SK143299A3 - BETA-AMYLOID PEPTIDE-BINDING PROTEINS AND POLYNUCLEOTIDESì (54) ENCODING THE SAME - Google Patents

Abstract

Novel proteins which bind human beta -amyloid peptide, polynucleotides which encode these proteins, and methods for producing these proteins are provided. Diagnostic, therapeutic, and screening methods employing the polynucleotides and polypeptides of the present invention are also provided.

Description

Technical field

The present invention relates to novel polynucleotides and proteins encoded by these polynucleotides, as well as to the therapeutic, diagnostic and research uses of these polynucleotides and proteins. In particular, the invention relates to polynucleotides and proteins encoded by such polynucleotides that bind to a β-amyloid peptide, one of the primary components of amyloid deposits accompanying Alzheimer's disease.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a progressive dementia of advanced age characterized by a number of structural abnormalities of the brain. Neurons in numerous areas of the central nervous system (CNS) become dysfunctional and die, resulting in changes in synaptic inputs. The cell bodies and proximal dendrites of these vulnerable neurons comprise a neurofibrillary network composed of connected spiral filaments, the main component of which is a phosphorylated microtubule-binding protein, in particular tau. One characteristic of the disease is the accumulation of amyloid-containing deposits in the brain called senile (or neuritic) plaques. The major component of amyloid plaques is the β-amyloid peptide (hereinafter called abbreviated BAP, also referred to in the literature as Αβ, βΑΡ, etc.), which forms dense aggregates during AD.

BAP is a 39-43 amino acid peptide resulting from proteolytic cleavage from an amyloid precursor protein (hereinafter referred to as APP) and composed of part of the transmembrane domain and the luminal / extracellular domain of APP. The 42 amino acid BAP peptide (BAP42) is believed to be a potentially more toxic aggregated form to humans. APP occurs as an isoform containing several BAPs. The major forms consist of 695, 751 and 770 amino acids, the latter two APPs comprising a domain having structural and functional homologies with Kunitz serine protease inhibitors. In normal individuals, BAP is not accumulated and is rapidly removed from circulating fluids. However, the peptide may form plates on the surface of dystrophic dendrites and axons, microglia, and reactive astrocytes. Aggregation and deposition (deposition) of BAP in neuritic plaques is believed to be one of the events that trigger AD. The study of the phenomena leading to the expression and importance of BAP and its individual role in AD is a major focus of neurological research. The discovery of BAP binding proteins is particularly crucial for advancing understanding of the pathogenesis of the disease and for the possible introduction of new therapeutic targets. Until the present invention, proteins and fragments thereof that bind to human BAP and have been implicated in the biological effects of BAP in AD have not been identified.

SUMMARY OF THE INVENTION

The present invention provides novel isolated polynucleotides that encode gene products that selectively bind the amino acid sequences of the human β-amyloid peptide (BAP).

In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:

(a) a polynucleotide comprising the nucleotide sequence set forth herein as SEQ ID NO: 1 (SEQ ID NO: 1);

(b) a polynucleotide comprising the nucleotide sequence of the β-amyloid peptide (BBP) binding protein (BBP) of the BBP1-fl clone deposited under accession number ATCC 98617;

(c) a polynucleotide encoding a β-amyloid peptide (BBP) binding protein encoded by the cDNA insert of the BBP1-fl clone deposited under ATCC accession number 98617;

d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO. no. 1 from nucleotide 202 to nucleotide 807,

(e) a polynucleotide containing the nucleotide sequence of the β-amyloid peptide (BBP) binding protein (BBP) of clone pEK196 deposited under ATCC accession number 98399;

(f) a polynucleotide encoding a β-amyloid peptide (BBP) binding protein encoded by the cDNA insert of clone pEK196 deposited under ATCC accession number 98399;

g) a polynucleotide encoding a protein comprising the amino acid sequence set forth herein as SEQ ID NO. no. 2

h) a polynucleotide encoding a protein comprising the fragment of the amino acid sequence of SEQ ID NO: 1; no. 2, which has binding activity for the human β-amyloid peptide, a fragment comprising the amino acid sequence from amino acid 68 to amino acid 269 of SEQ ID NO: 2; no. 2

(i) a polynucleotide which is an allelic variant of the polynucleotide of (a) to (f) above;

(j) a polynucleotide which encodes a species homolog of the protein of (g) to (i) above; and

(k) a polynucleotide capable of hybridizing under stringent conditions to each of the polynucleotides defined in (a) to (h).

These polynucleotides preferably include the nucleotide sequence set forth herein as SEQ ID NO: 1, the nucleotide sequence of the β-amyloid peptide (BBP) binding protein (BBP) of the BBP1-fl clone deposited under ATCC accession number 98617, or a polynucleotide encoding β-amyloid peptide (BBP) binding protein. cDNA insert of the BBP1-fl clone deposited under ATCC Accession No. 98617. Further elaboration provides a gene corresponding to cDNA sequence id. no. First

In other embodiments, the present invention provides a composition comprising a protein, said protein comprising an amino acid sequence selected from the group consisting of:

a) the amino acid sequence set forth herein as SEQ ID NO: 2; no. 2

b) the amino acid sequence of SEQ ID NO. no. 2 from amino acid 68 to amino acid 269,

(c) the amino acid sequence encoded by the cDNA insert of the clone BP1-fl deposited under accession number ATCC 98617, and

d) fragments of amino acid sequence id. no. 2, comprising the amino acid sequence from amino acid 185 to amino acid 217 of sequence id. no. Second

Preferably, these proteins comprise the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2. no. 2 from amino acid 68 to amino acid 269. Fusion proteins are also claimed in the present invention.

In certain preferred embodiments, 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 these polynucleotide compositions.

The invention also provides methods for producing BBP, which include;

(a) culturing a host cell culture according to claim 3 in a suitable culture medium; and

b) purifying the protein from the culture medium.

The present invention also provides compositions comprising an antibody that specifically reacts with these BBPs.

Methods and diagnostic procedures for detecting a disease state characterized by aberrant expression of human BAP are provided, as well as methods for identifying compounds that regulate BBP activity.

A further embodiment of the invention includes transgenic animals comprising a polynucleotide encoding a BBP operably linked to an expression control sequence.

Image overview

The following figures illustrate only certain embodiments of the invention. They are illustrative only and do not limit the invention described herein.

Figure 1: Scheme of the yeast two-hybrid test. The Y2H host strain expressing the Gal4 DNA binding domain fused to BAP ₄₂ (BAPBD, plasmid containing the TRP1 tag) and unfused BAP ₄₂ (BAP, a plasmid containing the URA3 tag) was transformed with the human fetal brain cDNA library in Y2H (plasmid containing the LEU2 tag) Gal4 activating domain proteins (unknown ^ ⁰ ) as described. The strains contained three episomal plasmids, indicated by rings, expressing said protein. Positive protein-protein interactions reconstituted Gal4 activity upstream of the activation sequence (GALUAS), thereby inducing transcription of the HIS3 reporter gene.

Figure 2: Evidence of BBP1 / BAP association. Y2H strains were tested for histidine prototrophy by making a decimal serial dilution and dropping 5 μΐ on synthetic agar lacking tryptophan, leucine, histidine and containing 25 mM 3-amino-triazole as described. All strains contained the expression plasmid pEK162 with the BAP fusion protein as indicated by the BAP description. The first columns (vector) contain independently derived strains carrying pEK162 and a vector pACT2 expressing an irrelevant fusion protein. These serve as a background measure for comparison with strains expressing the target proteins. The bars labeled BBPIDtm express the truncated BBP1 of pEK198 as described herein. The interaction between BAP fusion proteins and BBPIDtm reconstitutes Gal4 activity, resulting in the induction of the HIS3 reporter gene (see Figure

1), observed as increased prototrophic growth compared to control strains.

Figure 3: Biological assays demonstrating the interaction of BBP1 with protein G. The predicted intracellular domain of BBP1 was expressed as a Gal4 DNA binding domain with portions of rat Gas, Gao or Gai2 expressed as fusion proteins of the Gal4 activating domain. The Y2H responses of two independently derived clones of each strain were compared to those of cells lacking the protein G component (vector). The protocol is the same as in the legend to Figure 2.

Figure 4: Localization of interactions between BBP1 and BAP. BBP1Atm was divided into two overlapping segments as described herein. These proteins, BBP1AC and BBP1ÔN, were tested for interactions with BAP. The assay method and strains labeled with the vector or BBP1Atm are as in the legend to Figure 2. BBPlac or ΒΒΡ1ΔΝ labeled strains express the designated BBP1 segment as a fusion protein.

Figure 5: BBP1 mRNA expression in human tissues (A) and brain regions (B). Nylon membranes with 2 µg of transferred poly-A RNA isolated from the tissues, sized by size, were obtained from CLONTECH. The membranes were hybridized with a radiolabeled BBP1 probe cDNA as described. A predominant band of 1.25 kb (determined by molecular weight standard, not shown) was observed in all lanes. Higher molecular weight bands probably correspond to heteronuclear RNA, the BBP1 gene contains several introns. The probe was removed from the blots and hybridized again with β-actin as a control for integrity and amount.

