Vascular protein - 1 adhesion with aminoacylase activity

 

The invention relates to medicine and the application of vascular protein-1 adhesion with aminoacylase activity. The invention includes a method of manipulating the binding of endothelial cells to lymphocytes, mediated vascular protein-1 adhesion (VAP-1), including the inhibition or potentiation of enzyme activity aminoacids in these endothelial cells. The advantage of the invention is to develop a way of mediating early interaction in the processes of binding of lymphocytes to endothelial cells. 2 S. p. f-crystals, 6 ill., table 2.

Technical field the Invention relates to nucleic acid that encodes a new human protein adhesion to endothelial cells, labeled VAP-1, which has an adhesive function and aminoxide activity. The invention also relates to the use of nucleic acid and polypeptide VAP-1.

Background of the invention Continuous circulation of lymphocytes between blood and tissue is crucial for the functioning of the immune system. This process allows the lymphocytes to control all parts of the body in search of antigen, which allows them the donkey that lymphocytes can be separated to return to the site microenvironment in which they discovered an antigen that increases the selectivity of the immune response. Adhesive interactions between multiple receptors on circulating lymphocytes and their ligands expressed on the surface of endothelial cells in postcapillary venules, are as a means of migrating leukocytes and method to selectively control it. Although recently there has been significant progress in the description of the cascade of processes that are required for penetration into the fabric of circulating leukocytes from free flow, a lot in this process remains unclear. In particular, the currently known functions of adhesion molecules is not sufficient to explain all the ways of recycling and the binding specificity, the presence of which is shown both in normal conditions and in conditions of inflammation.

Currently available hypotheses come from multistep model of leukocyte adhesion to the endothelium, which is based on a cascade of sequential, but overlapping molecular interactions between multiple pairs of receptor-ligand (E. G. Butcher and Picker L., J. Science, 272:60-66 (1996); Springer, T. A., Cell, 76:301-314 (1994)). The original temporary and limited vzaimode tinavie ligands. At this stage, can participate integrins and other molecules. At different stages of rolling and testing may, at the appropriate signals to advance the next stage durable adhesion through the binding of activated integrins with their ligands of the Ig superfamily. Locally elevated levels of immobilized cytokines and other chemoattractants may be involved in the process of initiating a local activation, which is mediated by specific receptors and subsequent transmission of signals inside the cell. The final stage involves the process of migrating cells through the endothelial lining of the fabric through very unclear mechanism. Tissue-specific way circulation is based on regulating the expression of specific combinations of receptor-ligand adhesion molecules in the corresponding area of the body.

A previously described monoclonal antibody (MABS) 1B2, which recognizes a new molecule adhesion to endothelial cells and may block the binding of the lymphocyte with the top endothelial venules tonsils (HEV) in the analysis of frozen sections (M. Salmi and Jalkanen, S., Science, 257:1407-1409 (1992); U.S. Patent NoNo. 5512442 and 5580780 and application for U.S. Patent No. 08/447799, kotorie to determine vascular protein-1 adhesion (VAP-1). When inflammation is induced by the expression of surface VAP-1 and the molecules are on the surface of endothelial cells in the lumen of the vessels (M. Salmi et al., J. Exp. Med., 178: 2255-2260 (1993)). The method thus tissue of the tonsils in the presence of MAT 1B2 can be found two kinds of VAP-1 with different molecular weight: one of them has a mass of 90 kDa, and the second 170-180 kDa. However, the Western blot turns in nevosstanovlenie conditions leads to the predominance of molecules weighing 170-180 kDa (M. Salmi and Jalkanen , S., J. Exp. Med., 183:569-579 (1996)). Immunoreactive VAP-1 with slightly different molecular weights can also be found in other parts of the body, in particular in cells of smooth muscles of the vascular network, as well as in other tissues containing smooth muscle cells, although their function in these cells remains unclear. McNab and co-authors reported that VAP-1 is expressed on endothelial cells of sinusoidal liver and may mediate the binding of T-lymphocytes (G. McNab et al. , Gastroenterology, 110: 522-528 (1996)). Studies VAP-1 of tonsils by splitting specific glycosidase showed that VAP-1 is sialoglycoproteins containing, apparently, and N-and O-linked sugars in the presence of a large number of residues of sialic what about the VAP-1 mediates the binding of lymphocytes to HEV in lymphatic tissues under conditions of shear, moreover, in connection with sialic acid as desialylation molecule, as shown by studies of frozen sections, can no longer participate in the binding of lymphocytes (M. Salmi and Jalkanen , S., J. Exp. Med., 183:569-579 (1996)). The molecule undergoes positive regulation of inflammatory conditions in diseases of the skin, intestines, tonsils and synovial membrane, although the mediators that cause this phenomenon has not been identified (Arvilommi A.-M. et al., Eur. J. Immunol., 26: 825-833 (1996); M. Salmi et al., J. Exp. Med. , 178:2255-2260 (1993)). VAP-1 is different from adressen (PNAd) of peripheral lymph node (PLN), a recognized MAT MECA-79, although as VAP-1, PNAd can mediate the binding of lymphocytes to PLN under conditions of shear. However, in contrast to the PNAd, VAP-1 can act independently of L-selectin and to promote the binding of L-selectin-negative lymphocytes (M. Salmi and Jalkanen, S., J. Exp. Med., 183:569-579 (1996)). Thus, VAP-1 is a molecule with an important adhesive function in the new cascade, which acts independently from the known selectins and, apparently, able to mediate early interactions in the binding process of lymphocytes with PLN type HEV.

Summary of the invention the Invention relates to a purified nucleic acid molecule that encodes soudi the setup portion of the polypeptide, comprising the amino acid sequence shown on figure 1 (SEQ ID NO: 2); (b) purified nucleic acid molecule comprising a sequence encoding a VAP-1, from nucleotide sequence of VAP-1, is shown in figure 1 (SEQ ID NO: 1); (b) purified nucleic acid molecule that encodes a polypeptide VAP-1 comprising the amino acid sequence encoded by the cDNA clone VAP-1 stored in the Depository No. DSM 11536; (g) purified nucleic acid molecule comprising the coding VAP-1 sequence of the nucleotide sequence stored in the Depository No. DSM 11536; (d) purified nucleic acid molecule comprising a nucleotide sequence complementary to the nucleotide sequences VAP-1 specified in (a), (b), (C) or (d); (f) a purified nucleic acid molecule comprising a nucleotide sequence that differs from the coding sequence of the nucleic acid molecule (b) or (g), due to the degeneracy of the genetic code, and
(g) purified nucleic acid molecule comprising a nucleotide sequence that hybridizes with the molecule (d) and encodes VAP-1 having the amino acid sequence showing pnie relates to a method of creating a recombinant vector, including the introduction of a molecule containing a sequence of nucleic acid molecules for VAP-1 in the DNA sequence that can function as a vector. In addition, the invention relates to a recombinant vector comprising such DNA encoding VAP-1. The invention relates to a method for delivery of VAP-1 in the cell of the host, including the introduction of DNA that encodes a VAP-1 in the cell host. The invention relates, furthermore, to a recombinant cell host containing typed thus DNA. The invention also relates to a method for producing a polypeptide of the vascular protein-1 adhesion (VAP-1), comprising culturing the recombinant host cell containing a DNA encoding VAP-1 of the present invention, under conditions that allow expression of the encoded polypeptide VAP-1.

Further, the invention relates to a method of providing aminoxide activity of the host cell by transforming nucleic acid that encodes a VAP-1, host cell.

Further, the invention relates to a method of modifying the expression of vascular protein-1 adhesion (VAP-1), including: (a) introducing into the cell-master design DNA that contains: (i) the target sequence of VAP-1 and (ii) a regulatory sequence, with the tion of homologous recombination between the construct DNA and the endogenous sequence of VAP-1. The invention also relates to a cage-owner, in which the expression of VAP-1 was changed as indicated above.

The invention also relates to a recombinant cell host containing: (a) the target sequence of VAP-1 and (b) a regulatory sequence associated with the target sequence of VAP-1.

The invention relates to a method of oxidizing an amine, including the interaction of the amine with a polypeptide VAP-1 according to the present invention. Amin may represent, for example, benzylamine or methylamine. Polypeptide VAP-1 has the amino acid sequence at least 95% identical to a sequence selected from the group consisting of:
(a') a purified polypeptide comprising the amino acid sequence of VAP-1, is shown in figure 1 (SEQ ID NO: 2);
(b') a purified polypeptide comprising the amino acid sequence of VAP-1, encoded by the nucleotide sequence of VAP-1, is shown in figure 1 (SEQ ID NO:1):
(in') a purified polypeptide comprising the amino acid sequence encoded by the cDNA clone VAP-1 stored in the Depository No. DSM 11536;
(g) a purified polypeptide comprising the amino acid sequence of VAP-1, encoded by nucleotide sequences is -1, and has an amino acid sequence which shows at least 80% identity with the sequence shown in figure 1 (SEQ ID NO:2),
(d') a purified polypeptide comprising the amino acid sequence hepatopancrease part of the polypeptide according to paragraph (a) or (b').

The invention also relates to a method of inhibiting aminoxide activity, including the introduction of an effective amount of the inhibitor in the sample with aminoxide activity. The specified inhibitor may represent, for example, the formation of the hydroxylamine, propargylamine, isoniazid, nialamide, hydralazin, procarbazine, monomethylhydrazine, 3,5-ethoxy-4-aminomethylpyridine or MDL72145 ((E)-2-(3,4-acid)-3-fiorellini).

