KR100533911B1 - Vascular adhesion protein-1 having amine oxidase activity - Google Patents

Abstract

The present invention relates to VAP-1, wherein VAP-1 is an endothelial sialoglycoprotein and its cell surface expression is induced under inflammatory conditions. It has already been shown to mediate binding of recycle lymphocytes to human peripheral lymph node vascular endothelial cells in L-selectin independent form. VAP-1 cDNA was purified to encode an 84.6 KD type II transmembrane protein with a single transmembrane region located at the N-terminal end of the molecule. VAP-1 is located in the region of the cell with three putative O-glycosylation sites in vivo, mainly six dimers of 170-180KD dimer. VAP-1 does not have much similarity with any currently known adhesion molecule but is very identical to the copper containing amine oxidase family. Enzyme evaluation defines VAP-1 as membrane bound amine oxidase. Therefore, VAP-1 is a novel type of adhesion molecule having a dual function. Accurate inflammation settings, along with appropriate glycosylation, allow VAP-1 expression on the enema endothelial cell surface, which is in a location that mediates lymphocyte adhesion, to act as an adhesion receptor involved in a novel mechanism of lymphocyte return. The basic function at other positions depends on its inherent amine oxidase activity.

Description

VASCULAR ADHESION PROTEIN-1 HAVING AMINE OXIDASE ACTIVITY}

The present invention relates to a nucleic acid encoding a new human endothelial cell adhesion protein named VAP-1, which has cohesive function and amine oxidase activity. The invention also relates to the use of VAP-1 nucleic acids and polypeptides.

Continuous recycling of lymphocytes between blood and tissues is important for the function of the immune system. This process allows lymphocytes to be examined at every location in the body in antigen studies during the development and formation of responses to antigenic signs in specific microenvironmental tissue compartments. Lymphocytes can then return specifically to the point where they recognize the antigen and thus enhance the selectivity of the immune response. Cohesive interactions between complex receptors on circulating lymphocytes and their ligands expressed on the surface of endothelial cells in their posterior capillary vasculature provide both a means for migration and a method of selectively regulating it. Although recent advances have been made in explaining the chains required for circulating white blood cells to pass from free flowing blood into tissues, much remains to be found in this process. In particular, the function of recently known cohesive molecules is not sufficient to define all the individual recycling pathways and binding specificities that have been defined in normal and inflammatory coagulation to date.

The recent hypothesis suggests a multistage model of leukocyte adhesion to the endothelium that is sequentially dependent on continuous reactions, except for the repetitive interrelationships between several receptor-ligand pairs (Butcher, EG and Picker, LJ, Science 272: 60-66 (1996); Springer, TA Cell 76: 301-314 (1994). Initial, transient and tethering interactions between white blood cells and blood vessel walls in the blood stream are preferentially performed by selectin and glycoprotein ligands. Integrin and other molecules may also play a role at this stage. Rolling and sampling steps in the presence of an appropriate signal can lead to tight adhesion adjusted by binding of activated integrins to their Ig superfamily ligands. Partly enhanced steps of immobilized cytokines and other chemical attractants can be included when local activity is initiated, which is modulated by signals of continuous transduction with appropriate receptors in the cell. The final step involves translocation between bound cells through endothelial lining into tissue by mechanisms that are difficult to explain. Tissue specific recirculatory pathways are governed by controlled expression of specific adhesion molecule receptor-ligand bonds at appropriate locations.

We previously recognized a new human endothelial cell adhesion molecule and also a monoclonal antibody (MAb) capable of blocking lymphocytes bound to tonyllar high endotherial venules (HEV) by cryosplit analysis, 1B2 (Salmi, M. and Jalkanen, S., Science 257: 1407-1409 (1992); incorporated herein by reference; see US Pat. Nos. 5,512,442 and 5,580,780 and US Application 08 / 447,799). ). MAb 1B2 is specific for vascular adhesion protein-1 (VAP-1) and is defined as this. In the event of inflammation, surface expression of VAP-1 is induced and the molecule is found on the luminal surface of vascular endothelial cells (Salmi, M. et al . , J. Exp. Med. 178 : 2255-2260 (1993)). Two species of VAP-1 with different molecular masses can be detected by MAb 1B2 immunoprecipitation in tonsil tissue, one at 90 KD and the other at 170-180 KD. However, 170-180 KD species dominate after immunoblotting under non-reducing conditions (Salmi, M. and Jalkanen, S., J.EXP.Med. 183: 569-579 (1996)). . Although such functions remain to be measured in these cells, immunoreactive VAP-1 with somewhat different molecular weights can be found in smooth muscle cells of the vascular system as well as in other locations, especially in other smooth muscle-containing tissues. McNab et al. Reported that VAP-1 is expressed in the hepatic sinusoidal endothelium and can regulate T lymphocyte binding (McNab, G. et al., Gastroenterlolgy 1110: 522-528 (1996)). In the study of tonsils VAP-1, which allows digestion with specific glycosidase, N- and O have abundant sialic acid residues in which VAP-1 is bound to both α-2,3 and α-2,6. It shows that it is a sialoglycoprotein that can contain all of the bound sugars. VAP-1 inhibits the binding of lymphocytes to HEV when lymphocytic and sialic acid-dependent methods desialylated molecules can no longer support the binding of lymphocytes in frozen segment assays under shear conditions. Mediation (Salmi, M. and Jalkanen, S. J. Exp . Med. 183: 569-579 (1996)). Although the mediators of this phenomenon have not yet been demonstrated, VAP-1 is upregulated under certain immune conditions caused by disease in the skin, intestines, tonsils and synovial fluid (Arvilommi, A.-M et al., Eur J Immunol 26: 825-833 (1996; Salmi, M. et al., J. Exp, Ned. 178: 2255-2260 (1993)) Lymphocytes for PLN under shear conditions for both VAP-1 and PDAd. Although it can mediate binding, VAP-1 is distinct from the PLN (peripheral lymph nodule) addressin (PNAd) defined by MAb MECA-79. However, compared to PNAd, VAP-1 is L-selectin independent. Can operate under the L-selectin independent manner and support the binding of L-selectin negative lymphocytes (Salmi, M. and Jalkanen, S., J. EXP.Med. 183). : 569-579 (1996)) VAP-1 is therefore a molecule with important adhesion functions in a new pathway that acts independently of known selectins and lymphocyte formation for PLN type HEV. There is a tendency to adjust the initial interactions in the sum.

Summary of the Invention

  The present invention

    (a) a purified nucleic acid molecule encoding a polypeptide comprising the amino acid sequence (SEQ ID NO: 2) shown in FIG. 1;

    (b) a purified nucleic acid molecule comprising the VAP-1 coding sequence (SEQ ID NO: 1) of the VAP-1 base sequence in FIG. 1;

    (c) a purified nucleic acid molecule encoding a VAP-1 polypeptide comprising an amino acid sequence encoded by a VAP-1 cDNA clone contained in Accession No. DSM 11536;

    (d) a purified nucleic acid molecule comprising the coding sequence of the VAP-1 nucleotide sequence contained in Accession No. DSM 11536;

    (e) a purified nucleic acid molecule comprising a nucleotide sequence complementary to the VAP-1 nucleotide sequence in (a), (b), (c) or (d);

    (f) a purified nucleic acid molecule comprising a nucleotide sequence different from the coding sequence of the nucleic acid molecule of (b) or (d) due to denaturation of the genetic code; And

    (g) hybridizes the molecules of (e) and encodes VAP-1 having an amino acid sequence that exhibits at least 80% identity to the VAP-1 nucleotide sequence (SEQ ID NO: 2) in FIG. A purified nucleic acid molecule encoding vascular adhesion protein-1 (VAP-1), selected from the group consisting of purified nucleic acid molecules comprising nucleotide sequences.

    The present invention relates to a method for preparing a recombinant vector in which a molecule consisting of a nucleotide sequence of a VAP-1 nucleic acid molecule is inserted into a DNA sequence capable of acting as a vector. The present invention relates to a recombinant vector comprising such VAP-1 DNA. The present invention is directed to a method of providing VAP-1 to a host cell comprising introducing a DNA encoding such VAP-1. The present invention relates to a recombinant host cell containing such introduced DNA (introduced DNA). The invention produces vascular adhesion protein-1 (VAP-1) polypeptides comprising culturing recombinant host cells containing the VAP-1 encoding DNA of the invention under conditions such that the encoded VAP-1 polypeptide is expressed. It relates to a method for doing so.

    The present invention also relates to a method of providing amine oxidase activity to a host cell by transforming a nucleic acid encoding VAP-1 into the host cell.

    The present invention provides a composition comprising (a) (i) a VAP-1 targeting sequence; And (ii) introducing into the host cell a DNA construct comprising a regulatory sequence linked to a VAP-1 target sequence; (b) alter the expression of vascular adhesion protein-1 (VAP-1), including maintaining the host cell under conditions suitable for homogeneous recombination between the DNA construct and the endogeneous VAP-1 DNA sequence. It is about how to let. The invention also relates to host cells in which the expression of VAP-1 has been altered by the above method.

    The present invention (a) VAP-1 target sequence; And (b) relates to a recombinant host cell comprising a regulatory sequence bound to the VAP-1 target sequence.

    The present invention relates to a method for oxidizing an amine, comprising reacting an amine with a VAP-1 polypeptide of the invention. For example, the amine may be benzylamine or methylamine. VAP-1 polypeptide

(a ') a purified polypeptide comprising the VAP-1 amino acid sequence (SEQ ID NO: 2) in FIG. 1;

(b ') a purified polypeptide comprising a VAP-1 amino acid sequence (SEQ ID NO: 1) encoded by the VAP-1 sequence in FIG. 1;

(c ') a purified polypeptide comprising an amino acid sequence encoded by a VAP-1 cDNA clone contained in Accession No. DSM 11536;

(d ') hybridizes to the complement of DNA encoding the polypeptide of any one of (a')-(c ') and encodes VAP-1 and at least with the sequence (SEQ ID NO: 2) in FIG. A purified polypeptide comprising a VAP-1 amino acid sequence encoded by a nucleotide sequence having an amino acid sequence showing 80% identity; And

(e ') has an amino acid sequence that is at least 95% identical to a sequence selected from the group comprising a purified polypeptide comprising an amino acid sequence of an epitope-bearing portion of the polypeptide of (a') or (b ') .

The present invention relates to a method of inhibiting amine oxidase activity, comprising providing an effective amount of an inhibitor in a sample having said amine oxidase activity. For example, inhibitors may be semicarbazide, hydroxylamine, propargylamine, isoniazid, nialamide, hydrallazine, procarbazine ), Monomethylhydrazine, 3,5-ethoxy-4-aminomethylpyridine and MDL72145 ((E) -2 (3,4-dimethoxyphenyl) -3-fluoroallylamine).

The present invention provides for vascular adhesion protein-1 (VAP-1) -regulated binding of endothelial cells to lymphocytes, which involves altering the enzymatic activity of amine oxidase on endothelial cells by inhibition or potentiation. It is about how to adjust.