RNA, all lanes showed an equivalent signal (data not shown).

Figure 6: Expression of BBP1 and APP in hippocampus cells. In situ autoradiogram images of in situ hybridization showing the expression pattern of BBP1 (A) and APP (B) in the human hippocampus and entor a different Intex cortex. The sections used to make these images were taken from postmortem samples obtained from two different patients. Abbreviations: DG = dentate gyrus, CA1 = hippocampus area, EC = entorinal cortex.

Figure 7: Comparison of BBP1 interactions with human BAP or rodent BAP. Rodent BAP was constructed and expressed as a fusion protein as described herein. Strains designated human BAP are identical to those shown in Figure 2. Strains designated rodent BAP express rodent BAP as a fusion of the Gal4 DNA binding domain. The vector indicates control strains containing only the vector against the BAP fusion proteins, BBP1 indicates the strains expressing the BBP fusion protein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the isolation and cloning of human β-amyloid peptide (BBP1) binding protein. BBP1 was characterized in the yeast two-hybrid assay as a fusion protein that binds to a 42 amino acid fragment from BAP (BAP ₄₂ ). BBP1 expression has been demonstrated in human tissues and specific areas of the brain (see Figure 5). Importantly, BBP1 has been shown to selectively bind human BAP in a yeast two-hybrid system as opposed to rodent BAP. These findings support the hypothesis that BBP1 of the present invention can be used in diagnosis and treatment

Alzheimer's disease, as well as for the evaluation and testing of drugs for regulating the accumulation of amyloid-containing plaques in the brain.

BBP1 coding sequences

The human BBP1 starting clone (designated as clone 14) was obtained using a yeast two-hybrid (Y2H) genetic filter developed to identify proteins that interact with human BAP ₄₂ , a potentially more toxic form of BAP. BAP ₄₂ was expressed fused to the DNA binding domain of the yeast Gal4 gene and was also expressed as the free peptide (Figure 1). This strain was transformed with the human fetal brain cDNA Y2H library. One knee, marked no. 14, of about 10 ⁶ independent transformants, provided consistent activation of the reporter gene and contained an open reading frame related to the GAL4 domain frame. The cDNA insert contained 984 base pairs terminated with a poly-A region. This sequence encoded 201 amino acids (amino acid 68 to amino acid 269 of SEQ ID NO: 2) with two regions whose length and hydrophobicity were sufficient to cross the cell membrane. There are also potential glycosylation sites associated with asparagine. Strain 14 was designated as 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 showed that the BAP fusion protein specifically interacts with the protein of clone 14, although the reaction was weak. Since strongly hydrophobic protein domains, such as transmembrane regions, inhibit Y2H responses (Ozenberger, unpublished data), the clone 14 insert was truncated (BBP1 ™, see Table 2 below for further description) to remove the region of strongest hydrophobia and to test interactions again. with BAP. A much stronger Y2H response was observed with BBP1 ™, supporting the notion that the removed sequences encode a potential transmembrane (tm) anchor. Clone 14 identifies a novel BAP binding protein in the form of a fusion protein.

The BBP1 cDNA sequences contained in clone 14 have been shown to lack the 5 'end of the coding region of the protein because no potential initiation methionine codon is present. Numerous experiments with the conventional 5 'RAČE (rapid amplification of cDNA ends) method using standard reverse transcriptase resulted in only 27 nucleotides. Therefore, a genomic cloning procedure was used to isolate the 5 'end as described in Example 2 below.

Since the sequence encoding the 5 'end originated from a genomic library, there was a possibility that this region contained introns. This possibility was investigated by two methods as described in Example 2 below. The resulting data confirmed the sequences upstream (both genomic and cDNA) and the absence of introns in this region. The BBP1-fl plasmid containing the cDNA insert encoding the complete coding region of BBP1 was deposited with the American Type Culture Collection (ATCC) under accession number 98617. The complete coding region and the deduced protein sequence are shown in SEQ ID NOs. no. 1 and 2. The untranslated nucleotide sequences 3 'are contained in the original clone 14 (pEK196).

According to the present invention, nucleotide sequences that encode BBP1, fragments, fusion proteins, or functional equivalents thereof, can be used to produce recombinant DNA molecules that direct the expression of BBP1 or a functionally active peptide in the respective host cells. Alternatively, nucleotide sequences that hybridize to portions of the BBP1 sequence may be used for nucleic acid hybridization, Southern and northern analyzes, and the like.

The invention also includes polynucleotides with sequences complementary to those of the polynucleotides described herein. The present invention also encompasses polynucleotides capable of hybridizing under less stringent conditions, preferably under stringent conditions, and most preferably under high stringency conditions, with the polynucleotides described herein. Examples of stringent conditions are shown in the table below, highly stringent conditions are those that are at least stringent, such as conditions A- to F, stringent conditions are those stringent, at least such as conditions G to L, and weakly stringent conditions are those, which are at least stringent, such as conditions M to R.

Stringent conditions

stringent conditions Polynucleotide hybrid length hybrid (Bp) " hybridization temperature and buffer solution " Temperature to washing and buffer ' A DNA: DNA 50 65 [deg.] C .; lxSSC or 42 ° C; lxSSC, 50% formamide 65 ° C; 0,3xSSC B DNA: DNA <50 T _B *; x SSC T _B *; x SSC C DNA: RNA 50 67 ° C; lxSSC or 45 ° C; lxSSC, 50% formamide 67 ° C; 0,3xSSC D DNA: RNA <50 T _D *; x SSC T _D *; x SSC E RNA: RNA 50 65 ° C; lxSSC or 42 ° C; lxSSC, 50% formamide 70 ° C; 0,3xSSC F RNA: RNA <50 T _F *; x SSC T _F * J 1xSSC G DNAiDNA 50 65 ° C, 4xSSC or 42 ° C; 4xSSC, 50% formamide 65 ° C; x SSC H DNAiDNA <50 T _H *; T _h *; 4xSSC I DNAiRNA 50 67 ° C, 4xSSC or 45 ° C; 4xSSC, 50% formamide 67 ° C; x SSC J DNAiRNA <50 * That is, 4 x SSC * That is, 4 x SSC The RNAiRNA 50 70 ° C, 4xSSC or 50 ° C; 4xSSC, 50% formamide 67 ° C; x SSC L RNAiRNA <50 T ₁ *; 2xSSC T ₁ *; 2xSSC M DNAiDNA 50 50 ° C, 4xSSC or 40 ° C; 6xSSC, 50% formamide 50 ° C; 2xSSC

N DNA: DNA <50 T _n *; 6xSSC T _n *; 6xSSC ABOUT DNA: RNA 50 55 ° C, 4xSSC or 42 ° C; 6xSSC, 50% formamide 55C; 2xSSC P DNA: RNA <50 T _P *; 6xSSC Tp *, 6 * SSC Q RNA: RNA 50 60 ° C, 4xSSC or 45 ° C; 6xSSC, 50% formamide 60; 2xSSC R RNA: RNA <50 Tr *, 6 * SSC Tr *, 6 * SSC

The hybrid length is the length expected for the hybridized region of i) hybridizing polynucleotides. When a polynucleotide is hybridized to a target polynucleotide of an unknown sequence, the hybrid length is assumed to be equal to the length of the hybridizing polynucleotide. When polynucleotides of known sequence hybridize, the length of the hybrid can be determined by comparing the polynucleotide sequences and determining the region or regions by optimal sequence complementarity.

SSPE (1xSSPE is 0.15 M NaCl, 10 mM NaH 2 PO 4, and 1.25 mM EDTA, pH 7.4) can be replaced by SSC (1xSSC is 0.15 M NaCl and 15 mM sodium citrate) in hybridization and wash buffers, washes are performed 15 minutes after hybridization is complete. *

Tg - T _R : the hybridization temperature for hybrids expected to be less than 50 base pairs should be 5-10 ° C lower than the melting point T_ of the hybrid, where T _m is determined by the following equations. For hybrids shorter than 18 base pairs - T (= C) = 2 (base number A + T) + 4 (base number G + C). For hybrids between 18 and 49 base pairs in length - T _m (° C) = 81,5 + 16,6 (log ₁₀ [Na ⁺ ]) + 0,4l (% G + C) - (600 / N), where N is the number of bases in the hybrid, and [Na +] is the concentration of sodium ions in the hybridization buffer ([Na ⁺ ] for 1xSSC = 0.165 M).

Additional examples of stringent conditions for polynucleotide hybridization are provided in: Sambrook, J., Fritsch, EF, Maniatis, T., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, Chapters 9 and 11 , and Ausubel, FM et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference.

Each such hybridizing polynucleotide is preferably long such that it is at least 25% (more preferably at least 50% and most preferably at least 75%) as long as the polynucleotide of the present invention with which it hybridizes and has at least 60% identical sequence (more preferably at least 75%, most preferably at least 90% or 95%) with the polynucleotide of the present invention with which it hybridizes, wherein sequence identity is determined by comparing the sequences of hybridizing polynucleotides when aligned to maximize overlap and identity while minimizing sequence differences.

