WO1992018138A1 - Compositions et methodes d'utilisation de gap-43 de mammifere - Google Patents

Compositions et methodes d'utilisation de gap-43 de mammifere Download PDF

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WO1992018138A1
WO1992018138A1 PCT/US1992/003014 US9203014W WO9218138A1 WO 1992018138 A1 WO1992018138 A1 WO 1992018138A1 US 9203014 W US9203014 W US 9203014W WO 9218138 A1 WO9218138 A1 WO 9218138A1
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gap
cells
protein
antibody
cell
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Mark C. Fishman
Howard J. Federoff
Mauricio X. Zuber
Stephen M. Strittmatter
Dario Valenzuela
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The General Hospital Corporation
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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/38—Vector systems having a special element relevant for transcription being a stuffer
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

  • the present invention relates to the fields of molecular genetics and neurology. More particularly, the invention relates to the cDNA sequence and corresponding amino acid sequence of mammalian GAP-43, a neuronal growth-related protein. The present invention is further related to methods of regulating expression of GAP-43, thereby regulating axonal growth, and to methods of producing GAP-43 in prokaryotic or eukaryotic hosts cells or organisms. More particularly, the invention is related to a novel membrane-targeting peptide derived from GAP-43, which is capable of regulating membrane binding and growth cone enrichment of GAP-43, and is also capable of directing any desired protein or polypeptide to the membrane of neuronal or non-neuronal cells.
  • the invention further relates to the discovery that GAP-43 and biologically active peptides derived therefrom function as Internal Regulatory Proteins (IRPs), which can act intracellularly to modulate cell function.
  • IRPs Internal Regulatory Proteins
  • the present invention also is related to the clinical in vivo and in vitro diagnostic and therapeutic applications of GAP-43 and its regula ⁇ tory and membrane-targeting elements in. inter alia, neurological indica ⁇ tions in animals including humans.
  • GAP-43 is one of the proteins that specifically characterizes growing axons (Skene, Cell 37:697 (1984): Meiri, PNAS USA 83:3537 (1986)).
  • Axonally transported proteins are a small subset of total cellular proteins, and only a few of these vary such that their levels may be envisioned as directly mediating axonal growth (Skene and Williard. J. Cell. Biol. 89:86 (1981); Benowitz and Lewis. J. Neuroscience 3:2153 (1983): Skene, Cell 37:697 (1984): Meiri. PNAS USA 83:3537 (1986)).
  • GAP-43 is particularly attractive as a candidate since it is primarily a growth cone constituent where it is bound to the internal surface of growth cone membrane and serves as a substrate for protein kinase C.
  • its level of gene expression correlates well with axonal growth, both in cell culture and in vivo (Basi. Cell 49:785 (1987); Karns, Science 236:597 (1987): Neve, Molec. Brain Res. 2:177 (1987); de la Monte et al., unpublished).
  • CNS central nervous system
  • GAP-43 increases to high levels after injury in neurons capable of regeneration, such as toad or goldfish optic nerve or mammalian peripheral nerve, but not after similar injury to mammalian CNS neurons (Skene and Williard, J. Cell. Biol. 89:96 (1981)).
  • the present inventors have examined the role of GAP-43 in human CNS function and disease.
  • Human GAP -43 cDNA has been cloned, and its developmental and adult distribution examined by assay of post ⁇ mortem tissue.
  • the present inventors have discovered that GAP-43 expression persists in discrete regions of the adult, and unexpectedly, that acute ischemic injury is associated with heightened expression of GAP -43 even in areas where it is normally low.
  • GAP -43 Recognizing the potential importance of GAP -43 in mammalian CNS function and disease, the present inventors have succeeded in sequencing the mammalian GAP-43 gene by complimentary DNA (cDNA) cloning. The complete nucleotide sequence of the gene encoding rat GAP-43 and the amino acid sequence have been determined. cDNA for rat GAP -43 has been used as a probe to identify and clone cDNA for human GAP-43 from human brainstem and cerebellum libraries. The amino acid sequence for human GAP -43 also has been determined.
  • substantially pure mammalian GAP -43 protein or a functional derivative thereof.
  • rat and human GAP-43 proteins in substantially pure form, as well as the functional derivatives of these proteins.
  • Specific embodiments of the invention comprise substantially pure rat and human GAP-43 proteins and polypeptides having amino acid sequences corresponding to those shown in Figures 2 and 5A, respectively, and their functional derivatives.
  • Another embodiment of the invention provides for cDNA compris ⁇ ing a nucleotide sequence as shown in or substantially similar to that shown in Figures 2 or 5A, or functional derivatives thereof.
  • the cDNA of the invention may be incorporated into a suitable expression vector, such as a plasmid, and the vector may be used to transfect a prokaryotic or eukaryotic host cell, which may then express the cDNA under appropriate in vivo, in vitro or in situ conditions, all of which, together with the GAP-43 protein or polypeptide produced thereby, form additional embodiments of the invention.
  • yet another embodiment of the invention provides for a method of producing mammalian GAP-43 protein or polypeptide or a functional derivative thereof, comprising transfecting a prokaryotic or eukaryotic host cell with a vector comprising cDNA encoding mammalian GAP-43 protein or polypeptide, culturing said host cell in a suitable medium and under conditions permitting expression of said mammalian GAP-43 protein or polypeptide, and separating said mammalian GAP-43 protein or polypeptide, or their functional derivatives, from said medium.
  • the GAP-43 antibodies of the invention may be polyclonal or, preferably, monoclonal antibodies, and are suitable for a variety of preparative, diagnostic and therapeutic uses, which are to be understood as forming yet additional invention embodiments.
  • GAP -43 antigens and antibodies of the invention are well suited for appropriate labeling as. for example, with detectable or therapeutic labels, and for use with other active agents in compositions which may or may not be pharmaceutically acceptable, all of which may be determined as the particular preparative, diagnostic or therapeutic application may require.
  • Such labeled GAP-43 antigens, antibodies and their functional and chemical derivatives, as well as such compositions, comprise embodiments of the present invention.
  • GAP-43 antigens and, particularly, antibodies of the invention, together with their functional and chemical derivatives may be employed in various diagnostic methods known to those of skill. Such methods, including but not limited to immunocytochemical and immunometric methods, form additional embodiments of the invention. Accordingly, in one exemplary embodiment of the invention is provided a method of determining or detecting mammalian GAP-43 antigen or antibody in a sample, comprising contacting a sample suspected of containing GAP-43 antigen or antibody with detectably labeled GAP-43 antibody or antigen, respectively, incubating said sample with said antibody or antigen so as to allow the formation of a GAP-43 antigen- antibody complex, separating the complex thus formed from uncomplexed antigen or antibody, and detecting the labeled complexed antibody or antigen.
  • kits useful for the preparation, purification, isolation, determination or detection of GAP -43 antigen or antibody, or for therapeutic treatment with GAP -43 antigen or antibody, comprising carrier means being compartmentalized to receive in close confinement therein one or more container means, wherein one or more of said container means comprises preparatively. detectably or therapeutically labeled GAP-43 antigen or antibody, or their functional or chemical derivatives.
  • the present inventors also have evaluated GAP -43 expression in normal, as well as in damaged or diseased CNS tissue. It has been discovered that in vivo GAP-43 expression varies during development in neural tissue, and that regional variations in GAP-43 expression exist. Further, it has been discovered that GAP-43 expression undergoes significant changes as a result of damage to neural tissue.
  • GAP-43 expression may be enhanced or, if desired, inhibited.
  • GAP -43 expression is enhanced by nerve growth factor, and that this is inhibited by certain steroids.
  • the ability to modulate GAP-43 expression may be of great therapeutic utility in treating mammals, and particularly humans, suffering from damage to, or from disease or dysfunction of, the central or peripheral nervous system, the significance of these discoveries will be readily apparent.
  • the inventors by introducing into non-neural cells cDNA encoding GAP-43 or its functional derivative, the inventors have made the surprising discovery that even non-neural cells can form growth cone-like processes. Again, the potential therapeutic value of this discovery is profound.
  • the present invention comprises methods for evaluating or determining GAP-43 activity and expression in diseased or damaged CNS tissue, as well as in normal CNS tissue.
  • the present invention further comprises methods of treating mammals, including humans, suffering from damaged, diseased or dysfunctioning central or peripheral nervous tissue, and methods of modulating structural remodeling in normal CNS tissue in mammals including humans.
  • the invention comprises a method of inducing expression of GAP-43 in cells, comprising exposing said cells in vivo, in vitro or in situ to an effective amount of nerve growth factor.
  • exposing said cells in vivo, in vitro or in situ to an effective amount of nerve growth factor.
  • GAP -43 expression may be induced or enhanced by introducing into non-neural or neural cells cDNA encoding GAP-43. This may be accomplished in vivo, in vitro or in situ by a variety of means, including transfection, transduction and direct microinjection. all of which form intended non-limiting embodiments of the invention.
  • the cDNA of the invention may be introduced by means of a retroviral or viral vector, or may be attached to any number of cell surface receptor ligands and conveyed with such ligands into the cell. All of these methods, as well as the compositions and vectors comprising GAP-43 cDNA and its functional and chemical derivatives, form additional embodiments of the present invention.
  • yet additional embodiments of the present invention comprise methods of modulating structural remodeling, methods of modulating synaptic plasticity, and methods of modulating the microenvironment of cells, including neuronal and non-neuronal cells, comprising exposing said cells to an effective amount of one or more substances selected from the group consisting of nerve growth factor, steroid and their functional derivatives.
  • GAP -43 surprisingly contains a ten amino acid amino-terminus exon, and that this peptide is responsible for directing GAP-43 to the cell membrane, and especially to the growth cone regions of neuronal cells. It has further been discovered that this ten amino acid membrane-targeting peptide, and its functional derivatives, are capable of directing a desired protein or peptide to the cell membrane, when attached at or near the amino-terminus of such protein or peptide. This surprising discovery applies to proteins and peptides which are normally cytosolic, and not normally membrane-associated.
  • an additional embodiment of the present invention comprises a membrane-targeting peptide, or a functional derivative thereof, capable of directing any desired protein or peptide to the cell membrane of neuronal or non-ne ronal cells.
  • the membrane-targeting peptide of the invention, or the desired protein or peptide to which it is attached, may be diagnostically or therapeutically labeled.
  • Methods of diagnostic or therapeutic in vivo, in vitro or in situ treatment of neuronal or non- neuronal cells of animals, including humans, using the membrane- targeting peptide form additional embodiments of the present invention.
  • the present invention provides for genomic GAP-43, which has been isolated, and its intron-exon boundaries and transcriptional start sites mapped. Further, it has surprisingly been discovered that the GAP -43 promoter is quite unusual in its structure, and may be useful for the expression of other structural genes.
  • the present invention is directed to the surprising discovery that GAP -43 acts intracellularly to modify the binding capacity of other cell proteins, including that of G 0 .
  • the present invention thus provides for an important new class of internal regulatory proteins ("IRP"), of which GAP-43 is representative, comparable in effect and utility to external cell receptors.
  • IRP internal regulatory proteins
  • synthetic peptides comprising the amino terminus amino acids of GAP-43 also exhibit biological activity as internal regulatory proteins, and compositions comprising such proteins and their use constitute additional embodiments of the invention.
  • FIG. 1 Hybrid-selected translation of GAP-43 cDNA.
  • the EcoRI insert, GAP43-2 was used to select mRNA by the procedure of Ricciardi et al. PNAS 76:4927 (1979).
  • 0.5 mg of the GAP43-2 insert, or equivalent amounts of nonspecific DNA, the bacterial plasmid pSP65 were spotted onto nitrocellulose and hybridized with 17.5 mg of newborn rat brain polyadenylated [poIy(A) + ] RNA in a solution with 659c formamide, 400 mM NaCl, 10 mM 1,4- ⁇ iperazine diethanesulfonic acid (Pipes) pH 6.4 at 42 °C for 16 hours.
  • FIG. 1 Nucleotide sequence and predicted amino acid sequence of GAP-43.
  • the cDNA library was generated with RNA from dorsal root ganglia from embryonic day 17-18 rats. Total cellular RNA was isolated by the method of Chirgwin et al.. Biochemistry .18:5294 (1979), and poly(A) + RNA was selected with oligo-dT cellulose. Double-stranded cDNA was generated by the ribonuclease H method described by Gubler and Hoffman, Gene 25:263 (1983), ligated to EcoRI linkers, and ligated into the EcoRI site of the lambda phage cloning vectors, lgtlO and lgtll.
  • IPTG isopropyl b-D-thiogalactopyranoside
  • the cDNA inserts were subcloned into the EcoRI sites of M13 mpl8. Initial DNA sequence analysis of the two shorter clones revealed that they were included within the longest.
  • FIG. 3 Regulation of GAP-43 expression in PC12 cells.
  • PC12 cells were passaged in RPMI medium containing 109. horse serum and 59c fetal bovine serum. Forty hours after plating the cells, the medium was changed to include the different additives. After 4 days, the cells were photographed (A), then RNA was isolated from each cell culture. RNA (10 mg per sample) was denatured and run on a 1.2% agarose- formaldehyde gel, transferred to a GeneScreen nylon filter, bound to the filter by ultraviolet cross-linking, and probed with ⁇ P-labeled GAP43-2 (B). The final wash was SSC (x ⁇ .2). 0.1 SDS at 65°C.
  • the additives included were (i) none, (ii) 50 ng of NGF per milliliter, (iii) 10 M dibutyryl cAMP. and (iv) 50 ng of NGF per milliliter and lO ' ⁇ M dibutyryl cAMP.
  • the last lane is 10 mg of RNA from newborn brain run as a positive control for the blotting and hybridization procedure.
  • RNA was isolated from the designated rat tissues by a modification of the procedure of Chirgwin et al, Biochemistry 18:5294 (1979). Each RNA (10 mg) was denatured, underwent electrophoresis in a 1.2% agarose-form aldehyde gel, and was transferred to nitrocellulose. The filter was hybridized overnight at 42 °C with the EcoRI insert from lgtll GAP43-2 labeled with deoxycytidine 5 " - [a- P]triphosphate by nick translation. The final wash was done in SSC (x ⁇ .2), 0.1% SDS at 65 °C.
  • RNA samples (i) embryonic day 13 (E13) heart (H), (ii) E13 liver (L). (iii) E13 brain (B), (iv) E13 dorsal root ganglion (DRG), (v) to (viii) embryonic day 17 heart, liver, brain, and dorsal root ganglion; (ix) to (xii) newborn heart, liver, brain, and dorsal root ganglion; (xiii) to (xvi) adult heart, liver, brain and dorsal root ganglion.
  • the positions of the 18S and 28S ribosomal RNA are shown at the right.
  • a cDNA probe encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Piechacyk et al contend Cell 42:589 (1985)).
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • FIG. 5 Nucleotide sequence and deduced amino acid sequence of human GAP-43 cDNA. E: EcoRI; H: HaelH: M: Mspl. The coding region is denoted by thick bar. The scale is 100 bp. Arrows show the overlapping restriction fragments that were sequenced.
  • B Alignment of human, rat GAP-43 and mouse P-57 amino acid sequences. Vertical bars indicate identity, and colons show conservative substitutions. The amino acids are represented by IUPAC-IUB CBN one-letter symbols. The rat sequence is that of Figure 2. and the mouse sequence is from Cimler et al., J. Biol. Chem. 262:12158 (1987).
  • C Stem-loop structures in the 3 ? -untranslated region predicted by fold program of Zuker and Steigler, Nucl. Acids Res. 9:133 (1981).
  • FIG. 1 Northern blot showing the regional restriction of GAP-43 expression with maturation. Ten mg total RNA from 8-day-old, 16-year-old, and 64-year-old brain regions were loaded in each lane and the blot was probed with human GAP -43 probe Cla as described in Example II. The positions of 18S and 28S rRNA bands are indicated.
  • FIG. 7 Northern blot showing that GAP-43 expression increases in the wake of an ischemic event.
  • A Ten mg of RNA from different brain regions of a patient with a stroke in Area 17 (visual cortex). Expression in A17 has increased to levels comparable to the highest in the brain (All).
  • B Ten mg of RNA from Area 3,1,2.5 from three patients. all run and blotted on the same blot with an unrelated band excised between lanes 2 and 3. Lanes 1 and 2 were histologically normal, whereas 3 included a small stroke, and shows an increase in GAP-43 expression.
  • Figure 8. In situ hybridization reveals increased GAP-43 expression in regions adjacent to infarcts.
  • G The large bright foci are from areas of coagulative necrosis which also label nonspecifically with the sense probe (G).
  • G sense probe
  • GAP -43 E-brightfield and F-darkfield labeled with antisense probe.
  • H A control brightfield and
  • I darkfield labeled with the sense probe showing absence of specific GAP -43 binding (C-I xl60).
  • Figure 9 Enhanced GAP -43 expression in neurons of cerebellar cortex several days following a bout of severe hypotension and hypoxia. All sections were processed simultaneously.
  • A Normal adult cerebellar cortex showing absence of detectable GAP -43 labeling.
  • B Post-ischemic cerebellar cortex showing markedly increased expression of GAP -43 in the Purkinje cell and outer granule cell layers.
  • C A section adjacent to B hybridized with the sense strand probe as control (all x315).
  • the percentage of cells with processes was assayed by plating CHO cells onto poly-D-lysine-coated coverslips. Cells with processes longer than 20 microns were scored as positive. To ensure comparability, all assays were performed within the time window that extended from 30 to 45 minutes after plating. An important component of this assay was the time window selected, since, after longer plating times, or as cells reached confluence, processes were much less evident. As many cells as possible were counted during this time window, and all cells examined were included. The number of cells counted for the different lines was: IA, 406; IB, 408; 2A. 287; 2B, 303: 5E, 234; 4, 333; 12, 156; 14, 161. The proportion of cells with processes in GAP-43 expressing cell lines was significantly greater than in controls (p ⁇ 0.001).
  • FIG. 11 Schematic representation of experiments demonstrating that the amino-terminus exon is responsible for directing the GAP -43 protein to the cell membrane, and that it directs membrane targeting of chloramphenicol acetyl transferase.
  • the left column
  • CONSTRUCTION indicates the gene construction used for transfec- tion of COS, NIH 3T3, CHO or PC12 cells.
  • the right column (“MEM ⁇ BRANE”) indicates whether the expressed protein or fragment was membrane-associated (+) or not (-), as assayed by sub-cellular fractionation followed by Western blotting, direct immunofluorescence. or both.
  • the intact GAP -43 gene was significantly membrane- associated, as were GAP constructions lacking substantial portions of exon 2 (GAP(-intern.)) or the carboxy-terminus region of the GAP -43 gene (GAPtag). However, when the nucleotides encoding the first four amino acids of GAP-43 were deleted (GAP(- 1-4)), the expressed protein fragment was not membrane-associated.