The invention also relates to a method for manipulating the binding of endothelial cells to lymphocytes, mediated vascular protein-1 adhesion (VAP-1), including the change of enzyme activity oxidase in endothelial cells through inhibition or potentiation.

Brief description of drawings
Fig. 1. The cDNA sequence of VAP-1 isolated from a cDNA library isolated from the lung of a man, and predicted posledovatelnogo protein VAP-1, enclosed in the rectangle. Amino acid residues are highlighted in the sections outlined in the rectangle indicates that the specified cycle, when the sequencing of peptides cannot be attributed any amino acid residue. Potential sites of N-glycosylation sites are indicated by arrows with circles, whereas asparagine and presumptive sites of O-glycosylation sites are indicated by arrows with squares. Transmembrane domain between residues 5 and 27 marked darkening. The scale of the chart at the bottom of the figures indicates the position of the transmembrane domain (shown filled TMD region), and the relative location of the alleged sites of glycosylation in the extracellular part of the molecule is marked N or O to denote respectively the N - and O-linked sugar. Large arrow indicates 50 amino acids.

Fig. 2. The analysis method of sorting cells according to the intensity of fluorescence (FACS analysis) COS-7 cells, transfected with relatively VAP-1, to determine the orientation of the membrane and location of VAP-1 in the cell. A diagram showing the structure of the two plasmids the expression of VAP-1, used for transfection of COS-7 cells, shown in the upper part of the figure. VAP-1 is a native design sendtransaction, let's call it for brevity "pseudocontrol", provides a construction in which VAP-1 has the opposite orientation of the expression vector. Column 1: the Expression constructs used for transfection. Column 2: No permeabilization (-) or presence of permeabilization (+) transfected cells. Column 3: Negative control MAT staining. Column 4: Staining using MAT 1B2 against VAP-1. Column 5: Staining using MAT M2 against FLAG. Series A-F indicate the design expression or test "pseudocontrol" used for transfection and the results of staining of transfected cells. Arrows indicate positively the painted cell population. On nemerlebinding cells staining is observed with the use of MAT 1B2 against VAP-1 (rows b and C of the picture sorting cells according to the intensity of fluorescence, column 4). Test "pseudocontrol" transfected cells was negative (number And picture sorting cells according to the intensity of fluorescence, column 4). Not observed staining using MABS against FLAG in VAP-1 FLAG-transfected cells (row C, column 5). However, in the case permeabilizing cells, VAP-1-transfetsirovannyh cells are still positive on the cation MAT against FLAG (row F, column (5), which indicates that the permeability of cells in relation to the MAT against FLAG allows the specified antibodies to access the FLAG epitope, and this testifies to the fact that the epitope lies within the cell.

Fig. 3. Building representatives of the family of copper-containing aminoacids mammals for which data are available sequences, including VAP-1. The labels on the left are specific, located to the right of the squirrel. BSA denotes aminoxide of serum bull; VAP-1 denotes human vascular protein adhesion; PDO indicates placental diaminoksidazu person 1; PDO - placental diaminoksidazu man 2; TAO rats - rat diaminoksidazu. The numbers correspond to the first amino acid in each row are grouped proteins. Sequence taken from the latest available database and grouped using the GCG program Pileup. The remains characterized by identity with VAP-1, selected using the GCG program Box-shade.

Fig. 4. Processing sialidases VAP-1 expressed in COS-7 cells. Cell lysates of VAP-1-transfected and pseudotranslation COS-7 cells treated with sialidase (+) or not treated (-) before the LTO-PAA the eh using MAT 3G6 (tracks 5-8).

Fig. 5. A. Northern blot of mRNA VAP-1 with poly And+RNA isolated from smooth muscle of the intestine of man and tonsil stroma, from which the lymphocytes were partially removed by washing and compression. Gibadatovna mRNA size of 4.2 KB visible on both tracks. Century Northern-blotting mRNA VAP-1 from various human tissues. Northern-blotty get from Clon-tech Laboratories and each track is injected is equal to the amount of mRNA. All filters load32P-labeled cDNA probes VAP-1 containing the full coding sequence, and washed them under strict control (washing conditions after hybridization conditions: 0.1 × SSC, 0.1% of LTOs at 65oWith twice within 45 minutes).

Fig. 6. Ax cells, transfetsirovannyh cDNA VAP-1, mediates the adhesion of lymphocytes. A. Expression of VAP-1 on the surface of the Ah cells stably transfected with the cDNA VAP-1 or in the test for "pseudocontrol". On the histogram, the intensity of staining on a logarithmic scale is shown on the X-axis and the relative number of cells is shown on the y-axis. C. Increased levels of VAP-1-dependent binding of lymphocytes with VAP-1 transfectants. A significant larger number of PBL (small round sphere on top of the monolayer, examples of which are indicated by arrows) associated with VAP-1-cord the Quantitative determination of binding. The results of five independent experiments are presented as the average values ofSKO.

Detailed description of the invention
The interaction between receptors on the surface of cells and their ligands on endothelial cells of blood vessels is a crucial step in the mechanism controlling the movement of lymphocytes between blood and various lymphoid organs and is related to extravasation of leukocytes to sites of inflammation. Described a cDNA clone that encodes a vascular protein-1 adhesion (VAP-1), which mediates the reaction of the adhesion of leukocyte-endothelial cell. It turned out that the encoded VAP-1 has aminoxide activity. It is believed that in higher organisms aminoxide included in the metabolism of biogenic amines (McIntire W. and S. Hartmann, in Principles and Applications of Quinoproteins, V. L. Davidson, ed. , Marcel Dekker, Inc., N. Y., Chap. 6 (1992)).

The present invention relates to a purified nucleic acid molecule comprising polynucleotide encoding the polypeptide of vascular protein-1 adhesion (VAP-1), having the amino acid sequence shown in figure 1 (SEQ ID NO:2), which was determined by sequencing of clone cDNA having the nucleotide sequence shown on figure 1 (SEQ ID NO: 1). The clone with the CSO Treaty in International depositories (International Depository Authority of DSMZ-Deutsche Sammlung Von Mikroorganismen Und Zeilkulturen GmbH) at Mascheroder Weg 1b, D-38124 Braunschweig, Germany, may 7, 1997 under No. DSM 11536. The deposited clone contained in the plasmid pUC19.

On a column with a monoclonal antibody (MABS) for immunoaffinity separation were purified Monomeric and dimeric forms of VAP-1, size 90 and 170-180 kDa, respectively, which were isolated from the treated detergent lysates of smooth muscles in sufficient quantities in order to determine the internal peptide sequence after cleavage by trypsin and V8 protease. Part of the VAP-1 size 90 kDa treated N-terminal sequencing and used to construct a partially degenerate oligonucleotide primers for experiments in the framework of the polymerase chain reaction with reverse transcriptase RT-PCR using mRNA derived from the smooth muscle of the intestine of man.

One cDNA fragment of approximately 700 base pairs amplified using the indicated primers and clone into pUC18 to determine its sequence. For the detection of longer cDNA clones by PCR analyze a set of four human cDNA in a cDNA library to identify those that contain cDNA VAP-1, and libraries that demonstrate the highest signal is subjected to with what Biotec cDNA, isolated from the lung and the heart of man, lots of overlapping cDNA clones. Of these clones was obtained one cDNA size 2501 nucleotide pairs, described in figure 1 (SEQ ID NO:1), which contains a continuous open reading frame in size 2292 p. N. starting from the ATG-methioninamide codon, and then subcloned in pUC19. The thus determined nucleotide sequence of the cDNA VAP-1 contained in this clone and shown in figure 1 (SEQ ID NO:1), contains an open reading frame encoding a protein of 763 amino acid residues presented on figure 1 (SEQ ID NO:2), with the location of the initiating codon at the site 80-82 nucleotide sequence shown in figure 1 (SEQ ID NO:1) having a molecular weight of about 84,6 kDa.

VAP-1 according to the present invention possesses a strong identity to the family of enzymes related to copper to Minoxidil (EC 1.4.3.6.). Protein VAP-1, shown in figure 1 (SEQ ID NO:2), characterized by about 24% identity to the C-monoamine oxidase of Escherichia coli and has 41-81% similarity with the members of this family in mammals. The highest identity was shown with minoxidol from bovine serum (81%).

Unless otherwise specified, are listed in the op is rasenna A, G, C and T). "Nucleotide sequence" of a nucleic acid molecule or polynucleotide refers to the DNA molecule or polynucleotide consisting of a sequence of deoxyribonucleotides. In the case of RNA molecules or polynucleotide corresponding sequence consists of ribonucleotides (A, G, C and U), where each timidity deoxyribonucleotide (T) in a specific deoxyribonucleotide sequence is replaced by ribonucleotides uridine (U).

The nucleic acid molecules of the present invention can be an RNA, such as mRNA or DNA, including cDNA and genomic DNA obtained by cloning or during synthesis. The DNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may represent a coding strand, also known as semantic chain, or non-coding circuit, which is also called antisense chain, or complement.

