EA004256B1 - beta-AMINOLOID PEPTIDE BINDING PROTEIN, POLYNUCLEOTIDE ENCODING THEREOF AND METHODS FOR USING THE SAME - Google Patents

Abstract

1. A fragment of a cDNA molecule, comprising nucleotides 202-807 of the sequence SEQ ID NO:1, provided in the List of sequences, encoding a beta-amyloid peptide binding protein (BBP) fragment comprising amino acids 68-269 of SEQ ID NO:2, provided in said List, and also a variant of said cDNA fragment, obtained on the basis of degeneracy of the genetic code. 2. A fragment of a cDNA molecule according to claim 1, which is an insert of clone pEK 196 ATCC 98399. 3. A cDNA molecule having a nucleotide sequence SEQ ID NO:1, provided in the List of sequences, encoding a beta-amyloid peptide-binding protein (BBP) having an amino acid sequence of SEQ ID NO:2 provided in said List and also a variant of said cDNA molecule prepared on the basis of degeneracy of the genetic code. 4. A cDNA molecule according to claim 3, which is an insert of clone BBP1-f1 ATCC 98617. 5. A polynucleotide which hybridizes under stringent conditions to a cDNA molecule according to any one of claims 1-4 or the complementary molecule thereof, wherein said polynucleotide has at least 75% of the length of any one of said cDNA molecules and encodes a beta-amyloid peptide. 6. A probe or primer comprising a nucleotide sequence capable of hybridizing to nucleotides 172-194 of the cDNA molecule according to claim 3. 7. A polynucleotide comprising at least one expression control sequence operably linked to at least one polynucleotide selected from the group consisting of the cDNA molecule of claims 1 to 4. 8. A host cell strain transformed with the polynucleotide of claim 7 wherein said cell is a prokaryotic cell. 9. A host cell strain transformed with the polynucleotide of claim 7 wherein said cell is an eukaryotic cell. 10. A beta-amyloid peptide binding protein (BBP) fragment comprising amino acids 68-269 of SEQ ID NO:2 provided in the List of sequences encoded by a fragment of a cDNA molecule comprising nucleotides 202-807 of SEQ ID NO:1, provided in said List or is encoded by a variant of said cDNA fragment, obtained on the basis of degeneracy of the genetic code. 11. A beta-amyloid peptide binding protein (BBP) fragment encoded by a fragment of a cDNA molecule according to claim 1 which is an insert of clone pEK 196 ATCC 98399. 12. A beta-amyloid peptide binding protein (BBP) having an amino acid sequence of SEQ ID NO:2 provided in the List of sequences encoded by a cDNA molecule having a nucleotide sequence SEQ ID NO: 1 provided in said List or encoded by a variant of said cDNA molecule obtained on the basis of the degeneracy of the genetic code. 13. A protein BBP comprising an amino acid sequence encoded by a cDNA molecule according to claim 3, which is an insert of clone BBP1-f1 ATCC 98617. 14. A process for producing a protein according to any one of claims 12-13 or protein fragment according to any one of claims 10-11 comprising the steps of growing the host cell strain of claim 8 or 9 in appropriate culture medium and purifying the protein or a fragment thereof produced by said strain. 15. A protein produced according to the process of claim 14. 16. A fusion protein BBP comprising a protein according to any one of claims 12-13, 15 or protein fragment according to any one of claims 10-11 linked to a heterologous protein or peptide sequence. 17. An oligonucleotide being a BBP1 gene expression inhibitor comprising nucleotide sequence consisting of 15 to 35 nucleotides wherein said nucleotide sequence hybridizes under stringent conditions to a cDNA molecule according to any one of claims 1 to 4. 18. A method for determining the quantity of a polynucleotide encoding a beta-amyloid peptide-binding protein (BBP) in a sample comprising the steps of: (a) hybridizing to a sample a probe according to claim 6 under stringent conditions; and (b) determining the quantity of said probe bound to polynucleotides in the sample. 19. An antibody which binds specifically, to an extracellular region of a protein according to any one of claims 12-13, 15-16 or protein fragment according to any one of claims 10-11. 20. A method for detecting in a sample the quantity of a protein according to any one of claims 12-13, 15-16 or protein fragment according to any one of claims 10-11 comprising the steps of: a) incubating with a sample and a control, said control comprising varying amounts of known BBP protein or BBP protein fragment, an antibody according to claim 19 wherein said antibody is linked to a reporter molecule; b) determining the quantity of said antibody bound to said BBP protein or protein fragment in the sample; and c) calculating the quantity of said BBP protein or BBP protein fragment by comparing the quantity of antibody in b) to said controls. 21. A method for the treatment of a patient having need to inhibit beta-amyloid peptide accumulation in the brain comprising administering to the patient a therapeutically effective amount of a protein according to any one of claims 12-13, 15-16 or protein fragment according to any one of claims 10-11.

Description

This application discloses the benefits of provisional application US No. 60/064583, filed April 16, 1997, the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to new polynucleotides and proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research applications of these polynucleotides and proteins. In particular, the present invention relates to polynucleotides and proteins encoded by such polynucleotides that bind to a β-amyloid peptide, which is one of the primary components of amyloid deposits associated with the pathogenesis of Alzheimer's disease.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AO) is a disorder associated with progressive dementia in the elderly, characterized by a number of structural abnormalities of the brain. In multiple areas of the central nervous system (CNS), neurons become dysfunctional and die, resulting in a change in synaptic pathways. The bodies and proximal dendrites of these vulnerable neurons contain neurofibrillary tangles consisting of paired helical filaments, the main component of which is a phosphorylated, microtubule-binding protein, designated as a τ-protein (tau protein). One of the essential signs of this disease is the accumulation of amyloid deposits within the brain, called senile (or neuritic) plaques. The principal component of amyloid plaques is the β-amyloid peptide (hereinafter referred to as BAP, and in the literature also known as Av, BAP, etc.), which forms dense clusters during the development of AO.

BAP is a protein consisting of 39-43 amino acids that is formed as a result of proteolytic cleavage from the amyloid precursor polypeptide (hereinafter referred to as APP) and consisting of a portion of the transmembrane domain and the lumen domain of the endoplasmic reticulum APP. The 42-amino acid BAP peptide is believed to be a potentially more toxic aggregated form in humans. APP is found in the form of several BAP including isoforms. The main isoforms consist of 695, 751 and 770 amino acids, despite the fact that the last two APPs include a domain that has structural and functional homology with Kunitz serine protease inhibitors. In healthy individuals, BAP does not accumulate and is quickly removed from the circulating body fluids. However, the peptide is capable of forming plaques on the surface of abnormal dendrites and axons, microglia and reactive astrocytes. Aggregation and deposition of BAP in the form of neuritic plaques is considered as one of the triggering mechanisms for the development of Alzheimer's disease. The study of the processes leading to the expression and functions of BAP and their own role in the pathogenesis of AO takes significant attention in neurological studies. In particular, the discovery of proteins that can bind to the BAP protein is crucial in connection with an understanding of the pathogenetic pathways of this disease and leads to the emergence of new potential therapeutic targets.

Prior to the present invention, proteins and fragments thereof that would bind to the human BAP protein and which could be involved in the biological effects of BAP during the development of AO were not identified.

SUMMARY OF THE INVENTION

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

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

(a) a polynucleotide comprising the nucleotide sequence of 8EC GO N0 1;

(b) a polynucleotide comprising the nucleotide sequence of a β-amyloid peptide binding protein (BBP) of clone BBB1-11 introduced under ATCC 98617;

(c) a polynucleotide encoding a β-amyloid peptide binding protein (BBP) encoded by the cDNA insert of clone BBB1-11, inserted under ATCC 98617;

(b) a polynucleotide comprising the nucleotide sequence of 8ES GO N0 1 from the 202nd nucleotide to the 807th nucleotide;

(e) a polynucleotide comprising the nucleotide sequence of a β-amyloid peptide binding protein (BBP) of clone pEK196 introduced under ATCC 98399;

(1) a polynucleotide encoding a β-amyloid peptide binding protein (BBP) encoded by the cDNA insert of clone pEK196, inserted under ATCC 98399;

(e) a polynucleotide encoding a protein comprising the amino acid sequence of 8ES GO N0 2;

(11) a polynucleotide encoding a protein including an 8EO по N0 2 amino acid sequence fragment having binding activity to a β-amyloid peptide, i.e. a fragment comprising the amino acid sequence from the 68th amino acid to the 269th amino acid in accordance with 8EO ΙΌ N0 2;

(ί) a polynucleotide that is an allelic variant of a polynucleotide according to claims (a) to (£) described above;

(k) a polynucleotide that encodes a variant homologous to the protein according to (§) to (1) described above; and (l) a polynucleotide capable of hybridizing under stringent conditions with any polynucleotide according to (a) to (1).

Preferably, such a polynucleotide comprises a nucleotide sequence of 8E0 ΙΌ N0 1, a nucleotide sequence of a β-amyloid peptide binding protein (BBP) of clone BBB1-D introduced under ATCC deposit number 98617, or a polynucleotide encoding a protein (BBP) encoded by a β amyloid peptide encoded by insertion of cDNA of clone BBB1-D introduced under ATCC 98617. Another option is a gene corresponding to a cDNA sequence of 8E0 ΙΌ N0 1.

In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group which includes:

(a) the amino acid sequence of 8EO ΙΌ N0 2;

(b) the amino acid sequence of 8E0 ΙΌ N0 2 from the 68th amino acid to the 269th amino acid;

(c) the amino acid sequence encoded by the cDNA insert of clone BBB1-P. contributed by ATCC 96817; and (b) fragments of the amino acid sequence of 8E0 ΙΌ N0 2, including the amino acid sequence from the 185th amino acid to the 217th amino acid of 8E0 ΙΌ N0 2.

Preferably, such a protein comprises an amino acid sequence of 8E0 ΙΌ N0 2 or an amino acid sequence of 8E0 ΙΌ N0 2 from the 68th amino acid to the 269th amino acid. Fusion proteins are also formulated by the present invention.

In a number of preferred embodiments, the polynucleotide is operably linked to an expression control sequence. The present invention also provides a host cell, including bacterial, yeast, insect and mammalian cells, transformed with such polynucleotide compositions.

Also provided are methods for generating VVR, which include (a) growing a host cell culture according to claim 3 in a suitable culture medium; and (b) purification of the protein from the culture medium.

The present invention also provides compositions comprising an antibody specific for such VVR.

Methods and diagnostic techniques are also presented in connection with the identification of a disease characterized by abnormal expression of the human BAP protein, as well as methods for identifying compounds involved in the regulation of BBB activity.

Another embodiment of the present invention includes transgenic animals carrying a polynucleotide encoding BBB operably linked to an expression control sequence.

Brief Description of the Drawings

The following drawings characterize some variants of the present invention. They are illustrative only and in no way limit the present invention.

FIG. 1. Test for yeast dihybrid screening. The host strain Υ2Η expressing the DNA-binding domain of Oa14 fused to BAE42 (BAP ^BC ; plasmid carrying the TBP1 marker) and the non-fused BAP42 (BAP; plasmid carrying the ICA marker) was transformed with the Υ2H library of human fetal brain cells cDNA (plasmid, carrying the marker LFe2) expressing fusion proteins (iikothei ^AC ), including the activator domain of Oa14, as described in this text. Accordingly, strains carrying three extrachromosomal plasmids indicated by circles expressed the indicated protein. Positive protein-protein interactions restore Oa14 activity by the upstream activator sequence (0AbAi8), as a result of which transcription of the ΗΙ83 reporter gene is induced.