  • Point mutations were introduced into the sequences encoding the cysteines at positions three (C3) or four (C4) of the first exon. to result in expression of alahine in the resulting protein. Mutation of either C3 (GAP *C 3 ) or C4 (GAP *C 4 ) resulted in reduced membrane levels (+/-) as compared to intact GAP-43. Reduction was especially marked when C4 was altered. Mutation of both C3 and C4 (GAP *C 3 4 ) eliminated membrane association altogether.
  • CAT chloramphenicol acetyl transferase
  • CON had no GAP-43:
  • Brain membranes (BR) had GAP- 43 of the same molecular weight as that from the transfected gene.
  • A A linear depiction of the GAP-43 gene in the 5' to 3 ' orienta ⁇ tion. Representations of the phage inserts that were used for mapping are shown. The three exons are depicted as vertical bars. The sites shown are for restriction endonucleases BamHl (B), Kpnl (K), and Sad (S).
  • FIG. 14 Sequence of the GAP -43 promoter region. Nucleotide position + 1 denotes the A of the initiating ATG codon of the GAP-43 protein. This sequence includes the variably sized first exon which ends at +30. Major transcriptional start sites are denoted by arrows. Purine residues have been underscored by asterisks. The consensus Pit-1 binding site is overlined.
  • FIG. 15 Mobility shift in restriction fragments induced by H- DNA.
  • GAP-43 promoter fragments liberated by digestion of plasmid bsl.5RIX4 with the following enzymes: 1) Sspl, 603 bp; 2) Xbal/Sspl, 560bp; 3) Smal/Sspl, 490bp; 4) Sspl/Nhel, 409bp; 5) Sspl/Nsil, 284bp (contains region I), 319bp (contains regions II and III; 6) SspI/AccI, 314bp (region I), 289bp (regions II and III): 7) Xbal/Nhel, 360bp; and 8) Smal/Nhel, 295bp.
  • FIG. 16 Partial protein sequence for p34 and p38.
  • GAP-43 With different preparations of GAP-43, the range of EC50 was 150-800 nM (compare Fig.17D), perhaps reflecting partial inactivation of GAP-43 during purification.
  • MLCCMRRTKQVEKNDEDQKIEQDG. stimulates binding to the same level as with 1 uM GAP-43 (250%), and the addition of both GAP-43 and 1-24 peptide does not cause further stimulation (B. left). This peptide does not bind (35S)GTPuS in the absence of Go.
  • GAP (35 -53) ATKIQ ASFRGHITRKKLKD.
  • the stimulation of binding by the 1-24 peptide is saturable with an EC50 of 20 uM (B. right).
  • MLCCMRRTKQ. inhibits (35S)GTPuS binding to G 0 by 80 % at 1 mM (C, left).
  • a peptide with threonine in place of cysteine at positions 3 and 4 has no effect on (35S)GTPuS binding to Go.
  • the IC50 for the 1-10 peptide is 20 uM (C. right).
  • Panel D shows (35S)GTPuS binding to G 0 as a function of GAP-43 concentration in the presence or absence of 50 uM 1-10 peptide. Note that GAP-43 reverses inhibition by the peptide. and that the EC50 for GAP-43 is shifted from 600 nM to 12 uM by this concentration of peptide. This is consistent with direct competition between the 1-10 peptide and GAP -43. Methods. (35S)GTPuS (New England Nuclear, 2000 Ci/mmol) binding was determined as described previously (Huff, R.M. et al., J. Biol. Chem.
  • the concentration of (35S)GTPuS in these assays is approximately one tenth of the apparent KD (Huff, R.M. et al., J. Biol. Chem. 260: 10864-10871 (1985); Northrup, J.K. et al., J. Biol. Chem. 257: 11416-11423 (1982)), so that changes in both apparent KD and Bmax alter the level of binding. Total radioactivity bound was less than 109. of that added in all cases. Non-specific binding was determined in the presence of 10 uM unlabeled GTPuS and subtracted from total binding to determine specific binding. All assays were performed in duplicate.
  • An average assay tube contained 800.000 cpm and yielded 30,000 cpm total binding and 1500 cpm non-specific binding.
  • the addition of GAP -43 increased specific binding without effect on non-specific binding.
  • GAP-43 activated two different samples of G 0 and one of alphao, all prepared in Lubrol PX. However, the extent of the activation varied among the preparations. Activation by GAP -43 appears to be sensitive to the detergent used in the purification of G 0 and the composition of the binding buffer.
  • GAP -43 was purified by a modification of the method of Zwiers et al. (J. Neurochem. 44: 1083-1090 (1985)). and was greater than 97% pure by Coomassie blue staining of SDS-PAGE gels.
  • the protein was dialyzed extensively against the assay buffer and added to the GTPuS-G 0 incubations in the concentrations indicated above.
  • Peptides were synthesized at the Howard Hughes Medical Institute laboratory (Massachusetts General Hospital, Boston. MA), purified by reverse phase HPLC, and structure was confirmed by amino acid sequencing. The peptides were dissolved in assay buffer and added to the GTPuS-G 0 incubation at the indicated concentrations.
  • FIG. 18 Homology between GAP-43 amino terminus and the cytoplasmic tail of G-linked receptors. The first 10 amino acids of GAP-43 were compared to the sequence of the G-linked receptor family. Homology between the first 7 amino acids of GAP-43 and the amino terminal segment of the cytoplasmic tail of a number of G-linked receptors is shown. Residues which conform to the consensus sequence hydrophobic-leu-cys-cys-X-basic-basic are shaded.
  • cysteines illustrated in the receptors are located approximately 13 amino acids distal to the last transmembrane region, correspond to cys341 of the B2-adrenergic receptor, and are aligned as described in Higashijima, T. et al. (J. Biol. Chem. 263: 6491-6494 (1988)). The sequences shown are from human ( Figure 5) and fish (LaBate, M.E. & Skene. J.H.P. Neuron 3: 299-310 (1989)) GAP-43. human betal-adrenergic receptor (Bl-adrenergic, Frielle. T. et al.. Proc. Natl. Acad. Sci., U.S.A.
  • human beta2 adrenergic receptor B2-adrenergic, Koblika, B.K. et al.. Proc. Natl Acad. Sci. U.S.A. 84: 46-50 (1987)).
  • human rhodopsin rhodopsin, Nathans. J.D. & Hogness. D.S. Proc. Natl. Acad. Sci. U.S.A. 81: 4851-4855 (1984)
  • human blue-opsin blue-opsin, Nathans. J.
  • FIG. 19 Dose-dependent reduction by calmodulin of GAP-43- stimulated GTPgS binding to G 0 .
  • Vertical axis for both panels is GTPgS binding expressed as a percentage of control.
  • Left Panel. C calmodulin: G, GAP-43.
  • Addition of GAP-43 to G 0 (Lane 2) increased GTPgS binding to over 200% as compared to G Q alone (Lane 1).
  • Addition of calmodulin reduced this binding to control levels (Lane 5). No GTPgS binding was seen in the absence of G 0 , using GAP -43 alone (Lane 3) or GAP-43 plus calmodulin (Lane 4).
  • Right panel calmodulin concentration, uM, demonstrating the dose-dependency of the effect of calmodulin on GTPgS binding to G 0 .
  • cloning is meant the use of in vitro recombination techniques to insert a particular gene or other DNA sequence into a vector molecule.
  • in vitro recombination techniques to insert a particular gene or other DNA sequence into a vector molecule.
  • it is necessary to employ methods for generating DNA fragments, for joining the fragments to vector molecules, for introducing the composite DNA molecule into a host cell in which it can replicate, and for selecting the clone having the target gene from amongst the recipient host cells.
  • cDNA is meant complementary' or copy DNA produced from an RNA template by the action of RNA-dependent DNA polymerase (reverse transcriptase).
  • a cDNA clone means a duplex DNA sequence complementary to an RNA molecule of interest, carried in a cloning vector.
  • cDNA library is meant a collection of recombinant DNA molecules containing cDNA inserts which together comprise the entire genome of an organism.
  • a cDNA library may be prepared by- methods known to those of skill, and described, for example, in Maniatis et al.. Molecular Cloning: A Laboratory* Manual, supra.
  • RNA is first isolated from the cells of an organism from whose genome it is desired to clone a particular gene.
  • Preferred for the purposes of the present invention are mammalian, and particularly human, cell lines.
  • vector is meant a DNA molecule, derived from a plasmid or bacteriophage. into which fragments of DNA may be inserted or cloned.
  • a vector will contain one or more unique restriction sites, and may be capable of autonomous replication in a defined host or vehicle organism such that the cloned sequence is reproducible.
  • DNA expression vector is meant any autonomous element capable of replicating in a host independently of the host ' s chromosome, after additional sequences of DNA have been incorporated into the autonomous element's genome.
  • DNA expression vectors include bacterial plasmids and phages. Preferred for the purposes of the present invention is the lambda gtll expression vector.
  • substantially pure is meant any antigen of the present invention, or any gene encoding any such antigen, which is essentially free of other antigens or genes, respectively, or of other contaminants with which it might normally be found in nature, and as such exists in a form not found in nature.
  • functional derivative is meant the “fragments,” “variants,” “analogs,” or “chemical derivatives” of a molecule.
  • a “fragment” of a molecule, such as any of the cDNA sequences of the present inven ⁇ tion is meant to refer to any nucleotide subset of the molecule.
  • a “variant” of such molecule is meant to refer to a naturally occurring molecule substantially similar to either the entire molecule, or a fragment thereof.
  • An “analog” of a molecule is meant to refer to a non-natural molecule substantially similar to either the entire molecule or a fragment thereof.
  • a molecule is said to be "substantially similar” to another molecule if the sequence of amino acids in both molecules is substantially the same. Substantially similar amino acid molecules will possess a similar biological activity. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if one of the molecules contains additional amino acid residues not found in the other. __ -4
  • a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's solubility, absorption, biological half life, etc. The moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesir ⁇ able side effect of the molecule, etc. Moieties capable of mediating such effects are disclosed, for example, in Remington ' s Pharmaceutical Sciences. 16th ed.. Mack Publishing Co.. Easton. Penn. (1980).
  • a “functional derivative" of a gene of any of the antigens of the present invention is meant to include “fragments,” “variants,” or “analogues” of the gene, which may be “substantially similar” in nucleotide sequence, and which encode a molecule possessing similar activity.
  • a DNA sequence encoding GAP-43 or its functional derivatives, or the membrane-targeting peptide or functional derivatives thereof, may be recombined with vector DNA in accordance with conventional techni ⁇ ques, including blunt-ended or staggered-ended termini for ligation. restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed by Maniatis. T., et al, supra, and are well known in the art.
  • a nucleic acid molecule such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulator ⁇ ' information and such sequences are “operably linked” to nucleotide sequences which encode the polypeptide.
  • An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression.
  • regulatory regions needed for gene expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of protein synthesis.
  • promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of protein synthesis.
  • Such regions will normally include those 5'-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
  • the non-coding region 3' to the gene sequence coding for the protein may be obtained by the above-described methods.
  • This region may be retained for its transcriptional termination regulatory sequences, such as termination and polyadenylation.
  • the transcriptional termination signals may be provided. Where the transcriptional termination signals are not satisfactorily functional in the expression host cell, then a 3' region functional in the host cell may be substituted.
  • Two DNA sequences are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of the GAP-43 gene sequence, or (3) interfere with the ability of the GAP-43 gene sequence to be transcribed by the promoter region sequence.
  • a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.
  • the present invention encompasses the expression of the GAP-43 protein (or a functional derivative thereof) in either prokaryotic or eukaryotic cells.
  • Preferred prokaryotic hosts include bacteria such as E. coli. Bacillus, Streptomvces, Pseudomonas, Salmonella, Serratia, etc. The most preferred prokaryotic host is E. coli. Other enterobacterium such as Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species may also be utilized. Under such conditions, the GAP-43 will not be glycosylated.
  • the procaryotic host must be compatible with the replicon and control sequences in the expression plasmid.
  • GAP -43 protein (or a functional derivative thereof) in a prokaryotic cell (such as, for example. E. coli, B. subtilis, Pseudomonas. Streptomvces, etc.), it is necessary to operably link the GAP-43 encoding sequence to a functional prokaryotic promoter.
  • promoters may be either constitutive or. more preferably, regulatable (i.e.. inducible or derepressible).
  • constitutive promoters include the int promoter of bacteriophage 1. the bla promoter of the b-lactamase gene of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene of pBR325, etc.
  • inducible prokaryotic promoters examples include the major right and left promoters of bacteriophage 1 (P L and P R ), the trrj, recA. lacZ, lad, and gal promoters of E. coli, the a-amylase (Ulmanen, I., et al, J. Bacteriol. 162:176-182 (1985)) and the s-28-specific promoters of B. subtilis (Gilman. M.Z., et al.. Gene 32:11-20 (1984)), the promoters of the bacteriophages of Bacillus (Gryczan, T.J.. In: The Molecular Biology of the Bacilli. Academic Press, Inc.. NY (1982)), and Streptomvces promoters (Ward. J.M.. et al.. Mol Gen. 2.7
  • ribosome binding sites are disclosed, for example, by Gold, L., et al. (Ann- Rev. Microbiol 35:365-404 (1981)).
  • Most preferred hosts are eukaryotic hosts including yeast, insects, fungi, mammalian cells (especially human cells) either in vivo, or in tissue culture. Mammalian cells provide post-translational modifications to protein molecules including correct folding or glycosylation at correct sites. Mammalian cells which may be useful as hosts include cells of fibroblast origin such as VERO or CHO-K1, or cells of lymphoid origin, such as the hybridoma SP2/0-AG14 or the myeloma P3x63Sg8, and their derivatives. Preferred mammalian host cells include SP2/0 and J558L, as well as neuroblastoma cell lines such as IMR 332. that may provide better capacities for correct post-translational processing. COS cells also are convenient eukaiyotic hosts for GAP-43 expression, as well as for study of the regulation of GAP-43 expression, and are preferred for this purpose.
  • transcriptional and translational regulatory sequences may be employed, depending upon the nature of the host.
  • the transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus. bovine papilloma virus, Simian virus, or the like, where the regulatory signals are associated with a particular gene which has a high level of expression. Alternatively, 2%
  • Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the genes can be modulated.
  • regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical regulation, e.g.. metabolite.
  • Yeast provides substantial advantages in that it can also carry out post-translational peptide modifications including glycosylation.
  • Yeast recognizes leader sequences on cloned mammalian gene products and secretes peptides bearing leader sequences (i.e., pre-pep tides).
  • Any of a series of yeast gene expression systems incorporating promoter and termination elements from the actively expressed genes coding for glycolytic enzymes produced in large quantities when yeast are grown in mediums rich in glucose can be utilized.
  • Known glycolytic genes can also provide very efficient transcriptional control signals.
  • the promoter and terminator signals of the phosphoglycerate kinase gene can be utilized.
  • GAP -43 or functional derivatives thereof in insects can be achieved, for example, by infecting the insect host with a baculovirus engineered to express GAP -43 by methods known to those of skill.
  • sequences encoding GAP-43 may be operably linked to the regulatory regions of the viral polyhedron protein (Jasny, Science 238: 1653 (1987)). Infected with the recombinant baculovirus, cultured insect cells, or the live insects themselves, can M
  • GAP-43 protein in amounts as great as 20 to 50% of total protein production.
  • caterpillars are presently preferred hosts for large scale GAP-43 production according to the invention.
  • eukaryotic regulatory regions Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis.
  • Preferred eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer, D., et al, J. Mol Appl Gen. 1:273-288 (1982)); the TK promoter of Herpes virus (McKnight, S., Cell 31:355-365 (1982)); the SV40 early promoter (Benoist, C, et al.
  • yeast ga]4 gene promoter Johnston, S.A., et al, Proc. Natl Acad. Sci. (USA . 79:6971-6975 (1982): Silver, P.A., et al, Proc. Natl. Acad. Sci. fUSA . 81:5951-5955 (1984)).
  • the GAP -43 encoding sequence and an operably linked promoter may be introduced into a recipient prokaryotic or eukaryotic cell either as a non-replicating DNA (or RNA) molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since Bo
  • the expression of the GAP-43 protein may occur through the transient expression of the introduced sequence.
  • permanent expression may occur through the integration of the introduced sequence into the host chromosome.
  • a vector is employed which is capable of integrating the desired gene sequences into the host cell chromosome.
  • Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector.
  • the marker may provide for prototrophy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper or the like.
  • the selectable marker gene can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co- transfection. Additional elements may also be needed for optimal synthesis of single chain binding protein mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals. cDNA expression vectors incorporating such elements include those described by Okayama. H.. Mol Cel Biol 3:280 (1983).
  • the introduced sequence will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host.
  • a plasmid or viral vector capable of autonomous replication in the recipient host.
  • Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector: the number of copies of the vector which are desired in a particular host: and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
  • Preferred prokaryotic vectors include plasmids such as those capable of replication in E. coli (such as, for example, pBR322, ColEl, pSClOl, pACYC 184, TVX.
  • Such plasmids are, for example, disclosed by Maniatis, T., et al (In: Molecular Cloning. A Laboratory Manual Cold Spring Harbor Press, Cold Spring Harbor, NY (1982)).
  • Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids are disclosed by Gryczan, T. (In: The Molecular Biology of the Bacilli, Academic Press, NY (1982), pp. 307-329).
  • Suitable Streptomvces plasmids include pIJlOl (Kendall, K.J., et al, J.
  • Preferred eukaryotic plasmids include BPV, vaccinia, SV40, 2- micron circle, etc., or their derivatives.
  • Such plasmids are well known in the art (Botstein, D., et al, Miami Wntr. Sv p. 19:265-274 (1982); Broach, J.R., In: The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470 (1981): Broach, J.R., Cell 28:203-204 (1982); Bollon, D.P., et al, J. Clin. Hematol. Oncol 10:39-48 (1980); Maniatis. T.. In: Cell Biology: A Comprehensive Treatise. Vol. 3. Gene Expression, Academic Press, NY, pp. 563-608 (1980)).
  • the vector or DNA construct(s) may be introduced into an appropriate host cell by any of a variety of suitable means, including such biochemical means as transformation, transfection, conjugation, protoplast fusion, calcium phosphate-precipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran, and such mechanical means as electroporation, direct microinjection, and microprojectile (biolistic) bombardment (Johnston et al. Science
  • recipient cells After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells.
