag atccggtccc aggttatgac acacctccgt 1380 gtgatttatg agcgcatgaa tcagtctctc tccctgctct acaacgtgcc tgcagtggcc 1440 gaggagattc aggatgaagt tgatgagctg cttcagaaag agcaaaacta ttcagatgac 1500 gtcttggcca acatgattag tgaaccaagg atcagttacg gaaacgatgc tctcatgcca 1560 tctttgaccg aaacgaaaac caccgtggag ctccttcccg tgaatggaga gttcagcctg 1620 gacgatctcc agccgtggca ttcttttggg gctgactctg tgccagecaa cacagaaaac 1680 gaagttgagc ctgttgatgc ccgccctgct gccgaccgag gactgaccac tcgaccaggt 1740 tctgggttga caaatatcaa gacggaggag atctctgaag tgaagatgga tgcagaattc 1800 cgacatgact caggatatga agttcatcat caaaaattgg tgttctttgc agaagatgtg 1860 ggttcaaaca aaggtgcaat cattggactc atggtgggcg gtgttgtcat agcgacagtg 1920 atcttcatca ccttggtgat gctgaagaag aaacagtaca catccattca tcatggtgtg 1980 gtggaggttg acgccgctgt caccccagag gagcgccacc tgtccaagat gcagcagaac 2040 ggctacgaaa atccaaccta caagttcttt gagcagatgc agaactag 2088 <210> 14 <21l> 695 <212> PRT <213> Homo sapiens <400> 14 Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15 Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 25 30 G1n Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val G1n 40 45 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp 55 60 Thr Lys Glu Gly Ile Leu G1n Tyr Cys G1n Glu Val Tyr Pro Glu Leu 70 75 80 G1n Ile Thr Asn Val Val Glu Ala Asn G1n Pro Val Thr Ile G1n Asn 90 95 Trp Cys Lys Arg Gly Arg Lys G1n Cys Lys Thr His Pro His Phe Val 105 110 Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 120 125 Val Pro Asp Lys Cys Lys Phe Leu His G1n Glu Arg Met Asp Val Cys 135 140 1 x 2- Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu 150 155 160 Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile 170 175 Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 185 190 Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 200 205 Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220 Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240 Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255 Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265 270 Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285 Val Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu 290 295 300 Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe G1n Lys Ala Lys 305 310 315 320 Glu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser G1n Val Met Arg 325 330 335 Glu Trp Glu Glu Ala Glu Arg G1n Ala Lys Asn Leu Pro Lys Ala Asp 340 345 350 Lys.Lys Ala Val Ile G1n His Phe G1n Glu Lys Val Glu Ser Leu Glu 355 360 365 G1n Glu Ala Ala Asn Glu Arg G1n G1n Leu Val Glu Thr His Met Ala 370 375 380 Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn 385 390 395 400 Tyr Ile Thr Ala Leu G1n Ala Val Pro Pro Arg Pro Arg His Val Phe 405 410 415 Asn Met Leu Lys Lys Tyr Val Arg Ala Glu G1n Lys Asp Arg G1n His 420 425 430 Thr Leu Lys His Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala 435 440 445 Ala G1n Ile Arg Ser G1n Val Met Thr His Leu Arg Val Ile Tyr Glu 450 455 460 Arg Met Asn G1n Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala lib 465 470 475 480 Glu Glu Ile G1n Asp Glu Val Asp Glu Leu Leu G1n Lys Glu G1n Asn 485 490 495 Tyr Ser Asp Asp Val Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser 500 505 510 Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr 515 520 525 Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu G1n 530 535 540 Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn 545 550 555 560 Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr 565 570 575 Thr Arg Pro Gly Ser Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser 580 585 590 Glu Val Lys Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val 595 600 605 His His G1n Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys 610 615 620 Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val 625 630 635 640 Ile Phe Ile Thr Leu Val Met Leu Lys Lys Lys G1n Tyr Thr Ser Ile 645 650 655 His His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg 660 665 670 His Leu Ser Lys Met G1n G1n Asn Gly Tyr Glu Asn Pro Thr Tyr Lys 675 680 685 Phe Phe Glu G1n Met G1n Asn 690 695 <210> 15 <21l> 2094 <212> DNA <213> Homo sapiens <400> 15 atgctgcccg gtttggcact gctcctgctg gccgcctgga cggctcgggc gctggaggta 60 cccactgatg gtaatgctgg cctgctggct gaaccccaga ttgccatgtt ctgtggcaga 120 ctgaacatgc acatgaatgt ccagaatggg aagtgggatt cagatccatc agggaccaaa 180 acctgcattg ataccaagga aggcatcctg cagtattgcc aagaagtcta ccctgaactg 240 cagatcacca atgtggtaga agccaaccaa ccagtgacca tccagaactg 9tgcaagcgg 300 ggccgcaagc agtgcaagac ccatccccac tttgtgattc cctaccgctg cttagttggt 360 gagtttgtaa gtgatgccct tctcgttcct gacaagtgca aattcttaca ccaggagagg 420 atggatgttt gcgaaactca tcttcactgg cacaccgtcg ccaaagagac atgcagtgag 480 aagagtacca acttgcatga ctacggcatg ttgctgccct gcggaattga caagttccga 540 ggggtagagt ttgtgtgttg cccactggct gaagaaagtg acaatgtgga ttctgctgat 600 114 gcggaggagg atgactcgga tgtctggtgg ggcggagcag acacagacta tgcagatggg 660 agtgaagaca aagtagtaga agtagcagag gaggaagaag tggctgaggt ggaagaagaa 720 gaagccgatg atgacgagga cgatgaggat ggtgatgagg tagaggaaga ggctgaggaa 780 ccctacgaag aagccacaga gagaaccacc agcattgcca ccaccaccac caccaccaca 840 gagtctgtgg aagaggtggt tcgagttcct acaacagcag ccagtacccc tgatgccgtt 900 gacaagtatc tcgagacacc tggggatgag aatgaacatg cccatttcca gaaagccaaa 960 gagaggcttg aggccaagca ccgagagaga atgtcccagg tcatgagaga atgggaagag 1020 gcagaacgtc aagcaaagaa cttgcctaaa gctgataaga aggcagttat ccagcatttc 1080 caggagaaag tggaatcttt ggaacaggaa gcagccaacg agagacagca gctggtggag 1140 acacacatgg ccagagtgga agccatgctc aatgaccgcc gccgcctggc cctggagaac 1200 tacatcaccg ctctgcaggc tgttcctcct cggcctcgtc acgtgttcaa tatgctaaag 1260 aagtatgtcc gcgcagaaca gaaggacaga cagcacaccc taaagcattt cgagcatgtg 1320 cgcatggtgg atcccaagaa agccgctcag atccggtccc aggttatgac acacctccgt 1380 gtgatttatg agcgcatgaa tcagtctctc tccctgctct acaacgtgcc tgcagtggcc 1440 gaggagattc aggatgaagt tgatgagctg cttcagaaag agcaaaacta ttcagatgac 1500 gtcttggcca acatgattag tgaaccaagg atcagttacg gaaacgatgc tctcatgcea 1560 tctttgaccg aaacgaaaac caccgtggag ctccttcccg tgaatggaga gttcagcctg 1620 gacgatctcc agccgtggca ttcttttggg gctgactctg tgccagccaa.cacagaaaac 1680 gaagttgagc ctgttgatgc ccgccctgct gccgaccgag gactgaccac tcgaccaggt 1740 tctgggttga caaatatcaa gacggaggag atctctgaag tgaagatgga tgcagaattc 1800 cgacatgact caggatatga agttcatcat caaaaattgg tgttctttgc agaagatgtg 1860 ggttcaaaca aaggtgcaat cattggactc atggtgggcg gtgttgtcat agcgacagtg 1920 atcgtcatca ccttggtgat gctgaagaag aaacagtaca catccattca tcatggtgtg 1980 gtggaggttg acgccgctgt caccccagag gagcgccacc tgtccaagat gcagcagaac 2040 ggctacgaaa atccaaccta caagttcttt gagcagatgc agaacaagaa gtag 2094 <210> 16 <21l> 697 <212> PRT <213> Homo sapiens <400> 16 Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15 Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 25 30 G1n Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val G1n 40 45 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp 55 60 Thr Lys Glu Gly Ile Leu G1n Tyr Cys G1n Glu Val Tyr Pro Glu Leu 70 75 80 G1n Ile Thr Asn Val Val Glu Ala Asn G1n Pro Val Thr Ile G1n Asn 90 95 Trp Cys Lys Arg Gly Arg Lys G1n Cys Lys Thr His Pro His Phe Val 105 110 Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 120 125 Val Pro Asp Lys Cys Lys Phe Leu His G1n Glu Arg Met Asp Val Cys 135 140 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu 150 155 160 Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile 170 175 Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 185 190 Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 200 205 Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220 Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240 Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255 Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265 270 Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285 Val Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu 290 295 300 Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe G1n Lys Ala Lys 305 310 315 320 Glu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser G1n Val Met Arg 325 330 335 Glu Trp Glu Glu Ala Glu Arg G1n Ala Lys Asn Leu Pro Lys Ala Asp 340 345 350 Lys Lys Ala Val Ile G1n His Phe G1n Glu Lys Val Glu Ser Leu Glu 355 360 365 G1n Glu Ala Ala Asn Glu Arg G1n G1n Leu Val Glu Thr His Met Ala 370 375 380 Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn 385 390 395 400 Tyr Ile Thr Ala Leu G1n Ala Val Pro Pro Arg Pro Arg His Val Phe 405 410 415 Asn Met Leu Lys Lys Tyr Val Arg Ala Glu G1n Lys Asp Arg G1n His 420 425 430 Thr Leu Lys His Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala 435 440 445 Ala G1n Ile Arg Ser G1n Val Met Thr His Leu Arg Val lle Tyr Glu 450 455 460 Arg Met Asn G1n Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala 465 470 475 480 Glu Glu Ile G1n Asp Glu Val Asp Glu Leu Leu G1n Lys Glu G1n Asn 1 k 6, 485 490 495 Tyr Ser Asp Asp Val Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser 500 505 510 Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr 515 520 525 Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu G1n 530 535 540 Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn 545 550 555 560 Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr 565 570 575 Thr Arg Pro Gly Ser Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser 580 585 590 Glu Val Lys Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val 595 600 605 His His G1n Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys 610 615 620 Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val 625 630 635 640 Ile Val Ile Thr Leu Val Met Leu Lys Lys Lys G1n Tyr Thr Ser Ile 645 650 655 His His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg 660 665 670 His Leu Ser Lys Met G1n G1n Asn Gly Tyr Glu Asn Pro Thr Tyr Lys 675 680 685 Phe Phe Glu G1n Met G1n Asn Lys Lys 690 695 <210> 17 <21l> 2094 <212> DNA <213> Homo sapiens <400> 17 atgctgcccg gtttggcact gctcctgctg gccgcctgga cggctcgggc gctggaggta 60 cccactgatg gtaatgctgg cctgctggct gaaccccaga ttgccatgtt ctgtggcaga 120 ctgaacatgc acatgaatgt ccagaatggg aagtgggatt cagatccatc agggaccaaa 180 acctgcattg ataccaagga aggcatcctg cagtattgcc aagaagtcta ccctgaactg 240 cagatcacca atgtggtaga agccaaccaa ccagtgacca tccagaactg gtgcaagcgg 300 ggccgcaagc agtgcaagac ccatccccac tttgtgattc cctaccgctg cttagttggt 360 gagtttgtaa gtgatgccct tctcgttcct gacaagtgca aattcttaca ccaggagagg 420 atggatgttt gcgaaactca tcttcactgg cacaccgtcg ccaaagagac atgcagtgag 480 aagagtacca acttgcatga ctacggcatg ttgctgccct gcggaattga caagttccga 540 ggggtagagt ttgtgtgttg cccactggct gaagaaagtg acaatgtgga ttctgctgat 600 gcggaggagg atgactcgga tgtctggtgg ggcggagcag acacagacta tgcagatggg 660 agtgaagaca aagtagtaga agtagcagag gaggaagaag tggctgaggt ggaagaagaa 720 gaagccgatg atgacgagga cgatgaggat ggtgatgagg tagaggaaga ggctgaggaa 780 (C] ccctacgaag aagccacaga gagaaccacc agcattgcca ccaccaccac caccaccaca 840 gagtctgtgg aagaggtggt tcgagttcct acaacagcag ccagtacccc tgatgccgtt 900 gacaagtatc tcgagacacc tggggatgag aatgaacatg cccatttcca gaaagccaaa 960 gagaggcttg aggccaagca ccgagagaga atgtcceagg tcatgagaga atgggaagag 1020 gcagaacgtc aagcaaagaa cttgcctaaa gctgataaga aggcagttat ccagcatttc 1080 caggagaaag tggaatcttt ggaacaggaa gcagccaacg agagacagca gctggtggag 1140 acacacatgg ccagagtgga agccatgctc aatgaccgcc gecgcctggc cctggagaac 1200 tacatcaccg ctctgcaggc tgttcctcct cggcctcgtc acgtgttcaa tatgctaaag 1260 aagtatgtcc gcgcagaaca gaaggacaga cagcacaccc taaagcattt cgagcatgtg 1320 cgcatggtgg atcccaagaa agccgctcag atccggtccc aggttatgac acacctccgt 1380 gtgatttatg agcgcatgaa tcagtctctc tccctgetct acaacgtgcc tgeagtggcc 1440 gaggagattc aggatgaagt tgatgagctg cttcagaaag agcaaaacta ttcagatgac 1500 gtcttggcca acatgattag tgaaccaagg atcagttacg gaaacgatgc tctcatgcca 1560 tctttgaccg aaacgaaaac caccgtggag ctcctteccg tgaatggaga gttcagcctg 1620 gacgatctcc agccgtggca ttcttttggg gctgactctg tgccagccaa cacagaaaac 1680 gaagttgagc ctgttgatgc ecgccctgct gccgacegag gactgaccac tcgaccaggt 1740 tctgggttga caaatatcaa gacggaggag atctctgaag tgaatctgga tgcagaattc 1800 cgacatgact caggatatga agttcatcat caaaaattgg tgttctttgc agaagatgtg 1860 ggttcaaaca aaggtgcaat cattggactc atggtgggcg gtgttgteat agcgacagtg 1920 atcgtcatca ccttggtgat gctgaagaag aaacagtaca catccattca tcatggtgtg 1980 gtggaggttg acgccgctgt caccccagag gagcgccacc tgtccaagat gcagcagaac 2040 ggctacgaaa atccaaccta caagttcttt gagcagatgc agaacaagaa gtag 2094 <210> 18 <21l> 697 <212> PRT <213> Homo sapiens <400> 18 Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15 Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 25 30 G1n Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val G1n 40 45 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp 55 60 Thr Lys Glu Gly lle Leu G1n Tyr Cys G1n Glu Val Tyr Pro Glu Leu 70 75 80 G1n lle Thr Asn Val Val Glu Ala Asn G1n Pro Val Thr Ile G1n Asn 90 95 Trp Cys Lys Arg Gly Arg Lys G1n Cys Lys Thr His Pro His Phe Val 105 110 Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 120 125 Val Pro Asp Lys Cys Lys Phe Leu His G1n Glu Arg Met Asp Val Cys 135 140 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu 150 155 160 Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile 170 175 its Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 185 190 Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 200 205 Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220 Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240 Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255 Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265 270 Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285 Val Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu 290 295 300 Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe G1n Lys Ala Lys 305 310 315 320 Glu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser G1n Val Met Arg 325 330 335 Glu Trp Glu Glu Ala Glu Arg G1n Ala Lys Asn Leu Pro Lys Ala Asp 340 345 350 Lys Lys Ala Val Ile G1n His Phe G1n Glu Lys Val Glu Ser Leu Glu 355 360 365 G1n Glu Ala Ala Asn Glu Arg G1n G1n Leu Val Glu Thr His Met Ala 370 375 380 Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn 385 390 395 400 Tyr Ile Thr Ala Leu G1n Ala Val Pro Pro Arg Pro Arg His Val Phe 405 410 415 Asn Met Leu Lys Lys Tyr Val Arg Ala Glu G1n Lys Asp Arg G1n His 420 425 430 Thr Leu Lys His Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala 435 440 445 Ala G1n Ile Arg Ser G1n Val Met Thr His Leu Arg Val Ile Tyr Glu 450 455 460 Arg Met Asn G1n Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala 465 470 475 480 Glu Glu Ile G1n Asp Glu Val Asp Glu Leu Leu G1n Lys Glu G1n Asn 485 490 495 1 k OA Tyr Ser Asp Asp Val Leu Ala Asn Met Ile Ser Glu Pro Arg lle Ser 500 505 510 Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr 515 520 525 Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu G1n 530 535 540 Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn 545 550 555 560 Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr 565 570 575 Thr Arg Pro Gly Ser Gly Leu Thr Asn Ile Lys Thr Glu Glu lle Ser 580 585 590 Glu Val Asn Leu Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val 595 600 605 His His G1n Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys 610 615 620 Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val 625 630 635 640 Ile Val Ile Thr Leu Val Met Leu Lys Lys Lys G1n Tyr Thr Ser Ile 645 650 655 His His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg 660 665 670 His Leu Ser Lys Met G1n G1n Asn Gly Tyr Glu Asn Pro Thr Tyr Lys 675 680 685 Phe Phe Glu G1n Met G1n Asn Lys Lys 690 695 <210> 19 <21l> 2094 <212> DNA <213> Homo sapiens <400> 19 atgctgcccg gtttggcact gctcctgctg gccgcctgga cggctcgggc