FIG. 2. Detection of binding of BBP1 / BAP. The Υ2Η strains were tested for histidine prototrophy by obtaining 10-fold serial dilutions and digging in 5 μl onto synthetic agar medium lacking tryptophan, leucine, histidine and containing 25 mM 3-aminotriazole, as described in this text. All strains carry the plasmid pEK162 expressing the BAP fusion protein, which is indicated by the BAP tag. The first columns (vector) correspond to independently formed strains carrying plasmid pEK162 and vector pAST2 expressing an extraneous fusion protein. This option serves as a background for comparison with strains that express proteins of interest. Columns denoted by ΒΒΡΙΔΙιη. correspond to the expression of shortened BBB1 from plasmid pEK198 as described herein. The interaction between the BAP and ΒΒΡΙΔΒΒΡΙιη fusion proteins restores Ca14 activity, as a result of which the происходит83 reporter gene is induced (see Fig. 1), which is detected by increased prototrophic growth in comparison with control strains.

FIG. 3. Biotests showing the interaction of BBP1 with Ca proteins. The putative intracellular domain of the BBP1 protein was expressed as the Ca14 DNA-binding domain, coupled to parts of rat CaC, Cao or ΟαΟ2 rats expressed as fusion proteins including the Ca14 activator domain. The Υ2Η responses, representing two independently formed clones of each strain, were compared with the responses of cells lacking the C-protein component (vector column). The technique is described in the legend of FIG. 2.

FIG. 4. Mapping interactions between BBB1 and BAP. ΒΒΡ1 ΔΙιη was divided into two overlapping segments as described in this text. These proteins, designated VVRHS and ΒΒΡ1ΔΝ, were tested for their interaction with BAP. The test protocol and strains designated as vector and VVRSh1t are described in the legend of FIG.

2. The strains labeled as BBRS and ΒΒΡ1ΔΝ express the designated segment of BBB1 as a fusion protein.

FIG. 5. Expression of mRNA of BBP1 in human tissues (A) and brain regions (B). Nylon membranes, onto which 2 μg of size-fractionated polyadenylated RNA isolated from the designated tissues were applied, were obtained from C1oSce11. They were hybridized with a radioactively labeled cDNA-BBB1 probe as described herein. The main band corresponding to 1250 nucleotide pairs (determined by molecular weight markers: data not included here) was detected in all lines. Higher molecular weights appear to correspond to heteronuclear RNA; the BBP1 gene includes several introns. Blots were released and re-probed with β-actin in order to control the loading and RNA integration: all lines showed the same signal (data not included here).

FIG. 6. Expression of BBP1 and APP in hippocampal cells. The images of X-ray diffraction patterns obtained during hybridization of ίη κίΐιι and showing the expression parameters of BBP1 (A) and APP (B) in the hippocampus and in the entorhinal region of the human cerebral cortex are presented. Tissue sections used to obtain image data were taken during autopsy of two different patients. Abbreviations: IS - dentate gyrus of the cerebral cortex; CA1 - the lower region of the hippocampus; The EU is the entorhinal region of the cerebral cortex.

FIG. 7. Comparison of the interaction of VVR1 with human VAR and rodent. The rodent BAP was constructed and expressed as a fusion protein as described in this text. The strains labeled with human BAP are identical to those shown in FIG. 2. Strains labeled with rodent BAP express the rodent BAP as a fusion protein comprising the Ca14 DNA-binding domain. The vector defines control strains carrying only the vector as opposed to the BAP fusion proteins; BBB1 identifies strains expressing the BBPM! Fusion protein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the isolation and cloning of a protein (BBP1) that binds to a human β-amyloid peptide. BBP1 was characterized as a fusion protein in the yeast digibrid assay for binding to the 42 amino acid fragment of BAP (BAP42). The expression of BBP1 was demonstrated in human tissues and in specific parts of the brain (Fig. 5). It is important that for BBP1, selectivity in binding to human BAP in the system of yeast digibrid test is shown in comparison with rodent BAP. These data confirm the suggestion that the BBB1 of the present invention can be used in the diagnosis and treatment of Alzheimer's disease, as well as for the evaluation and screening of drugs designed to regulate the accumulation of amyloid plaques in the brain.

BBR1 coding sequence

The original human BBB1 clone (designated clone 14) was obtained using a yeast genetic hybrid test (Υ2Η) designed to identify proteins that interact with human BAP42, which is a potentially more toxic form of BAP. BAP42 was expressed by fusion with the DNA-binding domain of yeast Ca14 and was also expressed as a free peptide (Fig. 1). This strain was transformed with the Υ24 library of the human fetal brain cDNA. The only clone designated as No. 14, out of about a million independent transformants, caused distinct activation of the reporter gene and included an open coding frame that goes into the SAB4 domain. The cDNA insert included 984 base pairs and ended with the polyadenyl tract. This sequence encoded 201 amino acids (from the 68th amino acid to the 269th amino acid in accordance with 8EO ΙΌ N0 2) and had two sections characterized by a length

Ί and the level of hydrophobicity, sufficient for penetration through the cell membrane. It also contains potential glycosylation sites for asparagine residues (Ν-glycosylation). Clone 14 was designated as clone pEK196 and entered into the American Type Culture Collection under Depository Number ATCC 98399.

The plasmid derivative of the library was isolated from clone 14 and used to reconstruct the strains included in the Υ2Η test. Testing of these strains showed that the BAP fusion protein specifically interacts with the protein encoded by clone 14, although the response was rather weak. Considering the fact that protein domains possessing a high level of hydrophobicity, such as transmembrane regions, suppress the responses in the Υ2Η test (Oxspcrdsg unpublished data), clone 14 was shortened (to obtain the BBB1D1sh variant: see Table 2 below for further description) in order to remove the site of hard hydrophobicity with subsequent repeated testing for interaction with BAP. A significantly more powerful answer in the Υ2Η test was noted with the VVR1D1sh variant: this confirms the remark that the delegated sequences encoded a probable transmembrane anchor (1ш). Clone 14 identifies a new BAP-binding protein in the form of a fusion protein.

The BBP1 cDNA sequences located in clone 14 were identified as having lost the 5 ′ coding region, as indicated by the absence of a potential initiating codon of methionine. Numerous attempts to use the standard 5'CACE method (rapid amplification of cDNA ends) using conventional reverse transcriptase allowed a total of 27 nucleotides to be added. Thus, the genomic cloning technique described in Example 2 below was applied to isolate the 5'-terminal region.

Since the sequence encoding the 5'-terminal region is derived from the genomic library, there is a danger of the presence of an intron in this sequence. This probability was estimated using the two methods described below in Example 2. The data obtained as a result confirm that the accuracy of the isolation of the sequences lying above (from both genomic and cDNA sources) and the absence of introns in this region. Plasmid BBB1-P, including a cDNA insert encoding the full-sized protein BBB1, was enclosed in the American Type Culture Collection under Depository Number ATCC 98617. The complete coding region and the decoded polypeptide sequence are shown in 8EO ΙΌ N0 1 and 8E0 GO N0 2, respectively. The original clone (pEK196) includes 3'-untranslated nucleotide sequences.

In accordance with the present invention, nucleotide sequences that encode BBB1, fragments thereof, fusion proteins or functional equivalents can be used to form recombinant DNA molecules that direct expression of BBB1, or a functionally active peptide, in a suitable host cell. On the other hand, nucleotide sequences that hybridize with fragments of the BBP1 sequence can be used in nucleic acid hybridization tests, in Southern blotting, in Northern blotting, etc.

The present invention also provides polynucleotides whose sequences are complementary to the polynucleotides described herein.

The present invention also provides polynucleotides capable of hybridizing under conditions of reduced stiffness, more preferably under conditions of normal stiffness and most preferably under conditions of high stiffness with the polynucleotides described herein. Examples of hybridization stringency conditions are shown in the table below: conditions of high stringency are those conditions that are comparable to those given, for example, in columns AR; conditions of ordinary stringency are at least as stringent as shown, for example, in columns Cb; and conditions of reduced stiffness are at least as shown, for example, in columns MK.

Hardness conditions

Service- Wii stiffness Polynucleotide hybrid The length of the hybrid (nucleotide pairs) ¹ Temperature and Hybridization Buffer Temperature and Wash Buffer BUT DNA / DNA "fifty 65 ° С - 1х88С; or 42 ° С 1х88С; 50% formamide 65 ° C - 0,3x88C IN DNA / DNA <50 T _B * -1x88C T _B * -1x88C FROM DNA / RNA "fifty 67 ° С - 1х88С; or 45 ° С 1х88С; 50% formamide 67 ° C - 0,3x88C ϋ DNA / RNA <50 T _s * - 1x88C T _s * -1x88C E RNA / RNA "fifty 70 ° С - 1х88С; or 50 ° С 1х88С; 50% formamide 70 ° C - 0,3x88C R RNA / RNA <50 Tr * -1x88C T _in * -1x88C FROM DNA / DNA "fifty 65 ° С - 4х88С; or 42 ° C 4x88C; 50% formamide 65 ° С-1х88С N DNA / DNA <50 Tn * - 4x88C Tn * -4x88C I DNA / RNA "fifty 67 ° C - 4x88C; or 45 ° С 4х88С; 50% formamide 67 ° С-1х88С I DNA / RNA <50 T * 4x88C T ₃ * -4x88C

TO RNA / RNA "fifty 70 ° С - 4х88С; or 50 ° C 4x88C; 50% formamide 67 ° C -1x88C b RNA / RNA <50 T _b * -2x88C T _b * -2x88C M DNA / DNA "fifty 50 ° С - 4х88С; or 40 ° С 6х88С; 50% formamide 50 ° С-2х88С N DNA / DNA <50 Τν * - 6х88С T№-6x88C ABOUT DNA / RNA "fifty 55 ° С - 4х88С; or 42 ° C 6x88C; 50% formamide 55 ° С-2х88С R DNA / RNA <50 Tr * -6x88C Tr * -6x88C Ω RNA / RNA "fifty 60 ° С - 4х88С; or 45 ° С 6х88С; 50% formamide 60 ° С-2х88С IN RNA / RNA <50 T _B * - 4x88C T _B * -4x88C

Notes:

¹ .- the length of the hybrid refers to the length of the hybridizable portion (s) of the hybridizing polynucleotide. In the case when a polynucleotide is hybridized with a polynucleotide with an unknown sequence, the length of the hybrid implies the length of the entire initial hybridizing polynucleotide. When polynucleotides are hybridized with known sequences, the length of the hybrid can be determined by comparing the polynucleotide sequences and identifying the site or sites with optimal complementarity of the sequence.

^m - in the preparation of hybridization and washing buffers, 88C solution (1x88C contains 0.15 M NaCl, 15 mM sodium citrate) can be replaced by 88PE solution (1x88PE contains 0.15 M NaCl, 10 mM Ν; · ιΗ _; ΡΟ. · | and 1.25 mM EETL - pH = 7.4); washing is carried out for 15 minutes after completion of hybridization;

* T _in -T _in - the hybridization temperature for hybrids whose length is less than 50 nucleotide pairs should be 5-10 ° C lower than the melting temperature (T _t ) of this hybrid, despite the fact that T _t is determined in accordance with the following equations: for hybrids with a length of less than 18 pairs of nucleotides: T _t (° C) = 2x (number of bases A + T) + 4x (number of bases C + C). For hybrids with a length of 18 to 49 pairs of nucleotides: Tm (° C) = 81.5 + 16.6 (1 ° C | No. 1 '|) + 0.41 (% | C + C.' |) - (60 ( G). Where N is the number of bases in the hybrid, and | Ν; · ι '| is the concentration of sodium ions in the hybridization buffer (for a solution of 1x88C | Ν; ι' | = 0.165 M).