  • Expression of the cloned gene sequence(s) results in the production of the GAP-43 protein, or in the production of a fragment of this protein. This can take place in the transformed cells as such, or following the induction of these cells to differentiate (for example, by administration of bromo- deoxyuracil to neuroblastoma cells or the like).
  • the expressed protein may be isolated and purified in accordance with conventional conditions, such as extraction, precipitation, chromatography. affinity chromatography, electrophoresis. or the like.
  • the invention also relates to cloned genes which encode a fusion protein comprising GAP -43 or fragment thereof and a detectable enzyme such as beta-galactosidase. or any desired homologous or heterologous protein or peptide.
  • a detectable enzyme such as beta-galactosidase. or any desired homologous or heterologous protein or peptide.
  • the GAP-43, functional derivative thereof, or fusion protein comprising GAP-43 or fragment thereof and a detectable enzyme or desired protein or peptide may be isolated according to conventional methods known to those skilled in the art.
  • the cells may be collected by centrifugation, or with suitable buffers. Ivsed. and the protein isolated by column chromatography, for example, on DEAE-cellulose, phosphocellulose, polyribocytidylic acid-agarose, hydroxyapatite or by electrophoresis or immunoprecipitation.
  • the GAP -43 or functional derivative thereof, or fusion protein comprising GAP-43 and a detectable enzyme or desired protein or peptide may be isolated according to conventional methods known to those skilled in the art.
  • the cells may be collected by centrifugation, or with suitable buffers. Ivsed. and the protein isolated by column chromatography, for example, on DEAE-cellulose, phosphocellulose, polyribocytidylic acid-agarose, hydroxyapatite or by electrophor
  • anti-GAP-43' antibodies may be isolated by the use of anti-GAP-43' antibodies, or by the use of antibodies directed against the detectable enzyme or desired protein or peptide.
  • Such antibodies may be obtained by well-known methods, some of which as mentioned hereinafter.
  • the preparation of polyclonal rabbit anti-GAP-43 sera is disclosed in the examples portion of the present specification.
  • Another embodiment of the present invention comprises antibodies against the GAP-43 protein.
  • antibody (Ab) or “monoclonal antibody” (Mab) as used herein is meant to include intact molecules as well as fragments thereof (such as, for example. Fab and F(ab')-, fragments) which are capable of binding an antigen.
  • Fab and F(ab')-, fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al. J. Nucl Med. 24:316-325 (1983)).
  • the antibodies of the present invention may be prepared by any of a variety of methods.
  • cells expressing the GAP-43 protein, or a functional derivative thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies that are capable of binding GAP-43.
  • the antibodies of the present invention are monoclonal antibodies.
  • Such monoclonal antibodies can be prepared using hybridoma technology (Kohler et al. Nature 256:495 (1975); Kohler et al., Eur. J. Immunol 6:511 (1976): Kohler et al. Eur. -3 /,
  • the preferred monoclonal antibodies of this invention are those having the specificity of the monoclonal antibody designated MAb anti- GAP-43 (H5).
  • the invention comprises hybridoma strains which produce the monoclonal antibodies of the invention.
  • the preferred hybridoma cell line according to the invention is designated H-5, which produces monoclonal antibody designated MAb anti-GAP-43 (H5).
  • the H5 cell line has been deposited at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville. Maryland, USA 20851 on 21 December 1989. and given accession number ATCC HB 10316.
  • the antibodies of the present invention are well suited for use in standard immunodiagnostic assays known in the art, including such immunometric or "sandwich” assays as the forward sandwich, reverse sandwich, and simultaneous sandwich assays.
  • the antibodies of the present invention may be used in any number of combinations as may be determined by those of skill without undue experimentation to effect immunoassays of acceptable specificity, sensitivity, and accuracy for the GAP-43 antigen or equivalents thereof.
  • detecting it is intended to include determining the presence or absence of a substance or quantifying the amount of a substance.
  • the term thus refers to the use of the materials, compositions, and methods of the present invention for qualitative and quantitative determinations.
  • an anti-idiotypic antibody is an antibody which recognizes unique determinants present on the antibody produced by the clone of interest.
  • the anti-idiotypic antibody is prepared by immunizing an animal of the same strain used as the source of the monoclonal antibody with the monoclonal antibody of interest. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing antibody to these idiotypic determinants (anti-idiotypic antibody).
  • the anti-idiotypic antibody of the second animal which is specific for the monoclonal antibodies produced by a single clone, it is then possible to identify' other clones used for immunization. Idiotypic identity between the product of two clones demonstrates that the two clones are identical with respect to their recognition of the same epitopic determinants.
  • the anti-idiotypic antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti anti-idiotypic antibody which will be epitopically identical to the original MAb.
  • antibodies to the epitopic determinants of a monoclonal antibody it is possible to identify' other clones expressing antibodies of identical epitopic specificity.
  • idiotypic determinants are present in the hypervariable region yvhich binds to a given epitope.
  • the monoclonal antibodies of the present invention may be used to induce anti-idiotypic Abs in suitable animals, such as BALB/c mice. Spleen cells from these animals are used to produce anti- idiotypic hybridoma cell lines. Monoclonal anti-idiotypic Abs coupled to KLH are used as "immunogen" to immunize BALB/c mice. Sera from these mice will contain anti anti-idiotypic Abs that have the binding properties of the original Ab specific for the shared epitope. The anti- idiotypic MAbs thus have idiotopes structurally similar to the epitope being evaluated.
  • the hybrid cells may be cultivated both in vitro and in vivo. High in vivo production makes this the presently preferred method of culture. Briefly, cells from the individual hybrid strains are injected intraperitoneally into pristane-primed BALB/c mice to produce ascites fluid containing high concentrations of the desired monoclonal antibodies. Monoclonal antibodies of isotype IgM or IgG may be purified from cultured supernatants using column chromatography methods well known to those of skill in the art.
  • the antibodies of the present invention are particularly suited for use in immunoassays wherein they may be utilized in liquid phase or bound to a solid phase carrier.
  • the antibodies in these immunoassays can be detectably labeled in various ways.
  • labels and methods of labeling known in the art.
  • Examples of the types of labels which can be used in the present invention include, but are not limited to, enzymes, radioisotopes, fluorescent compounds, chemiluminescent compounds, bioluminescent compounds and metal chelates.
  • Those of ordinary skill in the art will know of other suitable labels for binding to antibodies, or will be able to ascertain the same by the use of routine experimentation.
  • the binding of these labels to antibodies can be accomplished using standard techniques commonly known to those of ordinary skill in the art.
  • antibodies of the present invention can be detectably labeled is by linking the antibody to an enzyme.
  • This enzyme when later exposed to its substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected as, for example, by spectrophotometric or fluorometric means.
  • enzymes which can be used to detectably label the antibodies of the present invention include malate dehydrogenase.
  • staphylococcal nuclease delta- V-steroid isomerase
  • yeast alcohol dehydrogenase alpha- glycerophosphate dehydrogenase
  • triose phosphate isomerase biotin-avidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease.
  • urease catalase, glucose- Vl-phosphate dehydrogenase, glucoamylase and acetylcholine esterase.
  • the presence of the detectably labeled antibodies of the present invention also can be detected by labeling the antibodies with a radioactive isotope which then can be determined by such means as the use of a gamma counter or a scintillation counter.
  • a radioactive isotope which are particularly useful for the purpose of the present invention are ' ⁇ , I. 32 P, 35 S, 14 C, 51 Cr, 36 CI, 57 Co, 58 Co, 59 Fe and 75 Se.
  • a fluorescently labeled antibody When a fluorescently labeled antibody is exposed to light of the proper wave length, its presence then can be detected due to the fluorescence of the dye.
  • cent labeling compounds are fluoroscein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin. o-phthaldehyde and fluorescamine.
  • the antibodies of the invention also can be detectably labeled using fluorescent emitting metals such as l ⁇ u. or others of the lanthanide series. These metals can be attached to the antibody molecule using such metal chelating groups as diethylenetriaminepentaacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA).
  • DTP A diethylenetriaminepentaacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • the antibodies of the present invention also can be detectably labeled by coupling them to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of the chemical reaction.
  • particularly useful chemiluminescent labeling compounds are luminal, isolumino theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound may be used to label the antibodies of the present invention.
  • Bioluminescence is a type of chemiluminescence found in biological systems in yvhich a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent antibody is determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for purposes of labeling include luciferin, luciferase and aequorin.
  • kits may comprise a carrier means being compartmentalized to receive in close confinement therewith one or more container means such as vials, tubes and the like, each of said container means comprising the separate elements of the assay to be used.
  • kits form are many, and include, for example, competitive and non-competitive assays.
  • Typical examples of assays which can utilize the antibodies of the invention are radioimmunoassays (RIA), enzyme immunoassays (EIA), enzyme-linked immunosorbent assays (ELISA), and immunometric, or sandwich, immunoassays.
  • RIA radioimmunoassays
  • EIA enzyme immunoassays
  • ELISA enzyme-linked immunosorbent assays
  • immunometric or sandwich, immunoassays.
  • immunometric assay or "sandwich immunoassay,” it is meant to include simultaneous sandwich, forward sandwich and reverse sandwich immunoassays. These terms are well understood by those skilled in the art. Those of skill will also appreciate that the antibodies of the present invention will be useful in other variations and forms of assays which are presently known or which may be developed in the future. These are intended to be included within the scope of the present invention.
  • nonrelevant antibodies of the same class or subclass (isotype) as those used in the assays can be used as "blockers.”
  • concentration of the "blockers” normally 1-100 microgs/microl is important, in order to maintain the proper sensitivity yet inhibit any unwanted interference by mutually occurring cross reactive proteins in human serum.
  • buffer system containing the "blockers” needs to be optimized.
  • Preferred buffers are those based on weak organic acids, such as imidazole, HEPPS, MOPS, TES, ADA, ACES.
  • HEPES HEPES, PIPES, TRIS, and the like, at physiological pH ranges.
  • Somewhat less preferred buffers are inorganic buffers such as phosphate, borate or carbonate.
  • known protease inhibitors should be added (normally at 0.01-10 microgs/ml) to the buffer which contains the "blockers.”
  • solid phase immunoadsorbents There are many solid phase immunoadsorbents which have been employed and which can be used in the present invention.
  • Well known immunoadsorbents include glass, polystyrene, polypropylene, dextran. nylon and other materials, in the form of tubes, beads, and microtiter plates formed from or coated with such materials, and the like.
  • the immobilized antibodies can be either covalently or physically bound to the solid phase immunoadsorbent, by techniques such as covalent bonding via an amide or ester linkage, or by adsorption.
  • suitable solid phase immunoadsorbents and methods for immobilizing antibodies thereon or will be able to ascertain such, using no more than routine experimentation.
  • labels such as radionuclides may be bound to the antibodies of the present invention either directly or by using an intermediary functional group.
  • An intermediary group which is often used to bind radioisotopes which exist as metallic cations to anti ⁇ bodies is diethylenetriaminepentaacetic acid (DTP A).
  • DTP A diethylenetriaminepentaacetic acid
  • Typical examples of metallic cations which are bound in this manner are: Tc, I, m IN. 131 I, 97 Ru, 67 Cu, 67 Ga and 68 Ga.
  • the antibodies of the invention can also be labeled with non-radioactive isotopes for purposes of diagnosis. Elements which are particularly useful in this manner are 157 Gd, 55 Mn, 162 Dy, 52 Cr and 56 Fe.
  • the antibodies of the present invention also may be used for immunotherapy in animals, including humans, having a disorder, such as a benign or cancerous neoplasia, which expresses the GAP-43 antigen with epitopes reactive with the antibodies of the present invention.
  • a disorder such as a benign or cancerous neoplasia, which expresses the GAP-43 antigen with epitopes reactive with the antibodies of the present invention.
  • the antibodies of the present invention When used for immunotherapy.
  • the antibodies of the present invention may be unlabeled or labeled with a therapeutic agent.
  • therapeutic agents which can be coupled to the antibodies of the invention for immunotherapy are drugs, radioisotopes, lectins and toxins.
  • Lectins are proteins, usually isolated from plant material, which bind to specific sugar moieties. Many lectins are also able to agglutinate cells and stimulate lymphocytes. Ricin is a toxic lectin which has been used immunotherapeutically. This use is accomplished by binding the alpha-peptide chain of ricin, which is responsible for toxicity, to the antibody molecule to enable site-specific delivery of the toxic defect. This is described, for example, in Vitetta et al. Science 238: 1098 (1987), and Pastan et al. Adv. Allergy 47: 641 (1986).
  • Toxins are poisonous substances produced by plants, animals or microorganisms that, in sufficient dose, are often lethal Diphtheria toxin, for example, is a protein produced by Corynebacterium diphtheria. This toxin consists of an alpha and a beta subunit which under proper conditions can be separated. The toxic alpha component can be bound to antibody and used for a site-specific delivery.
  • radioisotopes which can be bound to the antibodies of the present invention for use in immunotherapy are: - " 'Um, I, Y. 67 Cu, 217 Bi, 211 At, 212 Pb, 7 Sc and 109 Pd.
  • the expressed GAP -43 antigen normally is confined within the cell membrane. Accordingly, those of skill will recognize that in vivo diagnostic and therapeutic methods employing the antibodies of the invention may require some mechanism by which such antibodies can detect GAP-43 on the intracellular membrane.
  • One such method is to introduce the antibodies or fragments thereof into the cell ' s membrane or into the cell itself across the cell membrane. This may be accomplished, for example, by attaching the antibody to a ligand for yvhich the target cell contains receptor sites. The antibody can thus be transported into the cell membrane or across the cell membrane along with the ligand.
  • a carrier ligand will depend on several factors, as those of skill will appreciate. These include, for example, the kinetics of the ligand and its receptor, and of overall transport, which may include passive or active, with actively transported ligands preferred.
  • the means of attaching the antibody to the ligand also will vary within limits, and may be, for example, covalent or ionic, bearing in mind that such attachment should not unacceptably alter ligand-receptor affinity.
  • receptors suitable for such applications include the receptor for low density lipoprotein (LDL), which has been shown to contain all the information necessary for receptor endocytosis, Davis et al, J. Cell Biol. 107(63): Abstr. No. 3112 (1988), as well as known brain- specific receptors such as those for dopamine.
  • LDL low density lipoprotein
  • the ' ligand may itself be an antibody or fragment specific for the receptor, to which may be conjugated the antibody of the invention.
  • antibody fragments of the invention such as, for example. Fab or F(ab') 2 fragments
  • Single-chain antibodies may prove preferable for these and other reasons, as will be appreciated by those of skill.
  • an antibody When an antibody is to be transported into the cell's membrane or into the cell as described above, it will be preferred to diagnostically or therapeutically label the antibody in such a way that the label will be relatively more effective when the antibody is bound to its antigenic site on the GAP -43 protein. This may accomplished, for example, by employing a label which becomes active or detectable as a result of formation of the antigen-antibody complex. Alternatively, the antibody itself may be labeled in such a way that antigen-antibody complex formation induces a conformational change in the antibody to expose or more fully expose the previously unexposed or less fully exposed label All of the above criteria, and others, will be apparent to those of skill in carrying out these aspects of the invention.
  • liposomes having the antibodies of the present invention in their membrane to specifically deliver the antibodies to the target area.
  • These liposomes can be produced so that they contain, in addition to the antibody, such immunotherapy agents as drugs, radioisotopes, lectins and toxins, yvhich would be released at the target site.
  • the antibodies, and preferably, the GAP-43 encoding nucleotide sequences may be introduced into neural cells for diagnostic or therapeutic purposes is by the use of viral, including retroviral. vectors.
  • viral including retroviral. vectors.
  • suitable viruses may be mentioned the various herpes viruses.
  • Suitable retroviruses include human immunodeficiency virus (HIV).
  • HIV human immunodeficiency virus
  • Other suitable viruses and retroviruses are well knov n to those of skill.
  • an attenuated viral or retroviral strain For the purposes of the present invention, it may be preferred to employ an attenuated viral or retroviral strain.
  • retroviruses having attenuated cytopathicity such as HIV-2 ST (Kong et al. Science 240(4858): 1525 (1988)) or HIV-2 UC1 (Evans et al. Science 240(4858): 1523 (1988)), which enter neural cells by a CD4- dependent mechanism (Funke et al, J. Exp. Med. 165: 1230 (1987)).
  • the neurobiology of HIV infections is described, for example, in Johnson et al, FASEB J. 2(14): 2970 (1988).
  • CD4 is known to have a variant transcript in the human brain, with its highest content in forebrain (Maddon et al., Ce ⁇ 47: 333 (1986).
  • the DNA sequences which encode GAP -43, or a fragment thereof, may be used as DNA probes to isolate the corresponding antigen in humans according to the above-described methods for isolation of rat GAP-43 with labeled probes.
  • the human antigen genes may then be cloned and expressed in a host to give the human antigen. This human antigen may then be used in diagnostic assays for the corresponding autoantibody, and for therapeutic treatment of animals including humans.
  • the present inventors have undertaken experiments designed to elucidate the regulatory mechanisms which control expression of the GAP-43 gene. Modulation of GAP-43 expression offers a convenient and effective manner in which mammals, including humans, suffering from damaged, diseased or dysfunctioning central or peripheral nervous tissue, may be therapeutically treated. Further, methods of modulating structural remodeling in normal central or peripheral nervous tissue in mammals, including humans, according to the present invention, will be a significant aid to those of skill in further elucidating the mechanisms of neuron structure and function. The preclinical and clinical therapeutic use of the present invention in the treatment of neurological disease or disorders will be best accomplished by those of skill, employing accepted principles of diagnosis and treatment. Such principles are known in the art, and are set forth, for example, in Petersdorf, R.G.
  • the antigens, antibodies and compositions of the present invention, or their functional derivatives, are well suited for the preparation of pharmaceutical compositions.
  • the pharmaceutical compositions of the invention may be administered to any animal yvhich may experience the beneficial effects of the compounds of the invention. Foremost among such animals are humans, although the invention is not intended to be so limited.
  • compositions of the present invention may be administered by any means that achieve their intended purpose.
  • administration may be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal or buccal routes.
  • administration may be by the oral route.
  • the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the new pharmaceutical preparations may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the preparations particularly those preparations which can be administered orally and which can be used for the preferred type of administration, such as tablets, dragees. and capsules, and also preparations which can be administered rectally, such as suppositories, as well as suitable solutions for administration by injection or orally, contain from about 0.001 to about 99 percent, preferably from about 0.01 to about 95 percent of active compound(s), together with the excipient.
  • the dose ranges for the administration of the compositions of the present invention are those large enough to produce the desired effect, whereby, for example, the neoplastic tissue is reduced or eliminated or ameliorated.
