Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • 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
  • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• the present invention relates to a novel polynucleotides and proteins encoded by such polynucleotides, along with therapeutic, diagnostic, and research utilities for these polynucleotides and proteins.
• the invention relates to polynucleotides and proteins encoded by such polynucleotides which bind to b-amyloid peptide, one of the primary components of amyloid deposits associated with Alzheimer's Disease.
• AD Alzheimer's disease
• CNS central nervous system
• BAP is a 39-43 amino acid peptide derived by proteolytic cleavage of amyloid precursor protein (hereinafter "APP") and composed of a portion of the transmembrane domain and the luminal/extracellular domain of APP. It is thought that the BAP peptide comprising 42 amino acids (BAP42) is potentially the more toxic aggregated form in humans. APP occurs as several BAP-containing isoforms. The major forms are comprised of 695, 751 , and 770 amino acids, with the latter two APP containing a domain that shares structural and functional homologies with Kunitz serine protease inhibitors. In normal individuals, BAP does not accumulate and is rapidly removed from circulating fluids.
• APP amyloid precursor protein
• the peptide can form plaques on surfaces of dystrophic dendrites and axons, microglia, and reactive astrocytes.
• the aggregation and deposition of BAP in neuritic plaques is postulated as one of the initiating events of AD.
• Investigation of the events leading to the expression and consequences of BAP and their individual roles in AD is a major focus of neuroscience research.
• the discovery of proteins that bind BAP is critical to advance understanding of the pathogenesis of the disease and to potentially introduce novel therapeutic targets.
• proteins and fragments thereof which bind with human BAP and which may be involved in the biological effects of BAP in AD had not been identified. Summary of the Invention
• This invention provides novel isolated polynucleotides which encode gene products that selectively bind human ⁇ -amyloid peptide (BAP) amino acid sequences.
• BAP ⁇ -amyloid peptide
• the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) polynucleotide comprising the nucleotide sequence of SEQ
• a polynucleotide comprising the nucleotide sequence of a ⁇ - amyloid peptide-binding protein (BBP) of clone BBP1 -fl deposited under accession number ATCC 9861 7;
• BBP ⁇ - amyloid peptide-binding protein
• a polynucleotide comprising the nucleotide sequence of SEQ
• ID NO: 1 from nucleotide 202 to nucleotide 807;
• a polynucleotide comprising the nucleotide sequence of a ⁇ - amyloid peptide-binding protein (BBP) of clone pEK1 96 deposited under accession number ATCC 98399;
• BBP ⁇ - amyloid peptide-binding protein
• polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1 ; the nucleotide sequence of a ⁇ -amyloid peptide-binding protein (BBP) of clone BBP1 -fl deposited under accession number ATCC 9861 7; or a polynucleotide encoding a ⁇ -amyloid peptide-binding protein (BBP) encoded by the cDNA insert of clone BBP1 -fl deposited under accession number ATCC 9861 7.
• BBP ⁇ -amyloid peptide-binding protein
• Another embodiment provides the gene corresponding to the cDNA sequence of SEQ ID NO: 1 .
• the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
• fragments of the amino acid sequence of SEQ ID NO: 2 comprising the amino acid sequence from amino acid 1 85 to amino acid 21 7 of SEQ ID NO: 2.
• amino acid sequence of SEQ ID NO: 2 comprising the amino acid sequence from amino acid 1 85 to amino acid 21 7 of SEQ ID NO: 2.
• amino acid sequence of SEQ ID NO: 2 comprises the amino acid sequence of SEQ ID NO: 3
• the polynucleotide is operably linked to an expression control sequence.
• the invention also provides a host cell, including bacterial, yeast, insect, and mammalian cells, transformed with such polynucleotides compositions.
• Processes are also provided for producing a BBP which comprises (a) growing a culture of the host cell of claim 3 in a suitable culture medium; and (b) purifying the protein from the culture medium.
• compositions comprising an antibody which specifically reacts with such BBPs are also provided by the present invention.
• Another embodiment of the invention includes transgenic animals comprising a polynucleotide encoding a BBP operably linked to an expression control sequence.
• FIG. 1 Yeast 2-hvbrid screen design.
• a Y2H host strain expressing the Gal4 DNA-binding domain fused to BAP 42 (BAP BD ; plasmid containing TRP1 marker) and nonfusion BAP 42 (BAP; plasmid containing URA3 marker) was transformed with a Y2H human fetal brain cDNA library (plasmid containing LEU2 marker) expressing Gal4 activation domain fusion proteins (unknown AD ) as described. Therefore, strains contained three episomal plasmids, denoted by circles, expressing the indicated protein. Positive protein-protein interactions reconstituted Gal4 activity at the upstream activating sequence (GALUAS) thereby inducing transcription of the reporter gene HIS3.
• GLUAS upstream activating sequence
• Figure 2 Demonstration of BBP1 /BAP association.
• Y2H strains were assayed for histidine prototrophy by making 10-fold serial dilutions and spotting 5 ⁇ l on synthetic agar medium lacking tryptophan, leucine, histidine and containing 25 mM 3-amino-triazole as described. All strains contain the BAP fusion protein expression plasmid pEK1 62 as indicated by the label BAP.
• the first columns (vector) contain independently derived strains carrying pEK1 62 and the vector pACT2 expressing an irrelevant fusion protein. These serve as a measure of background for comparison with strains expressing target proteins.