Additional examples of stringency conditions for the polynucleotide hybridization are presented in Sambo 1., Btyksy E.R. & Mashaik

T., 1989, Mo1eci1ag Closid: A LabotaFgu Mapia1, Co1b 8rgd Nagbog Lab. Rgekk, So1b 8rtshd

Nagog, ΝΥ (chapters 9 and 11) and to the Shtep! Рог! Оос1к ίη Mo1esi1ag Vyuodu, 1995, Aikbе1 e! a1. (ebk.), to \ Uyeu & 8opk 1ps. (sections 2.10 and 6.3-6.4) included in this text in the form of bibliographic references.

Preferably, each of such hybridizing polynucleotides has a length of at least 25% (more preferably at least 50%, and most preferably at least 75%) of the length of the polynucleotide of the present invention with which it should be hybridized, and also characterized by at least 60% level of sequence identity (more preferably at least 75% level of identity, and most preferably at least 90% or 95% level of identity) with polynucleotide ohm of the present invention, with which it should be hybridized, despite the fact that the level of identity is determined by comparing the amino acid sequences in such a comparison that maximizes overlap and identity and minimizes sequence breaks.

Expression of WWR1

An isolated polynucleotide of the present invention may be operably linked to an expression control sequence, such as pMT2 or pEE expression vectors described by Kaufman et al. (KaiEtap e! A1., 1991, No. c1. As1bk Vek., 19, 4485-4490), in order to obtain the protein by recombinant means. Many expression control sequences are known in the art. The main methods for the expression of recombinant proteins are also well known: their examples can be found at Kaufman (KaiEtap, 1990, MeYubk En / ut1., 185, 53, 537-566). As used herein, the term “functionally linked” means that the isolated polynucleotide of the present invention and the expression control sequence are mutually arranged in a vector or in a cell so that the protein is expressed by the host cell that has been transformed (transfected) ligated to each other polynucleotide and expression control regulatory sequence.

Expression system for WWR1

A large number of cell types may be host cells suitable for expression of a particular protein. Mammalian host cells include, for example, green monkey CO 8 cells, Chinese hamster ovary CHO cells, human 293 kidney cells, human A431 epidermal cells, human Co1o205 cells, mouse 3T3 cells, SU-1 cells, other transformed primate cell lines, normal diploid cells, cells of lines isolated from cultured ίη νίΐΓΟ primary tissues, primary explants, HeLa cells, mouse cells L, BHK, Hb-60, I937 NaK or 1 IgK1 cells.

On the other hand, it is possible to synthesize protein in cells of lower eukaryotes, such as yeast, or in prokaryotes, such as bacteria. Potentially suitable yeast strains are strains of the species 8assyagotus cecidae, 8cChococagus nocicephidae, strains of Chroteus nigra, Sapb1ba or any other yeast strains capable of producing foreign proteins. Potentially suitable bacterial strains are the strains Exxepylaxis, Bacillus tuberosus, 8a1cope11a1ur1itipit, or any other bacterial strain capable of expressing heterologous proteins. If the protein has been synthesized in yeast or bacterial cells, it must be modified, for example, at suitable phosphorylation or glycosylation sites in order to obtain a functionally active protein. Such covalent attachment can be carried out using known chemical or enzymatic methods.

A protein can also be obtained by functional coupling the isolated polynucleotide of the present invention with suitable control sequences of one or more expression vectors for insect cells using an insect cell expression system. Materials and methods for implementing the baculovirus / insect cell expression system are available on a commercial basis in the form of standard kits manufactured, for example, by the company 1p - \ 'yogodep (8ap O | edo. CA, USA) - the MaxBac7 kit: such methods are well known in this areas of technology and are described, in particular, by Summers and Smith (8mshekk & 8ty, 1987, Techak Adgsi11. Ext. 81a1. Vi11., N 1555: included in this text as a bibliographic reference). As used herein, an insect cell capable of expressing a polynucleotide of the present invention is called transformed.

The protein of the present invention can be obtained by culturing the transformed host cells under conditions that are suitable for expression of the recombinant protein. The resulting expressed protein can then be purified from such a culture (for example, from a culture medium or from a cell extract) using known purification procedures such as gel filtration and ion exchange chromatography. Protein purification can also include chromatography on affinity columns containing agents that would bind to this protein: one or more purification steps on columns with affinity resins such as concanavalin A agarose, heparin-1ouoreag1-7, or sepharose-7 cyanachrome blue 3OA; one or more hydrophobic interaction chromatography steps using resins such as phenyl ether, butyl ether or propyl ether, or immunoaffinity chromatography.

On the other hand, the protein of the present invention can also be expressed in a form that would facilitate its purification. For example, it can be expressed as a fusion protein, such as a maltose-binding protein (MBP), glutathione-8 transferase (68T), or thyroidoxin (TECH). Standard kits for the expression and purification of such fusion proteins are commercially available, such as products of the companies, respectively, No. \ ν Epd1apb VbLaB (Beve ^ 1y, MA), Rnagtaaa (P1xa! A ^ ay, N1) and 1t'ygodep. Also, the protein can be labeled with an epitope and sequentially purified using a specific antibody directed at that epitope. One of these epitopes (E1ad) is available commercially at Kobak (Νον Наνеη, СТ).

Finally, for further protein purification, one or more high-performance liquid chromatography (HPLC) steps in the reverse phase can be used using a hydrophobic HPLC medium such as silica gel including a methyl or other aliphatic side chain. All or some of the above purification steps in various combinations can also be used to obtain a substantially homogeneous isolated recombinant protein. The protein thus purified is essentially free of other mammalian proteins and is defined as an isolated protein in accordance with the present invention.

The protein of the present invention can also be expressed as a product produced by a transgenic animal: for example, as a component of milk in transgenic cows, goats, pigs or sheep, which would be characterized by somatic or germ cells carrying a nucleotide sequence encoding a protein.

Protein can also be obtained using standard chemical synthesis. Synthetic methods for constructing the proteins of the present invention are well known to those skilled in the art. Synthetically created protein sequences due to the homology of the primary, secondary or quaternary structural and (or) conformational parameters can provide general biological properties, including protein activity. Thus, they can be used as biologically active or immunological substitutes for naturally purified proteins during screening of drug compounds and immunological processes associated with antibody formation.

The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins, but with naturally occurring or artificially created modifications. For example, modifications in peptide sequences or DNA sequences can be performed using methods well known to those skilled in the art. Modifications of protein sequences of interest may include substitutions, substitutions, insertions, or deletions of a selected amino acid residue as part of its coding sequence. For example, one or more cysteine residues can be delegated or substituted with another amino acid to change the conformation of a given molecule. Techniques for such substitutions, substitutions, insertions, or deletions are well known to those skilled in the art (see, for example, US Pat. No. 4,518,584). Preferably, such substitutions, substitutions, insertions and deletions retain the desired activity of the protein.

Other fragments and derivatives of protein sequences that are expected to retain the activity of the protein, in whole or in part, and thus can be used for screening or other immunological methods, can also be easily formed by specialists in the art based on what is described in this text. Such modifications are also encompassed by the scope of the present invention.

Hybrid Hybrid Yeast Test

Υ2Η tests showed that the binding of BAP to the BBP1 fusion protein is specific. The binding of BBB1 to BAP confirms that the activity of BBB1 may play a role in the pathogenesis of Alzheimer's disease.

The sequences of BBB1 were compared with SeiVaik data using a local search-matching program (VBA8T: A115s1sh1 e! A1., 1990). The BBP1 protein and translates of the expressed markers included in the database were compared, followed by searching for conservative sites and evaluating them using the program for searching for specific polypeptide motifs (ΜοδΤ: Ta! Igou e! A1., 1994). Such an analysis made it possible to establish the possibility of evolutionary kinship with the family of C-linked protein receptors (SRNS). Specifically, this analysis showed that BBP1 includes two potential transmembrane domains equivalent to the transmembrane domains 3 and 4 present in C-linked protein receptors. The hydrophilic loop located between them includes a well-known motif consisting of three amino acids - the remainder of aspartic (Ό) or glutamic acid, then arginine (I) and then the aromatic residue (Υ or P) (usually denoted as the sequence ΌΡΥ), which is almost conservative all members of this receptor family, and for it, participation in the molecular mechanism of mediating C-protein activation was previously shown (Asyatua & Katshk, 1996).

Test data Υ2Η (see Fig. 2-4) show that the BBB1 protein is a new protein, potentially including a functional module, common with representatives of the superfamily of C-linked protein receptors. Specifically, it is assumed that the BBP1 protein retains an important sequence of INR (from the 199th amino acid to the 201st amino acid in the composition of 8Εβ GO N0 2) between two predictable transmembrane domains and may have the potential to bind to the Protein involved in the regulation of the corresponding signaling mechanism.

Functional binding to Cao has been shown for APP (Νίδΐιίιηοίο e! A1., 1993; 18asha18ir e! A1., 1996), and BBP1 includes a structural motif, for which it is known that it is an activator sequence for Ca protein, being in the composition related C-linked protein receptors. In addition, the hypothesis based on the assumed location and orientation of transmembrane domains in BBP1 suggests that the portion of the protein that interacts with BAP should be topologically corresponding to the same position as BAP in APP.

Υ2 теста test strains were designed to evaluate the binding of the intracellular segment of BBB1 to Ca proteins. The putative intracellular sequences in BBP1 were expressed as a fusion protein and evaluated by their interaction with the C-terminal regions of the three Ca proteins. The protein segments that were used in these experiments are listed below in table. 2. The intracellular loop in the composition of BBB1 interacted with all three Ca proteins (Fig. 3), thereby confirming the assumption that BBB1 can function as a modulator of the activity of C proteins. These various Υ2H tests suggest an interesting model of the multiple protein complex, at least consisting of the interacting membrane proteins BBP1 and APP linked to a heterotrimeric C protein.

Table 2. Plasmids Used in Digibrid Yeast Assays

Expressing plasmid Protein Segment VAR REK162 (man) 1-42 REK240 (mouse) 1-42 WWR1 REK196 (clone 14) 68-269 REK198 (A1sh) 68-202 REK219 (DS) 68-175 REK216 (ΔΝ) 123-202 ρΟΖ339 (intracellular) 185-217 Οα ρΟΖ345 (Οα§) 235-394 ρΟΖ346 (Οαο) 161-302 ρΟΖ348 (Οαί2) 213-355

Further analysis of WWR1 was performed using Υ2Η tests. Two overlapping portions of the BBP1 sequences contained in the clone ΒΒΡΙΔΙιη. were amplified and cloned into the Υ24 vector of pAST2 (expressing plasmids pEK216 and pEK219 [Table 2] and their corresponding proteins ΒΒΡ1ΔΝ and BBRS [Fig. 4]). The ΔC construct is characterized by the absence of both transmembrane domains, and the ΔΝ construct encodes the first transmembrane domain plus 52 amino acids preceding it. These fusion proteins were evaluated using BAP fusion proteins, and the responses obtained were compared with the responses of strains expressing the longer version of the ΒΒΡ1Δ1111 protein. Protein ΒΒΡ1Δ11Π induces a weak response in the Υ2Η test (comparing the BBRI with the vector: Fig. 4), however, the ΒΒΡ1ΔΝ protein, including the first transmembrane domain and adjacent Ν -terminal sequences, results in a response that is only slightly weaker than that obtained for the protein ΒΒΡ1Δ1111 (Fig. 4). These data confirm that the main determinant of binding to BAP lies within the region of BBP1, which is supposedly topologically similar to BAP in the wild-type APP protein.