  • the doses should not be so large as to cause adverse side effects, such as unwanted cross reactions anaphalactic reactions and the like.
  • the dosage will vary with the age, condition, sex and extent of the disease in the patient. Counterindication, if any, immune tolerance and other variables will also affect the proper dosage.
  • the anti ⁇ bodies can be administered parenterally by injection or by gradual profusion over time.
  • the antibodies of the present invention also can be administered intravenously, intraparenterally. intramuscularly or subcutaneously.
  • compositions of the present invention are manufactured in a manner which is itself known, for example, by means of conventional mixing, granulating, dragee-making, dissolving, or lyophilizing processes.
  • pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding the resulting mixture and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee cores.
  • suitable excipients are, in particular, fillers such as saccharides, for example lactose or sucrose, mannitol or sorbitol.
  • cellulose preparations and/or calcium phosphates for example tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose. and/or polyvinyl pyrrolidone.
  • disintegrating agents may be added such as the above-mentioned starches and also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.
  • Dragee cores are provided with suitable coatings which, if desired, are resistant to gastric juices.
  • suitable coatings which, if desired, are resistant to gastric juices.
  • concentrated saccharide solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone. polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • suitable cellulose preparations such as acetyl- cellulose phthalate or hydroxypropymethyl-cellulose phthalate. are used.
  • Dye stuffs or pigments may be added to the tablets or dragee coatings, for example, for identification or in order to characterize combinations of active compound doses.
  • Other pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol.
  • the push-fit capsules can contain the active compounds in the form of granules which mav be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds are preferably dissolved or sus ⁇ pended in suitable liquids, such as fatty oils, or liquid paraffin.
  • stabilizers may be added.
  • Possible pharmaceutical preparations which can be used rectally include, for example, suppositories, which consist of a combination of one or more of the active compounds with a suppository base.
  • Suitable suppository bases are, for example, natural or synthetic triglycerides, or paraffin hydrocarbons.
  • gelatin rectal capsules which consist of a combination of the active compounds with a base.
  • Possible base materials include, for example, liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.
  • Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts.
  • suspensions of the active compounds as appropriate oily injection suspensions may be administered.
  • Suitable lipo- philic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran.
  • the suspension may also contain stabilizers.
  • the GAP-43 antigen of the present invention is unique to neuronal cells, and thus provides a convenient and useful marker. Accordingly, antibodies directed against GAP-43 may be used in various techniques well known to those of skill, to identify neuronal cells. Moreover, the antibodies of the present invention will allow detection, determination and therapeutic treatment of neoplasias and other disorders of neuronal origin, and, as such, offer a convenient and useful diagnostic and therapeutic method in vivo, in vitro or in situ, for preclinical and clinical evaluation and treatment of cancer and other disorders in animals including humans.
  • the antigen of the invention may be isolated in substantially pure form employing the antibodies of the present invention.
  • an embodiment of the present invention provides for substantially pure antigen GAP-43. said antigen characterized in that it is recognized by and binds to the antibodies of the present invention.
  • the present invention provides a method of isolating or purifying the GAP-43 antigen, by forming a complex of said antigen with one or more antibodies directed against GAP -43.
  • the substantially pure antigen GAP -43 of the present invention may in turn be used to detect or measure antibody to GAP-43 in a sample, such as cerebrospinal fluid, serum or urine.
  • a sample such as cerebrospinal fluid, serum or urine.
  • one embodi ⁇ ment of the present invention comprises a method of detecting the presence or amount of antibody to GAP -43 antigen in a sample, compris ⁇ ing contacting said sample containing said antibody to GAP-43 antigen with detectably labeled GAP-43, and detecting said label.
  • immunoreactive fractions and immunoreactive analogues of GAP-43 also may be used.
  • immunoreactive fraction is intended any portion of the GAP-43 antigen which demonstrates an equivalent immune response to an antibody directed against GAP -43.
  • immunoreactive analogue is intended a protein which differs from the GAP-43 protein by one or more amino acids, but which demons ⁇ trates an equivalent immunoresponse to an antibody of the invention.
  • the GAP -43 protein contains a novel membrane-targeting peptide domain which directs the GAP-43 protein to the cell membrane, and especially to the region of the growth cone of neuronal cells.
  • the structure of this membrane-targeting domain has been determined, and it has been shown that the peptide is effective in directing normally cytosolic proteins (which are not normally membrane-associated), to the cell membrane.
  • compositions and methods of this aspect of the invention it is possible, inter alia, to direct any desired protein to the cell membrane, including proteins which are not normally membrane- associated. Further, the compositions and methods of this aspect of the invention are of obvious utility in the therapeutic treatment of neurological damage and disorders in vitro, in vivo, and in situ, in animals. Those of skill will appreciate that the preceeding description of diagnostic and therapeutic methods is equally applicable to this embodiment of the invention. Further, it will be evident that the membrane-targeting peptide of the present invention will be of use in directing any desired protein or peptide to cell membranes, and will thus be of diagnostic and therapeutic utility in non-neurological indications as well. Examples of such indications include, but are not limited to, any applications wherein the membranes of cells may play an important role, such as immunological indications.
  • a cDNA library was generated from RNA of rat dorsal root ganglia from embryonic day 17 and cloned into the lgtll expression vector (Huynh et al, in "DNA Cloning A Practical Approach,” D.M. Glover. Ed. (IRL Press, Washington, D.C.. 1985) pp. 49-78). Three presumptive GAP-43 clones were identified with the antibody••• to GAP-43 described bv «-
  • GAP43-2 selected by hybridization a messenger RNA (mRNA) that directed the translation of a polypeptide that migrated in SDS- polyacrylamide gels with the expected mobility of native GAP-43, that is. a molecular size of about 43 kD.
  • mRNA messenger RNA
  • This in vitro translation product was selectively immunoprecipitated by antibody to GAP-43. The specificity of the immunoprecipitation was demonstrated by competition with unlabeled. purified GAP -43.
  • the complete nucleotide sequence of GAP43-2 and the predicted amino acid sequence are shoyvn in Fig. 2.
  • the reading frame includes the peptide fragment that was sequenced and is in the same reading frame as the b-galactosidase gene of lgtll.
  • a cDNA for rat GAP-43 was obtained independently by J.H.P. Skene and his colleagues (G. Basi. R. Jacobson. I. Virag, J.H.P. Skene, personal communication). Copies of the sequences were exchanged.
  • the predicted amino acid sequence of the present invention agrees perfectly with that provided by J.H.P.
  • the methionine identified as the start of the open reading frame is the first methionine after the in-frame stop codon (TAA) at nucleotide position 13 and is surrounded by eight of the nine nucleotide consensus sequences suggested by Kozak, Cell 44:283 (1986) to be the most favor ⁇ able context to initiate eukaryotic translation. This suggests that it is the first residue of the GAP -43 coding region. However, the information is insufficient to make this assignment unequivocally, and, therefore, the second methionine (amino acid 5) might play this role.
  • TAA in-frame stop codon
  • the predicted composition of GAP -43 is highly polar, without evident transmembrane domains or potential N-linked glycosylation sites. This composition is compatible with the observations that GAP-43 is membrane-associated but inaccessible to antibody recognition in the absence of membrane permeabihzation (Meiri et al, PNAS USA 83:3537 (1986)); thus it may be associated with the inner face of the membrane.
  • the predicted molecular size of the GAP-43 protein from the open reading frame is 24 kD, which is less than the 43 kD originally observed by Skene and Willard as the apparent molecular size of the molecule in SDS-polyacrylamide gels (Skene and Williard. J. Cell Biol 89:86 (1981). ibid., p. 96).
  • the molecular size has been uncertain because the apparent molecular size of GAP-43 depends on polyacrylamide concentration (Jacobson et al, J. Neurosci. 6:1843 (1986)), suggesting that this protein falls in the category of proteins that migrate anomalously on SDS-poly ⁇ acrylamide gels (Banker et al, J. Biol Chem.
  • GAP-43 RNA was synthesized from the cDNA in an in vitro transcription system yvith the use of the bacteriophage SP6 promoter, by the method of Melton et al. Nucleic Acids Res. 12:7035 (1984).
  • 800-base RNA was generated by transcribing the cDNA cut at the Sau3A site, 65 bases 3' of the end of the predicted open reading frame (Fig. 2). and a 1100-base RNA by truncating at the Hindlll site in the polylinker region at the 3' end of cDNA. Both the 800-base RNA and the 1100-base
  • RNA directed the synthesis of a polypeptide with an apparent molecular size of 40 kD when translated in vitro with reticulocyte lysate and analyzed on a 15% SDS-polyacrylamide gel.
  • the 40-kD translation product in both cases yvas immunoprecipitated yvith the antibody to
  • GAP-43 synthesized in vitro from newborn rat brain RNA comigrated with these translation products.
  • PC12 cells in which neurite outgrowth was promoted by nerve growth factor (NGF) and to a lesser extent by adenosine 3'.5 " -monophosphate
  • NGF nerve growth factor
  • cAMP cAMP
  • GAP-43 was expressed in a neural-specific manner (Fig. 4). At all ages, the major hybridizing band of about 1500 nucleotides is visible only in the neuronal tissues. The faint, large- molecular-size bands may correspond to unspliced precursor molecules, since the present inventors have discovered that the genomic GAP -43 gene contains intronic sequences.
  • GAP-43 mRNA in neuronal tissue is probably of neural rather than glial origin, since GAP-43 is localized in neurons (Meiri et al, PNAS USA 83:3537 (1986)) and no GAP-43 RNA was detected in the glioma cell line C6.
  • GAP-43 mRNA In neural tissues, the amount of GAP-43 mRNA varies with developmental stage. Peak concentrations occur in the perinatal period, with some delay in the central nervous system relative to the peripheral nervous system. The timing of expression accords well with periods of axon growth (Jacobson, Developmental Ne ⁇ ropathology (Plenum. New York, 1978)). However, the significant amount of GAP-43 RNA in adult neural tissues is in agreement with observations that GAP-43 protein persists in adult rat cortex, albeit in significantly lower amounts than during the perinatal period (Jacobson et al, J. Neurosci. 6:1843 (1986)). The persistence of GAP-43 expression suggests an ongoing role in the adult nervous system.
  • B-50 and Fl phosphoproteins electrophoretically and antigenically indistinguishable from GAP -43. have been assessed in adult neuronal tissue. These proteins serve as substrates for a protein kinase C-like enzyme, and their phosphorylation is regulated by neuropep tides, neurotransmitters, and during the course of long-term potentiation (Jacobson et al, J. Neurosci. 6:1843 (1986); Aloyo et al, J. Neurochem. 41:649 (1983): Zwiers et al, Progr. Brain Res. 56:405 (1982)). It is not known whether GAP -43 regulation in the adult also occurs by alterations in gene expression.
  • GAP -43 One model for the function of GAP -43 in the mature animal yvould include an ongoing role in synaptic turnover (Cotman et al. Science 225:1287 (1984)) and in other "plastic" changes of the nervous system, such as learning, that are accompanied by structural growth at the nerve terminal (Bailey and Chen, Science 220:91 (1983)).
  • Human brain tissue was harvested fresh at the time of autopsy and within 10 hours of death. Sections of brain no larger than 2x2x0.5 cm, obtained from specific regions yvere snap-frozen in isopentane (2-methyl butane) cooled with dry ice and then stored at -90°C. These specimens were used for in situ hybridization and immunocytochemistry. In addition, a small portion of tissue from the same regions, obtained fresh or from the frozen sample, was used for Northern blot analysis. Routine histopathology was performed on formalin-fixed, paraffin-embedded tissue immediately adjacent to the frozen blocks. cDNA cloning
  • the human GAP-43 cDNA was isolated from cDNA libraries of brainstem of a 1 day old and cerebellum of a 7 year old (both libraries were from American Type Culture Collection). These libraries yvere
  • Cryostat sections of human brain tissue were fixed in 4% paraformaldehyde, treated with 0.3% Triton X-100 followed by 1 mg/ml proteinase K, acetylated, and pre-hybridized in 509 formamide/2x SSC.
  • the tissue sections were then washed in 2x SSC with 10 mM dithiothreitol initially containing 50% formamide. then 50% formamide plus 0.1% Triton-X 100.
  • RNAase A Single stranded RNA was removed by treatment with 50 mg/ml RNAase A. The sections were further washed in 2x SSC with ImM DTT for 2 hours, then dehydrated through graded alcohols containing 0.3 M ammonium acetate. The radioactive signal was detected, using NTB2 Kodak emulsion. Emulsion-coated slides were counterstained with hema- toxylin.
  • Human GAP-43 is homologous to a mouse calmodulin binding protein As described in Example I, the cDNA for rat GAP-43 was cloned and used as a probe to identify related cDNA clones from human brainstem and cerebellum libraries. Overlapping clones were obtained from each of the libraries, which were identical in the overlapping regions. The sequence of the longest clone (Cla from the cerebellum library) is presented in Figure 5A, and a comparison with rat GAP-43 shown in Figure 5B. The identity of GAP -43 yvith that of a neural specific mouse calmodulin-binding protein, termed P-57. was described by Cimler et al. J. Biol. Chem.
  • GAP-43 expression persists in discrete regions of the adult human brain
  • RNA degradation brain tissue was obtained from patients with a postmortem interval of less than 10 hours. Adjacent sections were examined histopathologically. In two infants, 8 days and 1 month old, GAP -43 was uniformly and robustly expressed throughout the brain as assessed by Northern blots. The regions examined included the cerebellum, temporal cortex, temporal association cortex, frontal cortex, orbital frontal region (Area 11), hippocampus, visual cortex (Area 17/18), and spinal cord, some examples of which are shown in Figure 6. In contrast to the brain, levels in the spinal cord were low at these ages, which may be related to earlier maturation of this region (Anand and Hickey, New Engl. J. Med. 317:1321 (1987)).
  • GAP-43 was not expressed in any non-neuronal tissues examined either in the newborn or adult (including kidney, lung, liver, and adrenal). In the normal adult brain, GAP-43 expression varied markedly among different regions. For example, in three brains, levels comparable to the neonate were found in Broadman's Area 11 (orbital frontal gyrus) and much lower levels expressed in the visual cortex (Area 17/18). Levels were consistently low- in the hippocampus. The latter is of interest because the adult rat hippocampus is enriched in GAP-43. A similar distribution has been reported recently by Neve et al, Molec. Brain Res. 2:177 (1987).
  • the infarct was in the visual cortex (Area 17), and in the other, parietal lobe (Area 3,1,2,5).
  • the tissue utilized for Northern analysis and in situ hybridization included both the infarcted tissue and surrounding normal brain from the same Broadman's areas.
  • Figure 7A shows that after a stroke in Area 17, GAP-43 expression is increased to levels comparable to Area 11, the region normally most enriched in GAP-43 in the adult.
  • Figure 7B shows that GAP-43 levels in Area 3,1,2,5 from two normal brains were low (lanes 1 and 2) compared to another patient with a small stroke in that location (lane 3).
  • GAP -43 is neuron-restricted in its expression, and since neurons were absent in the infarcted regions ( Figure 8A1. B). most likely the heightened GAP-43 expression derived from the mor ⁇ phologically uninjured neurons.
  • in situ hybridization was used to study the distribution of GAP -43 expression in the region of infarction.
  • the detailed cellular anatomy of GAP -43 expres ⁇ sion is presented in Example III. Throughout most regions of the adult cerebral cortex, including the visual cortex (Area 17), as might be predicted from the Northern analysis, only a few scattered cells expressed GAP -43 (Figure 8C).
  • Growth cones are nerve terminal structures shared by developing and regenerating nerves (e.g., Ramon y Cajal, "Degeneration and
  • GAP -43 is one of the rapidly transported proteins which is notable for pronounced enrichment in axonal transport in developing and regenerating nerves.
  • the failure of mammalian CNS neurons to regenerate has been linked to the low and uninducible levels of GAP-43 in adult brain (Skene, Cell 37:697 (1984)).
  • GAP-43 is highly conserved betv een rat and human and clearly identical to a mouse protein recently identified as a calmodulin-binding protein which has the unusual property of releasing calmodulin when ambient calcium increases (Andreasen etal. Biochemistry 22:4615 (1983): Cimler et al. J. Biol. Chem. 260:10784 (1985): Alexander et al. J. Biol. Chem. 292:6108 (1987)).
  • one notion for its role in the growth cone might be that it regulates calmodulin activity, and that it does so by releasing it in focal cellular domains.
  • GAP-43 expression is highly regulated during development. In general, the highest levels correlate well with the periods of peak axonal elongation. However, its high level of expression in particular regions of the mature brain suggests that GAP-43 has an ongoing role in some adult neurons.
  • GAP-43 expression denotes cells actively engaged in remodeling their structure, especially at nerve terminals. Evidence for this is that in the rat the adult neurons which express GAP- 43 include most prominently hippocampal neurons and mitral cells of the olfactory bulb, neurons which do in fact remodel their terminals in the adult.
  • the human hippocampus expresses GAP -43 only at low levels, suggesting that, if GAP -43 is indeed an indicator of such structural remodeling, different regions of the human brain have retained this function.
  • GAP -43 The other function proposed for GAP -43 is learning, because its phosphorylation state changes in the yvake of long-term potentiation (Nelson and Routtenberg, Exp. Neurol 89:213 (1985)).
  • structural remodeling may be a facet of long-term learning (Chang and Greenough, Brain Res. 309:35 (1984): Goelet et al. Nature 322:419 (1983)), and growth of nerve terminal areas has been documented to accompany long- term learning in Aplysia (Bailey and Chan, Science 220:91 (1983)).
  • neurons of the adult human brain that use structural plasticity for long-term learning are those that express GAP-43.
  • NMDA N-methyl-D-aspartate
  • the inventors identify the cell populations that express GAP -43, and demonstrate that in the adult brain the widespread GAP-43- immunoreactive neurites emanate from a relatively small population of neurons, most of which are regionally restricted.
  • Tissues used for in situ hybridization and immunocytochemistry were obtained fresh and snap frozen in 2-methyl butane cooled with dry ice. Cryostat sections were fixed yvith the appropriate fixative immediately prior to use. Brain and spinal cord from embryonic (E) (days 12, 15, 18, and 20), postnatally developing (P) (days 1, 7 and 14), and adult rats were studied simultaneously. The tissue yvas fixed in 4% paraformaldehyde. treated with 0.3% Triton X-100 folloyved by 1 mg/ml proteinase K, acetylated, and pre-hybridized in 50% formamide/2x standard saline citrate (SSC). The probe was 1121 bases of GAP-43 antisense RNA. as described above.
  • Hybridization using 2 x 106 cpm per slide of ' ""S-labeled antisense or sense riboprobe was performed in a humidified chamber for 5 hours at 50°C.