BBP1 expression

The isolated polynucleotide of the invention may be operably linked to an expression control sequence, such as the expression vectors pMT2 or pED disclosed in Kaufman et al. (Nucleic Acids Res., 19, 4485-4490, 1991) to produce a recombinant protein. Many suitable expression control sequences are known in the art. General methods for the expression of recombinant proteins are also known and exemplified in Kaufman, R. (Methods in Enzymology, 185, 537-566, 1990). As defined herein, operably linked means that the isolated polynucleotide of the invention and expression control sequences are located in a vector or cell such that the protein is expressed by a host cell that has been transformed (transfected) with a ligated polynucleotide / expression control sequence.

Expression system for BBP1

A variety of cell types can serve as a suitable host cell for protein expression. Mammalian host cells include, for example, COS monkey cells, Chinese hamster ovary (CHO) cells, human kidney 293 cells, human 31431 epidermal cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cellular cells. strains derived from in vitro cultures of primary tissues, primary explants, HeLa cells, mouse L cells, BHK cells, HL-60 cells, U937 cells, HaK cells or Jurkat cells.

Alternatively, it is possible to produce the protein in lower eukaryotes, such as yeast, or in prokaryotes, such as bacteria. Possible suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida or any yeast strain capable of expressing heterologous proteins. Possible suitable strains of bacteria include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If it is a protein produced in yeast or bacteria, it may be necessary to modify the protein so produced, for example by phosphorylating or glycosylating the respective sites, to obtain a functional protein. Such covalent bonds may be performed using known chemical or enzyme methods.

The protein may also be produced by operably linking an isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors and using an insect expression system. Materials and methods for baculovirus / insect cell expression systems are commercially available as a kit, e.g. from Invitrogen, San Diego, California, U.S.A. (MaxBac7 kit) and these methods are well known in the art, as described in Summers and Smith (Texas Agricultural Experiment Station Bulletin No. 1555, 1987), incorporated herein by reference. According to terminology as used herein, an insect cell capable of expressing the polynucleotide of the present invention is transformed.

The protein of the invention can be prepared by growing transformed host cells under culture conditions suitable for expression of the recombinant protein. The resulting expressed protein is then purified from such a culture (i.e., tissue medium or cell extracts) using known purification procedures such as gel filtration and ion exchange chromatography. Protein purification may also include the use of an affinity column containing a protein binding agent, one or more use of a column with affinity resins such as concavalin A-agarose, heparin-toyopear17 or Cibacrom blue 3GA Sepharose7, one or more steps involving chromatography with hydrophobic interaction using resins such as phenylether, butyl ether or propyl ether, or immunoaffinity chromatography.

Alternatively, the protein of the invention may also be expressed in a form facilitating purification. For example, it may be expressed as a fusion protein, such as maltose binding protein (MBP), glutathione-S-transferase (GST), or thioredoxin (TRX). Kits for the expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen. An epitope can also be attached to the protein and the protein can then be purified using a specific antibody directed against that epitope. One such epitope (Flag) is commercially available from Kodak (New Haven, CT).

Finally, one or more reverse phase high performance liquid chromatography (RP-HPLC) procedures using hydrophobic RP-HPLC media, e.g. silica gel having attached methyl or other aliphatic groups. Some or all of the foregoing purification procedures in various combinations may also be used to obtain a substantially homogeneous isolated recombinant protein. The protein thus purified is substantially free of other mammalian proteins and according to the present invention is defined as an isolated protein.

The protein of the invention may also be expressed as the product of transgenic animals, e.g. as a milk component of transgenic cows, goats, pigs or sheep, characterized in that their somatic or germ cells contain a nucleotide sequence encoding a protein.

The protein may also be produced by known conventional chemical synthesis. Methods for constructing the proteins of the present invention by synthetic routes are known to those skilled in the art. Synthetically constructed protein sequences, by having a common primary, secondary or tertiary structure and / or conformational properties with proteins, can retain common biological capabilities, including protein activity. Thus, they can be used as biologically active or immunological substitutes for natural purified proteins for screening therapeutic compounds and in immunological procedures for antibody production.

The proteins of the invention also include proteins in which the characteristic amino acid sequences are similar to those of the purified proteins, but for which modifications occur naturally or deliberately created. One of ordinary skill in the art can create using known techniques, for example, modification of peptide sequences or DNA sequences. Desired protein sequence modifications may include alterations, substitutions, substitutions, insertions, or deletions of the selected amino acid residue in the coding sequence. For example, in order to alter the conformation of the molecule, one or more cysteine residues may be removed or replaced by another amino acid. Techniques for such substitution, substitution, insertion or deletion alterations are well known to those skilled in the art (see, e.g., U.S. Patent 4,518,584). Preferably, these alterations, substitutions, substitutions, insertions or deletions leave the desired protein activity.

Other fragments and derivatives of the protein sequences that would be expected to retain full or partial activity of the protein and thus could be useful for screening or other immunological methods can also be readily generated by one skilled in the art based on the present invention. It is therefore considered that these modifications are also encompassed by the present invention.

Yeast two-hybrid tests

Y2H assays have shown 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 sequences using a basic locator comparison program (BLAST, Altschul et al., 1990). BBP1 protein and translation of available short expressed marker tags were compared, screened for conserved segments, and evaluated using the MoST protein motif search algorithm (Tatusov et al., 1994). These analyzes revealed a possible evolutionary relationship to the family of protein G-coupled receptors (GPCR). More specifically, these analyzes have shown that BBP1 contains two potential transmembrane (tm) domains equivalent to the tm domains of 3 and 4 receptors associated with protein G. The intervening hydrophilic loop contains a well characterized three amino acid motif, aspartate (D) or glutamate followed by arginine (R) and aromatic residue (Y or F) (commonly referred to as the DRY sequence), this motif is retained by nearly all members of this receptor family and has been shown to serve as a molecular trigger for protein G activation (Acharya and Karnik, 1996).

Data from the Y2H assays (see Figures 2-4) show that BBP1 is a novel protein potentially containing a functional module in common with members of a large family of protein G-coupled receptors. More specifically, BBP1 shows a critical DRF sequence (amino acids 199 to 201 of 201). SEQ ID NO: 2), between the two predicted tm domains, and may have the potential to associate with the protein G-driven signaling pathway.

APP has also been shown to functionally associate with Gao (Nishimoto et al., 1993, Yamatsuji et al., 1996) and BBP1 contains a structural motif known to be a Ga protein activation sequence at related G protein-coupled receptors. In addition, the hypothesis based on the predicted position and orientation of the tm domain of BBP1 suggests that the protein region that interacts with BAP is topographically limited to the same location as BAP in APP.

Y2H assay strains were designed to evaluate the association of BBP1 intracellular region with Ga proteins. The predicted intracellular BBP1 sequences were expressed as a fusion protein and tested for interactions with the C-terminal regions of the three Ga proteins. The protein segments used in these experiments are listed in Table 2 below. The intracellular loop of BBP1 interacted with all three Ga proteins (Figure 3), supporting the assumption that BBP1 can act as a modulator of protein G activity. These various Y2H assays suggest a complex model of a composite protein complex composed of at least full membrane proteins BBP1 and APP attached to a heterotrimeric protein. G.

Table 2

Plasmids used in yeast two hybrid assays

Expression plasmid protein segment BAP pEK162 (Human) 1-42 pEK240 (Mouse) 1-42 BBP1 pEK196 (clone 14) 68-269 pEK198 (Atm) 68-202 pEK219 (AC) 68-175 pEK216 (AN) 123-202 pOZ339 (Intracellular) 185-217 ga pOZ345 (Gas) 235-394 pOZ346 (Gao) 161-302 pOZ348 (G i2) 213-355

Further BBP1 analysis was obtained using Y2H assays. The two overlapping portions of the BBP1 sequence contained in the BBP1 tm clone were amplified and cloned into the Y2H vector pACT2 (expression plasmids pEK216 and pEK219, Table 2 and the corresponding proteins ΒΒΡ1ΔΝ and BBP1AC, (Figure 4)). The AC construct lacked both tm domains, the construct ΔΝ encoded the first tm domain and the previous 52 amino acids. These fusion proteins were tested with the BAP fusion protein and the reactions were compared to those of strains expressing the larger BBP1Atm protein. BBP1AC protein elicited a weak Y2H response (compare BBP1AC to vector, Figure 4), but the proteín1ΔΝ protein containing the first tm domain and adjacent amino-proximal sequences produced a response only slightly weaker than that observed with BBP1 tm (Figure 4). These results suggest that the major determinant for BAP association is contained in the BBP1 region, which is believed to be topographically similar to BAP in the wild-type APP protein.