The Υ2Η system was used to show the selectivity and specificity of binding of BBB1 to human BAP compared to rodent BAP. The amino acid sequence of rodent BAP has three amino acid substitutions (C5K, Ε10Υ and Κ13 В) compared to the human BAP sequence. In the Υ2Υ test described in Example 6, the rodent protein exhibits reduced neurotoxicity and lack of binding to human brain tissue homogenates (Maddu c1 a1., 1992). It would be interesting, therefore, to assess the binding of the rodent BAP to BBB1 in the Υ2Η system. The sequence of human BAP in pEK162 was changed to encode a rodent peptide using oligonucleotide-mediated mutagenesis using PCR. The resulting plasmid pEK240 was identical to the plasmid expressing the human BAP fusion protein used in this experiment, with the exception of three codons producing three amino acid substitutions corresponding to the rodent peptide sequence. The interactions between the BBP1 fusion protein and the human and rodent BAP fusion proteins were compared using the Υ2Η biotest. Strains expressing rodent BBB1 and BAP did not show growth response (Fig. 7). These data confirm the assumption that VVR1 may be a specific mediator of the neurotoxic effect of VAR, and also indicates a mechanism explaining the reduced neurotoxicity of VAR rodent. It is also important that the obtained data demonstrate a high degree of specificity of the BBB1 / BAP interaction in the Υ2Η tests, while the substitutions of three amino acids were sufficient to completely disrupt such a relationship (Fig. 7).

Isolated BBB1 polypeptides

Proteins and protein fragments of the present invention include proteins whose amino acid sequences are at least 25% in length (more preferably at least 50%, and most preferably at least 75%) correspond to the length of the protein being presented, and which are characterized by at least 60% level of sequence identity (more preferably at least 75% level of identity, and most preferably at least 90% or 95% level of identity) with the sequence of the delivered protein, despite the fact that the level of identity is determined by comparing amino acid sequences with such a comparison that maximizes overlap and identity and minimizes sequence breaks. Also included in the present invention are proteins and protein fragments that comprise a segment, preferably comprising 8 or more (more preferably 20 or more, and most preferably 30 or more) amino acids arranged in a row that exhibit at least 75% sequence identity (more preferably at least 85% level of identity, and most preferably at least 95% level of identity) with any such segment of any protein presentable.

Also, the present invention provides species variants homologous to the claimed polynucleotides and proteins. As used in this text, a species homologous variant is a protein or polynucleotide that originates from a different species in comparison with a given protein or polynucleotide, but is characterized by a significant level of sequence similarity compared to this protein or polynucleotide. Preferably, the species homologous variant of the polynucleotide has at least 60% level of sequence identity (more preferably at least 75% level of identity, and most preferably at least 90% level of identity) compared to this polynucleotide, and the species homologous variant of the protein was characterized by at least 30% identity (more preferably at least 45% identity, and most preferably at least 60% m level of identity) compared with this protein, despite the fact that the level of sequence identity is determined by comparing the nucleotide sequences of the polynucleotides or amino acid sequences of the proteins with such a comparison that maximizes overlap and identity and minimizes sequence breaks. Species homologous variants can be isolated and identified by forming suitable probes or seeds based on the sequences presented here and screening a suitable source of nucleic acids corresponding to the desired species. Preferably, species homologous variants are isolated from mammalian species. More preferably, species homologous variants are isolated in mammalian species, such as, for example, chimpanzee Rap 1 year 1 obu 1sk. gorilla SogSha doga, orangutan Ropdo rudtayeyk, gibbon Nuooba! ek sopso1og, macaque Masasa tiaaaa, baboon Raryu raryu, hamadryl Rarya yatabguak, green monkey Segsyoriyesik ae1Shork, cactus biloba! mouse lemur Mugosebik type rush, house mouse Mik tiksi1ik, rat Ranik poguedudik, Chinese hamster Spseybik dpkeik, domestic cat EiNk sa! ik, American mink Mik! e1a U1kop, domestic dog Sashka GatShapk, rabbit Ogus! o1adik sorigsy 1 , domestic sheep Ou1k apek, domestic St. Nya 8iksGGGa and the horse Yeskssk saBa11ik, for which genetic maps have already been created, allowing one to evaluate the relations of synteny between the genomic organization of one species and the genomic organization of the corresponding genes in other species (O'Vpep & 8eyape /, 1988, App. Neu. Sepe !. , 22, 323-351; O'Vpep e! A1., 1993, Ya! Ige Sepe!., 3, 103-112; 1st appcop e! A1., 1995, Sepotkk, 25, 682-690; ., 1997, Ya! Ig Sepe!., 15, 47-56; O'Vpep e! A1., 1997, Tgepbk and Sepe!., 13 [10], 393-399; Sagueg & 8! Bk, 1997,

Sepot Nek., 7, 1123-1137: all articles are included here in the form of bibliographic references).

Also, the present invention provides allelic variants of the claimed polynucleotides or proteins: they are naturally occurring alternative forms of isolated polynucleotides that also encode proteins that are identical or have substantially similar sequences compared to those encoded by the claimed polynucleotides. Preferably, allelic variants are characterized by at least 60% level of sequence identity (more preferably at least 75% level of identity, and most preferably at least 90% level of identity) compared to this polynucleotide, while that the level of identity is determined by comparing the nucleotide sequences of the polynucleotides with such a comparison that maximizes overlap and identity and minimizes gaps lnostey. Allelic variants can be isolated and identified by using suitable probes or seeds based on the sequences presented here and screening a suitable source of nucleic acids from individuals of the corresponding species.

The present invention also provides polynucleotides, the sequences of which are complementary to the sequences of the polynucleotides claimed herein.

Applications

Proteins BBB1 of the present invention can be applied by methods that are standard for specialists in this field of technology based on what is set forth in this text. Specifically, BBB proteins can be used as immunogens to increase the amount of antibody produced that is specific for cloned polypeptides. Various procedures known in the art can be used to produce antibodies to BBB1 proteins. Such antibodies include, but are not limited to, polyclonal, monoclonal, fused, single chain antibodies, fragments of an EaB and an EaB expression library. In order to obtain antibodies of animal recipients of various species, including, but not limited to, rabbits, mice and rats, are injected with the BBP protein. In one embodiment, a polypeptide or fragment of a polypeptide having specific immune activity is coupled to an immunogenic carrier. Adjuvants may also be administered in combination with the polypeptide in order to enhance the immune response of the animal recipient. Examples of adjuvants that can be used include, but are not limited to, Freund's complete and incomplete19 complete adjuvants, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, high molecular weight pluronic alcohols, polyanions, peptides, oil emulsions, hemocyanin slug and dinitrophenol.

Monoclonal antibodies to the BBB1 proteins of the present invention can be obtained using any technique that would ensure the production of antibodies by a continuously cultured cell line. 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 Köhler and Milstein (Köyet & MPMate 1975, Ya! Ig, 256, 4202-4207), a hybridoma technique using human B cells described by Cosbor et al. (Kokot e! A1., 1983, 1ttypo1. Tobau, 4, 72), and based on the transformation of the Epstein-Barr virus hybrid method described by Cole et al. (Сеее! А1., Мопос1оп1 Apbobobek apb Sapseg Tyetaru, L.R. Ykk 1ps., Pp. 77-96).

Antibodies that react with the polypeptides of the present invention can then be used to screen for the presence and subcellular distribution of similar polypeptides in a biological sample. In addition, monoclonal antibodies specific for the BBB1 proteins of the present invention can also be used as drugs.

The BBB1 proteins can also be used as antigens useful in solid-state tests aimed at measuring the presence of antibodies that are immunoreactive in relation to the claimed peptides. Solid phase competition tests can be used for immunological quantitative analysis of clone-related 14 antigens in biological samples. Such an analysis is applicable not only to facilitate the complete characterization of the cellular function or functions of the polypeptides of the present invention, but can also be used to identify patients characterized by an abnormal amount of these proteins.

The BBP1 proteins of the present invention can also be used as trapping reagents in affinity chromatography to detect BAP itself and BAP aggregations, which are symptoms of Alzheimer's disease.

In addition, these BBB1 proteins are useful as reagents in a test designed to identify candidate molecules that affect the interaction of BAP and a cloned protein. Compounds that specifically block this interaction can be used to treat or prevent Alzheimer's disease.

Also, these BBB1 proteins are applicable in cell-free binding tests for ίη yugo, which determines the change in the influence of the binding compound of these proteins associated with β-amyloid peptides with BAP or BAP aggregations. Cell-free tests are especially applicable for screening a large number of compounds, since such tests are relatively economical and easy to perform in comparison with tests based on the use of living cells. Given the use of the polypeptides of the present invention, the development of such tests would be a routine for a person skilled in the art. In such tests, either BBB1 or BAP is labeled. Such labels include, but are not limited to, radioactive labels, antibodies, or fluorescent or ultraviolet labels. Binding of BBB1 to BAP or BAP aggregations is first detected in the absence of any of the test compounds. Then, compounds intended for testing are added to the test medium to determine whether these compounds influence this interaction.

Examples

Further, the present invention is described in the form of examples. The examples are presented solely in order to illustrate the invention by reference to specific options. These examples, illustrating some specific variations of the present invention, do not thereby introduce any limitations or scope to the scope of the present invention.

Hybrid yeast system (hereinafter referred to as Υ2Η). The expression plasmids for Υ2Η were constructed on the vectors pL82 and pACT2 (described by UabeNagreg e! A1., 1993) and pCIR (described by Oxepegdeg & Koynd, 1995). The yeast strain SU770 (Ocherbegdeg & Koynd, 1995) was used as the host strain in all of the 2G tests.