  • the tissue sections yvere then washed in 2x.SSC with 10 mM dithiothreitol initially containing 50% formamide, then 50% formamide plus 0.1% Triton-X 100.
  • Single stranded RNA yvas removed by treatment with 50 mg/ml RNAase A.
  • the sections were further washed in 2x SSC with 1 mM DTT for 2 hours, then dehydrated through graded alcohols containing 0.3 M ammonium acetate.
  • the radioactive signal was detected using NTB2 Kodak emulsion.
  • Emulsion coated slides were counterstained with hematoxylin and eosin.
  • GAP-43 protein was demonstrated in CNS tissue by immunohistochemical staining using the avidin-biotin horseradish peroxidase complex method (Hsu et al, J. Histochem. Cytochem. 29:577-580 (1981)) with 3.-3' diamino- benzidine as the chromagen and hematoxylin as a counterstain. Specificity of labeling was confirmed by pre-incubation of the primary antibody with native GAP-43 protein purified from neonatal rat brain.
  • GAP -43 expression during development assessed by in situ hybridization At embryonic days 12 and 15, GAP -43 mRNA expression in the CNS was low, but neurons of the dorsal root ganglia exhibited intense labeling, corresponding with their peak period of axonal growth.
  • GAP-43 levels were uniformly and strikingly high throughout the brain.
  • high-level expression persisted, but in contrast to the diffuse labeling observed at E20 and PI. in brains of P7 rats, discrete labeling over individual neurons could be appreciated due to expansion of the neuropil and growth of glial elements. As best as could be determined, all neurons were labeled at this age.
  • GAP-43 mRNA expression was diminished such that, the overall signal intensity was lower, and only 50-75% of cortical neurons were labeled.
  • GAP-43 was expressed in relatively few neurons such that throughout most of the cerebral cortex, only scattered cells were labeled, and the intensity of labeling was markedly reduced even compared to P14 brains.
  • the entorhinal cortex had moderately high densities of GAP-43-expressing neurons. While dense focal labeling of neurons was still present in the spinal cord, brainstem, and cerebellum of P14 rats, in adults GAP-43-expressing neurons were either absent, or present in very low densities in these regions. Hov/ever, in the adult, intense labeling of most neurons persisted in tyvo areas: the hippocampus and olfactory bulb.
  • the pattern of labeling in the hippocampus indicated that neurons throughout the dentate gyrus, CA1, and CA3 expressed GAP-43 mRNA. In the olfactory bulb, it was primarily the mitral cell region that contained high levels of GAP -43 mRNA.
  • GAP-43 is also in cell bodies (Alexander et al. J. Biol. Chem. 262:6108-6113 (1987)). Immunostaining with this antibody permitted localization of GAP-43-expressing cells, and comparison with the in situ hybridization data. The regional distribution and density of neurons containing immunoreactive GAP-43 mirrored the developmental pattern observed for its mRNA by in situ hybridization. GAP -43 immunolabehng was not detected in E12 embryos, and was present in only small amounts (manifested by faint immunohistochemical staining) in the E15 CNS.
  • GAP-43 immunoreactivity was more conspicuous, and at E20 it was detected at high levels in both somata and neurites of neural cells. This degree of immunostaining for GAP-43 protein persisted through P7. Subsequently, GAP -43 immunoreactivity diminished in most areas, thereby leaving a more restricted distribution of GAP-43 immunoreactivity. In adults, widespread but faint neuritic labeling was evident throughout the CNS. Neuronal perikarya were labeled heavily in the same regions identified by in situ hybridization as expressing high levels of GAP-43.
  • GAP-43 is expressed in all CNS neurons during the perinatal period. As development proceeds, its anatomical distribution becomes progressively restricted, such that, in the adult, GAP-43-containing neurons are inhomogeneously distributed, with the highest level expression largely limited to two discrete regions: the hippocampus and olfactory bulb.
  • the antibody generated by the present inventors to the ⁇ - gaIactosidase-GAP-43 fusion protein permitted intense labeling of neuronal perikarya. This difference from prior reports may be due to the chimeric nature of the antigen, which perhaps exposes some different epitopes to different degrees. Alternatively, the difference may reflect omission of aldehyde fixation, which yvas noted by the presnet inventors to diminish perikaryal labeling.
  • the procedure of the present invention allowed the present inventors to document that the site of GAP-43 gene expression mirrored that of the GAP -43 immunoreactivity.
  • the distribution of GAP-43 in the CNS differs between rats and humans. In the adult human brain, high levels persist in associative cortical regions more than in the hippocampus (Neve et al, Proc. Natl Acad. Sci. USA 85:3638-3642 (1988)), whereas in adult rats the highest levels of GAP-43 expression are in the hippocampus, olfactory bulb, and entorhinal cortex. The significance of this finding is unclear, but it may be related to species differences with respect to regional retention of neuronal plasticity. I t remains to be determined whether the subsets of CNS neurons which persistently express high levels of GAP-43 in the adult share biological features.
  • GAP-43 is expressed in cells involved in structural remodeling of synapses. Growth cones persist in certain regions of the adult brain (Sotelo et al. Lab. Invest. 25:653-671 (1971 . )) and direct visualization reveals ongoing synaptic rearrangements of single cells, at least in the peripheral nervous system (Purves et al, Nature 315:404-406 (1985)). In fact, there is evidence that such neuronal remodeling is integral to long-term learning (Chang et al, Brain Res. 309:35-46 (1984): Goelet et al. Nature 322:419-422 (1986): Horn et al.. J. Neurosci. 5:3161-3168 (1985)) and sexually dimorphic behavior (Kurz et al, Science 232:395-398 (1986)).
  • GAP-43 expression is high in both the olfactory nerve and its target, the mitral cells of the olfactory bulb. Since olfactory neurons continue a cycle of death and replacement throughout life (Graziadei et al. In: M. Jacobson (Ed.). Handbook of Sensory Physiology. Vol IX. Development of Sensory Systems. Berlin. Springer- Verlag pp. 55-83 (1978)), these synapses must be continuously changing.
  • Entorhinal neurons which express GAP-43 in the adult, can expand their peripheral fields by sprouting into denervated zones, although it is not clear that they remodel in the absence of injury'.
  • circuitry of the hippocampus is functionally plastic (Benowitz et al, J. Neurosci. 8:339-352 (1988); Cotman et al, Psvchol 33:371-401 (1982): Lee et al. In: G.A. Kerkut and H.V. Wheal (Eds). Electrophvsiology of Isolated Mammalian CNS Preparations. Academic, New York. 1981, pp. 189-212: Lee et al, J. Neurophysiol. 41:247-258.
  • the phenotype of an individual neuron depends upon its microenvironment.
  • Such "plasticity” is manifest, for example, in the choice of cell fate by precursor cells of the sympathoadrenal system, which assume a neuronal phenotype under the influence of nerve growth factor (NGF) or an endocrine, chromaffin cell phenotype in the presence of corticosteroids.
  • NGF nerve growth factor
  • chromaffin cell phenotype in the presence of corticosteroids.
  • Neurons additionally remodel their connections, a phenomenon termed synaptic plasticity, during normal development and in response to synaptic use (Easter et al. Science 230:507-511 (1985)).
  • Corticosteroids are necessary for normal development of the mammalian nervous system, influencing cell fate and neuronal structure and integrity (Doupe et al, J. Neurosci. 5:2119-2142 (1985); Doupe et al, J. Neurosci. 5:2143-2160 (1985); Anderson and Axel. Ce]i 47:1079-1090 (1986); Bohn and Lauder, Dev. Neurosci. 1:250-266 (1978): Scheff et al, Expt. Neurology 68:195-201 (1980); Scheff and Cotman, Expt. Neurology 76:644-654 (1982): Sapolsky et al, J. Neurosci. 5:1222-1227 (1985)).
  • neural crest lineage including small intensely fluorescent (SIF) cells and adrenal medullary cells, may exhibit either neuronal or chromaffin phenotypes.
  • SIF small intensely fluorescent
  • adrenal medullary cells may exhibit either neuronal or chromaffin phenotypes.
  • Corticosteroids cause them to assume chromaffin characteristics.
  • NGF neuronal growth factor
  • Cells of the clonal line PC12 which is derived from a rat adrenal medullary pheochromocytoma (Greene and Tischler, Proc. Nat'l Acad. Sci. USA 73:2424-2428 (1986); Greene and Tischler, in Advances in Cellular Neurobiology, Vol 3, pp. 373-413, Academic Press, New York (1982)), display a similar bipotential fate, becoming more neuronal with NGF and retaining chromaffin characteristics with exposure to corticosteroids.
  • corticosteroids are powerful negative regulators of GAP-43 gene expression in both PC12 cells and cultured sympathetic neurons. Further, it has been discovered that corticosteroids inhibit the stimulatory effect of NGF on GAP-43 expression.
  • Enzymes were purchased from Boehringer Mannheim, Neyv England Biolabs, or Bethesda Research Laboratories, and used as specified by the supplier. Tissue culture products yvere bought from Gibco. Radiochemicals were purchased from New England Nuclear-Du Pont. Agarose and cesium chloride were purchased from Bethesda Research Laboratories. Timed pregnant Sprague-Dawley rats were purchased from Charles River Rat. Steroids were bought from Sigma and NGF from Collaborative Research (2.5s form). All other chemicals were of the highest grade available.
  • PC12 cells were grown in Dulbecco ' s modified Eagles medium (DMEM) with 5% heat-inactivated horse serum and 109c fetal calf serum. Cells were used routinely when at approximately 20% confluence. Cortisol levels, determined by RIA, were 3 nM or less in the serum- containing medium. Cells were grown in a humidified incubator with 5% carbon dioxide at 37°C. Dissociated neurons from embryonic day 20 rat superior cervical ganglia were cultured in Ham's F12 medium supplemented with NGF (50 ng/ml), 0.6% glucose and 10% fetal calf serum. For steroid experiments, the compounds were usually dissolved in 95% ethanol. Controls performed with ethanol as a vehicle revealed no change in the abundance of GAP-43 or GAPDH RNA.
  • DMEM Dulbecco ' s modified Eagles medium
  • RNA was prepared from cultured cells by the guanidine isothiocyanate method (Chirgwin et al. Biochemistry 18:5294-5299 (1979)). Twenty micrograms of total RNA from each culture were electrophoresed through a 1.2% agarose gel containing 2.2 M formaldehyde, transferred by capillarity to Nytran (Schleicher and Schuell), and the nucleic acid immobilized by heat fixation. Prehybridization was done for at least 1 hour in a hybridization solution containing 50% formamide, 5xSSC (lxSSC: 150 mM sodium chloride, 15 mM sodium citrate, pH 7.0). lx Denhardt's solution, 1% sodium dodecyl- sulfate (SDS) and 100 mg/ml denatured salmon sperm DNA at 42°C.
  • 5xSSC lxSSC: 150 mM sodium chloride, 15 mM sodium citrate, pH 7.0
  • SDS sodium dodecyl-
  • Hybridization was performed for 10-12 hours at 42°C in the same solution, containing 1x105 cpm/ml of 32 P-labeled DNA probe prepared by random hexanucle ⁇ tide priming using the Klenoyv fragment of DNA polymerase I (Feinberg and Vogelstein, Anal. Biochem. 132:6-12 (1984)).
  • the probes yvere made from cloned cDNAs for GAP-43 as described herein or for GAPDH (Piechaczyk et al, Nucl. Acids Res. 12:6951-6963 (1984)). Blots were washed with 2xSSC at 65 °C tv ice.
  • PC12 cultures were split 48-60 hours prior to each experiment.
  • Cells were either left untreated or treated yvith either NGF (50 ng/ml) or dexamethasone (1 mM) for 6 hours.
  • NGF 50 ng/ml
  • dexamethasone 1 mM
  • Approximately ten million nuclei were prepared from each by the method of Greenberg (Greenberg et al. J. Biol Chem. 260:14101-14110 (1985)) yvith the following modifications. Lysis was in 10 mM sodium chloride, 10 mM Tris, pH 7.4, 3 mM calcium chloride and 200 units/ml RNAsin (Promega Biotec).
  • Nuclei were resuspended after washing with lysis buffer in 50 mM Tris, pH 8.3, 40% glycerol, 5 mM magnesium chloride, 0.1 mM ethylenediamine tetraacetic acid (EDTA), 2mM dithiothreitol and 200 units/ml RNAsin. Nuclei were counted and stored ' in liquid nitrogen at a concentration of 50 million/ml
  • Labeling of nascent chains in thawed nuclei was performed by adding to the suspended nuclei an equal volume of buffer containing 10 mM Tris, pH 8.0, 5 mM magnesium chloride. 0.3 M potassium chloride, and 10 mM each of adenosine, cytidine and guanosine nucleotide triphosphates. Three hundred mCi of ( 32 P) uridine triphosphate were then added (3000 Ci/mmol), and the nuclei labeled for 30 minutes at 30°C.
  • Nuclei were then digested with 100 units RQ1 DNAase (Promega Biotec) added in 600 ml of a buffer containing 60 mM Tris, pH 7.5, 15 M sodium chloride, 10 mM magnesium chloride, and 200 units/ml RNAsin for 45 minutes at 37 °C.
  • the labeled RNA was then digested with proteinase K (Boehringer Mannheim) as described (Greenberg et al, J. Biol. Chem. 260:14101-14110 (1985)).
  • the nucleic acids were ethanol-precipitated in the presence of sodium acetate.
  • the recovered labeled nucleic acid, which still contained DNA was subjected to another cycle of RQ1 DNAase (50 units enzyme in 250 ml buffer containing 50 mM Tris, pH 7.5, 10 mM sodium chloride, 7.5 mM magnesium chloride and 200 units RNAsin/ml) for 45 minutes at 37 °C, and then proteinase K (by adding 100 ml of buffer containing 5% SDS, 0.5M Tris, pH 7.4, 125 mM EDTA and 0.2 mg/ml proteinase K) for 30 minutes at 42°C.
  • RQ1 DNAase 50 units enzyme in 250 ml buffer containing 50 mM Tris, pH 7.5, 10 mM sodium chloride, 7.5 mM magnesium chloride and 200 units RNAsin/ml
  • proteinase K by adding 100 ml of buffer containing 5% SDS, 0.5
  • Plasmids containing cloned cDNAs for tyrosine hydroxylase (Lewis et al. J. Biol Chem. 285:14632-14637 (1983)), glyceraldehyde-3- ⁇ hosphate dehydrogenase (GAPDH), pBR322, and an 8 kilobase genomic fragment of the GAP-43 genei were linearized with appropriate restriction enzymes, phenol extracted, ethanol precipitated and recovered by centrifugation. DNA (250 mg/ml) was denatured by alkali (0.5 N sodium hydroxide) and neutralized by the addition of ten volumes of 1 M ammonium acetate.
  • alkali 0.5 N sodium hydroxide
  • Hybridization was performed in the same buffer containing labeled RNA at specific activities ranging from 1-3 x 106 cpm/ml for 4 days at 45°C. Washing and RNAase treatment yvere performed as described (Greenberg et al, J. Biol. Chem. 260:14101-14110 (1985)). Filters done in duplicate were counted after drying in a scintillation counter. Data are expressed as parts per million hybridized after subtracting background from vector containing filters.
  • NGF and glucocorticoids upon GAP -43 mRNA accumulation were measured by RNA blotting.
  • NGF addition resulted in a marked increase over the basal level, whereas dexamethasone caused a prominent diminution in GAP-43 mRNA levels.
  • Quantitation of GAP-43 RNA revealed that NGF caused a 3.5 fold increase, while dexamethasone lead to a 5.5 fold decrease.
  • Accumulation of GAP-43 mRNA in the presence of NGF was persistent, unlike that of c-fos (Greenberg et al, J. Biol Chem. 260:14101-14110 (1985)), which peaks within several hours and then rapidly declines despite the continued presence of NGF.
  • steroid concentration was 1 M for 48 hours of treatment.
  • the quantitation was derived by densitometry- and normalized for slight variations in RNA input.
  • Estradiol, testosterone, and pregnenolone had no effect on accumulated GAP-43 mRNA levels.
  • Dexamethasone, corticosterone, aldosterone and progesterone reduced the levels of GAP-43 RNA to 6%, 15%, 10% and 15% of control (defining the NGF-stimulated level of GAP -43 RNA as 100%). respectively.
  • These data suggest activation of either the mineralocorticoid or glucocorticoid receptors, although the progesterone effect may be mediated by its own receptor (Arriza et al. Science 237:268-275 (1987); Giguere et al. Cell 46:645-652 (1986)).
  • Corticosteroids block the NGF induction of a neuronal phenotype in both SIF and adrenal medullary cells (Doupe et al., J. Neurosci. 5:2119- 2142 (1985); Doupe et al, J. Neurosci. 5:2143-2160 (1985)).
  • PC12 cells were grown for 36 hours in the presence of NGF (50 ng/ml) and dexamethasone (1 mM). The results indicate that dexamethasone prevents the NGF-mediated increase in GAP -43 mRNA.
  • cycloheximide Larger sized transcripts were noted after treatment yvith 0.5 mg/ml cycloheximide. which may represent unspliced precursors. Dexamethasone suppression of GAP-43 expression was more pronounced with cycloheximide and dexamethasone than with dexamethasone alone.
  • NGF-treated PC12 cells are considered good models of differentiated neurons, it was desired to determine whether the effects of corticosteroids might be exerted upon primary neurons after they had achieved their fully differentiated state.
  • dissociated neurons of the rat superior cervical ganglion were cultured, to which 1 mM dexamethasone was added for 48 hours.
  • Total RNA was prepared, fractionated, blotted and probed as before.
  • Dexamethasone reduced the expression of GAP -43 RNA in sympathetic neurons.
  • the morphological appearance of the neurons in the dexamethasone-treated cultures was not different than that of the untreated cells. This suggests that neurite extension or maintenance over the short term may not depend upon the persistence of GAP-43 mRNA, but that long-term effects require evaluation since the GAP -43 gene product may have a long half-life.
  • GAP-43 gene* expression is subject to both positive and negative control: positive by NGF and negative by glucocorticoids. Both effects are direct, neither requiring new- protein synthesis.
  • cycloheximide was shown to further augment the dexamethasone suppression of GAP-43 mRNA. While not intending to be bound by a particular theory, this may be due to inhibition of synthesis on an mRNA-stabilizing protein.