The term molecule(s) "purified" nucleic acid refers to a nucleic acid molecule is DNA or RNA that is isolated from a native source or are synthetically. Purified RNA molecules include in vitro transcripts of RNA or DNA molecules according to the present issati, coding for the polypeptide VAP-1 having the amino acid sequence encoded by the cDNA clone contained in the plasmid deposited with a Depository may 7, 1997 under No. DSM 11536. The invention also relates to a purified molecule of nucleic acid having the nucleotide sequence shown in figure 1 (SEQ ID NO:1) or the nucleotide sequence of cDNA VAP-1, contained in the above-described deposited clone, or a nucleic acid molecule having a sequence complementary to one of the above sequences. Such isolated molecules, particularly DNA molecules, are used as probes for gene mapping by in situ hybridization with chromosomes, and for detecting expression of VAP-1 in human tissues, for example, by the method of the Northern-blot analysis.

Further, the present invention relates to fragments of molecules purified nucleic acids given in this description. The "fragment" of a molecule purified nucleic acid having a nucleic acid sequence of VAP-1 deposited cDNA or the nucleotide sequence of VAP-1, shown in figure 1 (SEQ ID NO:1) understand the fragments of a size of at least about 15 nucleotides, and more pradov and even more preferably, at least about 40 nucleotides in length, which are used as diagnostic probes and primers in accordance with the above description. Of course, larger fragments of length 50-1500 nucleotides can also be used within the present invention if they are fragments, corresponding mainly or wholly, the nucleotide sequence of the deposited cDNA or the sequence shown in figure 1 (SEQ ID NO:1). Since the gene VAP-1 was deposited, and the known nucleotide sequence shown in figure 1 (SEQ ID NO: 1), the creation of such DNA fragments is a routine procedure for specialists in this field. So, for example, to generate fragments of different lengths may be used such procedures as the splitting of restrictase, the destruction of dubbing or oligonucleotide synthesis.

In another aspect the invention relates to a purified molecule of nucleic acid, including polynucleotide that hybridizes under strict control hybridization with a part of polynucleotide in the nucleic acid molecule of the present invention, described above, for example, cDNA clone VAP-1 stored in the Depository No. DSM 11536. Under "with the amide, 5 x SSC (150 mm NaCl, 15 mm triacrylate salt), 50 mm sodium phosphate (pH of 7.6), 5 x denhardt's solution, 10% dextran sulfate and 20 g/ml denatured, destroyed DNA salmon sperm, followed by washing the filters in 0.1 x SSC at a temperature of approximately 65oC.

Under the nucleotide which hybridizes with "part" of polynucleotide understand polynucleotide (either DNA or RNA) hybridization, at least for 15 nucleotides (NT), and more preferably at least about 20 NT, still more preferably at least about 30 NT, and more preferably about 30-70 NT with a standard polynucleotide. They can be used as diagnostic probes and primers. Of course, polynucleotide, hybridization at greater during standard polynucleotides (for example, clone deposited cDNA), for example with a length of 50-750 NT or greater length with standard polynucleotides, can also be used as probes of the present invention, if they are polynucleotide, corresponding mainly or completely nucleotide sequence of VAP-1 deposited cDNA or the nucleotide sequence shown in highinitial DNA or as primers for amplification of a target sequence in a polymerase chain reaction (PCR), as described in the literature (e.g., in Molecular Cloning, A Laboratory Manual, 2nd edition, Sambrook J., Fritsch E. F. and Maniatis T., eds.. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.(1989)), which is incorporated into this description by reference.

Molecules purified nucleic acid of the present invention include DNA molecules that contain an open reading frame (ORF) with the position of the initiating codon at the site 80-82 on the nucleotide sequence of VAP-1, shown in figure 1 (SEQ ID NO:1) and DNA molecules which comprise a sequence that is fundamentally different from those described above but which, due to degeneracy of the genetic code can encode a protein VAP-1. Undoubtedly, the genetic code is well known to specialists in this field and for professionals with an average level of knowledge in this area obtaining the expressed variants is a routine procedure.

Further, the present invention relates to variants of the nucleic acid molecules of the present invention, which encode part, analogues or derivatives of protein VAP-1. Options can be natural, such as a natural allelic or splice-varia locus on a chromosome of an organism (Genes II, Lewin C. , ed., John Wiley & Sons, New York (1985)). Options not found in the natural state, can be obtained using mutagenesis techniques known to experts.

Such options include forms, obtained by carrying out the substitution of nucleotide deletions or insertions. Substitutions, deletions or insertions can include one or more nucleotides. Variants may contain alterations in the coding regions, non-coding regions, or both types of regions. Changes in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or insertions. Especially preferred among these are silent substitutions, insertions and deletions, which do not alter the properties and activities of protein VAP-1 or its parts. Also especially preferred in this regard are conservative substitutions.

Other embodiments of the present invention include molecules purified nucleic acid that contains polynucleotide having a nucleotide sequence identical to at least 90% and more preferably at least 95, 96, 97, 98 or 99% (a) a nucleic acid molecule that encodes a polypeptide comprising amino acids the th sequence of VAP-1 from the nucleotide sequence of VAP-1, shown in figure 1 (SEQ ID NO:1); (C) a nucleic acid molecule that encodes a polypeptide VAP-1 comprising the amino acid sequence encoded by clone ccnc VAP-1 stored in the Depository No. DSM 11536; (g) a nucleic acid molecule comprising the coding sequence of the nucleotide sequence of VAP-1 stored in the Depository No. DSM 11536; (d) a nucleic acid molecule comprising a nucleotide sequence complementary to the nucleotide sequences VAP-1, listed in paragraphs (a), (b), (C) or (d); (e) a nucleic acid molecule comprising a nucleotide sequence that differs from the coding sequence of the nucleic acid molecules according to (b) or (d) due to degeneracy of the genetic code, and (g) a nucleic acid molecule comprising a nucleotide sequence that hybridizes with the molecule (d) and encodes VAP-1, having an amino acid sequence that is at least 90% demonstrates the identity sequence VAP-1, is shown in figure 1 (SEQ ID NO: 2). The specified property does not depend on whether coded for a polypeptide VAP-1 activity. Because if even a molecule of nucleic acid is not how to use the nucleic acid molecule, for example, as hybridization probes or as primers in polymerase chain reaction (PCR). While the preferred nucleic acid molecules that have identical sequence at least 90, 95, 96, 97, 98, or 99% nucleic acid sequence VAP-1, shown in figure 1 (SEQ ID NO:1), or nucleic acid sequence VAP-1 deposited cDNA, which actually encodes a polypeptide having the activity of VAP-1 protein.

The phrase "polynucleotide, having a nucleotide sequence at least, for example, 90% "identical" standard nucleotide sequence that encodes a polypeptide VAP-1", mean that the nucleotide sequence of polynucleotide identical to a standard sequence except that the polynucleotide sequence may include up to ten point mutations per each 100 nucleotides of the standard nucleotide sequence that encodes a VAP-1 polypeptide. In the case when the molecule of any specific nucleic acid identical, at least 90, 95, 96, 97, 98 or 99%, for example, the nucleotide sequence of VAP-1, shown in figure 1 (SEQ ID NO:1), or nucleotide placenta is known computer programs, such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711).

Vectors and cells-host
The present invention also relates to vectors that include purified DNA molecule of the present invention, cells masters, which were constructed by genetic methods using DNA that encodes a VAP-1, and to obtain polypeptides VAP-1 or its fragments using recombinant techniques.

Polynucleotide can be connected with a vector containing a breeding marker for propagation in a cell host. In the General case, the plasmid vector is administered in the form of sediment, such as the precipitate of calcium phosphate or in complex with a charged lipid. If the vector is a virus, it can be assembled in vitro using the appropriate Assembly cell lines and transferred into the host cell by the method of transduction. Breeding markers include dihydrotetrazolo or resistance to neomycin cultures of eukaryotic cells, or the presence of genes for resistance to tetracycline or ampicillin in cultures of E. coli and other bacteria.

Preferred are vectors comprising CIS-active regulatory areas in respect of interest palynol nnogo vector or by considering the vector when introduced into a cell of the host. In some preferred in this regard, embodiments of the invention, the vectors provide for specific expression, which may be inducible and/or specific cell type. Particularly preferred among them are inducible under the influence of environmental factors vectors, which can be easily manipulated by such factors as temperature and food additives.

The expression vectors used in the present invention include vectors derived from chromosomes, Epsom and viruses, for example, vectors derived from bacterial plasmids, bacteriophage, yeast Epsom, chromosomal elements, yeast, viruses such as baculoviruses, papovavirus, viruses ospowiki, adenovirus, pox viruses of birds, the viruses pseudoleskeella and retroviruses, and vectors derived from combinations thereof, such as Comedy and phagemid.

The DNA insert should be operatively contact the appropriate promoter such as the promoter of phage lambdaLthe promoters of E. coli lac, trp and tac, early and late SV40 promoters and promoters of retroviral long repeats at the end of the genome (LTR), which to name a few. Specialists in this area known reply, its termination, and in the transcribed regions of the binding sites with ribosomes for broadcasting. The coding portion of the Mature transcripts expressed by these structures, preferably includes the site of translation initiation at the beginning and a termination codon (UAA, UGA or UAG), located respectively at the end of the coding sequence.

