EP0979271A1 - Vascular adhesion protein-1 having amine oxidase activity - Google Patents

Abstract

VAP-1 is an endothelial sialoglycoprotein whose cell surface expression is induced under inflammatory conditions. It has previously been shown to mediate the binding of recirculating lymphocytes to human peripheral lymph node vascular endothelial cells in an L-selectin independent fashion. A VAP-1 cDNA has been purified and shown to encode a type II transmembrane protein of 84.6 KD with a single transmembrane domain located at the very N-terminal end of the molecule. VAP-1 exists, in vivo, predominantly as a dimer of 170-180 KD. Six potential N-linked glycosylation sites are located in the extracellular domain, as are three putative O-glycosylation sites. VAP-1 has no significant similarity to any currently known adhesion molecules but has significant identity to the copper-containing amine oxidase family. Enzyme assays have defined VAP-1 as a membrane-bound amine oxidase. Thus, VAP-1 is a new type of adhesion molecule with dual functions. With the appropriate glycosylation, and in the correct inflammatory setting, VAP-1 expression on the lumenal endothelial cell surface in locations mediating lymphocyte adhesion allows it to function as an adhesion receptor involved in a novel mechanism of lymphocyte homing. Its primary function in other locations may depend on its inherent amine oxidase activity.

Description

Vascular Adhesion Protein- 1 Having Amine Oxidase Activity

Field ofthe Invention

The present invention is directed to nucleic acid encoding a novel human endothelial cell adhesion protein, designated VAP-1, having an adhesive function and an amine oxidase activity. The invention is also directed to uses of VAP-1 nucleic acid and polypeptide.

Background ofthe Invention

The continuous recirculation of lymphocytes between blood and tissue is critical for the functioning of the immune system. The process permits lymphocytes to patrol all locations in the body in search of antigen whilst allowing them to develop and formulate their response to antigenic threat in specialized microenvironmental tissue compartments. Thereafter lymphocytes can specifically return (home) to the site at which they encountered antigen, thus enhancing the selectivity of the immune response. The adhesive interactions between multiple receptors on the circulating lymphocytes and their ligands expressed on the surface of endothelial cells in post-capillary venules provide both the means for the emigration process and a way to selectively control it. Although considerable progress has recently been made in describing the cascade of events required for a circulating leukocyte to pass into the tissue from freely flowing blood, much in this process remains to be discovered. In particular, the functions of the currently known adhesion molecules are not sufficient to define all the individual recirculatory pathways and binding specificities that have so far been defined in normal and inflammatory settings.

Present hypotheses suggest a multistep model of leukocyte adhesion to endothelium that relies on a cascade of sequential but overlapping molecular interactions between several receptor-ligand pairs (Butcher, E. G., and Picker, L. J., Science 272:60-66 (1996); Springer, T.A., Cell 76:301-314 (1994)). The initial transient and tethering interactions between a leukocyte in the blood stream and the vessel wall are performed principally by the selectins and their glycoprotein ligands. Integrins and other molecules may also have a role in this phase. This rolling and sampling step can, in the presence of the appropriate signals, be followed by firm adhesion mediated by the binding of activated integrins to their Ig superfamily ligands. Locally elevated levels of immobilized cytokines and other chemoattractants might be involved in initiating this local activation which is mediated by the appropriate receptors and subsequent transduction of signals within the cell. The final stage involves the transmigration of the bound cell through the endothelial lining into the tissue by mechanisms which are poorly understood. Tissue specific recirculatory pathways rely on the regulated expression of particular adhesion molecule receptor-ligand combinations in the appropriate location.

We have previously described a monoclonal antibody (MAb), 1B2, which recognizes a novel human endothelial cell adhesion molecule and which can block lymphocyte binding to tonsillar high endothelial venules (HEV) in a frozen section assay (Salmi, M., and Jalkanen, S., Science 257:1407-1409 (1992); U.S. Patent Nos. 5,512,442 and 5,580,780, and U.S. Appl. No. 08/447,799, all incorporated herein by reference). MAb 1B2 is specific for, and thus defines, vascular adhesion protein- 1 (VAP-1). In inflammatory situations, surface expression of VAP-1 is induced and the molecule is found on the lumenal surface of vascular endothelial cells (Salmi, M., et al., J. Exp. Med. 775:2255-2260 (1993)). Two species of VAP-1 , of differing molecular mass, can be detected by MAb 1B2 immunoprecipitation of tonsil tissue, one of 90 KD and the other of 170-180 KD. However, after immunoblotting under non-reducing conditions, the 170-180 KD species is most prominent (Salmi, M., and Jalkanen, S., J. Exp. Med. 183:569-519 (1996)). Immunoreactive VAP-1, with slightly differing molecular masses, can also be found in other locations, particularly in the smooth muscle cells of the vasculature as well as in other smooth muscle-containing tissues, although its function in these cells remains to be determined. McNab et al. have reported that VAP-1 is expressed on hepatic sinusoidal endothelium and can mediate the binding of T lymphocytes (McNab, G., et al., Gastroenterology 770:522-528 (1996)). Studies of tonsillar VAP-1 using digestion with specific glycosidases have shown that VAP-1 is a sialoglycoprotein, probably containing both N- and O-linked sugars with abundant sialic acid residues of both the α2,3 and α.2,6 linked type. It has also been shown that VAP- 1 mediates lymphocyte binding to HEV in lymphatic tissues under conditions of shear and in a sialic acid dependent manner as the desialylated molecule can no longer support lymphocyte binding in the frozen section assay (Salmi, M., and Jalkanen, S. J. Exp. Med. 183:569-519 (1996)). The molecule is upregulated under certain inflammatory conditions caused by disease in the skin, gut, tonsil and synovium, although the mediators inducing this phenomenon have yet to be identified (Arvilommi, A.-M., et al., Eur. J. Immunol. 26:825-833 (1996); Salmi, M., et al, J. Exp. Med. 178:2255-2260 (1993)). VAP-1 is distinct from the PLN (peripheral lymph node) addressin (PNAd) defined by the MAb MECA-79, though both VAP-1 and PNAd can mediate lymphocyte binding to PLN under shear conditions. However, in contrast to PNAd, VAP-1 can operate in an L-selectin independent manner and support the binding of L-selectin negative lymphocytes (Salmi, M., and Jalkanen, S., J. Exp. Med. 183:569-519 (1996)). VAP-1 is therefore a molecule with an important adhesive function in a new pathway which operates independently of the known selectins and is likely to mediate early interactions in lymphocyte binding to PLN type HEV.

Summary ofthe Invention

The invention is directed to a purified nucleic acid molecule encoding a vascular adhesion protein- 1 (VAP-1), selected from the group consisting of:

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

(b) a purified nucleic acid molecule comprising the VAP-1 coding sequence of the VAP-1 nucleotide sequence in Figure 1 (SEQ ID NO:l); (c) a purified nucleic acid molecule encoding a VAP-1 polypeptide comprising the amino acid sequence encoded by the VAP-1 cDNA clone contained in Deposit Accession No. DSM 11536;

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

11536;

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

(f) a purified nucleic acid molecule comprising a nucleotide sequence that differs from the coding sequence of the nucleic acid molecule of (b) or (d) due to the degeneracy of the genetic code; and

(g) a purified nucleic acid molecule comprising a nucleotide sequence that hybridizes to a molecule of (e), and encodes a VAP-1 that has an amino acid sequence that shows at least 80% identity to the VAP- 1 sequence in Figure 1 (SEQ ID NO:2).

The invention is further directed to a method for making a recombinant vector comprising inserting a molecule comprising the sequence of the VAP-1 nucleic acid molecule into a DNA sequence that can act as a vector. The invention is further directed to a recombinant vector containing such VAP-1 DNA. The invention is directed to a method of providing a VAP-1 to a host cell, comprising introducing DNA encoding VAP-1 into a host cell. The invention is directed to a recombinant host cell containing such introduced DNA. The invention is also directed to a method for producing a vascular adhesion protein- 1 (VAP-1) polypeptide, comprising culturing a recombinant host cell containing the VAP-1 encoding DNA of the invention under conditions such that the encoded VAP-1 polypeptide is expressed.

The invention is further directed to a method of providing an amine oxidase activity to a host cell by transforming a nucleic acid encoding VAP-1 into the host cell. The invention is further directed to a method of altering the expression of vascular adhesion protein- 1 (VAP-1), comprising: (a) introducing into a host cell, a DNA construct comprising: (i) a VAP-1 targeting sequence; and (ii) a regulatory sequence linked to the VAP-1 targeting sequence; and (b) maintaining the host cell under conditions appropriate for homologous recombination between the DNA construct and the endogenous VAP-1 sequence. The invention is also directed to such host cell, wherein the expression of VAP-1 has been altered by the above method.

The invention is also directed to a recombinant host cell, containing: (a) a VAP-1 targeting sequence; and (b) a regulatory sequence linked to the VAP-1 targeting sequence. The invention is directed to a method of oxidizing an amine, comprising reacting the amine with the VAP-1 polypeptide of the invention. The amine can be, for example, benzylamine or methylamine. The VAP-1 polypeptide has an amino acid sequence at least 95% identical to a sequence selected from the group consisting of: (a') a purified polypeptide comprising the VAP- 1 amino acid sequence in Figure 1 (SEQ ID NO:2);

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

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

(d') a purified polypeptide comprising a VAP-1 amino acid sequence that is encoded by a nucleotide sequence that hybridizes to the complement of

DNA encoding the polypeptide of any one of (a') to (c'), and encodes a VAP-1 and has an amino acid sequence which shows at least 80% identity to a sequence in Figure 1 (SEQ ID NO:2); and

(e') a purified polypeptide comprising the amino acid sequence of an epitope-bearing portion of the polypeptide of (a') or (b').