Genetic screening. The polymerase chain reaction (PCR) method was used to amplify and modify sequences encoding the BAP protein. Oligonucleotides No. 1 (5'-SS ATS OLT OSA OLA TTS SOA S) and No. 3 (5'-AAOOSTTOTSOAS TTA SOS TATOAS AAS ACC OS) were used to amplify BAP using pCb621 as a template, which is a modified human APP clone ( 1ACOBEK E! A1., 1994). The amplified DNA includes codons 389-430 (which encode BAP42) of the APP precursor protein, subject to the following modifications. The semantic seed added the restriction 5'-site of Yco1 to the composition of the same broadcast open frame, in which there is a Yso1 site of pA82. Antisense seed added a stop codon and the sites ΗίηάΙΙΙ and 8aI, necessary for cloning. The products of this amplification were ligated into the TA cloning system (GpuGodspods Comp., CnHlAf CA), followed by removal using restriction enzymes LiCl and 8aI. The resulting fragment was cloned into the plasmid pA82 digested with restriction enzymes Lyso1 + 8a11. The resulting plasmid, pEK162, was confirmed by DNA sequencing at the site of the CA4 / BAP compound. The protein (BAP ⁸⁰ : FIG. 1) expressed from plasmid pEK162 includes a fusion protein containing the DNA-binding domain of the yeast Ca14 transcription activator protein (lacking functional activation sequences) with 42 BAP amino acids added from the C-terminus. The expression plasmid is formed in such a way that it mediates the expression of unmodified BAP42. Seed No. 2 (5'AASSTTAAS ATS CAT CAT SSA CAA TTS CCA S) was combined with seed No. 3 in the PCR method described above. The product obtained as a result of this amplification includes a 5 'located άΙΙΙηάΙΙΙ site and translation initiation signals optimized for expression in Sassyagossus sssssyas cells. Again, the DNA fragment was cloned into the TA system. Then it was isolated as a ΗίηάΙΙΙ fragment, which was cloned into the pCIR plasmid at the ΗίηάΙΙΙ site. The orientation of the BAP gene in the resulting plasmid pEK149 (BAP: Fig. 1) was confirmed by DNA sequencing. The BAP-expressing plasmids pK149 (using the HKA3 gene as a selective marker) and pEK162 (using the TBP1 gene as a selectable marker) were used to transform the yeast cells of the SU770 host (Oxfcrms & Wash. 1995). The strain carrying both of these plasmids was designated SU2091. A plasmid library, including cDNA fragments isolated from human fetal brain cells, cloned into the pAST2 vector (using the LFe2 gene as a selectable marker) for yeast yeast expression, was purchased from CIoxys baB. Ws (Ra1o Ayo, CA). The protein obtained from the library of FIG. 1 is designated as 411-11 (^ 41-1 ⁹¹ ° ^1. This library was used to transform SU2091. Samples were distributed in complete synthetic medium (8C) for culturing yeast lacking uracil, tryptophan and leucine to select cells carrying all three plasmids. Histidine was also absent in the medium and was present at a concentration of 25 mM 3-aminotriazole, an inhibitor of the product of the ΗΙ83 gene. 3-Aminotrizole was used to reduce the low constitutive activity of the ΗΙ83 reporter gene. The plates were incubated at 30 ° C for of 12 days. it was identified 24 colonies exhibiting enhanced prototrophy by histidine. Transformation controls indicated that the screening were tested June ¹⁰ individual clones. For efficient determination of the composition of positive clones was used the PCR method. Total DNA was isolated from each positive strain by means standard methods The resulting material was used as a template for PCR with seeds No. 4 (5 'TTTAATASS STASAAT CCA T) + No. 5 (5'TTTTTSASTAT STASSATTS T), which flank the cloning library segment pAST2 vector. DNA fragments were ligated into the TA system and tested by DNA sequencing. The library plasmid contained in clone No. 14 (as described above) was isolated by the bifunctional pathway to E. soi. The nucleotide sequence of the human cDNA was determined, which confirmed the sequence composition of the initial PCR product.

Biotests. Strains were grown overnight in 2 ml of 8C medium devoid of leucine and tryptophan until a density of approximately 7x10 ⁷ cells per 1 ml was reached. Cells were counted, and then 10-fold serial dilutions were prepared from 10 ⁴ to 10 ⁸ cells per 1 ml of sterile water. These samples were instilled in aliquots of 5 μl onto 8C medium lacking leucine, tryptophan and histidine and containing 25 mM 3-aminotriazole. The plates were incubated at 30 ° C for 2-3 days. Positive interprotein interactions were detected by an increase in prototrophic growth compared with control strains expressing a fusion protein with a Ca14 DNA binding domain plus an extraneous fusion protein with a transcription activation domain (or simply including a pAST vector that lacked any built-in sequences). These control strains were defined in the above figures as labeling vector. This test is characterized by a high level of reproducibility of the results and is suitable for detecting even weak growth induction mediated by a specific interaction between target proteins. The original BBP1 clone designated pEK196 and introduced under ATCC 98399 (designated clone 14 here) was used as a PCR template to shorten the protein product expressed by BBP1A1Sh. Semantic seed No. 6 (5'-TTTAATASS STASAATSS T) was annealed on the CAE4 sequence as part of pAST2. Antisense seed No. 7 (5'-STCCAS TTA AAA TCC ATS TCC TCC CAA SS) introduced a 3'-stop codon and HyoESite immediately from the 3'-side of the sequence encoding the EVE motif in BBP1. The PCR product was ligated into the cloning vector system TA followed by cutting with restriction enzymes EcoB1 + X1y1 and cloned into the plasmid pATC2. The hybrid product expressed by this plasmid (pEK198) was designated BBB1D1t. Similarly, seed No. 7 was combined with seed No. 8 (5'CAATT CCA AAA ATA AT CAC SST ACC) to construct plasmid pEK216 expressing BBP1AK. Again, the PCR product was ligated into the TA system, and the resulting plasmid processed with EcoB1 restriction enzymes + X1yE-bearing BBB1 fragment (codons 123-202) was ultimately ligated into pATC2 cleaved with the same restriction enzymes. The VVR1DS variant was formed using the RATS-specific primer No. 6 and the antisense primer No. 9 (5'-STCCAS TSA ACA TAT CCC CTT CAA AAA AC). After TA-cloning, isolation of the EcoB1-X1o1 fragment and cloning into pAST2, the resulting plasmid pEK219 expressed BBP1 from the 68th to 175th amino acid residue. The sequences encoding the intracellular loop of BBB1 were amplified using oligonucleotide probes No. 10 (5'CSTTCC ATC CAA STS CCA STC CCA TTC TST) + No. 11 (5'-AACASTC CAC TCA AAA CCC TAC AST CCA AAA C). This product, including the BBP1 codons from 185th to 217th, was digested with restriction enzymes Xco1 + X1u1 and cloned into pA82, cleaved with #o1 + 8a11 to obtain plasmid ρ0Ζ339. Plasmid constructs expressing all Sabelkov variants used the WatH1 site located near the center of each rat cDNA sequence (Kapd e1 ab, 1980) as a site for connection with pAST2. The sense seeds were annealed at sequences located on the 5'-side of the WatH1 site, and the antisense seeds were annealed at sequences of the 3'-stop codon, including the restriction site 8a11. The following seeds were used: Cao, semantic (No. 17) -5'-STSSATSSAS TSSTTSS-ACC AT, antisense (No. 18) - 5'STSSASSSST STATASASS ASAAS-ACC; Sak, semantic (No. 19) - 5'-STSSATSSAS TSSTTSAATS AT, antisense (No. 20) - 5'STSSASTAAA TTTSSSSSTT SSSTTSTT; Ca12, semantic (No. 21) - 5'-STSSATSSAS TSSTTTSASS ST, antisense (No. 22) - 5'STSSASSSSST TSTSTSSSSS SATSTTS. PCR products were cloned into TAvector. Ca gene sequences were isolated as BatH1-8a11 fragments, which were cloned into pAST2, cleaved by the restriction enzymes BatH1 + 8a11 (see Table 2 with the plasmid designation). Finally, oligonucleotide No. 23 was synthesized to convert a human BAP into a rodent sequence. This seed had the following sequence: 5'ATATSSSSATS CAT CCA CAA TTS CCA CAT CAC TCA CCA TTT CAA CTT CCT. Triplets represent the first 13 BAP codons: underlined are three nucleotides that have been modified to encode a rodent sequence. Oligonucleotide seed No. 23 was combined with seed No. 24 (5'TSACSTASAS SAAASASTTA), which was annealed to the участок2Η vector site located on the 3'-side of the cloning site, in PCR using pЕК.162 as a template. The resulting product was digested with restriction enzymes No. 1 + 8a11 and ligated into pA82 to obtain plasmid pEK240. The accuracy of the nucleotide sequence in the rodent coding segment was confirmed.

Genomic cloning: BACE method (rapid amplification of cDNA ends). The genomic clonothek on λ-phages (8! Ha! Adepe), corresponding to ^a 2.0 × 10 ⁶ rGik, was screened with a probe from a randomly primed EcoB1 / -C1a1 fragment corresponding to nucleotides 187-600 (Fig. 2). This probe was labeled ³² P-CTF using a reagent kit `` Ssskrpteg ΒίΙ in accordance with the manufacturer's recommendation (Ryagtas1a). The filters were hybridized under conditions of high stringency: 40 ° C in 50% formamide, 0.12 M NaΗP0 ₄ , 0.25 M No. C1, 7% 8–8 and 25 mg / ml of salmon sperm DNA irradiated with ultrasound and washed at 65 ° C in a 0.1x88C buffer containing 0.1% sodium dodecyl sulfate and scanned using a Dog VuMax M8 film. Clones of λ-phages hybridizing with the probe were purified by successful plating and re-screening. Ten positive clones were purified and subjected to further analysis by hybridization with a 45-nucleotide oligonucleotide probe corresponding to the 5'-sequence known in the original cDNA clone. This oligonucleotide was characterized by the reverse composition of nucleotides 157-201 (Fig. 2) and was characterized by the sequence δ'-ССССССССССС СССАТСТТСС

ASASSSSASAS TTTSTSSSSA STTSS. Λ-phage DNA was isolated using standard molecular biological techniques and subjected to direct sequencing using the Sanger method using an AB1-373 automatic sequencer.

BACE method. The synthesis of the first DNA strand was carried out using the heat-resistant polymerase system gT (H (Reg E1tag).

The following reagents were combined in a 1.5 ml tube to achieve a volume of 10 μl: 1x reverse transcriptase buffer, 1 mm MPS12, 1.6 mm dNTP (mixture), 2.5 units hTSH polymerase, 100 ng poly-A human hippocampal RNA (S1op1se11). 10 mM oligonucleotide (nucleotides 429-452 of FIG. 2: 5'-STTATSTTSS STSTS CAAA ACAC). The reaction mixture was incubated at 70 ° C for 15 minutes and immediately placed on ice. The Matayop cDNA synthesis kit (SopIesN) was used to form the second cDNA strand. The full volume (10 μl) of the reaction mixture of the first chain was supplemented with the following reagents: 1x buffer of the second chain, 0.8 mM dNTP mixture, 4x cocktail of the second chain (DNA polymerase I E.coN. DNA ligase E. soi, RNase-N E.coN) and distilled water to a volume of 80 μl. The tube was incubated at 16 ° C for one and a half hours, after which T4 phage DNA polymerase was added (10 units), and incubated for another 45 min at 16 ° C. To stop the reaction, 4 μl of a 20 x EETA / glycogen mixture (0.2 M EETA + 2 mg / ml glycogen) was added to the reaction mixtures, followed by extraction with a phenol / chloroform / iso-amyl alcohol mixture to remove enzymes and other impurities. DNA was precipitated by the addition of 0.1 × 3 M sodium acetate (pH = 5.2) and 2.5 × chemically pure ethanol with a temperature of -70 ° C. The DNA was washed once with 70% ethanol, dried and resuspended in 10 μl of distilled water. Half of the DNA was used for ligation with the Mata1yop adapter, followed by use in CASEPCR reactions in accordance with the S1op1ss11 protocol as follows: 5 μl of cDNA was added to 2 μl (10 mM) Matayyop (5'-STAATASSAS TSASTASASSS SSTSSASSSSSSSSSSSSSSSSeSSST DNA) ligation and 1 μl (1 unit) of T4 DNA ligase. The reaction mixture was incubated overnight at 16 ° C. The mixture was diluted 1:50 for the initial reaction. CASE was combined in a 0.2-ml PCR tube with the following reagents: 40 μl of distilled water, 1 μl of 10x K1ep1ac DNA polymerase. | (C1op1esy), 1 μl (10 mM) seed AR1 (5'CCATSSTAAT ASSASTSAST ATASSSS), 1 μl (10 mm) BBP1-specific seed (corresponding to nucleotides 187-209 - Fig. 2: 5'CESSASSSSSS SSSSSSSSSSS AT), 5 μl 10x K1ep1ad polymerase buffer, 1 μl of 10 mM dNTP mixture, 1 μl of diluted cDNA obtained in the previous reaction. The following cyclic conditions were carried out using a SepeAtr 2400 thermal cycler (Retksh E1teg): denaturation cycle at 94 ° C for 1 min followed by 5 cycles at 94 ° C for 30 s, at 72 ° C for 3 min, 5 cycles at 94 ° C 30 s, at 70 ° C 3 min, followed by 25 cycles at 94 ° C 30 s, at 68 ° C 3 min, with final chain completion at 72 ° C for 7 min. After this step, CACE-PCR was carried out with the seeds seeded according to the following scheme: 40 μl of distilled water, 1 μl (1 unit) 10x DNA polymerase At p1ac. | Co1b (Retksh E1teg), 1 μl (10 mM) of AP2 seed (5 'A-ASTASTA SSSSTSSASS SSS), 1 μl (10 mM) of BBP1-specific seed (corresponding to nucleotides 172-194 - Fig. 2: 5'-SSSSSSSATST TSSASASSSS SAS), 5 μl 10x Atr1yad polymerase buffer, 1 μl 10 mM dNTF mixture, 1 μl of the primary CACE product, the cyclic PCR conditions were as follows: the initial denaturation cycle was 9 min at 94 ° C, 25 cycles of 30 s at 94 ° C, 30 s at 68 ° C, 2 min at 72 ° C, followed by chain completion at 72 ° C for 7 min. The PCR product was transferred to a 1% agarose gel in 1 × TBE buffer. The resulting product, consisting of 350 pairs of nucleotides, was gel-purified and directly cloned using a TA Clone kit cloning kit (1 Puygodep). Ligation mixtures transformed Ope81U1 cells (1 Puitodep), which were plated on L -ampicillin agar (100 μg / ml) onto X-da1 plates. DNA mini-preparations were obtained and sequenced by the Sanger method on an AB1-373 automatic sequencer.

