WO1998026059A1 - Beta-secretase isolee a partir de cellules humaines 293 - Google Patents

Beta-secretase isolee a partir de cellules humaines 293 Download PDF

Info

Publication number
WO1998026059A1
WO1998026059A1 PCT/US1996/019549 US9619549W WO9826059A1 WO 1998026059 A1 WO1998026059 A1 WO 1998026059A1 US 9619549 W US9619549 W US 9619549W WO 9826059 A1 WO9826059 A1 WO 9826059A1
Authority
WO
WIPO (PCT)
Prior art keywords
secretase
app
activity
enzyme
amino
Prior art date
Application number
PCT/US1996/019549
Other languages
English (en)
Inventor
Susanna M. S. Chrysler
Sukanto Sinha
Pamela S. Keim
John P. Anderson
Original Assignee
Athena Neurosciences, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Athena Neurosciences, Inc. filed Critical Athena Neurosciences, Inc.
Priority to AU16840/97A priority Critical patent/AU1684097A/en
Priority to PCT/US1996/019549 priority patent/WO1998026059A1/fr
Publication of WO1998026059A1 publication Critical patent/WO1998026059A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6478—Aspartic endopeptidases (3.4.23)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates generally to the cleavage of /3-amyloid precursor protein to produce S-amyloid peptide. More particularly, the present invention relates to isolated and purified compositions containing an enzyme responsible for such cleavage (/3-secretase) and assays for identifying inhibitors of /3-secretase.
  • Alzheimer's disease is characterized by the presence of numerous amyloid plaques and neurofibrillary tangles (highly insoluble protein aggregates) present in the brains of Alzheimer's disease patients, particularly in those regions involved with memory and cognition.
  • 3-amyloid peptide is a major constituent of amyloid plaque which is produced by cleavage of 3-amyloid precursor protein. It is presently believed that a normal (non-pathogenic) processing of the 3-amyloid precursor protein occurs via cleavage by a putative "a-secretase" which cleaves between amino acids 16 and 17 of the /3-amyloid peptide region within the protein.
  • pathogenic processing occurs in part via a putative "/3-secretase" which cleaves at the amino-terminus of the /3-amyloid peptide region within the precursor protein.
  • /3-secretase cleaves at the amino-terminus of the /3-amyloid peptide region within the precursor protein.
  • novel enzymes can be used to catalyze reactions of a type associated with their class.
  • novel proteases can be used to cleave proteins for a variety of purposes, and the availability of new proteases provides unique capabilities.
  • identification, isolation, and purification of the putative /3-secretase enzyme would permit chemical modeling of a critical event in the pathology of Alzheimer's disease and would allow the screening of compounds to determine their ability to inhibit /3-secretase activity.
  • /3-secretase an enzyme responsible for the pathogenic cleavage of /3-amyloid precursor protein at the amino-terminus of the ⁇ -amyloid peptide region.
  • /3-secretase an enzyme responsible for the pathogenic cleavage of /3-amyloid precursor protein at the amino-terminus of the ⁇ -amyloid peptide region. It would be particularly desirable if such screening assays could be performed in a rapid format which would permit the screening of large numbers of test drugs in automated fashion.
  • /3-amyloid precursor protein is expressed in three differently-spliced forms of 695, 751, and 770 amino acids, and "normal" processing involves proteolytic cleavage at a site between residues Lys 16 and Leu 17 in the /3-amyloid peptide.
  • Soluble /3-amyloid peptide which has been cleaved at the putative /3-secretase site has also been found in the culture medium of non-diseased cells (Haass et al . (1992) Nature 359:322-325) and in CSF from healthy humans and animals (Seubert et al . (1992) Nature 359:325-327).
  • the possible existence of the putative ⁇ - secretase is discussed in, for example, Selkoe, "Cell Biology of the Amyloid /3-Protein and the Mechanism of Alzheimer's
  • the present invention provides a novel /3-secretase composition
  • a novel /3-secretase composition comprising an isolated and purified enzyme which cleaves /3-amyloid precursor protein (APP) at the amino- terminus of /3-amyloid peptide (/3AP) within APP, referred to hereinafter as "/3-secretase activity.
  • the compositions of the present invention will generally have a /3-secretase activity which is at least five-fold greater than that of a solubilized but unenriched membrane fraction from human 293 cells, preferably being at least ten-fold greater than that of the membrane fraction, and more preferably being at least 100- fold greater than that of the membrane fraction.
  • the /3-secretase enzyme is characterized by (1) an apparent molecular weight when glyosylated in the range from 260 kD to 300 kD when measured by gel exclusion chromatography in 9.2% hydrogenated Triton X-100, (2) a gel pattern as shown in Figs. 1 or 2 (discussed in more detail in the Experimental section hereinafter) , (3) a net negative charge at pH 5 and a net negative charge at pH 7.5, and (4) binding to wheat germ agglutinin.
  • /3-secretase is glycosylated. Methods for deglycosylation are described in the Experimental section hereinafter. The above apparent molecular weight is determined prior to any deglycosylation.
  • compositions of the present invention are generally useful as proteolytic chemical and specifically useful in assays for determining whether a test substance will inhibit proteolytic cleavage of APP resulting from the novel /3-secretase.
  • the method comprises exposing a polypeptide comprising the /3-secretase site of APP (located at the amino- terminus of the / SAP region within APP) to an at least partially purified /3-secretase in the presence of the test substance under conditions such that the /3-secretase would be expected to cleave the polypeptide into an amino-terminal fragment and a carboxy-terminal fragment in the absence of test substance which inhibits such cleavage.
  • Test substances which inhibit such cleavage are thus identified as having /3-secretase inhibition activity.
  • Such test methods preferably employ the /3-secretase compositions described above.
  • generation of the amino-terminal fragment and/or the carboxy- terminal amino acids of APP and may comprise a fusion polypeptide including an amino-terminal portion having a binding epitope.
  • Use of such a fusion polypeptide as the /3-secretase facilitates detection of cleavage by capture of the amino-terminal portion and labelling of the amino-terminal portion.