• BBPI Dtm express a truncated BBP1 from pEK1 98, as described in the text.
• the interaction between BAP and BBPI Dtm fusion proteins reconstitutes Gal4 activity, resulting in induction of a HIS3 reporter gene (see Figure 1 ), observed as enhanced prototrophic growth compared to the control strains.
• Figure 3 Bioassays demonstrating BBP1 interactions with G ⁇ proteins.
• the predicted intracellular domain of BBP1 was expressed as a Gal4 DNA-binding domain with portions of rat Gas, Gao, or Gai2 expressed as Gal4 activation domain fusion proteins.
• Y2H responses of two independently derived clones of each strain were compared to responses of cells lacking a G protein component (vector). The protocol is as described in the legend to Figure 2.
• FIG. 4 Localization of the interactions between BBP1 and BAP.
• BBPI ⁇ tm was divided into two overlapping segments as described in the text. These proteins, BBP1 ⁇ C or BBP1 ⁇ N, were assayed for interactions with BAP.
• the assay method and the strains labeled vector or BBPI ⁇ tm are as described in the legend to Figure 2.
• Strains labeled BBP1 ⁇ C or BBP1 ⁇ N express the indicated BBP1 segment as a fusion protein.
• FIG. 5 Expression of BBP1 mRNA in human tissues (A) and brain regions (B). Nylon membranes blotted with 2 ⁇ g size fractionated poly-A RNA isolated from the indicated tissues were obtained from CLONTECH. These were hybridized with a radiolabeled BBP1 cDNA probe as described. A predominant band corresponding to 1 .25 kb (determined from molecular weight markers, not shown) was observed in all lanes. Higher molecular weight bands likely correspond to heteronuclear RNA; the BBP1 gene contains several introns. Blots were stripped and reprobed with ⁇ -actin as a loading and RNA integrity control; all lanes exhibited equivalent signal (data not shown).
• Figure 7 Comparison of BBP1 interactions with human or rodent BAP. Rodent BAP was engineered and expressed as a fusion protein as described in the text. The strains labeled human BAP are identical to those shown in Figure 2.
• the strains labeled rodent BAP express rodent BAP as the Gal4 DNA-binding domain fusion.
• Vector indicates control strains containing only vector opposing the BAP fusion proteins;
• BBP1 indicates strains expressing the BBPI ⁇ tm fusion protein.
• the present invention relates to the isolation and cloning of a human ⁇ -amyloid peptide binding protein (BBP1 ).
• BBP1 has been characterized as a fusion protein in a yeast 2 hybrid assay as binding to the 42 amino acid fragment of BAP (BAP42).
• BAP42 the 42 amino acid fragment of BAP
• Expression of BBP1 has been shown in human tissues and in specific brain regions ( Figure 5).
• BBP1 has been demonstrated to selectively bind human BAP in a yeast 2 hybrid system as compared to rodent BAP.
• the initial human BBP1 clone (designated clone 14) was obtained by using a yeast 2-hybrid (Y2H) genetic screen developed to identify proteins which interact with human BAP 42 , a potentially more toxic form of BAP.
• BAP 42 was expressed fused to the yeast Gal4 DNA-binding domain and was also expressed as free peptide ( Figure 1 ).
• This strain was transformed with a human fetal brain cDNA Y2H library.
• a single clone, denoted #14 from approximately 10 6 independent transformants, produced consistent reporter gene activation and contained a substantial open reading frame continuous with that of the GAL4 domain.
• the cDNA insert comprised 984 base pairs, terminating in a poly-A tract.
• This sequence encoded 201 amino acids (amino acid 68 to amino acid 269 of SEQ ID NO: 2) with two regions of sufficient length and hydrophobicity to transverse a cellular membrane. There are also potential asparagine-linked glycosylation sites.
• Clone 14 was designated clone pEK196 and deposited as ATCC 98399 .
• the library-derived plasmid was isolated from clone 14 and used to reconstruct Y2H assay strains. Examination of these strains demonstrated that the BAP fusion protein specifically interacted with the clone 14 protein, although the response was weak.
• clone 14 insert was truncated (BBPI ⁇ tm; see Table 2 below for further description) to remove the region of strongest hydrophobicity and retested for interactions with BAP.
• BBPI ⁇ tm protein domains of strong hydrophobicity
• Clone 14 identifies a novel BAP binding protein in the form of a fusion protein.
• the BBP1 cDNA sequences contained in clone 14 were identified as lacking the 5' end of the protein coding region as no potential initiating methionine codon was present.
• Multiple attempts at conventional 5' RACE (rapid amplification of cDNA ends) utilizing a standard reverse- transcriptase only resulted in the addition of 27 nucleotides.
• a genomic cloning approach as described in Example 2, below, was used to isolate the 5' terminus.
• nucleotide sequences which encode BBP1 , fragments, fusion proteins or functional equivalents thereof may be used to generate recombinant DNA molecules that direct the expression of BBP1 , or a functionally active peptide, in appropriate host cells.
• nucleotide sequences which hybridize to portions of the BBP1 sequence may be used in nucleic acid hybridization assays, Southern and Northern blot assays, etc.
• the invention also includes polynucleotides with sequences complementary to those of the polynucleotides disclosed herein.
• the present invention also includes polynucleotides capable of hybridizing under reduced stringency conditions, more preferably stringent conditions, and most preferably highly stringent conditions, to polynucleotides described herein.