Northern blot analysis. Northern blots of mRNA from various human tissues and from tissues of various parts of the brain were obtained from C1op1es11 (Pa1o A11o, CA). The BBB1 sequences, extending from the initial fusion to the polyadenyl signal, were isolated into the EcoK1 fragment from the TA clone derived from the plasmid pEK196. Β-actin DNA was provided by the manufacturer. Radioactively labeled probes were obtained on the material of such DNA using random priming with the aim of introducing ³² R-dTCP (Ryagtaa Vuyesy, Rysayatau, N1). Hybridization was carried out according to the manufacturer's instructions (C1O1esy) in an Exteck Nui solution at 68 ° C. Blots were washed in 2x88C buffer (1x88C - 0.15 M sodium chloride, 0.015 M sodium citrate), 0.05% 8–8 at room temperature, followed by washing in 0.1x88C, 0.1% 8–8 at 50 ° C. Hybridization signals were visualized using a Kobak VuMax film.

Hybridization w 8Yi. DNA matrices for the synthesis of ribonuclein probes were obtained by PCR using a plasmid clone, including a full-sized human VVR cDNA. A single ribosonde was used that labeled the 3'-ITC (3 'untranslated segment) of this cDNA. The probe sequences were checked by comparing with the SepWap database to ensure that they only recognize suitable labels from all deposited sequences. To form ribosondes for BBP1, a pair of oligonucleotide seeds was constructed, which allow amplifying a region of 275 nucleotides from 3'-TEC cDNABPB1 and adding promoter sequences to T7 (sense) and T3 (antisense) polymerase. These primers had the following sequences: 5'-TAATAS6AST SASTAT 666 TTA6AA6AAA CA6ATTT6A6 (direct) and 5'-ATTAASSSTS ASTAAA6 66A CAA6T66CAA STT6STST6 (reverse). PCR products were purified on a 1.5% low-melting agarose gel, the product containing bands were excised, extracted with phenol and a phenol / chloroform mixture, and precipitated with ethyl alcohol. The precipitate was dried and resuspended in 1x TE buffer (10 mM TrisNS! 1 mM EETA - pH = 7.4). The APP ribosonde matrix included an EbeEEXEoE fragment from the coding sequence as described by Jacobsen et al. (1ACOBKEP1 AB, 1991). Fifty ng of the DNA template was used for the transcription reaction using the labeled ³⁵ 8-CTF (Ete Epnapb No. 1c1eag. Wok! Op, MA) and the Ribotonus Schortoe 6esch ™ (Rgoteda, Maikop, XVI).

Histochemical hybridization of ίη Kyi using sections of human hippocampal tissue obtained by rock! Toyota was carried out in accordance with the previously described (Kyobek, 1996). Slices were obtained with a thickness of 10 μm using the Naskeg-BrfK cryostat ultratome and, upon melting, were spread onto glass slides cooled to -20 ° C, coated with the Reagent-Reagent reagent (Uesug-Baib, Vigtdate, CA). All solutions were obtained in distilled water, treated with 0.1% (volume percent) diethyl pyrocarbonate and autoclaved. Sections were fixed by immersion in 4% paraformaldehyde in phosphate buffer (pH = 7.4), then they were successively immersed in 2x88C, distilled water and 0.1 M triethylamine (pH = 8.0). Then the sections were acetylated by immersion in 0.1 M triethanolamine containing 0.25% (volume percent) of acetic anhydride, washed in 0.2x88 ° C, dehydrated in 50%, 70% and 90% ethanol and quickly dried . One ml of a prehybridization solution containing 0.9 M NaCl, 1 mM EETA, 5x Dunhardt's solution, 0.25 mg / ml single-stranded herring sperm DNA (61LCO / BKE, 6yegkkegd, ME), 50% deionized formamide (EM 8c1epsek, 61bk1k p, N1) in 10 mM Tris (pH = 7.6) were pipetted onto each glass slide, which was then incubated for 3 hours at 50 ° C in a wet incubator. Then the sections were dehydrated by immersion in 50%, 70% and 90% ethanol and dried in air. Labeled ribosondes were added at a final concentration of 50,000 imp / min per 1 μl to a hybridization solution containing 0.9 M No. C1, 1 mM EETA, 1x Dunhardt solution, 0.1 mg / ml yeast tRNA, 0.1 mg / ml single chain Salmon sperm DNA, dextransulfate (10%), 0.08% bovine serum albumin (B8A), 10 mM dithiothreitol (Voyeppdet Mapple1t, 1pb1aparo11k, ΙΝ) and 50% deionized formamide in 10 mM Tris (pH = 7.6). Then the probes were denatured at 95 ° C (1 min), placed on ice (5 min) and dug into sections: they were left to hybridize overnight at 55 ° C in a wet incubator. Sections were washed sequentially once for 45 minutes at 37 ° C in 2x88C, including 10 mM dithiothreitol, then once for 30 minutes at 37 ° C in 1x88C containing 50% formamide, and once for 30 minutes at 37 ° C in 2x88C. Single-stranded and non-specifically hybridized ribosondes were digested by immersion in 10 mM Tris (pH = 8.0) containing bovine pancreatic RNase-A (Voyeppdet MappEepp; 40 mg / ml), 0.5 M No. 1C1 and 1 mM EETA. Sections were washed in 2x88C for 1 h at 60 ° C, then in 0.1x88C containing 0.5% (weight percent) of sodium thiosulfate for 2 hours at 60 ° C. Then the sections were dehydrated with 50-70-90% ethanol containing 0.3 M ammonium acetate, and dried. Slides were placed in radiographic cassettes and subjected to contact with Nuret Sht-Max (Atetkyat) for 14-30 days. When a satisfactory result was obtained, the slides were coated with an emulsion to detect nuclear tracks (ΝΊΒ2; Kobak) and tested for 7-21 days at 4 ° C. Emulsion autoradiograms were obtained and recorded in accordance with the manufacturer's instructions, and tissue sections located under the emulsion were stained with hematoxylin. To evaluate nonspecific labeling, a control probe was formed on the basis of a matrix taken from a set of Brtorte 6t1sh ™ 8uk1et ky (Rgoteda). This vector was linearized using restriction enzyme 8ca1 and transcribed using polymerase T3. In the obtained transcriptional reactions, two products were obtained - ribosondes, consisting of 250 and 1525 nucleotides, containing only the vector sequence. The mixture with this control probe was labeled as described above, added to the hybridization solution at a final concentration of 50,000 cpm in 1 μl. No specific hybridization was detected in the control sections, i.e., these sections produce a very weak uniform hybridization signal that does not correspond to any neuroanatomical markers (data not included).

Example 1. Cloning and selection

BAP-binding protein (BBP1).

The Hybrid Yeast Screening Method (Υ2Η) was developed to identify proteins that interact with

Human BAP42, a 42-amino acid proteolytic APP fragment that is potentially substantially more toxic in the aggregated form of BAP. BAP42 was expressed as a chimera in combination with the Ca1 4 DNA binding domain, and was also expressed as a free peptide (Fig. 1). This strain was transformed with the U2N library of the human fetal brain cDNA. The only clone designated, in accordance with the aforementioned, as clone 14, out of approximately one million independent transformants, showed significant activation of the reporter gene and essentially included an open coding frame that continuously transitions to the sequence encoding the CAB4 domain. The cDNA insert included 984 nucleotide pairs and terminated in the polyadenyl tract. The sequence encoded 201 amino acids (amino acid residues 68-269 in 8EO FROM N0 2) with the presence of two sites, characterized by a length and level of hydrophobicity, sufficient for penetration through the cell membrane.

The plasmid derivative of the library was isolated from clone 14 and used to reconstruct the strains included in the Υ2Η test. Testing of these strains showed that the BAP fusion protein specifically interacts with the protein encoded by clone 14, although the response was rather weak. Given that high-level hydrophobicity protein domains, such as transmembrane regions, suppress the responses in the Υ2Η test (Oxspcrdsg unpublished data), clone 14 was shortened (denoted as ΒΒΡ1Δ1111) in order to remove the region of hard hydrophobicity, followed by retesting interaction with BAP. A significantly more powerful answer in the Υ2Η test was noted with the ΒΒΡΙΔΙιη variant (Fig. 2): this confirms the assumption that the delegated sequences encoded a probable transmembrane anchor (1t). The nucleotide sequence of clone 14 was screened according to CspBap: as a result, a BAP-binding protein (BBP1) was identified as new.

Example 2. Isolation and confirmation of the 5'end of BP1.

The BBP1 cDNA sequences including clone 14 described in Example 1 were devoid of the 5'-coding region, as indicated by the absence of a potential initiating codon of methionine. Numerous attempts to use the standard 5'-CACE method (rapid amplification of cDNA ends) using conventional reverse transcriptase allowed a total of 27 nucleotides to be added. These sequences included the ATS triplet, but did not include the stop codon located above in the same broadcast coding frame, which confirms the accuracy of determining this triplet as the initiating codon. The genomic cloning technique was used to isolate the 5'-terminal portion of the BP1 gene.

Hybridization of the human genomic λ-library with a random priming probe corresponding to 400 pairs of nucleotides (bp) of the 5'-sequence of clone 14 led to the identification of 10 positive clones. Further characterization of these clones using a 45-dimensional oligonucleotide probe directed to the uppermost ΒΡΙ-sequence of clone 14 (and the corresponding 5′-top sequence 400 bp), this probe showed that 6 out of 10 isolated clones include terminal 5′- sequences within those that have been previously identified. It was found that the other four λ-clones represent other exons that are part of the original derivative of the cDNA probe obtained by random priming 400p. n (data not shown here).