The Y2H system was used to demonstrate the selectivity and specificity of BBP1 binding to human BAP as compared to rodent BAP. In the rodent BAP sequence there are three amino acid substitutions (G55R, F10Y and R13H) compared to the human sequence In the Y2H assay described in Example 6, the rodent peptide exhibits reduced neurotoxicity and the absence of binding to human brain homogenates (Maggio et al., 1992). It was therefore desirable to evaluate the association of rodent BAP with BBP1 in the Y2H system. The sequence of human BAP in pEK162 was altered to encode the rodent peptide by oligonucleotide-directed mutagenesis by PCR. The resulting plasmid, pEK240, is identical to the expression plasmid of the human BAP fusion protein used throughout this publication except for the three codons making amino acid substitutions in the rodent peptide sequence. The interactions between the BBP1 fusion protein and the human BAP fusion protein and the rodent BAP fusion protein were compared in the Y2H bioassay. Strains expressing BBP1 and rodent BAP did not respond to growth (Figure 7). This finding supports the assumption that BBP1 may serve as a specific mediator of the neurotoxic effect of BAP and provides a mechanism explaining the decreased neurotoxicity of rodent BAP. Importantly, these data also serve to illustrate the high degree of specificity of the BBP1 / BAP interaction in the Y2H assays, since the substitution of the three amino acids was sufficient to completely abrogate the association (Figure 7).

Isolated BBP1 polypeptides

The proteins and protein fragments of the present invention include proteins having an amino acid sequence long such that it is at least 25% (more preferably at least 50% and most preferably at least 75%) long than the described protein, and has at least 60% identical sequence (more preferably at least 75%, most preferably at least 90% or 95%) of the disclosed protein, wherein the identity of the sequences is determined by comparing the amino acid sequences of the proteins, while being aligned to maximize overlap and identity while minimizing sequence differences. Also included in the present invention are proteins and protein fragments that comprise a segment consisting preferably of 8 or more (more preferably 20 or more, most preferably 30 or more) adjacent amino acids having at least 75% identical sequence (more preferably at least 85) %, most preferably at least 95%) with any such segment of each of the disclosed proteins.

The present invention also provides species homologs of the disclosed polynucleotides and proteins. As used herein, a species homolog is a protein or polynucleotide originating from a different species than that of a given protein or polynucleotide, but with significant sequence similarity to a given protein or polynucleotide. Polynucleotide species homologues preferably have at least 60% identical sequence (more preferably at least 75%, most preferably at least 90%) with said polynucleotide, and protein species homologues preferably have at least 30% identical sequence (more preferably at least 45%, most preferably at least 45%). 60%) with a given protein, wherein the identity of the sequences is determined by comparing the nucleotide sequences of the polynucleotides or amino acid sequences of the proteins when aligned to maximize overlap and identity while minimizing sequence differences. Species homologues can be isolated and recognized by generating appropriate probes or primers from the sequences provided herein and testing a suitable source of nucleic acids of the desired species, preferably species homologues are isolated from mammalian species. More preferably, the species homologs are isolated from certain mammalian species such as Pan troglodytes, Gorilia gorilia, Pongo pygmaeus, Hylobates concolor, Macaca mulatta, Papio papio, Papio hamadryas, Cerco22 pithecus aethiops, Cebus capucinus, Aotus trivirceus, Musculus, Rattus norvegicus, Cricetulus griseus, Felis catus, Mustela vison, Canis familiaris, Oryctolagus cuniculus, Bos taurus, Ovis aries, Sus scrofa, and Eguus caballus, for which genetic maps have been created to identify a synthetic relationship between genome genes species and genomic organization of related genes in another species (O'Brien and Seuanez, Ann. Rev. Genet., 22, 323-351, 1988, O'Brien et al., Nature Genetics, 3, 103-112, 1993, Johansson et al., Genomics, 25, 682-690, 1995; Lyons et al., Nature Genetics, 15, 47-56,

1997, O'Brien et al., Trends in Genetics, 13 (10), 393-399; Carver and Stubbs, Genome Research, 7, 1123-1137, 1997).

The invention also encompasses allelic variants of the disclosed polynucleotides or proteins, i. naturally occurring alternative forms of isolated polynucleotides that also encode proteins that are identical or have significantly similar sequences to the proteins encoded by the disclosed polynucleotides. Allelic variants preferably have at least 60% identical sequence (more preferably at least 75%, most preferably at least 90%) with a given polynucleotide, wherein the identity of the sequences is determined by comparing the nucleotide sequences of the polynucleotides when aligned to maximize overlap and identity while minimized sequence differences. Allelic variants can be isolated and recognized by generating appropriate probes or primers from the sequences provided herein and testing a suitable nucleic acid source of individuals of the species concerned.

The invention also includes polynucleotides with sequences complementary to those of the polynucleotides described herein.

Application

The BBP1 proteins of the present invention can be used in the present invention in a variety of applications common to the skilled artisan. More specifically, BBPs can be used as immunogens to generate antibodies that are specific for cloned polypeptides. Various methods known in the art can be used to generate antibodies against BBP1 proteins. Such antibodies include, but are not limited to, monoclonal, polyclonal, chimeric, single chain antibodies, Fab fragments, and a Fab expression library. BBP injections are administered to various host animals, including but not limited to rabbits, mice, and rats. In one embodiment, a polypeptide or fragment of a polypeptide having a specific immunoactivity capability is conjugated to an immunogenic carrier. Adjuvants may also be administered in conjunction with the polypeptide to enhance the immunological response of the host animal. Examples of adjuvants that may be used include, but are not limited to, complete and incomplete Freund's adjuvant, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, Pluronic polyols, polyanions, peptides, oil emulsions, hemocyanin from a suppository and dinitrophenol.

Monoclonal antibodies to the BBP1 proteins of the present invention can be prepared using any procedure that ensures the production of antibodies by a continuous cell line in tissue culture. These procedures are well known to those skilled in the art and include, but are not limited to, the hybridoma technology originally described by Kohler and Milstein (Nature, 256, 4202497, 1975), the human B cell hybridoma method described by Kosbor et al. (Immunology Today, 4, 72, 1983) and the EBVhybridoma method described by Col 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 test for the occurrence and subcellular distribution of similar polypeptides in biological samples. In addition, monoclonal antibodies specific for BBP1 proteins of the present invention can be used as medicaments.

BBP1 proteins can also serve as antigens useful in solid-phase assays measuring the presence of antibodies that immunologically react with the claimed peptides. Solid phase competition assays can be used to measure immunological amounts of antigen related to clone 14 in biological samples. This determination is not only applicable to facilitate complete characterization of the cellular function or functions of the polypeptides of the present invention, 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 to detect BAP and BAP aggregates as markers for AD.

In addition, BBP1 are useful as reagents in the assay to identify candidate molecules that interact with BAP and cloned protein. Compounds that specifically block this association could be useful in the treatment or prevention of AD.

BBP1 are also useful in cell-free in vitro binding assays, where the alteration of the binding of β-amyloid peptide-associated proteins to BAP or BAP aggregates by a particular compound is determined. Cell-free assays are particularly useful in screening a relatively large number of compounds since these assays are cost-effective and easier to perform than assays using live cells. Following the description of the 25 polypeptides of the present invention, the development of these assays is routine to one of ordinary skill in the art. In these assays, it is labeled with either BBP1 or BAP. These labels include, but are not limited to, radiolabels, antibodies, and fluorescent or fluorescent labels

Even ultraviolet marks. Binding of BBP1 to BAP or BAP aggregates is first determined in the absence of test compound. Test compounds are then added to the assay to determine if this compound alters the interaction of interest.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is further described by way of the following examples. The examples serve only to illustrate the invention and demonstrate preferred embodiments of the invention. These preferred embodiments, while illustrating certain specific aspects of the invention, do not constitute a limitation of the invention nor limit the scope of the invention.

Yeast two-hybrid system (Y2H)

Y2H expression plasmids were constructed in the vectors pAS2 and pACT2 (described by WadeHarper et al., 1993) and pCUP (described by Ozenberger and Young, 1995). Yeast strain CY770 (Ozenberger and Young, 1995) served as a host for all Y2H assays.

Genetic filter

The polymerase chain reaction (PCR) method was used to amplify and modify BAP coding sequences. Oligonucleotides no. 1 (5'-CC ATG GAT GCA GAA TTC CGA C) and no. 3 (5'-AAGCTTGTCGAC TTA CGC TATGAC AAC ACC GC) were used to amplify BAP using pCLL621, a modified human APP clone (Jacobsen et al., 1994) as a template. The amplified DNA consisted of codons 389 to 430 (which code for BAP ₄₂ ) ^{from the} precursor protein APP with the following modifications.

The sense strand primer added a 5 'NcoI restriction site to the same translational reading frame as the NcoI site in pAS2. The antisense strand primer added a stop codon and HindIII and SalI sites for cloning. The product from this amplification was ligated into the TA cloning system (Invitrogen Corp., Carlsbad, CA) and then removed by digestion with NcoI and SalI. This fragment was cloned into NcoI and SalI digested pAS2. The resulting plasmid pEK162 was verified by DNA sequencing through the GAL4 / BAP junction. The protein (BAP ^BD , Figure 1) expressed from pEKI62 contained a fusion protein comprising a DNA binding domain of a yeast transcriptional activation protein Gal4 (lacking functional activation sequences) with the addition of 42 amino acids from BAP to the carboxy terminus. An expression plasmid has been developed that mediates the expression of unmodified BAP ₄₂ . 2 (5'-AAGCTTAAG ATG GAT GCA GAA TTC CGA C) was paired with oligonucleotide no. 3 in PCR as described above. The product of this amplification contained a 5 'HindIII site and translation initiation signals optimized for expression in Saccharomyces cerevisiae.