• stringency conditions are shown in the table below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R.
• the hybrid length is that anticipated for the hybridized region(s) of the hybridizing polynucleotides.
• the hybrid length is assumed to be that of the hybridizing polynucleotide.
• the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity.
• each such hybridizing polynucleotide has a length that is at least 25%(more preferably at least 50%, and most preferably at least 75 %) of the length of the polynucleotide of the present invention to which it hybridizes, and has at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with the polynucleotide of the present invention to which it hybridizes, where sequence identity is determined by comparing the sequences of the hybridizing polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps.
• the isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res. _9, 4485-4490 ( 1 991 ), in order to produce the protein recombinantly.
• an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res. _9, 4485-4490 ( 1 991 ), in order to produce the protein recombinantly.
• Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 1 85, 537-566 (1 990).
• operably linked means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.
• Mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 ceils, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
• monkey COS cells Chinese Hamster Ovary (CHO) cells
• human kidney 293 ceils human epidermal A431 cells
• human Colo205 cells human Colo205 cells
• CV-1 cells other transformed primate cell lines
• normal diploid cells cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
• yeast in lower eukaryotes such as yeast or in prokaryotes such as bacteria.
• yeast strains include Saccharomyces cerevisiae,
• the protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system.
• kits form Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac7 kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1 555 (1987), incorporated herein by reference.
• an insect cell capable of expressing a polynucleotide of the present invention is "transformed.”
• the protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein.
• the resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography.
• the purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl7 or Cibacrom blue 3GA Sepharose7; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.
• the protein of the invention may also be expressed in a form which will facilitate purification.
• it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transf erase (GST) or thioredoxin (TRX) .
• Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively.
• the protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope.
• One such epitope (“Flag") is commercially available from Kodak (New Haven, CT).
• RP-HPLC reverse-phase high performance liquid chromatography
• hydrophobic RP-HPLC media e.g., silica gel having pendant methyl or other aliphatic groups
• RP-HPLC media e.g., silica gel having pendant methyl or other aliphatic groups
• Some or ail of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein.
• the protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated protein.
• the protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.
• the protein may also be produced by known conventional chemical synthesis. Methods for constructing the proteins of the present invention by synthetic means are known to those skilled in the art.
• the synthetically-constructed protein sequences by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.
• the proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered.
• modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques.
• Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence.
• one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule.
• Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., USP No. 4,51 8,584).
• such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.
• Y2H assays demonstrated that the association of BAP with the BBP1 fusion protein is specific.
• the association of BBP1 with BAP suggests that BBP1 activity may have a defined role in the pathogenesis of Alzheimer's disease.
• BBP1 sequences were compared to Genbank using the basic local alignment search tool (BLAST; Altschul et al., 1 990).
• the BBP1 protein and translations of available expressed sequence tags were aligned, searched for conserved segments, and evaluated by the MoST (Tatusov et al., 1 994) protein motif search algorithm.
• GPCR G protein-coupled receptor
• the intervening hydrophilic loop contains a well-characterized three amino acid motif, aspartate (D) or glutamate followed by arginine (R) and an aromatic residue (Y or F) (commonly referred to as the DRY sequence), that is conserved in almost all members of this receptor family and has been shown to serve as a molecular trigger for G protein activation (Acharya and Karnik, 1 996).
• BBP1 represents a novel protein potentially containing a functional module shared with members of the G protein-coupled receptor superfamily. Specifically, it appears that BBP1 retains the critical DRF sequence (amino acids 1 99 to amino acids 201 of SEQ ID NO: 2), between two predicted tm domains, and may have the potential to couple to a G protein regulated signaling pathway.
• BBP1 contains a structural motif known to be a G ⁇ protein activating sequence in the related G protein-coupled receptors. Additionally, a hypothesis based on the predicted position and orientation of BBP1 tm domains suggests that the region of the protein that interacts with BAP would be topographically constrained to the same location as BAP in APP. Y2H assay strains were engineered to evaluate the association of the BBP1 intracellular region with G ⁇ proteins. The predicted intracellular sequences of BBP1 were expressed as a fusion protein and assayed for interactions with C-terminal regions of three G ⁇ proteins.
• BBP1 BBP1 ⁇ N and BBP1 ⁇ C, ( Figure 4)).
• the ⁇ C construct lacked both tm domains; the ⁇ N construct encoded the first tm domain plus the preceding 52 amino acids.
• BBP1 ⁇ C protein induced a weak Y2H response (compare BBP1 ⁇ C to vector, Figure 4), but the BBP1 ⁇ N protein, containing the first tm domain and adjacent amino-proximal sequences produced a response only slightly weaker than that observed with BBPI ⁇ tm ( Figure 4).
• the Y2H system was utilized to demonstrate the selectivity and specificity of BBP1 binding to human BAP as compared to rodent BAP.
• the rodent peptide demonstrates reduced neurotoxicity and an absence of binding to human brain homogenates (Maggio et al., 1 992). It was of interest, therefore, to evaluate the association of rodent BAP with BBP1 in the Y2H system.
• the sequence of human BAP in pEK1 62 was changed to encode the rodent peptide by oligonucleotide directed mutagenesis by PCR.