  • Fig. 1 shows the elution profile of /3-secretase purified from human 293 cells produced by gel exclusion chromatography on Superdex 200.
  • Fig. 2 is a similar elution profile shown for /3-secretase purified from human brain.
  • Fig. 3 is an electrophoretic gel elution profile of (3-secretase activity.
  • Fig. 4 is a schematic illustration of an APP- containing fusion peptide useful in performing the screening assays of the present invention, having a binding epitope derived from maltose-binding protein (MBP) .
  • An assay is run by exposing the fusion polypeptide to /3-secretase which cleaves the 125 amino acid portion of APP (APP C-125) at the amino-terminus of the /3AP .
  • the MBP portion may then be captured, and the carboxy-terminus of the APP fragment which is exposed by cleavage with /3-secretase may be identified with 192 antibody specific for said terminus.
  • SW-192 antibody bound to a reporter is utilized, which antibody recognizes the carboxy-terminus of the Swedish mutation of APP.
  • Fig. 5 illustrates APP 638 which is a recombinantly expressed form of APP truncated after /3AP (A/3) .
  • APP 638 may be used in a /3-secretase assay where the /3AP peptide is cleaved and the carboxy-terminus of the amino-terminal fragment of APP 638 recognized by 192 antibody in either a Western blot or ELISA assay.
  • the carboxy terminal /3AP fragment can also be measured using a 3D6/266 assay.
  • Fig. 6 is the complete amino acid sequence of the Swedish mutation of the fusion polypeptide of maltose-binding protein and APP fragment utilized in the Experimental section hereinafter (SEQ ID No.: 3).
  • Fig. 7 is a standard curve generated for the ⁇ - secretase ELISA described in detail in the Experimental section below.
  • Fig. 8 is an anionic exchange column elution profile obtained for deglycosylated /3-secretase enzyme.
  • Fig. 9 is a Western blot of the deglycosylated ⁇ - secretase enzyme probed with 238B antisera.
  • Fig. 10 is an elution profile of the /3-secretase enzyme from a cationic exchange column.
  • the present invention provides a novel protease which cleaves the /3-amyloid precursor protein (APP) at the amino-terminus of the /3-amyloid peptide (/3AP) therein. It is believed that this protease is the putative /3-secretase responsible for the pathogenic processing of APP to produce / SAP in /3AP-related conditions, such as Alzheimer's disease, Down's syndrome, HCHWA-D, and the like. Thus, the novel protease of the present invention will be referred to hereinafter as "/3-secretase . " The /3-secretase of the present invention will be useful as a protease in in vi tro and in vivo systems where proteases may generally find use.
  • (8-secretase may be used to cleave or attempt to cleave proteins in order to characterize, process, modify, or otherwise react with the protein as a substrate.
  • /3-secretase will have general utility as a proteolytic chemical reagent in a wide variety of chemical reactions and systems.
  • the /3-secretase of the present invention will have a specific utility in the performance of screening assays to identify /3-secretase inhibitors, i.e., test compounds which are able to inhibit the proteolytic cleavage of APP in the presence of / 8-secretase. Such assays will be described in detail below.
  • APP /3-amyloid precursor protein
  • APP refers to 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 a / SAP region (defined below) 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.
  • a 751-amino acid polypeptide has been described by Ponte et al . (1988) Nature 331:525-527 and Tanzi et al . (1988) Nature 331:528-530.
  • a 770-amino acid isotype of APP is described in Kitaguchi et al. (1988) Nature 331:530-532.
  • a number of specific variants of APP have also been described having point mutations which can differ in both position and phenotype . A general review of such mutations is provided in Hardy (1992) Nature Genet . 1:233-234.
  • a mutation of particular interest is designated the "Swedish” mutation where the normal Lys-Met residues at positions 595 and 596 are replaced by Asn-Leu. This mutation is located directly upstream of the normal /3-secretase cleavage site of APP, which occurs between residues 596 and 597 of the 695 isotype.
  • /3-amyloid peptide refers to the approximately 43 amino acid peptide which comprises residues 597-640 of the 695 amino acid isotype of APP.
  • BAP is produced by processing of the APP including cleavage at both the amino-terminus and carboxy-terminus of the region. It is believed that the /3-secretase of the present invention is responsible for cleavage of APP at the amino-terminus of / SAP in normal and pathogenic processing of APP in human cells.
  • /3-secretase has been characterized in a number of respects, as described in detail in the Experimental section below.
  • /3-secretase has an apparent molecular weight in the range from 260 kD to 300 kD, with a tentative molecular weight of 280 kD, as determined by gel exclusion chromatography in 0.2% hydrogenated Triton X-100. /3-secretase will bind to wheat germ agglutinin but not to concanavalin A. It has been found to have a net negative charge at pH 4.75 and pH 5 (where it does not bind to a cationic exchange material) and a net negative charge at pH 7.5 (where it binds to an anion exchange material) .
  • the /3-secretase of the present invention will cleave both wild-type (normal) and the Swedish mutation of APP at the putative /3-secretase cleavage site on the immediate amino-terminal side of the /3AP fragment, and has been found to have a higher proteolytic activity with respect to the Swedish form of APP. Proteolytic activity appears to be at its peak at a pH from 5 to 5.5, with very low activity at pH 7.5 and above. /3-secretase is resistant to many known protease inhibitors (see Table 1 in the Experimental section below) .
  • /3-secretase appears to recognize only those polypeptide substrates which have retained a substantial number of residues upstream and downstream from the cleavage site (from either the wild-type, Swedish, or other mutated form) of APP, with an oligopeptide analog including 17 residues upstream and 16 residues downstream from the cleavage site (with a total of 33 amino acids) being resistant to cleavage by /3-secretase.
  • the /3-secretase of the present invention will be provided in an isolated and purified form.