gctggaggta 60 cccactgatg gtaatgctgg cctgctggct gaaccccaga ttgccatgtt ctgtggcaga 120 ctgaacatgc acatgaatgt ccagaatggg aagtgggatt cagatccatc agggaccaaa 180 acctgcattg ataccaagga aggcatcctg cagtattgcc aagaagtcta ccctgaactg 240 cagatcacca atgtggtaga agccaaccaa ccagtgacca tccagaactg gtgcaagcgg 300 ggccgcaagc agtgcaagac ccatccccac tttgtgattc cctaccgctg cttagttggt 360 gagtttgtaa gtgatgccct tctcgttcct gacaagtgca aattcttaca ccaggagagg 420 atggatgttt gcgaaactca tcttcactgg cacaccgtcg ccaaagagac atgcagtgag 480 aagagtacca acttgcatga ctacggcatg ttgctgccct gcggaattga caagttccga 540 ggggtagagt ttgtgtgttg cccactggct gaagaaagtg acaatgtgga ttctgctgat 600 gcggaggagg atgactcgga tgtctggtgg ggcggagcag acacagacta tgcagatggg 660 agtgaagaca aagtagtaga agtagcagag gaggaagaag tggctgaggt ggaagaagaa 720 gaagccgatg atgacgagga cgatgaggat ggtgatgagg tagaggaaga ggctgaggaa 780 ccetacgaag aagccacaga gagaaccacc agcattgeca ccaccaccac caccaccaca 840 gagtctgtgg aagaggtggt tcgagttcct acaacagcag ccagtacccc tgatgccgtt 900 gacaagtatc tcgagacacc tggggatgag aatgaacatg cccatttcca gaaagccaaa 960 1 2-C2 gagaggcttg aggccaagca ccgagagaga atgtcccagg tcatgagaga atgggaagag 1020 gcagaacgtc aagcaaagaa cttgcctaaa gctgataaga aggcagttat ccagcatttc 1080 caggagaaag tggaatcttt ggaacaggaa gcagccaacg agagacagca gctggtggag 1140 acacacatgg ccagagtgga agccatgctc aatgaccgcc gccgcctggc cctggagaac 1200 tacatcaccg ctctgcaggc tgttcctcct cggcctcgtc acgtgttcaa tatgctaaag 1260 aagtatgtcc gcgcagaaca gaaggacaga cagcacaccc taaagcattt cgagcatgtg 1320 cgcatggtgg atcccaagaa agccgctcag atccggtccc aggttatgac acacctccgt 1380 gtgatttatg agcgcatgaa tcagtctctc tccctgctct acaacgtgcc tgcagtggcc 1440 gaggagattc aggatgaagt tgatgagctg cttcagaaag agcaaaacta ttcagatgac 1500 gtcttggcca acatgattag tgaaccaagg atcagttacg gaaacgatgc tctcatgcca 1560 tctttgaccg aaacgaaaac caccgtggag ctccttcccg tgaatggaga gttcagcctg 1620 gacgatctcc agccgtggca ttcttttggg gctgactctg tgccagecaa cacagaaaac 1680 gaagttgagc ctgttgatgc ccgccctgct gccgaccgag gactgaccac tcgaccaggt 1740 tctgggttga caaatatcaa gacggaggag atctctgaag tgaagatgga tgcagaattc 1800 cgacatgact caggatatga agttcatcat caaaaattgg tgttctttgc agaagatgtg 1860 ggttcaaaca aaggtgcaat cattggactc atggtgggcg gtgttgtcat agcgacagtg 1920 atcttcatca ccttggtgat gctgaagaag aaacagtaca catccattca tcatggtgtg 1980 gtggaggttg acgccgctgt caccccagag gagcgCcacc tgtccaagat gcagcagaac 2040 ggctacgaaa atccaaccta caagttcttt gagcagatgc agaacaagaa gtag 2094