The DNA fragment was again cloned into the TA system. It was then isolated as a HindIII fragment and cloned into pCUP digested with HindIII. The orientation of the BAP gene in the resulting plasmid pEK149 (BAP, Figure 1) was verified by DNA sequencing. Expression of the BAP plasmids pEK149 (which uses URA3 as the selection marker) and pEK162 (which uses TRP1 as the selection marker) were transformed into the yeast host CY770 (Ozenberger and Young, 1995). The strain containing both plasmids was called CY2091. By Clontech Laboratories, Inc. (Palo Alto, CA) was purchased a plasmid library consisting of cDNA fragments isolated from human fetal brain cloned into the yeast two-hybrid expression vector pACT2 (which uses LEU2 as a selectable marker). The protein derived from the library is indicated as unknown in Figure 1. This library was used to transform CY2091. The samples were dispersed on synthetic complete (SC) yeast growth medium without uracil, tryptophan and leucine to select cells containing all three plasmids. Also, there was no histidine in the medium and contained 3-aminotriazole, an inhibitor of the yeast HIS3 gene product at a concentration of 25 mM. 3-Amino-triazole was used to reduce the constitutive expression activity of the low level of the HIS3 reporter gene. Plates were incubated at 30 ° C for 12 days. Twenty-four colonies were shown to exhibit increased histidine prototrophy. Transformation controls showed that 10 ⁶ individual clones were passed through the filter. A PCR approach was used to quickly determine the content of positive clones. Total DNA was isolated from each positive strain by standard methods. This material was used as a template for PCR using oligonucleotides no. 4 (5'-TTTAATACCA CTACAATGGA T) and no. 5 (5'-TTTTCAGTAT CTACGATTCA T) that flank the vector cloning region of the pACT2 library. The DNA fragments were ligated into the TA system and screened for DNA sequencing. The plasmid library contained in clone no. 14 (as described above) was isolated after transfer to E. coli. The nucleotide sequence of the human cDNA sequences was determined, which verified the sequence of the starting PCR product.

Biological tests

The strains were grown overnight in 2 ml SC medium lacking leucine and tryptophan to a density of approximately 7x10 ⁷ cells per ml. Cells were counted and a series of decimal dilutions from 10 ⁴ to 10 ⁸ cells per ml were diluted with sterile water. These samples were added dropwise at 5 μΐ on SC medium lacking tryptophan, leucine and histidine and containing 25 mM 3-amino-triazole. Plates were incubated for 2-3 days at 30 ° C. Positive protein / protein interactions were recognized by increased prototrophic growth compared to control strains expressing a Gal4 DNA binding domain fusion protein and an irrelevant transcriptional activation domain fusion protein (or simply containing a pACT vector without inserted sequences). These control strains were designated as vector in the figures described above. This assay method was highly reproducible and ensured the detection of even a small growth induction mediated by a specific interaction between the target proteins. The original BBP1 clone, designated pEK196 and deposited as ATCC 98399 (referred to herein as clone 14), was used as a PCR template to truncate the protein product for BBP1 expression. Sense primer no. 6 (5 ¹ - TTTAATACCA CTACAATGGA T) was inserted on the GAL4 sequences in pACT2. Antisense prajmer Br. 7 (CTCGAG TTA AAA TCG ATC TGC TCC CAA CC) inserted a 3 'stop codon and an XhoI site immediately 3' to the sequences encoding the DRF motif of BBP1. The PCR product was ligated into the TA cloning vector and then digested with EcoR1 + XhoI and cloned into pACT2. The hybrid product expressed by this plasmid (pEK198) was designated BBP1tm. Similarly, primer no. 7 paired with primer no. 8 (5'-GAATT CCA AAA ATA AAT GAC GCT ACG) for the construction of the BBPII expression plasmid pEK216. The PCR product was ligated again into the TA system and the resulting plasmid digested with EcoR1 +

The Xhol with the BBP1 fragment (codons 123-202) was finally ligated into pACT2 digested with the same enzymes. BBPIAC was generated using pACT2-specific oligonucleotide no. 6 with antisense oligonucleotide no. 9 (5 ' -CTCGAG TCA AGA TAT GGG CTT GAA AAA AC). After cloning TA, isolating the EcoRI-XhoI fragment and cloning into pACT2, the resulting plasmid pEK219 expressed BBP1 from residue 68 to 175. The sequences encoding the BBP1 intracellular loop were amplified using oligonucleotides # 1. 10 (5 '—CCTTCC ATG GAA GTG GCA GTC GCA TTG TCT) and No. 11 (5AACACTCGAG TCA AAA CCC TAC AGT GCA AAA C). This product containing BBP1 codons 185-217 was digested with NcoI + XhoI and cloned into pcoI digested with NcoI + SalI to give pOZ339. The construction of all Ga plasmid expression plasmids used a BamHI site near the center of each rat cDNA sequence (Kang et al., 1990) as a fusion site in pACT2. Sense primers were annealed to sequences 5 'from the BamHI site, antisense primers were annealed to sequences 3' from the stop codon, and included a SalI restriction site. The primers were: Gao, sense (# 17) = 5 '

-GTGGATCCAC TGCTTCGAGG AT, antisense (# 18) = 5'-GTCGACGGTT GCTATACAGG ACAAGAGG, Gas, sense (# 19) = 5'-GTGGATCCAG TGCTTCAATG AT, antisense (# 20) = 5'-GTCGACTAAG TTTGGTTGTTTGTTTGTTTTTGTTTGTTTTGTTTGTTT , sense (# 21) = 5'-GTGGATCCAC TGCTTTGAGG GT, antisense (# 22) =

5'-GTCGACGGTC TTCTTGCCCC CATCTTCC. PCR products were cloned into TA vector. Sequences G were isolated as BamHISal I fragments and cloned into BamHI + SalI digested pACT2. See Table 2 for plasmid designation. Finally, oligonucleotide no. 23 for the conversion of human BAP into a rodent sequence. The primer had the sequence 5'-ATATGGCCATG GAT GCA GAA TTC GGA CAT GAC TCA GGA TTT GAA GTT CGT. Triplets represent the first 13 BAP codons, the three nucleotides that have been altered to produce a rodent sequence are underlined. Oligonucleotide no. 23 was paired with no. 24 (5'-TGACCTACAG GAAAGAGTTA) flanking the region of the Y2H vector that is 3 'of the cloning site in PCR using pEK162 as a template. The product was digested with Ncol +

SalI and ligated into pAS2 to give pEK240. The nucleotide sequence of the rodent BAP coding segment was verified.

Genomic cloning

Rapid Amplification of cDNA Ends: the human lambda genomic library (Stratagene), corresponding to 2.0 x 10 ⁶ pfu, was tested with a probe generated from an EcoRI / Cla1 fragment generated using random primers corresponding to nucleotides 187 to 187 600 (Figure 2). The probe was labeled with [ ³² P] -CTP using the T ^ QuickPrimer Kit according to the manufacturer's protocol (Pharmacia). Filters were hybridized under high stringency conditions: 40 ° C in 50% formamide, 0.12 M NaHPO ₄ , 0.25 M NaCl, 7% SDS and 25 mg / ml sonicated salmon sperm DNA and washed at 65 ° C at 0 ° C. 1xSSC containing 0.1% sodium dodecylsulfate and exposed to Kodak BioMax MS. Lambda phage clones hybridizing to the probe were purified from the coatings by successive screening and re-screening. Ten positive clones were purified and subjected to further analysis by hybridization with a 45 nucleotide probe directed against most of the 5 'sequences known from the original cDNA clone. This oligonucleotide was reverse complementary to nucleotides 157-201 (Figure 2) and had the sequence 5'-CCAGGCGGCC GCCATCTTGG AGACCGACAC TTTCTCGCCA CTTCC. The lambda phage DNA was isolated by standard molecular biology techniques and subjected to direct sequencing using fluorescent dideoxynucleotide cyclic sequencing on an ABI 373 sequencer.