Direct sequencing of λ-phage DNA from clones corresponding to the 5′-end of BBB1 represents about 500 nucleotides located on top and overlapping with known clone 14. This additional sequence allegedly encodes 62 amino acids located in the Ν side with respect to the previously characterized methionine, identified before the detection of MET, preceding the intra-frame stop codon. Although there are two other lower methionine residues in relation to the upper MET, using standard approaches, the applicants experimentally determined the sequences of the “terminal part of the human BBB1 gene as including the first of the 5'-methionine codons following the intra-frame stop codon. The complete coding segment and the decoded amino acid sequence are shown in 8EO ΙΌ N0 1 and 2. A plasmid (designated ΒΒΡ1-Π) including this amino acid sequence was added to the American Type Culture Collection under Depository Number ATCC 98617.

Since the 5'-coding sequences originated from the genomic clonotheque, it is likely that this segment includes introns. This probability was estimated using two methods. First, the direct seed corresponding to the 5'MET segment and the reverse seed corresponding to the region within clone 14 were used to amplify the sequences of the brain cDNA, as well as genomic DNA. Products of identical size were obtained from both samples: this indicates the absence of introns within this region and confirms the relationship of the upper sequence with the original sequence. Secondly, cDNA sequences were isolated in experiments based on the modified 5'-BACE method (see Materials and Methods section), which turned out to be identical to those that were isolated from the genomic clone. These data confirmed the accuracy of the establishment of the upper sequences (from the sources of both the cDNA library and the genomic clon library) and the absence of introns in this segment.

Example 3. Characteristics of WWR1.

The sequences of VVR1 were compared with SeiVaik data using a local matching program (VBA8T: L115syi1 e! A1., 1990). We identified two genomic sequences of the free-living nematode Saeogia bibcidae8 and one genomic sequence of the Drosophila Igoopor1a te1aodaieg, as well as a large number of expressed markers of the genomes of humans, mice, and other mammalian species. However, no complete cDNA sequences were found, nor were any functional data associated with the gene. The BBP1 protein and translation products of the expressed expressing markers were compared, tested for the presence of conserved segments and evaluated using the Mo8T program (Ta! And e! A1., 1994), aimed at searching for specific protein motifs. These analyzes showed a likely evolutionary link to the C-linked protein receptor family. Specifically, these analyzes showed that BBP1 includes two potential transmembrane domains equivalent to the transmembrane domains 3 and 4 present in C-linked protein receptors. The hydrophilic loop located between them includes a well-known motif that includes three amino acids - aspartic (Ό) or glutamic acid, then arginine (B) and then an aromatic residue (Υ or E) (usually denoted as sequence ΌΒΥ), which is conservative in almost all members this receptor family, and for it, participation in the molecular mechanism of mediating C-protein activation was previously shown (Asyagua & Kagshk, 1996).

These data indicate that the BBP1 protein is a new protein that potentially includes a functional module that is common with members of the superfamily of C-linked protein receptors. Specifically, it is believed that the BBB1 protein retains a critical sequence ΌΒΡ between its two putative domains so that it can be a potential ligand of C-linked proteins involved in the regulation of the signaling pathway.

Structural analysis of the BBP1 protein showed that it includes a structural motif known as the activator sequence of the Ca protein in the composition of related C-linked protein receptors. Hybrid yeast tests showing the interaction of BBB1 with various members of the C-linked protein receptor family are illustrated in FIG. 3. Based on structural predictions, the BBP1 protein is defined as a protein that penetrates twice through the membrane at both ends of the membrane into the lumen component. But other orientations of this protein cannot be completely rejected. The potential protein interactions described above were investigated in the Υ2Η tests. Two overlapping segments of the BBB1 sequences included in the BBB1D1t clone were amplified and cloned into the А2Η vector pAST2 (expressing plasmids pK216 and pKK219-table 2; and the corresponding proteins BBB1ΔN and BBB1DS - Fig. 4). The DS construct loses both transmembrane domains, and the DI construct encodes the first transmembrane domain plus the previous 52 amino acids. These fusion proteins were tested for interaction with BAP fusion proteins, and the responses obtained were compared with those of strains expressing the larger BBB1D1t protein. The results confirm that the main determinant of binding to the BAP protein lies within the segment of the BBP1 protein, which is supposedly topologically similar to the segment of the BAP within the wild-type APP precursor.

Example 4. Tissue distribution of expression of human BBB1.

The expression of BBR1 mRNA was investigated as the initial step in detecting the activity of this gene and its encoded product. A basic transcript of 1250 nucleotides in length was found in all tissues (Fig. 5A). A high level of expression in the heart was noted. The whole brain was characterized by an average level of expression. All samples of individual parts of the brain showed expression of BBB1 (Fig. 5B). Interestingly, all limbic regions were characterized by a relatively large number of BBP1 mRNA. There were areas of the brain in which initially there were aggregations of BAP and the associated manifestation of neurotoxicity. An analysis of autologous X-ray diffraction patterns of ίη b1i obtained using a specific BBB1 ribosonde showed that, in the human hippocampus, the entorhinal part of the cerebral hemispheres, BBB1 mRNA was expressed in medium to large sized cells, consistent with expression in neurons as opposed to glial cells (Fig. 6 ) Moreover, BBB1 mRNA is virtually expressed in all hippocampal and entorhinal neurons, since it was not possible to identify any real or laminar differences in the intensity of the hybridization signal. Interestingly, the parameters of BBB1 expression turned out to be substantially similar to the parameters detected using a ribosonde directed to an mRNA encoding an amyloid protein precursor, APP (Fig. 6). In general, BBR1 mRNA was detected in all tissues and all tested segments of the brain. Analysis of the expression of BBP1 mRNA expression also showed the presence of a high level of expression in the hippocampus segment.

Example 5. Distribution of expression of BBP1 in cell lines.

The expression of BBP1 was also investigated in a number of cell lines, and data were also taken from bBE8T, a collection of expressed markers available at the National Center for Biotechnological Information. Reverse polymerase chain reaction (RT-PCR) methods were used to quantify the expression of BBP1 mRNA in cell lines commonly used for expression of recombinant proteins, as well as in a number of tumor cell lines. The BBP1 protein was found in Chinese hamster ovary cells and human HEK293 cells. Signals were detected in the No. 2-embryonic stem cell line and the 1MK82 and 8Κ-Ν-8Η neuroblastoma lines. The expression of BBP1 was detected in the lines of tumor cells originating from the following tissues: colon (Cx-1, Co1o205, M1P101, 8 No. 948, CaCO, 8 No. 620, B8174T), ovaries (A27808, A2780IIIR), mammary gland (MCP- 7, 8KVg-3, T47-I, B7474), lungs (Lx-1, A5439), melanoma (Lox, 8kte130), leukemia (L60, CEM), prostate (BJAR, Ii 145, RS-3). All samples showed expression of BBP1 using Northern blotting with mRNA isolated from the following tumor cell lines: promyelocytic leukemia (ΗΕ-60), carcinoma (IeBa 83), chronic myelogenous leukemia (K-562), lymphoblastoid leukemia (MOBT-4) , Burkitt's lymphoma (Katz), colorectal adenocarcinoma (8 \ U480), lung carcinoma (A549) and melanoma (C361).

Example 6. Selective interaction of VVR1 with human VAR as opposed to rodent VAR.

In the rodent BAP, there are three amino acid substitutions (C5K, Υ10Υ and Κ13Η) compared to the human sequence. The rodent peptide exhibits reduced neurotoxicity and lack of binding to human brain homogenates (Maddu e1 a1, 1992). Thus, it was interesting to evaluate the binding of the rodent BAP to the BBP1 protein in the Υ2Η test system. The sequence of human BAP in pEK162 was changed to encode a rodent peptide using oligonucleotide directed mutagenesis using PCR described above. The resulting plasmid pEK240 was identical to the plasmid expressing the human BAP fusion protein used in the present invention, with the exception of three codons producing three amino acid substitutions corresponding to the rodent amino acid sequence. The interactions between the BBP1 fusion protein and the human and rodent BAP fusion proteins were compared using the Υ2Η biotest. Strains expressing rodent BBB1 and BAP did not show the ability to respond in terms of growth rate (Fig. 7). These data confirm the assumption that VVR1 may be a specific mediator of the neurotoxic effect of VAR, and also indicates a mechanism explaining the reduced neurotoxicity of VAR rodent. It is also important that the obtained data demonstrate a high degree of specificity of the BBB1 / BAP interaction in the Υ2Η tests, while the substitutions of three amino acids were sufficient to completely disrupt such a relationship (Fig. 7).

It is clear that the present invention can be practiced in other ways in comparison with those specific options that are available in the above descriptions and examples. Various modifications and variations of the present invention are possible in light of the descriptions and, accordingly, they fall within the scope of the attached claims.

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8! Ehp, Ό. (1997). Ap t! Gase11i1ag rgo! Et! Ya! Т ат ату атуууууууу ре рррррр ап ап ап ап ап ап ап ап ап ап ап ап !екекекек

Akyeteg'k bjake. ΝρΙιιιό 389, 689-695.

List of Sequences (1) GENERAL INFORMATION (ί) APPLICANT: Ogepegdeg Vgas1 A.

Bassop D.5.

Kadkozyk ENEEP.

(ϋ) TITLE OF THE INVENTION: Proteins binding to a β-amyloid peptide and polynucleotides encoding them (ϋί) NUMBER OF SEQUENCE: 2 (ίν} CORRESPONDENCE ADDRESS:

(A) ADDRESS: Ateghsap Note Rhois (B) STREET: Opera Satriz Οτίνθ (C) CITY: Ragzgrappu (ϋ) STATE: N6 (E) COUNTRY: United States of America (D) Zip Code: 07054 (V) FORM, PRIGE COMPUTER (A) MEDIA TYPE: floppy disk (B) COMPUTER: 1VM-Compatible (C) OPERATING SYSTEM: PC-005 / M5-005 (O) PROGRAM: Razepyp Be1ease 1.0, νετδ. 1.30 (νί) CURRENT APPLICATION PARAMETERS (A) APPLICATION NUMBER: 03 (B) DELIVERY DATE:

(C) CLASSIFICATION:

(νϋί) PATENT ATTORNEY INFORMATION:

(A) NAME: SALES APZGEA S.