  • the GAP-43 gene is highly regulated. To-date it has been reported only in neurons. The present inventors, hoyvever, have obtained data which support low level expression in other cells that derive from the neural crest. Peak levels in the animal are achieved at the time of neurite growth, relating either to normal development or to regeneration. The molecular regulators of its cell-specific and groyvth-related expression have not yet been elucidated. Nerve groyvth factor directly increases expression of several genes, such as c-fos, NGFIA, NGFIB. beta actin and a cloned cDNA related to intermediate filaments (Greenberg et al, J. Biol Chem. 260:14101-14110 (1985): Milbrandt, Science 238:797-799 (1987): Milbrandt, Neuron 1:183-188 (1988); Leonard et al. J. Cell Biol 106:181- 193 (1988)).
  • NGF induction is rapid, exerted within minutes, and declining after several hours. This is in contrast to the NGF effect on GAP-43 expression, which is slower in onset and persistent.
  • c-fos include calcium entry, other growth factors and serum withdrayval and repletion, and these effects are seen in a variety of cell types (Greenberg and Ziff, Nature 311:433-438 (1984)).
  • Genes such as c-fos have been likened to the immediate-early genes of DNA viruses (Goelet et al. Nature 322:419-422 (1986)), some of which themselves encode transcriptional regulators that reprogram the cellular machinery' to a dedicated function.
  • GAP-43 The delayed response of GAP-43 to NGF suggests that it may fall into a different class of NGF-regulated genes than do c-fos, NGFIA, NGFIB, etc., and may play a role in longer-term adaptation rather than in immediate responses. Unlike these other genes, although NGF does cause a large increase in GAP-43 mRNA accumulation, its effect upon transcription rate is negligible. It is therefore likely that its action is mediated through a post-transcriptional mechanism.
  • corticosteroids The effects of corticosteroids upon the nervous system are widespread (for review see McEwen et al, Physiological Rev. 66:1121- 1188 (1986)). Since steroids act through their receptors as transcriptional regulators, it is important to determine yvhich neuronal genes are regulated by corticosteroids. In considering cells of neural crest lineage, it is of particular interest to determine whether the antagonistic effects of corticosteroids and NGF on cell phenotype are mirrored at the level of gene expression, i.e., whether the same gene may be bimodally regulated by the two agents.
  • the present inventors have shoyvn that GAP-43 transcription is suppressed by corticosteroids. and that the concomitant presence of NGF does not prevent this suppression. This is similar to the glucocorticoid inhibitory effect on NGF-mediated neuronal differentiation of cultured chromaffin cells (Doupe et al, J. Neurosci. 5:2119-2142 (1985)).
  • GAP-43 is dually regulated by NGF and corticosteroids in a manner at least compatible with the known divergent effects of these modulators of cell fate.
  • SCG 10 another neural-specific gene, designated SCG 10
  • SCG 10 gene expression is stimulated by NGF and repressed by glucocorticoids, although the levels of control differ somewhat between the two genes.
  • expression vectors encoding rat GAP-43 are introduced into several types of non-neuronal cells.
  • Expression vectors were constructed using rat GAP-43 cDNA (Karns, L.R. et al. Science 236:597 (1987)) inserted into plasmids containing the SV40 origin of replication under the control of the adenovirus major late promoter, the SV40 early promoter, or the cytomegalovirus promoter. The results were similar using all of these vectors.
  • COS 7 cells (Gluzman, Y., Cell 23:175 (1981)) were transfected as described (Zuber, M.X. et al. Science 234:1258 (1986)) and examined for GAP-43 immunoreactivity using rabbit anti-GAP antibody as described above. Control transfections were done identically using a similar vector expressing the T-cell-specific membrane protein CDS (Seed. B. et al, Proc. Natl. Acad. Sci. USA 84:3365 (1987)). Cells were examined 1 hour after plating.
  • Control COS cells were essentially round after immunofluorescent labeling of the CD8-transfected cells with antibody to CD8.
  • GAP-43 transfected cells GAP-43 immunoreactivity was prominent in about 5-20% of the cells, depending upon transfection efficiency.
  • Cells that expressed high levels of GAP -43 had a distinctive structure with many cells extending processes from their cell perimeter. To ensure that this was not due to better visualization of the cell surface by antibody to GAP -43, the surface of all cells y vas labeled by rhodaminated wheat germ agglutinin.
  • CD8-transfected cells, mock trans ⁇ fected cells, or cells in the same GAP -43 transfected dish that did not express GAP -43 did not have these extensive processes, although shorter or single processes did occur. Long, thin processes appeared to be associated only with high level GAP -43 expression.
  • GAP-43 a series of clonal, stably transformed CHO cell lines was generated that constitutively expressed GAP-43. Control cell lines 30 minutes after plating were generally round. In GAP-43-expressing lines, GAP -43 expression clearly correlated yvith process extension. Many cells expressing GAP-43 extended filopodial processes that were narrow and between 20 and 75 mm in length. In both control and cell lines expressing GAP -43, the perimeter often included broad, thin, ruffled lamellipodia.
  • Process formation was assessed from four independent lines transfected with a control plasmid and four independent lines expressing the highest amounts of GAP -43. as determined by Western blot. Living cells were examined by the use of Nomarski optics. All CHO cell lines with GAP-43 immunoreactivity had a greater tendency to extend processes than did control cell lines. In addition, cells expressing GAP -43 often had multiple processes (ranging between 6% and 11 of cells) as compared to control cell lines (from 0.5 to 1%), and the process length was longer than in the control cells.
  • the neuronal protein GAP-43 therefore causes a change in the shape of these non-neuronal cells. Filopodia and lamellipodia extend directly from the cell soma, such that the cell protrusions resemble growth cones.
  • GAP-43 In non-neuronal cells. GAP-43 is removed from its normal biological context, and is expressed in a deregulated fashion, so the changes observed here may not mimic the effect of GAP-43 in its neuronal context. However, there is evidence that GAP-43 is, in fact, related to growth cone function, in that it is enriched in growth cones (Katz, F. et al, J. Neurosci. 5:1402 (1985): DeGraan, P.N.E. et al, Neurosci. 61:235 (1985): Meiri. K.F. et al. Proc. Natl Acad. Sci. USA 83:3537 (1986); Skene, J.H.P. et al.
  • GAP-43 a neuron-specific molecule
  • GAP-43 interacts with more general mechanisms that control cell shape (Bray. D. et al. Science 239:883 (1988); Smith, S.J., Science 242:708 (1988)).
  • gro y vth cone structure has been suggested to utilize cellular mechanisms, such as floyv of cortical actin and selective adhesion, that may be used as a general means to impart cellular motion (Bray, D. et al. Science 239:883 (1988): Smith, S.J., Science 242:708 (1988)). How GAP-43 might interact with such machinery remains to be determined.
  • the GAP-43 protein contains a novel membrane-targeting peptide domain which directs the GAP-43 protein to the cell membrane, and especially to the region of the growth cone of neuronal cells.
  • the structure of this membrane-targeting domain has been determined, and it has been shown that the peptide is effective in directing normally cytosolic proteins (which are not normally membrane-associated), to the cell membrane.
  • compositions and methods of this aspect of the invention it is possible, inter alia, to direct any desired protein to the cell membrane, including proteins which are not normally membrane- associated. Further, the compositions and methods of this aspect of the invention are of obvious utility in the therapeutic treatment of neurological damage and disorders in vitro, in vivo, and in situ, in animals.
  • GAP -43 While it is associated with cell membranes, and especially with the growth cones of developing or regenerating neuronal cells, lacks any such highly hydrophobic region.
  • the GAP -43 protein is encoded in three exons, as shown in Figure 2.
  • the short (10 amino acid residues) amino-terminus exon has surprisingly been discovered to encode a membrane-targeting peptide domain.
  • Experiments in which large portions of the second GAP-43 exon were removed did not affect membrane binding of the remaining protein.
  • a synthetic GAP -43 gene lacking the initial four amino acids (MET LEU CYS CYS). and beginning at the MET of position five failed to bind to the membranes of neuronal or non-neuronal host cells (see Figure 11), indicating that the first exon is responsible for this membrane- targeting function.
  • membrane-targeting peptide any amino acid sequence as follows:
  • LYS GLN or a functional derivative thereof, which, when attached at or near the amino-terminus end of a desired protein or peptide, will effect the direction of said protein or peptide to the cell membrane.
  • the membrane-targeting peptide of the invention may be attached to a desired protein or peptide by yvell knoyvn methods, including but not limited to direct synthesis by manual or, preferably, automated methods.
  • An alternate preferred method by yvhich the membrane-targeting peptide of the invention may be attached to the desired protein or peptide involves modifying the gene encoding the desired protein or peptide. so that the expressed gene product will include the membrane-targeting peptide at its amino-terminus end. This may be accomplished by yvell- known methods, including but not limited to blunt-ended or sticky-ended ligation methods as described herein.
  • the present invention provides for cDNA coding for a membrane-targeting domain comprising the nucleotide sequence atg ctg tgc tgt atg aga aga ace aaa cag or a functional derivative thereof.
  • non-neuronal cells including COS cells, NIH 3T3 cells, and CHO cells
  • neuronal cells PC12 cells
  • plasmids containing the GAP -43 gene in which mutations were introduced into the nucleotide sequence of the cysteines at positions three (C3) or four (C4), to result instead in expression of alanine at those positions ( Figure 11).
  • the invention comprises an amino acid sequence comprising
  • peptides including the first five, six, seven, eight or nine amino acids of exon 1 also will alloyv membrane binding when attached to a desired protein or peptide.
  • peptides including the first five, six, seven, eight or nine amino acids of exon 1 also will alloyv membrane binding when attached to a desired protein or peptide.
  • the first GAP -43 exon described above was ligated at the amino-terminus end of the gene encoding chloramphenicol acetyl transferase (CAT), a protein which is normally cytosolic, and not membrane-associated. Plasmids containing this sequence were used to transfect neuronal and non-neuronal cells ( Figure 11). Immunofluorescence assay revealed that the expressed CAT protein was membrane-associated in transfected cells. This demonstrates that the amino acids of the first GAP-43 exon are sufficient to accomplish membrane targeting of a desired protein or peptide.
  • CAT chloramphenicol acetyl transferase
  • GAP-43 will direct CAT to the same location as GAP-43 in transfected PC12 cells. These cells resemble neuronal cells in putting out long processes tipped by groyvth cones. GAP -43 is normally especially enriched in neuronal cell groyvth cones, and data suggest that the membrane- targeting peptides of the present invention are responsible for this observed growth cone enrichment.
  • the present inventors employed mutational analysis and laser scanning confocal microscopy of fusion proteins that included regions of GAP -43 and chloramphenicol acetyltransferase (CAT).
  • CAT chloramphenicol acetyltransferase
  • Plasmids were constructed that encode fusion proteins of the first 10 amino acids of GAP-43, MLCCMRRTKQ, fused to the amino terminus to the complete CAT protein (GAP10CAT), or the first 40 amino acids of GAP-43 fused to CAT (GAP40CAT).
  • Immunoblotting was carried out as follows: Chimeric proteins with the amino terminus of GAP-43 fused to CAT associate with COS cell membranes. CAT, GAP10CAT and GAP40CAT were transiently expressed in COS cells. Immunoblots of membrane (M) and cytosolic (C) fractions from each transfection were prepared using anti-CAT antibody. In the CAT-transfected cells, immunoreactivity is found only in the cytosolic fraction and co-migrates with purified CAT protein. In the GAP40CAT and GAP10CAT cells, nearly all of the immunoreactivity is membrane-associated and migrates more slowly than CAT, as expected for fusion proteins with Mr 4000 or 1000 greater than CAT. Molecular weight standards of 116. 84. 58, 48.5. 36.5 and 26.6 kilodaltons were used.
  • Membrane association of GAP and GAP40CAT was evaluated in PC12 cells. Stably transfected PC12 cells expressing CAT, GAP40CAT, or GAP were selected as described herein. Immunoblots of membrane (M) and cytosolic (C) fractions were stained yvith anti-CAT or anti-GAP antibodies. CAT-transfected cells (CAT) contained immunoreactivity in the cytosolic, but not in the membrane fraction, and this immunoreactive CAT co-migrated with purified CAT. In contrast, GAP40CAT transfected cells (G40CAT) contained membrane-associated CAT immunoreactivity which migrated more slowly. Fractions from rat brain (BR) demonstrated that most, but not all, endogenous GAP-43 immunoreactivity is membrane-associated. In transfected PC12 cells over-expressing GAP-43. nearly all of the GAP-immunoreactivity is membrane-associated and co-migrates with purified GAP -43.
  • M membrane
  • C cytosolic
  • the GAP -43 coding sequence replaced the stuffer at the Xba I sites of the CDM8 plasmid described by Seed, B. Nature 329:840-846 (1987).
  • the inserted GAP-43 sequence included the entire coding sequence of rat GAP -43, from the NIa III site at the start of translation to the Sau 3AI site 68 bp downstream from the termination codon, as described herein.
  • pCAT the Hind III to Bam HI fragment containing the CAT coding sequence and polyadenylation site from pSV2CAT (Gorman, CM.. Moffat, L.F. & Howard. B.H.
  • pGAP40CAT and pGAPlOCAT include the first forty or ten amino acids of GAP-43, respectively, fused in-frame yvith CAT in pCAT by the use of polylinkers.
  • DEAE dextran and chloroquine was used as described (Zuber, M.X.. Simpson, E.R. & Waterman, M.R. Science 234:1258-1261 (1986)).
  • a neomycin resistance plasmid co-transfected with the plasmid of interest on a 1 to 10 ratio was used as described herein.
  • 400 ug/ml of active Geneticin (GIBCO) were used.
  • Transient transfection of PC12 cells was performed by electroporation with the Bio-Rad Inc. electroporation system using 300 volts and 960 microfarad. After 8 hour the medium was changed. Twenty-four hours after electroporation the cells were plated on poly-D-lysine-coated coverslips in the presence of 50 ng/ml NGF and analyzed 24 hours later.
  • rabbit anti-GAP-43 antibodies were made by immunizing rabbits against four peptides including aa 1 to 24, aa 35 to 53, aa 53 to 69, and aa 212 to 228 of rat GAP-43.
  • Anti-GAP-43 antibody was affinity-purified on GAP peptide agarose.
  • Anti-GAP antibody was bound to a resin that contained 10 mg/ml of each peptide coupled to agarose by the cyanogen bromide method and the antibody was eluted at pH 3.5.
  • Rabbit anti-CAT antibodies were obtained from 5 Prime-3 Prime, Inc. Secondary antibodies were obtained from Organon Teknika, Jackson Immunologicals. and Vector labs.
  • COS or PC12 cells were scraped from 100 mm confluent petri dishes and pelleted at 2000 x g for 10 minutes.
  • the pelleted cells were homogenized by Polytron in 10 mM Tris- HCl. ImM EDTA, pH 7.6 (300 ul/dish) and centrifuged at 250,000 x g for 30 minutes at 4°C. The supernatant was collected as the cytosol fraction.
  • the pellet was washed by homogenization and centrifugation in the same buffer, and then resuspended to the same volume as the cytosol fraction.
  • Rat brain was obtained from 1 day old rats and homogenized by Polytron in 10 M Tris-HCl, ImM EDTA, pH 7.6 (10 ml/gram wet weight tissue).
  • the cytosol and washed membrane fractions were prepared by centrifugation as described for the cell extracts.
  • GAP-43 protein was purified from rat brain by a modification of the method of Andreasen et al, (Biochemistry 22: 4615-4618 (1982)) and used as a positive control for immunostaining.
  • the same volume of cytosol or membrane fraction was electrophoresed on polyacrylamide gels (Laemmli, Nature 227: 680-685 (1970)).
  • Proteins were electrophoretically transferred to nitrocellulose and excess sites were blocked with 4% BSA. Membranes were then incubated for 24 hour at 4°C with 40 ug/ml affinity purified anti-GAP, or a 1:1000 dilution of anti-CAT antibodies. Bound antibody was detected using anti-rabbit Vectastain horseradish peroxidase method according to the manufacturer ' s instructions. Tetramethyl benzidine (Kirkegaard and Perry, Gaithersburg, MD) was employed as peroxidase substrate.
  • GAP-43 and the GAP-CAT chimeric proteins in NGF-treated transfectants of PC12 cells yvas investigated by confocal microscopy in order to determine yvhether the amino terminus accounts for the growth cone enrichment of GAP-43 in neuronal cells.
  • CAT remains cytosolic, whereas GAP -43 is distributed in a punctate pattern with notable enrichment in growth cones, a pattern similar to that of native GAP-43_ in neurons.
  • the amino terminus of GAP-43 fused to CAT caused the resulting fusion protein to acquire a distribution that closely resembled that of GAP-43 itself.
  • Perinuclear labeling for both GAP -43 and the chimeric protein was detected at a low level, and may be due to localization to the Golgi, as has been observed for native GAP-43 (Van Hooff, C.O.M., Holthuis, CM., Oestreicher, A.B., Boonstra, J., De Graan, P.N.E. & Gispen, W.H. J. Cell Biol. 108:1115-1125 (1989)).
  • Glutaraldehyde fixation provided better histologic preservation of the finer processes of the growth cones, and revealed that the chimeric protein accumulates especially within filopodia.
  • Anti-CAT antibody was used for CAT, GAP40CAT and GAP10CAT, whereas anti-GAP-43 antibody was used for GAP-43.
  • Control PC12 cells of this variant expressed undetectable levels of GAP-43 and CAT immunoreactivity.
  • PC12 cells were transferred to poly-D-lysine coated coverslips 24 hours before immunofluorescence in the presence of 50 ug/ml nerve growth factor (NGF). Fixed with 3.7% formaldehyde for 7 minutes, and permeabilized with 0.1 % Triton-X-100 for 3 minutes.
  • NGF nerve growth factor
  • GAP40CAT Localization of GAP40CAT within the growth cone of a PC12 cell was demonstrated using a higher power comparison of PC12 cells expressing GAP40CAT viewed with Nomarski optics and scanning confocal immunofluorescence, labeled with anti-CAT antibodies. Cells had been treated with NGF for seven days. One growth cone appeared brightly labeled, but a smaller one did not. Unequal labeling of different growth cones, even of the same cells, occurs for native GAP -43 in neurons (Goslin, K., Schreyer, D.J., Skene, J.H.P. & Banker, G. Nature 336: 672-674 (1988)) as well. Comparison of the Nomarski and immunoflu orescent images shoyved that filopodia were especially labeled. Similar results were seen for GAP10CAT.
  • the growth cone membrane is also distinctive in its protein make-up.
  • the growth cone membrane has binding sites that recognize and bind the palmitylated amino terminus of GAP -43. While the present inventors do not intend to be bound by any particular theory, it seems less likely that the palmitylated residues interact with the lipid bilayer directly, because that would likely cause a more uniform membrane distribution for GAP-43.