<210> 20 <21l> 697 <212> PRT <213> Homo sapiens <400> 20 Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15 Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 25 30 G1n Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val G1n 40 45 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp so 55 60 Thr Lys Glu Gly Ile Leu G1n Tyr Cys G1n Glu Val Tyr Pro Glu Leu 70 75 80 G1n Ile Thr Asn Val Val Glu Ala Asn G1n Pro Val Thr Ile G1n Asn 90 95 Trp Cys Lys Arg Gly Arg Lys G1n Cys Lys Thr His Pro His Phe Val 105 110 Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 120 125 Val Pro Asp Lys Cys Lys Phe Leu His G1n Glu Arg Met Asp Val Cys 135 140 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu 150 155 160 Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile 170 175 Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 185 190 kL Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 200 205 Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220 Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240 Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255 Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265 270 Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285 Val Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu 290 295 300 Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe G1n Lys Ala Lys 305 310 315 320 Glu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser G1n Val Met Arg 325 330 335 Glu Trp Glu Glu Ala Glu Arg G1n Ala Lys Asn Leu Pro Lys Ala Asp 340 345 350 Lys Lys Ala Val Ile G1n His Phe G1n Glu Lys Val Glu Ser Leu Glu 355 360 365 G1n Glu Ala Ala Asn Glu Arg G1n G1n Leu Val Glu Thr His Met Ala 370 375 380 Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn 385 390 395 400 Tyr Ile Thr Ala Leu G1n Ala Val Pro Pro Arg Pro Arg His Val Phe 405 410 415 Asn Met Leu Lys Lys Tyr Val Arg Ala Glu G1n Lys Asp Arg G1n His 420 425 430 Thr Leu Lys His Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala 435 440 445 Ala G1n Ile Arg Ser G1n Val Met Thr His Leu Arg Val Ile Tyr Glu 450 455 460 Arg Met Asn G1n Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala 465 470 475 480 Glu Glu Ile G1n Asp Glu Val Asp Glu Leu Leu G1n Lys Glu G1n Asn 485 490 495 Tyr Ser Asp Asp Val Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser 500 505 510 i I-LTyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr 515 520 525 Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu G1n 530 535 540 Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn 545 550 555 560 Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr 565 570 575 Thr Arg Pro Gly Ser Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser 580 585 590 Glu Val Lys Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val 595 600 605 His His G1n Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys 610 615 620 Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val 625 630 635 640 Ile Phe Ile Thr Leu Val Met Leu Lys Lys Lys G1n Tyr Thr Ser Ile 645 650 655 His His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg 660 665 670 His Leu Ser Lys Met G1n G1n Asn Gly Tyr Glu Asn Pro Thr Tyr Lys 675 680 685 Phe Phe Glu G1n Met G1n Asn Lys Lys 690 695 <210> 21 <21l> 1341 <212> DNA <213> Homo sapiens <400> 21 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccacccagca cggcatccgg 60 ctgcccctgc gcagcggcct ggggggcgcc cccctggggc tgcggctgcc ccgggagacc 120 gacgaagagc ccgaggagcc cggccggagg ggcagctttg tggagatggt ggacaacctg 180 aggggcaagt cggggcaggg ctactacgtg gagatgaccg tgggcagccc cccgcagacg 240 ctcaacatcc tggtggatac aggcagcagt aactttgcag tgggtgctgc cccccacccc 300 ttcctgcatc gctactacca gaggcagctg tccagcacat accgggacct ccggaagggt 360 gtgtatgtgc cctacaccca gggcaagtgg gaaggggagc tgggcaccga cctggtaagc 420 atcccccatg gccccaacgt cactgtgcgt gccaacattg ctgccatcac tgaatcagac 480 aagttcttca tcaacggctc caactgggaa ggcatcctgg ggctggccta tgctgagatt 540 gccaggcctg acgactccct ggagcctttc tttgactctc tggtaaagca gacccacgtt 600 cccaacctct tctccctgca cctttgtggt gctggcttcc ccctcaacca gtctgaagtg 660 ctggcctctg tcggagggag catgatcatt ggaggtatcg accactcgct gtacacaggc 720 agtctctggt atacacccat ccggcgggag tggtattatg aggtcatcat tgtgcgggtg 780 gagatcaatg gacaggatct gaaaatggac tgcaaggagt acaactatga caagagcatt 840 gtggacagtg gcaccaccaa ccttcgtttg cccaagaaag tgtttgaagc tgcagtcaaa 900tccatcaagg cagcctcctc cacggagaag ttccctgatg gtttctggct aggagagcag 960 ctggtgtgct ggcaageagg caccacccct tggaacattt tcccagtcat ctcactctac 1020 ctaatgggtg aggttaccaa ccagtccttc cgcatcacca tccttccgca gcaatacctg 1080 12,1 cggccagtgg aagatgtggc cacgtcccaa gacgactgtt acaagtttgc catctcacag 1140 tcatccacgg gcactgttat gggagctgtt atcatggagg gcttctacgt tgtctttgat 1200 cgggcccgaa aacgaattgg ctttgctgtc agcgcttgcc atgtgcacga tgagttcagg 1260 acggcagcgg tggaaggccc ttttgtcacc ttggacatgg aagactgtgg ctacaacatt 1320 ccacagacag atgagtcatg a 1341 <210> 22 <21l> 446 <212> PRT <213> Homosapiens <400> 22 Met Ala Ser Met Thr Gly Gly G1n G1n Met Gly Arg Gly Ser Thr G1n 1 5 10 is His Gly Ile Arg Leu Pro Leu Arg Ser Gly Leu Gly Gly Ala Pro Leu 25 30 Gly Leu Arg Leu Pro Arg Glu Thr Asp Glu Glu Pro Glu Glu Pro Gly 40 45 Arg Arg Gly Ser Phe Val Glu Met Val Asp Asn Leu A-rg Gly Lys Ser so 55 60 Gly Gin Gly Tyr Tyr Val Glu Met Thr Val Gly Ser Pro Pro G1n Thr 70 75 80 Leu Asn Ile Leu Val Asp Thr Gly Ser Ser Asn Phe Ala Val Gly Ala 90 95 Ala Pro His Pro Phe Leu His Arg Tyr Tyr G1n Arg G1n Leu Ser Ser 105 110 Thr Tyr Arg Asp Leu Arg Lys Gly Val Tyr Val Pro Tyr Thr G1n Gly 120 125 Lys Trp Glu Gly Glu Leu Gly Thr Asp Leu Val Ser Ile Pro His Gly 135 140 Pro Asn Val Thr Val Arg Ala Asn Ile Ala Ala Ile Thr Glu Ser Asp ' 150 155 160 Lys Phe Phe Ile Asn Gly Ser Asn Trp Glu Gly Ile Leu Gly Leu Ala 170 175 Tyr Ala Glu Ile Ala Arg Pro Asp Asp Ser Leu Glu Pro Phe Phe Asp 185 190 Ser Leu Val Lys G1n Thr His Val Pro Asn Leu Phe Ser Leu His Leu 200 205 Cys Gly Ala Gly Phe Pro Leu Asn G1n Ser Glu Val Leu Ala Ser Val 210 215 220 Gly Gly Ser Met lle Ile Gly Gly Ile Asp His Ser Leu Tyr Thr Gly 225 230 235 240 Ser Leu Trp Tyr Thr Pro Ile Arg Arg Glu Trp Tyr Tyr Glu Val Ile 245 250 255 Ile Val Arg Val Glu Ile Asn Gly Gin Asp Leu Lys Met Asp Cys Lys 260 265 270 12-1-1- Glu Tyr Asn Tyr Asp Lys Ser Ile Val Asp Ser Gly Thr Thr Asn Leu 275 280 285 Arg Leu Pro Lys Lys Val Phe Glu Ala Ala Val Lys Ser Ile Lys Ala 290 295 300 Ala Ser Ser Thr Glu Lys Phe Pro Asp Gly Phe Trp Leu Gly Glu G1n 305 310 315 320 Leu Val Cys Trp G1n Ala Gly Thr Thr Pro Trp Asn Ile Phe Pro Val 325 330 335 Ile Ser Leu Tyr Leu Met Gly Glu Val Thr Asn G1n Ser Phe Arg Ile 340 345 350 Thr Ile Leu Pro G1n G1n Tyr Leu Arg Pro Val Glu Asp Val Ala Thr 355 360 365 Ser G1n Asp Asp Cys Tyr Lys Phe Ala Ile Ser G1n Ser Ser Thr Gly 370 375 380 Thr Val Met Gly Ala Val Ile Met Glu Gly Phe Tyr Val Val Phe Asp 385 390 395 400 Arg Ala Arg Lys Arg Ile Gly Phe Ala Val Ser Ala Cys His Val His 405 410 415 Asp Glu Phe Arg Thr Ala Ala Val Glu Gly Pro Phe Val Thr Leu Asp 420 425 430 Met Glu Asp Cys Gly Tyr Asn Ile Pro G1n Thr Asp Glu Ser 435 440 445 <210> 23 <21l> 1380 <212> DNA <213> Homo sapiens <400> 23 atggctagca tgactggtgg acagcaaatg ggtcgcggat cgatgactat ctctgactct 60 ccgcgtgaac aggacggatc cacccagcac ggcatccggc tgcccctgcg cagcggcctg 120 gggggcgccc ccctggggct gcggctgccc cgggagaccg acgaagagcc cgaggagccc 180 ggccggaggg gcagctttgt ggagatggtg gacaacctga ggggcaagtc ggggcagggc 240 tactacgtgg agatgaccgt gggcagcccc ccgcagacgc tcaacatcct ggtggataca 300 ggcagcagta actttgcagt. gggtgctgcc ccccacccct tcctgcatcg ctactaccag 360 aggcagctgt ccagcacata ccgggacctc cggaagggtg tgtatgtgcc ctacacccag 420 ggcaagtggg aaggggagct gggcaccgac ctggtaagca tcccccatgg ccccaacgtc 480 actgtgcgtg ccaaCattgc tgccatcact gaatcagaca agttcttcat caacggctcc 540 aactgggaag gcatcctggg gctggcctat gctgagattg ccaggcctga cgactccctg 600 gagcctttct ttgactctct ggtaaagcag acccacgttc ccaacctctt ctccctgcac 660 ctttgtggtg ctggcttccc cctcaaccag tctgaagtgc tggcctctgt. cggagggagc 720 atgatcattg gaggtatcga ccactcgctg tacacaggca gtctctggta tacacccatc 780 cggcgggagt ggtattatga ggtcatcatt gtgcgggtgg agatcaatgg acaggatctg 840 aaaatggact gcaaggagta caactatgac aagagcattg tggacagtgg caccaccaac 900 cttcgtttgc ccaagaaagt gtttgaagct gcagtcaaat ccatcaaggc agcctcctcc 960 acggagaagt tccctgatgg tttctggcta ggagagcagc tggtgtgctg gcaagcaggc 1020 accacccctt ggaacatttt cccagtcatc tcactctacc taatgggtga ggttaccaac 1080 cagtccttcc gcatcaccat ccttccgcag caatacctgc ggccagtgga agatgtggcc 1140 acgtcccaag acgactgtta caagtttgcc atctcacagt catccacggg cactgttatg 1200 ggagctgtta tcatggaggg cttctacgtt 9tctttgatc gggcccgaaa acgaattggc 1260 tttgctgtca gcgcttgcca tgtgcacgat gagttcagga cggcagcggt ggaaggccct 1320 tttgtcacct tggacatgga agactgtggc tacaacattc cacagacaga tgagtcatga 1380 <210> 24 <21l> 459 <212> PRT <213> Homo sapiens <400> 24 Met Ala Ser Met Thr Gly Gly G1n G1n Met Gly Arg Gly Ser Met Thr 1 5 10 15 Ile Ser Asp Ser Pro Arg Glu G1n Asp Gly Ser Thr G1n His Gly Ile 25 30 Arg Leu Pro Leu Arg Ser Gly Leu Gly Gly Ala Pro Leu Gly Leu Arg 40 45 Leu Pro Arg Glu Thr Asp Glu Glu Pro Glu Glu Pro Gly Arg Arg Gly 55 60 Ser Phe Val Glu Met Val Asp Asn Leu Arg Gly Lys Ser Gly G1n Gly 70 75 80 Tyr Tyr Val Glu Met Thr Val Gly Ser Pro Pro G1n Thr Leu Asn Ile 90 95 Leu Val Asp Thr Gly Ser Ser Asn Phe Ala Val Gly Ala Ala Pro His 105 110 Pro Phe Leu His Arg Tyr Tyr G1n Arg G1n Leu Ser Ser Thr Tyr Arg 120 125 Asp Leu Arg Lys Gly Val Tyr Val Pro Tyr Thr G1n Gly Lys Trp Glu 135 140 Gly Glu Leu Gly Thr Asp Leu Val Ser Ile Pro His Gly Pro Asn Val 150 155 160 Thr Val Arg Ala Asn Ile Ala Ala Ile Thr Glu Ser Asp Lys Phe Phe 170 175 Ile Asn Gly Ser Asn Trp Glu Gly Ile Leu Gly Leu Ala Tyr Ala Glu 185 190 Ile Ala Arg Pro Asp Asp Ser Leu Glu Pro Phe Phe Asp Ser Leu Val 200 205 Lys G1n Thr His Val Pro Asn Leu Phe Ser Leu His Leu Cys Gly Ala 210 215 220 Gly Phe Pro Leu Asn G1n Ser Glu Val Leu Ala Ser Val Gly Gly Ser 225 230 235 240 Met Ile Ile Gly Gly lle Asp His Ser Leu Tyr Thr Gly Ser Leu Trp 245 250 255 Tyr Thr Pro Ile Arg Arg Glu Trp Tyr Tyr Glu Val Ile Ile Val Arg 260 265 270 Val Glu Ile Asn Gly G1n Asp Leu Lys Met Asp Cys Lys Glu Tyr Asn 275 280 285 Tyr Asp Lys Ser Ile Val Asp Ser Gly Thr Thr Asn Leu Arg Leu Pro 290 295 300 Lys Lys Val Phe Glu Ala Ala Val Lys Ser Ile Lys Ala Ala Ser Ser 305 310 315 320 Thr Glu Lys Phe Pro Asp Gly Phe Trp Leu Gly Glu G1n Leu Val Cys 325 330 335 Trp G1n Ala Gly Thr Thr Pro Trp Asn Ile Phe Pro Val Ile Ser Leu 340 345 350 Tyr Leu Met Gly Glu Val Thr Asn G1n Ser Phe Arg Ile Thr Ile Leu 355 360 365 Pro G1n G1n Tyr Leu Arg Pro Val Glu Asp Val Ala Thr Ser G1n Asp 370 375 380 Asp Cys Tyr Lys Phe Ala Ile Ser G1n Ser Ser Thr Gly Thr Val Met 385 390 395 400 Gly Ala Val Ile Met Glu Gly Phe Tyr Val Val Phe Asp Arg Ala Arg 405 410 415 Lys Arg Ile Gly Phe Ala Val Ser Ala Cys His Val His Asp Glu Phe 420 425 430 Arg Thr Ala Ala Val Glu Gly Pro Phe Val Thr Leu Asp Met Glu Asp 435 440 445 Cys Gly Tyr Asn Ile Pro G1n Thr Asp Glu Ser 450 455 <210> 25 <21l> 1302 <212> DNA <213> Homo sapiens <400> 25 atgactcagc atggtattcg tctgccactg cgtagcggtc tgggtggtgc tccactgggt 60 ctgcgtctgc cccgggagac cgacgaagag cccgaggagc ccggccggag gggcagcttt 120 gtggagatgg tggacaacct gaggggcaag tcggggcagg gctactacgt ggagatgacc 180 gtgggcagcc ccccgcagac gctcaacatc ctggtggata caggcagcag taactttgca 240 gtgggtgctg ceccccaccc cttcctgcat cgctactacc agaggcagct gtccagcaca 300 taccgggacc tccggaaggg tgtgtatgtg ccctacaccc agggcaagtg ggaaggggag 360 ctgggcaccg acctggtaag catcccccat ggccccaacg tcactgtgcg tgccaacatt 420 gctgccatca ctgaatcaga caagttcttc atcaacggct ccaactggga aggcatcctg 480 gggctggcct atgctgagat tgccaggcct gacgactccc tggagccttt ctttgactct 540 ctggtaaagc agacccacgt tcccaacctc ttctccctgc acctttgtgg tgctggcttc 600 cccctcaacc agtctgaagt gctggcctct gtcggaggga gcatgatcat tggaggtatc 660 gaccactcgc tgtacacagg cagtctctgg tatacaccca tccggcggga gtggtattat 720 gaggtcatca ttgtgcgggt ggagatcaat ggacaggatc tgaaaatgga ctgcaaggag 780 tacaactatg acaagagcat tgtggacagt ggcaccacca accttcgttt gcccaagaaa 840 gtgtttgaag ctgeagtcaa atccatcaag gcagcctcct ccacggagaa gttccctgat 900 ggtttctggc taggagagca gctggtgtgc tggcaagcag gcaccaccce ttggaacatt 960 ttcccagtca tctcactcta cctaatgggt gaggttacca accagtcctt ccgcatcacc 1020 atccttccgc agcaatacct gcggccagtg gaagatgtgg ccacgtccca agacgactgt 1080 tacaagtttg ccatctcaca gtcatccacg ggcactgtta tgggagctgt tatcatggag 1140 1 l-k ggcttctacg ttgtctttga tcgggcecga aaacgaattg gctttgctgt cagcgcttgc 1200 catgtgcacg atgagttcag gacggcagc9 gtggaaggce cttttgtcac cttggacatg 1260 gaagactgtg gctacaacat tccacagaca gatgagtcat ga 1302 <210> 26 <21l> 433 <212> PRT <213> Homo sapiens <400> 26 Met Thr G1n His Gly Ile Arg Leu Pro Leu Arg Ser Gly Leu Gly Gly 1 5 10 15 Ala Pro Leu Gly Leu Arg Leu Pro Arg Glu Thr Asp Glu Glu Pro Glu 25 30 Glu Pro Gly Arg Arg Gly Ser Phe Val Glu Met Val Asp Asn Leu Arg 40 45 Gly Lys Ser Gly G1n Gly Tyr Tyr Val Glu Met Thr Val Gly Ser Pro 55 60 Pro G1n Thr Leu Asn Ile Leu Val Asp Thr Gly Ser Ser Asn Phe Ala 70 75 80 Val Gly Ala Ala Pro His Pro Phe Leu His Arg Tyr Tyr G1n Arg G1n 90 95 Leu Ser Ser Thr Tyr Arg Asp Leu Arg Lys Gly Val Tyr Val Pro Tyr 105 110 Thr G1n Gly Lys Trp Glu Gly Glu Leu Gly Thr Asp Leu Val Ser Ile 120 125 Pro His Gly Pro Asn Val Thr Val Arg Ala Asn Ile Ala Ala Ile Thr 135 140 Glu Ser Asp Lys Phe Phe Ile Asn Gly Ser Asn Trp Glu Gly Ile Leu 150 155 160 Gly Leu Ala Tyr Ala Glu lle Ala Arg Pro Asp Asp Ser Leu Glu Pro 170 175 Phe Phe Asp Ser Leu Val Lys G1n Thr His Val Pro Asn Leu Phe Ser 185 190 Leu His Leu Cys Gly Ala Gly Phe Pro Leu Asn G1n Ser Glu Val Leu 200 205 Ala Ser Val Gly Gly Ser Met Ile Ile Gly Gly Ile Asp