• the resultant plasmid, pEK240 is identical to the human BAP fusion protein expression plasmid utilized throughout this report except for the three codons producing the amino acid substitutions for the rodent peptide sequence.
• Interactions between BBP1 fusion protein and rodent and human BAP fusion proteins were compared by Y2H bioassay. Strains expressing BBP1 and the rodent BAP failed to produce a growth response ( Figure 7). This finding supports the premise that BBP1 may serve as a specific mediator of the neurotoxic effects of BAP, and provides a mechanism to explain the reduced neurotoxicity of the rodent BAP.
• Proteins and protein fragments of the present invention include proteins with amino acid sequence lengths that are at least 25 %(more preferably at least 50%, and most preferably at least 75 %) of the length of a disclosed protein and have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with that disclosed protein, where sequence identity is determined by comparing the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while minimizing sequence gaps.
• proteins and protein fragments that contain a segment preferably comprising 8 or more (more preferably 20 or more, most preferably 30 or more) contiguous amino acids that shares at least 75 % sequence identity (more preferably, at least 85 % identity; most preferably at least 95% identity) with any such segment of any of the disclosed proteins.
• Species homologues of the disclosed polynucleotides and proteins are also provided by the present invention.
• a species homologue is a protein or polynucleotide with a different species of origin from that of a given protein or polynucleotide, but with significant sequence similarity to the given protein or polynucleotide.
• polynucleotide species homologues have at least 60% sequence identity (more preferably, at least 75 % identity; most preferably at least 90% identity) with the given polynucleotide, and protein species homologues have at least 30% sequence identity (more preferably, at least 45% identity; most preferably at least 60% identity) with the given protein, where sequence identity is determined by comparing the nucleotide sequences of the polynucleotides or the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while minimizing sequence gaps.
• Species homologues may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.
• species homologues are those isolated from mammalian species. Most preferably, species homologues are those isolated from certain mammalian species such as, for example, Pan troglodytes, Gorilla gorilla, Pongo pygmaeus, Hylobates concolor, Macaca mulatta, Papio papio, Papio hamadryas, Cercopithecus aethiops, Cebus capucinus, Aotus trivirgatus, Sanguinus oedipus, Microcebus murinus, Mus musculus, Rattus norvegicus, C ⁇ cetulus griseus, Felis catus, Mu stela vison, Can is familiaris, Oryctolagus cuniculus, Bos taurus, Ovis aries, Sus scrofa, and Equus caballus, for which genetic maps have been created allowing the identification of syntenic relationships between the genomic organization of genes in one species and the genomic organization of the related genes in another species
• allelic variants of the disclosed polynucleotides or proteins that is, naturally-occurring alternative forms of the isolated polynucleotides which also encode proteins which are identical or have significantly similar sequences to those encoded by the disclosed polynucleotides.
• allelic variants have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% identity) with the given polynucleotide, where sequence identity is determined by comparing the nucleotide sequences of the polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps.
• Allelic variants may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from individuals of the appropriate species.
• the invention also includes polynucleotides with sequences complementary to those of the polynucleotides disclosed herein.
• BBP1 proteins of the present invention can be used in a variety of applications routine to one of skill in the art based upon this disclosure.
• the BBPs can be used as immunogens to raise antibodies which are specific to the cloned polypeptides.
• Various procedures known in the art may be used for the production of antibodies to BBP1 proteins.
• Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments and an Fab expression library.
• various host animals including, but not limited to rabbits, mice, and rats, are injected with a BBP.
• the polypeptide or a fragment of the polypeptide capable of specific immunoactivity is conjugated to an immunogenic carrier.
• Adjuvants may also be administered in conjunction with the polypeptide to increase the immunologic response of the host animal.
• adjuvants include, but are not limited to, complete and incomplete Freund's, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
• Monoclonal antibodies to BBP1 proteins of the present invention can be prepared using any technique which provides for the production of antibodies by continuous cell line in culture. Such techniques are well known to those of skill in the art and include, but are not limited to, the hybridoma technology originally described by Kohler and Milstein (Nature 1 975, 256,4202-497), the human B-cell hybridoma technique described by Kosbor et al. (Immunology Today 1 983, 4, 72) and the EBV- hybridoma technique described by Cole et al. (Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp 77-96).
• Antibodies immunoreactive to the polypeptides of the present invention can then be used to screen for the presence and subcellular distribution of similar polypeptides in biological samples.
• monoclonal antibodies specific to the BBP1 proteins of the present invention can be used as therapeutics.
• the BBP1 proteins can also serve as antigens useful in solid phase assays measuring the presence of antibodies which immunoreact with the claimed peptides.
• Solid phase competition assays can be used to measure immunological quantities of clone 14-reiated antigen in biological samples. This determination is not only useful in facilitating the complete characterization of the cellular function or functions of the polypeptides of the present inventions, but can also be used to identify patients with abnormal amounts of these proteins.
• BBP1 proteins of the present invention can also be used as capture reagents in affinity chromatography for the detection of BAP and BAP aggregates as markers for AD.
• BBP1 s are useful as reagents in an assay to identify candidate molecules which effect the interaction of BAP and the cloned protein. Compounds that specifically block this association could be useful in the treatment or prevention of AD.
• BBP1 s are also useful in acellular in vitro binding assays wherein alteration by a compound in the binding of these beta amyloid peptide associated proteins to BAP or BAP aggregates is determined.