  • the fatty acid moiety of another acylated protein, N-myristylated VP4 of poliovirus has been shown by X-ray diffraction to interact with specific amino acid residues of other viral proteins and not with the lipid bilayer (Schultz, A.M., Henderson. L.E.
  • GAP-43 and GAP-CAT fusion proteins bind to the membrane of non-neuronal cells, similar or identical binding sites must be present in other cell types, but because GAP-43 is neuron-specific, these sites would presumably be targets for different proteins in non-neuronal cells.
  • GAP-43 causes enrichment especially in filopodia. This is the normal location of GAP-43 in these cells, as evidenced by electron microscopy (Van Hooff, C.O.M., Holthuis. CM., Oestreicher, A.B.. Boonstra. J., De Graan. P.N.E. & Gispen. W.H. J. Cell Biol. 108:1115-1125 (1989)). Given the obseivation that transfected GAP-43 enhances the propensity of non-neuronal cells to extend filopodia as described herein, it will be of interest to correlate GAP-43 location with motile activity of particular filopodia.
  • the present invention provides, in another aspect, for a method of introducing a desired protein or peptide into the membrane region of a neuronal or non-neuronal cell, and for a method of directing a desired protein or peptide to the growth cone areas of neuronal cells.
  • a method for directing a desired protein or peptide to the membrane of a cell comprising
  • step (b) introducing the resulting protein or peptide comprising said membrane- targeting domain into a cell wherein the resulting protein or peptide of step (b) is directed to said membrane of said cell by said membrane-targeting domain.
  • the present invention provides nucleotide sequences encoding the membrane-targeting peptide comprising the above amino acid sequences or their functional or chemical derivatives, as well as the addition of these sequences by well known methods to nucleotide sequences encoding proteins or peptides other than GAP-43 (as well as GAP-43 itself), and the expression of the resulting sequences in prokaryotic of eukaryotic hosts by methods well known to those of skill.
  • the desired protein or peptide may be diagnostically or therapeutically labeled, and the utility of the composition and methods of this aspect of the invention will be apparent to those of skill, and may be readily utilized for in vitro, in vivo, or in situ diagnostic or therapeutic purposes in animals including humans with the exercise of merely routine skill
  • GAP-43 regulation occurs at the level of gene expression.
  • cis or trans-acting elements that might regulate its expression.
  • elements of the GAP -43 gene that confer its responsiveness to growth factors, cause cellular restriction of expression, and regulate the gene during development of the nervous system.
  • the entire rat genomic DNA of GAP-43 has been cloned.
  • genomic GAP -43 has been isolated, and its intron- exon boundaries and transcriptional start sites have been mapped. It has surprisingly been discovered that the promoter is quite unusual in its structure, containing a repetitive sequence capable of forming unusual conformations, and lacking some canonical promoter components. Transcription can initiate from more than one site, and some of the start sites are utilized differently in the central and peripheral nervous systems. Further, the inventors have investigated whether the GAP-43 promoter contains regions recognized by brain-specific nuclear proteins. Regions of the GAP promoter have been examined by gel electrophoresis mobility shifts, and a domain which binds protein(s) present in brain but not in liver nuclear extracts has been identified. The binding activity diminishes with brain maturation.
  • the binding site is limited to a stretch of about 20 nucleotides, which also is specifically protected in DNase protection assays by brain nuclear extracts and not by liver extracts.
  • the region has a sequence similar to binding sites recognized by a class of DNA binding proteins known as POU.
  • Genomic clones containing the three GAP-43 exons were isolated from a library constructed by inserting size fractioned SauIIIA partial digests of rat genomic DNA into the BamHl site of bacteriophage EMBL-3.
  • the library was initially screened on Colony Plaque Screen filters (DuPont/NEN) following standard protocols with random primed GAP-43 cDNA, as described hereinabove. To find exon I. the library was replated and duplicate lifts were probed sequentially with three oligonucleotides complementary to the 5' most region of the cDNA (#4, -68 to -39: #2, -38 to -9 and #5, + 1 to +20 in Figure 14). Clones positive for at least two oligonucleotides were selected for further analysis. Inserts from positive phage were subcloned into the Sail site of the pBluescript vector (Stratagene) for mapping with a variety of restriction enzymes.
  • the GAP-43 promoter was sequenced by the dideoxy method of Sanger et al. Proc. Natl Acad. Sci. USA 74:5463-5467(1977) using Sequenase as described by the supplier (USB). Subclones of the bacteriophage clones containing the first exon were constructed by- standard methods in pBluescript (Stratagene) for double stranded sequencing and in M13 vector (Messing. Meth. Enzvmol 101:20-78 (19- 83)) for single-stranded sequencing.
  • RNAse protection analysis yvas done as described in Krieg and Melton, Meth. Enzvmol. 155:397-415 (1987).
  • a genomic piece of GAP-43 from the Xbal site at -475 from the translation start site to the Sspl site at +83 (in the first intron) was cloned into the Xbal and EcoRV sites of pSP72 (Promega).
  • RNAse protection analysis showed three major GAP-43 transcripts at -474S. -51/52 and -78 bases from the translational start site. Protections were performed on tRNA. RNA prepared from newborn rat lung, dorsal root ganglia, and cerebral cortex. The probe extended 475 bases upstream from the translational start site (Xbal site). An over exposure showed additional longer transcripts which were much more abundant in the cerebral cortex as opposed to the DRG. Markers were MSPII digested pBR322.
  • RNAse protection analyses were carried out showing the heterogeneity of GAP -43 transcripts in different areas of the nervous system and in PC12 cells.
  • RNA from control PC12 cells was compared with that obtained from NGF treated PC12 cells.
  • RNA samples derived from the CNS had a higher proportion of the longer transcripts than samples from DRG or PC12 cells.
  • RNA samples from newborn rat heart, liver, lung, cerebellum, spinal cord, cortex, hippocampus, and dorsal root ganglia were used.
  • the longer upstream start sites as a group constituted the start sites of a significant fraction of RNA in the central nervous system tissues but not in the . dorsal root ganglia.
  • a rat genomic library was screened with probes derived from the GAP-43 cDNA, as described herein above.
  • Initial screening with radiolabeled full length cDNA provided two classes of phage. which subsequent analysis showed to correspond to the second and third exons of the gene. Because the first exon proved to be small, and hence underrepresented in the cDNA probe, additional rounds of screening using three oligonucleotide probes derived from the 5' most region of the cDNA were necessary in order to obtain clones containing the 5' end of the gene.
  • a map of the GAP-43 gene is shovm in Figure 13a, with represen ⁇ tations of the phage used to map it. The gene spans at least 50 kb and contains 3 small exons.
  • the first is about 80 bp (see below for a description of the variability of the 5 ' end), the second is 565 bp, and the third is 672 bp, and they are separated by 2 introns of greater than 24 kb and 20 kb, respectively.
  • the first exon contains the 5' untranslated sequences of the mRNA and encodes the first 10 amino acids of the protein. This short amino terminal region of the protein contains the "sorting sequence” that directs binding of GAP-43 (and heterologous fusion proteins) to growth cone membranes, as described hereinabove.
  • the second exon encodes the bulk of the protein and includes a region identified by Alexander et al. J. Biol Chem. 263:7544-7549 (1988) as the calmodulin binding site.
  • the third exon encodes the carboxy-terminal 28 amino acids and contains 587 bases of untranslated sequence and the poly-A addition site.
  • the sequence of the 5' region of the gene is displayed in Figure 14. It contains no TATA or CAAT boxes, but does contain a sequence, TATTCATG (overlined). yvhich is identical to the consensus Pit-1 binding site. This octamer binds a class of proteins thought to regulate transcription of several genes, including prolactin and growth hormone (Bodner et al. Cell 55:505-518 (1988); Ingraha et al. Cell 55:519-529 (1988)).
  • a striking feature of the promoter sequence is that more than 80% of the coding strand is composed of purines (underscored by asterisks in the figure), with two uninterrupted purine homopolymer stretches spanning from -118 to -188, and from -238 to -370, respectively. Some areas of these homopolymer stretches that are not simply alternating G and A contain tandem repeats, which possess some mirror symmetry (for example -168 to -118). Hairpin forming palindromes centered at -112, -232 and -509 flank the homopolymer regions and may influence secondary structure.
  • H-DNA triple stranded conformation
  • Htun and Dahlberg devised a simple gel system to demonstrate that H-DNA will introduce a severe kink in DNA. Their assay is based upon the enhanced stability of H-DNA at low pH. When fragments of DNA which contain an H-forming region are electrophoresed at low pH. an H-DNA induced kink yvill retard mobility as compared to the mobility at a pH not favoring H-DNA formation (Htun and Dahlberg, (1988), supra. The present inventors exploited this mobility shift to demonstrate that the purine homopolymer region from -240 to -370 in the GAP-43 promoter is capable of forming stable H-DNA structures in linear DNA in vitro.
  • Figure 15 is a representation of the restriction digest fragments of the GAP-43 promoter which were analyzed by gel electrophoresis, as described hereinbelow. The potential H-DNA forming homopurine- homopyrimidine regions are shown as thickened lines.
  • aliquots of the digests represented in Figure 15 were loaded on 1.4% agarose gels that had been equilibrated with Tris-acetate at either pH 7.4 or 4.0 and run in parallel. Bands that shifted at pH 4 exhibited smearing that may result from the B to H transition (Htun and Dahlberg, Science 241:1791-1795 (1988)).
  • GAP-43 upstream sequence Another notable feature of the GAP-43 upstream sequence is the absence of the TATA motif. Genes that lack a TATA sequence to direct initiation of transcription often have multiple mRNA start sites. This proved to be true for GAP-43.
  • RNAse protection analysis was used to determine the transcriptional start sites for GAP-43 in several tissues. RNA from lung, dorsal root ganglia (DRG) and cerebral cortex (CTX) was analyzed with a probe extending to -475 bases from the translation start site. Using this probe, three major bands were protected, corresponding to transcriptional start sites at -47/-48, -51/-52, and -78. These same sites were identified by primer extension. Additional minor bands become visible after longer exposure.
  • RNA from the cerebral cortex as compared to the dorsal root ganglia. This is interesting in light of observations that suggest the regulation of GAP-43 gene expression in the central and peripheral nervous system is different (Skene et al, J. Cell Biol 89:86-95; J. Cell Biol. 89:96-103 (1981)). Hence. RNA from other areas of the CNS, as well as from PC12 cells (which are believed to derive from sympatho- adrenal precursor cells), was analyzed.
  • RNA from the hippocampus, cortex and cerebellum has a higher proportion of the transcripts initiating from the area around -230 than RNA from DRG or PC12 cells, although the amount of each of these longer messages is relatively small.
  • a probe was used that pools all messages that start beyond -234, the difference between start sites in the CNS and PNS becomes more apparent.
  • This analysis showed that the longer GAP-43 transcripts together actually account for a significant fraction of the total GAP-43 transcripts, and that these transcripts are much more prevalent in RNA from the CNS than that from the DRG or PC12 cells.
  • the 5'end of GAP -43 mRNA is heterogeneous, and upstream start sites are used more commonly in the central nervous system as compared to the peripheral nervous system.
  • the present embodiment of the invention is directed to the isolation and characterization of genomic sequences containing the GAP-43 gene.
  • Three small exons corresponding to the ⁇ 1.5 kb mRNA are separated by introns of at least 24 and 20 kb. respectively.
  • the promoter region is rather unusual. There are several long homopurine- homopyrimidine stretches in the upstream region yvhich are potentially capable of forming triple stranded "H-DNA" (Wells et al. FASEB J. 2:2939-2949 (1988)). It is here demonstrated that one of these regions does, in fact, form H-DNA in vitro.
  • the promoter lacks a canonical TATA box, and has multiple transcription initiation sites. The utilization of some of these sites differs in various parts of the nervous system.
  • the rat GAP -43 gene is a single copy gene that consists of three exons and two introns spanning at least 50 kb.
  • the present inventors have obtained some evidence that the exons correspond to functional domains in the protein.
  • the first exon which encodes only the first 10 amino terminal residues, contains the stretch responsible for membrane targeting of GAP-43.
  • Cysteines at positions 3 and 4 in the protein are acylated and may be involved in membrane binding, as described hereinabove.
  • the amino terminus is necerney for membrane binding of GAP -43, and contains sufficient information to target heterologous fusion proteins to the same membrane domains as GAP-43, including those of the growth cone, as described hereinabove.
  • the second exon includes the calmodulin binding region from amino acid 43 to 51 (Alexander et al, J. Biol Chem. 263:7544-7549 (1988)) as well as a serine at position 41 that is a substrate for protein kinase C (Coggins and Zwiers, Soc. Neurosci. Abstract (1988)).
  • Exons I and II contain regions that are well conserved between fish and several mammalian GAP -43 proteins (Labate and Skene, (1989)).
  • the promoter of GAP -43 is unusual in sequence and structure.
  • the lack of a TATA box and consequent use of multiple start sites cause the GAP-43 promoter to resemble promoters of constitutively expressed housekeeping genes.
  • the GAP-43 promoter lacks the consensus Sp-1 binding sites (GGGCGGG) that have been correlated with the promoters of housekeeping genes (Dynan, Trends Genet. 2:196-197 (1986)).
  • GGGCGGG consensus Sp-1 binding sites
  • GAP-43 is regulated differently in the central and peripheral nervous systems. For example, axotomy of mammalian central neurons does not cause increased GAP -43 expression and transport, whereas axotomy of a peripheral nerve does (Skene and Willard; J. Cell Biol. 89:96-103 (1981). As described hereinabove, GAP-43 does not appear to be irreversibly repressed in the CNS, and may play a role in plasticity other than in axonal growth (Benoyvitz and Routtenberg, T.I.N.S. 10:527-532 (1987)), but it is clear that there is a difference in regulation centrally and peripherally. Hence, the usage of different start sites suggests the possibility that the mRNA from different neurons may differ at the 5' end, in turn regulating ribosome binding or stability.
  • Thy-1 a gene expressed in. although not limited to. neurons, has been demonstrated to be expressed in a developmental- ⁇ - regulated, tissue-specific fashion at the transcriptional level, and also lacks a TATA box and Sp-1 binding sites (Spanopoulou et al, Molec. Cell Biol. 8:3847-3856 (1988)). Also. like GAP-43. the choice of transcriptional start sites in the Thy-1 promoter can vary between expressing tissues, with upstream start sites being more prominent in the brain (ibid.). This suggests an additional level of control in brain versus other tissues for both GAP -43 and Thy-1.
  • a potential upstream regulatory element present in the GAP-43 promoter is the consensus Pit-1 binding site (TATTCATG). This and related sequences are recognized and bound by transcription factors known collectively as POU proteins.
  • TATTCATG consensus Pit-1 binding site
  • This group originally included Pit-1, Oct-1 and Oct-2 in mammals, and unc86 in nematodes (revieyved in Herr et al. Genes Develop. 2:1513-1516 (1988)), and has recently been expanded by the finding of cDNAs encoding proteins that share the two peptide regions that characterize this family (He et al, Nature 340:35-42 (1989)). As described in the following example, the present inventors have identified and cloned brain-specific proteins that bind this region of GAP-43 and may regulate its transcription.
  • GAP-43 promoter Another remarkable feature of the GAP-43 promoter is the presence of long homopurine-homopyrimidine stretches. These are interesting because they may bind proteins specific to GAGA stretches (Biggin and Tjian, Ce]l 53:699-711 (1988); Gilmour et al. Science 245:1487-1490 (1989)), and because they have the potential to take on a triple stranded conformation called H-DNA. Such homopolymer regions have been found to be overrepresented in the 5' ends of eukaryotic and eukaryotic viral genes, leading to the speculation that they may somehow be involved in transcriptional control (Wells et al, FASEB J.
  • Htun and Dahlberg Science 243:1571-1576 (1989)).
  • adoption of the H configuration perhaps stabilized by protein interactions, would cause a kink in the DNA.
  • This kink could phase nucleosomes by exclusion from the kinked region, thereby making the DNA around the kink more accessible to tran ⁇ scriptional factors (Htun and Dahlberg, Science 241:1791-1795 (1988); Han and Grunstein, Cell 55:1137-1145 (1988)).
  • H-DNA could serve as a repressor of transcription by directly blocking access to DNA in its immediate vicinity (Maher et al. Science 245:725-730 (1989)).
  • additional embodiments of the present invention comprise, inter alia, a nucleotide sequence as shown in Figure 13 encoding genomic GAP-43, or a functional or chemical derivative thereof, as well as a nucleotide sequence as shown in Figure 14 encoding the GAP-43 promoter, or a functional or chemical derivative thereof.
  • this aspect of the invention is directed to a promoter substantially as shown in Figure 14, characterized in that it contains multiple start sites and a consensus Pit-1 binding site, but lacks a TATA box and consensus Sp-1 binding sites, and further characterized in that it comprises long homopurine-homopurimidine stretches capable of taking on triple stranded (H-DNA) conformation.
  • DNA expression vectors comprising the structural gene as described above, host cells transfected with these vectors, and the proteins produced thereby.
  • GTP Binding Protein G 0
  • the neuronal growth cone contains specialized transduction machinery which converts signals from the microenvironment into directed growth of axons or dendrites.
  • Subcellular fractions fiom neonatal rat brain that are enriched in growth cone membranes have simple and distinctive protein composition.
  • the two major non-cytoskeletal proteins in growth cone membrane preparations have molecular weights of 40,000 and 35,000. By electrophoretic, immunologic and partial protein sequence criteria, these proteins have been identified as the alpha and beta subunits of the GTP binding protein, G 0 .
  • Immunohistologic staining of neuronally differentiated rat pheochromocytoma cells demonstrates high concentrations of the alpha subunit of G 0 at the distal tips of cellular processes.
  • the mechanisms by which axons transduce information form their extracellular milieu into directed growth are poorly understood.
  • the distal tip of a neuronal axon has a unique ultrastructure termed the growth cone, which is thought to be critical for this process (Bray, D., et al, Ann. Rev. Cell Biol. 4:43 (1988)).
  • the membrane of the axonal growth cone, and therefore its transduction system, can be fractionated from other neuronal constituents (Pfenninger, K.H., et al.
  • a growth cone membrane fraction was prepared from neonatal rat brain (Pfenninger, K.H., et al. Cell 35:573 (1983); Ellis, L., et al, J. Cell Biol !0_i:1977 (1985); Simkowitz, P., et al, J. Neurosci: 9:1004 (1989); Cheng, N., et al, J. Biol. Chem. 2__3:3935 (1988)).
  • This preparation has a simple protein composition by SDS-PAGE (Ellis, L., et al, J. Cell Biol 101:1977 (1985): Simkowitz, P., et al, J. Neurosci.