  • isolated and purified it is meant that the /3-secretase has been either (1) isolated and at least partially purified from a natural source, such as human brain tissue or human 293 cells (as described in detail in the Experimental section below) or (2) is produced recombinantly and synthetically.
  • a natural source such as human brain tissue or human 293 cells (as described in detail in the Experimental section below) or (2) is produced recombinantly and synthetically.
  • a natural source such as human brain tissue or human 293 cells (as described in detail in the Experimental section below)
  • /3-secretase be obtained from cellular sources using known protein purification techniques.
  • Contaminating proteins may be removed from the / 8-secretase co-positions by specific techniques, including serial lectin chromatography on agarose-bound succinylated-wheat germ agglutinin (SWGA) and agarose-bound lentil lectin (LCA) . These lectins, although partly binding /3-secretase activity, preferentially bind other contaminating proteins in the purified fractions, and thus allow increased enrichment of the /3-secretase activity. The 3-secretase will be isolated and purified to an extent sufficient to increase the /3-secretase activity in the resulting composition to a useful level.
  • SWGA succinylated-wheat germ agglutinin
  • LCDA lentil lectin
  • the /3-secretase preparations of the present invention will have sufficient activity to cleave APP and APP-containing polypeptides as described in the Experimental section below.
  • the /3-secretase compositions of the present invention will have an activity which is at least 10-fold greater than that of a solubilized but unenriched membrane fraction from human 293 cells. More preferably, the compositions will have a / 8-secretase activity which is at least about 100-fold greater than that of the solubilized membrane fraction from human 293 cells.
  • a specific method for determining /3-secretase activity in units of "ng m '1 h "1 " is described in detail in the Experimental section below (see footnote 1 to Table 3) .
  • compositions comprising a naturally- occurring /3-secretase molecule
  • the present invention comprises compositions including polypeptides comprising fragments of /3-secretase as well as polypeptides substantially homologous to the intact /3-secretase or a portion thereof.
  • the /3-secretase polypeptides of the present invention will generally exhibit at least about 80% sequence homology with the naturally-occurring sequence of the full-length
  • the /3-secretase polypeptides will exhibit at least about 85% sequence homology, more usually at least about 95% sequence homology, and often 99% sequence homology or higher.
  • the length of comparison sequences will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues.
  • the (8-secretase polypeptides of the present invention may also be in the form of fusion polypeptides where an active fragment of the /3-secretase molecule is joined to all or a portion of another protein. Fusions may be generated with heterologous proteins (for example, a reporter polypeptide, a binding polypeptide, or the like) . Fusion polypeptides may be formed either recombinantly or by synthetic methods which are well-known in the art.
  • the (8-secretase polypeptides of the present invention may also have amino acid residues which have been chemically modified by known techniques, such as phosphorylation, sulfonation, biotinylation or the addition of other moieties.
  • the modifications will be useful for labelling reagents, purification targets, affinity ligands targeting, or the like.
  • the /3-secretase polypeptides of the present invention may be used to prepare polyclonal and/or monoclonal antibodies using conventional techniques with the /3-secretase as an immunogen.
  • the intact /3-secretase molecule, or fragments thereof, optionally coupled to a carrier molecule may be injected into small vertebrates, with monoclonal antibodies being produced by well-known methods. See, for example, Harlow and Lane (1988) Antibodies : A Laboratory- Manual , Cold Spring Harbor Press, New York, and Goding (1986) Monoclonal Antibodies : Principles and Practice (2nd ed.) Academic Press, New York. Antibodies produced from (S-secretase will be useful for performing conventional immunoassays to detect /3-secretase in biological and other specimens .
  • /3-secretase compositions described above will be particularly useful for performing in vi tro assays for detecting /3-secretase inhibitors, where at least partially purified /3-secretase is combined with the polypeptide substrate comprising the /3-secretase cleavage site of APP in the presence of the test substrate. Conditions are maintained such that the /3-secretase would cleave the polypeptide substrate into an amino-terminal fragment and a carboxy- terminal fragment in the absence of a substance which inhibits such cleavage.
  • Cleavage of the polypeptide substrate in the presence of the test compound is compared with that in the absence of the test compound, and those test substances which provide significant inhibition of the cleavage activity (usually at least about 25% inhibition, more usually at least about 50% inhibition, preferably at least about 75% inhibition, and often at least about 90% inhibition or higher) are considered to be /3-secretase inhibitors.
  • Such / 8-secretase inhibitors may then be subjected to further in vi tro and/or in vivo testing to determine if they inhibit the production of (SAP in cellular and animal models. Suitable in vivo and in vi tro tests are described in copending application Serial Nos . 07/965,972 and 07/831,722, the full disclosures of which are incorporated herein by reference.
  • Suitable substrate polypeptides will include a region of the APP molecule which is recognized and cleaved by /3-secretase.
  • the substrate polypeptide will include at least about 15 amino acid residues, preferably at the least about 25 amino acids, on the amino-terminal side of the cleavage site (located between amino acids 596 and 597 in the 695-amino acid isomer) and at least about 28 amino acids, preferably at least about 42 amino acids, on the carboxy- terminal side of the cleavage site.
  • the cleavage site will typically comprise the Met-Asp or the Leu-Asp cleavage site characteristic of the wild-type and Swedish forms of /3APP.
  • APP APP
  • polypeptide including both wild-type and mutant forms of APP, particularly including the Swedish mutation of APP.