(B) REGISTRATION NUMBER: 34988 (C) REGISTRATION NUMBER: E8126 (ίχ) TELECOMMUNICATION INFORMATION:

(A) PHONE: (1-973) 683-2169 (B) FAX: (1-973) 683-4117 (2) INFORMATION FOR Zeo Yu О 1 (ί) CHARACTERISTIC OF THE SEQUENCE (A) LENGTH: .810 nucleotides (B ) TYPE: nucleic acid (C) NUMBER OF THREADS: single-stranded (ϋ) TOPOLOGY: linear (ϋ) MOLECULAR TYPE: mRNA (ίχ) PROPERTIES:

(A) NAME / KEY: segment '(B) LOCALIZATION: 1 ... 807 (χί) DESCRIPTION OF THE SEQUENCE: ZEO Ιϋ ΝΟ I

ATS SAT ATT TTA AAA SSS TST SSS AAT STS ATT SSS SSS SST SAS SSS48

Me Nhz Not Ly Yuz S1y Zeg Rgo Azp Wa1 11e Rgo Agd Akha Nkhz C1u 15 1015

SAS AAS AAS ACC SSA ASA SAS SSA AST SSS STS TAT SST ATS SSS SST96

С1п Luz Azp Thg Agd Ahd Azr C1y Thg C1u Ley Tug Rgo Mes AgdS1u

2530

SSS TTT AAS AAS STS SSS STS TTS SSS TTS TSS STS SSS STS STS SSS144

Rgo Rye Luz Azp Ley A1a Lye Ley Rgo Rye Zeg Ley Rgo Lye Leyshu

4045

CCA CCC OSA ACC OSA AST CCC CAC AAA STS CCC CCC CCC AAC CCC192

С1у С1у С1у Зег СХу Зег С1у О1и Luz Wah Zeg Wah Xeg Zuz MebA1a

55 .60

SSS SSS TSS SSS TST SST SSS TST SST SSS SAS SSS STS SSS SSS ASA240

A1a A1a Tgr Rgo Zeg SXu Rgo Zeg AXa Rgo SXi A1a UaX Thg AXaAgd

- 70 7580

STS STT SST STS STS TSS TTS STS TSA STS AST ASA OSA SSS TSS SSS268

Ley Va1 Cxv Va1 Ley Tgr Rb Va1 Zeg Va1 Thr Thr Cxu Rgo TgrS1u '85 9095

SST STT SSS SSS SSS SSS SSS SSS SSS SAS SAS SSS SST SAS SSS SAS SAS SAS336

A1a Va1 A1a Tg Zeg A1a C1u C1u C1u C1i Zeg Ley Zuz Suz C1iAzr

100 105 110

STS AAA STS SSA SAA TAT ATT TOT AAA SAT SSA AAA ATA AAT SAS SST384

Ley Luz Va1 S1y S1n Tug Not Suz Luz Azr Rgo Luz Ne Azp AzrA1a

115 120 125

ACC SAA CAA SSA STT AAS TST ASA AAS TAS ASA SST SAT STT TSS TST432

Tyr C1l C1i Rgo Va1 Azp Suz Tyr Azp Tug Tig A1a Nhz Wa1 zegSuz

130 135140

TTT SSA OSA SSS AAS ATA AST TOT AAS SAT TSS AST SSS AAT SAA ASA480

Рее Рgo А1а Рго Азп Not Тг Суз ьз Азр Зег Зег С1у Азп С1иТг

145 150 155160

SAT TTT AST OCO AAS CAA OTT SOT TTT TTS AAO SSS ATA TST TSS SSA528

Nhz Pb Thb O1y Azp C1i Ya1 C1u Pbb Pbb Rg Ne Zeg SuzAgd. 165 170175

AAT STA AAT SOS TAT TSS TAS AAA-STS OSA STS SSA TTS TST STT TTT576

Azp ν »1 Azp C1u Tug Run Tug bz Va1 A1a Va1 A1a Lye Zeg Leyer

180 '185190

STT SSA TSS TTS yOA SSA SAT SSA TTT TAS STT SSA TAS SST OST TTS624

Lei C1u Tgr Lei O1u A1a Azr Agd Rye Tug Lei C1u Tug Rgo A1aie

195 200205

OST TTS TTA AAS TTT TSS AST STA SSS TTT TST SSA ATT OSS ASS STA672

С1у Ьеи ёи ЛУ РЬе Суз Тьг Уа1 С1у Рее Суз С1у 11е С1у ЗегЬей

210 215220

ATT SAT TTS ATT STT ATT TSA ATS SAS ATT STT SSA SST TSA SAT SOA720

Not Azr Pye Not Nei Not Zeg MeB SHn 11e Wa1 SHu Rgo Zeg AzrO1u

225 230 235240

AOT AOT TAS ATT ATA SAT TAS TAT OSA ACC ASA STT ASA ASA STO AST768

Zeg Zeg Tug 1He 11e Azr Tug Tut SHU Thg Agd Ley Thg Agd Ley Zeg

245 250255

ATT AST AAT OAA ASA TTT ASA AAAA ACC SAA TTA TAT CCA TAA810

Not Thr Azp Cl and Thr Rye Agd Luz Thl Cln Ley Thug Pr

260 '265 (2) INFORMATION FOR 8E <2 Ιϋ ΝΟ 2 (ί) SEQUENCE CHARACTERISTIC (A) LENGTH: 269 amino acids (B) TYPE: amino acid (ϋ) TOPOLOGY: linear.

(ίί) MOLECULAR TYPE: protein (χί) DESCRIPTION OF THE SEQUENCE: 8E <2 GO ΝΟ 2

Me Nhs Not Yee C1u Run Rgo Azp UaH Non Rgo Agd AXa N1z C1u one • · 5 10 fifteen C1p Bz Azp Tht twenty Agd Agd Avr C1u Tht 25 C1u Bie Tug Rgo MeC thirty ^Age C1u Rgo Pb Bz 35 Azp Bie Aha Bie Bie 40 Rgo Pb Run Bie Rgo 45 Bie Bie C1u <31u O1u fifty C1u Zeg C1u Zeg C1u 55 C1 and B _y = Ya1 Run Ya1 60 Zeg Bz Me Aha A1a 65 A1a Tgr Rgo Zeg C1u 70 Rgo Run A1a Rgo C1 and 75 A1a Ya1 Tht Aha Agd 80 Bie Ya1 C1u Ya1 Bj 85 Tgr Pb Ya1 Run Ya1 90 Tht Tht C1u Rgo Tgr 95 C1u A1a Ya1 A1a Tht one hundred Run A1a C1u C1u SHi 105 C1 and Run Bie Bz suz 110 C1 and AZR Bie Bz Ya1 115 O1u C1p Tug Not Suz 120 Lou Avr Rgo Bz Not 125 A8P Azr A1a Bx C1p 130 O1i Rgo Ya1 Azp Suz 135 Tht Azp Tug Tht A1a 140 Nhz Y & 1 Run Suz Pb 145 Rgo A1a Rgo Azp 11th 150 Bx Suz bz Avr Run 155 Run C1u Azp C1 and Tht 160 Nhz Pb Tht C1u Azp 165 C1 and Wah C1u pb Pb 170 Bz Rgo Not Run Suz 175 Agd Azp Wah Azp C1u 180 tug Zeg Here Bz Wah 185 A1a Ya1 A1a Bie Run 190 Bie Pb

Lei <31u Tgr Lei O1u A1a Azr Agd Rye Tug Lei C1u Tug Rgo A1a Lei 195 200205

С1у Ьеи ёи ЛУВ Ре Суз Тьг Уа1 С1у Рее Суз С1у Не 61у Зег бей 210 215220

11th Azr Rye 11e Lei 11th Zeg MeC C1n Ne Wa1 (31u Rgo Zeg AzrS1u

225 230 235240

Zeg Zeg Tug Not Not Azr Tug Tukh * C1u Tgr Agd Ley Thg Agd Leyzeg

245 250255

11th Trg Azp O1 and Thn Pye Agd bz Thb C1n bie Tug Rgo

260265

Claims (21)

CLAIM 1. A cDNA molecule fragment comprising nucleotides 202-807 of the sequence 8E0 GO N0: 1, shown in the List of sequences, encoding a fragment of a protein that binds to the β-amyloid peptide (BBP), including amino acids 68-269 of the sequence 8E0 GO N0: 2, shown in this List, as well as a variant of the specified cDNA fragment, obtained on the basis of the degeneracy of the genetic code. 2. A fragment of the cDNA molecule according to claim 1, which represents the insert of the clone pEK 196 ATSS 98399. 3. A cDNA molecule having the nucleotide sequence 8E0 GO N0: 1, listed in the Sequence List, encoding a protein that binds to the β-amyloid peptide (BBP), having the amino acid sequence 8E0 GO N0: 2, listed in this List, as well as the variant indicated cDNA molecules derived from the degeneracy of the genetic code. 4. The cDNA molecule according to claim 3, which is an insert of the BBR1-J ATCC clone 98617. 5. A polynucleotide that hybridizes under stringent conditions with a cDNA molecule according to any one of claims 1 to 4 or with a molecule complementary to it, despite the fact that said polynucleotide has a length of at least 75% of the length of the indicated cDNA molecule and encodes β- amyloid peptide. 6. A probe or primer comprising a nucleotide sequence capable of hybridizing with the nucleotides 172-194 of the cDNA molecule of claim 3. 7. Polynucleotide comprising at least one sequence that regulates the expression, functionally associated with at least one polynucleotide selected from the group consisting of a cDNA molecule according to any one of paragraphs.1-4. 8. A host cell strain transformed by a polynucleotide according to claim 7, wherein said cell is a prokaryotic cell. 9. A host cell strain transformed by a polynucleotide according to claim 7, wherein said cell is a eukaryotic cell. 10. A fragment of the protein binding to the β-amyloid peptide (VVR), comprising amino acids 68-269 of sequence 8E0 GO N0: 2, listed in the List of sequences, encoded by a fragment of the cDNA molecule, including nucleotides 202-807 of sequence 8E0 GO N0: 1, given in this A list, or encoded version of the specified cDNA fragment, obtained on the basis of the degeneracy of the genetic code. 11. A fragment of the protein binding to the β-amyloid peptide (BBP), encoded by a fragment of the cDNA molecule according to claim 1, which is an insert of the clone pEK 196 ATCC 98399. 12. Protein binding to β-amyloid peptide (VVR), having the amino acid sequence 8E0 GO N0: 2, listed in the Sequence List, encoded by a cDNA molecule, having a nucleotide sequence 8E0 GO N0: 1, listed in this List, or encoded by a variant of the cDNA molecule indicated , obtained on the basis of the degeneracy of the genetic code. 13. The VVR protein comprising the amino acid sequence encoded by the cDNA molecule according to claim 3, which is an insert of the BBR1-J ATCC 98617 clone. 14. The method of obtaining a protein according to any one of paragraphs.12-13 or a fragment of this protein according to any one of paragraphs.10-11, which consists in growing a host cell strain according to any one of paragraphs.8-9 in a suitable culture medium and purify produced by the specified strain protein or its fragment. 15. Protein, obtained in accordance with the method according to 14. 16. The VVR fusion protein, comprising a protein according to any one of claims 12-13, 15, or a fragment of this protein according to any one of claims 10-11, associated with a heterologous protein or peptide sequence. 17. Oligonucleotide, which is an inhibitor of the expression of the BBR1 gene, comprising a nucleotide sequence consisting of 15-35 nucleotides, which hybridizes under harsh conditions with the cDNA molecule according to any one of claims 1 to 4. 18. A method for determining the amount of a polynucleotide encoding a protein binding to a β-amyloid peptide (VVR) in a sample, comprising the following steps: (a) probe hybridization according to claim 6 with a sample under stringent conditions; and (b) determining the amount of said probe bound to polynucleotides in the sample. 19. An antibody that specifically binds to the extracellular region of a protein according to any one of claims 12-13, 15-16, or a fragment of a given protein according to any one of claims 10-11. 20. The method of determining in the sample the amount of protein according to any one of paragraphs. 12-13, 15-16 or a fragment of this protein according to any one of paragraphs.1011, comprising the following stages: a) incubating the antibody according to claim 19 with a sample and a control, while said control includes different amounts of a known VVR protein or a fragment of the VVR protein and where said antibody is associated with a reporter molecule; b) determining the amount of said antibody bound to said VVR protein or protein fragment in the sample; and c) quantitative determination of the specified VVR protein or a fragment of the VVR protein by comparing the amount of antibody specified in b) with the indicated controls. 21. A method for treating a patient in need of inhibiting the accumulation of a β-amyloid peptide in the brain, comprising administering to the patient a therapeutically effective amount of a protein according to any one of claims 12-13, 1516 or a fragment of this protein according to any one of claims 10-11.