His Ser Leu 210 215 220 Tyr Thr Gly Ser Leu Trp Tyr Thr Pro Ile Arg Arg Glu Trp Tyr Tyr 225 230 235 240 Glu Val Ile Ile Val Arg Val Glu Ile Asn Gly G1n Asp Leu Lys Met 245 250 255 Asp Cys Lys Glu Tyr Asn Tyr Asp Lys Ser Ile Val Asp Ser Gly Thr 260 265 270 Thr Asn Leu Arg Leu Pro Lys Lys Val Phe Glu Ala Ala Val Lys Ser 12-1 275 280 285 Ile Lys Ala Ala Ser Ser Thr Glu Lys Phe Pro Asp Gly Phe Trp Leu 290 295 300 Gly Glu G1n Leu Val Cys Trp G1n Ala Gly Thr Thr Pro Trp Asn Ile 305 310 315 320 Phe Pro Val Ile Ser Leu Tyr Leu Met Gly Glu Val Thr Asn G1n Ser 325 330 335 Phe Arg Ile Thr Ile Leu Pro G1n G1n Tyr Leu Arg Pro Val Glu Asp 340 345 350 Val Ala Thr Ser G1n Asp Asp Cys Tyr Lys Phe Ala Ile Ser G1n Ser 355 360 365 Ser Thr Gly Thr Val Met Gly Ala Val Ile Met Glu Gly Phe Tyr Val 370 375 380 Val Phe Asp Arg Ala Arg Lys Arg Ile Gly Phe Ala Val Ser Ala Cys 385 390 395 400 His Val His Asp Glu Phe Arg Thr Ala Ala Val Glu Gly Pro Phe Val 405 410 415 Thr Leu Asp Met Glu Asp Cys Gly Tyr Asn Ile Pro G1n Thr Asp Glu 420 425 430 Ser <210> 27 <21l> 1278 <212> DNA <213> Homo sapiens <400> 27 atggctagca tgactggtgg acagcaaatg ggtcgcggat cgatgactat ctctgactct 60 ccgctggact ctggtatcga aaccgacgga tcctttgtgg agatggtgga caacctgagg 120 ggcaagtcgg ggcagggcta ctacgtggag atgaccgtgg gcagcccccc gcagacgctc 180 aacatcctgg tggatacagg cagcagtaac tttgcagtgg gtgctgcccc ccaccccttc 240 ctgcatcgct actaccagag gcagctgtcc agcacatacc gggacctccg gaagggtgtg 300 tatgtgccct acacccaggg caagtgggaa ggggagctgg gcaccgacct ggtaagcatc 360 ccccatggcc ccaacgtcac tgtgcgtgcc aacattgctg ccatcactga atcagacaag 420 ttcttcatca acggctccaa ctgggaaggc atcctggggc tggcctatgc tgagattgcc 480 aggcctgacg actccctgga gcctttcttt, gactctctgg taaagcagac ccacgttccc 540 aacctcttct ccctgcacct ttgtggtgct ggcttccccc tcaaccagtc tgaagtgctg 600 gcctctgtcg gagggagcat gatcattgga ggtatcgacc actcgctgta cacaggcagt 660 ctctggtata cacccatccg gcgggagtgg tattatgagg tcatcattgt gcgggtggag 720 atcaatggac aggatctgaa aatggactgc aaggagtaca actatgacaa gagcattgtg 780 gacagtggca ccaccaacct tcgtttgccc aagaaagtgt ttgaagctgc agtcaaatcc 840 atcaaggcag cctcctccac ggagaagttc cctgatggtt tctggctagg agagcagctg 900 gtgtgctggc aagcaggcac caccccttgg aacattttcc cagtcatctc actctaccta 960 atgggtgagg ttaccaacca gtccttccgc atcaccatcc ttccgcagca atacctgcgg 1020 ccagtggaag atgtggccac gtcccaagac gactgttaca agtttgccat ctcacagtca 1080 tccacgggca ctgttatggg agctgttatc atggagggct tctacgttgt ctttgatcgg 1140 gcccgaaaac gaattg9Ctt tgctgtcagc gcttgccatg tgcacgatga gttcaggacg 1200 gcagcggtgg aaggcccttt tgtcaccttg gacatggaag actgtggcta caacattcca 1260 cagacagatg agtCatga 1278 1-2-0k <210> 28 <21l> 425 <212> PRT <213> Homo sapiens <400> 28 Met Ala Ser Met Thr Gly Gly G1n G1n Met Gly Arg Gly Ser Met Thr 1 5 10 15 Ile Ser Asp Ser Pro Leu Asp Ser Gly Ile Glu Thr Asp Gly Ser Phe 25 30 Val Glu Met Val Asp Asn Leu Arg Gly Lys Ser Gly G1n Gly Tyr Tyr 40 45 Val Glu Met Thr Val Gly Ser Pro Pro G1n Thr Leu Asn Ile Leu Val 55 60 Asp Thr Gly Ser Ser Asn Phe Ala Val Gly Ala Ala Pro His Pro Phe 70 75 80 Leu His Arg Tyr Tyr G1n Arg G1n Leu Ser Ser Thr Tyr Arg Asp Leu 90 95 Arg Lys Gly Val Tyr Val Pro Tyr Thr G1n Gly Lys Trp Glu Gly Glu 105 110 Leu Gly Thr Asp Leu Val Ser Ile Pro His Gly Pro Asn Val Thr Val 120 125 Arg Ala Asn Ile Ala Ala Ile Thr Glu Ser Asp Lys Phe Phe Ile Asn 135 140 Gly Ser Asn Trp Glu Gly Ile Leu Gly Leu Ala Tyr Ala Glu Ile Ala 150 155 160 Arg Pro Asp Asp Ser Leu Glu Pro Phe Phe Asp Ser Leu Val Lys G1n 170 175 Thr His Val Pro Asn Leu Phe Ser Leu His Leu Cys Gly Ala Gly Phe 185 190 Pro Leu Asn G1n Ser Glu Val Leu Ala Ser Val Gly Gly Ser Met Ile 200 205 Ile Gly Gly lle Asp His Ser Leu Tyr Thr Gly Ser Leu Trp Tyr Thr 210 215 220 Pro Ile Arg Arg Glu Trp Tyr Tyr Glu Val Ile Ile Val Arg Val Glu 225 230 235 240 Ile Asn Gly G1n Asp Leu Lys Met Asp Cys Lys Glu Tyr Asn Tyr Asp 245 250 255 Lys Ser Ile Val Asp Ser Gly Thr Thr Asn Leu Arg Leu Pro Lys Lys 260 265 270 Val Phe Glu Ala Ala Val Lys Ser Ile Lys Ala Ala Ser Ser Thr Glu 275 280 285 Lys Phe Pro Asp Gly Phe Trp Leu Gly Glu G1n Leu Val Cys Trp G1n 290 295 300 Ala Gly Thr Thr Pro Trp Asn Ile Phe Pro Val Ile Ser Leu Tyr Leu 305 310 315 320 Met Gly Glu Val Thr Asn G1n Ser Phe Arg Ile Thr Ile Leu Pro G1n 325 330 335 G1n Tyr Leu Arg Pro Val Glu Asp Val Ala Thr Ser G1n Asp Asp Cys 340 345 350 Tyr Lys Phe Ala Ile Ser G1n Ser Ser Thr Gly Thr Val Met Gly Ala 355 360 365 Val Ile Met Glu Gly Phe Tyr Val Val Phe Asp Arg Ala Arg Lys Arg 370 375 380 Ile Gly Phe Ala Val Ser Ala Cys His Val His Asp Glu Phe Arg Thr 385 390 395 400 Ala Ala Val Glu Gly Pro Phe Val Thr Leu Asp Met Glu Asp Cys Gly 405 410 415 Tyr Asn Ile Pro G1n Thr Asp Glu Ser 420 425 <210> 29 <21l> 1362 <212> DNA <213> Homo sapiens <400> 29 atggcecaag ccctgccctg gctcctgctg tggatgggcg cgggagtgct gcctgcccac 60 ggcacccagc acggcatccg gctgcccctg cgcagcggcc tggggggcgc ccccctgggg 120 ctgcggctgc cccgggagac cgacgaagag cccgaggagc ccggccggag gggcagcttt, 180 gtggagatgg tggacaacct gaggggcaag tcggggcagg gctactacgt ggagatgacc 240 gtgggcagcc ccccgcagac gctcaacatc ctggtggata caggcagcag taactttgca 300 gtgggtgctg ccccccaccc cttcctgcat cgctactacc agaggCagct gtccagcaca 360 taccgggacc tccggaaggg tgtgtatgtg ccctacaccc agggcaagtg ggaaggggag 420 ctgggcaccg acctggtaag catcccccat ggccccaacg tcactgtgcg tgccaacatt 480 gctgccatca ctgaatcaga caagttcttc atcaacggct ccaactggga aggcatcctg 540 gggctggcct atgctgagat tgccaggcct gacgactccc tggagccttt ctttgactct 600 ctggtaaagc agacccacgt tcccaacctc ttctccctgc acctttgtgg tgctggcttc 660 cccctcaacc agtctgaagt gctggcctct gtcggaggga gcatgatcat tggaggtatc 720 gaccaetcgc tgtacacagg cagtctctgg tatacaccca tccggcggga gtggtattat 780 gaggtcatca ttgt9cgggt ggagatcaat ggacaggatc tgaaaatgga ctgcaaggag 840 tacaactatg acaagagcat tgtggacagt ggcaccacca accttcgttt gcccaagaaa 900 gtgtttgaag ctgcagtcaa atccatcaag gcagcctcct ccacggagaa gttccctgat 960 ggtttctggc taggagagca gctggtgtgc tggcaagcag gcaccacccc ttggaacatt 1020 ttcccagtca tctCactcta cctaatgggt gaggttacca accagtcctt ccgcatcacc 1080 atccttccgc agcaatacct gcggccagtg gaagatgtgg ccacgtccca agacgactgt 1140 tacaagtttg ccatctcaca gtcatccacg ggcactgtta tgggagctgt tatcatggag 1200 ggcttctacg ttgtctttga tcgggcccga aaacgaattg gctttgctgt cagcgcttgc 1260 catgtgcacg atgagttcag gacggcagcg gtggaaggcc cttttgtcac cttggacatg 1320 gaagactgtg gctacaacat tccacagaca gatgagtcat ga 1362 <210> 30 <21l> 453 <212> PRT <213> Homo sapiens <400> 30 (3 Met Ala Gin Ala Leu Pro Trp Leu Leu Leu Trp Met Gly Ala Gly Val 1 5 10 is Leu Pro Ala His Gly Thr G1n His Gly Ile Arg Leu Pro Leu Arg Ser 25 30 Gly Leu Gly Gly Ala Pro Leu Gly Leu Arg Leu Pro Arg Glu Thr Asp 40 45 Glu Glu Pro Glu Glu Pro Gly Arg Arg Gly Ser Phe Val Glu Met Val so 55 60 Asp Asn Leu Arg Gly Lys Ser Gly G1n Gly Tyr Tyr Val Glu Met Thr 70 75 80 Val Gly Ser Pro Pro G1n Thr Leu Asn Ile Leu Val Asp Thr Gly Ser 90 95 Ser Asn Phe Ala Val Gly Ala Ala Pro His Pro Phe Leu His Arg Tyr 105 110 Tyr G1n Arg Gin Leu Ser Ser Thr Tyr Arg Asp Leu Arg Lys Gly Val 120 125 Tyr Val Pro Tyr Thr Gin Gly Lys Trp Glu Gly Glu Leu Gly Thr Asp 135 140 Leu Val Ser Ile Pro His Gly Pro Asn Val Thr Val Arg Ala Asn Ile 150 155 160 Ala Ala Ile Thr Glu Ser Asp Lys Phe Phe Ile Asn Gly Ser Asn Trp 170 175 Glu Gly Ile Leu Gly Leu Ala Tyr Ala Glu lle Ala Arg Pro Asp Asp 185 190 Ser Leu Glu Pro Phe Phe Asp Ser Leu Val Lys G1n Thr His Val Pro 200 205 Asn Leu Phe Ser Leu Gin Leu Cys Gly Ala Gly Phe Pro Leu Asn Gin 210 215 220 Ser Glu Val Leu Ala Ser Val Gly Gly Ser Met lle Ile Gly Gly Ile 225 230 235 240 Asp His Ser Leu Tyr Thr Gly Ser Leu Trp Tyr Thr Pro Ile Arg Arg 245 250 255 Glu Trp Tyr Tyr Glu Val Ile Ile Val Arg Val Glu Ile Asn Gly Gin 260 265 270 Asp Leu Lys Met Asp Cys Lys Glu Tyr Asn Tyr Asp Lys Ser Ile Val 275 280 285 Asp Ser Gly Thr Thr Asn Leu Arg Leu Pro Lys Lys Val Phe Glu Ala 290 295 300 Ala Val Lys Ser Ile Lys Ala Ala Ser Ser Thr Glu Lys Phe Pro Asp 305 310 315 320 Gly Phe Trp Leu Gly Glu G1n Leu Val Cys Trp Gin Ala Gly Thr Thr 132 325 330 335 Pro Trp Asn lle Phe Pro Val Ile Ser Leu Tyr Leu Met Gly Glu Val 340 345 350 Thr Asn G1n Ser Phe Arg Ile Thr Ile Leu Pro G1n G1n Tyr Leu Arg 355 360 365 Pro Val Glu Asp Val Ala Thr Ser G1n Asp Asp Cys Tyr Lys Phe Ala 370 375 380 Ile Ser G1n Ser Ser Thr Gly Thr Val Met Gly Ala Val Ile Met Glu 385 390 395 400 Gly Phe Tyr Val Val Phe Asp Arg Ala Arg Lys Arg Ile Gly Phe Ala 405 410 415 Val Ser Ala Cys His Val His Asp Glu Phe Arg Thr Ala Ala Val Glu 420 425 430 Gly Pro Phe Val Thr Leu Asp Met Glu Asp Cys Gly Tyr Asn Ile Pro 435 440 445 G1n Thr Asp Glu Ser 450 <210> 31 <211> 1380 <2125 DNA <213> Homo sapiens <400> 31 atggcccaag ccctgccctg gctcctgctg tggatgggcg cgggagtgct gcctgcccac 60 ggcacccagc acggcatccg gctgcccctg cgCagcggcc t99ggggcgc ccccctgggg 120 ctgcggctgc cccgggagac cgacgaagag cccgaggagc ccggccggag gggcagcttt 180 gtggagatgg tggacaacct gaggggcaag tcggggcagg gctactacgt ggagatgacc 240 gtgggcagcc ccccgcagac gctcaacatc ctggtggata caggcagcag taactttgca 300 gtgggtgctg ccccccaccc cttcctgcat cgctactacc agaggcagct gtccagcaca 360 taccgggacc tccggaaggg tgtgtatgtg ccctacaccc agggcaagtg ggaaggggag 420 ctgggcaccg acctggtaag catcccccat ggccccaacg tcactgtgcg tgccaacatt 480 gctgccatca ctgaatcaga caagttcttc atcaacggct ccaactggga aggcatcctg 540 gggctggcct atgctgagat tgecaggcct gacgactccc tggagccttt ctttgactct 600 ctggtaaagc agacccacgt tcccaacctc ttctccctgc acctttgtgg tgctggcttc 660 cccctcaacc agtctgaagt gctggcctct gtcggaggga gcatgatcat tggaggtatc 720 gaccactcgc tgtacacagg cagtctctgg tatacaccca tccggcggga gtggtattat 780 gaggtcatca ttgtgcgggt ggagatcaat 9gacaggatc tgaaaatgga ctgcaaggag 840 tacaactatg acaagagcat tgtggacagt ggcaccacca accttcgttt gcccaagaaa 900 gtgtttgaag ctgcagtcaa atccatcaag gcagcctcct ccacggagaa gttcectgat 960 ggtttctggc taggagagca gctggtgtgc tggcaagcag gcaccacccc ttggaacatt 1020 ttcccagtca tctcactcta cctaatgggt gaggttacca accagtcctt ccgcatcacc 1080 atccttccgc agcaatacct gcggccagtg gaagatgtgg ccacgtccca agacgactgt 1140 tacaagtttg ccatctcaca gtcatccacg ggCactgtta tgggagctgt tatcatggag 1200 ggcttctacg ttgtctttga tcggg=ga aaacgaattg gctttgctgt cagcgcttgc 1260 catgtgcacg atgagttcag gacggcageg gtggaaggcc cttttgtcac cttggacatg 1320 gaagactgtg gctacaacat tccacagaca gatgagtcac ageagcagca geagcagtga 1380 <210> 32 <21l> 459 <212> PRT <213> Homo sapiens 113 <400> 32 Met Ala G1n Ala Leu Pro Trp Leu Leu Leu Trp Met Gly Ala Gly Val 1 5 10 15 Leu Pro Ala His Gly Thr G1n His Gly Ile Arg Leu Pro Leu Arg Ser 25 30 Gly Leu Gly Gly Ala Pro Leu Gly Leu Arg Leu Pro Arg Glu Thr Asp 40 45 Glu Glu Pro Glu Glu Pro Gly Arg Arg Gly Ser Phe Val Glu Met Val 55 60 Asp Asn Leu Arg Gly Lys Ser Gly G1n Gly Tyr Tyr Val Glu Met Thr 70 75 80 Val Gly Ser Pro Pro G1n Thr Leu Asn Ile Leu Val Asp Thr Gly Ser 90 95 Ser Asn Phe Ala Val Gly Ala Ala Pro His Pro Phe Leu His Arg Tyr 105 110 Tyr G1n Arg G1n Leu Ser Ser Thr Tyr Arg Asp Leu Arg Lys Gly Val 120 125 Tyr Val Pro Tyr Thr G1n Gly Lys Trp Glu Gly Glu Leu Gly Thr Asp 135 140 Leu Val Ser Ile Pro His Gly Pro Asn Val Thr Val Arg Ala Asn Ile 150 155 160 Ala Ala Ile Thr Glu Ser Asp Lys Phe Phe Ile Asn Gly Ser Asn Trp 170 175 Glu Gly Ile Leu Gly Leu Ala Tyr Ala Glu Ile Ala Arg Pro Asp Asp 185 190 Ser Leu Glu Pro Phe Phe Asp Ser Leu Val Lys G1n Thr His Val Pro 200 205 Asn Leu Phe Ser Leu G1n Leu Cys Gly Ala Gly Phe Pro Leu Asn G1n 210 215 220 Ser Glu Val Leu Ala Ser Val Gly Gly Ser Met Ile Ile Gly Gly Ile 225 230 235 240 Asp His Ser Leu Tyr Thr Gly Ser Leu Trp Tyr Thr Pro Ile Arg Arg 245 250 255 Glu Trp Tyr Tyr Glu Val Ile Ile Val Arg Val Glu Ile Asn Gly G1n 260 265 270 Asp Leu Lys Met Asp Cys Lys Glu Tyr Asn Tyr Asp Lys Ser Ile Val 275 280 285 Asp Ser Gly Thr Thr Asn Leu Arg Leu Pro Lys Lys Val Phe Glu Ala 290 295 300 Ala Val Lys Ser Ile Lys Ala Ala Ser Ser Thr Glu Lys Phe Pro Asp 305 310 315 320 Gly Phe Trp Leu Gly Glu G1n Leu Val Cys Trp G1n Ala Gly Thr Thr 325 330 335 Pro Trp Asn Ile Phe Pro Val Ile Ser Leu Tyr Leu Met Gly Glu Val 340 345 350 Thr Asn Gin Ser Phe Arg Ile Thr Ile Leu Pro G1n Gin Tyr Leu Arg 355 360 365 Pro Val Glu Asp Val Ala Thr Ser Gin Asp Asp Cys Tyr Lys Phe Ala 370 375 380 Ile Ser G1n Ser Ser Thr Gly Thr Val Met Gly Ala Val Ile Met Glu 385 390 395 400 Gly Phe Tyr Val Val Phe Asp Arg Ala Arg Lys Arg Ile Gly Phe Ala 405 410 415 Val Ser Ala Cys His Val His Asp Glu Phe Arg Thr Ala Ala Val Glu 420 425 430 Gly Pro Phe Val Thr Leu Asp Met Glu Asp Cys Gly Tyr Asn Ile Pro 435 440 445 Gin Thr Asp Glu Ser His His His His His His 450 455 <210> 33 <21l> 25 <212> PRT <213> Homo sapiens <400> 33 Ser Glu Gin Gin Arg Arg Pro Arg Asp Pro Glu Val Val Asn Asp Glu 1 5 10 15 Ser Ser Leu Val Arg His Arg Trp Lys 25 <210> 34 <21l> 19 <212> PRT <213> Homo sapiens <400> 34 Ser Glu Gin Leu Arg Gin G1n His Asp Asp Phe Ala Asp Asp Ile Ser 1 5 10 is Leu Leu Lys <210> 35 <21l> 29 <212> DNA <213> Homo sapiens <400> 35 gtggatccac ccagcacggc atccggctg 29 <210> 36 <21l> 36 <212> DNA <213> Homo sapiens <400> 36 gaaagctttc atgactcatc tgtctgtgga atgttg 36 <210> 37 <21l> 39 <212> DNA <213> Homo sapiens <400> 37 gatcgatgac tatctctgac tctccgcgtg aacaggacg 39 <210> 38 <21l> 39 <212> DNA <213> Homo sapiens <400> 38 gatccgtcct gttcacgcgg agagtcagag atagtcatc 39 <210> 39 <21l> 77 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Hu-Asp2 <400> 39 cggcatccgg ctgcccctgc gtagcggtct gggtggtgct ccactgggtc tgcgtctgcc 60 ccgggagacc gacgaag 77 <210> 40 <21l> 77 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Hu-Asp2 <400> 40 cttcgtcggt ctcccggggc agacgcagac ccagtggagc accacceaga ecgctacgca 60 ggggcagccg gatgccg 77 <210> 41 <21l> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Caspase 8