  • Use of fusion substrate polypeptides is often preferred, where an affinity region can be fused to the /3-secretase cleavage site of APP, producing a molecule whose cleavage can be conveniently monitored in solid phase test systems.
  • the screening assays of / 8-secretase and suitable substrate polypeptide are conveniently performed using "sandwich" assays where the amino-terminal or the carboxy- terminal fragment produced by cleavage is captured on a solid phase. The captured fragment may then be detected using an antibody specific for the end of the fragment exposed by / 8-secretase cleavage.
  • the polypeptide substrate is a fusion polypeptide combining maltose-binding protein and a 125-amino acid carboxy-terminal fragment of APP.
  • the assay uses anti-maltose-binding protein antibody to capture the amino-terminal cleavage product, where the carboxy-terminus of the cleavage product is detected by an antibody specific thereto.
  • An exemplary antibody is 192 antibody or SW-192 antibody, described in more detail in copending application 08/143,697, filed on October 27, 1993, the full disclosure of which is incorporated herein by reference.
  • the binding of the antibody to the cleaved fusion polypeptide is detected using conventional labelling systems, such as horseradish peroxidase or other detectable enzyme labels, which are bound to the antibody directly (covalently) , or indirectly through intermediate linking substances, such as biotin and avidin.
  • Frozen tissue (293 cell paste or human brain) was cut into pieces and combined with five volumes of homogenization buffer (20 mM Hepes, pH 7.5, 0.25 M sucrose,
  • the suspension was homogenized using a blender and centrifuged at 1000 x g (10 min, 4°C) to produce a post- nuclear supernatant which was saved on ice.
  • the pellets were resuspended in fresh homogenizing buffer at the original volume, and the centrifugation step was repeated.
  • the second supernatant was combined with the first one, and the supernatant pool ("PNS") was centrifuged at 16,000 x g for
  • the pellets were suspended in extraction buffer (20 mM MES, pH 6.0 , 0.5% Triton X-100, 150 mM NaCl, 2 mM EDTA, 5 ⁇ g/ml leupeptin, 5 ⁇ g/ml E64, 1 ⁇ g/ml pepstatin, 0.2 mM PMSF) at the original volume. After vortex-mixing, the extraction was completed by agitating the tubes at 4°C for a period of one hour. The mixtures were centrifuged as above at 16,000 x g, and the supernatants were pooled. The pH of the extract was adjusted to 7.5 by adding -1% (v/v) of 1 M Tris base (not neutralized) .
  • the neutralized extract was loaded onto a wheat germ agglutinin-agarose (WGA-agarose) column pre-equilibrated with 10 column volumes of 20 mM Tris, pH 7.5 , 0.5% Triton X-100,
  • N-acetylglucosamine elution buffer were then used to elute the column, followed by five column volumes of 10% chitin hydrolysate in 20 mM Tris, pH 7.5 , 0.5% Triton X-100, 2 mM EDTA. All of the above eluates were combined (pooled WGA- eluate) .
  • the pooled WGA-eluate was diluted 1:4 with 20 mM NaOAc, pH 5.0, 0.5% Triton X-100, 2 mM EDTA.
  • the pH of the diluted solution was adjusted to 5.0 by adding a few drops of glacial acetic acid while monitoring the pH.
  • This "SP load" was passed through a 5-ml Pharmacia HiTrap SP-column equilibrated with 20 mM NaOAc, pH 5.0 , 0.5% Triton X-100, 2 mM EDTA, at 4 ml/min at 4°C.
  • /3-Secretase activity was present in the flow-through fraction, which was neutralized by adding enough 1 M Tris (not neutralized) to bring the pH up to 7.5.
  • the enzyme solution was then loaded onto a 1-ml Pharmacia HiTrap Q-column equilibrated with approximately 10 column volumes of 20 mM Tris, pH 7.5, 0.2% hydrogenated Triton X-100, 2 mM EDTA, at 1.5 ml/min at 4°C.
  • the column was washed with 10 column volumes of 20 mM Tris, pH 7.5 , 0.2% hydrogenated Triton X-100, 50 mM NaCl, 2 mM EDTA. Protein was eluted using a linear gradient from 50 mM TO 350 Mm NaCl over 30 minutes at a flow-rate of 1 ml/min at 4°C.
  • the protein concentrations in the HiQ fractions were measured using a BioRad colorimetric protein assay, and the / 8-secretase activity was measured using the MBP-C125 cleavage assay at pH 5.5.
  • the fractions in the ascending portion of the protein peak have the highest specific activity and were pooled for further purification of the enzyme .
  • the pooled fractions from the HiTrap Q were then applied to a column of concanavalin A-agarose (10% v/v of pool) equilibrated with 10 column volumes of 20 mM Tris, pH 7.5, 0.2% hydrogenated Triton X-100, 150 mM NaCl, 2 mM EDTA.
  • the Con A flow-through was then loaded onto a Superdex 200 (26/60) gel exclusion chromatography column, which was eluted with phosphate buffered saline, pH 7.4 , 0.2% hydrogenated Triton X-100, 2 mM EDTA, at 1 ml/min, collecting 3 min/fraction.
  • Fractions containing /3-secretase activity were identified using the MBP-C125 cleavage assay.
  • the apparent molecular weight of the / 8-secretase activity eluting from the Superdex column was estimated from the peak elution volume (relative to that of standard proteins) to be 280,000 ⁇ 9800 (average of two runs for 293 cells, and two runs for human brain) .
  • Results from a large-scale preparation of the enzyme from human brain tissue is shown in Table 1 below.
  • the Sp . Act . is thus expressed as ng of protein produced per ⁇ g of /3-secretase per hour.
  • Fig. 1 shows the protein pattern of the Superdex 200 (26/60) elution profile for /3-secretase purified from 293 cells
  • Fig. 2 shows the elution profile for /3-secretase purified from human brain. A number of protein bands are visible in the fractions with / 8-secretase activity.
  • an aliquot of substantially purified / 8-secretase was electrophoresed under non-denaturing conditions into a 6% separating-4% stacking native gel system, according to the method of Laemmli (Nature, (1970) 227:680), except that the SDS is replaced by 0.2% hydrogenated triton X-100.
  • the gel is soaked for -30 minutes in 0.1 M sodium acetate, pH 5.5.
  • the lane of electrophoresed protein (7 cm long x 1 cm wide x 1 mm thick) is then cut into -2.5 mm pieces using a clean razor blade, sequentially from the top of the stacker to the bottom of the separating gel.
  • Each of these slices is combined with 60 ⁇ l water and 10 ⁇ l 1 M sodium acetate in a microcentrifuge tube, then homogenized using a Kontes Deltaware motorized pellet pestle microhomogenizer .
  • MBP-C125 SW is added to the desired concentration (0.5-0.7 ⁇ g/ml), and the mixture incubated overnight.
  • the samples are then diluted with Specimen Diluent (defined below) 50-fold, and analyzed by the /3-secretase activity ELISA assay. The results are shown in Fig. 3.