The technology, based on recombinant DNA, also includes methods of recombination, as described in Treco et al. (Treco et al., WO 94/12650 and Treco et al. WO 95/31560), which is given in the present description by reference, and these methods can be used to alter the expression of VAP-1 and the activity of the oxidase in the cells. Specific regulatory areas (e.g., promoters) can be introduced into the host genome with the aim of changing the expression of endogenous VAP-1.

Thus, the invention relates to a method of obtaining recombinants host cell with altered expression of vascular protein-1 adhesion (VAP-1), including: (a) introducing into host cell design DNA, comprising: (i) the target sequence of VAP-1 and (ii) a regulatory sequence associated with the target sequence of VAP-1, and (b) the location of the host cell in conditions pottu VAP-1. The invention also relates to recombinant host cell in which expression of VAP-1 was changed to the above method.

The invention also relates to a method of modifying gene expression of VAP-1 due to, for example, activation of the expression of VAP-1 in the cell that normally expresses or is not normally expresses in a desirable environmental conditions or hormonal conditions. Changes in the expression of VAP-1 can also include the use sequence of the present invention for the purpose of inactivation of the expression of the native gene. Homologous recombination or site-directed action is used to replace or violations of the regulatory area, in normal conditions linked with the endogenous gene VAP-1, with a regulatory sequence that leads to expression of a gene at a higher level than in the corresponding nitrostilbene cells, or creates a picture of gene expression that differs from that observed in the corresponding nitrostilbene cells from the point of view of regulation and induction. In this regard, the invention relates to a method of obtaining proteins through activation of endogenous gene that encodes the desired product in kliknete and preferably also a regulatory sequence. As described in the works of Treco et al. (Treco et al., WO 94/12650, and Treco et al., WO 95/31560), are often added to these are other structural components, such as breeding markers, amplificatoare markers or exon and is not associated splice-donor sites. DNA in this design can be treated as exogenous DNA, which is injected into the host cell by the method of the present invention. Exogenous DNA may contain a sequence that is identical to or different from endogenous DNA that was present in the cell prior to transfection.

The target sequence or sequence design DNA are DNA sequences that allow permitted homologous recombination in the genome of the selected cells containing the gene of VAP-1 in the locus of VAP-1. Target sequences are, for example, DNA sequences homologous to the DNA sequences VAP-1, which are normally present in the genome of the received cells and are mainly located on both ends of the regulatory sequence. Use(s) target(s) sequence(s) selected to meet customers VAP-1, in which the design of the DNA to be inserted.

Regulatory sequence construkction joining matrix, negative regulatory elements, binding sites with transcription factors or their combinations.

The site of introduction of the DNA sequences found mainly inside the endogenous gene VAP-1 or in the direction reverse transcription, or website, affecting the function of the gene VAP-1. DNA sequences that alter the expression of endogenous gene VAP-1 can be introduced into the host cell in the form of a single design DNA or in the form of specific sequences of DNA in the genome transtitional cells become physically bound state. When this DNA can be introduced in the form of a linear, double stranded DNA in the presence of single-stranded sections on one or both ends, or without them, or the DNA may be introduced in the form of circular DNA. After the introduction of regulatory DNA in the host cell specified by the cell is placed in a condition suitable for the implementation of homologous recombination between the genomic DNA and a part of the introduced DNA. Homologous recombination between the genomic DNA and introduced DNA results in homologous recombinant host cell in which the sequence that modifies the expression of the endogenous gene VAP-1, operatively linked to a gene VAP-1. Obtained in this method the LCA VAP-1, which leads to the production of the protein VAP-1 in vitro, or cell can be used for delivery of in vivo protein VAP-1 (e.g., gene therapy).

Purposeful procedure may be a simple introduction of the regulatory sequence, the location of the endogenous gene VAP-1 under the control of the new regulatory sequence (for example, when inserting a promoter or enhancer, or both, in the opposite direction during the transcription of the endogenous gene). Purposeful procedure may be a simple deletion of a regulatory element, such as a tissue-specific deletion of a negative regulatory element. Purposeful procedure may lead to the replacement of the existing item; for example, tissue-specific enhancer can be replaced with another enhancer with greater or lesser specificity in relation to cell type than the natural elements, or with a different pattern of regulation or induction that is different from the corresponding nerafinirovannoe the cell.

Purposeful impact on gene can be used to replace the existing regulatory area VAP-1 regulatory sequence isolated from another gene, or a new regulatory consistently what It leads to disruption of endogenous sequences, which control expression of the endogenous gene VAP-1 either by replacing all or part of the endogenous (genomic) sequences, or by other effects, interfering with the endogenous sequence.

Design DNA, which include exogenous DNA and optionally a DNA encoding breeding marker, along with additional sequences necessary for the expression of exogenous DNA into a recipient host cells used for transfection of cells in which you plan to change products VAP-1. In the works of Treco et al. described in more detail methods of homologous recombination used to modify the expression of the endogenous gene (Treco et al. WO 94/12650, and Treco et al., WO 95/31560), which is given in the present description as a reference.

DNA or recombinant constructs can be introduced into cells using various methods, including transformation, transfection, electroporation, microinjection, transduction, the precipitation of calcium phosphate, as well as the content that is associated with the participation of the liposomes, polybrene or DEAE-dextran. These methods are described in many laboratory manuals, such as Davis et al. , Basic Methods In Molecular Biology (1986)). Alternative ruses, herpes, adenovirus associated with adenovirus factor, mumps and polio.

Examples of suitable host cells include, but are not limited to, bacterial cells, such as cells of E. coli, Streptomyces and Salmonella typhimurium, fungal cells such as yeast cells, insect cells such as cells of Drosophila S2 and Spodoptera Sf9, animal cells such as cells of the Ah and CRL 1998 (both are endothelial cells, melanoma cells, Cho, COS and Bowes, as well as plant cells. The appropriate environment for the cultivation and culturing conditions described above host cells known in the art.

Suitable prokaryotic and eukaryotic cells are well known to specialists in this field.

Known bacterial promoters suitable for use in the present invention include the promoters of E. coli and lacZ, the T3 and T7 promoters, the gpt promoter, the lambda promoters PRand PLand the trp promoter. Suitable eukaryotic promoters include the early promoter of CMV, timeintensity the HSV promoter, the early and late SV40 promoters, the promoters of long repeats RNA at the ends of the genome of retroviruses (LTR), such as the rous sarcoma virus (RSV), and metallothionein promoters, such as the Oia, higher eukaryotes may be increased by inserting enhancer sequence into the vector. Enhancers are CIS-active DNA elements, components usually from 10 to 300 nucleotide pairs in length, which can enhance the transcriptional activity of the promoter in this type of the host cell. Examples of enhancers include the SV40 enhancer, which is located on the late side of the site of replication initiation in the area from 100 to 270 p. N., early enhancer promoter and cytomegalovirus enhancer of polyoma located on the late side of the site of replication initiation, and adenovirus enhancers.

Polypeptide VAP-1 can be expressed in a modified form, such as a protein, and may include not only secretion signals but also additional heterologous functional areas. So, for example, may be added to the area of additional amino acids, particularly charged amino acids to the N-Termini of the polypeptide VAP-1 to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. In addition, for easier cleaning to polypetide VAP-1 can be added to the peptide part. Such areas can be removed at the final stage Perea excretion, to improve stability and to facilitate cleaning is a well-known and routine to those of other used for this purpose methods.

Protein VAP-1 can be isolated and purified from cultures of recombinant cells using known methods, including precipitation with ammonium sulfate or ethanol, acid extraction, anion - or cation-exchange chromatography, chromatography on phosphocellulose, chromatography based on hydrophobic interaction, affinity chromatography, chromatography on hydroxylapatite and chromatography on the lectin. The polypeptides of the present invention include natural purified products, products of chemical synthesis and foods produced using recombinant techniques from a prokaryotic or eukaryotic host cells, including, for example, the cells of bacteria, yeast, higher plants, insects and mammals. Depending on the nature of the host cell used in the process of obtaining recombinant, polypeptides of the present invention can be a glycosylated or deglycosylated products.

Polypeptides VAP-1 and fragments thereof
Installed and shown in figure 1 (SEQ ID NO:1) the nucleotide sequence is the R in figure 1 (SEQ ID NO:2), with the position of the initiating codon at the site 80-82 nucleotide sequence shown in figure 1 (SEQ ID NO:1), which is characterized by a molecular weight 84,6 kDa. Protein has 6 potential sites of N-glycosylation and 3 alleged site of O-glycosylation sites per monomer (Fig.1) (definition carried out using to predict the site of O-glycosylation of the E-mail server NetO-glic@cbs.dtu.dk (Hansen et al., Biochem. J., 308:801-813 (1995)).

Thus, the present invention also relates to a purified polypeptide VAP-1 having the amino acid sequence encoded by the deposited cDNA, or the amino acid sequence shown in figure 1 (SEQ ID NO: 2), or to a peptide or polypeptide comprising a portion of the above polypeptides. The terms "peptide" and "Oligopeptide" are treated as synonyms (as is commonly the case), and each of these terms can be used for any of the described cases, depending on the context to indicate the circuit containing at least two amino acids linked by peptide bond.

In engineering it is known that certain amino acid sequence of the polypeptide VAP-1 can be changed without making a serious impact on structuroscopy significant activity of VAP-1. Such mutants include deletions, insertions, inversions, repeats, and substitutions of similar residues. Small changes such "neutral" amino acid substitutions generally have little effect on activity. Rules to determine which amino acid changes are likely to be phenotypically silent (for example, should not have a pronounced deleterious effect on the function), can be found in Bowie et al. (Bowie, J. U. et al. , Science, 247:1306-1310 (1990)). Changes are preferably of a minor nature and include, for example, conservative substitutions of amino acids, which do not impact significantly on the folding or activity of the protein VAP-1.