Cleavage Site <400> 41 gatcgatgac tatctctgac tctccgctgg actctggtat cgaaaccgac g 51 <210> 42 <21l> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Caspase 8

Cleavage Site <400> 42 gatecgtcgg tttcgatacc agagtccagc ggagagtcag agatagtcat c 51 <210> 43 <21l> 32 <212> DNA <213> Homo sapiens <400> 43 aaggatcctt tgtggagatg gtggacaacc tg 32 <210> 44 <21l> 36 <212> DNA <213> Homo sapiens <400> 44 gaaagctttc atgactcatc tgtctgtgga atgttg 36 <210> 45 <21l> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 6-His tag <400> 45 gatcgcatca tcaccatcac catg 24 <210> 46 <21l> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: 6-His tag <400> 46 gatccatggt gatggtgatg atgc 24 <210> 47 <21l> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 47 gactgaccac tc9accaggt tc 22 <210> 48 <21l> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 48 cgaattaaat tccagcacac tggctaettc ttgttctgca tctcaaagaa c 51 <210> 49 <21l> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 49 cgaattaaat tccagcacac tggcta 26 <210> 50 <21l> 1287.<212> DNA <213> Artificial.Sequence <220> <223> Description of Artificial Sequence: Hu-Asp2(b) delta TM <400> 50 atggcccaag ccctgccctg gctcctgetg tggatgggcg cgggagtgct gcctgcccac 60 ggcacccagc acggcatccg gctgcccctg cgcagcggec tggggggcgc ccccctgggg 120 ctgcggctgc cccgggagac cgacgaagag cccgaggagc ccggccggag gggcagcttt 180 gtggagatgg tggacaacct gaggggcaag tcggggcagg gctactacgt ggagatgacc 240 gtgggcagcc ccccgcagac gctcaacatc ctggtggata caggcagcag taactttgca 300 gtgggtgctg ccccccaccc cttcctgcat cgctactacc agaggcagct gtccagcaca 360 taccgggacc tccggaaggg tgtgtatgtg ccctacaccc agggeaagtg ggaaggggag 420 ctgggcaccg acctggtaag catcccccat ggccccaacg tcactgtgcg tgccaacatt 480 gctgccatca ctgaatcaga caagttcttc atcaacggct ccaactggga aggcatcctg 540 gggctggcct atgctgagat tgccaggctt tgtggtgctg gcttccccct caaccagtct 600 gaagtgctgg cctctgtcgg agggagcatg atcattggag gtatcgacca ctcgctgtac 660 acaggcagtc tctggtatac acccatccgg cgggagtggt attatgaggt catcattgtg 720 cgggtggaga tcaatggaca ggatctgaaa atggactgca aggagtacaa ctatgacaag 780 agcattgtgg acagtggcac caccaacctt cgtttgccca agaaagtgtt tgaagctgca 840 gtcaaatcca tcaaggcagc ctcctccacg gagaagttcc ctgatggttt ctggctagga 900 gagcagctgg tgtgctggca agcaggcacc accccttgga acattttccc agtcatctca 960 ctctacctaa tgggtgaggt taccaaccag tccttccgca teaccatcct tccgcagcaa 1020 tacctgcggc cagtggaaga tgtggccacg tcccaagacg actgttacaa gtttgccatc 1080 tcacagtcat ccacgggcac tgttatggga gctgttatca tggagggctt ctacgttgtc 1140 tttgatcggg cccgaaaacg aattggcttt gctgtcagcg cttgccatgt gcacgatgag 1200 ttcaggacgg cagcggtgga aggccctttt gtcaccttgg acatggaaga ctgtggctac 1260 aacattccac agacagatga gtcatga 1287 <210> 51 <21l> 428 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Hu-Asp2(b) delta TM

* 13k <400> 51 Met Ala G1n Ala Leu Pro Trp Leu Leu Leu Trp Met Gly Ala Gly Val 1 5 10 15 Leu Pro Ala His Gly Thr G1n His Gly Ile Arg Leu Pro Leu Arg Ser 25 30 Gly Leu Gly Gly Ala Pro Leu Gly Leu Arg Leu Pro Arg Glu Thr Asp 40 45 Glu Glu Pro Glu Glu Pro Gly Arg Arg Gly Ser Phe Val Glu Met Val so 55 60 Asp Asn Leu Arg Gly Lys Ser Gly G1n Gly Tyr Tyr Val Glu Met Thr 70 75 80 Val Gly Ser Pro Pro G1n Thr Leu Asn Ile Leu Val Asp Thr Gly Ser 90 95 Ser Asn Phe Ala Val Gly Ala Ala Pro His Pro Phe Leu His Arg Tyr 105 110 Tyr G1n Arg G1n Leu Ser Ser Thr Tyr Arg Asp Leu Arg Lys Gly Val 120 125 Tyr Val Pro Tyr Thr G1n Gly Lys Trp Glu Gly Glu Leu Gly Thr Asp 135 140 Leu Val Ser Ile Pro His Gly Pro Asn Val Thr Val Arg Ala Asn Ile 150 155 160 Ala Ala Ile Thr Glu Ser Asp Lys Phe Phe Ile Asn Gly Ser Asn Trp 170 175 Glu Gly Ile Leu Gly Leu Ala Tyr Ala Glu Ile Ala Arg Leu Cys Gly 185 190 Ala Gly Phe Pro Leu Asn G1n Ser Glu Val Leu Ala Ser Val Gly Gly 200 205 Ser Met Ile Ile Gly Gly Ile Asp His Ser Leu Tyr Thr Gly Ser Leu 210 215 220 Trp Tyr Thr Pro Ile Arg Arg Glu Trp Tyr Tyr Glu Val Ile Ile Val 225 230 235 240 Arg Val Glu Ile Asn Gly G1n Asp Leu Lys Met Asp Cys Lys Glu Tyr 245 250 255 Asn Tyr Asp Lys Ser Ile Val Asp Ser Gly Thr Thr Asn Leu Arg Leu 260 265 270 Pro Lys Lys Val Phe Glu Ala Ala Val Lys Ser Ile Lys Ala Ala Ser 275 280 285 Ser Thr Glu Lys Phe Pro Asp Gly Phe Trp Leu Gly Glu G1n Leu Val 290 295 300 Cys Trp G1n Ala Gly Thr Thr Pro Trp Asn Ile Phe Pro Val Ile Ser 305 310 315 320 i lo Leu Tyr Leu Met Gly Glu Val Thr Asn G1n Ser Phe Arg Ile Thr Ile 325 330 335 Leu Pro G1n G1n Tyr Leu Arg Pro Val Glu Asp Val Ala Thr Ser G1n 340 345 350 Asp Asp Cys Tyr Lys Phe Ala Ile Ser G1n Ser Ser Thr Gly Thr Val 355 360 365 Met Gly Ala Val Ile Met Glu Gly Phe Tyr Val Val Phe Asp Arg Ala 370 375 380 Arg Lys Arg Ile Gly Phe Ala Val Ser Ala Cys His Val His Asp Glu 385 390 395 400 Phe Arg Thr Ala Ala Val Glu Gly Pro Phe Val Thr Leu Asp Met Glu 405 410 415 Asp Cys Gly Tyr Asn Ile Pro G1n Thr Asp Glu Ser 420 425 <210> 52 <21l> 1305 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Hu-Asp2(b) delta TM <400> 52 atggcccaag ccctgccctg gctcctgctg tggatgggcg cgggagtgct gcctgcccac 60 ggcacccagc acggcatccg gctgcccctg cgcagcggcc tggggggcgc ccccctgggg 120 ctgcggctgc cccgggagac cgacgaagag ccegaggagc ccggccggag gggcagcttt 180 gtggagatgg tggacaacct gaggggcaag tcggggeagg gctactacgt ggagatgacc 240 gtgggeagcc cecegcagac gctcaacatc ctggtggata caggcagcag taactttgca 300 gtgggtgctg ccccccaccc cttcctgcat cgctactacc agaggcagct gtecagcaca 360 taccgggacc tccggaaggg tgtgtatgtg ccctacaccc agggcaagtg ggaaggggag 420 ctgggcaccg acctggtaag catcccccat ggccccaacg tcactgtgcg tgccaacatt 480 gctgccatca ctgaatcaga caagttcttc atcaacggct ccaactggga aggcatcctg 540 gggctggcct atgctgagat tgccaggctt tgtggtgctg gcttccccct caaccagtct 600 gaagtgctgg cctctgtcgg agggagcatg atcattggag gtatcgacca ctcgctgtac 660 acaggcagtc tctggtatac acccatccgg cgggagtggt attatgaggt catcattgtg 720 cgggtggaga tcaatggacaggatctgaaa atggactgca aggagtacaa ctatgacaag 780 agcattgtgg acagtggcac caccaacctt cgtttgccca agaaagtgtt tgaagctgca 840 gtcaaatcca tcaaggcagc ctcctccacg gagaagttcc ctgatggttt ctggctagga 900 gagcagctgg tgtgctggca agcaggcacc accccttgga acattttccc agtcatetca 960 ctctacctaa tgggtgaggt taccaaccag tccttccgca tcaccatcct tccgcagcaa 1020 tacctgcggc cagtggaaga tgtggccacg tcccaagacg actgttacaa gtttgccatc 1080 tcacagtcat ccacgggcactgttatggga gctgttatca tggagggctt ctacgttgtc 1140 tttgatcggg cccgaaaacg aattggcttt gctgtcagcg cttgccatgt gcacgatgag 1200 ttcaggacgg cagcggtgga aggccctttt gtcaccttgg acatggaaga ctgtggctac 1260 aacattccac agacagatga gtcacagcag cagcagcagc agtga 1305 <210> 53 <21l> 434 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Hu-Asp2(b) delta TM <400> 53 Met Ala G1n Ala Leu Pro Trp Leu Leu Leu Trp Met Gly Ala Gly Val 1 5 10 15 Leu Pro Ala His Gly Thr G1n His Gly Ile Arg Leu Pro Leu Arg Ser 25 30 Gly Leu Gly Gly Ala Pro Leu Gly Leu Arg Leu Pro Arg Glu Thr Asp 40 45 Glu Glu Pro Glu Glu Pro Gly Arg Arg Gly Ser Phe Val Glu Met Val so 55 60 Asp Asn Leu Arg Gly Lys Ser Gly G1n Gly Tyr Tyr Val Glu Met Thr 70 75 80 Val Gly Ser Pro Pro G1n Thr Leu Asn Ile Leu Val Asp Thr Gly Ser 90 95 Ser Asn Phe Ala Val Gly Ala Ala Pro His Pro Phe Leu His Arg Tyr 105 110 Tyr G1n Arg G1n Leu Ser Ser Thr Tyr Arg Asp Leu Arg Lys Gly Val 120 125 Tyr Val Pro Tyr Thr G1n Gly Lys Trp Glu Gly Glu Leu Gly Thr Asp 135 140 Leu Val Ser Ile Pro His Gly Pro Asn Val Thr Val Arg Ala Asn Ile 150 155 160 Ala Ala Ile Thr Glu Ser Asp Lys Phe Phe Ile Asn Gly Ser Asn Trp 170 175 Glu Gly Ile Leu Gly Leu Ala Tyr Ala Glu Ile Ala Arg Leu Cys Gly 185 190 Ala Gly Phe Pro Leu Asn G1n Ser Glu Val Leu Ala Ser Val Gly Gly 200 205 Ser Met Ile Ile Gly Gly Ile Asp His Ser Leu Tyr Thr Gly Ser Leu 210 215 220 Trp Tyr Thr Pro Ile Arg Arg Glu Trp Tyr Tyr Glu Val Ile Ile Val 225 230 235 240 Arg Val Glu Ile Asn Gly G1n Asp Leu Lys Met Asp Cys Lys Glu Tyr 245 250 255 Asn Tyr Asp Lys Ser Ile Val Asp Ser Gly Thr Thr Asn Leu Arg Leu 260 265 270 Pro Lys Lys Val Phe Glu Ala Ala Val Lys Ser Ile Lys Ala Ala Ser 275 280 285 Ser Thr Glu Lys Phe Pro Asp Gly Phe Trp Leu Gly Glu G1n Leu Val 290 295 300 Cys Trp G1n Ala Gly Thr Thr Pro Trp Asn Ile Phe Pro Val Ile Ser 305 310 315 320 1 k-kA Leu Tyr Leu Met Gly Glu Val Thr Asn G1n Ser Phe Arg Ile Thr Ile 325 330 335 Leu Pro G1n G1n Tyr Leu Arg Pro Val Glu Asp Val Ala Thr Ser G1n 340 345 350 Asp Asp Cys Tyr Lys Phe Ala Ile Ser G1n Ser Ser Thr Gly Thr Val 355 360 365 Met Gly Ala Val Ile Met Glu Gly Phe Tyr Val Val Phe Asp Arg Ala 370 375 380 Arg Lys Arg Ile Gly Phe Ala Val Ser Ala Cys His Val His Asp Glu 385 390 395 400 Phe Arg Thr Ala Ala Val Glu Gly Pro Phe Val Thr Leu Asp Met Glu 405 410 415 Asp Cys Gly Tyr Asn Ile Pro G1n Thr Asp Glu Ser His His His His 420 425 430 His His <210> 54 <21l> 2310 <212> DNA <213> Homo sapiens <400> 54 atgctgcccg gtttggcact gctcctgctg gccgcctgga cggctcgggc gctggaggta 60 cccactgatg gtaatgctgg cctgctggct gaaccccaga ttgccatgtt ctgtggcaga 120 ctgaacatgc acatgaatgt ccagaatggg aagtgggatt cagatccatc agggaccaaa 180 acctgcattg ataccaagga aggcatcctg cagtattgcc aagaagtcta ccctgaactg 240 cagatcacca atgtggtaga agccaaccaa ccagtgacca tccagaactg gtgcaagcgg 300 ggccgcaagc agtgcaagac ccatccccac tttgtgattc cctaccgctg cttagttggt 360 gagtttgtaa gtgatgccct tctcgttcct gacaagtgca aattcttaca ccaggagagg 420 atggatgttt gcgaaactca tcttcactgg cacaccgtcg ccaaagagac atgcagtgag 480 aagagtacca acttgcatga ctacggcatg ttgctgccct gcggaattga caagttccga 540 ggggtagagt ttgtgtgttg cccactggct gaagaaagtg acaatgtgga ttctgctgat 600 gcggaggagg atgactcgga tgtctggtgg ggcggagcag acacagacta tgcagatggg 660 agtgaagaca aagtagtaga agtagcagag gaggaagaag tggctgaggt ggaagaagaa 720 gaagccgatg atgacgagga cgatgaggat ggtgatgagg tagaggaaga ggctgaggaa 780 ccctacgaag aagccacaga gagaaccacc agcattgcca ccaccaccac caccaccaca 840 gagtctgtgg aagaggtggt tcgagaggtg tgctctgaac aagccgagac ggggccgtgc 900 cgagcaatga tctcccgctg gtactttgat gtgactgaag ggaagtgtgc cccattcttt 960 tacggcggat gtggcggcaa ccggaacaac tttgacacag aagagtactg catggccgtg 1020 tgtggcagcg ccatgtccca aagtttactc aagactaccc aggaacctct tggccgagat 1080 cctgttaaac ttcctacaac agcagccagt acccctgatg ccgttgacaa gtatctcgag 1140 acacctgggg atgagaatga acatgcccat ttccagaaag ccaaagagag gcttgaggcc 1200 aagcaccgag agagaatgtc ccaggtcatg agagaatggg aagaggcaga acgtcaagca 1260 aagaacttgc ctaaagctga taagaaggca gttatccagc atttccagga gaaagtggaa 1320 tctttggaac aggaagcagc caacgagaga cagcagctgg tggagacaca catggccaga 1380 gtggaagcca tgctcaatga ccgccgccgc ctggccctgg agaactacat caccgctctg 1440 caggctgttc ctcctcggcc tcgtcacgtg ttcaatatgc taaagaagta tgtccgcgca 1500 gaacagaagg acagacagca caccctaaag catttcgagc atgtgcgcat ggtggatccc 1560 aagaaagccg ctcagatccg gtcccaggtt atgacacacc tccgtgtgat ttatgagcgc 1620 atgaatcagt ctctctccct gctctacaac gtgcctgcag tggccgagga gattcaggat 1680 gaagttgatg agctgcttca gaaagagcaa aactattcag atgacgtctt ggccaacatg 1740 1(+2-- attagtgaac caaggatcag ttacggaaac gatgctctca tgccatcttt gaccgaaacg 1800 aaaaccaccg tggagctcct tcccgtgaat ggagagttca gcctggacga tctccagccg 1860 tggcattctt ttggggctga ctctgtgcca gccaacacag aaaacgaagt tgagcctgtt 1920 gatgcccgcc ctgctgccga ccgaggactg accactcgac caggttctgg gttgacaaat 1980 atcaagacgg aggagatctc tgaagtgaag atggatgcag aattccgaca tgactcagga 2040 tatgaagttc atcatcaaaa attggtgttc tttgcagaag atgtgggttc aaacaaaggt 2100 gcaatcattg gactcatggt gggcggtgtt gtcatagcga cagtgatcgt catcaccttg 2160 gtgatgctga agaagaaaca gtacacatcc attcatcatg gtgtggtgga ggttgacgcc 2220 gctgtcaccc cagaggagcg ccacctgtcc aagatgcagc agaacggcta cgaaaatcca 2280 acctacaagt tctttgagca gatgcagaac 2310 <210> 55 <21l> 770 <212> PRT <213> Homo sapiens <400> 55 Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15 Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 25 30 G1n Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val G1n 40 45 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp 55 60 Thr Lys Glu Gly Ile Leu G1n Tyr Cys G1n Glu Val Tyr Pro Glu Leu 70 75 80 G1n Ile Thr Asn Val Val Glu Ala Asn G1n Pro Val Thr Ile G1n Asn 90 95 Trp Cys Lys Arg Gly Arg Lys G1n Cys Lys Thr His Pro His Phe Val 105 110 Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 120 125 Val Pro Asp Lys Cys Lys Phe Leu His G1n Glu Arg Met Asp Val Cys 135 140 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu 150 155 160 Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile 170 175 Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 185 190 Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 200 205 Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220 Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240 11+3 Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255 Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265 270 Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285 Glu Val Cys Ser Glu G1n Ala Glu Thr Gly Pro Cys Arg Ala Met Ile 290 295 300 Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe 305 310 315 320 Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr 325 330 335 Cys Met Ala Val Cys Gly Ser Ala Met Ser G1n Ser Leu Leu Lys Thr 340 345 350 Thr G1n Glu Pro Leu Ala Arg Asp Pro Val Lys Leu Pro Thr Thr Ala 355 360 365 Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp 370 375 380 Glu Asn Glu His Ala His Phe G1n Lys Ala Lys Glu Arg Leu Glu Ala 385 390 395 400 Lys His Arg Glu Arg Met Ser G1n Val Met Arg Glu Trp Glu Glu Ala 405 410 415 Glu Arg G1n Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile 420 425 430 G1n His Phe G1n Glu Lys Val Glu Ser Leu Glu G1n Glu Ala Ala Asn 435 440 445 Glu Arg G1n G1n Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met 450 455 460 Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu 465 470 475 480 G1n Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys 485 490 495 Tyr Val Arg Ala Glu G1n Lys Asp A-rg G1n His Thr Leu Lys His Phe 500 505 510 Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala G1n Ile Arg Ser 515 520 525 G1n Val Met Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn G1n Ser 530 535 540 Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile G1n Asp 545 550 555 560 Glu Val Asp Glu Leu Leu G1n Lys Glu G1n Asn Tyr Ser Asp Asp Val fkt-k'- 565 570 575 Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala 580 585 590 Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro 595 600 605 Val Asn Gly Glu Phe Ser Leu Asp Asp Leu G1n Pro Trp His Ser Phe 610 615 620 Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val 625 630 635 640 Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser 645 650 655 Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp 660 665 670 Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His G1n Lys Leu 675 680 685 Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly 690 695 700 Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu 705 710 715 720 Val Met Leu Lys Lys Lys G1n Tyr Thr Ser Ile His His Gly Val Val 725 730 735 Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met 740 745 750 G1n G1n Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu G1n Met 755 760 765 G1n Asn 770 <210> 56 <21l> 2253 <212> DNA <213> Homo sapiens <400. 56 atgctgcccg gtttggcact gctcctgctg gccgcctgga cggctcgggc gctggaggta 60 cccactgatg gtaatgctgg cctgctggct gaaccccaga ttgccatgtt ctgtggcaga 120 ctgaacatgc acatgaatgt ccagaatggg aagtgggatt cagatccatc agggaccaaa 180 acctgcattg ataccaagga aggcatcctg cagtattgcc aagaagtcta ccctgaactg 240 cagatcacca atgtggtaga agccaaccaa ccagtgacca tccagaactg gtgcaagcgg 300 ggccgcaagc agtgcaagac ccatccccac tttgtgattc cctaccgctg cttagttggt 360 gagtttgtaa gtgatgccct tctcgttcct gacaagtgca aattcttaca ccaggagagg 420 atggatgttt gcgaaactca tcttcactgg cacaccgtcg ccaaagagac atgcagtgag 480 aagagtacca acttgcatga ctaeggcatg ttgctgccct gcggaattga caagttccga 540 ggggtagagt ttgtgtgttg cccactggct gaagaaagtg acaatgtgga ttctgctgat 600 gcggaggagg atgactcgga tgtctggtgg ggcggagcag acacagacta tgcagatggg 660 agtgaagaca aagtagtaga agtagcagag gaggaagaag tggctgaggt ggaagaagaa 720 gaagccgatg atgacgagga cgatgaggat ggtgatgagg tagaggaaga ggctgaggaa 780 ccctacgaag aagccacaga gagaaccacc agcattgcca ccaccaccac caccaccaca 840 gagtctgtgg aagaggtg9t tegagaggtg tgctctgaac aagccgagac ggggccgtgc 900 cgagcaatga tctcccgctg gtactttgat gtgactgaag ggaagtgtgc cccattcttt 960 tacggcggat gtggcggcaa ccggaacaac tttgacacag aagagtactg catggccgtg 1020 tgtggcagcg ccattcctac aacagcagcc agtacccctg atgccgttga caagtatctc 1080 gagacacctg gggatgagaa tgaacatgcc catttccaga aagccaaaga gaggcttgag 1140 gccaagcacc gagagagaat gtcccaggtc atgagagaat gggaagaggc agaacgtcaa 1200 gcaaagaact tgcctaaagc tgataagaag gcagttatcc agcatttcca ggagaaagtg 1260 gaatctttgg aacaggaagc agccaacgag agacagcagc tggtggagac acacatggcc 1320 agagtggaag ccatgctcaa tgaccgccgc cgcctggccc tggagaacta catcaccgct 1380 ctgcaggctg ttcctcctcg gcctcgtcac gtgttcaata tgctaaagaa gtatgtccgc 1440 gcagaacaga aggacagaca gcacacccta aagcatttcg agcatgtgcg catggtggat 1500 cccaagaaag ccgctcagat ccggtcccag gttatgacac acctccgtgt gatttatgag 1560 cgcatgaatc agtctctctc cctgctctac aacgtgcctg cagtggccga ggagattcag 1620 gatgaagttg atgagctgct tcagaaagag caaaactatt cagatgacgt cttggccaac 1680 atgattagtg aaccaaggat cagttacgga aacgatgctc tcatgccatc tttgaccgaa 1740 acgaaaacca ccgtggagct ccttcccgtg aatggagagt tcagcctgga cgatctccag 1800 ccgtggcatt cttttggggc tgactctgtg ccagccaaca cagaaaacga agttgagcct 1860 gttgatgccc gccctgctgc cgaccgagga ctgaccactc gaccaggttc tgggttgaca 1920 aatatcaaga cggaggagat ctctgaagtg aagatggatg cagaattccg acatgactca 1980 ggatatgaag ttcatcatca aaaattggtg ttctttgcag aagatgtggg ttcaaacaaa 2040 ggtgcaatca ttggactcat ggtgggcggt gttgtcatag cgacagtgat cgtcatcacc 2100 ttggtgatgc tgaagaagaa acagtacaca tceattcatc atggtgtggt ggaggttgac 2160 gccgctgtca ccccagagga gcgccacctg tccaagatgc agcagaacgg ctacgaaaat 2220 ccaacctaca agttctttga gcagatgcag aac 2253 <210> 57 <21l> 751 <212> PRT <213> Homo sapiens <400> 57 Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15 Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 25 30 G1n Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val G1n 40 45 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp 55 60 Thr Lys Glu Gly Ile Leu G1n Tyr Cys G1n Glu Val Tyr Pro Glu Leu 70 75 80 G1n Ile Thr Asn Val Val Glu Ala Asn G1n Pro Val Thr Ile G1n Asn 90 95 Trp Cys Lys Arg Gly Arg Lys G1n Cys Lys Thr His Pro His Phe Val 105 110 Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 120 125 Val Pro Asp Lys Cys Lys Phe Leu His G1n Glu Arg Met Asp Val Cys 135 140 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu 150 155 160 Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile 170 175 Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 185 190 Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 200 205 Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220 Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240 Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255 Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265 270 Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285 Glu Val Cys Ser Glu G1n Ala Glu Thr Gly Pro Cys Arg Ala Met Ile 290 295 300 Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe 305 310 315 320 Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr 325 330 335 Cys Met Ala Val Cys Gly Ser Ala Ile Pro Thr Thr Ala Ala Ser Thr 340 345 350 Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp Glu Asn Glu 355 360 365 His Ala His Phe G1n Lys Ala Lys Glu Arg Leu Glu Ala Lys His Arg 370 375 380 Glu Arg Met Ser G1n Val Met Arg Glu Trp Glu Glu Ala Glu Arg G1n 385 390 395 400 Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile G1n His Phe 405 410 415 G1n Glu Lys Val Glu Ser Leu Glu G1n Glu Ala Ala Asn Glu Arg G1n 420 425 430 G1n Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met Leu Asn Asp 435 440 445 Arg Arg A-rg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu G1n Ala Val 450 455 460 Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys Tyr Val Arg 465 470 475 480 Ala Glu G1n Lys Asp Arg G1n His Thr Leu Lys His Phe Glu His Val 1,.