  • APA jequirity bean
  • con A jack bean
  • CSA scotch broom
  • DSA ji son weed
  • ECA coral tree
  • AIA Jacalin (AIA) lentil (LCA) horseshoe crab (LPA) tomato (LEL) maackia ( AA) peanut (PNA) pokeweed (POA) castor bean (RCA1) potato (STL) wheat germ - succinylated (S GA)
  • TKA China gourd
  • UDA stinging nettle
  • UEAI gorse
  • UEAI I hairy vetch
  • WGA wheat germ
  • a potentially contaminating protein was detected in the / S-secretase preparations.
  • Antisera were raised against a synthetic peptide sequence (NH 2 -CGGEAKGAEDAPDADTA-CONH 2 [SEQ ID NO. 4] ) corresponding to a 14-amino-acid sequence in the predicted carboxy-terminal end of this new polypeptide with a CGG linker at the amino-terminal end of the peptide.
  • This antisera (238B) detected a family of polypeptides with an indistinguishable pattern as seen previously with other antisera. However, 238B showed greater sensitivity on Western blots, and can be used to detect these polypeptides in the purified enzyme preparations.
  • the further purification of the /3-secretase enzymatic activity was achieved by developing a protocol to substantially deglycosylate the glycoprotein (s) present in the purified enzyme preparation, while maintaining enzymatic activity.
  • the removal of the Asn-linked carbohydrate residues was effected by treatment with the enzyme PNGase F.
  • the deglycosylated enzyme preparation was subjected to sequential anion-exchange and cation-exchange chromatography . Enzymatic deglycosylation was performed as follows.
  • the eluate from the DEAE-Sepharose beads (-250:1) was then treated with 1/20 (v/v) of recombinant PNGase F (Glyko, 2.5 U/ml) for 4-5 d at 37°C, in the presence of 3 mM (S-mercaptoethanol .
  • This treatment removed Asn-linked carbohydrate chains from glycoproteins .
  • Cation-exchange chromatography was used to further purify the enzymatic activity.
  • the peak activity fractions from the MiniQ elution were diluted 1:5 in SP Buffer A (20 mM NaOAc, pH 4.75, 2 mM EDTA, 0.2% R-TX100) in order to alter the pH as well as lower the ionic strength prior to cation exchange chromatography.
  • the diluted enzyme pool was mixed with 50:1 of a 50% slurry of Pharmacia SP-Sepharose ® beads. Following overnight mixing at 4°C, the beads were collected by low speed centrifugation, and the supernatant ("SP Flow- Through”) was saved for further analysis.
  • the SP-Sepharose beads were eluted stepwise with 100 mM NaCl increments in SP Buffer A, up to 600 mM NaCl. These fractions (E1-E6, -0.45 ml each) , along with the SP Flow-Through, and an aliquot of the SP Load, were analyzed for both (S-secretase activity as well as immunoreactivity for the previously identified polypeptide. The results are shown in Fig 10. All of the immunoreactive bands bind to the SP-Sepharose, since they are quantitatively depleted in the SP-Flow Through, and elute in fractions E2-E4. In contrast, the /3-secretase enzymatic activity quantitatively flows through (95% recovered in the SP Flow-Through) , and thus is separated from the contaminating polypeptides.
  • Recombinant proteins were generated with both the wild-type APP sequence (MBP-C125 WT) at the cleavage site (..Val-Lys-Met-Asp-Ala..) [SEQ ID No.:l] or the "Swedish" double mutation (MBP-C125 SW) ( ..Val-Asn-Leu-Asp-Ala.. ) [SEQ ID No.: 2] .
  • the entire sequence of the recombinant protein with the Swedish sequence is given in Fig. 6 [SEQ ID No.: 3] . As shown in Fig.
  • cleavage of the intact MBP-fusion protein results in the generation of a truncated amino-terminal fragment, with the new SW-192 Ab-positive epitope uncovered at the carboxy terminus.
  • This amino-terminal fragment can be recognized on Western blots with the same Ab, or, quantitatively, using an anti-MBP capture-biotinylated SW-192 reporter sandwich format, as shown in Fig. 4.
  • Anti-MBP polyclonal antibodies were raised in rabbits (Josman Labs, Berkeley) by immunization with purified recombinantly expressed MBP (New England Biolabs) . Antisera were affinity purified on a column of immobilized MBP. MBP- C125 SW and WT substrates were expressed in E. coli , then purified using affinity binding to and elution from immobilized amylose-agarose matrix (New England Biolabs) . The purified substrates were stored frozen at -40 °C in 3 M guaninide-HCl and 1% Triton X-100, @ -0.7 mg/ml .
  • Microtiter 96 -well plates were coated with purified anti-MBP antibody (@ 5-10 ⁇ g/ml) , followed by blocking with human serum albumin.
  • S-secretase solution (1-10 ⁇ l) is mixed with substrate (0.5 ⁇ l) in a final volume of 50 ⁇ l, with a final buffer composition of 20 mM sodium acetate, pH 5.5, 0.03% Triton X-100, in individual wells of 96-well microtiter plates, and incubated at 37°C for 2 h.
  • Specimen Diluent 0.2 g/1 sodium phosphate monobasic, 2.15 g/1 sodium phosphate dibasic, 0.5 g/1 sodium azide, 8.5 g/1 sodium chloride, 0.05% Triton X-405, 6 g/1 BSA
  • Biotinylated SW192 antibodies were used as the reporter.
  • SW192 polyclonal antibodies were biotinylated using NHS-biotin (Pierce) , following the manufacturer's instruction.
  • the biotinylated antibodies were used at about 240 ng/ml, the exact concentration varying with the lot of antibodies used.
  • the ELISA was developed using streptavidin-labeled alkaline phosphatase (Boeringer-Mannheim) and 4 -methyl-umbelliferyl phosphate as fluorescent substrate. Plates were read in a Cytofluor 2350 Fluorescent Measurement System. Recombinantly generated MBP-26SW (product analog) was used as a standard to generate a standard curve (Fig. 7) , which allowed the conversion of fluorescent units into amount of product generated.
  • This assay protocol was used to screen for inhibitor structures, using "libraries” of compounds assembled onto 96- well microtiter plates. Compounds were added, in a final concentration of 20 ⁇ g/ml in 2% DMSO, in the assay format described above, and the extent of product generated compared with control (2% DMSO only) / 8-secretase incubations, to calculate "% inhibition.” “Hits” were defined as compounds which result in >35% inhibition of enzyme activity at test concentration. Using this system, 70 “hits” were identified out of the first 6336 compounds tested, a “hit” rate of -1.1%. Thus, the assay has been shown to be capable of distinguishing "non-inhibitors" (the majority of compounds) from “inhibitors. "
  • MOLECULE TYPE peptide (xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 2 :
  • MOLECULE TYPE DNA (genomic)