• Acellular assays are extremely useful in screening sizable numbers of compounds since these assays are cost effective and easier to perform than assays employing living cells.
• the development of these assays would be routine to the skilled artisan.
• either BBP1 or BAP is labeled.
• Such labels include, but are not limited to, radiolabels, antibodies, and fluorescent or ultraviolet tags. Binding of a BBP1 to BAP or BAP aggregates is first determined in the absence of any test compound. Compounds to be tested are then added to the assay to determine whether such compounds alter this interaction.
• Y2H Y2H expression plasmids were constructed in vectors pAS2 and pACT2 (described in
• PCR polymerase chain reaction
• Oligonucleotides #1 (5' - CC ATG GAT GCA GAA TTC CGA C) and #3 (5' - AAGCTTGTCGAC TTA CGC TATGAC AAC ACC GC) were used to amplify BAP using pCLL621 , a modified human APP clone (Jacobsen et al., 1 994), as template.
• the amplified DNA consists of codons 389 to 430 (which encodes BAP 42 ) of the APP precursor protein with the following modifications.
• the sense strand primer added a 5' Ncol restriction site in the same translational reading frame as the Ncol site in pAS2.
• the antisense strand primer added a stop codon and Hindlll and Sail sites for cloning.
• the product from this amplification was ligated into the TA cloning system (Invitrogen Corp., Carlsbad, CA), and subsequently removed by digestion with Ncol and Sail. This fragment was cloned into pAS2 cleaved with Ncol plus Sail.
• the resultant plasmid, pEK1 62 was confirmed by DNA sequencing through the GAL4/BAP junction.
• the protein (BAP B ; Figure 1 ) expressed from pEK162 comprised a fusion protein containing the DNA-binding domain of the yeast transcriptional activation protein Gal4 (lacking functional activation sequences) with the addition of the 42 amino acids of BAP to the carboxy-terminus.
• An expression plasmid was developed that mediates the expression of unmodified BAP 42 .
• Oligo #2 (5' - AAGCTTAAG ATG GAT GCA GAA TTC CGA C) was paired with oligo #3 in a PCR as described above.
• the product of this amplification contains a 5' Hindlll site and translation initiation signals optimized for expression in Saccharomyces cerevisiae. Again, the DNA fragment was cloned into the TA system.
• a plasmid library consisting of cDNA fragments isolated from human fetal brain cloned into the yeast 2- hybrid expression vector pACT2 (which used LEU2 as the selection marker) was purchased from Clontech Laboratories, Inc. (Palo Alto, CA). The library-derived protein is depicted in Figurel as unknown AD .
• This library was used to transform CY2091 .
• the samples were spread on synthetic complete (SC) yeast growth medium lacking uracil, tryptophan, and leucine to select cells containing all three plasmids.
• the medium also lacked histidine and contained 3-amino-triazole, an inhibitor of the product of the yeast HIS3 gene, at a concentration of 25 mM.
• 3-Amino-triazole was utilized to reduce activity from low-level constitutive expression of the HIS3 reporter gene. Plates were incubated at 30°C for 1 2 days. Twenty-four colonies exhibiting increased histidine prototrophy were isolated. Transformation controls indicated that the screen assayed 10 6 individual clones.
• a PCR approach was utilized to quickly determine the content of positive clones. Total DNA was isolated from each positive strain by standard methods. This material was used as template for PCRs using oligos #4 (5' - TTTAATACCA CTACAATGGA T) plus #5 (5' - TTTTCAGTAT CTACGATTCA T) which flank the cloning region of the library vector pACT2. DNA fragments were ligated into the TA system and examined by DNA sequencing. The library plasmid contained in clone #14 (as described above) was isolated by shuttle into E. coli. The nucleotide sequence of the human cDNA sequences was determined, confirming the sequence of the initial PCR product.
• Bioassays Strains were grown overnight in 2ml SC medium lacking leucine and tryptophan to a density of approximately 7 x 10 7 cells per ml. Cells were counted and 1 0-fold serial dilutions made from 1 0 4 to 10 8 cells per ml in sterile water. These samples were spotted in 5 ⁇ l aliquots on SC medium lacking leucine, tryptophan and histidine and containing 25 mM 3-amino-triazole. Plates were incubated at 30°C for 2 to 3 days.
• Sense primer #6 (5'- TTTAATACCA CTACAATGGA T) annealed to GAL4 sequences in pACT2.
• the antisense primer #7 (5'-CTCGAG TTA AAA TCG ATC TGC TCC CAA CC) incorporated a 3' stop codon and Xhol site immediately 3' to the sequences encoding the DRF motif of BBP1 .
• the PCR product was ligated into the TA cloning vector and subsequently digested with EcoRI + Xhol and cloned into pACT2.
• the hybrid product expressed from this plasmid (pEK1 98) was denoted BBPI ⁇ tm.
• primer #7 was paired with primer #8 (5'-GAATT CCA AAA ATA AAT GAC GCT ACG) to engineer the BBP1 ⁇ N expression plasmid pEK216.
• the PCR product was ligated into the TA system and the resultant plasmid digested with fcoRI + Xhol with the BBP1 fragment (codons 1 23-202) finally ligated into pACT2 digested with the same enzymes.
• BBP1 ⁇ C was made by using the pACT2-specific oligo #6 with antisense oligo #9 (5'- CTCGAG TCA AGA TAT GGG CTT GAA AAA AC).