1. Sequence of VAP-1 cDNA isolated from human lung cDNA library and expected sequence of VAP-1 protein. V8 peptides that have been purified and sequenced from N-terminal, trypsin and immunopurified VAP-1 proteins are boxed. Italic amino acid residues within the box range indicate that no amino acid can correspond to the circulation in the peptide sequence. Potential N-glycosylation sites are indicated by asparagine indicated by arrows and circles, and the expected O-glycosylation sites are indicated by arrowed squares. The transmembrane region between residues 5 and 27 is indicated by shading. The diagram at the bottom of the figure shows the location of the transmembrane region (shown as the filled TMD site) and the relative positions of the expected glycosylation sites at the cell site of the molecule are represented by N and O-linked sugars, respectively. Is indicated by O. Indicator bars represent 50 amino acids.

Figure 2. FACS analysis of VAP-1 infected with COS-7 cells to confirm membrane orientation and cell position of VAP-1. A diagram showing the structure of two VAP-1 expressing plasmids used to infect COS-7 cells is shown at the top of the figure. VAP-1 is a native VAP-1 expression construct. VAP-1 FLAG is a FLAG epitope labeled VAP-1. Negative control for mock cell infection provided a construct in which VAP-1 was in the reverse direction in the expression vector. Column 1: expression constructs used for cell infection. Column 2: impermeability (-) and permeability (+) of the infected cells. Column 3: Negative Control MAb Staining. Column 4: anti-VAP-1 MAb 1B2 staining. Column 5: anti-FLAG MAb M2 staining. A-F rows represent the staining morphology resulting from the expression constructs used in the infection or mock control and the infected cells. Arrows indicate positively stained cell populations. Staining in opaque cells appears to have anti-VAP-1 MAb 1B2 (FACS panel B and column C, column 4). The mock control that infected the cells is negative (FACS panel A column, column 4). No staining appears to have anti-FLAG MAb in VAP-1 FLAG cell infected cells (column C, column 5). However, while VAP-1 infecting cells in the infiltrated cells is still positive (column E, column 4), the VAP-1 FLAG infected cells at this time are positively stained for anti-FLAG MAb (column F, Column 5) Permeability of cells to anti-FLAG MAbs allows access to FLAG epitopes and thus indicates that these epitopes are placed within the cells.

 FIG. 3 is a complex arrangement of mammalian members of the copper amine oxidase family from which sequence data is available, including VAP-1. The label on the left indicates the specific protein arranged in each column. : BSAO, bovine serum amine oxidase; VAP-1, human vascular adhesion protein-1; PDAO1, human placental diamine oxidase 1; PDAO2, human placental diamine oxidase 2; Rat DAO, rat diamine oxidase. The numbers correspond to the first amino acid in each row of the arranged protein. Sequences were obtained from the latest available database and arranged using GCG Pileup. Residues with identity to VAP-1 were highlighted using a GCG dark box.

4 is a sialidase treatment of VAP-1 expressed in COS-7 cells. Cell lysates from infected VAP-1 and mock infected COS-7 cells were either VAP-1 MAb 1B2 (lanes 1-4) or negative control MAb 3G6 (lanes 5-8) with SDS-PAGE, immunoblotting Treatment with sialidase (+) or (-) prior to immunoblottiong and probing.

FIG. 5 shows Northern blot analysis of VAP-1 mRNA in poly A ⁺ RNA extracted from human gut smooth muscle and tonsil matrix, in which A. lymphocytes were partially removed by washing and compression. 4.2 kb hybridizing mRNA is shown on both tracks. B. Northern blot analysis of VAP-1 mRNA in other human tissues. Northern blots were obtained from Clontech laboratories and the same amount of mRNA loaded into each lane. All filters were probed with ³² P-labeled VAP-1 cDNA probes containing the entire coding sequence and washed at high temperature (post-combination wash conditions were 0.1 × SSC, 0.1% SPS for 2 × 45 minutes at 65 ° C.). ).

Figure 6 Ax cells transfected with VAP-1 cDNA regulatory lymphocyte adhesion. A. Expression of VAP-1 on the cell surface of Ax cells stably transfected with VAP-1 cDNA or mock control. The intensity of staining at log scale in the bar graph is shown on the x-axis and the relative cell numbers are shown on the y-axis. B. Increased VAP-1 due to non-independent binding of lymphocytes to VAP-1 cell infectious agents. A significant amount of PBL (small round sphere at the top of a single layer, for example indicated by an arrow) binds more to the VAP-1 cell infector (left) than to a mock cell infector (right). Phase-contrast micrographs, magnification × 100. C. Combine amount. Five independent experimental results are expressed as mean ± SEM.

  The interaction between leukocyte surface receptors and their ligands in vascular endothelial cells precisely regulates lymphocyte migration between blood and various lymphoid organs, as well as the overflow of white blood cells into the site of inflammation. A cDNA clone encoding vascular adhesion protein-1 (VAP-1) that regulates leukocyte-endothelial cell adhesion response is described. Encoded VAP-1 surprisingly also has amine oxidase activity. In higher organisms, amine oxidase is believed to be involved in the metabolism of bio-based amines (McIntire, W. and C. Hartmann, Principles and Applications of Quinoproteins, VL Davisson, ed., Marcel Dekker, Inc. NY, Chap 6 (1992).

VAP-1 cDNA Bolt

The present invention provides a purified nucleic acid molecule comprising a polynucleotide encoding a vascular adhesion protein-1 (VAP-1) polypeptide having the amino acid sequence (SEQ ID NO: 2) shown in FIG. It was determined by sequencing cDNA clones providing the nucleotide sequences shown (SEQ ID NO: 1). Clones containing nucleotide sequences were deposited on 7 May 1997 under the International Depository Authority of DSMA-Deutsche Sammlung Von Mikroorganismen Und Zwllkulturen GmvHdp Budapest Treaty, D-38124 Braunschweig, Mascheroder Weglb, and assigned accession number DSM 11536. The deposited clones are contained in the pUC19 plasmid.

 90 and 170-180KD, monomeric and dimeric forms of VAP-1 using monoclonal antibody (MAb) immunoaffinity columns were digested with trypsin and V8 protease, respectively, and then internal peptide sequences Purified from detergent lysates of smooth muscle in an amount sufficient to obtain. The 90KD VAP-1 moiety was used to prepare partially modified oligonucleotide primers for RT-PCR experiments in mRNA prepared from N-terminal sequencing and human intestinal smooth muscle.

 Approximately 700 bp of single cDNA particles were amplified using these primers and cloned into pUC18 to confirm their sequencing. In order to find longer cDNA clones, panels of 10 human cDNA libraries were analyzed by PCR to identify those containing VAP-1 cDNAs and libraries providing strong signals were screened with PCR-generated VAP-1 cDNA fragments. It became. In this method many duplicate cDNA clones were isolated from human lung and cardiac cDNA libraries. 2501 bp single cDNA was derived from these clones and recloned into pUC19 as described in FIG. (subcloning). The measured nucleotide sequence of the VAP-1 cDNA inserted into this clone in FIG. 1 (SEQ ID No: 1) is approximately 84.6 from the initiation codon at positions 80-82 of the nucleotide sequence (SEQ ID No: 2) in FIG. It includes an open reading frame that encodes a protein of 763 amino acid residues in FIG. 1 (SEQ ID NO: 1) with a molecular weight of KD.

VAP-1 of the present invention has the same identity as the type of enzyme called copper containing amine oxidase (EC1.4.3.6). The VAP-1 protein shown in FIG. 1 (SEQ ID NO: 2) is about 24% identical to Escherichia Coli Cu-monoamine oxidase and about 41-81% similar to this class of mammalian member. The highest identity found is confirmed in bovine serum amine oxidase (81%).

Unless otherwise indicated, each "base sequence" presented herein is represented as a sequence of deoxyribonucleotides (abbreviated A, G, C and T). However, the base sequences of deoxyribonucleotides are listed by nucleic acid molecules and polynucleotide sequences in the case of DNA molecules and polynucleotides. In the case of RNA molecules or polynucleotides, the corresponding sequences are those of ribonucleotides (A, G, C and U), where each thymidine deoxyribonucleotide (T) in the deoxyribonucleotide sequence specified is Ribonucleotide uridine (U).

 The nucleic acid molecule of the present invention may be in the form of RNA, such as mRNA, or DNA, including genomic DNA obtained from or cloned with cDNA. DNA can be double stranded or single stranded. The single stranded DNA or RNA may be a coding strand known as the sense strand or may be a non-coding strand, also referred to as an anti-sense strand or supplement.

Nucleic acid molecules, DNA or RNA are denoted by "purified" nucleic acid molecules, which are taken from the natural environment or produced synthetically. Purified RNA molecules include RNA transcription in vitro of the DNA molecules of the invention.

In another aspect, the present invention provides a purified nucleic acid molecule encoding a VAP-1 polypeptide having an amino acid sequence encoded by a cDNA clone contained in a plasmid designated Accession Number DSM 11536 on May 7, 1997. do. The invention further relates to a purified nucleic acid molecule having the depicted nucleotide sequence (SEQ ID NO: 1) in FIG. 1 or to a nucleotide sequence of VAP-1 cDNA contained in the designated clone described above or one of said sequences. It provides a nucleic acid molecule having a nucleotide sequence. Such isolated molecules, particularly DNA molecules, are useful as probes for detecting the expression of the VAP-1 gene in human tissues by gene mapping by in situ hybridization with chromosomes and, for example, by Northern blot analysis. .

The present invention relates to fragments of the purified nucleic acid molecules described herein. Fragments of purified nucleic acid molecules having the VAP-1 nucleotide sequence of the deposited cDNA or the VAP-1 nucleotide sequence (SEQ ID NO: 1) shown in FIG. 1 are at least about the lengths useful as the diagnostic probes and primers described herein. 15nt, more preferably at least about 20nt, even more preferably at least 30nt, even more preferably at least 40nt. Of course even longer fragments of 50-1500nt are useful in accordance with the present invention, such as, but not all, the nucleotide sequence of most deposited cDNAs, or fragments corresponding to the sequence shown in FIG. 1 (SEQ ID NO: 1). It will be common for one skilled in the art to generate such DNA particles since the VAP-1 gene has been deposited and provided with the sequencing (SEQ ID NO: 1) shown in FIG. 1. For example, cleavage of restriction nucleases, shear or oligonucleotide synthesis by sonication can be readily used to produce particles of various sizes.

In another aspect, the invention hybridizes a portion of a polynucleotide to a VAP-1 cDNA clone contained in Accession No. DSM 11536 in the nucleic acid molecule of the invention described above under strong stringent hybridization conditions. Provided are purified nucleic acid molecules comprising polynucleotides. 50% formamide, 5x SSC (150 mM NaCl, 15 mM trisodium citrate), 10% dextran sulphate and 50 mM sodium phosphate (pH7.6), 5x denhalt solution by "stringent hybridizaion conditions" The solution containing 20 g / ml denatured and sheared salmon sperm DNA was incubated overnight at 42 ° C. and washed with a filter at 0.1 × SSC at 65 ° C.

Polynucleotides (DNA or RNA) are shown that hybridize to a “part” of polynucleotides to a reference polynucleotide of at least about 15 nucleotides (nt), more preferably about 30-70 nt. These are useful as diagnostic probes and primers. Of course, even polynucleotides that hybridize to longer portions of the cited polynucleotides (e.g., designated cDNA clones), e.g., 50-750nt in length or to the full length of the cited polynucleotides, are also VAP-1 of the deposited cDNA. It is also useful as a probe according to the present invention because it is a polynucleotide corresponding to most of the nucleotide sequence or the nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1). Some of these are described as Molecular Cloning, A Laboratory Marual, 2nd Edition, Sambrook, J., Fritsch, E.F. as probes or according to universal DNA hybridization techniques. And as primers for amplification of target sequences by polymerase chain reaction (PCR) as described in Maniatis, T., eds., Cold Spring Harbor Laboratory press, Cold Spring Harbor, N.Y. (1989).