The invention is also directed to a method of inhibiting amine oxidase activity, comprising providing an effective amount of an inhibitor to a sample possessing the amine oxidase activity. The inhibitor can be, for example, semicarbazide, hydroxylamine, propargylamine, isoniazid, nialamide, hydrallazine, procarbazine, monomethylhydrazine, 3,5-ethoxy-4-aminomethyl- pyridine, or MDL72145 ((E)-2-(3,4-dimethoxyphenyl)-3-fluoroallylamine).

The invention is further directed to a method of manipulating vascular adhesion protein- 1 (VAP-l)-mediated binding of endothelial cells to lymphocytes, comprising altering the enzymatic activity of amine oxidase in the endothelial cells, by inhibition or potentiation.

Brief Description ofthe Figures

Fig. 1. Sequence of the VAP-1 cDNA isolated from a human lung cDNA library and the predicted sequence of VAP-1 protein. The N-terminal, tryptic and V8 peptides that were purified and sequenced from immunopurified VAP-1 protein are boxed. An italicized amino acid residue within a boxed region indicates that no amino acid could be assigned to that cycle in the peptide sequencing. Potential N-glycosylation sites are indicated by arrowed and circled asparagines and the putative O-glycosylation sites by an arrowed square. The transmembrane domain between residues 5 and 27 is indicated by shading. The scale diagram at the foot of the figure indicates the location of the transmembrane domain (shown by the filled TMD region) and the relative location of the putative glycosylation sites in the extracellular portion of the molecule is indicated by N and O to indicate N- and O-linked sugar, respectively. The scale bar represents 50 amino acids.

Fig. 2. FACS analysis of VAP-1 transfected COS-7 cells to determine the membrane orientation and cellular location of VAP- 1. A diagram indicating the structure of the two VAP-1 expression plasmids used to transfect the COS-7 cells is shown at the top of the Figure. VAP-1 is the native VAP-1 expression construct. VAP-1 FLAG is the FLAG epitope tagged VAP-1. The negative control for mock transfections was provided by a construct in which VAP-1 is in an inverse orientation in the expression vector. Column 1 : the expression construct used in the transfection. Column 2: no permeabilization (-) or permeabilization (+) of the transfected cells. Column 3: negative control MAb staining. Column 4: anti-VAP-1 MAb 1B2 staining. Column 5: anti-FLAG MAb M2 staining. Rows A-F indicate the expression construct or mock control used in transfection and the resulting staining pattern of the transfected cells. Arrows indicate the positively staining cell population. On non-permeabilized cells staining is seen with the anti-VAP-1 MAb 1B2 (FACS panel row B and C, column 4). Mock control transfected cells were negative (FACS panel row A, column 4). No staining is seen with the anti-FLAG MAb in VAP-1 FLAG transfected cells (row C, column 5). However, in permeabilized cells, VAP-1 transfected cells were still positive (row E, column 4) but now VAP-1 FLAG transfected cells also stained positively with the anti-FLAG MAb (row F, column 5), indicating that permeabilization of the cells to the anti-FLAG MAb has permitted its access to the FLAG epitope and therefore that this epitope lies within the cell. Fig. 3. Multiple alignment of the mammalian members of the copper amine oxidase family for which sequence data are available, including VAP-1. Labels on the left refer to the particular protein aligned in each row: 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. Numbers correspond to the first amino acid in each row of the aligned proteins. Sequences were extracted from the most recent available database and aligned using GCG Pileup. Residues having identity with VAP-1 were highlighted using GCG Boxshade.

Fig. 4. Sialidase treatment of VAP-1 expressed in COS-7 cells. Cell lysates from VAP-1 transfected and mock transfected COS-7 cells were treated with sialidase (+) or not (-) before SDS-PAGE, immunoblotting and probing with the VAP-1 MAb 1B2 (lanes 1-4) or negative control MAb 3G6 (lanes 5-8).

Fig. 5. A. Northern blot analysis of VAP-1 mRNA in poly A⁺ RNA extracted from human gut smooth muscle and tonsil stroma from which lymphocytes have been partially removed by washing and squeezing. A 4.2 kb hybridizing mRNA can be seen in both tracks. B. Northern blot analysis of VAP-1 mRNA in different human tissues. Northern blots were obtained from Clontech Laboratories and equal amounts of mRNA were loaded in each lane. All the filters were probed with a ³²P-labeled VAP-1 cDNA probe containing the entire coding sequence and washed at high stringency (post-hybridization washing conditions were 0.1 x SSC, 0.1 % SDS at 65 °C for 2 x 45 min).

Fig. 6. Ax cells transfected with the VAP-1 cDNA mediate lymphocyte adhesion. A. Expression of VAP-1 on the cell surface of Ax cells stably transfected with VAP-1 cDNA or mock control. In the histograms the intensity of staining on a log scale is shown on the x-axis and the relative number of cells is shown on the y-axis. B. Increased VAP-1 dependent binding of lymphocytes to VAP-1 transfectants. Considerably more PBL (small round spheres on top of the monolayer, examples of which are arrowed) are bound to the VAP-1 transfectants (left) than to mock- transfectants (right). Phase-contrast micrographs, magnification x 100. C. Quantitation of the binding. Results of five independent experiments are presented as mean ± SEM.

Detailed Description ofthe Invention

Interactions between leukocyte surface receptors and their ligands on vascular endothelial cells critically control lymphocyte traffic between the blood and various lymphoid organs, as well as extravasation of leukocytes into sites of inflammation. A cDNA clone encoding a vascular adhesion protein- 1 (VAP-1) which mediates the leukocyte-endothelial cell adhesion reaction is described. The encoded VAP-1 surprisingly also has an amine oxidase activity. In higher organisms, amine oxidase is thought to be involved in the metabolism of biogenic amines (Mclntire, W. and C. Hartmann, in Principles and Applications of Quinoproteins, V.L. Davidson, ed., Marcel Dekker, Inc., N.Y., Chap. 6 (1992)). VAP-1 cDNA

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 shown in Figure 1 (SEQ ID NO:2), which was determined by sequencing a cDNA clone giving the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1). A clone containing the nucleotide sequence was deposited under the terms of the Budapest Treaty with the International Depository Authority of DSMZ-Deutsche Sammlung Von Mikroorganismen Und Zellkulturen GmbH at the address of Mascheroder Weg lb, D-38124 Braunschweig, Germany, on May 7, 1997, and given Deposit Accession No. DSM 11536. The deposited clone is contained in the pUC19 plasmid.

Using a monoclonal antibody (MAb) immunoaffinity column, the 90 and 170- 180 KD monomeric and dimeric forms, respectively, of VAP- 1 were purified from detergent lysates of smooth muscle in sufficient quantities to obtain the internal peptide sequence after digestion with trypsin and V8 protease. A portion of the 90 KD VAP-1 was subjected to N-terminal sequencing and used to design partially degenerate oligonucleotide primers for RT-PCR experiments on mRNA prepared from human gut smooth muscle.

A single cDNA fragment of approximately 700 bp was amplified using these primers and cloned into pUC18 in order to determine its sequence. To find longer cDNA clones, a panel of 10 human cDNA libraries was analyzed by PCR in order to identify those containing VAP-1 cDNAs and the libraries giving the strongest signal were screened with the PCR-generated VAP- 1 cDNA fragment. In this manner a number of overlapping cDNA clones were isolated from human lung and heart cDNA libraries. A single cDNA of 2501 bp as described in Figure 1 (SEQ ID NO:l) containing a continuous open reading frame of 2292 bp starting at an ATG methionine codon was derived from these clones and subcloned into pUC19. The determined nucleotide sequence of the VAP-1 cDNA contained in this clone in Figure 1 (SEQ ID NO: 1) contains an open reading frame encoding a protein of 763 amino acid residues of Figure 1 (SEQ ID NO:2), with an initiation codon at positions 80-82 of the nucleotide sequence in Figure 1 (SEQ

ID NO: 1) and a molecular weight of about 84.6 KD.

The VAP-1 of the present invention has significant identity to a family of enzymes termed the copper-containing amine oxidases (EC 1.4.3.6). The VAP-1 protein shown in Figure 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 family. The highest identity found was with bovine serum amine oxidase

(81 %). Unless otherwise indicated, each "nucleotide sequence" set forth herein is presented as a sequence of deoxyribonucleotides (abbreviated A, G , C and T).

However, by "nucleotide sequence" of a nucleic acid molecule or polynucleotide is intended, for a DNA molecule or polynucleotide, a sequence of deoxyribonucleotides. For an RNA molecule or polynucleotide, the corresponding sequence is that of ribonucleotides (A, G, C and U), where each thymidine deoxyribonucleotide (T) in the specified deoxyribonucleotide sequence is replaced by the ribonucleotide uridine (U).

The nucleic acid molecules of the present invention may be in the form of RNA. such as mRNA, or in the form of DNA, including cDNA and genomic DNA obtained by cloning or produced synthetically. The DNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand or complement.

By "purified" nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment or produced synthetically. Purified RNA molecules include in vitro RNA transcripts of the DNA molecules of the present invention.

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

The present invention is further directed to fragments of the purified nucleic acid molecules described herein. By a fragment of a purified nucleic acid molecule having the VAP-1 nucleotide sequence of the deposited cDNA or the VAP-1 nucleotide sequence shown in Figure 1 (SEQ ID NO: l) is intended fragments at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 50-1500 nt in length are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in Figure 1 (SEQ ID NO: l). Since the VAP-1 gene has been deposited and the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1) is provided, generating such DNA fragments would be routine to the skilled artisan. For example, restriction endonuclease cleavage, shearing by sonication, or oligonucleotide synthesis could easily be used to generate fragments of various sizes.