• G ⁇ protein expression plasmids utilized the Bam ⁇ site near the center of each rat cDNA sequence (Kang et al., 1 990) as the site of fusion in pACT2.
• Sense primers annealed to sequences 5' of the BamVW site; antisense primers annealed to sequences 3' of the stop codon and included a Sail restriction site.
• PCR products were cloned into the TA vector. G ⁇ sequences were isolated as BamYW-Sall fragments and cloned into pACT2 digested with Bam ⁇ + Sail. See Table 2 for plasmid designations.
• oligonucleotide #23 was synthesized for the conversion of human BAP to the rodent sequence.
• This primer has the sequence 5'- ATATGGCCATG GAT GCA GAA TTC GGA CAT GAC TCA GGA TJT GAA GTT CGT.
• the triplets represent the first 1 3 codons of BAP; the three nucleotides that were changed to produce the rodent sequence are underlined.
• Oligo #23 was paired with #24 (5'-TGACCTACAG GAAAGAGTTA) which anneals to a region of the Y2H vectors that is 3' of the cloning site in a PCR using pEK1 62 as the template.
• the product was cleaved with Ncol + Sail and ligated into pAS2 to produce pEK240.
• the nucleotide sequence of the segment encoding rodent BAP was confirmed.
• This oligonucleotide was the reverse complement of nucleotides. 1 57-201 ( Figure 2) and has the sequence 5'-CCAGGCGGCC GCCATCTTGG AGACCGACAC TTTCTCGCCA CTTCC. Lambda phage DNA was isolated by standard molecular biology techniques and subjected to direct sequencing using fluorescent dideoxy cycle sequencing on an ABl 373 sequencer.
• RACE First strand DNA synthesis was performed using the xTth thermal-stable polymerase system (Perkin Elmer). The following reagents were combined in a 1 .5 mL tube to give a 10 microliter volume: 1 X reverse transcription buffer, 1 mM MnCI 2 , 1 .6 mM dNTP mix, 2.5U rTth polymerase, 100ng human hippocampus poly A + RNA (Clontech), 10mM oligonucleotide (nt 429-452, Figure 2; 5'-GTTATGTTGG GTGCTGGAAA ACAG). The reaction was incubated at 70°C for 1 5 minutes and immediately placed on ice.
• the Marathon cDNA synthesis kit (Clontech) was used for second strand cDNA generation.
• the entire 10 vl from the first strand reaction was combined with the following reagents: 1 X second strand buffer, 0.8 mM dNTP mix, 4X second strand cocktail (E.coli DNA polymerase I, E.coli DNA ligase, E.co/i RNaseH), and dH 2 0 up to a volume of 80 ⁇ l.
• the tube was incubated at 1 6-C for 1 .5 hours after which time T4 DNA polymerase ( 10U) was added and incubated for an additional 45 minutes at 1 6-C.
• T4 DNA polymerase 10U
• EDTA/glycogen (0.2M EDTA/2mg/ml glycogen) was added to the reaction mixes followed by a phenol/chloroform/isoamyl alcohol extraction to remove enzymes and other impurities.
• the DNA was precipitated by adding 0.1 X volume 3M Na acetate pH 5.2 and 2.5X volume reagent grade EtOH and place at -70-C. The DNA was washed once with 70% EtOH, dried down and resuspended in 10 ⁇ l dH 2 0.
• the PCR cycling conditions were an initial denaturing cycle of 9' at 94°C, 25 cycles of 30" at 94°C, 30" at 68°C, 2' at 72°C, followed by a 72°C extension for 7'.
• the PCR product was run on a 1 % agarose gel in 1 XTBE buffer.
• the resulting 350 base pairs product was gel purified and directly cloned using the TA Cloning Kit (Invitrogen) .
• Ligation mixes were transformed into OneShot Cells (Invitrogen) and plated on LB-ampicillin ( l OO ⁇ g/ml) agar plates containing X-gal. Mini-prep DNA was obtained and examined by fluorescent dideoxy cycle sequencing on an ABI 373 sequencer.
• oligonucleotide primers were designed to amplify a 275 base pairs region from the 3' UTR of the BBP1 cDNA and, in addition, add the promoter sequences for T7 (sense) and T3 (antisense) polymerase. These primers contained the following sequences: 5'-TAATACGACT CACTATAGGG TTAGAAGAAA CAGATTTGAG (forward); 5'-ATTAACCCTC ACTAAAGGGA CAAGTGGCAA CTTGCCTTTG (reverse) .
• PCR products were gel purified on 1 .5% low-melt agarose gels, and bands containing the products were excised, phenol and phenol-chloroform extracted, and ethanol precipitated. Pellet were dried and resuspended in 1 X TE buffer ( 10 mM Tris-HCI, 1 mM EDTA, pH 7.4).
• the APP riboprobe template consisted of a Ddel-Xhol fragment from the protein coding region, as described by Jacobsen et al. ( 1 991 ). Fifty ng of DNA template was used for transcription reactions using ( 35 S)-CTP (New England Nuclear, Boston, MA) and the Riboprobe GeminiTM System (Promega, Madison, Wl).