485 490 495 Arg Met Val Asp Pro Lys Lys Ala Ala G1n lle Arg Ser G1n Val Met 500 505 510 Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn G1n Ser Leu Ser Leu 515 520 525 Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile G1n Asp Glu Val Asp 530 535 540 Glu Leu Leu G1n Lys Glu G1n Asn Tyr Ser Asp Asp Val Leu Ala Asn 545 550 555 560 Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala Leu Met Pro 565 570 575 Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro Val Asn Gly 580 585 590 Glu Phe Ser Leu Asp Asp Leu G1n Pro Trp His Ser Phe Gly Ala Asp 595 600 605 Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val Asp Ala Arg 610 615 620 Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser Gly Leu Thr 625 630 635 640 Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp Ala Glu Phe 645 650 655 Arg His Asp Ser Gly Tyr Glu Val His His G1n Lys Leu Val Phe Phe 660 665 670 Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val 675 680 685 Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu Val Met Leu 690 695 700 Lys Lys Lys G1n Tyr Thr Ser Ile His His Gly Val Val Glu Val Asp 705 710 715 720 Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met G1n G1n Asn 725 730 735 Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu G1n Met G1n Asn 740 745 750 <210> 58 <21l> 2316 <212> DNA <213> Homo sapiens <400> 58 atgctgcccg gtttggcact gctcctgctg gccgcctgga cggctcgggc gctggaggta 60 cccactgatg gtaatgctgg cctgctggct gaaccccaga ttgccatgtt ctgtggcaga 120 ctgaacatgc acatgaatgt ccagaatggg aagtgggatt cagatccatc agggaccaaa 180 acctgcattg ataccaagga aggcatcctg cagtattgcc aagaagtcta ccctgaactg 240 cagatcacca atgtggtaga agccaaCcaa ccagtgacca tccagaactg gtgcaagcgg 300 ggccgcaagc agtgcaagac ccatccccac tttgtgattc cctaccgctg cttagttggt 360 gagtttgtaa gtgatgccct tctcgttcct gacaagtgca aattcttaca ccaggagagg 420 atggatgttt gcgaaactca tcttcactgg cacaccgtcg ccaaagagac atgcagtgag 480 aagagtacca acttgcatga ctacggcatg ttgctgccct gcggaattga caagttccga 540 ggggtagagt ttgtgtgttg cccactggct gaagaaagtg acaatgtgga ttetgctgat 600 gcggaggagg atgactcgga tgtctggtgg ggcggagcag acacagacta tgcagatggg 660 agtgaagaca aagtagtaga agtagcagag gaggaagaag tggctgaggt ggaagaagaa 720 gaagccgatg atgacgagga cgatgaggat ggtgatgagg tagaggaaga ggctgaggaa 780 ccctacgaag aagccacaga gagaaccacc agcattgcca ccaccaccac caccaccaca 840 gagtctgtgg aagaggtggt tcgagaggtg tgctctgaac aagccgagac ggggccgtgc 900 cgagcaatga tctcccgctg gtactttgat gtgactgaag ggaagtgtgc cccattcttt 960 tacggcggat gtggcggcaa ccggaacaac tttgacacag aagagtactg catggccgtg 1020 tgtggeagcg ccatgtccca aagtttactc aagactaccc aggaacctct tggccgagat 1080 cctgttaaac ttcctacaac agcagccagt acccctgatg ccgttgacaa gtatctcgag 1140 acacctgggg atgagaatga acatgcccat ttccagaaag ccaaagagag gcttgaggcc 1200 aagcaccgag agagaatgtc ccaggtcatg agagaatggg aagaggcaga acgtcaagca 1260 aagaacttgc etaaagctga taagaaggca gttatccagc atttccagga gaaagtggaa 1320 tctttggaac aggaagcagc caacgagaga cagcagctgg tggagacaca catggccaga 1380 gtggaagcca tgctcaatga ccgccgccgc ctggccctgg agaactacat caccgctctg 1440 caggctgttc ctcctcggcc tcgtcacgtg ttcaatatgc taaagaagta tgtccgcgca 1500 gaacagaagg acagacagca caccctaaag catttcgagc atgtgcgcat ggtggatccc 1560 aagaaagccg ctcagatccg gtcccaggtt atgacacacc tccgtgtgat ttatgagcgc 1620 atgaatcagt ctctctccct gctctacaac gtgcctgcag tggccgagga gattcaggat 1680 gaagttgatg agctgcttca gaaagagcaa aactattcag atgacgtctt ggccaacatg 1740 attagtgaac caaggatcag ttacggaaac gatgctctca tgccatcttt gaccgaaacg 1800 aaaaccaccg tggagctcct tcccgtgaat ggagagttca gcctggaega tctccagccg 1860 tggcattctt ttggggctga ctctgtgcca gecaacacag aaaacgaagt tgagcctgtt 1920 gatgcccgcc ctgctgccga ccgaggactg accactcgac caggttctgg gttgacaaat 1980 atcaagacgg aggagatctc tgaagtgaag atggatgcag aattccgaca tgactcagga 2040 tatgaagttc atcatcaaaa attggtgttc tttgcagaag atgtgggttc aaacaaaggt 2100 gcaatcattg gactcatggt gggcggtgtt gtcatagcga cagtgatcgt catcaccttg 2160 gtgatgctga agaagaaaca gtacacatcc attcatcatg gtgtggtgga ggttgacgcc 2220 gctgtcaccc cagaggagcg ccacctgtcc aagatgcagc agaacggeta cgaaaatcca 2280 acctacaagt tctttgagca gatgcagaac aagaag 2316 <210> 59 <21l> 772 <212> PRT <213> Homo sapiens <400> 59 Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15 Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 25 30 G1n Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val G1n 40 45 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp so 55 60 Thr Lys Glu Gly Ile Leu G1n Tyr Cys G1n Glu Val Tyr Pro Glu Leu 70 75 80 G1n Ile Thr Asn Val Val Glu Ala Asn G1n Pro Val Thr Ile G1n Asn 90 95 Trp Cys Lys Arg Gly Arg Lys G1n Cys Lys Thr His Pro His Phe Val 105 110 Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 120 125 Val Pro Asp Lys Cys Lys Phe Leu His G1n Glu Arg Met Asp Val Cys 135 140 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu 150 155 160 Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile 170 175 Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 185 190 Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 200 205 Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220 Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240 Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255 Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265 270 Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285 Glu Val Cys Ser Glu G1n Ala Glu Thr Gly Pro Cys Arg Ala Met lle 290 295 300 Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe 305 310 315 320 Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr 325 330 335 Cys Met Ala Val Cys Gly Ser Ala Met Ser G1n Ser Leu Leu Lys Thr 340 345 350 Thr G1n Glu Pro Leu Ala Arg Asp Pro Val Lys Leu Pro Thr Thr Ala 355 360 365 Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp 370 375 380 Glu Asn Glu His Ala His Phe G1n Lys Ala Lys Glu Arg Leu Glu Ala 385 390 395 400 Lys His Arg Glu Arg Met Ser G1n Val Met Arg Glu Trp Glu Glu Ala 405 410 415 Glu Arg G1n Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile 420 425 430 G1n His Phe G1n Glu Lys Val Glu Ser Leu Glu G1n Glu Ala Ala Asn 435 440 445 Glu Arg G1n G1n Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met 450 455 460 Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu 465 470 475 480 G1n Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys 485 490 495 Tyr Val Arg Ala Glu G1n Lys Asp Arg G1n His Thr Leu Lys His Phe 500 505 510 Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala G1n Ile Arg Ser 515 520 525 G1n Val Met Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn G1n Ser 530 535 540 Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile G1n Asp 545 550 555 560 Glu Val Asp Glu Leu Leu G1n Lys Glu G1n Asn Tyr Ser Asp Asp Val 565 570 575 Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala 580 585 590 Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro 595 600 605 Val Asn Gly Glu Phe Ser Leu Asp Asp Leu G1n Pro Trp His Ser Phe 610 615 620 Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val 625 630 635 640 Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser 645 650 655 Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp 660 665 670 Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His G1n Lys Leu 675 680 685 Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly 690 695 700 Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu 705 710 715 720 Val Met Leu Lys Lys Lys G1n Tyr Thr Ser Ile His His Gly Val Val 725 730 735 Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met 740 745 750 G1n G1n Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu G1n Met Isk 755 760 765 G1n Asn Lys Lys 770 <210> 60 <21l> 2259 <212> DNA <213> Homo sapiens <400> 60 atgctgcecg gtttggcact gctcctgctg gccgcctgga c99ctegggc gctggaggta 60 cccactgatg gtaatgctgg cctgctggct gaaccccaga ttgccatgtt ctgtggcaga 120 ctgaacatgc acatgaatgt ccagaatggg aagtgggatt cagatecatc agggaccaaa 180 acctgcattg ataccaagga aggcatcctg cagtattgcc aagaagtcta ccctgaactg 240 cagatcacca atgtggtaga agccaaccaa ccagtgacca tccagaactg gtgcaagcgg 300 ggccgcaagc agtgcaagac ccatccccac tttgtgattc cctaccgctg cttagttggt 360 gagtttgtaa gtgatgccct tctcgttcct gacaagtgca aattcttaca ccaggagagg 420 atggatgttt gcgaaactca tcttcactgg cacaccgtcg ccaaagagac atgcagtgag 480 aagagtacca acttgcatga ctacggcatg ttgctgccct gcggaattga caagttccga 540 ggggtagagt ttgtgtgttg cccactggct gaagaaagtg acaatgtgga ttetgctgat 600 gcggaggagg atgactcgga tgtctggtgg ggcggagcag acacagacta tgcagatggg 660 agtgaagaca aagtagtaga agtagcagag gaggaagaag tggctgaggt ggaagaagaa 720 gaagccgatg atgaegagga cgatgaggat ggtgatgagg tagaggaaga ggctgaggaa 780 ccctacgaag aagecacaga gagaaccacc agcattgcca ccaccaccac caccaccaca 840 gagtctgtgg aagaggtggt tcgagaggtg tgetctgaac aagccgagac ggggccgtgc 900 cgagcaatga tct=gctg gtactttgat gtgactgaag ggaagtgtgc cccattcttt 960 tacggcggat gtggcggcaa ccggaacaac tttgacacag aagagtactg catggccgtg 1020 tgtggcagcg ccattcctac aacagcagcc agtacccctg atgccgttga caagtatctc 1080 gagacacctg gggatgagaa tgaacatgcc catttccaga aagccaaaga gaggcttgag 1140 gccaagcacc gagagagaat gtcceaggtc atgagagaat gggaagaggc agaacgtcaa 1200 gcaaagaact tgcctaaagc tgataagaag gcagttatcc agcatttcca ggagaaagtg 1260 gaatctttgg aaCaggaagc agccaacgag agacagcage tggtggagac acacatggcc 1320 agagtggaag ccatgctcaa tgaccgccgc cgcctggecc tggagaacta catcaccgct 1380 ctgcaggctg ttcctcctcg gcctcgtcac gtgttcaata tgctaaagaa gtatgtccgc 1440 gcagaacaga aggacagaca gcacacccta aagcatttcg agcatgtgcg catggtggat 1500 cccaagaaag ccgctcagat ccggtcccag gttatgacac acctccgtgt gatttatgag 1560 cgcatgaatc agtctctctc cctgctctac aacgtgcctg cagtggccga ggagattcag 1620 gatgaagttg atgagctgct tcagaaagag caaaactatt cagatgacgt cttggccaac 1680 atgattagtg aaccaaggat cagttacgga aacgatgctc tcatgccatc tttgaccgaa 1740 acgaaaacca ccgtggagct cctteccgtg aatggagagt tcagcctgga cgatctccag 1800 ccgtggcatt cttttggggc tgactctgtg ccagccaaca cagaaaacga agttgagcct 1860 gttgatgccc gccctgctgc cgaccgagga ctgaccaetc gaccaggttc tgggttgaca 1920 aatatcaaga cggaggagat ctctgaagtg aagatggatg cagaattccg acatgactca 1980 ggatatgaag ttcatcatca aaaattggtg ttctttgeag aagatgtggg ttcaaacaaa 2040 ggtgcaatca ttggactcat ggtgggcggt gttgtcatag cgacagtgat cgtcatcacc 2100 ttggtgatge tgaagaagaa acagtacaca tccattcatc atggtgtggt ggaggttgac 2160 gccgctgtca ccccagagga gcgccacctg tccaagatgc agcagaacgg ctacgaaaat 2220 ccaacctaca agttctttga gcagatgcag aacaagaag 2259 <210> 61 <21l> 753 <212> PRT <213> Homc sapiens <400> 61 Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15 Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 25 30 G1n Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val G1n 40 45 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp 55 60 Thr Lys Glu Gly Ile Leu G1n Tyr Cys G1n Glu Val Tyr Pro Glu Leu 70 75 80 G1n Ile Thr Asn Val Val Glu Ala Asn G1n Pro Val Thr Ile G1n Asn 90 95 Trp Cys Lys Arg Gly Arg Lys G1n Cys Lys Thr His Pro His Phe Val 105 110 Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 120 125 Val Pro Asp Lys Cys Lys Phe Leu His G1n Glu Arg Met Asp Val Cys 135 140 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu 150 155 160 Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile 170 175 Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 185 190 Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 200 205 Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220 Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240 Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255 Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265 270 Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285 Glu Val Cys Ser Glu G1n Ala Glu Thr Gly Pro Cys Arg Ala Met Ile 290 295 300 Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe 305 310 315 320 Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr 325 330 335 Cys Met Ala Val Cys Gly Ser Ala Ile Pro Thr Thr Ala Ala Ser Thr 340 345 350 153 Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp Glu Asn Glu 355 360 365 His Ala His Phe G1n Lys Ala Lys Glu Arg Leu Glu Ala Lys His Arg 370 375 380 Glu Arg Met Ser G1n Val Met Arg Glu Trp Glu Glu Ala Glu Arg G1n 385 390 395 400 Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val lle G1n His Phe 405 410 415 G1n Glu Lys Val Glu Ser Leu Glu G1n Glu Ala Ala Asn Glu Arg G1n 420 425 430 G1n Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met Leu Asn Asp 435 440 445 Arg Arg Arg Leu Ala Leu Glu Asn Tyr lle Thr Ala Leu G1n Ala Val 450 455 460 Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys Tyr Val Arg 465 470 475 480 Ala Glu G1n Lys Asp Arg G1n His Thr Leu Lys His Phe Glu His Val 485 490 495 Arg Met Val Asp Pro Lys Lys Ala Ala G1n Ile Arg Ser G1n Val Met 500 505 510 Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn G1n Ser Leu Ser Leu 515 520. 