Purified nucleic acid molecules of the present invention are described first with DNA molecules that constitute an open reading frame (ORF) having an initiation codon at positions 80-82 of the VAP-1 nucleotide sequence (SEQ ID NO: 1) shown in FIG. It contains a DNA molecule that encodes a VAP-1 protein because it contains a sequence that is substantially different from that but due to a decrease in the genetic code. Of course, genetic code is well known in the art. Thus, creating degenerative variants is common to those skilled in the art.

The present invention relates to a variant of a nucleic acid molecule of the invention that encodes a protein, homologue or derivative of a VAP-1 protein. The variant may occur naturally, such as a natural allele or a conjugate variant. "Allelic variant" refers to one of several selective forms of genes that occupy a given position on the chromosome of an organism. Gene II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants can be generated using prior art mutagenic techniques.

Such variants include those produced by nucleotide substitution, deletion or addition. Substitutions, deletions or additions may comprise one or more nucleotides. The variant may be modified in the coding region, the non-coding region or both. Modifications in the coding region may also produce traditional, non-traditional amino acid substitutions, removals or additions. Particularly preferred of these are silent substitutions, additions and removals that do not alter the properties and activities of the VAP-1 protein or some of them. Of particular note here is the traditional substitution.

 A further embodiment of the invention includes (a) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence (SEQ ID NO: 2) in FIG. 1; (b) a nucleic acid molecule comprising the VAP-1 coating sequence of the VAP-1 nucleotide sequence (SEQ ID NO: 1) in FIG. 1; (c) a nucleic acid molecule encoding a VAP-1 polypeptide comprising an amino acid sequence encoded by a VAP-1 cDNA clone contained in Accession No. DSM11536; (d) a nucleic acid molecule comprising the coding sequence of the VAP-1 nucleotide sequence contained in Accession No. DSM11536; (e) a nucleic acid molecule comprising a nucleotide sequence complementary to a VAP-1 nucleotide sequence in (a), (b), (c) or (d); (f) a nucleic acid molecule wherein (b) or (d) due to a decrease in the genetic code comprises a base sequence different from the coding sequence of the nucleic acid molecule; And (g) a nucleotide sequence encoding VAP-1 having an amino acid sequence that hybridizes to the molecule of (e) and exhibits at least 90% identity with the VAP-1 sequence (SEQ ID NO: 2) in FIG. 1. Nucleic acid molecules and the like include purified nucleic acid molecules comprising polynucleotides that are at least 90% identical and more preferably have at least 95%, 96%, 97%, 98% or 99% identical nucleotide sequences. It ignores whether they encode polypeptides with VAP-1 activity. This can be achieved by one of ordinary skill in the art, even where the particular nucleic acid molecule does not encode a polypeptide having VAP-1 activity, such as as a hybridization probe or polymerase chain reaction (PCR) primer. Because, I know if you use. However, better yet, at least 90% of the VAP-1 nucleic acid sequence shown in FIG. 1 (SEQ ID NO: 1) or the VAP-1 nucleic acid sequence of the designated cDNA encoding a polypeptide having virtually VAP-1 protein activity. , 95%, 96%, 97%, 98% or 99% identical nucleic acid molecules.

A 10 point mutation for each 100nt of each of the cited sequences encoding the VAP-1 polypeptide is a polynucleotide sequence having at least 90% "identical" sequencing for the reference nucleotide sequence encoding the VAP-1 polypeptide. point mutations) are identified as identical to the cited sequence, except that they may contain more than one point mutation. Any particular nucleic acid molecule is for example at least 90%, 95%, 96%, 97%, 98% or 99 in the VAP-1 nucleotide sequence shown in FIG. 1 (SEQ ID NO: 1) or the nucleotide sequence of a designated cDNA clone. Percent identity can typically be measured using a known computer program, such as the Bestfit program (Sisconsin Sequence Analysis Package, Version 8 for Unix, Geneties Computer Group, University Research Park 575 Science Drive, Madison, WI 53711).

Vectors and Host Cells

The present invention is directed to the production of VAP-1 polypeptides or particles thereof by recombinant techniques, as well as vectors comprising purified DNA molecules of the invention, host cells genetically constructed with DNA encoding VAP-1.

The polynucleotide can be linked to a vector containing a marker that can be selected for propagation in the host. Generally, plasmid vectors are introduced into complexes with precipitated or filled lipids, such as calcium phosphate precipitates. If the vector is a virus, it can be packaged in vitro using appropriate packaging cell lines and then transduced into host cells. Selectable markers include dihydrofolate reductase or neomycin resistance genes for eukaryotic cell cultures and tetracycline or ampicillin resistance genes for culturing E. coli and other bacteria.

Even better is a vector comprising a cis-acting control region for the polynucleotide of interest. Suitable trans-acting factors can be supplied by the host, by the complementary vector, or by the vector itself upon introduction into the host. A better specific example in this regard is that the vector is specifically expressed, which may be inductively morphologically specific to the cell. Particularly preferred among these vectors are those that can be induced by easy-to-process environmental factors such as temperature and nutritional additives.

Expression vectors useful in the present invention include chromosomal episomal- and virus-derived vectors such as bacterial plasmids, bacteriophages, yeast episomes, yeast chromosomal components, bacuroviruses, vaccinia virus, adenoviruses, poxviruses, gastric rabies Viruses derived from viruses such as RNA and RNA tumor viruses and vectors derived from combinations thereof such as cosmids and phagemids.

DNA insertion should, in some name, be effectively bound to suitable promoters such as the phage lambda PL promoter, the E. coli lac, trp tac promoter, the early SV40 and late promoters, and the RNA tumor viral LTR _S promoter. Other suitable promoters will be well known to the skilled artisan. The expression construct will further contain a site at the initiation, termination, and transcriptional region, at the ribosomal binding site for metastasis. The coding portion of the mature transcript expressed by the structure preferably comprises an initial initiation of the coding sequence and an end codon (UAA, UGA or UAG) suitably located at the end of the coding sequence.

All such recombinant DNA techniques are described in Treco et al., WO94 / 12560 and Treco et al. The recombinant methods described in WO95 / 31560 also include and can be applied to modify VAP-1 expression and amine oxidase activity in cells. In particular, regulatory regions (eg, promoters) can be introduced into the host genome to alter the expression of endogenous VAP-1.

Accordingly, the present invention is directed to altering the expression of vascular adhesion protein- (VAP-1) and (i) VAP-1 target sequences; (Ii) introducing into a host cell, a DNA structure comprising a regulatory sequence bound to a VAP-1 target sequence; (b) a method for producing a recombinant host cell comprising maintaining the host cell between the DNA structure and the endogenous VAP-1 sequence under conditions suitable for similar recombination. The present invention relates to recombinant host cells in that the expression of VAP-1 has been altered by the invention. Altering the expression of VAP-1 also includes using the sequences of the present invention to inactivate the expression of native genes. Similar recombination or targets are used to replace or incapacitate regulatory regions normally associated with endogenous VAP-1, and the regulatory sequences allow genes to be expressed at a higher level than evidence in cells not infected with the corresponding cells or infected with the corresponding cells. Evidences and other regulatory or induction patterns should be provided in non-cellular cells. Accordingly, the present invention relates to a method for producing a protein by activating an endogenous gene that encodes a desired product in a cell.

If the DNA construct is targeted, it should contain at least the target sequence and preferably the regulatory sequence ... Selectable markers, expandable markers or exons and non-as described in Treco et al. WO94 / 12650 and Treco et al. WO / 31560dp. There are often additional structures, such as junction donor sites. In this structure, DNA may be referred to as exogenous DNA, which is introduced into a host cell by the method of the present invention. Exogenous DNA can process the same or different sequences as exogenous DNA present in cells prior to infection.

The target sequence of the DNA structure is a DNA sequence that allows suitable similar recombination into the genome of a selected cell containing the VAP-1 gene at the VAP-1 site. The target sequence is, for example, a DNA sequence similar to the VAP-1 DNA sequence normally present in the genome of the cell that is obtained and located at both ends of the regulatory sequence. The target sequence used is selected for the VAP-1 site into which the DNA structure is to be inserted.

The regulatory sequence of the DNA structure may consist of one or more promoters, enhancers, skeletal-adhesion regions or interstitial attachment sites, negative regulatory components, transcriptional soluble binding sites, or a combination thereof.

The site of introduction of the DNA sequence will generally be a site that affects the function of or within the endogenous VAP-1 gene or VAP-1 gene. DNA sequences that alter the expression of the endogenous VAP-1 gene can be introduced into the host cell as a single DNA structure or as separate DNA sequences that are physically bound in the genome of the infected cell. Moreover, the DNA can be introduced as linear, double-stranded DNA with or without a single stranded region at one or both ends, or the DNA can be introduced as circular DNA. After the regulatory DNA is introduced into the host cell, the cells are maintained under conditions suitable for similar recombination that occurs between the genomic DNA and a portion of the introduced DNA. Similar recombination between genomic DNA and introduced DNA results in similar recombinant host cells in which sequences that alter the expression of endogenous VAP-1 genes are effectively bound to the VAP-1 gene. The resulting similar recombinant host cell produced by the method can be cultured under conditions suitable for the expression of the VAP-1 protein by producing the VAP-1 protein in vitro or the cell can be grown in vivo with the VAP-1 protein. It can be used for in vivo delivery (ie gene therapy).

The target result may be a simple insertion of a regulatory sequence, locating an endogenous VAP-1 gene under the control of a new regulatory sequence (eg, by manipulation of an endogenous gene, to a promoter or enhancer or both). The target result may be a simple removal of regulatory components, such as the removal of tissue-specific negative regulatory components. Targeted results can replace the components present; For example, crude-specific enhancers may be replaced by enhancers that have broader or different cell-type specificities than naturally occurring components and exhibit different regulatory or induction patterns than the corresponding uninfected cells.

Genetic targets can be used to replace existing regulatory regions of VAP-1 with regulatory sequences isolated from other genes or new regulatory sequences synthesized by genetic processing methods. According to the present invention, the introduction of exogenous DNA replaces all or part of the endogenous (genomic) sequence or otherwise damages the endogenous sequence that regulates expression of the endogenous VAP-1 gene by destroying the function of the endogenous sequence. Bring it.

DNA structures comprising exogenous DNA, DNA encoding an optional marker, along with additional sequences necessary for the expression of exogenous DNA in the host cell to be used are used to transfect cells in which VAP-1 production is altered. . Moreover, details of similar recombination methods for altering endogenous gene expression are all provided in Treco et al. WO94 / 12650 and Tresco et al., WO95 / 31560, incorporated herein by reference.

DNA or recombinant constructs are introduced into cells by a variety of methods including transformation, infection, electroporation, microinjection, transduction, calcium phosphate precipitation and liposome-, polybrene- or DEAE dextran-mediated cell infection. Can be. This method is described in many standard experimental guides, such as in Davis et al., Basic Method In Molecular Biology (1986). Alternatively, infectious vectors such as RNA tumor virus, herpes, adenovirus, adenovirus based, mumps and polio virus vectors can be used to introduce DNA structures.