In another aspect, the invention provides a purified nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, the VAP- 1 cDNA clone contained in Deposit Accession No. DSM 1 1536. By "stringent hybridization conditions" is intended overnight incubation at 42° C in a solution comprising: 50% formamide, 5x SSC (150 mM NaCl, 15mM trisodium citrate), 50 raM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 x SSC at about 65 °C. By a polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 nt of the reference polynucleotide. These are useful as diagnostic probes and primers. Of course, polynucleotides hybridizing to a larger portion of the reference polynucleotide (e.g., the deposited cDNA clone), for instance, a portion 50-750 nt in length, or even to the entire length of the reference polynucleotide, are also useful as probes according to the present invention, as are polynucleotides corresponding to most, if not all, of the VAP-1 nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in Figure 1 (SEQ ID NO: l ). Such portions are useful diagnostically either as a probe according to conventional DNA hybridization techniques or as primers for amplification of a target sequence by the polymerase chain reaction (PCR), as described, for instance, in Molecular Cloning, A Laboratory Manual, 2nd. edition, Sambrook, J., Fritsch, E.F. and Maniatis, T., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), incorporated herein by reference.

Purified nucleic acid molecules of the present invention include DNA molecules comprising an open reading frame (ORF) with an initiation codon at positions 80-82 of the VAP- 1 nucleotide sequence shown in Figure 1 (SEQ ID NO:l) and DNA molecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the VAP-1 protein. Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate the degenerate variants.

The invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of the VAP-1 protein. Variants may occur naturally, such as a natural allelic or splice variant. By an "allelic variant" is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants may be produced using art-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions, deletions or additions. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the VAP-1 protein or portions thereof. Also especially preferred in this regard are conservative substitutions.

Further embodiments of the invention include purified nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical to (a) a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence in Figure 1 (SEQ ID NO:2); (b) a nucleic acid molecule comprising the VAP-1 coding sequence of the VAP-1 nucleotide sequence in Figure 1 (SEQ ID NO: 1); (c) a nucleic acid molecule encoding a VAP-1 polypeptide comprising the amino acid sequence encoded by the VAP-1 cDNA clone contained in Deposit Accession No. DSM 11536; (d) a nucleic acid molecule comprising the coding sequence of the VAP-1 nucleotide sequence contained in Deposit Accession No. DSM 11536; (e) a nucleic acid molecule comprising a nucleotide sequence complementary to the VAP-1 nucleotide sequences in (a), (b), (c) or (d); (f) a nucleic acid molecule comprising a nucleotide sequence that differs from the coding sequence of the nucleic acid molecule of (b) or (d) due to the degeneracy of the genetic code; and (g) a nucleic acid molecule comprising a nucleotide sequence that hybridizes to a molecule of (e), and encodes a VAP-1 that has an amino acid sequence that shows at least 90% identity to the VAP-1 sequence in Figure 1 (SEQ ID NO:2). This is irrespective of whether they encode a polypeptide having VAP-1 activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having VAP-1 activity, one of skill in the art would still know how to use the nucleic acid molecule, for example, as a hybridization probe or a polymerase chain reaction (PCR) primer. Preferred, however, are nucleic acid molecules having sequences at least 90%, 95%, 96%, 97%, 98% or 99% identical to the VAP-1 nucleic acid sequence shown in Figure 1 (SEQ ID NO:l) or to the VAP-1 nucleic acid sequence of the deposited cDNA which do, in fact, encode a polypeptide having VAP-1 protein activity.

By a polynucleotide having a nucleotide sequence at least, for example, 90% "identical" to a reference nucleotide sequence encoding a VAP-1 polypeptide is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to ten point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the VAP-1 polypeptide. Whether any particular nucleic acid molecule is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the VAP-1 nucleotide sequence shown in Figure 1 (SEQ ID NO:l) or to the nucleotides sequence of the deposited cDNA clone can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 5371 1).

Vectors and Host Cells

The present invention also relates to vectors which include the purified DNA molecules of the present invention, host cells which are genetically engineered with the DNA encoding VAP-1, and the production of VAP-1 polypeptides or fragments thereof by recombinant techniques.

The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells. Selectable markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria.

Preferred are vectors comprising cis-acting control regions to the polynucleotide of interest. Appropriate trans-acting factors may be supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host. In certain preferred embodiments in this regard, the vectors provide for specific expression, which may be inducible and/or cell type-specific. Particularly preferred among such vectors are those inducible by environmental factors that are easy to manipulate, such as temperature and nutrient additives.

Expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors, e.g., vectors derived from bacterial plasmids, bacteriophage, yeast episomes, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as cosmids and phagemids.

The DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda Pi promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the coding sequence.

Such recombinant DNA technology also includes the recombination methods described in Treco et al, WO 94/12650, and Treco et al, WO 95/31560, both incorporated herein by reference, which can be applied to alter VAP-1 expression and amine oxidase activity in cells. Specifically, regulatory regions (for example, promoters) can be introduced into the host genome to alter the expression of the endogenous VAP-1. Thus, the invention relates to a method of making a recombinant host cell wherein the expression of vascular adhesion protein- 1 (VAP-1) is altered, comprising: (a) introducing into a host cell, a DNA construct, comprising: (i) a VAP-1 targeting sequence; and (ii) a regulatory sequence linked to the VAP-1 targeting sequence; and (b) maintaining the host cell under conditions appropriate for homologous recombination between the DNA construct and the endogenous VAP-1 sequence. The invention also relates to a recombinant host cell, wherein the expression of VAP-1 has been altered by the above method.

The invention also relates to a method of altering expression of the VAP-1 gene by, for example, activating expression of VAP-1 in a cell that does not normally express it, or does not normally express it under a desired environment or hormonal condition. Altering the expression of VAP-1 may also include using the sequences of the invention to inactivate expression of the native gene. Homologous recombination or targeting is used to replace or disable the regulatory region normally associated with the endogenous VAP-1 gene, with a regulatory sequence which causes the gene to be expressed at levels higher than evident in the corresponding nontransfected cell, or causes the gene to display a pattern of regulation or induction that is different than evident in the corresponding nontransfected cell. The invention, therefore, relates to a method of making proteins by activating an endogenous gene which encodes the desired product in cells.

If the DNA construct is to be targeted, it must include at least a targeting sequence and preferably also a regulatory sequence. As described in Treco et al, WO 94/12650, and Treco et al, WO 95/31560, there frequently are additional construct components, such as selectable markers, amplifiable markers, or an exon and unpaired splice-donor site. The DNA in such construct may be referred to as exogenous DNA, which is introduced into a host cell by the method of the present invention. Exogenous DNA can possess sequences identical to or different from the endogenous DNA present in the cell prior to transfection. The targeting sequence or sequences of the DNA construct are DNA sequences which permit legitimate homologous recombination into the genome of the selected cell containing the VAP-1 gene at the VAP-1 locus. Targeting sequences are, for example, DNA sequences which are homologous to VAP- 1 DNA sequences normally present in the genome of the cells as obtained, and located, generally, at both ends of the regulatory sequence. The targeting sequence or sequences used are selected with reference to the VAP-1 site into which the DNA construct is to be inserted.

The regulatory sequence of the DNA construct can be comprised of one or more promoters, enhancers, scaffold-attachment regions or matrix attachment sites, negative regulatory elements, transcription factor binding sites, or combinations thereof.

The site of introduction of the DNA sequence will generally be within or upstream of the endogenous VAP-1 gene or at a site that affects the VAP-1 gene function. The DNA sequences which alter the expression of the endogenous VAP-1 gene can be introduced into the host cell as a single DNA construct, or as separate DNA sequences which become physically linked in the genome of a transfected cell. Further, the DNA can be introduced as linear, double stranded DNA, with or without single stranded regions 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 cell is maintained under conditions appropriate for homologous recombination to occur between the genomic DNA and a portion of the introduced DNA. Homologous recombination between the genomic DNA and the introduced DNA results in a homologously recombinant host cell in which sequences which alter the expression of endogenous VAP-1 gene is operatively linked to a VAP-1 gene. The resulting homologous recombinant host cells produced by this method can be cultured under conditions suitable for the expression of the VAP-1 protein, thereby producing the VAP-1 protein in vitro, or the cells can be used for in vivo delivery of a the VAP-1 protein (i.e., gene therapy).

The targeting event can be a simple insertion of a regulatory sequence, placing the endogenous VAP-1 gene under the control of the new regulatory sequence (e.g., by insertion of either a promoter or an enhancer, or both, upstream of the endogenous gene). The targeting event can be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element. The targeting event can replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally-occurring elements, or displays a pattern of regulation or induction that is different from the corresponding nontransfected cell.

Gene targeting can be used to replace the existing regulatory region of VAP-1 with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods. According to this method, introduction of the exogenous DNA results in disablement of the endogenous sequences which control expression of the endogenous VAP-1 gene, either by replacing all or a portion of the endogenous (genomic) sequence or otherwise disrupting the function of the endogenous sequence. DNA constructs, which include exogenous DNA and, optionally, DNA encoding a selectable marker, along with additional sequences necessary for expression of the exogenous DNA in recipient host cells, are used to transfect cells in which the VAP-1 production is to be altered. Further details of the homologous recombination methods to alter endogenous gene expression is provided in Treco et al, WO 94/12650, and Treco et al, WO 95/31560, both incorporated herein by reference.