Abstract

L'invention concerne des compositions comportant une protéase isolée à partir de cellules humaines 293 ou des fragments de polypeptide présentant une homologie de séquence d'environ 85 % avec ladite protéase. La protéase native est appelée β-secrétase et coupe une protéine précurseur β-amyloïde du côté aminé terminal du peptide de β-amyloïde. La β-secrétase présente un poids moléculaire apparent compris entre 260-300 kD environ lorsqu'elle est glycosylée, et fixe l'agglutinine de germe de blé. Une technique de sélection destinée à identifier des inhibiteurs de β-secrétase montre que ladite β-secrétase n'est pas inhibée par des inhibiteurs habituels de sérine, cystéine, aspartyl et métalloprotéases.
PCT/US1996/019549 1996-12-11 1996-12-11 Beta-secretase isolee a partir de cellules humaines 293 WO1998026059A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU16840/97A AU1684097A (en) 1996-12-11 1996-12-11 Beta-secretase isolated from human 293 cells
PCT/US1996/019549 WO1998026059A1 (fr) 1996-12-11 1996-12-11 Beta-secretase isolee a partir de cellules humaines 293

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1996/019549 WO1998026059A1 (fr) 1996-12-11 1996-12-11 Beta-secretase isolee a partir de cellules humaines 293

Publications (1)

Publication Number Publication Date
WO1998026059A1 true WO1998026059A1 (fr) 1998-06-18

Family

ID=22256256

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/019549 WO1998026059A1 (fr) 1996-12-11 1996-12-11 Beta-secretase isolee a partir de cellules humaines 293

Country Status (2)

Country Link
AU (1) AU1684097A (fr)
WO (1) WO1998026059A1 (fr)

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19856261C1 (de) * 1998-12-07 2000-03-30 Hoechst Marion Roussel De Gmbh Aß-Peptid Screening Assay
WO2000023576A2 (fr) * 1998-10-16 2000-04-27 Hook Vivian Y H Secretases liees a la demence d'alzheimer
WO2000047618A2 (fr) * 1999-02-10 2000-08-17 Elan Pharmaceuticals, Inc. Compositions a base d'enzyme beta-secretase et procedes
WO2000058479A1 (fr) * 1999-03-26 2000-10-05 Amgen Inc. Genes et polypeptides beta secretase
WO2000017369A3 (fr) * 1998-09-24 2000-11-23 Upjohn Co Secretase de la maladie d'alzheimer
US6245884B1 (en) 1998-10-16 2001-06-12 Vivian Y. H. Hook Secretases related to alzheimer's dementia
WO2001049098A2 (fr) * 1999-10-13 2001-07-12 Michael Jerome Bienkowski Secretase associee a la maladie d'alzheimer, substrats app et utilisations correspondants
US6545127B1 (en) 1999-06-28 2003-04-08 Oklahoma Medical Research Foundation Catalytically active recombinant memapsin and methods of use thereof
GB2389182A (en) * 1999-02-10 2003-12-03 Elan Pharm Inc Method of purifying b-secretase
EP1445263A1 (fr) * 1999-02-10 2004-08-11 Elan Pharmaceuticals, Inc. Enzyme humaine de beta-secretease, inhibiteurs et leurs compositions et utilisations.
US6844148B1 (en) 1998-09-24 2005-01-18 Pharmacia & Upjohn Company Alzheimer's disease secretase, APP substrates therefor, and uses therefor
US6870030B2 (en) 1997-01-28 2005-03-22 Smithkline Beecham Corporation Asp2
US7115410B1 (en) 1999-02-10 2006-10-03 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
US7335632B2 (en) 2001-10-23 2008-02-26 Comentis, Inc. Beta-secretase inhibitors and methods of use thereof
EP1939297A1 (fr) * 1998-09-24 2008-07-02 Pharmacia & Upjohn Company LLC Sécrétase de la maladie d'Alzheimer
US7456007B1 (en) 1998-12-31 2008-11-25 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
US7514408B1 (en) 1999-12-02 2009-04-07 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
US8772457B2 (en) 2010-11-10 2014-07-08 Genentech, Inc. BACE1 antibodies
US10882920B2 (en) 2014-11-19 2021-01-05 Genentech, Inc. Antibodies against BACE1 and use thereof for neural disease immunotherapy
US11008403B2 (en) 2014-11-19 2021-05-18 Genentech, Inc. Anti-transferrin receptor / anti-BACE1 multispecific antibodies and methods of use

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991013904A1 (fr) * 1990-03-05 1991-09-19 Cephalon, Inc. Proteases analogues a la chymotrypsine et leurs inhibiteurs
US5424205A (en) * 1990-10-05 1995-06-13 Athena Neurosciences, Inc. Amyloidin protease and uses thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991013904A1 (fr) * 1990-03-05 1991-09-19 Cephalon, Inc. Proteases analogues a la chymotrypsine et leurs inhibiteurs
US5424205A (en) * 1990-10-05 1995-06-13 Athena Neurosciences, Inc. Amyloidin protease and uses thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ADVANCES IN BEHAVIORAL BIOLOGY, NEW YORK: PLENUM PRESS, 1995, Vol. 44, GLENNER et al., "Alzheimer's and Parkinson's Diseases: Recent Developments", pages 127-130. *
BIOCHEM. BIOPHYS. RES. COMM., 1991, Vol. 177, No. 1, TAGAWA et al., "Alzheimer's Disease Amyloid beta-Clipping Enzyme (APP Secretase): Identification, Purification and Characterization of the Enzyme", pages 377-387. *
JOURNAL OF NEUROCHEM., 1993, Vol. 61, No. 2, NELSON et al., "Identification of a Chymotrypsin-Like Mast Cell Protease in Rat Brain Capable of Generating the N-Terminus of the Alzheimer Amyloid beta-Protein", pages 567-577. *
JOURNAL OF NEUROSCI. RES., 1992, Vol. 33, SAMBAMURTI et al., "Evidence for Intracellular Cleavage of the Alzheimer's Amyloid Precursor in PC12 Cells", pages 319-329. *