Representative examples of suitable hosts include , but are not limited to , bacterial cells such as E. Coli, Streptomyces and Salmonella typhimurium cells, fungal cells such as yeast cells, insect cells such as Drosophila S2 and Spodoptera sf9 cells, Ax and CRL 1998 cells (all internal Animal cells), CHO, COS and Bowes melanoma cells, and plant cells. Suitable culture medium and conditions for the host cells described above are known in the art.

Suitable prokaryotic and eukaryotic vectors will be readily apparent to those skilled in the art.

Among the known bacterial promoters suitable for use in the present invention include E. coli lac I and lac Z promoters, T3 and T7 promoters, gpt promoters, lambda P _R and P _L promoters and trp promoters. Suitable eukaryotic promoters include metals such as the CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, promoters of RNA tumor virus LTRs and promoters of Rous sarcoma virus (RSV) and mouse metallothionein-I promoters. Rotionine promoters.

Transcription of the DNA encoding the VAP-1 polypeptides of the invention by higher eukaryotic cells can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10-300 bp, which act to increase the transcriptional activity of the promoter in a given host cell form. Examples of enhancers include SV40 enhancers located behind the replication organs at bp100-270, cytomegalovirus early promoter enhancers, polyoma enhancers behind the replication organs, and adeno virus enhancers.

VAP-1 polypeptides can be expressed in modified forms such as soluble proteins and can include additional heterologous functional regions as well as secretory signals. For example, additional amino acid regions, particularly charged amino acids, can be added to the N-terminus of the VAP-1 polypeptide to improve stability and persistence in the host cell during purification or subsequent operation and storage. In addition, peptide molecules may be added to the VAP-1 polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of polypeptide molecules to produce secretion or excretion, to enhance stability and to facilitate purification, among others, is a familiar and general skill in the art.

VAP-1 protein may be prepared by ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, affinity chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxyllapatite chromatography and lectin chromatography. Can be recovered and purified from recombinant cell culture by well known methods, including. Polypeptides of the present invention are naturally purified products, products of chemical synthesis processes and products produced by recombinant techniques from prokaryotic or eukaryotic hosts, including, for example, bacteria, yeast, higher plants, insects and mammalian cells. Include. Depending on the host used in the recombinant production process, the polypeptides of the invention can be glycosylated or aglycosylated.

VAP-1 Polypeptides and Fragments

The predetermined nucleotide sequence of the VAP-1 cDNA of FIG. 1 (SEQ ID NO: 1) is shown in FIG. 1 (SEQ ID NO: 1) having a molecular weight of 84.6 KD and an initiation codon at positions 80-82 of the nucleotide sequence. An open reading frame encoding the protein of the 763 amino acid residue of SEQ ID NO: 2).

Proteins were determined using six potential N-glycosylation sites per monomer and three putative 0-glycosylation sites (0-glycosylation site prediction email server, NetOoglyc@cbs.dtu.dk (Hansen et al., Biochem, J. 308: 801-813 (1995).

Accordingly, the present invention provides for the purification of a purified VAP-1 polypeptide having an amino acid sequence encoded by a designated cDNA, a peptide or polypeptide comprising an amino acid sequence at 1 (SEQ ID NO: 2) or a portion of the polypeptide. The terms "peptide" and "oligopeptide" are regarded as synonyms (as generally accepted) and each term may be used interchangeably as necessary to refer to at least two amino acid chains bound by peptide bonds. .

It will be appreciated in the art that some amino acid sequences of the VAP-1 polypeptide can be varied without significant impact on the structure or function of the protein. Thus, the present invention includes variants of VAP-1 polypeptides that exhibit essentially the activity of a VAP-1 polypeptide. Such mutations include elimination insertions, conversions, repeats and substitutions of similar residues. Small changes or such "neutral" amino acid substitutions generally have little effect on activity. Guidance on amino acid changes that are morphologically asymptomatic (ie, unlikely to have a serious detrimental effect on function) can be found in Bowie, J. U. et al., Science 247; 1306-1310 (1990). Changes are minimal properties such as general amino acid substitutions that do not seriously affect the folding or activity of the VAP-1 protein.

Amino acids in the VAP-1 protein of the present invention that are basic in function can be demonstrated by methods known in the art, such as sited-directed mutagenesis and removal analysis. The resulting mutant molecule is tested for biological activity such as amine oxidase activity or cohesive function.

Polypeptides of the invention are preferably provided in substantially purified form. Recombinantly generated versions of the VAP-1 polypeptide can be purified in fact by the one step method described, for example, in Smith and Johnson, Gene 67: 31-40 (1998).

Polypeptides of the present invention include polypeptides having at least 90% similarity, more preferably at least 95% similarity, and more preferably 96%, 97%, 98% or 99% similarity to those described above. Polypeptides of the invention are at least 90% or 95%, even more preferably 96%, 97%, 98 with the VAP-1 polypeptide encoded by the designated cDNA or the VAP-1 polypeptide of FIG. 1 (SEQ ID NO: 2) Also included are portions of such polypeptides comprising% or 99% identical polypeptides and having at least 30 amino acids and more preferably at least 50 amino acids.

For example, a polypeptide having at least 90% identical amino acid sequence to a reference amino acid sequence of a VAP-1 polypeptide may cause the polypeptide sequence to comprise no more than 10 amino acid changes per 100 amino acids of each of the reference amino acids of the VAP-1 polypeptide. Except for the amino acid sequence of the polypeptide is identical to the reference sequence.

Polypeptides of the invention can be used to generate polyclonal and monoclonal antibodies, which can be used as assays and antagonists capable of enhancing or inhibiting VAP-1 protein function or to assess VAP-1 protein expression. useful. Details of the monoclonal antibodies raised against VAP-1 are all incorporated by reference in Salmi, M. and Jalkanen, S., Science 257: 1407-1409 (1992), US Pat. Nos. 5512442 and 5580780, and US Described in application 08/44799. Moreover, such polypeptides can be used in two yeast hybridization systems to "capture" the VAP-1 protein, which binds a candidate agonist and an antagonist protein (Fields and Song, Nature 340: 245-246 (1989).

As used herein, the term “antibody (Ab) or“ monoclonal antibody ”(MAb) refers to antibody particles (eg, Fab and F (ab) 2 particles) that can specifically bind to the VAP-1 protein. Fab and F (ab`) 2 particles are deficient in Fc particles of the complete antibody and are easily removed from the circulation and may have less non-specific tissue binding than complete antibodies ( WahI et al., J, Nucl. Med: 316-325 (1983)), therefore, these particles are preferred.

Antibodies of the invention can be prepared by one of a variety of methods. For example, cells expressing VAP-1 protein or antigenic particles thereof can be administered to the animal to cause production of serum containing polyclonal antibodies. In a better way, the preparation of the VAP-1 protein is made and purified to be virtually free of natural contaminants. Such preparations are introduced into animals to produce monoclonal antisera of greater specific activity.

In the most preferred method, the antibodies of the invention are monoclonal antibodies (or VAP-1 proteins that bind their particles). Such monoclonal antibodies can be prepared using hybridoma technology. (kohler et al. nature 256: 495 (1975): kohler et al., Eur. J. Immunol. 6: 511 (1976); Kohler et al., Eur. J. Immunol. 6: 292 (1976): Hammerling et al. In: Monoclonal Antibodies ard T-cell Hybridomas.Elesvier NY (1981) pp. 563-681

Fed. It is contemplated that F (ab ') 2 and other particles of the antibodies of the invention can be used according to the methods described herein. Such particles are typically produced by proteolytic cleavage using enzymes such as papain (to produce Fab particles) or pepsin (to produce F (ab ′) 2 particles). Alternatively, VAP-1 protein binding particles can be produced through the application of recombinant DNA technology or synthetic chemistry.

"Humanized" chimeric monoclonal antibodies can be generated using genetic constructs derived from hybridoma cells that produce the monoclonal antibodies described above. Methods for generating fancy antibodies are known in the prior art. See Morrison, Science 229: 1202 (1985): et al., Biotechniques 4: 214 (1986): Cabilly et al. US Pat. No. 4,816,567: Tonguchi et al., EP 171,496.

VAP-1 Usage

VAP-1 of the present invention has considerable identity with the class of enzymes referred to as copper-containing amine oxidases (enzyme order classification, EC 1.4.3.6). The VAP-1 protein shown in FIG. 1 (SEQ ID NO: 2) is about 24% identical to Escherichia coli Cu-monoamine oxidase and about 41-81% similar to mammalian members of this class. The highest identity found is in bovine serum amine oxidase (81%). Important amine oxidase enzyme activity using benzylamine, monobenzylamine oxidase (MAO) substrates is detected in VAP-1 expressing CHO cells but not in mock cell infected cells. In the presence of inhibitors of copper containing monoamine oxidase, semicarbazide and hydroxyl amine, VAP-1 has no activity against benzylamine.

Thus, peptides encoded by the DNA of the present invention may be used to enzymatically oxidize amines by reacting amines with VAP-1 polypeptides having amine oxidase activity. The amines can be, for example, aromatic amines which are particular endogenous and xenobiotic. The amine is best benzyl amine.

The amine oxidase assay conditions of benzylamine as amine oxidase substrates are described in detail in the Material and Method section, infra . Sodium phosphate buffer is added at a final volume of 0.2-5 ml, preferably 0.5 ml, for a final condition of 0.05-0.2 mM, preferably 0.1 mM. Unlabeled benzylamine is added at a concentration of 1-100 nmol, preferably 80 nmol and ¹⁴ C-benzylamine is added so that the activity is 0.1-1.0 μCi, preferably 0.4 μCi. 0.05-0.5 ml, preferably 0.1 ml of cytosolic sample can be added for evaluation. After incubation at 37 ° C. for 1 hour, the aldehyde product of the amine oxidase reaction is extracted with toluene containing 0.1-0.5 g / l, preferably 0.35 g / l diphenyl oxazole. Further extraction can be carried out using a toluene / diphenyl oxazole mixture. The first extraction (and the second extraction) is calculated at ¹⁴ C on the liquid scintillation counter.

The present invention relates to a method of inhibiting VAP-1 amine oxidase activity by providing an effective amount of an amine oxidase inhibitor. Inhibitors are for example semicarbazide, hydroxylamine, propargylamine, isoniazid, nialamide, hydralazine, procarbazine, monomethylhydrazine, 3,5-ethoxy-4-aminomethylpyridine or MDL 72146 ( (E) -2- (3,4-dimethoxyphenyl) -3-fluoroarylamine), preferably semicarbazide or hydroxyl amine. Semicarbazide may be provided at a concentration of 10-1000 μM. The hydroxylamine may be provided at a concentration of 0.1-100 μM or here in a range such as 1-10 or 2-8 μM, preferably at a concentration of 5 μM.

In accordance with the present invention endothelial vascular adhesion protein-1 (VAP-1) -mediated binding to lymphocytes may be mediated by providing inhibitors or enhancers of amine oxidase to the host. The host can be a mammalian cell line, animal or human. The present invention can be used in vitro or in vivo for treatment.

Although the present invention will be described in general, the same will be more readily understood through examples provided by the description and not by way of limitation.