  • p34 and p38 have similar molecular weights to the alpha and beta subunits of the GTP -binding protein, G d (Stryer, L., et al, Ann. Rev. Cell Biol. 2:391 (1986): Gilman. A.G., Ann. Rev. Biochem. 56:615 (1987)).
  • Co-electrophoresis of the growth cone membranes with purified bovine grain G 0 demonstrated that p34 co- migrates with the beta subunit, and p38 with the alpha subunit of G 0 .
  • Immunoblotting demonstrated that p34 reacts yvith an anti-beta subunit antiserum, and that p38 reacts yvith an anti-alpha subunit G 0 antiserum. Furthermore, the predominant protein species of p38 must be alpha 0 , because equal protein concentrations of p38 and alpha Q , as determined by Coomassie blue staining, exhibited identical immunoreactivity. The same was true for p34 and the beta subunit of G Q . Alpha; subunit was about 10-fold less reactive than alpha 0 with this antiserum (Gilman. A.G.. Ann. Rev. Biochem. 56:615 (1987)). so that it cannot account for a major percentage of the alpha 0 immunoreactivity.
  • G olf a related G protein, G olf , is localized to the terminal region of primary olfactory neurons in the adult (Jones, D.T., et al. Science 244:790 (1989)), and that G 0 stains through ⁇ out cultured primary neurons but is concentrated at regions of cell-cell contact (Jones, D.T., et al. Science 244:790 (1989)).
  • G olf a related G protein
  • the neuronal protein GAP -43 is enriched in these groyvth cones as it is in those of primary neurons (Van Hooff, C.O.M., et al, J. Cell Biol 108:1115 (1989)).
  • Alpha Q immunofluorescence was highly concentrated in these growing tips of PC12 cells (although it was not found exclusively there).
  • protease inhibitors were employed throughout the procedure: 100 ug/ml soybean trypsin inhibitor. 1 ug/ml pepstatin A. 30 uM leupeptin. and 1 mM PMSF.
  • the crude brain homogenate was layered over a step gradient of sucrose at 0.75 M. 1.0 M and 2.2 M. The gradient was centrifuged at 250.000 x g for 40 min. and the 0.32/0.75 M interface was collected as the groyvth cone particle fraction. This fraction was lysed in 5 mM Tris-HCl. pH 7.6. and the membranes were collected by centrifugation at 250.000 x g for 40 min.
  • the membranes were washed by resuspension in 20 ug/ml saponin and 0.3
  • PC12 cells were grown on poly-D-lysine treated coverslips for 48 hours in the presence of 100 ng/ml nerve groyvth factor.
  • the cells were fixed with 3.7% formaldehyde in phosphate buffered saline (PBS), and then permeabilized with 0.1% Triton X-100. After incubation with 5 mg/ml bovine serum albumin in PBS, the cells were incubated with 1:1000 anti-bovine brain al ⁇ ha 0 antiserum for 1 hour at 23 °C, rinsed with PBS. and incubated with 0.3% H 2 0. for 15 minutes to reduce background. Bound rabbit immunoglobulin was detected by use of the Texas red conjugated donkey anti-rabbit IgG (Jackson Immunologicals).
  • Proteins of the axonal groyvth cone membrane were identified by SDS-PAGE. Two separate preparations of axonal groyvth cone membranes, purified bovine brain G Q , and crude brain homogenate were electrophoresed through a 10% polyacrylamide gel in the presence of SDS and stained with Coomassie blue. Enrichment of two bands, termed p34 and p38, in the growth cone membrane preparation relative to crude brain was observed. These proteins comigrated with the alpha and beta subunits of purified G Q . Under these conditions, actin and GAP-43 comigrated with an apparent Mr of 43.000. Anti-G 0 immunoblots of growth cone membrane show G Q reactivity
  • Immunoblotting revealed alpha 0 immunoreactivity migrating at the position of alpha Coomassie blue staining in both the purified G 0 preparation and the growth cone membrane preparation.
  • the gels were loaded such that Coomassie blue labeling of alpha 0 was identical to that of P38, and similarly matched for other pairs.
  • the Coomassie stained gels were run in parallel. Note that the pairs also were immunostained to the same degree, as the total protein was increased, demonstrating that p38 is as immunoreactive as authentic alpha 0 with this antiserum. This suggests that most or all of p38 is alpha Q .
  • the partial protein sequence for p34 and p38 is identical to that of G Q .
  • the partial protein sequence for p34 and p38 is shown in Figure 16.
  • the sequence of three peptides from p38 matches the sequence of three peptides from alpha Q from rat brain (Goh. J.W., Science 244:980 (1989)).
  • the sequence of three peptides from p34 is compared to that of beta j and beta 2 subunits from bovine brain (Fong, H.K.W., et al, Proc. Natl. Acad. Sci. USA 84:3792 (1987)). Note that two peptides are identical to regions in which beta j and beta 2 are identical.
  • the other peptide contains a mixture of the sequences for beta j and beta-..
  • Alpha 0 immunoreactivity is concentrated in the tips of PC12 processes
  • Alpha 0 staining of PC12 cells differentiated with nerve growth factor revealed high concentrations of the antigen at the distal tips of the cellular processes.
  • There yvas also a lower level of diffuse staining in the region of the cell body surrounding the nucleus.
  • the specificity of the antibody has been demonstrated (Huff, R.M., et al, J. Bio Chem. 260:10864 (1985): Worley. P.F., et al. Proc. Natl. Acad. Sci.
  • GAP-43 stimulates GTP B S binding, GTPase activity and GDP
  • GAP-43 differs from receptors in that it acts on isolated alpha 0
  • Pertussis toxin blocks receptor/G 0 interaction, but not GAP-43/G 0 interaction.
  • GAP-43 does not alter its potency for stimulating G t) .
  • Calmodulin can bind to GAP-43. but 20 uM calmodulin does not alter the GAP-43 effect
  • G Q may integrate extracellular and intraneuronal growth clues in taa
  • GAP-43 could be an upstream regulator of G 0 activity
  • G 0 is both a major component of the growth cone membrane
  • G protein specifically G 0 , is a major constituent of the growth cone
  • G proteins in the growth cone membrane.
  • G proteins in general, couple
  • G Q can interact with a number of cell
  • cAMP is transduced via a G protein (Snaar-Jagalska. B.E., et al, F.E.B.S.
  • N-CAM cell adhesion molecules
  • GAP -43 enriched molecule, GAP -43, also exists in discrete regions of the adult brain (Benowitz, L.I., et al. Trends Neurosci. 10:527 (1987); Skene,
  • G 0 may transduce regulatory' signals for axonal extension during neuronal development and for synaptic plasticity
  • GAP-43 is a Novel Internal Regulator of Protein Binding
  • the present invention is directed to the surprising discovery that GAP -43 acts within the cell to modify- the binding capacity of other cell proteins, including that of G 0 .
  • GAP-43 acts within the cell to modify- the binding capacity of other cell proteins, including that of G 0 .
  • IRP internal regulatory proteins
  • GAP-43 comprising the amino terminus amino acids of GAP-43 duplicate exactly the modulation in GTP binding by G Q that is caused by the intact GAP-43 protein.
  • a peptide comprising the first 24 amino acids of GAP-43 stimulates GTPuS binding to the same level as GAP -43, acting .27
  • Shorter peptides for example, a peptide
  • a further aspect of the invention is directed to the discovery that
  • a consensus amino acid sequence is found in GAP-43 and beta adrenergic receptors, said sequence comprising
  • hydrophobic-leu-cys-cys-x-basic-basic or functional derivatives thereof hydrophobic-leu-cys-cys-x-basic-basic or functional derivatives thereof. It will be further appreciated from the
  • invention may be prone to palmitylation.
  • the IRP proteins and peptides of the invention the target protein activity
  • the desired protein is* preferably a G protein, and, most preferably, G Q .
  • the preferred binding substrate is GTP, and GTPgS is most preferred.
  • the environment is preferably that
  • a living cell which may be a central or peripheral neural cell.
  • compositions and methods of the invention are directed, inter alia, to mechanisms involved in neuronal growth and synaptic plasticity. It may be desirable, for a given medicinal indication, to reduce as well as enhance neural groyvth or plasticity. This may be accomplished, for example, by administering antibodies directed against the IRP peptides of the invention, or against the sites at which such IRP peptides have their physiological effect.
  • the invention is directed to antibodies, preferably monoclonal antibodies, directed against the IRP peptides of the invention, and to functional or chemical derivatives thereof, said antibodies or their said derivatives being optionally detectably or therapeutically labeled.
  • the invention is directed to pharmaceutical
  • compositions comprising the IRP peptide of the invention, together with a pharmaceutically acceptable carrier, and optionally comprising one or more therapeutically effective agents, as well as to pharmaceutical compositions comprising an antibody directed against the IRP peptides of the invention, together with a pharmaceutically acceptable carrier, and
  • compositions of the invention will be accomplished by those of skill wihtout undue experimentation, keeping in mind those
  • the invention is directed to a method of
  • the GAP-43 sequences of the invention have been used to isolate a G-like protein from neural cells.
  • a protein of MW 39,000 has been found to bind specifically a GAP-43 with high affinity.
  • Cell extracts were introduced into columns containing GAP-43 in a buffer comprising
  • the protein elutes in a single band with equimolar EDTA buffer. The protein does not react
  • inventions may be produced by any known means, for example, using recombinant genetic methods as described hereinabove, and that ' ⁇ I ?
  • nucleotide sequences encoding the IRP peptides of the invention may be deduced and optomized for a desired host expression system with the exercise of merely routine skill
  • GAP-43 there is a family of other molecules that contain sequences similar to the amino terminus of GAP-43, and hence regulate G 0 from inside the cell. It is of great interest that the amino terminus of GAP-43 bears a significant resemblance to the cytosolic domains of several G protein-linked receptors (such as the beta receptor). This suggests that GAP-43 may interact with G 0 in a similar place in the molecule as do the cytosolic domains of the G protein-linked receptors. It will further be appreciated that the novel modulatory effects of
  • IRP peptides herein may be, for example, stimulatory or inhibitory.
  • Therapeutics 7th ed., Macmillan Publishing Co., New York (1985), or the current editions thereof. As such, they may act as agonists, partial agonists, reverse agonists, antagonists, etc. Those of skill will recognize
  • desired protein comprising introducing into an environment comprising said desired protein and its binding substrate a substance which it is
  • the desired protein may be a G protein, and, preferably-, is G 0 .
  • GAP-43 Stimulates GTP 1 —g 'S ' " Binding ⁇ * to G U...
  • GAP-43 itself does not bind (35S)GTPuS, but GAP-43
  • G 0 is known to be partially inactivated during
  • a GAP-43 Decapeptide Interacts With G Q .
  • decapeptide which are critical for membrane association, are also required for G 0 regulation, a decapeptide with threonines substituted for
  • neurotransmitter receptor structure e.g., a large extracellular region
  • B2-adrenergic receptor linkage with G s is interrupted most specifically by a point mutation at a palmitylated
  • cysteine in the cytoplasmic tail of the protein (O ' Doyvd, B.F. et al.. J. Biol.
  • GAP-43 yvhich are required for G Q regulation are also subject to
  • GAP-43 is not homologous to this peptide.
  • adrenergic receptor with G s is interrupted most specifically by a point mutation at a palmitylated cysteine in the cytoplasmic tail of the protein.
  • cysteines in the amino terminus of GAP -43 are also palmitylated. There is a consensus sequence shared by GAP-43 and these receptors
  • GAP -43 has previously been shown to bind calmodulin, an interaction
  • the present data provide a growth cone mechanism for the
  • alpha polypeptide exists in a GDP-bound state until an
  • agonist-receptor complex causes the exchange of GTP for GDP.
  • GTP-bound activated alpha subunit then exerts its action on second
  • G 0 is predominantly expressed in brain, where it is the major form of G protein. In adults, it is found in the neuropil (-).
  • G 0 may respond to a
  • GAP-43 is expressed only in neurons, and the protein is enriched in the growth cone. Therefore, the present inventors knew whether GAP-43 might interact with G 0 .
  • GAP-43 enhances GTPgS binding to G G .
  • a small region of GAP-43. defined by a synthetic decapeptide. exerts this action. Stimulation of GTPgS binding by GAP-43 is similar to that by agonist-receptor complexes, and the decapeptide sequence has homology with these receptors.
  • GAP-43 mimics transmembrane receptors and activates G Q , creating a GTP -bound alpha subunit which then triggers an intracellular second messenger system.
  • GAP-43 binding to G Q might function primarily to disrupt G 0 -receptor or G 0 -effector interactions. It is also conceivable that GAP -43 is an effector of G 0 activation by receptor in some as yet unknown manner.
  • GAP -43 provides a mechanism to integrate extracellular signals yvith an intracellular program for neuronal groyvth. Further regulation of the system could occur via other modifications, such as phosphorylation of the receptor by receptor kinases such as BARK, or phosphorylation of GAP-43 by protein kinase C. In this model, GAP-43 might synergistically enhance the response of G 0 to extracellular ligands,
  • GAP-43 is unique among G-protein regulators in that it is an intracellular protein with no presently known capacity to respond directly
  • GAP protein, GAP, Despite the similarity in their names. GAP and GAP-43 are
  • palmitylated state The relative ability of palmitylated versus non-
  • GAP-43 also exists in discrete regions of the adult
  • G 0 /G j antagonist pertussis toxin, blocks long-term potentiation, perhaps implicating G 0 in this process as well (Goh, J.W..
  • G 0 may transduce intracellular

Abstract

ADNc GAP-43 de mammifère cloné. L'invention porte sur les séquences de nucléotides et les séquences amino-acides correspondantes de GAP-43 humain et de GAP-43 du rat. Les séquences pratiquement pures visées par la présente invention peuvent s'exprimer chez des hôtes procaryotes et eucaryotes; elles présentent une utilité pour le suivi et la régulation de la croissance neuronale chez les animaux et chez l'être humain. L'invention porte sur des peptides nouveaux ciblés sur des membranes qui sont capables de diriger toute protéine ou tout peptide désiré sur la membrane cellulaire de cellules neuronales ou non neuronales. L'invention révèle en outreque des dérivés de GAP 43 et de peptide agissent comme des protéines et des peptides de régulation interne de protéines G, tels que Go. Les séquences pratiquement pures décrites par la présente invention sont utilisées à des fins thérapeutiques et diagnostiques.
PCT/US1992/003014 1991-04-10 1992-04-10 Compositions et methodes d'utilisation de gap-43 de mammifere WO1992018138A1 (fr)

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EP0673385A1 (fr) * 1992-08-13 1995-09-27 The General Hospital Corporation Compositions concernant gap-43 des mammiferes et procedes d'utilisation
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US10596223B2 (en) 2011-12-21 2020-03-24 Xigen Inflammation Ltd. JNK inhibitor molecules for treatment of various diseases
US10624948B2 (en) 2013-06-26 2020-04-21 Xigen Inflammation Ltd. Use of cell-permeable peptide inhibitors of the JNK signal transduction pathway for the treatment of various diseases
US11331364B2 (en) 2014-06-26 2022-05-17 Xigen Inflammation Ltd. Use for JNK inhibitor molecules for treatment of various diseases
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Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0673385A1 (fr) * 1992-08-13 1995-09-27 The General Hospital Corporation Compositions concernant gap-43 des mammiferes et procedes d'utilisation
EP0673385A4 (fr) * 1992-08-13 1995-11-29 Gen Hospital Corp Compositions concernant gap-43 des mammiferes et procedes d'utilisation.
WO1995015765A1 (fr) * 1993-12-07 1995-06-15 The General Hospital Corporation Peptides permettant de surmonter l'inhibition de la croissance nerveuse
US5543498A (en) * 1993-12-07 1996-08-06 The General Hospital Corporation Peptides to overcome inhibition of nerve growth
US8183339B1 (en) 1999-10-12 2012-05-22 Xigen S.A. Cell-permeable peptide inhibitors of the JNK signal transduction pathway
US8236924B2 (en) 1999-10-12 2012-08-07 Xigen Sa Cell-permeable peptide inhibitors of the JNK signal transduction pathway
US8278413B2 (en) 1999-10-12 2012-10-02 Xigen Sa Cell-permeable peptide inhibitors of the JNK signal transduction pathway
US8569447B2 (en) 1999-10-12 2013-10-29 Xigen Sa Cell-permeable peptide inhibitors of the JNK signal transduction pathway
US8748395B2 (en) 2005-09-12 2014-06-10 Xigen Inflammation Ltd. Cell-permeable peptide inhibitors of the JNK signal transduction pathway
US9290538B2 (en) 2005-09-12 2016-03-22 Xigen Inflammation Ltd. Cell-permeable peptide inhibitors of the JNK signal transduction pathway
US9006185B2 (en) 2008-05-30 2015-04-14 Xigen Inflammation Ltd. Use of cell-permeable peptide inhibitors of the JNK signal transduction pathway for the treatment of various diseases
US9180159B2 (en) 2008-05-30 2015-11-10 Xigen Inflammation Ltd. Use of cell-permeable peptide inhibitors of the JNK signal transduction pathway for the treatment of chronic or non-chronic inflammatory digestive diseases
US9610330B2 (en) 2008-05-30 2017-04-04 Xigen Inflammation Ltd. Use of cell-permeable peptide inhibitors of the JNK signal transduction pathway for the treatment of various diseases
US10023615B2 (en) 2008-12-22 2018-07-17 Xigen Inflammation Ltd. Efficient transport into white blood cells
US8981052B2 (en) 2010-06-21 2015-03-17 Xigen Inflammation Ltd. JNK inhibitor molecules
US9624267B2 (en) 2010-06-21 2017-04-18 Xigen Inflammation Ltd. JNK inhibitor molecules
US9150618B2 (en) 2010-10-14 2015-10-06 Xigen Inflammation Ltd. Use of cell-permeable peptide inhibitors of the JNK signal transduction pathway for the treatment of chronic or non-chronic inflammatory eye diseases
US10596223B2 (en) 2011-12-21 2020-03-24 Xigen Inflammation Ltd. JNK inhibitor molecules for treatment of various diseases
US10624948B2 (en) 2013-06-26 2020-04-21 Xigen Inflammation Ltd. Use of cell-permeable peptide inhibitors of the JNK signal transduction pathway for the treatment of various diseases
US11779628B2 (en) 2013-06-26 2023-10-10 Xigen Inflammation Ltd. Use of cell-permeable peptide inhibitors of the JNK signal transduction pathway for the treatment of various diseases
US11331364B2 (en) 2014-06-26 2022-05-17 Xigen Inflammation Ltd. Use for JNK inhibitor molecules for treatment of various diseases

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