Amino acids in the protein VAP-1 of the present invention, which are necessary for its functioning, can be determined by methods known in the art, such as site-directed mutagenesis and deletion analysis. The obtained mutant molecules are then tested for the presence of biological activity, such as aminosilane activity or adhesive function.

The polypeptides of the present invention are preferably provided in purified form. The obtained recombinant method version polypeptide VAP-1 may be essentially cleansing the

The polypeptides of the present invention include polypeptides that have at least 90% similarity, more preferably at least 95% similarity, and still more preferably at least 96, 97, 98 or 99% similarity with the above-described polypeptides. Other polypeptides of the present invention include polypeptides that are identical, at least 90%, more preferably identical, at least 90 or 95% and even more preferably, identical, at least 96, 97, 98 or 99% of the polypeptide VAP-1, encoded by the deposited cDNA, or the polypeptide VAP-1, is shown in figure 1 (SEQ ID NO:2), and also include portions of such polypeptides containing at least 30 amino acids and more preferably, at least 50 amino acids.

Under the polypeptide having the amino acid sequence at least, for example, 90% "identical" standard amino acid sequence of VAP-1, imply that the amino acid sequence of the polypeptide is identical to the standard sequence except that the polypeptide sequence may include up to ten amino acid changes per 100 amino acids in the standard amino acid nab the production of monoclonal antibodies, used to detect the expression of protein VAP-1 or agonists or antagonists, can enhance or inhibit the functioning of the protein VAP-1. In more detail the procedure of increasing the production of monoclonal antibodies against VAP-1 as described in Salmi and Jalkanen (Salmi, M. and Jalkanen, S., Science, 257: 1407-1409 (1992), U.S. patent NoNo. 5512442 and 5580780 and in the application for U.S. Patent No. 08/447799, which are given in the present description as a reference. Further, such polypeptides can be used in two-hybrid system yeast (Fields and Song, Nature, 340:245-246 (1989)) to "capture" a protein, binding protein VAP-1, which can also be agonists and antagonists of the present invention.

In the context of the present invention, the term "antibody" (AT) or "monoclonal antibody" (MAT) includes intact molecules as well as antibody fragments (such as Fab and F(ab')2fragments), which are able to specifically bind the protein VAP-1. Fragments Fab and F(ab')2do not contain the Fc fragment of intact antibody, which is quickly removed from circulation, and in this regard they may have less ability to communicate with non-specific tissues than an intact antibody (Wahl et al., J. Nucl. Med., 24:316-325 (1983)). Thus, these Masegosa of many methods. For example, cells expressing the protein VAP-1 or antigenic fragment can be introduced into the body of an animal to induce formation of sera containing polyclonal antibodies. Within the preferred method to receive and purify protein VAP-1 to obtain essentially free of natural impurities of the drug. Then this drug is injected into the animal organism for the production of polyclonal antisera with a higher specific activity.

Under the most preferred method, the antibodies of the present invention are monoclonal antibodies (or fragments thereof that bind to the protein VAP-1). Such monoclonal antibodies can be produced using hybridoma technology (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol., 6:511 (1976); Kohler et al., Eur. J. Immunol. , 6:292 (1976); Hammerling et al., In:Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N. Y. (1981), pp.563-681).

It should be borne in mind that Fab, F(ab')2and other fragments of the antibodies of the present invention can also be used in accordance with the methods disclosed in the present description. Such fragments typically receive when proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (s using the techniques of recombinant DNA or chemical synthesis.

"Humanized" chimeric monoclonal antibodies can be obtained using genetic structures formed by hybridoma cells that produce the above-described monoclonal antibodies. Methods of producing chimeric antibodies are known in the art (see review, Morrison, Science, 229:1202 (1985); Oi et al., BioTechniques, 4:214 (1986); Cabilly et al. U.S. patent No. 4816567; Taniguchi et al., EP 171496.

The use of VAP-1
VAP-1 according to the present invention has a significant level of identity with a family of enzymes related to the copper-containing Minoxidil (code according to the classification of enzymes EC 1.4.3.6). Protein VAP-1, is shown in figure 1 (SEQ ID NO:2), about 24% identical Cu-aminoxide Escherichia coli and approximately 41-81% similar to the representatives of this family in mammals. The highest level of identity is shown when compared with minoxidol from the serum of a bull (81%). Using benzylamine substrate monoamine oxidase (MAO) was found significant enzymatic activity of the oxidase in expressing VAP-1 cells SNO, but not in pseudotranslation cells. In the presence of inhibitors of copper-containing monoamine oxidase - semicarbazide and hydroxylamine was shown that VAP-1 is not active against benzylamine.

oxidation of the amine in the reaction of amine with a polypeptide VAP-1, with aminoxide activity. The amine may, for example, to treat a range of endogenous and xenobiotics aromatic amines. In addition, the amine may be treated to some endogenous and xenobiotics aliphatic amines such as methylamine. Preferably the amine is benzylamine.

The conditions determining aminoxide activity when using benzylamine as a substrate for the oxidase described in detail below in the section Materials and methods. In the final volume of 0.2 to 5 ml, preferably in a final volume of 0.5 ml, add sodium phosphate buffer to a final concentration of 0.05-0.2 mm, preferably 0.1 mm. Then add the unlabeled benzylamine to a concentration of 1-100 nmol, preferably 80 nmol, and enter14S-benzylamine, so that activity was 0,1-1,0 MX, preferably of 0.4 MX. To test add a sample of cell lysate volume of 0.05-0.5 ml, preferably of 0.1 ml After incubation at 37oC for 1 hour aldehyde product aminosilane reaction is extracted with toluene containing 0.1-05 g/l, preferably 0.35 g/l, diphenyloxazole. More may be added extraction using toluene/diphenyloxazole sessionnum the counter.

The invention also relates to a method of inhibiting aminoxide activity of VAP-1 with the introduction of an effective amount of amine oxidase inhibitor. The inhibitor may represent, for example, the formation of the hydroxylamine, propargylamine, isoniazid, nialamide, hydralazin, procarbazine, monomethylhydrazine, 3,5-ethoxy-4-aminomethylpyridine or MDL72145 ((E)-2-(3,4)acid)-3-fiorellini), preferably the formation or hydroxylamine. The formation may be injected at a concentration of 10-1000 μm, or in another range of concentrations, such as 50-1000 or 80-500 μm, preferably in a concentration of 100 μm. The hydroxylamine may be injected in a concentration of 0.1-100 μm, or in another range of concentrations, such as 1-10 μm or 2-8 μm, preferably in a concentration of 5 μm.

In the framework of the present invention the binding of endothelial cells to lymphocytes, mediated vascular protein-1 adhesion (VAP-1) may be modified by the introduction into the host cell inhibitor or amplifier oxidase. The host cell may be a cell line of mammalian - human or animal. The invention can be used for treatment in vitro or in vivo.

Provides a General description of the invention poyasnyaetsya description.

Example 1
The selection of cDNA VAP-1
Smooth muscles of the intestine of man, in whom VAP-1 is highly expressed, produced from material taken during surgical procedures, using it as a source for further purification VAP-1 protein. On immunoaffinity column with monoclonal antibody b of the processed detergent lysates smooth muscle purified forms of VAP-1 size 90 and 170-180 kDa in sufficient quantities to define the internal peptide sequence after cleavage of their trypsin and V8 protease. In addition, conduct direct sequencing of the N-end section of the VAP-1 size of 90 kDa. The profile of elution of the peptide from the HPLC column used for the purification of peptides from both forms of VAP-1, are identical, as the peptide sequence in the respective peaks, indicating that the protein is a dimer consisting of 2 subunits size of 90 kDa.

Some of the peptide sequences VAP-1 is used to generate a partially degenerate oligonucleotide primers for experiments in the framework of RT-PCR using mRNA derived from smooth muscle of the intestine of man. A single cDNA fragment of approximately 700 p. N. amplified using primers and clone into pUC18 to determine the et protein, containing some of the sequences of the peptides purified from Emunim way material treated with trypsin and V8, and this confirms the fact that the amplification touched upon the desired cDNA fragments. For the selection of longer cDNA clones analyze a set of 10 libraries of human cDNA by PCR to identify those that contain cDNA VAP-1, and libraries that demonstrate the highest signal, is subjected to screening cDNA fragment VAP-1 obtained by PCR. In this way produce many overlapping cDNA clones from the cDNA libraries of the lung and heart of man. Of these clones selected cDNA size 2501 p. N. , which contains a continuous open reading frame size 2292 p. N., beginning with methioninol codon ATG, and subcloning in pUC19. After the start codon should peptide sequence found in the N-end of the purified protein VAP-1 size of 90 kDa, and related open reading frame encoding all VAP-1 peptides after treatment with trypsin and V8, which were identified by sequencing of the protein (Fig.1). This clone contains untranslated 5'-plot size 80 p. N. and noncoding 3'-plot 129 p. N. following the TAG stop kodnovania put, which native mRNA VAP-1 may be longer than in the situation with the selected cDNA.