Cited By (69)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6870030B2 (en) 1997-01-28 2005-03-22 Smithkline Beecham Corporation Asp2
EP0855444B1 (fr) * 1997-01-28 2006-09-20 SmithKline Beecham plc Proteinase aspartique 2
US7368536B2 (en) 1998-09-24 2008-05-06 Pharmacia & Upjohn Company Alzheimer's disease secretase, APP substrates therefor, and uses therefor
CN1300320C (zh) * 1998-09-24 2007-02-14 法玛西雅厄普约翰美国公司 阿尔茨海默氏疾病分泌酶
US7812123B2 (en) 1998-09-24 2010-10-12 Elan Pharmaceuticals, Inc. Alzheimer's disease secretase
WO2000017369A3 (fr) * 1998-09-24 2000-11-23 Upjohn Co Secretase de la maladie d'alzheimer
US7041473B1 (en) 1998-09-24 2006-05-09 Pharmacia & Upjohn Company Alzheimer's disease secretase, APP substrates therefor, and uses thereof
US6835565B1 (en) 1998-09-24 2004-12-28 Pharmacia & Upjohn Company Alzheimer's disease secretase
US7456269B2 (en) 1998-09-24 2008-11-25 Pharmacia & Upjohn Company Alzheimer's disease secretase, APP substrates thereof, and uses thereof
US6844148B1 (en) 1998-09-24 2005-01-18 Pharmacia & Upjohn Company Alzheimer's disease secretase, APP substrates therefor, and uses therefor
US6828117B2 (en) 1998-09-24 2004-12-07 Pharmacia & Upjohn Company Alzheimer's disease secretase, APP substrates therefor, and uses therefor
US6420534B1 (en) 1998-09-24 2002-07-16 Pharmacia & Upjohn Company Alzheimer's disease secretase, APP substrates therefor, and uses thereof
US6440698B1 (en) 1998-09-24 2002-08-27 Mark E. Gurney Alzheimer's disease secretase, APP substrates therefor, and uses therefor
EP1939297A1 (fr) * 1998-09-24 2008-07-02 Pharmacia & Upjohn Company LLC Sécrétase de la maladie d'Alzheimer
US6500667B1 (en) 1998-09-24 2002-12-31 Pharmacia & Upjohn Company Aspartyl protease 2 (Asp2) antisense oligonucleotides
US6867018B1 (en) 1998-09-24 2005-03-15 Pharmacia & Upjohn Company Alzheimer's disease secretase, APP substrates therefor, and uses thereof
US7378511B2 (en) 1998-09-24 2008-05-27 Pharmacia & Upjohn Company Alzheimer's disease secretase, APP substrates therefor, and uses therefor
US7375187B2 (en) 1998-09-24 2008-05-20 Pharmacia & Upjohn Company Alzheimer's disease secretase, APP substrates therefor, and uses therefor
US6825023B1 (en) 1998-09-24 2004-11-30 Pharmacia & Upjohn Company Alzheimer's disease secretase, APP substrates therefor, and uses therefor
US6797487B2 (en) 1998-09-24 2004-09-28 Pharamcia & Upjohn Company Polynucleotides encoding alzheimer's disease secretase
EA009216B1 (ru) * 1998-09-24 2007-12-28 Фармация Энд Апджон Компани Способы идентификации средств, модулирующих активность аспартилпротеазы asp2 человека
KR100768381B1 (ko) * 1998-09-24 2007-10-18 파마시아 앤드 업존 캄파니 엘엘씨 알츠하이머병 세크레타제
US6699671B1 (en) 1998-09-24 2004-03-02 Pharmacia & Upjohn Company Alzheimer's disease secretase, APP substrates therefor, and uses therefor
US6706485B1 (en) 1998-09-24 2004-03-16 Pharmacia & Upjohn Company Method of identifying agents that inhibit APP processing activity
US6727074B2 (en) 1998-09-24 2004-04-27 Pharmacia & Upjohn Company Alzheimer's disease secretase, APP substrates therefor, and uses therefor
US6737510B1 (en) 1998-09-24 2004-05-18 Pharmacia & Upjohn Company Alzheimer's disease secretase, APP substrates therefor, and uses thereof
US6753163B2 (en) 1998-09-24 2004-06-22 Pharmacia & Upjohn Company Alzheimer's disease secretase, APP substrates therefor, and uses therefor
US6913918B2 (en) 1998-09-24 2005-07-05 Pharmacia & Upjohn Company Alzheimer's disease secretase, APP substrates therefor, and uses therefor
US6790610B2 (en) 1998-09-24 2004-09-14 Pharmacia & Upjohn Company Alzheimer's disease, secretase, APP substrates therefor, and uses therefor
WO2000023576A2 (fr) * 1998-10-16 2000-04-27 Hook Vivian Y H Secretases liees a la demence d'alzheimer
WO2000023576A3 (fr) * 1998-10-16 2000-09-28 Vivian Y H Hook Secretases liees a la demence d'alzheimer
US6313268B1 (en) 1998-10-16 2001-11-06 Vivian Y. H. Hook Secretases related to Alzheimer's dementia
US6245884B1 (en) 1998-10-16 2001-06-12 Vivian Y. H. Hook Secretases related to alzheimer's dementia
DE19856261C1 (de) * 1998-12-07 2000-03-30 Hoechst Marion Roussel De Gmbh Aß-Peptid Screening Assay
US7109027B2 (en) 1998-12-07 2006-09-19 Sanofi-Aventis Deutschland Gmbh Aβ-Peptide screening assay
US7456007B1 (en) 1998-12-31 2008-11-25 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
WO2000047618A2 (fr) * 1999-02-10 2000-08-17 Elan Pharmaceuticals, Inc. Compositions a base d'enzyme beta-secretase et procedes
GB2389182B (en) * 1999-02-10 2004-02-04 Elan Pharm Inc Method of purifying b-secretase
GB2389182A (en) * 1999-02-10 2003-12-03 Elan Pharm Inc Method of purifying b-secretase
US7067271B1 (en) 1999-02-10 2006-06-27 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
WO2000047618A3 (fr) * 1999-02-10 2001-09-07 Elan Pharm Inc Compositions a base d'enzyme beta-secretase et procedes
US7109017B1 (en) 1999-02-10 2006-09-19 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
US7427478B2 (en) 1999-02-10 2008-09-23 Elan Pharmaceuticals, Inc. Beta-secretase enzyme compositions and methods
US7115410B1 (en) 1999-02-10 2006-10-03 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
EP1445263A1 (fr) * 1999-02-10 2004-08-11 Elan Pharmaceuticals, Inc. Enzyme humaine de beta-secretease, inhibiteurs et leurs compositions et utilisations.
GB2364059B (en) * 1999-02-10 2004-01-14 Elan Pharm Inc Beta-Secretase enzyme compositions and methods
US7252963B2 (en) 1999-02-10 2007-08-07 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
US7262043B2 (en) 1999-02-10 2007-08-28 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
US7276349B2 (en) 1999-02-10 2007-10-02 Elan Pharmaceuticals, Inc. Beta-secretase enzyme compositions and methods
US6627739B1 (en) 1999-02-10 2003-09-30 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
US7579180B2 (en) 1999-03-26 2009-08-25 Amgen Inc. Beta secretase polypeptides
US7582465B2 (en) 1999-03-26 2009-09-01 Amgen Inc. Beta secretase genes
WO2000058479A1 (fr) * 1999-03-26 2000-10-05 Amgen Inc. Genes et polypeptides beta secretase
US7244708B2 (en) 1999-06-28 2007-07-17 Oklahoma Medical Research Foundation Inhibitors of memapsin 2 and use thereof
US6545127B1 (en) 1999-06-28 2003-04-08 Oklahoma Medical Research Foundation Catalytically active recombinant memapsin and methods of use thereof
EP1249498A3 (fr) * 1999-09-23 2004-09-22 PHARMACIA & UPJOHN COMPANY Sécrétase de la maladie d'Alzheimer, substrats de la protéine précurseur d'amylase (APP) et procédés d'utilisation
EP1249498A2 (fr) * 1999-09-23 2002-10-16 PHARMACIA & UPJOHN COMPANY Sécrétase de la maladie d'Alzheimer, substrats de la protéine précurseur d'amylase (APP) et procédés d'utilisation
WO2001050829A3 (fr) * 1999-10-13 2003-12-04 Michael Jerome Bienkowski Secretase de la maladie d'alzheimer, substrats app destines a celle-ci et utilisation de cette derniere
WO2001049098A2 (fr) * 1999-10-13 2001-07-12 Michael Jerome Bienkowski Secretase associee a la maladie d'alzheimer, substrats app et utilisations correspondants
WO2001049098A3 (fr) * 1999-10-13 2003-11-20 Michael Jerome Bienkowski Secretase associee a la maladie d'alzheimer, substrats app et utilisations correspondants
WO2001050829A2 (fr) * 1999-10-13 2001-07-19 Michael Jerome Bienkowski Secretase de la maladie d'alzheimer, substrats app destines a celle-ci et utilisation de cette derniere
US7514408B1 (en) 1999-12-02 2009-04-07 Elan Pharmaceuticals, Inc. β-secretase enzyme compositions and methods
US7335632B2 (en) 2001-10-23 2008-02-26 Comentis, Inc. Beta-secretase inhibitors and methods of use thereof
US8772457B2 (en) 2010-11-10 2014-07-08 Genentech, Inc. BACE1 antibodies
US9453079B2 (en) 2010-11-10 2016-09-27 Genentech, Inc. Methods and compositions for neural disease immunotherapy
US9879094B2 (en) 2010-11-10 2018-01-30 Genentech, Inc. Nucleic acid molecules encoding for BACE1 antibodies
US10882920B2 (en) 2014-11-19 2021-01-05 Genentech, Inc. Antibodies against BACE1 and use thereof for neural disease immunotherapy
US11008403B2 (en) 2014-11-19 2021-05-18 Genentech, Inc. Anti-transferrin receptor / anti-BACE1 multispecific antibodies and methods of use
US11746160B2 (en) 2014-11-19 2023-09-05 Genentech, Inc. Antibodies against BACE1 and use thereof for neural disease immunotherapy