The DNA or recombinant constructs may be introduced into cells by a variety of methods, including transformation, transfection, electroporation, microinjection, transduction, calcium phosphate precipitation, and liposome-, polybrene-, or DEAE dextran-mediated transfection. Such methods are described in many standard laboratory manuals, such as Davis et al, Basic Methods In Molecular Biology (1986). Alternatively, infectious vectors, such as retroviral, herpes, adeno-virus, adenovirus-associated, mumps and poliovirus vectors, can be used to introduce the DNA construct. Representative examples of appropriate 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, animal cells such as Ax and CRL 1998 cells (both endothelial cells), CHO, COS and Bowes melanoma cells, and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.

Suitable prokaryotic and eukaryotic vectors will be readily apparent to the skilled artisan.

Among known bacterial promoters suitable for use in the present invention include the E. coli lacl and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda P_R and P_L promoters and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV), and metallothionein promoters, such as the mouse metallothionein-I promoter. Transcription of the DNA encoding the VAP-1 polypeptides of the present invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

The VAP-1 polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the VAP-1 polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties 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 peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.

The VAP-1 protein can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or non-glycosylated.

VAP-1 Polypeptides and Fragments

The determined nucleotide sequence of the VAP-1 cDNA of Figure 1 (SEQ ID NO: 1) contains an open reading frame encoding a protein of 763 amino acid residues of Figure 1(SEQ ID NO:2), with an initiation codon at positions 80- 82 of the nucleotide sequence in Figure 1 (SEQ ID NO: 1) and a molecular weight of 84.6 KD. The protein has 6 potential N-glycosylation sites and 3 putative O-glycosylation sites (determined using the O-glycosylation site prediction Email server, NetOglyc@cbs.dtu.dk (Hansen et al, Biochem. J. JOS: 801-813 (1995)) per monomer (Fig. 1). Thus, the invention further provides a purified VAP-1 polypeptide having the amino acid sequence encoded by the deposited cDNA, or the amino acid sequence in Figure 1 (SEQ ID NO:2), or a peptide or polypeptide comprising a portion of the above polypeptides. The terms "peptide" and "oligopeptide" are considered synonymous (as is commonly recognized) and each term can be used interchangeably as the context requires to indicate a chain of at least two amino acids coupled by a peptidyl linkage. It will be recognized in the art that some amino acid sequences of the VAP-1 polypeptide can be varied without significant effect of the structure or function of the protein. Thus, the invention further includes variations of the VAP-1 polypeptide which show substantial VAP-1 polypeptide activity. Such mutants include deletions, insertions, inversions, repeats, and substitutions of similar residues. Small changes or such "neutral" amino acid substitutions will generally have little effect on activity. Guidance concerning which amino acid changes are likely to be phenotypically silent (i.e., are not likely to have a significant deleterious effect on a function) can be found in Bowie, J.U., et al, Science 247:1306-1310 (1990). Changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the VAP-1 protein.

Amino acids in the VAP-1 protein of the present invention that are essential for function can be identified by methods known in the art, such as site- directed mutagenesis and deletion analysis. The resulting mutant molecules are then tested for biological activity such as amine oxidase activity or adhesive function.

The polypeptides of the present invention are preferably provided in a substantially purified form. A recombinantly produced version of the VAP-1 polypeptide can be substantially purified, for example, by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988).

The polypeptides of the present invention include polypeptides which have at least 90% similarity, more preferably at least 95% similarity, and still more preferably at least 96%, 97%, 98% or 99% similarity to those described above. Further polypeptides of the present invention include polypeptides at least 90% identical, more preferably at least 90% or 95% identical, still more preferably at least 96%, 97%, 98% or 99% identical to the VAP-1 polypeptide encoded by the deposited cDNA or the VAP-1 polypeptide of Figure 1 (SEQ ID NO:2), and also include portions of such polypeptides with at least 30 amino acids and more preferably, at least 50 amino acids. By a polypeptide having an amino acid sequence at least, for example, 90% "identical" to a reference amino acid sequence of a VAP-1 polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to ten amino acid alterations per each 100 amino acids of the reference amino acid of the VAP-1 polypeptide.

The polypeptides of the present invention can also be used to raise polyclonal and monoclonal antibodies, which are useful in assays for detecting VAP-1 protein expression or as agonists and antagonists capable of enhancing or inhibiting VAP-1 protein function. Details of monoclonal antibodies raised against VAP-1 is described in Salmi, M., and Jalkanen, S., Science 257: 1407- 1409 (1992), U.S. Patent Nos. 5,512,442 and 5,580,780, and U.S. Appl. No. 08/447,799, all incorporated herein by reference. Further, such polypeptides can be used in the yeast two-hybrid system (Fields and Song, Nature 340:245-246 (1989)) to "capture" VAP-1 protein binding proteins which are also candidate agonist and antagonist according to the present invention.

As used herein, the term "antibody" (Ab) or "monoclonal antibody" (MAb) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')₂ fragments) which are capable of specifically binding to VAP-1 protein. Fab and F(ab')₂ fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al, J. Nucl. Med. 24:316-325 (1983)). Thus, these fragments are preferred.

The antibodies of the present invention may be prepared by any of a variety of methods. For example, cells expressing the VAP-1 protein or an antigenic fragment thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation of VAP-1 protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity. In the most preferred method, the antibodies of the present invention are monoclonal antibodies (or VAP-1 protein binding fragments thereof). 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 and T-Cell Hybridomas, Elsevier, N.Y., (1981) pp. 563-681).

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

"Humanized" chimeric monoclonal antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. See, for review, Morrison, Science 229:1202 (1985); Oi et al, BioTechniques 4:214 (1986); Cabilly et al, U.S. Patent No. 4,816,567; Taniguchi et al., EP 171496.

VAP-1 Uses

The VAP-1 of the present invention has significant identity to a family of enzymes termed the copper-containing amine oxidases (Enzyme Commission classification, EC 1.4.3.6). The VAP-1 protein shown in Figure 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 family. The highest identity found was with bovine serum amine oxidase (81%). Using benzylamine, a monoamine oxidase (MAO) substrate, significant amine oxidase enzymatic activity was detected in the VAP-1 expressing CHO cells but not in mock transfected cells. In the presence of inhibitors of copper-containing monoamine oxidases, semicarbazide and hydroxylamine, VAP-1 had no activity against benzylamine.

Thus, the peptides encoded by the DNA of the invention may be used to enzymatically oxidize an amine, by reacting the amine with the VAP-1 polypeptide having amine oxidase activity. The amine can be, for example, certain endogenous and xenobiotic aromatic amines. The amine can also be, for example, certain endogenous and xenobiotic aliphatic amines such as methylamine. The amine is, preferably, benzylamine.

Amine oxidase assay conditions of benzylamine as an amine oxidase substrate are described in detail in the Materials and Methods section, infra. In a final volume of 0.2-5 ml, preferably at a final volume of 0.5 ml, sodium phosphate buffer is added for a final concentration of 0.05-0.2 raM, preferably 0.1 raM. Unlabeled benzylamine is added to a concentration of 1-100 nmol, preferably 80 nmol, and ¹⁴C-benzylamine is added such that the activity is 0.1-1.0 μCi, preferably 0.4 μCi. A cell lysate sample of 0.05-0.5 ml can be added for the assay, preferably 0.1 ml. Following 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/1, preferably 0.35 g/1, of diphenyl oxazole. An additional extraction can be performed using the toluene/diphenyl oxazole mixture. The first extraction (and second extraction) is counted for ¹⁴C in a liquid scintillation counter.

The invention further relates to a method of inhibiting the VAP-1 amine oxidase activity by providing an effective amount of an inhibitor of amine oxidase. The inhibitor can be, for example, semicarbazide, hydroxylamine, propargylamine, isoniazid, nialamide, hydrallazine, procarbazine, monomethylhydrazine, 3,5-ethoxy-4-aminomethylpyridine, or MDL72145 ((E)-2-(3,4-dimethoxyphenyl)-3-fluoroallylamine), preferably, semicarbazide or hydroxylamine. Semicarbazide may be provided at a concentration of 10-1000 μM, or any range therein, such as 50-1000 or 80-500 μM, preferably, at a concentration of 100 μM. Hydroxylamine may be provided at a concentration of 0.1-100 μM, or any range therein, such as 1-10 or 2-8 μM, preferably, at a concentration of 5 μM. According to the invention, vascular adhesion protein- 1 (VAP-1)- mediated binding of endothelial cells to lymphocytes can be modified by providing an inhibitor or potentiator of amine oxidase to the host. The host can be mammalian cell line, animal or human. The invention can be used for treatment in vitro or in vivo.

Having generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration and not intended to be limiting.

Examples

Example I

Isolation of a VAP-1 cDNA

Human gut smooth muscle, in which VAP-1 is strongly expressed, was obtained from material removed in surgical procedures and was used as a source from which to purify VAP-1 protein. Using a MAb immunoaffinity column, the 90 and 170-180 KD forms of VAP-1 were purified from detergent lysates of smooth muscle in sufficient quantities to obtain internal peptide sequence after digestion with trypsin and V8 protease. In addition, a portion of the 90 KD VAP-1 was subjected to N-terminal sequencing directly. The peptide elution profile from the HPLC column used to purify the peptides from both forms of VAP-1 was identical, as were the peptide sequences of the corresponding peaks, indicating that the protein is a dimer composed of two 90 KD subunits.