525 Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile G1n Asp Glu Val Asp 530 535 540 Glu Leu Leu G1n Lys Glu G1n Asn Tyr Ser Asp Asp Val Leu Ala Asn 545 550 555 560 Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala Leu Met Pro 565 570 575 Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro Val Asn Gly 580 585 590 Glu Phe Ser Leu Asp Asp Leu G1n Pro Trp His Ser Phe Gly Ala Asp 595 600 605 Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val Asp Ala Arg 610 615 620 Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser Gly Leu Thr 625 630 635 640 Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp Ala Glu Phe 645 650 655 Arg His Asp Ser Gly Tyr Glu Val His His G1n Lys Leu Val Phe Phe 660 665 670 Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile lle Gly Leu Met Val 154 675 680 685 Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu Val Met Leu 690 695 700 Lys Lys Lys G1n Tyr Thr Ser Ile His HisGly Val Val Glu Val Asp 705 710 715 720 Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met G1n G1n Asn 725 730 735 Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu G1n Met G1n Asn Lys 740 745 750 Lys <210> 62 <21l> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic <400> 62 Leu Glu Val Leu. Phe G1n Gly Pro 1 5 <210> 63 <21l> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic <400> 63 Ser Glu Val Asn Leu Asp Ala Glu Phe Arg 1 5 10 <210> 64 <21l> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic <400> 64 Ser Glu Val Lys Met Asp Ala Glu Phe Arg 1 5 10 <210> 65 <21l> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic <400>65 Arg Arg Gly Gly Val Val Ile Ala Thr Val Ile Val Gly Glu Arg 1 5 10 is <210> 66 <21l> 518 <212> PRT <213> Homo sapiens <400> 66 Met Gly Ala Leu Ala Arg Ala Leu Leu Leu Pro Leu Leu Ala G1n Trp 1 5 10 15 Leu Leu Arg Ala Ala Pro Glu Leu Ala Pro Ala Pro Phe Thr Leu Pro 25 30 Leu Arg Val Ala Ala Ala Thr Asn Arg Val Val Ala Pro Thr Pro Gly 40 45 Pro Gly Thr Pro Ala Glu Arg His Ala Asp Gly Leu Ala Leu Ala Leu so 55 60 Glu Pro Ala Leu Ala Ser Pro Ala Gly Ala Ala Asn Phe Leu Ala Met 70 75 80 Val Asp Asn Leu G1n Gly Asp Ser Gly Arg Gly Tyr Tyr Leu Glu Met 90 95 Leu Ile Gly Thr Pro Pro Gin Lys Leu Gin Ile Leu Val Asp Thr Gly 105 110 Ser Ser Asn Phe Ala Val Ala Gly Thr Pro His Ser Tyr Ile Asp Thr 120 125 Tyr Phe Asp Thr Glu Arg Ser Ser Thr Tyr Arg Ser Lys Gly Phe Asp 135 140 Val Thr Val Lys Tyr Thr G1n Gly Ser Trp Thr Gly Phe Val Gly Glu 150 155 160 Asp Leu. Val Thr Ile Pro Lys Gly Phe Asn Thr Ser Phe Leu Val Asn 170 175 Ile Ala Thr Ile Phe Glu Ser Glu Asn Phe Phe Leu Pro Gly Ile Lys 185 190 Trp Asn Gly Ile Leu, Gly Leu Ala Tyr Ala Thr Leu Ala Lys Pro Ser 200 205 Ser Ser Leu Glu Thr Phe Phe Asp Ser Leu Val Thr G1n Ala Asn Ile 210 215 220 Pro Asn Val Phe Ser Met G1n Met Cys Gly Ala Gly Leu Pro Val Ala 225 230 235 240 Gly Ser Gly Thr Asn Gly Gly Ser Leu Val Leu Gly Gly Ile Glu Pro 245 250 255 Is(--Ser Leu Tyr Lys Gly Asp Ile Trp Tyr Thr Pro Ile Lys Glu Glu Trp 260 265 270 Tyr Tyr G1n Ile Glu Ile Leu Lys Leu Glu Ile Gly Gly G1n Ser Leu 275 280 285 Asn Leu Asp Cys Arg Glu Tyr Asn Ala Asp Lys Ala Ile Val Asp Ser 290 295 300 Gly Thr Thr Leu Leu Arg Leu Pro G1n Lys Val Phe Asp Ala Val Val 305 310 315 320 Glu Ala Val Ala Arg Ala Ser Leu Ile Pro Glu Phe Ser Asp Gly Phe 325 330 335 Trp Thr Gly Ser G1n Leu Ala Cys Trp Thr Asn Ser Glu Thr Pro Trp 340 345 350 Ser Tyr Phe Pro Lys Ile Ser Ile Tyr Leu Arg Asp Glu Asn Ser Ser 355 360 365 Arg Ser Phe Arg Ile Thr Ile Leu Pro G1n Leu Tyr Ile G1n Pro Met 370 375 380 Met Gly Ala Gly Leu Asn Tyr Glu Cys Tyr Arg Phe Gly Ile Ser Pro 385 390 395 400 Ser Thr Asn Ala Leu Val Ile Gly Ala Thr Val Met Glu Gly Phe Tyr 405 410 415 Val Ile Phe Asp Arg Ala G1n Lys Arg Val Gly Phe Ala Ala Ser Pro 420 425 430 Cys Ala Glu Ile Ala Gly Ala Ala Val Ser Glu Ile Ser Gly Pro Phe 435 440 445 Ser Thr Glu Asp Val Ala Ser Asn Cys Val Pro Ala G1n Ser Leu Ser 450 455 460 Glu Pro Ile Leu Trp Ile Val Ser Tyr Ala Leu Met Ser Val Cys Gly 465 470 475 480 Ala Ile Leu Leu Val Leu Ile Val Leu Leu Leu Leu Pro Phe Arg Cys 485 490 495 G1n Arg Arg Pro Arg Asp Pro Glu Val Val Asn Asp Glu Ser Ser Leu 500 505 510 Val Arg His Arg Trp Lys 515 <210> 67 <21l> 475 <212> PRT <213> Homo sapiens <400> 67 Met Gly Ala Leu Ala Arg Ala Leu Leu Leu Pro Leu Leu Ala G1n Trp 1 5.10 15 Leu Leu Arg Ala Ala Pro Glu Leu Ala Pro Ala Pro Phe Thr Leu Pro 25 30 Leu Arg Val Ala Ala Ala Thr Asn Arg Val Val Ala Pro Thr Pro Gly 40 45 Pro Gly Thr Pro Ala Glu Arg His Ala Asp Gly Leu Ala Leu Ala Leu 55 60 Glu Pro Ala Leu Ala Ser Pro Ala Gly Ala Ala Asn Phe Leu Ala Met 70 75 80 Val Asp Asn Leu G1n Gly Asp Ser Gly Arg Gly Tyr Tyr Leu Glu Met 95 Leu Ile Gly Thr Pro Pro G1n Lys Leu G1n Ile Leu Val Asp Thr Gly 105 110 Ser Ser Asn Phe Ala Val Ala Gly Thr Pro His Ser Tyr lle Asp Thr 120 125 Tyr Phe Asp Thr Glu Arg Ser Ser Thr Tyr Arg Ser Lys Gly Phe Asp 135.140 Val Thr Val Lys Tyr Thr G1n Gly Ser Trp Thr Gly Phe Val Gly Glu 150 155 160 Asp Leu Val Thr Ile Pro Lys Gly Phe Asn Thr Ser Phe Leu Val Asn 170 175 Ile Ala Thr Ile Phe Glu Ser Glu Asn Phe Phe Leu Pro Gly Ile Lys 185 190 Trp Asn Gly lle Leu Gly Leu Ala Tyr Ala Thr Leu Ala Lys Pro Ser 200 205 Ser Ser Leu Glu Thr Phe Phe Asp Ser Leu Val Thr G1n Ala Asn Ile 210 215 220 Pro Asn Val Phe Ser Met G1n Met Cys Gly Ala Gly Leu Pro Val Ala 225 230 235 240 Gly Ser Gly Thr Asn Gly Gly Ser Leu Val Leu Gly Gly lle Glu Pro 245 250 1 255 Ser Leu Tyr Lys Gly Asp Ile Trp Tyr Thr Pro Ile Lys Glu Glu Trp 260 265 270 Tyr Tyr G1n lle Glu lle Leu Lys Leu Glu lle Gly Gly G1n Ser Leu 275 280 285 Asn Leu Asp Cys Arg Glu Tyr Asn Ala Asp Lys Ala Ile Val Asp Ser 290 295 300 Gly Thr Thr Leu Leu Arg Leu Pro G1n Lys Val Phe Asp Ala Val Val 305 310 315 320 Glu Ala Val Ala Arg Ala Ser Leu lle Pro Glu Phe Ser Asp Gly Phe 325 330 335 Trp Thr Gly Ser G1n Leu Ala Cys Trp Thr Asn Ser Glu Thr Pro Trp 340 345 350 Ser Tyr Phe Pro Lys Ile Ser Ile Tyr Leu Arg Asp Glu Asn Ser Ser 355 360 365 Arg Ser Phe Arg Ile Thr Ile Leu Pro G1n Leu Tyr Ile G1n Pro Met 370 375 380 Met Gly Ala Gly Leu Asn Tyr Glu Cys Tyr Arg Phe Gly Ile Ser Pro 385 390 395 400 Ser Thr Asn Ala Leu Val Ile Gly Ala Thr Val Met Glu Gly Phe Tyr 405 410 415 Val Ile Phe Asp Arg Ala G1n Lys Arg Val Gly Phe Ala Ala Ser Pro 420 425 430 Cys Ala Glu Ile Ala Gly Ala Ala Val Ser Glu Ile Ser Gly Pro Phe 435 440 445 Ser Thr Glu Asp Val Ala Ser Asn Cys Val Pro Ala G1n Ser Leu Ser 450 455 460 Glu Pro Ile Leu Trp His His His His His His 465 470 475 <210> 68 <21l> 413 <212> PRT <213> Homo sapiens <400> 68 Ala Leu Glu Pro Ala Leu Ala Ser Pro Ala Gly Ala Ala Asn Phe Leu 1 5 10 15 Ala Met Val Asp Asn Leu G1n Gly Asp Ser Gly Arg Gly Tyr Tyr Leu 25 30 Glu Met Leu Ile Gly Thr Pro Pro G1n Lys Leu G1n Ile Leu Val Asp 40 45 Thr Gly Ser Ser Asn Phe Ala Val Ala Gly Thr Pro His Ser Tyr Ile 55 60 Asp Thr Tyr Phe Asp Thr Glu Arg Ser Ser Thr Tyr Arg Ser Lys Gly 70 75 80 Phe Asp Val Thr Val Lys Tyr Thr G1n Gly Ser Trp Thr Gly Phe Val 90 95 Gly Glu Asp Leu Val Thr Ile Pro Lys Gly Phe Asn Thr Ser Phe Leu 105 110 Val Asn Ile Ala Thr Ile Phe Glu Ser Glu Asn Phe Phe Leu Pro Gly 120 125 Ile Lys Trp Asn Gly Ile Leu Gly Leu Ala Tyr Ala Thr Leu Ala Lys 135 140 Pro Ser Ser Ser Leu Glu Thr Phe Phe Asp Ser Leu Val Thr G1n Ala 150 155 160 Asn Ile Pro Asn Val Phe Ser Met G1n Met Cys Gly Ala Gly Leu Pro 170 175 Val Ala Gly Ser Gly Thr Asn Gly Gly Ser Leu Val Leu. Gly Gly Ile 185 190 Glu Pro Ser Leu Tyr Lys Gly Asp Ile Trp Tyr Thr Pro Ile Lys Glu 200 205 Glu Trp Tyr Tyr G1n Ile Glu Ile Leu Lys Leu Glu Ile Gly Gly G1n 210 215 220 Ser Leu Asn Leu Asp Cys Arg Glu Tyr Asn Ala Asp Lys Ala Ile Val 225 230 235 240 Asp Ser Gly Thr Thr Leu Leu Arg Leu Pro G1n Lys Val Phe Asp Ala 245 250 255 Val Val Glu Ala Val Ala Arg Ala Ser Leu Ile Pro Glu Phe Ser Asp 260 265 270 Gly Phe Trp Thr Gly Ser G1n Leu Ala Cys Trp Thr Asn Ser Glu Thr 275 280 285 Pro Trp Ser Tyr Phe Pro Lys Ile Ser Ile Tyr Leu A-rg Asp Glu Asn 290 295 300 Ser Ser Arg Ser Phe Arg Ile Thr Ile Leu Pro G1n Leu Tyr Ile G1n 305 310 315 320 Pro Met Met Gly Ala Gly Leu Asn Tyr Glu Cys Tyr Arg Phe Gly Ile 325 330 335 Ser Pro Ser Thr Asn Ala Leu. Val Ile Gly Ala Thr Val Met Glu Gly 340 345 350 Phe Tyr Val Ile Phe Asp Arg Ala G1n Lys Arg Val Gly Phe Ala Ala 355 360 365 Ser Pro Cys Ala Glu Ile Ala Gly Ala Ala Val Ser Glu Ile Ser Gly 370 375 380 Pro Phe Ser Thr Glu Asp Val Ala Ser Asn Cys Val Pro Ala G1n Ser 385 390 395 400 Leu Ser Glu Pro Ile Leu Trp His His His His His His 405 410 <210> 69 <21l> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Peptide <400> 69 Gly Leu Ala Leu Ala Leu Glu Pro 1 5 <210> 70 fk:;,v <21l> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Peptide <400> 70 Glu Val Lys Met Asp Ala Glu Phe 1 5 <210> 71 <21l> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Peptide <400> 71 Glu Val Asn Leu Asp Ala Glu Phe 1 5 <210> 72 <21l> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Peptide <400> 72 Leu Val Phe Phe Ala Glu Asp Val 1 5 <210> 73 <21l> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Peptide <400> 73 Lys Leu Val Phe Phe Ala Glu Asp 1 5 <210> 74 <21l> 38 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 74 cgctttaagc ttgccaccat gggcgcactg gcccgggcg 39 <210> 75 (61 <21l> 57 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 75 cgctttctcg agctaatggt gatggtgatg gtgccacaaa atgggctcgc tcaaaga 57 <210> 76 <21l> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Peptide <400> 76 Arg Arg Gly Gly Val Val Ile Ala Thr Val Ile Val Gly Glu Arg 1 5 10 is