Transient expression of VAP-1 in COS-7 cells arising after transfection with expression vector containing cDNA leads to strong staining of the cell surface in a population of cells using VAP-1 b, IB2 (Fig.2, the picture sorting cells according to the intensity of fluorescence, the number In column 4). Control transfected cells was negative (Fig.2, the picture sorting cells according to the intensity of fluorescence, row a, column 4). Thus, it was concluded that isolated cDNA encoding immunoreactive VAP-1, which is expressed on the cell surface of transfected cDNA VAP-1.

VAP-1 protein
The open reading frame contained in cDNA VAP-1, encodes a protein of 763 amino acids in size 84,6 kDa. Search in the database of acceptable protein sequences showed that VAP-1 has substantial identity to the family of enzymes related to copper to Minoxidil (classification of enzymes EC 1.4.3.6). The degree of identity varies from about 24% level, characteristic for C-oxidase in Escherichia coli, to 41-81% in the case of representatives of this family in mammals. Higher identichnost eratively throughout the length of the protein, except for a short portion at the N-end of the molecule. In Fig. 3 shows for comparison VAP-1 and 4 other representative copper-bearing aminoxide mammals in relation to sequences which relevant information is available.

VAP-1 does not show significant identity with any of the currently known adhesion molecules and contains no protein domains from among those that are occasionally found in such proteins, although we noted the presence of RGD motifs on the site between residues 726-728. Unknown, however, the functional significance of this common motif from the point of view of binding with integrin, if this process takes place. Protein has 6 potential sites of N-glycosylation and 3 alleged site of O-glycosylation sites per monomer (Fig.1) (definition carried out using to predict the site of O-glycosylation of the E-mail server Netglic@cbs.dtu.dk (Hansen et al., Biochem. J., 308:801-813 (1995)).

Examination of the protein sequence revealed the presence of sites with characteristics of membrane domains, except for the portion 23 of the molecule, composed mainly of hydrophobic amino acids in the far N end (mod who drove secretion, because it contains the potential cleavage site at position 19, which was established by the method of von Heine (von Hejine G., Nucl. Acids. Res., 14: 4683-4690 (1986)), either transmembrane domain. N-terminal protein sequence of the protein VAP-1 size 90 kDa indicates that this hydrophobic region was not cut from the material that we had cleared, and it is unlikely it can function as a normal split the signal sequence for secretion. In addition, the charged residues flanking the hydrophobic region, suggests that this may be the membrane domain of the membrane protein type II (E. Hartmann et al., Proc. Natl. Acad. Sci. USA, 86:5786-5790 (1989)), with N-end in the cytoplasm and With the end of the extracellular domain.

To determine whether the specified hydrophobic region to function as a transmembrane domain and to determine the orientation of VAP-1 in the membrane, the authors have created designs for the expression of cDNA VAP-1, in which the FLAG epitope recognized by a commercially available MAT M2, was framed reading after initiating methioninamide codon in the predicted cytoplasmic side of the proposed transmembrane domain. The specified design and control design, in which DVS is the legacy of the cells according to the intensity of fluorescence unstable expression of FLAG and VAP-1 epitopes, recognized accordingly MAT M2 and 1B2 (Fig. 2) using as permeabilizing and not permeabilizing cells. Staining under the action of the MAT against the FLAG was only on permeabilizing cells (Fig.2, row F, column 5), whereas the staining of VAP-1 was observed as the population permeabilizing and not permeabilizing cells (Fig. 2, rows C and F, column 4). Control transfetsirovannyh cells were negative on the MAT and against the FLAG, and against VAP-1 (Fig.2, rows a and D, columns 4 and 5). These data suggest that N-terminal FLAG epitope is located on the cytoplasmic side of the cell membrane, the hydrophobic region captures the lipid bilayer and a large C-terminal extracellular domain that recognizes MAT 1B2, blocking adhesion. All estimated glycosylation sites are located in the extracellular part of the molecule.

To determine the size and glycosylation of recombinant VAP-1, temporarily expressed in COS-7 cells, the authors conducted in-ordinator-SDS page using MAT 1B2 immunoblotting divided cell extracts VAP-1 and pseudotranslation cells when processing them before this sialidases and in its absence (Fig.4). The obvious increase of the mole the first VAP-1, as native VAP-1, represent sialoglycoprotein, thus decreasing the negative charge by removing negatively charged sialic acids reduces the mobility of VAP-1 LTO-PAG. A similar effect when sialidases processing was also observed in the case of VAP-1 from tonsils (M. Salmi and Jalkanen, S., J. Exp. Med., 183:569-570(1996)).

The expression of VAP-1
Northern blot analysis of mRNA isolated from smooth muscle of the intestine of man and exhausted by lymphocytes stroma tonsils, shows that the cloned cDNA VAP-1 hybridized in both tissues with mRNA size 4.2 thousand p. N. (Fig.5A). Further Northern-blotting showed that mRNA VAP-1 size 4.2 thousand p. N. is expressed in a wide range of human tissues. When this messenger is not detected in peripheral blood leukocytes or the brain, but is highly expressed in lung, small intestine and the Appendix in comparison with other tissues. And only a small number of mRNA VAP-1 are detected in the spleen, thymus, testis, liver, pancreas, kidney, bone marrow and fetal liver. An intermediate level of expression observed in the prostate gland, the ovary, the mucosa of the large intestine, heart, placenta, skeletal muscle and simpatica the

Materials and methods
Antibodies and reagents. The literature describes a murine monoclonal antibodies MAT 1B2 against VAP-1 and 3G6 against T cells chickens (M. Salmi and Jalkanen , S., Science, 257:1407-1409 (1992)). MAT M2, discriminating FLAG peptide (DYKDDDDK), obtained from KEVO Lab, Espoo, Finland.

Purification and sequencing of VAP-1. When abdominal surgery cut normal samples of the colon, separating them from their own plates of the lamina propria. The smooth muscle wall is crushed into small pieces and are lysed overnight in lyse buffer (150 mm NaCl, 10 mm Tris-base, pH 2.7, and 1.5 mm MgCl2, 1% NP-40, 1% Aprotinin and 1 mm PMSF). After clarification of the supernatant of the lysate sequentially applied to the column for immunoaffinity chromatography, containing 5 ml Spug-activated beads Sepharose, including normal rat serum, does not bind to the monoclonal antibodies and MABS against VAP-1 (3 mg/ml of beads). Specific column is washed with lytic buffer and VAP-1 antigens elute 50 mm triethylamine. The eluate immediately frozen and then lyophilizer. After that, the sample is dissolved in a non buffer for sample Lemly (Laemmli) and share in 5-12,5% of the LTO-SDS page gel.

After moving to polyvinylidenedifluoride membrane (Applied Boisystems Inc. , Foster City, CA, USA) method elect-180 kDa and a piece of material the size of 90 kDa cleaved by trypsin (purity for sequencing, Boehringer Mannheim GmbH, Mannheim, Germany) as described in the work of Fernandez et al. (J. Fernandez et al., Anal. Biochem. , 201: 255-262 (1992)). Erwerbende peptides separated by HPLC (150A; Applied Biosystems) on a column of Vydac C18(2.1 x 150 mm). Analysis of the N-terminal sequence is performed on the sequencing machine proteins (477 A; Applied Biosystems), equipped with on-line amino acid analyzer based phenylthiohydantoin (120 A; Applied Biosystems). The remaining part of the material the size of 90 kDa is subjected to analysis to determine the N-terminal sequence before splitting. After cleavage by trypsin membrane newly cleaved by V8 protease (purity for sequencing, Boehringer Mannheim) and erwerbende peptides clean and is sequenced as described above. Data sequence number of peptides confirmed using laser desorption method of mass spectrometry matrix in order to confirm the predicted mass (Lasermat, Finnigan).

The technique of molecular biological research. The selection of the E. coli small and large plasmids, splitting restrictase, plaque hybridization, purification of phage lambda and the extraction ragovoy DNA, transformation of E. coli, the cDNA synthesis and construction of plasmids carried out in accordance with standard technology research (Sambrook J. et al., Molodet using a set of QIAEX (QIAEGEN, Hilden, Germany) according to the attached manufacturer's recommendations. The DNA probes are prepared using labeled DNA Amersham Multiprime and [32-P]dCTP with the activity of 3000 Ci/mmol (Amersham, Bucks, UK). The autoradiography is performed using intensifying screens, using, if necessary, hyperplane Amersham Hyperfilm MP. The PCR fragments do stupid and clone into pUC18 using a set of SweClone (Pharmacia, Uppsala, Sweden). Plasmid DNA is sequenced using the Sequenase kit version 2.0 (United States Biochemical Corporation, Cleveland, HE, USA), in accordance with the manufacturer's recommendations, or on the device for DNA sequencing (University of Turku, Department of Medical Genetics). The Assembly sequence and the analysis is performed using the Wisconsin Package, version 8.1-UNIX in Genetics Computer Group. Comparison of existing databases is performed using BLAST Email or WWW server of the National Centre for information in biotechnology (National Center for Biotechnology Information) (http://www.ncbi.nlm.nih. gov/). Oligonucleotide primers for sequencing and PCR receive in commercial mode from Kebo Lab (Espoo, Finland). PCR carried out using Dynazyme polymerase (Finnzymes, Espoo, Finland) under conditions recommended by the manufacturer, and variabledispenser to amplify a cDNA fragment VAP-1 of smooth muscle mRNA RT-PCR, represent N2; gctgtgatcacmatyttygc (SEQ ID NO:3), constructed from the N-terminal AVITIFA sequence of the protein VAP-1 (SEQ ID NO: 4) (residues 13 to 19 in full protein) and T4; ccggccctgrtagaasac (SEQ ID NO: 5), is constructed from treated trypsin peptide sequence VFYQGR (SEQ ID NO:6) (residues 264 to 269). For amplification with these primers using the following conditions: 94oC, 1 minute; 55oC, 1 minute; 72oWith 2 minutes for 30 cycles. Total RNA derived from cell lines and tissues using device Ultraspec (Biotecx, Houston, TX, USA), in accordance with the manufacturer's recommendations. Multiple Northern-blotty of human tissues receive from Clontech Laboratories (Palo Alto, CA, USA) and hybridized with32P-labeled samples in accordance with the manufacturer's recommendations. A set of libraries of human cDNA, and the cDNA library of light (catalog No. HL3004a) and heart (catalog No. HL3026a) are obtained from Clontech Laboratories.