RACE

First strand DNA synthesis was performed using the thermostable rTth polymerase system (Perkin Elmer). The following reagents were mixed in a 1.5 ml tube to give a volume of 10 μΐ: 1 x reverse transcription buffer, 1 mM MnCl ₂ , 1.6 mM dNTP, 2.5 U rTth polymerase, 100 ng poly A ⁺ RNA human hippocampus (Clontech), 10 mM oligonucleotide (nucleotides 429-452, Figure 2, 5'-GTTATGTTGG GTGCTGGAAA ACAG). The reaction was incubated for 15 minutes at 70 ° C and immediately put on ice. A Marathon cDNA synthesis kit (Clontech) was used to generate a second strand of cDNA. The total volume of 10 μΐ of the first strand reaction was mixed with the following reagents: 1 x second strand buffer, 0.8 mM dNTP mix, 4 x second strand cocktail (E. coli DNA polymerase I, E. coli DNA ligase, E. coli RNase H) and Η ₂ θ for a volume of 80 μΐ. The tube was incubated for 1.5 hours at 16 ° C, then T4 DNA polymerase (10U) was added and incubated for an additional 45 minutes at 16C. To terminate the reaction, 4 μΐ 20 x EDTA / glycogen (0.2 M EDTA (2 mg / ml glycogen)) was added to the reaction mixtures, followed by phenol / chloroform / isoamyl alcohol extraction to remove enzymes and other impurities. by adding 0.1 x volume of 3 M sodium acetate pH 5.2 and 2.5 x volume of standard purity EtOH and placed at -70 ° C. The DNA was washed once with 70% EtOH, dried and resuspended in 10 μΐ dH ₂ O Half of the DNA was used for ligation with the Marathon adapter and was used in the following Clontech RAČE PCR reactions: 5 μΐ cDNA was added to 2 μΐ (10 mM) Marathon (5'-CTAATACGAC TCACTATAGG GCTCGAGCGG CCGCCCGGGC AGGT), 1 x DNA ligated solution and 1 μΐ (1 U) T4 DNA ligase The reaction mixture was incubated overnight at 16 ° C. The mixture was diluted 1:50 for the initial RAČE reaction and mixed with the following reagents in a 0.2 ml PCR tube: 40 μΐ dH ₂ O, μΐ 10 x Klentaq DNA polymerase (Clontech), 1 μΐ (10mM) of AP1 primer (5'-CCATCCTAAT ACGACTCACT ATAGGGC), 1 μΐ (10mM) of BBP1 specific primer (corresponding to nucleotides 187 to 209, Figure 2, 5'-CCAGACGGCCA GGCGGCCGCC AT), 5 μΐ 10 x tmm solution. Klentaq, 1 μΐ of 10 mM dNTP, 1 μΐ of diluted cDNA from the above reaction. Using a GenAmp PCR System (Perkin Elmer), the cycles were run under the following conditions: denaturation cycle 94 ° C, 1 minute, then 5 cycles 30 '' at 94'C, 3 'at 72'C, 5 cycles 30'' at 94 ° C, 3 'at 70 ° C, followed by 25 cycles of 30''at 94 ° C, 3' at 68 ° C, with a final extension of 7 'at 72 ° C. This was followed by a nested RAC PCR reaction: 40 μΐ dH ₂ O, 1 μΐ (1 U) 10 x AmplitaqGold DNA polymerase (Perkin Elmer), 1 μΐ (10mM) of AP2 primer (5'-ACTCACTATA GGGCTCGAGC GGC), 1 μΐ (10 mM) primer specific for BBP1 (corresponding to nucleotides 172-194, Figure 2, 5'-GCCGCCATCT TGGAGACCGA CAC), 5 μΐ of 10x Amplitaq polymerase buffer, 1 μΐ of 10 mM dNTP, 1 μΐ of primary RAČE product. The cycling conditions for PCR were: an initial denaturation cycle of 9 'at 94'C, 25 cycles of 30''at94'C,30''at68'C,2' at 72'C, followed by a final extension of 7 'at 72'C . The PCR product was run on a 1% agarose gel in 1x TBE buffer. The resulting 350 base pair product was gel purified and directly cloned using the TA Cloning Kit (Invitrogen). The ligation mixtures were transformed into OneShot cells (Invitrogen) and plated on LB-Agar ampicillin (100 µg / ml) plates containing X-gal. DNA was isolated by miniprep and checked by direct sequencing using fluorescent dide32 oxynucleotide cyclic sequencing on an ABI 373 sequencer.

Northern analysis

Northern blots (membranes) with mRNA samples from various human tissues and various brain tissues were obtained from Clontech (Palo Alto, CA). BBP1 sequences located from the original fusion junction to the poly-A region were isolated using the EcoRI fragment from the TA clone derived from pEK196. Βactin DNA was provided by the manufacturer. Radiolabeled probes were generated from these DNAs using a random primer method incorporating ³² P-dCTP (Pharmacia Biotech, Piscataway,

NJ). Hybridizations were performed according to the manufacturer's instructions (Clontech) in Express Hyb at 68'C. Blots were washed in 2 x SSC (1 X SSC is 0.15 M sodium chloride, 0.015 M sodium citrate), 0.05% SDS at room temperature, followed by two washes in 0.1 x SSC, 0.1% SDS at 50 ° C. Hybridization signals were visualized by exposure to Kodak BioMax film.

In situ hybridization

DNA templates for ribosone synthesis (RNA probes)) were prepared by PCR using a plasmid clone containing the complete human BBP cDNA. One ribon probe directed against the 3 'UTR of the cDNA was used. To ensure that the probe from all stored sequences only recognizes the target, the probe sequences were checked against the GenBank database. A pair of oligonucleotide primers was designed to generate ribosomes for BBP1 to amplify a region of 275 base pairs from 3 '

UTR from BBP1 cDNA, and additionally added promoter sequences for T7 (sense) and T3 (antisense) polymerase. These primers contained the following sequences: 5'-TAATACGACT CACTATAGGG TTAGAAGAAA CAGATTTGAG (forward primer), 5'-ATTAACCCTC ACTAAAGGGA CAAGTGGCAA CTTGCCTTTG (reverse primer). PCR products were purified on 1.5% low melting agarose agarose gels, and the bands containing the products were excised, extracted with phenol and phenol-chloroform, and precipitated with ethanol. The pellets were dried and resuspended in 1 x TE buffer (10 mM TrisHCl, 1 mM EDTA, pH 7.4). The APP ribosome template consisted of a DdeI-XhoI fragment from the coding region of the protein as described by Jacobsen et al. (1991). 50 ng of DNA template was used for transcriptional reactions using (? ⁵ S) -CTP (New England Nuclear, Boston, MA) and the Riboprobe Gemini ™ System kit (Promega, Madison, WI).

The histochemical procedure of in situ hybridization using posthumous sections of the human hippocampus was performed as described previously (Rhodes, 1996). 10 µπι sections were cut on Hacker-Brights and melted onto cold (-20 ° C) Vectabond coated slides (Vector Labs, Burlingame, CA). All solutions were prepared from dH ₂ O treated with 0.1% (v / v) diethylpyrocarbonate and autoclaved. Sections were fixed by immersion in 4% paraformaldehyde in PBS (pH 7.4) and then immersed sequentially in 2 x SSC, dH ₂ O and 0.1 M triethanolamine containing 0.25% (v / v) acetic anhydride, washed in 0 , 2 x SSC, dehydrated in 50%, 70% and 90% ethanol and dried rapidly. One ml prehybridization solution containing 0.9M NaCl, 1 mM EDTA, 5x Denhardt's solution, 0.25 mg / ml single strand salmon sperm DNA (GIBCO / BRL, Gaithersberg, MD), 50% deionized formamide (EM Sciences, Gibbstown, NJ) in 10 mM Tris (pH 7.6), was pipetted onto each slide and incubated for 3 hours at 50 ° C in a humidified chamber. The sections were then dehydrated by immersion in 50%, 70% and 90% ethanol and air dried. Labeled ribosondes were added at a final concentration of 50,000 cpm / μΐ to a hybridization solution containing 0.9 M NaCl, 1 mM EDTA, 1x Denhardt's solution, 0.1 mg / ml yeast tRNA, 0.1 mg / ml single stranded salmon sperm DNA, dextran sulfate (10%), 0.08% BSA, 10 mM DTT (Boehringer Mannheim, Indianapolis, IN) and 50% deionized formamide in 10 mM Tris (pH 7.6). The probes were then denatured at 95 ° C (1 minute), placed on ice (5 minutes) and pipetted on sections and allowed to hybridize overnight at 55 ° C in a humidified chamber. Sections were then washed 1x 45 minutes at 37 ° C in 2x SSC containing 10 mM DTT, followed by 1x 30 minutes at 37 ° C in 1x SSC containing 50% formamide and 1x 30 minutes at 37 ° C in 2x SSC. The single stranded and nonspecific hybridized ribosome was digested by immersion in 10 mM Tris pH 8.0 containing bovine pancreas RNase A (Boehringer Mannheim, 40 mg / ml), 0.5 M NaCl and 1 mM EDTA. Sections were washed for 1 hour in 2x SSC at 60 ° C, then for 2 hours in 0.1x SSC containing 0.5% (w / v) sodium thiosulfate at 60 ° C. The sections were then dehydrated in 50%, 70% and 90% ethanol containing 0.3M ammonium acetate and dried. Slides were placed in X-ray cassettes and left for 14-30 days against Hyperfilm b-Max (Amersham). When a satisfactory image was obtained, slides were coated with nuclear-track emulsion (NTB-2, Kodak) and exposed for 7-21 days at 4'C. Autoradiograms were developed from the emulsion and fixed according to the manufacturer's instructions, and tissue sections below the emulsion were stained with hernátoxylin. To determine non-specific labeling, a control probe was generated from the template provided in the Riboprobe Gemini ™ System Kit (Promega). This vector was linearized using Seal and transcribed using T3 polymerase. The resulting transcriptional reaction gave rise to two products, 250 and 1525 base pair ribosomes containing only the vector sequence. The mixture with this control probe was labeled as described above and added to the hybridization solution at a final concentration of 50,000 cmp / μΐ. No specific hybridization was observed in the control sections. these sections gave a very weak uniform hybridization signal that did not follow neuroanatomic landmarks (data not shown).