Claims

What is claimed is:

1 A method for assaying hu-Asp 1 a-secretase activity comprising the steps of:

(a) contacting hu-Asp I protein with an amyloid precursor protein (APP) substrate, wherein said substrate contains an a-secretase cleavage site; and (b) measuring cleavage of the APP substrate at the a-cleavage site, thereby assaying hu-Aspl a-secretase activity.

2. A method according to claim 1, wherein the hu-Asp 1 protein comprises a polypeptide produced in cell transformed or transfected with a polynucleotide comprising a nucleotide sequence that encodes hu-Asp 1 or a fragment thereof that retains Aspl a-secretase activity.

3. A method of claim 2, wherein the hu-Asp l protein is purified and isolated from said cell.

4. A method according to claims 2 or 3, wherein the nucleotide sequence encodes a polypeptide that comprises the hu-Asp l amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof, wherein said fragment retains a-secretase activity.

5. A method according to any one of claims 2-4, wherein the polynucleotide sequence encodes a hu-Asp 1 amino acid sequence lacking the transmembrane an-dno acids 469-492 of SEQ ID NO: 2.

6 A method according to claim 5, wherein the polynucleotide sequence encodes a hu-Aspl amino acid sequence further lacking the cytoplasmic domain amino acids 493-518 of SEQ H) NO: 2.

7. A method according to any one of claims 1-6, wherein the hu-Asp I anfino acid sequence lacks amino terminal amino acids 1-62 of SEQ ID NO: 2.

8. A method according to claims 1 or 2, wherein the contacting step comprises growing a cell transfected or transformed with a polynucleotide encoding huAsp l protein or a fragment thereof that retains hu-Asp l asecretase activity, wherein the cell is grown under conditions in which the cell expresses the hu-Asp 1 protein in the presence of the APP substrate.

9. A method of claim 8, wherein said cell expresses a polynucleotide encoding an APP substrate containing an a-secretase cleavage site, and wherein the contacting step comprises growing the cell under conditions in which the cell expresses the hu-Asp 1 protein and the APP substrate.

10. A method of any one of claims 1-9, wherein the APP substrate asecretase cleavage site comprises the amino acid sequence LWFAEDF or KLWFAED.

11. A method of any one of claims 1-10, wherein the APP substrate comprises a human APP isoform. and further comprises a carboxy-terminal di-lysine.

12. A method of claims 10 or 11, wherein the APP substrate comprises a detectable label.

13. A method of claim 12, wherein the detectable label is selected from the group consisting of radioactive labels, chemiluminescent labels, enzymatic labels, chemiluminescent labels and flourescent labels.

14. A method of any one of claims 1-13, wherein the APP substrate comprises a human APP isoform and the determining step comprises measuring the production of arnyloid alpha peptide (sAPPa).

15. A method of any one of claims 1-14, wherein the method further comprises the steps of (a) determining the level of hu-Aspl a-secretase activity in the presence and absence of a modulator of hu-Asp I a-secretase activity; and (b) comparing the hu-Asp I a-secretase activity'in the presence and absence of the modulator, wherein modulators that increase hu-Aspl a- secretase activity are identified as candidate Alzheirner's disease therapeutics.

16. A method of claim 15, wherein the method further comprises a step of treating Alzheirner's disease with said candidate Alzheimer's disease therapeutic.

17. A composition comprising a candidate Alzheimer's disease therapeutic identified by the method of claim 15.

18. A hu-AspI protease substrate peptide or fragment thereof, wherein said peptide comprises an amino acid sequence consisting of fifty or fewer amino acids, said amino acid sequence including the hu-AspI cleavage site having the amino acid sequence GLALALEP

19. The substrate of claim 18, wherein the substrate comprises a detectable label.

20. The substrate of claim 19, wherein the detectable label is selected from the group consisting of radioactive labels, enzymatic labels, cherniluminescent labels and floureseent labels.

21. A method for assaying hu-Asp 1 proteolytic activity comprising the steps of:

(a) contacting hu-Aspl protein with an hu-Aspl substrate according to claim 18, 19, or 20 under acidic conditions, and (b) determining the level of hu-Aspl proteolytic activity.

22. A method according to claim 20, wherein the hu-Asp I protein comprises a polypeptide produced in cell transformed or transfected with a polynucleotide comprising the nucleotide sequence that encodes hu-Asp 1.

23. A method of claim 2 1, wherein the hu-Asp 1 protein is purified and isolated from said cell.

24. A method according to claims 22 and 23, wherein the nucleotide sequence encodes a polypeptide that comprises the hu-Asp I amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof, wherein said fragment retains proteolytic activity.

25. A purified polynucleotide comprising a nucleotide sequence encoding a polypeptide that comprises a fragment of a hu-Asp- 1 protein, wherein said nueleotide sequence lacks the sequence that encodes the transmembrane domain of said hu-Asp l protein, and wherein the hu-Asp l polypeptide fragment encoded by said polynucleotide has hu-Aspl a- secretase activity.

26. A polynucleotide of claim 25, wherein the polypeptide comprises a fragment of the hu-Asp l amino acid sequence set forth in SEQ ID NO: 2, and wherein the polypeptide lacks the transmembrane domain amino acids 469-492 of SEQ ID NO: 2.

27. A polynucleotide of claim 26 wherein the polypeptide further lacks cytoplasmic domain amino acids 493-518 of SEQ ID NO: 2.

28. A purified polynucleotide comprising a nucleotide sequeene encoding a polypeptide that comprises a fragment of a hu-Asp l protein, wherein said nucleotide sequence lacks the sequence that encodes amino acids 1-62 of SEQ ID NO: 2, and wherein the polypeptide has hu-Asp 1 a-secretase activity.

29. A polypeptide of claim 28, wherein said polypeptide lacks amino terminal amino acids 23-62 of SEQ H) NO: 2.

30. A purified polynucleotide comprising a nucleotide sequence that hybridizes under stringent conditions to the non-coding strand complementary to SEQ ID NO: 1, wherein the nucleotide sequence encodes a polypeptide having Asp 1 proteolytic activity and wherein the polynucleotide lacks nucleotides encoding a transmembrane domain.

31. A purified polynucleotide comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid according to claim 30, wherein the nucleotide sequence encodes a polypeptide further lacking a pro-peptide domain corresponding to aniino acids 23-62 of SEQ ID NO: 2.

32. A vector comprising the polynucleotide of any one of claims 25-3 1.

33. A host cell transformed or transfected with a vector of claim 32.

34 A host cell transformed or transfected with a polymicleotide of any one of claims 25-3 1.

35. A purified polypeptide comprising a fragment of a hu-Asp l protein, wherein said polypeptide lacks the hu-Asp l transmembrane domain of said hu-Asp l protein and retains hu-Aspl a-secretase activity.

36. A polypeptide of claim 35, wherein said polypeptide comprises a fragment of hu-Asp l having the amino acid sequence set forth in SEQ ID NO: 2 and wherein said polypeptide lacks the transmembrane domain amino acids 469-492 of SEQ ID NO: 2.

37. A polypeptide according to claim 35 or 36, wherein said polypeptide further lacks cytoplasmic domain amino acids 493-518 of SEQ ID NO: 2.

38. A polypeptide according to any one of claims 35-37, which further lacks amino terminal amino acids 1-62 of SEQ ID NO: 2.

39. A polypeptide comprising a fragment of hu-Asp l having the amino acid sequence set forth in SEQ ID NO: 2 and wherein said polypeptide lacks the amino terminal amino acids 1-62 of SEQ H) NO: 2 and retains APP processing activity.

40. A polypeptide comprising an amino acid sequence at least 95% identical to a fragment of hu-Asp l protein, wherein said polypeptide and said fragment lack a transmemebrane domain and retain hu-Asp l a- secretase activity.

41. A polypeptide comprising an amino acid sequence at least 95% identical to a fragment of hu-Asp l protein, wherein said polypeptide and said fragment lack the amino termal amino acids corresponding to the propeptide domain of hu-Asp 1 and retain APP processing activity.