Example 1

Isolation of VAP-1 cDNA

 Human organ smooth muscle with strong expression of VAP-1 was used as a purification source of VAP-1 protein obtained from the material removed during surgery. VAP-1 in the form of 90 and 170-180KD was digested with trypsin and V8 protease using a Mab immunoaffinity column and purified from the washing eluate of smooth muscle in an amount sufficient to obtain internal peptide sequences. In addition, some 90KD VAP-1 were directly N-terminally sequenced. Peptide elution profiles from the HPLC column used to purify peptides from both forms of VAP-1 were the same as in the peptide sequence of the corresponding peak, which means that the dimer of the protein consists of two 90KD subunits. (dimer).

Some VAP-1 peptide sequences were used to design partially modified oligonucleotide primers for RT-PCR experiments on RNA prepared from human organ smooth muscle. A single cDNA fragment of about 700 bp was amplified using these primers and cloned into pUC18 to confirm the rm sequence. This cDNA sequence contained a continuous open reading frame encoding the protein containing the trypticity of the immunopurified VAP-1 material and a portion of the sequence of the V8 peptide, thereby confirming that the correct cDNA fragment was amplified. Panels of 10 human cDNA libraries were analyzed by PCR to isolate longer cDNA clones to identify those containing VAP-1 cDNA's and the libraries showing the strongest signals were screened with PCR-producing VAP-1 cDNA fragments.

In this way, multiple redundant cDNA clones were isolated from human lung and cardiac cDNA libraries. 2501 bp cDNA containing 2292 bp continuous open reading frame starting from ATG methionine codon was derived from these clones and subcloned into pUC19. The initiation codon is followed by the peptide sequence found at the N-terminus of the purified 90KD VAP-1 protein and the adjacent open reading frame encodes both VAP-1 tryptic and V8 peptides identified by protein sequencing (FIG. 1). De There are 80 bp 5 ′ untranslated region and 120 bp 3 ′ untranslated region following the TAG stop codon in the clone. Poly A sequences as well as polyadenylation signals were not found at the 3 ′ end of the cDNA, suggesting that native VAP-1 mRNA is longer than indicated by the isolated cDNA.

Transient expression of VAP-1 in COS-7 cells by transfection with expression vectors containing cDNA showed strong surface staining of cell populations with VAP-1 MAb, 1B2 (FIG. 2, FACS panel B column). , Column 4). Infected cell controls were negative (FIG. 2, FACS panel B column duf, column 4). It is therefore concluded that cDNA is isolated and encodes VAT-1 of an immune response expressed on the surface of VAP-1 cDNA transfected cells.

VAP-1 Protein

An open reading frame was contained in the VAP-1 cDNA encoding 84.6 KD of 763 amino acid proteins. Investigations of the available protein sequence database showed that VAP-1 was highly identical to the enzymatic family of copper-containing amine oxidase (Enzyme Commission Classification, EC1.4.3.6). This identity varies from 24% for E. coli Cu-monoamine oxidase to 41-81% for the mammalian members of this family. The highest identity found was in bovine serum amine oxidase (81%), which showed significant conservation over the entire length of the protein except for the short region at the N-terminus of the molecule. The VAP-1 various families in four different mammalian members of the copper containing amine oxidase family, whose sequence data are available, are shown in FIG. 3.

VAP-1 has no significant identity with any currently known adhesion molecule and contains no protein domains often found in such proteins despite knowing the occurrence of RGD motifs between residues 726-728. The functional significance of these commonly occurring motifs with integrin binding is not yet known. Proteins were identified as 6 potential N-glycosylation sites and 3 putative 0-glycosylation sites per monomer (as determined using the 0-glycosylation site prediction, Email server, NeOglyc @ cbs.dtu, dk hansen et al., Biochem. J. 308: 801-813 (1995) (FIG. 1).

Experiments with protein sequences show distinct regions with membrane spanning domain properties at the most N-terminus (residual 5-27) of the molecule shown in FIG. 1 except for the predominantly hydrophobic, 23 amino acid position. Did. This area is located in G., Nucl. Von Heijne. Acids Res. 14: 4683-4690 (1986), because it contains a potential cleavage site at position 19, it can be interpreted as either separable evidence or as a permeable membrane domain. The N-terminal protein sequence of the 90KD VAP-1 protein indicated that this hydrophobic region was not cleaved from the material we purified and could not serve as a normal cleavage signal sequence for secretion. In addition, the charged properties on the hydrophobic region are characterized by the type II membrane proteins having longitudinal N-terminus and C-terminus (Hartman, E et al., Proc. Natil Acad. Dci. USA 86: 5786- 5790 (1986)).

To demonstrate that the hydrophobic region has the same function as the transmembrane domain and to ensure the orientation of VAP-1 in the membrane, we use a commercially available and cytoplasmic part of the putative transmembrane domain where FLAG epitopes recognized by MAb M2 are predicted. At the beginning of the initial methionine codon, the VAP-1 cDNA expressing substances were transferred where they were transferred into the frame. VAP-1 cDNA control constructs reversed in this construct and carrier were transferred to COS-7 cells, and transient expression of FLAG and VAP-1 epitopes recognized by M2 and 1B2, respectively, resulted in permeated and non-transfected cells. It was analyzed by FACS analysis (FIG. 2). Staining of FLAG appeared only in permeated cells, whereas VAP-1 staining appeared in both permeated and non-permeable (Fig. 2. F line, column 5). Cell infected groups did not show both FLAG and VAP-1 MAbs (FIG. 2, lines A and D, column 4.5). This is a huge C-terminal extracellular moiety that is recognized by MAb 1B2 where the N-terminal FLAG epitope is located on the cytoplasmic side of the cell membrane, the hydrophobic portion spans the bilipid layer and interferes with adhesion. All potential glycosylation sites are located outside the cell of the molecule.

To confirm the size and glycosylation status of recombinant VAP-1 transiently expressed in COS-7 cells, we transformed the cells with and without pretreatment with sialidase using MAb1B2 and cell extracts of isolated VAP-1. And immunoblotting. Sialoglycoprotein is the same as the original VAP-1 in Sialidase treatment, which increases the molecular weight of VAP-1 from 170-180kD to about 180-190kD and removes negative sialic acids, which is the cause of decrease in VAP-1 fluidity in SDS-PAGE. This decreases in the total cathode. Similar results for sialidase treatment have been found for tonsil VAP-1. (Salmi, M., and Jalkanen, S., J. Exp. Med. 183: 569-579 (1996)).

VAP-1 expression

Northern Blot analysis of mRNA isolated from human intestinal smooth muscle and leukocyte depleted Tonsil stroma showed that the cloned VAP-1 cDNA hybridized to 4.2 kb mRNA in both tissues (FIG. 5A). Northern blot analysis showed better results with 4.2kb VAP-1 mRNA being widely expressed in human tissues. This message could not be detected in other blood leukocytes, but was intensely expressed in the lungs, intestines and cecum compared to other tissues. Small amounts of VAP-1 mRNA were detected in the spleen, thymus, testes, liver, pancreas, bone marrow and fetal liver. Intermediate stages of expression were shown in the prostate, ovary, and large intestinal villus, heart, placenta, skeletal muscle and lymphocytes (FIG. 5B). Other mRNA types were not detected after prolonged autoradiography.

Materials and methods

Mouse MAbs 1B2 against Abs and reagents VAP-1, 3G6 against T cells in chickens are described in Salmi, M., and Jalkanen, S., Science 257: 1407-9 (1992)) have. MAb M2 recognizing FLAG peptide (DYKDDDDK) was obtained from KEBO Laboratories, Espoo, Finland.

Normal intestinal samples obtained from VAP-1 purification and sequencing abdominal surgery were dissected from lamina propria. Smooth muscle cell wall was cut and digested into small pieces overnight in lysis buffer (150mNaCl, 100mM Tris-base, pH 7.2, 1.5mM MgCl2.1% NP-40, Aprotinin and 1mM PM5F). The supernatant digested after clarification was placed in an immunoaffinity column with 5 ml containing CnBr activated Sepharose beads prepared with normal rat serum, unbound MABs and anti-VAP-1 MAb (3 mg / ml heads). .

This specific column was washed with lysis buffer and the VAP-1 antigens were eluted with 50 mM trityl amine. This elutate was immediately frozen and immediately freeze-dried. Samples were dissolved in non-reducing Laemmlis sample buffer and separated on 5-12.5% SDS-PAGE gel. After transfer to the polyvinylidine ditluride membrane by electroblotting, the membrane was stained with coomassie bule, and 90, 170-180 kD bands were excised. All 170-180 kD and 90 kD portions were trypsinized (Sequencing grade, Boehringer Mannheim, GmbH, Mannheim, Germany). Like Fernandez et. As explained by al. (Fernandez, J., et. al.) Anal. Biochem. 201: 251-262 (1992). Elute peptides were separated by HPLC (150 A; Applied Biosystems) on a Vydac C18 column (2.1 × 150 mm), and N-terminal sequencing was performed on a protein analyzer equipped with an on-line phenylthiohydantoin amino acid analyzer (120A; Applied Biosystems). 477A; Applied Biosystems).

The remaining portion of 90 KD was subjected to N-terminal sequencing without prior digestion and trypsinized, and the membranes were retreated with V8 protease (Sequencing grade, Boehringer Mannheim), and the isolated peptides were purified and sequenced. lost. Sequences of several peptides were identified using a laser equipped with matrix desorption mass spectrometry to identify their expected mass (Lasermat, Finnigan).

Molecular biological techniques. Isolation of small and large scale Plasmids from E. coli, restriction enzyme digestion, Plaque hybridization, purification of λ phage and extraction of phage DNA, transformation of E. coli, cDNA synthesis and plasmid construction are performed by standard techniques (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory.Cold Spring Harbor, NY (1989)). DNA fragments isolated from agarose gels were performed using the QIAEX kit. (QIAEGEN, Hilden, Germany). DNA probes were performed using an Amersham Multiprime DNA labeling kit and [α- ³² P] dCTP (Amersham, Bucks, UK) below 3000 Ci / mMol. Automatic radiographic graphics were performed using intense screens in Amersham Hyperfilm MP if necessary. PCR fragments are blunt ends cloned into pUC18 using the Sureclone kit (Pharmacia, Uppsala, Sweden). Plasmid DNA was sequenced using version 2.0kit in the author's knowledge and in the DNA sequencing facility of Turkueogkr's Medical Genetics (United States Biochemical Corporation, Cleveland, OH, USA). Sequence sets and analysis were performed using the Version 8.1-UNIX Wiscosin Package from Genetic Computer Group. Comparative analysis of the data was made using BLAST email or NCBI's WWW server (http://www.ncbi.nlm.nih.gov/). Oligonucleotide primers used for sequencing and PCR were purchased from kebo lab (Espoo, Filand). PCR was carried out using Dynazyme polymerase under various suggested amplification constants and manufacturer's suggested conditions according to the characteristics of PCR primers. Primers for amplifying cDNA fragments of VAP-1 from smooth muscle mRNA by RT-PCR were constructed with the VAP-1 n-terminal protein sequence AVITIFA (SEQ I1) NO: 4) (SEQ ID NOS: 13-19) N2; gctgtgatcacmatyttygc (SEQ II). And ccggccctgrtagaasac produced from the T4 Tryptic peptide sequence VFYQGR (SEQ; ID NO: 6) (base sequences 264 to 269). The amplification conditions of these primers were run for 30 minutes at 94 ° C for 1 minute, 55 ° C for 1 minute, and 72 ° C for 2 minutes. Total RNA was harvested from cell lines and tissues using Ultraspec (Biotecx, Houston, TX, USA) to the manufacturer's knowledge. Human multistage tissue Northern blots were obtained from Clontech Laboratories (Palo Alto, CA, USA). It was hybridized with probes labeled ³² P as suggested by the manufacturer. Human cDNA aggregates and lungs (catalog No. HL 3004a) and heart (catalog No. HL 3026a) cDNA aggregates were obtained from Clontech Laboratories.