Example 1

Cloning and Isolation of BAP Binding Protein (BBP1)

A yeast two-hybrid (Y2H) genetic filter has been developed to screen for and identify proteins that interact with human BAP ₄₂ , a 42 amino acid proteolytic fragment of APP that is considered to be a potentially more toxic aggregated form of BAP. BAP ₄₂ was expressed fused to the yeast Gal4 DNA binding domain and was also expressed as the free peptide (Figure 1). This strain was transformed with the human fetal brain cDNA Y2H library. One clone, designated clone 14 as defined above, of about 10 ⁶ independent transformants, produced consistent activation of the reporter gene and contained a substantial open reading frame related to the GAL4 domain framework. The cDNA insert contained 984 base pairs terminated with a poly-A region. This sequence encoded 201 amino acids (SEQ ID NO: 2, amino acid residues 68-269) with two regions of length and hydrophobia sufficient to cross the cell membrane.

The library-derived plasmid was isolated from clone 14 and used to reconstruct Y2H assay strains. Examination of these strains showed that the BAP fusion protein specifically interacts with the protein of clone 14, although the reaction was weak. Because strongly hydrophobic protein domains, such as transmembrane regions, inhibit Y2H responses (Ozenberger, unpublished data), the insert of clone 14 was truncated (hereinafter BBP1 ™) to remove the region of strongest hydrophobia and again tested for BAP interactions. With BBP1 tm (Figure 2), a much stronger Y2H response was observed, supporting the notion that the removed sequences encode a potential transmembrane (tm) anchor. The nucleotide sequence of clone 14 was compared to GenBank records, showing that the BAP binding protein (BBP1) thus identified is novel.

Example 2

Isolation and confirmation of the 5 'end of the BBP1 cDNA sequence of BBP1 contained in clone 14 described in Example 1 above lacking the 5' end of the coding region of the protein, since no potential initiation methionine codon is present. Numerous experiments with the conventional 5 'RAČE (rapid amplification of cDNA ends) method using standard reverse transcriptase resulted in only 27 nucleotides. These sequences contained ATG, but there was no upstream stop codon in the same translation reading frame to ensure this was the initiation codon. A genomic cloning procedure was introduced to isolate the 5 'end of the BBP1 gene.

Hybridization of the human lambda genomic library with a random primer primer corresponding to 400 base pairs (bp) of the 5 'sequence of clone 14 resulted in the identification of 10 positive clones. Further characterization of these clones using a 45 base oligonucleotide probe directed against the most upstream sequences of BBP1 clone 14 (and the corresponding 5 'upstream 400 bp probe sequence) revealed that 6 of these 10 clones contained the 5' end sequences contained in the sequences identified already more likely. The other 4 lambda clones were found to represent other exons that were contained in the original 400 base pairs of cDNA-derived probe and generated using random primers (data not shown). Direct cyclic DNA sequencing of lambda phage representative clones corresponding to the 5 'end of BBP1 revealed 500 nucleotides upstream that overlap with the sequence known for clone 14. This additional sequence potentially encodes 62 additional amino acids upstream from the previously characterized MET before reaching MET preceded by stopcodone within. Although there are two MET residues downstream of the 37 most distant upstream MET, by standard convention we have tentatively defined the amino terminus sequence of the human BBP1 gene to include the first 5 'MET that follows the stop codon. The complete coding region and the deduced protein sequence is shown in SEQ ID NOs. no. 1 and 2. The plasmid (designated BBP1-fl) containing this amino acid sequence was deposited with the American Type Culture Collection under accession number ATCC 98617).

Since the 5 'coding sequences were derived from a genomic library, there was a possibility that this region contained introns. This possibility was investigated by two methods. First, the forward primer directed to the 5 'MET region and the reverse primer of the original clone 14 were used to amplify sequences from brain cDNA, as well as from genomic DNA. Both samples yielded products of the same size, indicating the absence of introns in this region and confirmed the binding of the upstream sequence to the original sequence. Second, cDNA sequences were isolated in modified 5 'RAČE experiments (see Materials and Methods, supra) that were identical to sequences obtained from a genomic clone. These findings verified upstream sequences (from both genomic and cDNA sources) and the lack of introns in this region.

Example 3

Characterization of BBP1

BBP1 sequences were compared to GenBank using a basic search program for local comparison (BLAST, Altschul et al., 1990). They coincided with two genomic sequences of Caenorhabditis elegans and one genomic sequence of Drosophila melanogaster and a large number of human, mouse and other mammalian expressed expressed marker sequences. However, no complete cDNA sequences or functional data attributed to the gene were available. BBP1 protein and translation of available short expressed sequences were compared, scanned for the occurrence of conserved segments, and evaluated using the MoST protein motif search algorithm (Tatusov et al., 1994). These analyzes revealed a possible evolutionary relationship to the family of G protein-coupled receptors. More specifically, these analyzes showed that BBP1 contains two potential transmembrane (tm) domains equivalent to the tm domains of 3 and 4 receptors associated with protein G. The intervening hydrophilic loop contains a well characterized three amino acid motif, aspartate (D) or glutamate followed by arginine (R) and aromatic residue (Y or F) (commonly referred to as DRY sequences), this motif is retained by almost all members of this receptor family and has been shown to serve as a molecular trigger for protein G activation (Acharya and Karnik, 1996). These data indicate that BBP1 is a novel protein potentially containing a functional module shared with members of a large family of protein G-coupled receptors. More specifically, BBP1 shows that it retains a critical DRF sequence between two predicted dark domains so that it may have the potential to link to a signaling pathway proteinom G.

Structural analysis of BBP1 showed that it contains a structural motif known to be the Ga protein activation sequence of related G-coupled receptors. Y2H assays demonstrating the interaction of BBP1 with various members of the G-coupled receptors are shown in Figure 3. based on structural predictions, BBP1 is depicted as a double-permeable membrane with both ends in the luminal compartment. Other orientations cannot be completely excluded. The potential protein interactions described above were investigated in Y2H assays. Two overlapping portions of the BBP1 sequences contained in the BBP1 clone were amplified and cloned into the Y2H vector of pACT2 (expression plasmids pEK216 and pEK219, Table 2 and the corresponding proteins ΒΒΡ1ΔΝ and BBP1AC, Figure 4). The construct lacked both tm domains, the construct oval encoded the first tm domain and the previous 52 amino acids. These fusion proteins were tested with the BAP fusion protein and the reactions were compared to those of strains expressing the larger BBP1 ™ protein. These results suggest that the major determinant for BAP association is contained in the BBP1 region, which is believed to be topographically similar to BAP in the wild-type APP protein.

Example 4

Tissue distribution of human BBP1 expression

BBP1 mRNA expression was evaluated as an initial step in elucidating the activity of the gene and its product. A 1.25 kb master transcript was observed in all tissues (Figure 5A). There was a high level of expression in the heart. The whole brain showed moderate expression levels. All samples originating from separate areas of the brain expressed BBP1 expression (Figure 5B). Interestingly, the limbic regions contained a relatively larger amount of BBP1 mRNA. These are areas of the brain where BAP aggregation and associated neurotoxicity first occur. Analysis of in situ hybridization autoradiograms obtained using a BBP1-specific ribosome showed that in the human hippocampus and entorinal cortex, BBP1 mRNA is expressed in medium to large cells in a pattern consistent with neuronal expression and in contrast to glial cell pattern (Figure 6). In addition, BBP1 mRNA is expressed in virtually all hippocampal and entorinal neurons, i. there are no significant or laminar variations in the intensity of the hybridization signal. Interestingly, the distribution of BBP1 expression was markedly similar to that observed using ribosondes directed against the amyloid APP precursor protein mRNA (Figure 6). Collectively, BBP1 mRNA was observed in all tissues examined and all areas of the brain. In situ analysis of BBP1 mRNA expression also revealed significant expression in the hippocampus region.