Some of the VAP-1 peptide sequences were used to design partially degenerate oligonucleotide primers for RT-PCR experiments on mRNA prepared from human gut smooth muscle. A single cDNA fragment of approximately 700 bp was amplified using these primers and cloned into pUC18 in order to determine its sequence. The cDNA sequence contained a continuous open reading frame that encoded a protein containing some of the sequences of the tryptic and V8 peptides of the immunopurified VAP-1 material, thereby confirming that the correct cDNA fragments had been amplified. To isolate longer cDNA clones a panel of 10 human cDNA libraries were analyzed by PCR in order to identify those containing VAP-1 cDNA's and the libraries giving the strongest signal were screened with the PCR-generated VAP-1 cDNA fragment. In this manner a number of overlapping cDNA clones were isolated from human lung and heart cDNA libraries. A cDNA of 2501 bp which contained a continuous open reading frame of 2292 bp starting at an ATG methionine codon was derived from these clones and subcloned into pUC19. The start codon was followed by the peptide sequence found at the N-terminal of the purified 90 KD VAP-1 protein and the contiguous open reading frame encoded all of the VAP-1 tryptic and V8 peptides identified by protein sequencing (Fig. 1). A 5' untranslated region of 80 bp and a 3' untranslated region of 129 bp following the TAG stop codon was present in the clone. Neither a polyadenylation signal nor a poly A sequence was found at the 3' end of the cDNA, suggesting that the native VAP-1 mRNA may be longer than indicated by the cDNA isolated here.

Transient expression of VAP-1 in COS-7 cells by transfection with an expression vector containing the cDNA resulted in strong surface staining of a population of cells with the VAP-1 MAb, 1B2 (Fig. 2, FACS panel row B, column 4). Control transfected cells were negative (Fig. 2, FACS panel row A, column 4). Thus, it was concluded that a cDNA was isolated, encoding immunoreactive VAP-1 which is expressed on the surface of VAP-1 cDNA transfected cells.

VAP-1 Protein

The open reading frame contained in the VAP-1 cDNA encoded a 763 amino acid protein of 84.6 KD. Searching of the available protein sequence databases revealed that VAP-1 has significant identity to a family of enzymes termed the copper-containing amine oxidases (Enzyme Commission classification, EC 1.4.3.6). This identity varied from between 24%, for Escherichia coli Cu-monoamine oxidase, to 41-81% for mammalian members of this family. The highest identity found was with bovine serum amine oxidase (81 %) which exhibited significant conservation throughout the entire length of the protein except for a short region at the N-terminal end of the molecule. A multiple alignment of VAP- 1 with the four other mammalian members of the copper-containing amine oxidase family for which sequence data are available is shown in Fig. 3.

VAP-1 has no significant identity to any currently known adhesion molecules and contains none of the protein domains sometimes found within such proteins, although we note the occurrence of an RGD motif between residues 726-728. The functional significance with regard to integrin binding, if any, of this commonly occurring motif is unknown. The protein has 6 potential N-glycosylation sites and 3 putative O-glycosylation sites (determined using the O-glycosylation site prediction Email server, NetOglyc@cbs.dtu.dk (Hansen et al, Biochem. J. 505:801-813 (1995)) per monomer (Fig. 1). Examination of the protein sequence revealed no obvious areas with characteristics of membrane spanning domains except for a region of 23, predominantly hydrophobic, amino acids at the very N-terminal end (residues 5 to 27) of the molecule that are indicated in Fig. 1 (SEQ ID NO:2). This region could be interpreted as either a cleavable secretion signal, because it contained a potential cleavage site at position 19 as determined by the method of von Heijne, G., Nucl. Acids Res. 74:4683-4690 (1986), or a transmembrane domain. The N-terminal protein sequence of the 90 KD VAP-1 protein showed that this hydrophobic region was not cleaved off in the material we had purified and was thus unlikely to function as a normal cleavable signal sequence for secretion. In addition, the charge characteristics of the residues flanking the hydrophobic region suggested that it could be the membrane spanning domain of a type II membrane protein (Hartmann, E., et al, Proc. Natl. Acad. Sci. USA 56:5786-5790 (1989)) having a cytoplasmic N-terminus and a C-terminal extracellular domain. In order to determine if this hydrophobic region could function as a transmembrane domain and ascertain the orientation of VAP-1 in the membrane, we made VAP-1 cDNA expression constructs in which a FLAG epitope, recognized by the commercially available MAb M2, was placed in frame after the initiating methionine codon on the predicted cytoplasmic side of the putative transmembrane domain. This construct, and a control construct in which the VAP-1 cDNA was placed in an inverse orientation in the vector, were transfected into COS-7 cells and the transient expression of the FLAG and VAP-1 epitopes, recognized by MAbs M2 and 1B2 respectively, was analyzed by FACS analysis of permeabilized and non-permeabilized cells (Fig. 2). Staining with the FLAG MAb was seen on permeabilized cells only (Fig. 2, row F, column 5), whereas VAP-1 staining was seen in both permeabilized and non-permeabilized cell populations (Fig. 2, rows C and F, column 4). Control transfected cells were negative with both the FLAG and VAP-1 MAbs (Fig. 2, rows A and D, columns 4 and 5). This suggests that the N-terminal FLAG epitope is located on the cytoplasmic side of the cell membrane, the hydrophobic region spans the lipid bilayer and that there is a large C-terminal extracellular domain recognized by the adhesion blocking MAb 1B2. All the putative glycosylation sites are located in the extracellular portion of the molecule.

To determine the size and glycosylation status of recombinant VAP-1 transiently expressed in COS-7 cells, we immunoblotted SDS-PAGE separated cell extracts of VAP-1 and mock transfected cells with and without prior sialidase digestion (Fig. 4) using MAb 1B2. The increase in apparent molecular mass of VAP-1 from 170-180 KD to approximately 180-190 KD upon sialidase treatment indicates that recombinant VAP-1, like native VAP-1, is a sialoglycoprotein and the reduction in the net negative charge due to removal of the negatively charged sialic acids causes a decrease in the mobility of VAP-1 in SDS-PAGE. A similar effect upon sialidase treatment has been observed with tonsillar VAP-1 (Salmi, M., and Jalkanen, S., J. Exp. Med. 183:569-519 (1996)). VAP-1 Expression

Northern blot analysis of mRNA isolated from human gut smooth muscle and lymphocyte-depleted tonsil stroma showed that the cloned VAP-1 cDNA hybridizes to a 4.2 kb mRNA in both tissues (Fig. 5A). Further Northern blot analysis showed that a 4.2 kb VAP-1 mRNA is expressed in a wide range of human tissues. The message was not detectable in peripheral blood leukocytes or brain but was strongly expressed in lung, small intestine and appendix when compared with other tissues. Only low amounts of the VAP-1 mRNA were detected in spleen, thymus, testis, liver, pancreas, kidney, bone marrow and fetal liver. An intermediate level of expression was seen in prostate, ovary, the mucosal lining of the colon, heart, placenta, skeletal muscle and lymph node (Fig. 5B). No other mRNA species were detected even after prolonged autoradiography.

Materials and Methods

Ab's and Reagents. Mouse MAbs 1B2 against VAP-1 and 3G6 against chicken T cells have been described Salmi, M., and Jalkanen, S., Science 257: 1407-9 (1992)). The MAb M2 recognizing the FLAG peptide (DYKDDDDK) was obtained from KEBO Lab, Espoo, Finland.

VAP-1 Purification and Sequencing. Normal gut samples obtained from abdominal surgery were dissected free from the lamina propria. The smooth muscle wall was minced into small pieces and lysed in a lysis buffer (150 mM NaCl, 10 mM Tris-base, pH 7.2, 1.5 mM MgCl₂, 1 % NP-40, 1 % Aprotinin and 1 mM PMSF) overnight. After clarification, the lysate supernatant was sequentially applied to immunoaffinity columns containing 5 ml of CnBr activated Sepharose beads armed with normal rat serum, non-binding MAbs and an anti-VAP-1 MAb (3 mg/ml beads). The specific column was washed with the lysis buffer and the VAP-1 antigens eluted with 50 mM triethylamine. The eluate was immediately frozen and subsequently lyophilized. The sample was then dissolved in non-reducing Laemmli's sample buffer and separated on a 5-12.5% SDS-PAGE gel.

After transfer to polyvinylidine difluoride membrane (Applied Biosystems Inc., Foster City, CA, USA) by electroblotting, the membrane was stained with Coomassie blue and the 90 and 170-180 KD bands excised. The whole of the 170-180 KD band and a portion of the 90 KD material was digested with trypsin (sequencing grade, Boehringer Mannheim GmbH, Mannheim, Germany) as described by Fernandez et al. (Fernandez, J., et al, Anal Biochem. 201:255-262 (1992)). The eluted peptides were separated by HPLC (150 A; Applied Biosystems) on a Vydac Cj₈ column (2.1 x 150 mm). N-terminal sequence analysis was performed on a protein sequencer (477 A; Applied Biosystems) equipped with an on-line phenylthiohydantoin amino acid analyzer (120A; Applied Biosystems). The remaining portion of the 90 KD material was subjected to N-terminal sequence analysis without prior digestion. Following trypsin digestion, the membranes were redigested with V8 protease (sequencing grade, Boehringer Mannheim) and the eluted peptides purified and sequenced as above. The sequences of some of the peptides were confirmed using laser assisted matrix desorption mass spectrometry to confirm their predicted mass (Lasermat, Finnigan). Molecular Biology Techniques. Small and large scale plasmid isolation from E. coli, restriction enzyme digestion, plaque hybridization, lambda phage purification and phage DNA extraction, E. coli transformation, cDNA synthesis and plasmid construction were performed according to standard techniques (Sambrook, J., et al, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)). Isolation of DNA fragments from agarose gels was performed using a QIAEX kit (QIAEGEN, Hilden, Germany) following instructions supplied by the manufacturer. DNA probes were prepared using an Amersham Multiprime DNA labeling kit and [α-³²P]dCTP at -3000 Ci/mMol (Amersham, Bucks, UK). Autoradiography was performed using intensifying screens, if required, on Amersham Hyperfilm MP. PCR fragments were blunt end cloned into pUC18 using a SureClone kit (Pharmacia, Uppsala, Sweden). Plasmid DNA was sequenced using a Sequenase version 2.0 kit (United States Biochemical Corporation, Cleveland, OH, USA) according to the manufacturer's instructions or in the DNA sequencing facility of the University of Turku, Department of Medical Genetics. Sequence assembly and analysis was performed using the Wisconsin Package version 8.1 -UNIX of the Genetics Computer Group. Database comparisons were made using the BLAST Email or WWW server of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Oligonucleotide primers for sequencing and PCR were obtained commercially from Kebo Lab (Espoo, Finland). PCR was performed using Dynazyme polymerase (Finnzymes, Espoo, Finland) under conditions recommended by the manufacturer and variable amplification parameters chosen with regard to the characteristics of the PCR primers. The primers used for amplifying the VAP-1 cDNA fragment from smooth muscle mRNA by RT-PCR were N2; gctgtgatcacmatyttygc (SEQ ID NO: 3), designed from the VAP-1 N-terminal protein sequence AVITIFA (SEQ ID NO:4) (residues 13 to 19 of the complete protein) and T4; ccggccctgrtagaasac (SEQ ID NO:5), designed from the tryptic peptide sequence VFYQGR (SEQ ID NO:6) (residues 264 to 269). Amplification conditions with these primers were 94°C, 1 min; 55°C, 1 min; 72°C, 2 min for 30 cycles. Total RNA was prepared from cell lines and tissues using Ultraspec (Biotecx, Houston, TX, USA) in accordance with the manufacturer's instructions. Human multiple tissue Northern blots were obtained from Clontech Laboratories (Palo Alto, CA, USA) and hybridized with ³²P labeled probes as recommended by the manufacturer. The human cDNA library panel and lung (catalogue No. HL3004a) and heart (catalogue No. HL3026a) cDNA libraries were 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) obtained from the ATCC (Rockville, MD) were used as hosts for transfection and expression of VAP-1. COS-7 and CRL 1998 cells were grown in RPMI 1640 (Gibco BRL) supplemented with 10% fetal calf serum (PAA-Linz, Austria), 2 mM glutamine (Biological Industries, Israel), and 128 U/ml penicillin and 128 μg/ml streptomycin. CHO cells were grown in alpha-MEM plus CHO nucleosides with the same supplements. Expression plasmids consisting of VAP-1 cDNAs subcloned into the expression vector pcDNA3 (Invitrogen, San Diego, CA) were used for transient COS-7 cell transfections and generation of stably transfected CHO cell lines. Expression plasmids (20 μg) were used to transfect cells by electroporation with a Bio-Rad Gene Pulser apparatus (0.3 kV, 960 μF, 0.4 cm cuvette in RPMI plus 1 mM Na-pyruvate, 2 mM L-glutamine, without serum). Transiently transfected cells were assayed 3 days post-transfection. Stably transfected cells were selected by culturing in the presence of 0.5 mg/ml Geneticin (Gibco BRL) for 4 weeks.

Example 2

VAP-1 Enzymatic Activity

The finding that VAP-1 has significant identity to the copper-containing amine oxidase family, and in particular to secreted bovine serum amine oxidase (BSAO), led us to examine if VAP-1 possessed amine oxidase activity. The copper-containing amine oxidases are distinguished by the presence of an unusual quinone cofactor, enzyme bound copper and activity only against primary polyamines or monoamines. Thus, they are distinct from the FAD-containing intracellular (mitochondrial) monoamine oxidases (Mclntire, W.S. and Hartmann, C, in Principles and Applications of Quinoproteins, p. 97 (V.L. Davidson, ed., Dekker, New York ( 1993)).

To determine which type of activity was possessed by VAP-1, we generated stable CHO cell transfectants expressing VAP-1 protein. Sonicated lysates of these cells were assayed for diamine oxidase (DAO) activity using putrescine as a substrate, or monoamine oxidase (MAO) activity, using benzylamine as a substrate. As positive controls, commercially available DAO and MAO were assayed and negative controls were provided by mock plasmid transfected CHO cell lysates. The results showed that the VAP-1 expressing cells had negligible activity towards putrescine but significant activity was detected using the MAO substrate benzylamine (Table 1). However, using benzylamine, a monoamine oxidase (MAO) substrate, significant activity was detected in the VAP-1 expressing CHO cells and the positive control of commercially available bovine plasma MAO (Table 1). Mock CHO cell lysates showed insignificant enzyme activity. In the presence of 100 μM semicarbazide and 10 μM hydroxylamine, specific inhibitors of copper-containing monoamine oxidases (Lyles, Intl. J. Biochem. Cell Biol 28:254-214 (1996)), VAP-1 had no activity against benzylamine (Table 1).

In addition, we demonstrated that adhesion-competent VAP-1 expressed in Ax cells also possessed MAO activity against benzylamine which was inhibitable by semicarbazide and hydroxylamine (Table 1).. Ax cells themselves appear to possess a native MAO activity against benzylamine which is not inhibitable by semecarbazide or hydroxylamine as a low level of activity was detected in mock control cells (Table 1).

To confirm that MAO activity is found in VAP-1 in vivo, we immunoaffinity purified VAP-1 from tonsil and assayed the material in triplicate. Tonsillar VAP-1, like VAP-1 from transfected CHO cells, demonstrated activity against benzylamine with an A₄₉₀ increase per hour of 0.09 at 37° C under the assay conditions used, which was 4.5 times greater than that of boiled sample. It was not possible to measure specific activities due to the very low yield of tonisllar VAP-1 protein obtained.

Benzylamine, while commonly used as a substrate for measuring amine oxidase activity, is not found in vivo. Thus a number of biologically occurring endogenous amines were tested to see whether they could be utilised by VAP-1

(Table 2). Methylamine at 1 mM appeared to be readily used by VAP-1 , however, no other amines tested demonstrated reactivity. The lower specific activities observed with benzylamine in these cell lysates compared with the lysates assayed in Table 1 reflects the variation in VAP-1 expression found in different batches of cells. The presence of a quinone cof actor in VAP-1 was shown by separating a portion of the immunopurified tonsillar VAP-1 material by SDS-PAGE under reducing conditions and transferring the material to nitrocellulose. The nitrocellulose filter was then stained with nitroblue tetrazolium/Na glycinate under redox cycling conditions to specifically stain quinone moieties in the protein (Paz, M.A., et al, J. Biol. Chem. 266:689-92 (1991)). The stain reacted with both monomeric 90 KD and dimeric 180 KD VAP-1, showing that tonsillar VAP-1 probably has a quinone cofactor in each subunit.

Materials and Methods

Amine Oxidase Assays. Confluent CHO or Ax cells (10-15 x 10⁶ per flask) stably transfected with a VAP-1 cDNA expression plasmid were scraped from a flask into 10 ml of 100 mM phosphate buffer, pH 7.2, centrifuged and the cell pellet washed with a further 10 ml of buffer. The cell pellet was finally resuspended in 1.5 ml of phosphate buffer and the cells lysed by a 2 x 10 sec sonication at medium power on ice (Braun sonicator). Sonicated lysates were used directly in enzyme assays (50-100 μl per assay) or after storage at -20 °C.

Total protein concentrations were measured by the Bradford method using bovine gamma globulin as a standard and a Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Hercules, CA, USA).

Amine oxidase activities were measured according to previously described techniques using as substrates, ¹⁴C labeled putrescine (Amersham) by the method of D'Agostino, L., et al, Biochem. Pharmacol. 38:41-49 (1989) (incorporated herein by reference), ³H histamine (New England Nuclear) by the method of Baylin, S.B., and Margolis, S., Biochem. Biophys. Acta 397:294-306 (1975) (incorporated herein by reference), and ¹⁴C benzylamine (Amersham) assayed in a similar manner to putrescine except that 0.4 μCi of the label was used per reaction which contained 80 nmol of unlabeled benzylamine substrate. A 0.5 ml reaction volume contained 100 mM sodium phosphate buffer, pH 7.2, 0.4 μCi ¹⁴C benzylamine (Amersham), 80 nmol of unlabeled benzylamine and sample up to a maximum of 100 μl of crude cell lysate per reaction. Following incubation at 37 °C for 1 hour, the aldehyde product of the reaction was extracted by adding 900 μl of toluene containing 0.35 g/1 of diphenyloxazole and shaking vigorously. After allowing the phases to separate, 700 μl of the toluene layer was removed and an additional extraction performed with 700 μl of toluene/diphenyl oxazole, 500 μl of this was removed, added to the first extraction and counted for ^l4C in a liquid scintillation counter. All enzyme assays were performed in the presence of blanks containing boiled sample (10 min, 100°C) or non-expressing sample in duplicate or triplicate and the results are expressed as pmol substrate used min^"1 mg protein^"1. Controls of crude porcine kidney DAO and bovine plasma MAO were obtained from the Sigma Chemical Company (St. Louis, MO, USA).

Discussion

Prominent staining with the MAb 1B2 recognizing VAP-1 is found in the endothelial cells of vessels in several locations, particularly in PLN type lymphoid tissues. However, as the total levels of VAP-1 found at these locations is relatively low, it proved difficult to isolate and purify sufficient quantities of endothelial VAP-1 from which to obtain protein sequence information. Of the other tissues in which VAP-1 is found, it is most abundant in the smooth muscle of the vasculature and gut-associated smooth muscle (Salmi, M., et al, J. Exp. Med. 178:2255-2260 (1993)). VAP-1 from these sources has a marginally different molecular mass, probably due to glycosylation differences, but otherwise resembles the form previously analyzed in tonsil and PLN type tissues. Using protein sequence information obtained from VAP-1 purified from gut smooth muscle, a cDNA encoding this adhesion molecule was isolated. The evidence for this is based on the following: Firstly, protein sequence obtained from immunopurified VAP-1 was found in the predicted protein sequence of the VAP-1 cDNA clone subsequently isolated. Secondly, transfected cells expressing the VAP-1 cDNA could be stained on their surface with the MAb 1B2 which was originally used to define VAP-1 (Salmi, M., and Jalkanen, S., Science 257:1407-1409 (1992)) and VAP-1 immunoprecipitated from these cells had a similar molecular mass, 170-180 KD, to that found in vivo. .

VAP-1 is a large, dimeric, type π transmembrane protein having a membrane spanning domain located at the N-terminal end of the molecule. The intracellular domain is particularly small, being only 4 amino acids in length, leaving a large glycosylated extracellular domain of some 163 KD per dimer. All the potential glycosylation sites, 12 N-linked and 6 putative O-linked per dimer, are located in the extracellular domain. Although it is currently not known if all of these are utilized, previous data suggest that VAP-1 contains both N- and O-linked sugars and numerous sialic acid residues which could be presented either on the terminal residues of the N-linked carbohydrate or as part of the O-linked sugar. As carbohydrates, and sialic acids in particular, are thought to play an important part in the adhesive function of VAP- 1 protein (Salmi, M., and Jalkanen, S., J. Exp. Med. 183:569-519 (1996)), it will be necessary to determine which glycosylation sites are used and what carbohydrates are presented on them.

Since no VAP-1 mRNA species of significantly different size was seen in any of the tissues examined by Northern blotting, and all VAP-1 cDNA clones and PCR fragments analyzed contained similar sequences, there is no conclusive evidence to suggest that there are forms of VAP-1 encoded by variant mRNAs. Thus it seems likely that the inventors have cloned a cDNA encoding the predominant form of VAP- 1 that has been studied previously by immunoblotting and immunoprecipitation. However, as almost all of these tissues examined contain smooth muscle, either from vascular tissue or other sources, as well as vascular endothelium, it is impossible to distinguish between a smooth muscle and endothelial VAP-1 mRNA if very similar mRNA types exist in these tissues. It is thus conceivable that there might be other form(s) of VAP-1 encoded by an mRNA differing slightly from that already isolated.

Interestingly, the N-terminal region of VAP- 1 with the transmembrane region is the part of the molecule that diverges most from its homologue BS AO. BSAO is known to be a secreted protein found at high levels in serum and has a secretion signal sequence at its N-terminus which is removed by proteolytic processing in the secretory pathway (Mu, D., et al, J. Biol. Chem. 269:9926-9932 (1994)). Thus BSAO and VAP-1 may not have similar physiological properties. Preliminary evidence suggests that mouse VAP-1 has a transmembrane domain similar to that found in human VAP-1 and thus may have the same functions as the human molecule. The extracellular domain of VAP-1 contains the amine oxidase enzyme activities and thus VAP-1 can function as an ecto-enzyme. The copper-containing amine oxidases are a diverse class of enzymes with widely differing substrate specificities, but they can be broadly classed into two types, those with activity against polyamines such as putrescine and histamine and those, such as VAP-1, with monoamine oxidase activities. The physiological substrates of the monoamine oxidases are unclear, although there are several candidates.

Another characteristic of these enzymes is the presence of an unusual carbonyl-containing cofactor, the chemical identity of which has been the subject of some controversy but which has been identified in BSAO and pea amine oxidase as topaquinone (6-hydroxy dopa) and in lysyl oxidase as a cross-linked lysine tyrosylquinone. In several mammalian species, including humans, a copper-containing monoamine oxidase found both in the cell membrane and in a soluble form in serum has been identified. This activity is sensitive to inhibition by carbonyl reactive agents such as semicarbazide and hydroxylamine (Lyles, G.A., Intl. J. Biochem. Cell Biol. 28:259-214 (1996)) and the enzyme responsible is commonly referred to as a semicarbazide-sensitive amine oxidase (SSAO). We have shown that VAP-1 contains a covalently bound quinone which, from studies of other copper-containing monoamine oxidases, is likely to be formed by self-processing in a reaction containing enzyme-bound copper. These results, and the sensitivity of VAP-1 enzyme activity to the carbonyl-reactive agents semicarbazide and hydroxylamine, show that VAP-1 is a membrane bound SSAO.

The physiological roles of SSAOs have been difficult to define since little is known of their in vivo substrates and their substrate specificities vary considerably between species. Metabolism of endogenous and xenobiotic primary monoamines would appear to be one candidate function and it is possible that this is the function of the VAP-1 found in non-endothelial locations such as smooth muscle. Whether the SSAO activity of VAP-1 has any role in its adhesive properties is unclear at present. Preliminary in vitro adhesion experiments performed using VAP-1 transfected CRL 1998 cells and PBL (peripheral blood lymphocytes) in the presence of semicarbazide and hydroxylamine have suggested that the enzymatic activity may have a negative effect on the adhesive interactions between these cells as, in the presence of these inhibitors, the numbers of PBL bound to the VAP-1 transfected cells increase. It is not known whether this effect is a primary one, caused by the enzymatic activity having some direct and specific effect on the adhesive mechanism used by the cells, or secondary, in which the physiological state of the cells is adversely affected by a product of the monoamine oxidase activity thus reducing their adhesive potential.

Example 3

Adhesion assay

The VAP-1 cDNA in pcDNA3 was used to transfect Ax cells, a rat HEV derived endothelial cell line which probably provides a more natural functional environment for VAP-1 than other potential hosts such as CHO cells. Stable transfectants were obtained which expressed VAP-1 on their cell surface as determined by FACS analysis (Fig. 6A) and these were used in lymphocyte adhesion assays. When analysed under rotatory conditions, PBL bound to VAP-1 transfected Ax cells 25.6 times better than to mock transfected cells (Fig. 6B and 6C). The enhanced binding to VAP-1 transfectants was statistically significantly inhibited, although not completely abolished, by anti-VAP-1 mAb treatment (inhibition 29.6% ± 10.7%, P = 0.05). These adhesion results are pooled from five independent experiments, in which 2-3 parallel transfectant monolayers were analysed each time using three independently transfected cell lines and PBL from six different donors. Thus these data show that the VAP-1 cDNA encodes a functional adhesion molecule which is located on the cell surface of transfected cells and which, when expressed in Ax cells, can directly mediate the binding of PBL.

Materials and Methods

Ax-cells in which VAP-1 was stably expressed or mock control transfectants were plated within wax -pen circles drawn on gelatin-precoated microscope slides (20 000 cells per 2 cm diameter circle). The cells were allowed to grow to confluence and after two washings, 100 μ of RPMI 1640 medium containing 10% FCS and 10 mM HEPES (the assay medium) was added within each wax -pen circle to evenly cover the adherent cell monolayer. Meanwhile, PBL were isolated from freshly drawn blood using Ficoll- centrifugation and adjusted to a concentration of 40x 10⁶ cells/ml in the assay medium. Thereafter the slides were transferred to an orbital shaker operating at 60 rpm at 7°C, and 5 μl of the PBL suspension was applied onto each wax- pen circle. The assay was continued for 30 min with constant rotation. The slides were carefully decanted and dipped once in cold RPMI to remove non- adherent cells. Adherent cells were fixed to the sections by incubating the slides vertically in ice-cold PBS containing 1% glutaraldehyde overnight. The number of adherent cells was counted using an ocular grid (magnification x 200). The grid covers an area of 0.25 mm². Nine predefined areas of 0.25 mm² at the center of the circle where the transfectants formed a confluent monolayer were counted on each slide. Two slides per sample (total area = 4.5 mm²) were counted in each of five independent experiments. In certain experiments the adhesion assay was performed in the presence of function- blocking mAbs against VAP-1 (a combination of 1B2 and TK8-14, both at 50 μg/ml diluted in the assay medium) or class-matched negative control mAbs (a combination of 3G6 and 7C7 both at 50 μg/ml). The mAbs were preincubated with the transfectant monolayer on the slides for 30 min at 7°C before the labelled lymphocytes were added. The number of adherent cells was counted in five independent experiments as described above.

Discussion

The adhesion assays performed on VAP-1 expressing Ax cells indicated that the cDNA encodes a functional VAP-1 which can support interactions with its ligand on PBL and lead to stable binding of the PBL to the Ax cells. However, complete inhibition of this increased adhesion with anti- VAP-1 mAbs 1B2 and TK8-14 was not observed, suggesting that the VAP-1 molecule in rat-derived Ax cells is not functioning exactly as it does in its native environment. It maybe that the carbohydrate modifications of the protein in Ax cells, the local membrane environment or VAP- 1 conformation is sufficiently different from that in human HEV such that the mAb can no longer block all VAP-1 interactions with its ligand. It is also possible that a subpopulation of VAP-1 molecules may exist on transfectants in a form lacking one or other of the epitopes recognised by mAb 1B2 and TK8-14. Finally, we are left with the possibility that long term over expression of VAP- 1 in stable transfectants alters the expression of other adhesion molecule(s) on the Ax cell surface. The function of this other putative adhesion molecule would not, of course, be inhibitable by anti VAP-1 mAb. In HEV binding assays VAP-1 has been shown to function independently of lymphocyte L- selectin and it mediates the binding of CD8⁺ PBL much better than CD4⁺ PBL. Ax VAP-1 cDNA transfectants reproduce these observations since analysis of immunomagnetically purified L-selectin negative cells and CD8⁺ and CD4⁺ cells showed that L-selectin was not necessary for efficient binding to Ax transfectants and that the CD8⁺ subset of PBL adhered several fold better to VAP-1 transfectants than CD4⁺ cells (data not shown).

Table 1. Assay of Amine Oxidase Activity in CHO and Ax Cells Expressing VAP-1.

Cell type enzyme activity in nmol product min^"1 mg^"1 protein

Substrate CHO VAP- 1 CHO Mock D Diiaammiinnee Monoamine Ax VAP- 1 Ax Mock 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±0.80 benzylamine + SC 0.61±0.17 1.32+0.26 ND ND 0.35±0.09 1.OO±O.OO benzylamine + HA 0.35±0.17 1.32+0.26 ND ND 0.3110.04 0.90+0.10 ______^

Each assay was done in triplicate and the mean specific activity ±SEM is shown. ND, not done.

Table 2. Substate Specificity of VAP-1 Amine Oxidase Activity

Cell type enzyme activity in nmol product min^"1 mg^"1 protein

Substrate CHO VAP- 1 CHO Mock

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 tryptamine 0.07±0.04 0.35±0.62 β-phenylethylamine 0.10±0.03 0.26+0.00 histamine 0.05±0.03 0.20+0.00

The experiment was performed twice on different samples with comparable results and the results of one representative experiment is shown. Each assay was done in triplicate and the mean specific ±SEM is given. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

The disclosure of all references, patent applications, and patents cited herein, if any, 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) : F N-20520

(ii) TITLE OF INVENTION: 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: Patentin 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 Gin Lys Thr lie Leu Val Leu Leu lie Leu Ala Val lie Thr 1 5 10 15

He Phe Ala Leu Val Cys Val Leu Leu Val Gly Arg Gly Gly Asp Gly 20 25 30

Gly Glu Pro Ser Gin Leu Pro His Cys Pro Ser Val Ser Pro Ser Ala 35 40 45

Gin Pro Trp Thr His Pro Gly Gin Ser Gin Leu Phe Ala Asp Leu Ser 50 55 60

Arg Glu Glu Leu Thr Ala Val Met Arg Phe Leu Thr Gin Arg Leu Gly 65 70 75 80

Pro Gly Leu Val Asp Ala Ala Gin Ala Arg Pro Ser Asp Asn Cys Val 85 90 95

Phe Ser Val Glu Leu Gin 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 He Val 115 120 125

Phe Phe Gly Arg Gin Pro Gin 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 Gin Glu Tyr 165 170 175

Leu Asp He Asp Gin Met He Phe Asn Arg Glu Leu Pro Gin 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 Gin Ser Gly Asp Arg Ala 210 215 220 Thr Trp Phe Gly Leu Tyr Tyr Asn He 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 He Gin Lys Val Phe Tyr Gin Gly Arg Tyr Tyr Asp 260 265 270

Ser Leu Ala Gin Leu Glu Ala Gin Phe Glu Ala Gly Leu Val Asn Val 275 280 285

Val Leu He 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 Gin Phe Tyr Pro Gin Gly 305 310 315 320

Pro Arg Phe Ser Val Gin 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 He Phe Asp Val 340 345 350

Arg Phe Gin Gly Glu Arg Leu Val Tyr Glu He Ser Leu Gin Glu Ala 355 360 365

Leu Ala He 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 Gin Ala Pro Lys Thr He Arg Asp Ala Phe Cys Val Phe 420 425 430

Glu Gin Asn Gin 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 He Glu He Arg Phe Tyr Ala Thr Gly Tyr He Ser 485 490 495

Ser Ala Phe Leu Phe Gly Ala Thr Gly Lys Tyr Gly Asn Gin 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 Gin Leu Gin 545 550 555 560

Arg Leu Gin Val Thr Arg Lys Leu Leu Glu Met Glu Glu Gin 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 He Gin Met Leu 595 600 605

Ser Phe Ala Gly Glu Pro Leu Pro Gin Asn Ser Ser Met Ala Arg Gly 610 615 620

Phe Ser Trp Glu Arg Tyr Gin Leu Ala Val Thr Gin Arg Lys Glu Glu 625 630 635 640

Glu Pro Ser Ser Ser Ser Val Phe Asn Gin Asn Asp Pro Trp Ala Pro 645 650 655

Thr Val Asp Phe Ser Asp Phe He Asn Asn Glu Thr He Ala Gly Lys 660 665 670

Asp Leu Val Ala Trp Val Thr Ala Gly Phe Leu His He Pro His Ala 675 680 685

Glu Asp He 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 He Tyr Phe Arg Gly Asp Gin Asp Ala Gly Ala Cys Glu Val 725 730 735

Asn Pro Leu Ala Cys Leu Pro Gin 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

What ls Claimed Is:

1 A purified nucleic acid molecule encoding a vascular adhesion protein- 1 (VAP-1), selected from the group consisting of: (a) a purified nucleic acid molecule encoding a polypeptide comprising the amino acid sequence in Figure 1 (SEQ ID NO:2);

(b) a purified nucleic acid molecule comprising the VAP-1 coding sequence of the VAP-1 nucleotide sequence in Figure 1 (SEQ ID NO:l);

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

(d) a purified nucleic acid molecule comprising the coding sequence of the VAP-1 nucleotide sequence contained in Deposit Accession No. DSM 11536; (e) a purified nucleic acid molecule comprising a nucleotide sequence complementary to the VAP-1 nucleotide sequences in (a), (b), (c) or (d);

(f) a purified nucleic acid molecule comprising a nucleotide sequence that differs from the coding sequence of the nucleic acid molecule of (b) or (d) due to the degeneracy of the genetic code; and (g) a purified nucleic acid molecule comprising a nucleotide sequence that hybridizes to a molecule of (e), and encodes a VAP-1 that has an amino acid sequence which shows at least 80% identity to the VAP-1 sequence in Figure 1 (SEQ ID NO:2).

2. The nucleic acid molecule of claim 1 wherein the encoded VAP-1 has amine oxidase activity.

3. The nucleic acid molecule of claim 1 which has the VAP-1 nucleotide sequence in Figure 1 (SEQ ID NO: l).

4. The nucleic acid molecule of claim 1 which has the VAP-1 nucleotide sequence in Figure 1 (SEQ ID NO:l) encoding the VAP-1 polypeptide having the amino acid sequence in Figure 1 (SEQ ID NO:2).

5. The nucleic acid molecule of claim 1 which has the nucleotide sequence of the VAP-1 cDNA clone contained in Deposit Accession No. DSM 1 1536.

6. The nucleic acid molecule of claim 1 which has the nucleotide sequence encoding the VAP-1 polypeptide having the amino acid sequence encoded by the VAP-1 cDNA clone contained in Deposit Accession No. DSM 1 1536.

7. The nucleic acid molecule of claim 1 which is a cDNA molecule.

8. The nucleic acid molecule of claim 1 which is a RNA molecule.

9. A method for making a recombinant vector comprising inserting the purified nucleic acid molecule of claim 1 into a vector.

10. A recombinant vector produced by the method of claim 9.

11. A method of providing a VAP-1 to a host cell, comprising introducing the purified nucleic acid molecule of claim 1 into said host cell.

12. A recombinant host cell containing the purified nucleic acid molecule of claim 1.

13. A method for producing a vascular adhesion protein- 1 (VAP-1) polypeptide, comprising culturing the recombinant host cell of claim 12 under conditions such that said VAP-1 polypeptide is expressed, and recovering said VAP-1 polypeptide.

14. A method of providing an amine oxidase activity to a host cell by introducing the nucleic acid of claim 2 into said host cell.

15. A method of altering the expression of vascular adhesion protein- 1 (VAP-1), comprising:

(a) introducing into a host cell, a DNA construct comprising: (i) a VAP- 1 targeting sequence; and

(ii) a regulatory sequence linked to said VAP-1 targeting sequence; and

(b) maintaining said host cell under conditions appropriate for homologous recombination between said DNA construct and the endogenous VAP-1 DNA sequence.

16. The method of claim 15, wherein said regulatory sequence prevents the expression of said VAP-1 under a desired condition.

17. The method of claim 15, wherein said regulatory sequence increases the expression of said VAP-1 under a desired condition.

18. A recombinant host cell, containing:

(a) a VAP-1 targeting sequence; and (b) a regulatory sequence linked to said VAP-1 targeting sequence.

19. A method of oxidizing an amine, comprising reacting said amine with a vascular adhesion protein- 1 (VAP-1) polypeptide having amine oxidase activity.

20. The method of claim 19, wherein said VAP-1 polypeptide has an amino acid sequence at least 95% identical to a sequence selected from the group consisting of:

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

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

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

(d') a purified polypeptide comprising a VAP-1 amino acid sequence that is encoded by a nucleotide sequence that hybridizes to the complement of DNA encoding the polypeptide of any one of (a') to (c'), and encodes a VAP-1 and has an amino acid sequence which shows at least 80% identity to a sequence in Figure 1 (SEQ ID NO:2); and

(e') a purified polypeptide comprising the amino acid sequence of an epitope-bearing portion of the polypeptide of (a') or (b').

21. The method of claim 19, wherein said amine is selected from the group consisting of benzylamine and methylamine.

22. A method of inhibiting amine oxidase activity, comprising providing an effective amount of an inhibitor to a sample possessing said amine oxidase activity.

23. The method of claim 22, wherein said inhibitor is selected from the group consisting of semicarbazide, hydroxylamine, propargylamine, isoniazid, nialamide, hydrallazine, procarbazine, monomethylhydrazine, 3,5-ethoxy-4- aminomethylpyridine, and MDL72145 ((E)-2-(3,4-dimethoxyphenyl)-3- fluoroallylamine).

24. A method of manipulating vascular adhesion protein- 1 (VAP-1 )- mediated binding of endothelial cells to lymphocytes, comprising inhibiting the enzymatic activity of amine oxidase in said endothelial cells.

25. A method of manipulating vascular adhesion protein- 1 (VAP- 1 )- mediated binding of endothelial cells to lymphocytes, comprising potentiating the enzymatic activity of amine oxidase in said endothelial cells.