Cell Culture and Expression of VAP-1 cDNAs in Mammalian Cells. COS-7 (monkey fibroblasts), CHO (Chinese hamster ovary cells), and CRL1998 (human immortalized umbilical vein endothelial cells) from ATCC (Rockville, MD) were used as hosts for transformation and VAP-1 expression. . COS-7, CRL 1998 cells were treated with 10% fetal calf plasma (PAA-Linz, Austria), 2 mM glutamine (Biological Industries, Israel), and RPMI 1640 supplemented with 128 U / ml penicillin and 128 μg / ml streptomycin. Gibco BRL). CHO cells were grown in α-MEM with the addition of CHO nucleotides. Expression plasmids composed of VAP-1 cDNAs subclone to the expression vector pcDNA3 (Invitrogen, San Diego, Calif.) Were used for transient COS-7 cell transformation and production of stably transformed CHO cell lines. Expression plasmids (20 μg) were transformed by Electrophoration with a Bio-Rad Gene Pulser instrument (1 mM Na-pyruvate, 2 mM L-glutamaine and 0.3kv, 960㎌, 0.4cm Cuvette in RPMI without plasma). Transiently transformed cells were then analyzed for 3 days. Stable transformed cells were selected by incubation for 4 weeks in the presence of 0.5 mg / ml Geneticin (Gibco BRL).

Example 2

VAP-1 Enzymatic Activity

VAP-1 is importantly the same for amine oxidant aggregates containing copper and has been used for the fact that VAP-1 has amine oxidase activity in secreted Bovine Serum Amine Oxidase (BSAO). Amine oxidants, including copper, were distinguished by the presence of unique quinone coenzymes, enzymes bound to copper, and activity corresponding to basic monomeric or multimeric amines. So they are distinguished from intracellular (mitochondrial) monomeric amine oxidases that contain FAD. McIntire, W.S., and Hartmann, C., in Principles and Applications of Quinoproteins, P. 97 (V.L. Davidson, ed., Deckker, New York (1993)).

To identify what activity was induced by VAP-1, we produced stable CHO cell transformants expressing VAP-1 protein. The sonicated eluate of these cells was analyzed for monomer amine oxidant (DAO) activity using putrescine as substrate or monomer amine oxidant (MAO) activity using benzylamine. As positive controls, commercially available DAO and MAO were analyzed and negative controls provided by the mock plasmid of transformed CHO cells. These results showed that VAP-1 expressing cells had weak activity against putrescine, but this important activity was detected using the substrate benzylamine of MAO (Table 1). However, significant activity with benzylamine, the substrate of monomeric amine oxidant (MAO), was measured in the positive control of Bovine plasma MAO (Table 1) commercially available with CHO cells expressing VAP-1. Mock CHO cell eluates showed insignificant enzymatic activity. In the presence of 100 μM semicarbazide and 10 μM hydroxylamine, VAP-1, a specific inhibitor of copper-containing monomeric amines (Lyles, Intl. J. Biochem, Cell Biol. 28: 254-274 (1996)) It did not have activity for 1).

In addition, we demonstrated that adhesion-competent VAP-1 expressed in Ax cells possessed MAO activity against benzylamine that could be inhibited by semicarbazide and hydroxylamine (Table 1). Low levels of Ax cells show corresponding natural MAO activity against benzylamine that is not inhibited by semecarbazide or hydroxylamine when detected in mock control cells (Table 1). In the laboratory, they appear themselves to take up MAO activity in VAP-1.

To confirm the discovery of MAO activity in VAP-1 in vitro , we purified VAP-1 from Tonsil furnace immunologically and analyzed the material three times. Tonsillar VAP-1, such as VAP-1 of transformed CHO cells, demonstrated an increase in activity for benzylamine under analytical conditions at 0.09 per hour at 37 ° C. at absorbance 490 nm, which is 4.5 times greater than that of boiled samples. To have. The tonsillar VAP-1 protein obtained was unable to measure specific activity because of its very low efficiency.

When generally used as a substrate for measuring amine oxidant activity, no benzyl amine has been found in the laboratory. Thus, many of the biologically endogenous amines that were generated were examined by VAP-1 (Table 2) to see if they could be used. At 1 mM methylamine was readily used by VAP-1, but the other amines tested did not react. The low specific activity observed with benzylamine in the lysate of these cells compared to the lysates analyzed in Table 1 shows a change in VAP-1 expression found in other cultures of the cells.

The presence of quinone cofactors in VAP-1 was confirmed by reducing the conditions and transferring the materials to nitrocellulose to separate portions of the VAP-1 material of the immunosaturated tonsils by SDS-PAGE. The nitrocellulose filter was then stained with nitroblue tetrazolium / Na glycine under cyclic redox conditions to specifically stain the quinone moiety in the protein (Paz, MA, et al., J. Biol. Chem. 266: 689- 92 (1991). The staining shows that monomer 90KD and dimer 180KD VAP-1, tonsil VAP-1 each have quinone cofactors in the subunits.

Materials and methods

Amine Oxidase Assay. Large amounts of CHO or Ax cells (10-15 × 10 ⁶ per flask) stably infected with the VAP-1 cDNA expressing plasmid were scraped from the flask into 10 ml of 100 mM phosphorylation buffer, pH 7.2 and the cell precipitate after centrifugation. Wash by adding 10 ml of buffer. The cell precipitate was finally remixed in 1.5 ml of phosphorylation buffer and the cells were digested with a 2 x 10 second sonicator on ice on medium power. Sonicated lysates were used directly in enzyme assays (50-100 μl per assay) or stored at −20 ° C.

Total protein concentration was determined by the technique described previously using the following substrates, which were determined by D'Agostino, L et al., Biochem. Pharmacol. Putrescine (Amersham) labeled by ¹⁴ C by the method of 38: 47-49 (1989) (described in reference), Baylin, SB, and Margolis, S., Biochem. Biophys. ³ H histamine (described in reference) by the method of Acta 397: 294-306 (1975), labeled 0.4 ¹⁴ C bezylalamine (Amersham), analyzed in a similar manner for putrescine, except that Ci is used for each reaction containing an unlabeled benylamine substrate.

0.5 ml of the reaction solution is 100 mM sodium phosphate buffer, pH7.2, 0.4 Ci ¹⁴ Cbenzylamine (Amersham), contains 80 nmol of unlabeled benzylamine. In addition, 100 μl of the lysate cell extract for each reaction is sampled to the maximum.

After incubation at 37 ° C. for one hour, 900 μl of toluene with 0.35 g / l diphenyloxazole is added and vigorously shaken to extract the aldehyde product of the reaction. After separating the phases, remove 700 μl of toluene layer, perform further extraction with 700 μl of toluene / diphenyl oxazole, remove 500 μl of this toluene / diphenyl oxazole layer and remove Collect and measure ¹⁴ C with a liquid scintillation counter. All enzymatic assays were performed in the presence of blanks containing sample solution boiled at 100 ° C. for 10 minutes, or samples not represented in the same batch of 2 or 3 batches, and the results were determined with pmol substrate / (minmg-protein). It is expressed in form. Controls of unrefined porcine kidney DAO and bovine plasma MAO were obtained from Sigma Chemical Company (St. Louis, MO, USA).

evaluation

Predominant staining with MAb 1B2 recognizing VAP-1 was obtained in endothelial tissues of blood vessels, particularly in PLN type lymphocytes. However, since the overall level of VAP-1 detected in these tissues is not relatively high, it has proved difficult to identify and purify sufficient amounts to obtain protein sequence information. Among the other tissues where VAP-1 was found, it was the most abundant in the smooth muscles of the vasculature and the intestinal related muscles (Salmi, M., et al., J. Exp. Med. 178: 2255). -2260 (1993). VAP-1 obtained from different materials has a slightly different molecular weight due to differences in glycolysis. However, they are similar to those analyzed in tonsils and PLN type tissues.

When protein sequence information obtained from VAP-1 purified from intestinal soft muscle was used, the cDNA encoding the adhesion molecule was isolated. This conclusion is based on several facts. First, protein sequences obtained from immunologically purified VAP-1 were found in the predicted protein sequences of serially isolated VAP-1 cDNA clones. Second, the transformed cells expressing VAP-1 cDNA were surfaced with MAb 1B2 originally used to identify VAP-1 (Salmi, M., and Talkanen, S., Science 257: 1407-1409 (1992)). VAP-1, which could be stained and immunoprecipitated from their cells, had a similar molecular weight of 170-180 KD in the laboratory.

VAP-1 is a type II membrane protein with a large, dimeric, membrane-located domain located at the N-terminal end of the molecule. The intracellular domain is uniquely small and has only 4 amino acids in length, leaving a large, glycosylated extracellular domain of 163 KD per dimer. All potential glycosylation sites with 12 N-linked and 6 potential O-linked sites per dimer are located in the extracellular domain. Although it is not currently known that all of these are used, previous data included that VAP-1 contains N- and O-linked sugars and a significant number of sialic acids that appear as terminal residues of carbohydrates linked to N or as part of O-linked sugars. It is shown. Especially when carbohydrates and sialic acids are thought to be important parts of the adhesion function of VAP-1 protein (Salmi, M., and Jalkanen, S., J. Exp. Med. 183: 569-579 (1996), Glycosylation moieties will be needed to determine which carbohydrates are used and which carbohydrates are present in. VAP-1 mRNA species of very different sizes are not seen in any tissues tested by Northern blotting, and all VAP-1 cDNA clones There is no conclusive evidence to show the VAP-1 form encoded by variant mRNAs when the analyzed PCR fragments contain similar sequences, so the inventors dominate the VAP-1 studied by previous immunoblotting and immunoprecipitation. It seemed to have replicated the form-encoding cDNA, but almost all of these tissues studied were smooth muscle obtained from vascular endothelial or other tissues as well as vascular tissues. When included, it is impossible to distinguish between smooth muscle and endothelial VAP-1 if very similar mRN forms exist in this tissue, so other forms of VAP-1 encoded by mRNA that differ slightly from those already isolated. It is thought to be.

Interestingly, the N-terminal region of VAP-1 with the permeable membrane region was part of the molecules that diversified most from the similar BSAO. BSAO is known as a secreted protein found at high levels in plasma and has a secreted signal sequence at the N-terminus that has been removed by the proteolytic process in the secretory pathway (Mu, D., et al. , J. Biol. Chem. 269: 9926-9932 (1994). Thus, BSAO and VAP-1 do not have similar physiological properties. Preliminary evidence suggested that murine VAP-1 has a permeable membrane domain similar to that found in human VAP-1, and thus has the same function as human molecules. The VAP-1 extracellular domain includes amine oxidase activity so that VAP-1 can function as an ecto-enzyme. Amine oxidases, including copper, are a variety of enzymes with specificities of a wide variety of different substrates. However, they can be categorized into two forms: those that have activity against polyamines, such as putrescine, histamine, and VAP-1 types of monomeric amine oxidant activity. Although there are some, the physiological substrate of the monomeric amine oxidant is not clear.