Cell culture and expression of cDNA VAP-1 in mammalian cells. COS-7 (monkey fibroblasts), SNO (cells Chinese hamster ovary) and CRL1998 (an immortalized endothelial cells from umbilical veins person), obtained from ATS (Rockville, MD) is used as host organisms for transference (PAA-Linz, Austria), 2 mm glutamine (Biological Industries, Israel) and 128 IU/ml penicillin and 128 µg/ml streptomycin. Cho cells grown in medium alpha MEM plus SNO with nukes and the same additives. The expression plasmids comprising cDNA VAP-1, subcloning in the expression vector pcDNA3 (Invitrogen, San Diego, CA) and used for temporary transfection of COS-7 cells to generate stably transfected Cho cell lines. The expression plasmids (20 μg) used for transfection of cells by means of electroporation on the instrument BIO-Rad Gene Pulser (0,3 kV, 960 μf, 0.4 cm cuvette in RPMI medium, containing 1 mm Na-pyruvate, 2 mm L-glutamine, without serum). Temporarily transfetsirovannyh cells examined after 3 days after the transfection. Stable transfetsirovannyh cells are selected under cultivation in the presence of 0.5 mg/ml geneticin (Geneticin, Gibco BRL) for 4 weeks.

Example 2
Animatechnica activity of VAP-1
Finding that VAP-1 possesses a strong identity with the family of copper-containing aminoacids, in particular with Sekretareva minoxidol bovine serum (BSA), made the logical study aminoxide of activity VAP-1. Copper-containing amine oxidase characterized by the presence of unusual chimonobambusa, they differ from the FAD-containing intracellular (mitochondrial) monoamine oxidase (McInture W. S. and S. Hartmann, in Principles and Applications of Quinoproteins, p. 97 (V. L. Davidson. ed., Dekker, New York (1993)).

To determine what type of activity has a VAP-1, the authors obtain stable transfectants of Cho cells expressing VAP-1 protein. Voiced lysates of these cells are examined for the presence of dimenoxadol (DAO) activity using as a substrate of putrescine or monoaminoxidase activity (MAO) using as substrate benzylamine. As a positive control using commercially available preparations TAO and MAO, and as negative controls using lysates of Cho cells, transfetsirovannyh pseudoplasmodia. The results show that cells expressing VAP-1 have negligible activity against putrescine, however, when using benzylamine as a substrate of MAO detected significant activity (table 1). In the case of using benzylamine as substrates of monoamine oxidase (MAO) there is considerable activity in Cho cells expressing VAP-1, and in a positive control, representing a commercially available the practical activity. In the presence of 100 μm of semicarbazide and 10 μm hydroxylamine as specific inhibitors of copper-containing monoamine oxidase (Lyies, Intl. J. Biochem. Cell Biol., 28:254-274 (1996)) VAP-1 is inactive with benzylamine (table 1).

In addition, the authors have shown that competent in adhesion VAP-1 is expressed in Ah cells, also has MAO activity against benzylamine, which is inhibited by semicarbazide and hydroxylamine (table 1). Apparently, Ah cells themselves have a native MAO activity with benzylamine, which is not inhibited by semicarbazide or hydroxylamine as "pseudocontrol" cells have a low level of activity (table 1).

To confirm that MAO activity is present in VAP-1 in vivo, the authors performed a clean VAP-1 of tonsils immunoaffinity method and triple repeat the analysis of the obtained material. VAP-1 of tonsils and VAP-1 from transfected Cho cells, demonstrates activity benzylamino with hourly increase And4900.09 at 37oWith the existing conditions of the test, which is 4.5 times larger than the specified parameter in the case of the boiled sample. However, due to the very low yield obtained from tonsils white is as a substrate for determining aminoxide activity not found in vivo. Thus, it was tested many biologically available endogenous amines from the point of view of establishing their ability to be disposed of VAP-1 (table 1). Apparently, methylamine concentration of 1 mm can be easily disposed of VAP-1, however, none of the studied amines does not show such reactivity. In these cell lysates have lower specific activity with benzylamine, if it be compared with data on lysates, are shown in Table 1, which reflects variations in the expression of VAP-1, available in various series of cells.

The presence of quinone factor in VAP-1 is shown in the separation of the LTO-page in reducing conditions of the immuno-purified material VAP-1 of tonsils with subsequent transfer of the material onto the nitrocellulose. Then the nitrocellulose filter paint nitroblue-tetrazolium/glycinato Na in redox conditions cyclization with specific staining of quinone groups of the protein (Paz M. A. et al., J. Biol. Chem., 266: 689-692 (1991)). The paint reacts with Monomeric plot VAP-1 size of 90 kDa and dimeric plot size 180 kDa, which indicates that VAP-1 of tonsils has, apparently, quinone cofactor in each su is 10-15106on the flask), stable transfetsirovannyh plasmid cDNA in the expression of VAP-1, soskrebajut with flasks in 10 ml of 100 mm phosphate buffer, pH 7,2, centrifuged and the cell sediment was washed with 10 ml buffer. Sediment cells finally resuspended in 1.5 ml phosphate buffer and cells are lysed by sound pulses, the average power of 2 times for 10 seconds on ice (ultrasound device Braun sonicator). Voiced lysates are used directly in the enzymatic tests (50-100 µl per test) or after storage at -20oC. Total protein concentration was measured by the method of Bradford using standard bovine gamma globulin and set Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA).

Minoxidol activity measured in accordance with the procedures described above using as substrates14C-labelled putrescine (Amersham) according to method D'agostino et al. (D'agostino L. et al., Biochem. Pharmacol. , 38: 47-49 (1989), included in the description by reference),3H histamine (New England Nuclear) according to the method Beilina and Margolis (Baylin S. B. and S. Margolis , Biochem. Biophys. Acta, 397:294-396 (1975), included in the description by reference), and14C - benzylamine (Amersham) and spend a definition similar to the way with putrescine, except that the quality is of Ilumina. 0.5 ml reaction mixture containing 100 mm sodium phosphate buffer, pH 7,2, 0,4 MX14C-benzylamine (Amersham), 80 nmol of it benzylamine and a sample of the crude cell lysate volume of a maximum of 100 ál per reaction. After incubation at 37oC for 1 hour aldehyde reaction product is extracted by adding 900 μl of toluene containing 0.35 g/l of diphenyloxazole, and vigorously shake the mixture. After separation of the phases selected 700 ál toluene layer and perform additional extraction using 700 μl of toluene/diphenyloxazole selected 500 ál of the specified layer, add to the first extracted portion and calculate the level14With the meter for liquid-scintillation counting. All enzymatic tests carried out in the presence of the blind controls containing boiled sample (10 minutes, 100oC) or inexpressibly sample, double or triple repetition and the results expressed as pmol substrate used min-1mg protein-1. The untreated controls TAO of kidney pig and MAO from plasma ox receive from the company Sigma Chemical Company (St. Louis, MO, USA).

Discussion
Pronounced staining using MAT 1B2 detection of VAP-1, showing, is since the overall level of VAP-1, found in these areas is relatively low, it has proved difficult to isolate and purify sufficient quantities of endothelial VAP-1 in order to obtain the necessary information about the protein sequence. If you look at other fabrics in which was discovered VAP-1, the most richly represented in smooth muscle vascular and smooth muscle related to the intestine (M. Salmi et al. , J. Exp. Med., 178:2255-2260 (1993)). VAP-1 from these sources is characterized by a slightly different molecular masses, which is probably related to differences in glycosylation, and yet similar to the form previously obtained from tonsils and tissues PLN type.

Using data on protein sequences obtained for VAP-1, isolated and purified from smooth muscle of the intestine, isolated cDNA encoding the indicated adhesion molecule. Proof of this is the following: firstly obtained for immunoscience VAP-1 protein sequence found in the predicted protein sequence subsequently selected cDNA clone VAP-1. And secondly, transfetsirovannyh cells expressing cDNA YAP-1, painted surface using MAT 1B2, which was originally ispolzuet close to that found in vivo protein molecular weight (170-180 kDa).