• Labeled riboprobes were added at a final concentration of 50,000 cpm///l to hybridization solution containing 0.9M NaCl, I mM EDTA, 1 x Denhardt's, 0.1 mg/ml yeast tRNA, 0.1 mg/ml single-stranded salmon sperm DNA, dextran sulfate ( 10%), 0.08% BSA, 10mM DTT (Boehringer Mannheim, Indianapolis, IN), and 50% deionized formamide in 10mM Tris (pH 7.6). The probes were then denatured at 95 °C ( 1 min), placed on ice (5 min), and pipetted onto the sections and allowed to hybridize overnight at 55 °C in a humidified chamber.
• the sections were washed in 2XSSC for 1 hr at 60°C, followed by 0.1 XSSC containing 0.5% (w/v) sodium thiosulfate for 2 hrs. at 60°C.
• the sections were then dehydrated in 50, 70, 90% ethanol containing 0.3M ammonium acetate, and dried.
• the slides were loaded into X-ray cassettes and opposed to Hyperfilm b-Max (Amersham) for 14- 30 days. Once a satisfactory exposure was obtained, the slides were coated with nuclear-track emulsion (NTB-2; Kodak) and exposed for 7-21 days at 4°C.
• NTB-2 nuclear-track emulsion
• emulsion autoradiograms were developed and fixed according to the manufacturer's instructions, and the underlying tissue sections were stained with hematoxylin.
• a control probe was generated from a template provided in the Riboprobe GeminiTM System kit (Promega) . This vector was linearized using Seal and transcribed using T3 polymerase. The resulting transcription reaction generates two products, a 250 base and a 1 ,525 base riboprobe, containing only vector sequence.
• This control probe mixture was labeled as described above and added to the hybridization solution at a final concentration of 50,000 cpm/ ⁇ l. No specific hybridization was observed in control sections, i.e., these sections gave a very weak uniform hybridization signal that did not follow neuroanatomical landmarks (data not shown).
• Example 1 Cloning and Isolation BAP-binding protein (BBP1 ).
• a yeast 2-hybrid (Y2H) genetic screen was developed to identify proteins which interact with human BAP 42 , a 42 amino acid proteolytic fragment of APP which is considered to potentially be the more toxic aggregated form of BAP.
• BAP 42 was expressed fused to the yeast Gal4 DNA-binding domain and was also expressed as free peptide ( Figure 1 ).
• This strain was transformed with a human fetal brain cDNA Y2H library.
• the cDNA insert comprised 984 base pairs, terminating in a poiy-A tract. This sequence encoded 201 amino acids (SEQ ID NO: 2; amino acid residues 68 to 269) with two regions of sufficient length and hydrophobicity to transverse a cellular membrane.
• the library-derived plasmid was isolated from clone 14 and used to reconstruct Y2H assay strains. Examination of these strains demonstrated that the BAP fusion protein specifically interacted with the clone 14 protein, although the response was weak. Since protein domains of strong hydrophobicity, such as transmembrane regions, inhibit Y2H responses (Ozenberger, unpublished data), clone 14 insert was truncated (hereinafter BBPI ⁇ tm) to remove the region of strongest hydrophobicity and retested for interactions with BAP. A much more robust Y2H response was observed with BBPI ⁇ tm (Figure 2), supporting the notion that the deleted sequences encode a potential transmembrane ("tm”) anchor.
• the nucleotide sequence of Clone 14 was searched against GenBank; the BAP binding protein (BBP1 ) thus identified appeared to be novel.
• Example 2 Isolation and confirmation of the 5' terminus of BBP1 .
• Hybridization of a human genomic lambda library with a randomly- primed probe corresponding to 400 base pairs (bps) of the 5' sequence of clone 14 resulted in identification of 10 positive clones. Further characterization of these clones using a 45-base oligonucleotide probe directed to the most upstream BBP1 sequence of clone 14 (and corresponding to the 5' upstream sequence of the 400 base pairs probe revealed that 6 of the 10 clones included the terminal 5' sequences contained within those previously identified. It was determined that the other 4 lambda clones represented other exons which were contained within the original 400 base pairs randomly-primed cDNA-derived probe (data not shown).
• BBP1 sequences were compared to Genbank using the basic local alignment search tool (BLAST; Altschul et al., 1 990) . Two Caenorhabditis elegans and one Drosophila melanogaster genomic sequence and a large number of human, mouse and other mammalian expressed sequence tags were identified. However, no complete cDNA sequences were available nor were any functional data attributed to the gene.
• the BBP1 protein and translations of available expressed sequence tags were aligned, searched for conserved segments, and evaluated by the MoST (Tatusov et al., 1 994) protein motif search algorithm. These analyses revealed a potential evolutionary relationship to the G protein-coupled receptor family.
• BBP1 contains two potential transmembrane (tm) domains equivalent to tm domains 3 and 4 of G protein-coupled receptors.
• the intervening hydrophilic loop contains a well-characterized three amino acid motif, aspartate (D) or glutamate followed by arginine (R) and an aromatic residue (Y or F) (commonly referred to as the DRY sequence), that is conserved in almost all members of this receptor family and has been shown to serve as a molecular trigger for G protein activation (Acharya and Karnik, 1 996).
• D aspartate
• R arginine
• Y or F aromatic residue
• BBP1 represents a novel protein potentially containing a functional module shared with members of the G protein-coupled receptor superfamily.
• BBP1 retains the critical DRF sequence between two predicted tm domains, so may have the potential to couple to a G protein regulated signaling pathway.