Example 5

Distribution of BBP1 expression in cell lines

BBP1 expression was also screened in numerous cell lines and data was extracted from dbEST, the National Expression of Biotechnology Information, NCBI, short expression database. Reverse polymerase polymerase chain reaction (RT-PCR) methods were used to qualitatively determine the expression of BBP1 mRNA in cell lines generally used for expression of recombinant proteins, as well as in a variety of cancer cell lines. BBP1 was observed in hamster CHO cells and human HEK293 cells. Signals were observed in the Ntera-2 embryonic stem cell line and the IMR32 and SK-N-SH neuroblastoma lines. BBP1 expression was observed in cancer cell lines that originated from the following tissues: colon (Cx-1, Colo205, MIP101, SW948, CaCo, SW620, LS174T), ovaries (A2780S, A2780DDP), breast (MCF-7, SKBr- 3, T47-D, B7474), lung (Lx-1, A5439), melanoma (Lox, Skmel30), leukemia (HL60, CEM), prostate (LNCAP, Dul45, PC-3). Northern blot testing with mRNA from the following cancer cell lines showed BBP1 expression in all samples: promyelocytic leukemia (HL-60), carcinoma (HeLa S3), chronic myeloid 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

Compared to the human sequence, there are three amino acid substitutions (G5R, F10Y and R13H) in the rodent BAP sequence. The rodent peptide exhibited reduced neurotoxicity and did not bind to human brain homogenates (Maggio et al., 1992). It was therefore interesting to evaluate the association of rodent BAP with BBP1 in the Y2H system. The sequence of human BAP in pEK162 was altered to encode the rodent peptide by oligonucleotide-directed mutagenase by the PCR described above. The resulting plasmid pEK240 was identical to the expression plasmid of the human BAP fusion protein used throughout the present invention except for the three codons in which there were amino acid substitutions to generate the rodent peptide sequence. Interactions between BBP1 fusion protein and rodent BAP fusion proteins and human BAP were compared using the Y2H bioassay. Strains expressing BBP1 and rodent BAP did not generate a growth response (Figure 7). This finding supports the assumption that BBP1 may serve as a specific mediator of the neurotoxic effects of BAP and provides a mechanism to explain the reduced toxicity of rodent BAP. Importantly, these data also serve to illustrate the high degree of specificity of BBP1 / BAP interaction in the Y2H assays, since the substitution of three amino acids was sufficient to completely abrogate association (Figure 7).

The skilled artisan will appreciate that the invention may be practiced otherwise than as specifically described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible on the basis of the above teachings and are therefore within the scope of the appended claims.

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Claims (24)

PATENT CLAIMS An isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising a nucleotide sequence with identification number 1 (SEQ ID NO: 1), (b) a polynucleotide comprising the nucleotide sequence of the β-amyloid peptide (BBP) binding protein (BBP) of the BBP1-fl clone deposited under accession number ATCC 98617; (c) a polynucleotide encoding a β-amyloid peptide (BBP) binding protein encoded by the cDNA insert of the BBP1-fl clone deposited under ATCC accession number 98617; d) a polynucleotide comprising a nucleotide sequence from nucleotide 202 to nucleotide 807 of SEQ ID NO. no. 1 (e) a polynucleotide containing the nucleotide sequence of the β-amyloid peptide (BBP) binding protein (BBP) of clone pEK196 deposited under ATCC accession number 98399; (f) a polynucleotide encoding a β-amyloid peptide (BBP) binding protein encoded by the cDNA insert of clone pEK196 deposited under ATCC accession number 98399; g) a polynucleotide encoding a protein comprising the amino acid sequence of id. no. 2 h) a polynucleotide encoding a protein comprising the fragment of amino acid sequence id. no. 2, having binding activity to the human β-amyloid peptide, wherein the fragment comprises an amino acid sequence from amino acid 68 to amino acid 269 of SEQ ID NO: 2; no. 2 (j) a polynucleotide which is an allelic variant of the polynucleotide of (a) to (f) above; (k) a polynucleotide which encodes a species homolog of the protein of (g) to (h) above; and l) a polynucleotide capable of hybridizing under stringent conditions to each of the polynucleotides defined in a) to h). The polynucleotide of claim 1, which is operably linked to at least one expression control sequence. The host cell transformed with the polynucleotide of claim 2. The host cell of claim 3, wherein the cell is a prokaryotic or eukaryotic cell. A method of producing a protein encoded by the polynucleotide of claim 2, comprising the steps of a) culturing the host cell tissue culture of claim 3 in a suitable culture medium, and b) purifying the protein from the culture medium. Protein prepared by the method of claim 5. 7. A protein comprising an amino acid sequence selected from the group consisting of: a) the amino acid sequence of SEQ. no. 2 b) an amino acid sequence from amino acid 68 to amino acid 269 of SEQ ID NO: 2; no. 2 (c) the amino acid sequence encoded by the cDNA insert of the BBP1-fl clone deposited under ATCC accession number 98617, and d) fragments of amino acid sequence id. no. 2, comprising the amino acid sequence from amino acid 185 to amino acid 217 of sequence id. no. Second The protein of claim 7, which comprises the amino acid sequence of SEQ ID NO: 2. no. Second A fusion protein comprising BBP1 linked to a heterologous protein or peptide sequence. The fusion protein of claim 9 wherein BBP1 has amino acid sequence id. no. Second An oligonucleotide that encodes an antisense sequence complementary to a portion of the BBP1 sequence of SEQ ID NO. no. 1, and which inhibits BBP1 gene expression. 12. A method for determining a polynucleotide encoding a β-amyloid peptide (BBP) binding protein in a sample, comprising the steps of: (a) hybridizing the sample to a probe specific for said polynucleotide under conditions for said probe effective to hybridize specifically to said polynucleotide; and b) examining hybridization of said probe with polynucleotides in the sample, wherein said probe comprises a nucleic acid sequence having a region of 20 or more base pairs at least 90% identical to polynucleotide sequence id. no. First 13. A method for determining a polynucleotide encoding a β-amyloid peptide (BBP) binding protein in a sample comprising the steps of: (a) hybridizing the sample to a probe specific for said polynucleotide under conditions for said probe effective to hybridize specifically to said polynucleotide; and b) examining hybridization of said probe with polynucleotides in the sample, wherein said probe comprises a nucleic acid sequence having a region of 20 or more base pairs at least 90% identical to the polynucleotide sequence of the cDNA insert of ATCC 98617 or ATCC 98399. An antibody that specifically binds to a polypeptide comprising a region of sequence at least 90% identical to amino acid sequence id. no. Second An antibody that specifically binds to a polypeptide comprising a region of sequence at least 90% identical to the amino acid sequence of a β-amyloid peptide (BBP) binding protein encoded by the cDNA insert of ATCC 98617. A method of detecting a polypeptide comprising a region at least 90% identical to amino acid sequence id. no. 2 in a sample, comprising steps (a) incubating the sample with a reagent that binds specifically to said polypeptide under conditions effective for specific binding; and b) assaying the binding of the reagent to the polypeptide in the sample. A method for detecting a polypeptide comprising a region of the sequence at least 90% identical to the amino acid sequence of a β-amyloid peptide (BBP) binding protein encoded by the cDNA insert of ATCC 98617, comprising the steps of: (a) incubating the sample with a reagent that binds specifically to said polypeptide under conditions effective for specific binding; and b) assaying the binding of the reagent to the polypeptide in the sample. A method for diagnosing a disease characterized by aberrant expression of human β-amyloid peptide (BAP), comprising the steps of: (a) incubating a sample suspected of aberrant expression of the human β-amyloid peptide with a reagent comprising a polypeptide that comprises a region at least 90% identical to amino acid sequence id. no. 2 under conditions effective for specific binding of the reagent to the human β-amyloid peptide, and b) assaying the binding of the reagent to the peptide in the sample. 19. A method for diagnosing a disease characterized by aberrant expression of a human β-amyloid peptide, comprising: a) incubating a sample suspected of aberrant expression of human β-amyloid peptide with a reagent comprising a polypeptide comprising a region at least 90% identical to the amino acid sequence of the β-amyloid peptide binding protein encoded by the cDNA insert of ATCC 98617 under conditions effective to specifically bind the human? amyloid peptide, and b) assaying the binding of the reagent to the peptide in the sample. 20. The diagnostic method, wherein the method comprises analyzing the presence of the polynucleotide of claim 1 in a host-derived sample. 21. A method for identifying compounds that regulate the activity of a β-amyloid peptide binding protein comprising: a) incubating a sample comprising a human β-amyloid peptide in an assay medium containing the test compound and a reagent comprising a polypeptide that comprises a region at least 90% identical to amino acid sequence id. no. 2 under conditions effective to specifically bind the reagent to the human β-amyloid peptide, and b) comparing the binding of the reagent to the peptide in the sample in the presence and absence of the test compound; and c) relating the difference between the binding in step b) to a test compound regulating the activity of the β-amyloid peptide binding protein. 22. A method for identifying compounds that regulate the activity of a β-amyloid peptide binding protein comprising: (a) incubating a sample containing the human β-amyloid peptide in an assay medium containing the test compound and a reagent comprising a polypeptide comprising a region at least 90% identical to the amino acid sequence of the β-amyloid binding protein encoded by the cDNA insert of ATCC 98317 under conditions effective for specific binding human β-amyloid peptide reagents, a b) comparing the binding of the reagent to the peptide in the sample in the presence and absence of the test compound; and c) referring the difference between the binding in step b) to the test compound regulating the activity of the βamyloid peptide binding protein. 23. A method of treating a patient in need of inhibiting the accumulation of β-amyloid peptide in the brain, comprising administering to the patient a therapeutically effective amount of the polypeptide of claim 7. A transgenic or chimeric animal comprising the polynucleotide of claim 2.