Other properties of these enzymes are discussed in the discussion, except for the presence of cofactors containing unique carbonyl groups and their identification in BSAO and pea amine like topaquinone (6-Hydroxydopa) and in lysyl oxidase like cross-linked lysine tyrosylquinone. The chemical identity that has been the subject. In some species of stunts, including humans, the monomeric amine oxidase containing copper found in both soluble forms of cell membranes and plasma has been found to be the same. This activity is sensitive and responsive to inhibition by carbonyl reaction mediators such as Semicabazide, hydroxylamine (Lyles, G, A., Intl .. J. Biochem. Cell Biol 28: 259-274 (1996)). It is usually referred to as semicarbazide-sensitive amine oxidant (SSAO). We obtained from studies of monoamine amine oxidizers in which VAP-1 contains other copper and contains covalently bound quinones that are shaped by self-processing during enzymatic reactions with copper. It is shown from the study of other copper containing monoamine oxidase. These results and the sensitivity of VAP-1 enzyme activity to carbonyl-related mediators Semicarbazide and Hydroxylamine show that VAP-1 is a membrane bound to SSAO. The physiological roles of SSAOs were difficult to define because few substrates were known in the laboratory and their substrate specificities varied greatly among species. Metabolism of the early endogenous and Xenobiotic monoamine amines showed a supportive function, which is a function of VAP-1 found in the endianpian position, like smooth muscle. It is not clear until now what role SSAO activity of VAP-1 plays in adhesive properties. Preliminary in vitro adhesion experiments using VAP-1 and PBL (external vascular lymphocytes) transformed with CRL 1998 cells in the presence of semicarbazide and hydroxylamin revealed that VAP-1 infected in the presence of inhibitors of enzymatic activity. Increasing the number of PBLs bound to cells suggests that they may have a negative effect on cohesive interactions between these cells. These results are the first to be caused by enzymatic activity that has a direct and specific binding to the adhesion mechanism used in the cell, or the physiological state of the cell is adversely affected by the product of the mono or oxidase activity, thus their adhesion potential. I don't know if this is the second to shrink.

Example 3

Adhesion analysis

In pcDNA3, VAP-1 cDNA was used to transform cells, and mouse HEV was extracted from endothelial cell lines, which might provide a more natural functional environment for VAP-1 than in other potential hosts such as CHO cells. It was. Stable transformants were obtained with those expressing VAP-1 on their cell surface as determined by FACS analysis (Fig. 6A) and used in their lymphocyte adhesion assays. When analyzed under rotatory conditions, PBLs bound to VAP-1 were 25.6 times higher than transformed into mock cells (Fig. 6B and 6C).

Although enhanced binding to VAP-1 transformants is not complete, the results show that the inhibition is clearly inhibited by anti-VAP-1 single antibody (mAb) treatment (lower than 29.6%). 10.7%, P = 0.05).

These adhesion results were pooled in five independent experiments, in which 2-3 parallel transgenic monolayers separated PBLs from three independently transformed cell lines and six different donors. It was analyzed several times using.

Therefore, these results show that VAP-1 cDNA is located on the surface of transformed cells and encodes a functional cohesive molecule that can directly regulate PBL binding when expressed in Ax cells.

Materials and methods

Ax-cells and mock control transformants stably expressing VAP-1 were plated on wax-pen circles drawn on gelatin pretreated microscope slides. (20 000 cell / 2 cm diameter circle)

 These cells are allowed to grow sufficiently and after two washings, 100 μl of RPMI 1640 medium containing 10% FCS and 10 mM HEPES (neonatal medium) is added to each wax-pen circle to evenly cover the adherent cell membrane.

PBL, on the other hand, was isolated from freshly collected blood using Ficollcentrifugation and Adjust the concentration to 10 ⁶ cell / ml. The slides are then transferred to a rotary shaker operating at 60 rpm at 7 degrees. 5 μl of PBL solution is added to each wax-pen circle. This analysis is maintained at constant rotation for 30 minutes. The slides carefully follow the top solution and soak once in cold RPMI to drop adhered cells. Adherent cells are fixed in zones by incubating the slides vertically overnight in cold PBS containing 1% glutaraldehyde.

The number of adhered cells is counted using the eyepiece grating (magnification x200). The size of the grid is 0.25 mm ² . The transformant counts on each slide nine predetermined areas of 0.25 mm ² at the center of the circle forming the rich monolayer. Two slides per sample solution (total area = 4.5 mm ² ) are counted in five independent experiments. In some experiments, adhesion assays were performed under conditions that showed the ability to block MAb against VAP-1 (a combination of 1B2 and TK8-14, both diluted in autopsy medium at 50 μg / ml), or class-matched. It may also be performed under conditions of negative mAbs (a combination of 3G6 and 7C7, all diluted to 50 μg / ml).

MAb is pre-incubated with the transformant monolayer at 7 degrees for 30 minutes before labeled lymphocytes are added. The number of adherent cells is performed by the five independent experiments described above.

evaluation

Adhesion assays performed on VAP-1 expressed in Ax cells indicate that cDNA can maintain functional interactions with its ligands on PBLs and encode functional VAP-1 that can lead to stable binding of PBLs to Ax cells. Can be. However, no complete inhibition of anti-VAP-1 mAbs 1B2 and TK8-14 and this increased adhesion was observed and that VAP-1 molecules do not necessarily function in rat Ax cells as it did in its original environment. Hints. Perhaps the carbohydrate modification, membrane environment, or VAP-1 modification of a protein in Ax cells is different from that in human HEVs where mAn no longer interferes with all its VAP-1 interactions with its ligand. It is also possible that a subpopulation of VAP-1 molecules may be present in the transformant in a form lacking one or more epitopes recognized by MAb 1B2 and TK8-14. After all, long-term overexpression of VAP-1 in stable transformants remains a possibility for altering the expression of other adhesion molecules on the Ax-cell surface. These putative adhesion molecules will not be inhibited by anti-VAP-1 MAb. In HEV binding assays, VAP-1 functions independently of lymphocyte L selectin and mediates the binding of CD8 + PBL more than CD4 + PBL. Analysis of immunomagnetically purified L-selection negative cells and CD8 + and CD4 + cells is unnecessary for sufficient binding of L-selection to Ax-transformers and that CD8 +, a PBL constituent, is more VAP than CD4 +. The Ax VAP-1 cDNA transformant reproduces this observation when showing that the −1 transformant is better attached to several folded constructs (data not shown).

Evaluation of Amine Oxidase in CHO and Ax Cells Expressing VAP-1 Cellular Enzyme Activity in Nm Product min- ¹ mg- ¹ Protein Substrate CHO VAP-1 CHO Simulated Diamine Monoamine Ax VAP-1 Ax Simulated Oxidase Oxidase Putrescine 0.87 ± 0.17 1.45 ± 0.13 4.87 ± 0.10 0.29 ± 0.00 ND ND Benzylamine 26.94 ± 0.70 0.13 ± 0.13 0.57 ± 0.10 8.83 ± 0.00 2.15 ± 0.04 0.80 ± 0.80benzylamine + SC 0.61 ± 0.17 1.32 ± 0.26 ND ND 0.35 ± 0.09 1.00 ± 0.00benzylami + HA 0.35 ± 0.17 1.32 ± 0.26 ND ND 0.31 ± 0.04 0.90 ± 0.10

Each assessment is made three times and the average specific activity ± SEM is shown. ND, not performed.

Substrate Specificity of VAP-1 Amine Oxidase Activity Cellular Enzyme Activity in Nm Product min- ¹ mg- ¹ Protein      Substrate CHO VAP-1 CHO Simulation      Benzylamine 4.57 ± 0.29 0.18 ± 0.62 Methylamine 4.28 ± 0.22 0.18 ± 0.44 Tyramine 0.04 ± 0.10 0.09 ± 0.35 Trytamine 0.07 ± 0.04 1.35 ± 0.62 β-phenylethylamine 0.10 ± 0.03 0.26 ± 0.00 Histamine 0.05 ± 0.03 0.20 ± 0.00

Run the experiment twice on different samples with similar results and plot the results of a representative experiment.

It's time. Each assessment is made three times and an average specificity ± SEM is provided.

While the present invention has been described in connection with specific embodiments, it may be further modified, and the present application generally does not depart from the spirit of the invention within the art to which the invention pertains within the spirit of the invention without departing from the scope of the appended claims It should also be understood to cover the application or the like.

All references, patent applications, and descriptions of patents cited herein are hereby incorporated by reference in their entirety.

SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: BioTie Therapies Ltd. (B) STREET: Tykistonkatu 6 (C) CITY: Turku (E) COUNTRY: Finland (F) POSTAL CODE (ZIP): FIN-20520 (ii) TITLE OF A: Vascular Adhesion Protein-1 Having Amine Oxidase Activity (iii) NUMBER OF SEQUENCES: 2 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patent In Release # 1.0, Version # 1.30 (EPO) (vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08 / 862,433 (B) FILING DATE: 23-MAY-1997 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2501 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GCCAACAGAG CCCCCGTCTT GCTGGCGTGA GAATACATTG CTCTCCTTTG GTTGAATCAG 60 CTGTCCCTCT TCGTGGGAAA ATGAACCAGA AGACAATCCT CGTGCTCCTC ATTCTGGCCG 120 TCATCACCAT CTTTGCCTTG GTTTGTGTCC TGCTGGTGGG CAGGGGTGGA GATGGGGGTG 180 AACCCAGCCA GCTTCCCCAT TGCCCCTCTG TATCTCCCAG TGCCCAGCCT TGGACACACC 240 CTGGCCAGAG CCAGCTGTTT GCAGACCTGA GCCGAGAGGA GCTGACGGCT GTGATGCGCT 300 TTCTGACCCA GCGGCTGGGG CCAGGGCTGG TGGATGCAGC CCAGGCCCGG CCCTCGGACA 360 ACTGTGTCTT CTCAGTGGAG TTGCAGCTGC CTCCCAAGGC TGCAGCCCTG GCTCACTTGG 420 ACAGGGGGAG CCCCCCACCT GCCCGGGAGG CACTGGCCAT CGTCTTCTTT GGCAGGCAAC 480 CCCAGCCCAA CGTGAGTGAG CTGGTGGTGG GGCCACTGCC TCACCCCTCC TACATGCGGG 540 ACGTGACTGT GGAGCGTCAT GGAGGCCCCC TGCCCTATCA CCGACGCCCC GTGCTGTTCC 600 AAGAGTACCT GGACATAGAC CAGATGATCT TCAACAGAGA GCTGCCCCAG GCTTCTGGGC 660 TTCTCCACCA CTGTTGCTTC TACAAGCACC GGGGACGGAA CCTGGTGACA ATGACCACGG 720 CTCCCCGTGG TCTGCAATCA GGGGACCGGG CCACCTGGTT TGGCCTCTAC TACAACATCT 780 CGGGCGCTGG GTTCTTCCTG CACCACGTGG GCTTGGAGCT GCTAGTGAAC CACAAGGCCC 840 TTGACCCTGC CCGCTGGACT ATCCAGAAGG TGTTCTATCA AGGCCGCTAC TACGACAGCC 900 TGGCCCAGCT GGAGGCCCAG TTTGAGGCCG GCCTGGTGAA TGTGGTGCTG ATCCCAGACA 960 ATGGCACAGG TGGGTCCTGG TCCCTGAAGT CCCCTGTGCC CCCGGGTCCA GCTCCCCCTC 1020 TACAGTTCTA TCCCCAAGGC CCCCGCTTCA GTGTCCAGGG AAGTCGAGTG GCCTCCTCAC 1080 TGTGGACTTT CTCCTTTGGC CTCGGAGCAT TCAGTGGCCC AAGGATCTTT GACGTTCGCT 1140 TCCAAGGAGA AAGACTAGTT TATGAGATAA GCCTCCAAGA GGCCTTGGCC ATCTATGGTG 1200 GAAATTCCCC AGCAGCAATG ACGACCCGCT ATGTGGATGG AGGCTTTGGC ATGGGCAAGT 1260 ACACCACGCC CCTGACCCGT GGGGTGGACT GCCCCTACTT GGCCACCTAC GTGGACTGGC 1320 ACTTCCTTTT GGAGTCCCAG GCCCCCAAGA CAATACGTGA TGCCTTTTGT GTGTTTGAAC 1380 AGAACCAGGG CCTCCCCCTG CGGCGACACC ACTCAGATCT CTACTCGCAC TACTTTGGGG 1440 GTCTTGCGGA AACGGTGCTG GTCGTCAGAT CTATGTCCAC CTTGCTCAAC TATGACTATG 1500 TGTGGGATAC GGTCTTCCAC CCCAGTGGGG CCATAGAAAT ACGATTCTAT GCCACGGGCT 1560 ACATCAGCTC GGCATTCCTC TTTGGTGCTA CTGGGAAGTA CGGGAACCAA GTGTCAGAGC 1620 ACACCCTGGG CACGGTCCAC ACCCACAGCG CCCACTTCAA GGTGGATCTG GATGTAGCAG 1680 GACTGGAGAA CTGGGTCTGG GCCGAGGATA TGGTCTTTGT CCCCATGGCT GTGCCCTGGA 1740 GCCCTGAGCA CCAGCTGCAG AGGCTGCAGG TGACCCGGAA GCTGCTGGAG ATGGAGGAGC 1800 AGGCCGCCTT CCTCGTGGGA AGCGCCACCC CTCGCTACCT GTACCTGGCC AGCAACCACA 1860 GCAACAAGTG GGGTCACCCC CGGGGCTACC GCATCCAGAT GCTCAGCTTT GCTGGAGAGC 1920 CGCTGCCCCA AAACAGCTCC ATGGCGAGAG GCTTCAGCTG GGAGAGGTAC CAGCTGGCTG 1980 TGACCCAGCG GAAGGAGGAG GAGCCCAGTA GCAGCAGCGT TTTCAATCAG AATGACCCTT 2040 GGGCCCCCAC TGTGGATTTC AGTGACTTCA TCAACAATGA GACCATTGCT GGAAAGGATT 2100 TGGTGGCCTG GGTGACAGCT GGTTTTCTGC ATATCCCACA TGCAGAGGAC ATTCCTAACA 2160 CAGTGACTGT GGGGAACGGC GTGGGCTTCT TCCTCCGACC CTATAACTTC TTTGACGAAG 2220 ACCCCTCCTT CTACTCTGCC GACTCCATCT ACTTCCGAGG GGACCAGGAT GCTGGGGCCT 2280 GCGAGGTCAA CCCCCTAGCT TGCCTGCCCC AGGCTGCTGC CTGTGCCCCC GACCTCCCTG 2340 CCTTCTCCCA CGGGGGCTTC TCTCACAACT AGGCGGTCCT GGGATGGGGC ATGTGGCCAA 2400 GGGCTCCAGG GCCAGGGTGT GAGGGATGGG GAGCAGCTGG GCACTGGGCC GGCAGCCTGG 2460 TTCCCTCTTT CCTGTGCCAG GACTCTCTTT CTTCCACTAC C 2501 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 763 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Asn Gln Lys Thr Ile Leu Val Leu Leu Ile Leu Ala Val Ile Thr 1 5 10 15 Ile Phe Ala Leu Val Cys Val Leu Leu Val Gly Arg Gly Gly Asp Gly 20 25 30 Gly Glu Pro Ser Gln Leu Pro His Cys Pro Ser Val Ser Pro Ser Ala 35 40 45 Gln Pro Trp Thr His Pro Gly Gln Ser Gln Leu Phe Ala Asp Leu Ser 50 55 60 Arg Glu Glu Leu Thr Ala Val Met Arg Phe Leu Thr Gln Arg Leu Gly 65 70 75 80 Pro Gly Leu Val Asp Ala Ala Gln Ala Arg Pro Ser Asp Asn Cys Val 85 90 95 Phe Ser Val Glu Leu Gln Leu Pro Pro Lys Ala Ala Ala Leu Ala His 100 105 110 Leu Asp Arg Gly Ser Pro Pro Pro Ala Arg Glu Ala Leu Ala Ile Val 115 120 125 Phe Phe Gly Arg Gln Pro Gln Pro Asn Val Ser Glu Leu Val Val Gly 130 135 140 Pro Leu Pro His Pro Ser Tyr Met Arg Asp Val Thr Val Glu Arg His 145 150 155 160 Gly Gly Pro Leu Pro Tyr His Arg Arg Pro Val Leu Phe Gln Glu Tyr 165 170 175 Leu Asp Ile Asp Gln Met Ile Phe Asn Arg Glu Leu Pro Gln Ala Ser 180 185 190 Gly Leu Leu His His Cys Cys Phe Tyr Lys His Arg Gly Arg Asn Leu 195 200 205 Val Thr Met Thr Thr Ala Pro Arg Gly Leu Gln Ser Gly Asp Arg Ala 210 215 220 Thr Trp Phe Gly Leu Tyr Tyr Asn Ile Ser Gly Ala Gly Phe Phe Leu 225 230 235 240 His His Val Gly Leu Glu Leu Leu Val Asn His Lys Ala Leu Asp Pro 245 250 255 Ala Arg Trp Thr Ile Gln Lys Val Phe Tyr Gln Gly Arg Tyr Tyr Asp 260 265 270 Ser Leu Ala Gln Leu Glu Ala Gln Phe Glu Ala Gly Leu Val Asn Val 275 280 285 Val Leu Ile Pro Asp Asn Gly Thr Gly Gly Ser Trp Ser Leu Lys Ser 290 295 300 Pro Val Pro Pro Gly Pro Ala Pro Pro Leu Gln Phe Tyr Pro Gln Gly 305 310 315 320 Pro Arg Phe Ser Val Gln Gly Ser Arg Val Ala Ser Ser Leu Trp Thr 325 330 335 Phe Ser Phe Gly Leu Gly Ala Phe Ser Gly Pro Arg Ile Phe Asp Val 340 345 350 Arg Phe Gln Gly Glu Arg Leu Val Tyr Glu Ile Ser Leu Gln Glu Ala 355 360 365 Leu Ala Ile Tyr Gly Gly Asn Ser Pro Ala Ala Met Thr Thr Arg Tyr 370 375 380 Val Asp Gly Gly Phe Gly Met Gly Lys Tyr Thr Thr Pro Leu Thr Arg 385 390 395 400 Gly Val Asp Cys Pro Tyr Leu Ala Thr Tyr Val Asp Trp His Phe Leu 405 410 415 Leu Glu Ser Gln Ala Pro Lys Thr Ile Arg Asp Ala Phe Cys Val Phe 420 425 430 Glu Gln Asn Gln Gly Leu Pro Leu Arg Arg His His Ser Asp Leu Tyr 435 440 445 Ser His Tyr Phe Gly Gly Leu Ala Glu Thr Val Leu Val Val Arg Ser 450 455 460 Met Ser Thr Leu Leu Asn Tyr Asp Tyr Val Trp Asp Thr Val Phe His 465 470 475 480 Pro Ser Gly Ala Ile Glu Ile Arg Phe Tyr Ala Thr Gly Tyr Ile Ser 485 490 495 Ser Ala Phe Leu Phe Gly Ala Thr Gly Lys Tyr Gly Asn Gln Val Ser 500 505 510 Glu His Thr Leu Gly Thr Val His Thr His Ser Ala His Phe Lys Val 515 520 525 Asp Leu Asp Val Ala Gly Leu Glu Asn Trp Val Trp Ala Glu Asp Met 530 535 540 Val Phe Val Pro Met Ala Val Pro Trp Ser Pro Glu His Gln Leu Gln 545 550 555 560 Arg Leu Gln Val Thr Arg Lys Leu Leu Glu Met Glu Glu Gln Ala Ala 565 570 575 Phe Leu Val Gly Ser Ala Thr Pro Arg Tyr Leu Tyr Leu Ala Ser Asn 580 585 590 His Ser Asn Lys Trp Gly His Pro Arg Gly Tyr Arg Ile Gln Met Leu 595 600 605 Ser Phe Ala Gly Glu Pro Leu Pro Gln Asn Ser Ser Met Ala Arg Gly 610 615 620 Phe Ser Trp Glu Arg Tyr Gln Leu Ala Val Thr Gln Arg Lys Glu Glu 625 630 635 640 Glu Pro Ser Ser Ser Ser Val Phe Asn Gln Asn Asp Pro Trp Ala Pro 645 650 655 Thr Val Asp Phe Ser Asp Phe Ile Asn Asn Glu Thr Ile Ala Gly Lys 660 665 670 Asp Leu Val Ala Trp Val Thr Ala Gly Phe Leu His Ile Pro His Ala 675 680 685 Glu Asp Ile Pro Asn Thr Val Thr Val Gly Asn Gly Val Gly Phe Phe 690 695 700 Leu Arg Pro Tyr Asn Phe Phe Asp Glu Asp Pro Ser Phe Tyr Ser Ala 705 710 715 720 Asp Ser Ile Tyr Phe Arg Gly Asp Gln Asp Ala Gly Ala Cys Glu Val 725 730 735 Asn Pro Leu Ala Cys Leu Pro Gln Ala Ala Ala Cys Ala Pro Asp Leu 740 745 750 Pro Ala Phe Ser His Gly Gly Phe Ser His Asn 755 760

Claims (25)

delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete Semicarbazide, hydroxylamine, propargylamine, isoniazide, nialamide, hydralazine, procarbazine, monomethylhydrazine, 3,5-ethoxy-4-aminomethylpyridine and A method of inhibiting amine oxidase activity, comprising providing an effective amount of inhibitor selected from the group consisting of MDL72145 ((E) -2 (3,4-dimethoxyphenyl) -3-fluoroallylamine). delete A method of modulating vascular adhesion protein-1 (VAP-1) mediated binding of endothelial cells to lymphocytes, comprising inhibiting the enzymatic activity of amine oxidase in endothelial cells. A method of regulating vascular adhesion protein-1 (VAP-1) mediated binding of endothelial cells to lymphocytes, comprising imparting enzymatic activity of amine oxidase in endothelial cells.