VAP-1 is a large dimeric transmembrane protein type II, with the membrane domain located at the N-end of the molecule. The intracellular domain is especially small and contains only 4 amino acids in length, whereas a large glycosylated extracellular domain is specified in the dimer size of about 163 kDa. All potential glycosylation sites per dimer, namely 12 N-linked and presumably 6 O-linked, are located in the extracellular domain. Although it is currently unknown whether all they are, obtained earlier data suggest that VAP-1 contains both N-and O-linked sugars, as well as many residues of sialic acid that may be present either on the terminal residues of N-linked carbohydrate, or as part of O-linked sugars. As carbohydrates, in particular sialic acids are believed to play an important role in the realization of the adhesive function of VAP-1 protein (Salmi, M. and Jalkanen, S., J. Exp. Med., 183: 569-579 (1996)), it is necessary to identify which sites of glycosylation are used and what kind of carbs they contain.

Because the analysis method Northern-bathing not revealed in any of the tissue types of mRNA VAP-1 with significantly different reason to believe, there are forms of VAP-1, encoded by the various variants of mRNA. Thus, it is possible that the inventors cDNA clone encoding a dominant form of VAP-1, which was previously investigated by the method of immunoblotting and method immunosurgery. However, since all investigated tissue contains smooth muscles, originating either from the vascular tissue, or from other sources, as well as vascular endothelium, it is impossible to distinguish between mRNA VAP-1 of smooth muscle and endothelium, when, as is the case in these tissues, they are very similar types of mRNA. Thus, it is possible that it may(may) be the other(s) form(s) VAP-1 encoded(s) mRNA, slightly different from the already selected type.

It is interesting that the N-terminal site of the VAP-1 transmembrane region represents the portion of the molecule that is most different from that of the homologous BSA. It is known that BSA is a secretory protein that is found in high quantities in the serum, which is a signal secretion sequence at its N end, removed during proteolytic processing during secretion metabolism (Mu D. et al., J. Biol. Chem., 269:9926-9932 (1994)). Thus, BSA and VAP-1 VAP-1 has a transmembrane domain, similar to that was found in human VAP-1 and, thus, may have the same functions as the human molecule. The extracellular domain of VAP-1 has activity oxidase, so that VAP-1 can function as ectoenzyme. Copper-containing amine oxidase are a diverse class of enzymes with very different substrate specificity, which can be very roughly divided into two types: those that possess activity against polyamines, such as putrescine and histamine, and those, such as VAP-1, which have monoaminoxidase activity. Physiological substrates of aminoxide unknown, although there is an assumption concerning a few of these possible connections.

Another characteristic feature of these enzymes is the presence of unusual carbonyl-containing cofactor, relative to the chemical nature of which were obtained contradictory data, which was identified in BSA and aminoxide peas as tapaninen (6-hydroxy DOPA), and lysyl oxidase as cross stitched disintigration. In some mammalian species, including humans, copper-containing monoamine oxidase found in the cell membrane, and in SIV and, such as the formation and hydroxylamine (Lyies G. A., Intl. J. Biochem. Cell Biol., 28:259-274 (1996)), and such enzymes are referred to semicarbazides Minoxidil (SCAO). The authors have shown that VAP-1 contains covalently-bound quinone, which, taking into account the available data on other copper-containing the monoamine oxidase, is formed, probably, the self-processing reaction, including associated with copper enzyme. These results, as well as the sensitivity of the enzymatic activity of VAP-1 to carbonylation agents semicarbazide and hydroxylamine - indicates that VAP-1 is associated with the membrane of SCO.

The physiological role of SCO difficult to determine because very little is known about their substrates in vivo, and their substrate specificity largely varies in different species. One such feature may be the metabolism of endogenous and xenobiotics primary monoamines, and it is possible that to this genus belongs the function of VAP-1, detected at the positions different from endothelial tissues such as smooth muscle.

It is currently unknown whether SCO activity of VAP-1 have a role to play in the implementation of adherent cells. Preliminary experiments on adhesion in vitro, providesecurity and hydroxylamine, suggest that the enzymatic activity can have a negative impact on adhesive interactions between these cells, since the presence of these inhibitors, the number of PBL associated with transfitsirovannykh VAP-1 cells increases. It is unknown, however, whether this effect is primary caused by direct and specific influence of enzymatic activity on adhesive mechanism used by cells, or it is secondary, in which the physiological state of the cells adversely affects the product monoaminoxidase activity, reducing, thus, their adhesive potential.

Example 3
Adhesion test
cDNA VAP-1 in pcDNA3 used for transfection Ah cells and endothelial cell lines, derived from HEV
rats, which are thought to create a more natural functional environment for VAP-1 than other potential host cell, such as SNO. Get stable transfectants expressing on the cell surface of VAP-1, which was determined by the method of sorting cells according to the intensity of fluorescence (Fig.6 (a), which are then used in tests on the adhesion of lymphocytes. In the study in terms of vasami (Fig. 6B and 6C). Increased binding to VAP-1 transfectants statistically significantly inhibited, although not completely eliminated, when processing a monoclonal antibody against VAP-1 inhibition by 29.6%10,7%, P=0.05). These results in adhesion combine of five independent experiments, in which the analyzed 2-3 parallel monolayer of transfectants, each time using three independently transfected cell lines and PBL from six different donors. The data obtained show that cDNA VAP-1 encodes a functional adhesion molecule located on the cell surface of transfected cells and which, upon expression in Ah cells may be a direct mediator of binding to PBL.

Materials and methods
Ah cells, in which VAP-1 stably expressed, or "pseudocontrol" transfectants placed on a contoured wax circles covered with gelatin microscopic plates (20,000 cells on a circle of diameter 2 cm). Cells are grown to confluence and after two washes in each delineated wax circle by observing the uniformity of the coating of adhesive monolayer of cells, add 100 ál of RPMI medium 1640 containing 10% serum amniotic body of the collet allocate PBL and bring their concentration to a value of 40106cells/ml test environment. After that, the plate is transferred to the corner shaker operating at a speed of 60 revolutions per minute, 7oWith, and each wax circle put 5 μl of the suspension PBL. The test is continued for 30 minutes with constant rotation. The plate is carefully decanted and immersed at once in cold RPMI medium to remove unattached cells. Adherent cells fixed on the slices during incubation of the plates overnight in a vertical position into a chilled on ice, FBI, containing 1% glutaraldehyde. Count the number of adhered cells using ocular grid (magnification x 200). The grid covers an area of about 0.25 mm2. On each glass count nine specified regions of size 0.25 mm2in the center of the circle, where the transfectants formed flushed monolayer. In each of five independent experiments count on two plates on the sample (the total area is equal to 4.5 mm2). In some experiments, the adhesion test is carried out in the presence of the MAT, blocking the function of VAP-1 (combination 1B2 and TC-14, both used at a concentration of 50 μg/ml with dilution medium for the test) or negative control class-specific MAT (combination 3G6 and S, both ol the 7oSince before adding the labeled lymphocytes. The number of adhered cells counted in five independent experiments, as described above.

Discussion
The adhesion tests conducted with Ah cells expressing VAP-1, show that the cDNA encodes a functional VAP-1, which can support the interaction with its ligand on PBL and leads to stable binding of PBL with Ah cells. However, not observed complete inhibition of such increased adhesion with MAT against VAP-1 (1B2 and TC-14), suggesting that the molecule VAP-1 in Ah cells of rats does not function exactly as native environment. Perhaps modification of carbohydrates to proteins Ah cells, the local membrane environment or the conformation of VAP-1 differ sufficiently from those in human HEV, so that the MAT can no longer block all interaction between VAP-1 with its ligand. It is also possible that a subpopulation of molecules VAP-1 can exist in the transfectants as devoid of one or the other of the epitopes recognized by MABS 1B2 and TC-14. Finally, the authors have focused on the possibility that long-term overexpression VAP-1 in stable transfectants modifies the expression of others(Oh) molecules(s) adhesion on the surface of the Ah cells. The function d is aniu HEV shows that VAP-1 is functioning independently from the lymphocyte L-selectin and mediates the binding of CD8+PBL is much better than CD4+PBL. Ah cells obtained by transfection of cDNA VAP-1 reproduces these properties, because the analysis immunomagnetically treated negative for L-selectin cells and CD8+and CD4+cells showed that L-selectin is not required for efficient binding with the Ah-transfectants and that a subset of CD8+cells in PBL is characterized by several times the best tack to VAP-1 transfectants than in CD4+cells (data not shown).

Although the invention is described relative to a specific implementation options, it is obvious that it may be subject to other modifications and this application covers in General all possible ways of using or adapting the above description, the principles of the invention and includes such departures from the present disclosure that are already known or commonly practiced in the technique to which this invention relates and which can be applied to the essential features specified in the description and which are covered by the framework of the above claims.

The disclosure of all references saoc.


Claims

1. The way to manipulate the process of binding of endothelial cells to lymphocytes, mediated vascular protein-1 adhesion (VAP-1), with aminoacylase activity having the amino acid sequence shown in Fig. 1 (SEQ ID NO: 2), characterized in that the inhibition of enzyme activity aminoacids carried out by introducing into the cell-master inhibitor aminoacids.

2. The way to manipulate the process of binding of endothelial cells to lymphocytes, mediated vascular protein-1 adhesion (VAP-1), with aminoacylase activity having the amino acid sequence shown in Fig. 1 (SEQ ID NO: 2), characterized in that the potentiation of enzyme activity aminoacids carried out by introducing into the cell-master potentiator aminoacids.

 

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