• BBP1 Structural analysis of BBP1 indicated it contained a structural motif known to be a G ⁇ protein activating sequence in the related G protein- coupled receptors.
• Y2H assays demonstrating the interaction of BBP1 with various members of the G protein coupled receptors are illustrated in Figure 3. Based on structural predictions, BBP1 is depicted as transversing a membrane twice with both termini in the lumenal compartment. Other orientations cannot be entirely ruled out. The potential protein interactions described above were investigated in Y2H assays.
• Example 4 Tissue distribution of human BBP1 expression.
• BBP1 mRNA was evaluated as an initial step in elucidating the activity of the gene and its product. A major transcript of 1 .25 kb was observed in all tissues (Figure 5A). There was a high level of expression in heart. Whole brain exhibited an intermediate level of expression. Samples derived from separate brain regions all exhibited BBP1 expression ( Figure 5B). Interestingly, limbic regions contained relatively greater amounts of BBP1 mRNA. These are the regions of the brain where BAP aggregation and associated neurotoxicity initially occur.
• BBP1 mRNA is expressed in medium to large cells in a pattern consistent with expression in neurons as opposed to glial cells ( Figure 6) .
• BBP1 mRNA is expressed in virtually all hippocampal and entorhinal neurons, i.e., there do not appear to be any real or laminar differences in the intensity of the hybridization signal.
• the pattern of BBP1 expression was strikingly similar to the pattern observed using a riboprobe directed against mRNA for the amyloid precursor protein APP (Figure. 6).
• BBP1 mRNA was observed in all tissues and all brain regions examined. In situ analysis of BBP1 mRNA expression also revealed extensive expression in the hippocampus region.
• Example 5 Cell line distribution of BBP1 expression.
• BBP1 expression was also investigated in numerous cell lines and data were extracted from dbEST, the collection of expressed sequence tags from the National Center for Biotechnology Information.
• Reverse- transcription polymerase chain reaction (RT-PCR) methods were utilized to qualitatively assess BBP1 mRNA expression in cell lines commonly utilized for recombinant protein expression as well as a variety of cancer cell lines.
• BBP1 was observed in hamster CHO and human HEK293 cells. Signals were observed in the embryonic stem cell line Ntera-2 and neuroblastoma lines IMR32 and SK-N-SH.
• BBP1 expression was observed in cancer cell lines representing the following tissue origins: colon (Cx-1 , Colo205, MIP101 , SW948, CaCo, SW620, LS1 74T), ovarian (A2780S, A2780DDP), breast (MCF-7, SKBr-3, T47-D, B7474), lung (Lx-1 , A5439), melanoma (Lox, Skmel30), leukemia (HL60, CEM), prostate (LNCAP, Du 145, PC-3).
• a Northern blot probing mRNA isolated from the following cancer cell lines demonstrated BBP1 expression in all samples: promyelocytic leukemia (HL-60), carcinoma (HeLa S3), chronic myelogenous leukemia (K-562), lymphoblastic leukemia (MOLT-4), Burkitt's lymphoma (Raji), colorectal adenocarcinoma (SW480), lung carcinoma (A549), and melanoma (G361 ).
• Example 6 Selective interaction of BBP1 with human BAP versus rodent BAP There are three amino acid substitutions (G5R, F10Y and R1 3H) in the rodent BAP sequence compared to the human sequence.
• the rodent peptide demonstrated reduced neurotoxicity and an absence of binding to human brain homogenates (Maggio et al., 1 992). It was of interest, therefore, to evaluate the association of rodent BAP with BBP1 in the Y2H system.
• the sequence of human BAP in pEK1 62 was changed to encode the rodent peptide by oligonucleotide directed mutagenesis by PCR, described above.
• the resultant plasmid, pEK240 was identical to the human BAP fusion protein expression plasmid utilized throughout the present invention except for the three codons producing the amino acid substitutions for the rodent peptide sequence.
• Alzheimer's disease Nature 382, 685-691 .
• Jacobsen, J, Muenkel, H, Blume, A, and Vitek, M ( 1 991 ).
• Kang, Y.-S. Kane, J., Kurjan, J., Stadel, J., and Tipper, D. ( 1 990). Effects of expression of mammalian Ga and hybrid mammalian-yeast Ga proteins on the yeast pheromone response signal transduction pathway. Mol Cell Biol 10, 2582-2590. LaFerla, F., Tinkle, B., Bieberich, C, Haudenschild, C, and Jay, G. (1 995).
• the Alzheimer's A ⁇ peptide induces neurodegeneration and apoptotic cell death in transgenic mice. Nature Genetics 9, 21 -30.
• Alzheimer amyloid protein precursor complexes with brain GTP-binding protein Go. Nature 362, 75-79.
• Alzheimer's disease Nat Med 2, 864-870. Selkoe, D. ( 1 997). Alzheimer's Disease: Genotypes, phenotype, and treatments. Science 275, 630-631 .
• Amyloid ⁇ protein gene cDNA, mRNA distribution and genetic linkage near the Alzheimer locus. Science 235, 880-884.
• Cip1 is a potent inhibitor of G 1 cyclin-dependent kinases. Cell 75, 805-81 6.
• Ser Ser Tyr lie lie Asp Tyr Tyr Gly Thr Arg Leu Thr Arg Leu Ser 245 250 255 lie Thr Asn Glu Thr Phe Arg Lys Thr Gin Leu Tyr Pro 260 265

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