Also Published As

Publication number Publication date
AU1684097A (en) 1998-07-03

Similar Documents

Publication Publication Date Title
US5744346A (en) β-secretase
US6329163B1 (en) Assays for detecting β-secretase inhibition
WO1998026059A1 (fr) Beta-secretase isolee a partir de cellules humaines 293
JP3937459B2 (ja) 新規なアミロイド前駆体タンパク質及びその使用法
Bennett et al. The syntaxin family of vesicular transport receptors
US6221645B1 (en) β-secretase antibody
EP0871720A2 (fr) Beta-secretase, anticorps diriges contre la beta-secretase et dosages permettant de detecter l'inhibition de la beta-secretase
US5292652A (en) Amyloidin protease and uses thereof
AU643835B2 (en) Proteases causing abnormal degradation of amyloid beta-protein precursor
CA2388660A1 (fr) Secretases liees a la demence d'alzheimer
NZ247356A (en) Method, kit and chromogenic substrate for diagnosing alzheimers disease
Xia et al. FAD mutations in presenilin-1 or amyloid precursor protein decrease the efficacy of a γ-secretase inhibitor: evidence for direct involvement of PS1 in the γ-secretase cleavage complex
US5968764A (en) Glucose transporter vesicle aminopeptidase
CA2452832A1 (fr) Essai de criblage in vitro de la .gamma.-secretase
Herr et al. Purification and microsequencing of the intra-acrosomal protein SP-10. Evidence that SP-10 heterogeneity results from endoproteolytic processes
NZ247575A (en) Identifying proteolytic enzymes with specificity for the precursor to alzheimers disease beta amyloid protein (especially cathepsin d) and their use in regulating beta-amyloid formation
CA2151927A1 (fr) Cathepsine d, protease amyloidogene dans la maladie d'alzheimer
Abramovitz et al. Inhibition of superoxide production in human neutrophils by purified soybean polypeptides. Re-evaluation of the involvement of proteases.
US20030022251A1 (en) Gamma-secretase in vitro screening assay
JP2003532413A (ja) γ−セクレターゼ活性の変調
US20050065076A1 (en) Gamma three protease
Chang et al. A Novel Brain Cysteine Protease Forms an SDS Stable Complex with the β‐Amyloid Precursor Protein a
Dupont et al. A new approach to monitoring proteolysis phenomena using antibodies specifically directed against the enzyme cleavage site on its substrate
Fine et al. Identification of the human albumin variant “Gainesville” with proalbumin “Christchurch”
WO1993022676A1 (fr) IDENTIFICATION DE L'ANTICHYMOTRYPSINE α1 CEREBRALE ET METHODES PERMETTANT DE DIAGNOSTIQUER LA MALADIE FONDEE SUR SA DETECTION

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE HU IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG US UZ VN AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase