Pergamon PII: s0161-5890(!37)00060-6 Molecular Immunology, Vol. 34, No. 16-17. pp. 1221-1236, 1997 K;I 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0161-5890/97 $17.00 +O.OO A MOUSE MOLECULAR MIMIC OF HUMAN VASCULAR ADHESION PROTEIN-l MARK0 SALMI,*$ DAVID J. SMITH,* PETRI BONO,* TAINA LEU,” JUKKA HELLMAN,? MARJA-TERTTU MATIKAINEN? and SIRPA JALKANEN* *National Public Health Institute and MediCity Research Laboratory, University of Turku, 20520 Turku, Finland; tCentre of Biotechnology, University of Turku, 20520 Turku, Finland (First received 6 January 1997; accepted in rerised,fbrm 23 April 1997) Abstract-Human vascular adhesion protein-l (VAP-I) is an endothelial sialoglycoprotein which exists in forms of M, 90000 and 170000 and mediates lymphocyte binding to vessels under shear. VAP-I is functionally defined by an inhibitory mouse mAb lB2. A large-scale immunoaffinity purification of VAP-1 from human tonsil lysates was performed to determine the protein sequence for VAP-1 cDNA cloning. A dominant protein of molecular weight 90000 was obtained which yielded an N-terminal sequence of 20 amino acids which bore no significant identity to any protein sequence in the data banks. A mouse mAb (5Bll) against a synthetic peptide from this sequence was raised and found to stain tissues in an identical manner to mAb lB2, to inhibit lymphocyte adhesion to endothelial cells and to recognize VAP-1. Later, the N-terminal sequence obtained from the lB2 immunoprecipitations was found to be identical to a mouse cyclophilin C associated protein (mCy- CAP) subsequently published by others. We show here by several criteria at the protein and DNA level that VAP-1 is distinct from mCyCAP. Moreover, we elucidate the mechanism which results in binding of mCyCAP to mAb 1 B2 during antibody synthesis in hybridoma cells and the sequelae of co-precipitation of mCyCAP during the immunoaffinity chromatography. Binding of mCyCAP to a mouse mAb has not been described before and suggests a new function for this molecule in immuno- globulin synthesis and/or secretion. Moreover, these data indicate that the N-terminal peptide of mCyCAP is a molecular mimic of a functionally important epitope of VAP-1. K: 1997 Elsevier Science Ltd. All rights reserved. Kej‘ words: adhesion molecules, leukocyte trafficking, monoclonal antibodies, epitopes, SRCR- proteins 1. INTRODUCTION Lymphocyte trafficking between blood and the lymphatic organs is vital to normal immunosurveillance and for mounting an adequate response to different types of anti- genie insults in the setting of inflammation. Lymphocytes exit from the blood into tissues by binding to adhesion molecules expressed on the luminal surface of endothelial cells of blood vessels (Bevilacqua, 1993; McEver et al., 1995; Picker and Butcher, 1992; Shimizu et al., 1992; Springer, 1994). Vascular adhesion protein- 1 (VAP- 1) is one of these molecules which mediate lymphocyte- endothelial cell interactions in man (Salmi and Jalkanen, IAuthor to whom correspondence should be addressed at: MediCity Research Laboratory, University of Turku, Tykist(ikatu 6, 20520 Turku, Finland. Ab6re~~iation.s: CsA = cyclosporin A, GST = glutathione-s- transferase. HEV = high endothelial venule(s), MaQBP= Mac-2 binding protein, mCyC = mouse cyclophilin C, mCy- CAP = mouse cyclophilin C associated protein, SRCR = scavenger receptor cysteine rich-repeat, VAP-1 = vascular adhesion protein- 1 1992). VAP-1 was initially characterized by a mouse mAb lB2 which inhibits lymphocyte binding to high endo- thelial venules (HEV), the predilected site of lymphocyte trafficking, and to purified VAP-1 protein in vitro. VAP- 1 is a homodimer of two subunits of M, 90000 which is expressed on HEV, smooth muscle cells and dendritic cells of germinal center (Salmi and Jalkanen, 1992). It is a highly sialylated molecule and mediates oligosaccharide (sialic acid)-dependent, selectin-independent binding of lymphocytes to HEV under conditions of shear (Salmi and Jalkanen, 1996). To clone VAP-1 cDNA, a dominant protein of M, 90 000 was purified with immunoaffinity chromatography using mAb lB2. The novel N-terminal sequence of this molecule was used to raise antibodies which displayed all the expected anti-VAP-1 characteristics. However, the N- terminal amino acid sequence obtained turned out to be identical to that of a mouse molecule called mCyCAP identified later (Chicheportiche and Vassalli, 1994; Friedman et al., 1993). Nevertheless, VAP-1 is not ident- ical to mCyCAP, or its probable human homolog Mac- 2 binding protein (Mac2BP) (Koths et al.. 1993; Ullrich 1227