Different forms of human vascular adhesion protein-1 (VAP-1) in blood vessels in vivo and in cultured endothelial cells: implications for lymphocyte-endothelial cell adhesion models.
Vascular endothelium plays a pivotal role in controlling leukocyte extravasation from the blood into the tissues. Vascular adhesion protein-1 (VAP-1) is a novel endothelial cell molecule which mediates lymphocyte binding to the vascular lining (Salmi, M., and Jalkanen, S., Science 1992. 257:1407). In this study, we analyzed endothelial cell type-specific differences of VAP-1. In vivo, VAP-1 is a 90/170-kDa molecule which is mainly expressed on the lumenal surface and in cytoplasmic granules of peripheral lymph node-type postcapillary venules (high endothelial venules, HEV). In tonsil HEV, VAP-1 is modified with abundant sialic acids. VAP-1 is also detectable in the cytoplasm of human umbilical vein endothelial cells (HUVEC) and in an endothelial cell hybrid EaHy-926, although both cell types lack detectable surface VAP-1. Cultured endothelial cells do not express MECA-79-defined peripheral lymph node addressins either. VAP-1 was not translocated onto the endothelial cell surface after stimulation with multiple cytokines, mitogens or secretagogues which induced expression of other known endothelial adhesion molecules. Biochemical analyses revealed that VAP-1 is a approximately 180-kDa protein in these endothelial cell types. Digestions with neuraminidase, O-glycanase and N-glycanase, as well as treatment of cells with tunicamycin and benzyl-N-acetylgalactosaminide, did not alter the molecular mass of VAP-1 in EaHy-926. Pulse-chase experiments showed that VAP-1 is directly synthesized as a 180-kDa molecule without any detectable precursors. Thus, in cultured endothelial cells, VAP-1 is a 180-kDa protein which is devoid of post-translational modifications, and in particular, lacks the sialic acids crucial for the function of VAP-1 in tonsil vessels. Notably, the endothelial cell types commonly used as a model in studying lymphocyte-endothelial cell interactions lack surface expression of VAP-1 and peripheral node addressins, and hence are inherently of limited use in analyses of the initial adhesion of lymphocytes.
Salmi
M
M
National Public Health Institute, Turku, Finland.
Jalkanen
S
S
D000116:Acetylgalactosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D000581:Amidohydrolases / Q000494:pharmacology; D006631:Amine Oxidase (Copper-Containing); D001593:Benzyl Compounds / Q000494:pharmacology; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004730:Endothelium, Vascular / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D006031:Glycosylation / Q000187:drug effects; D006596:Hexosaminidases / Q000494:pharmacology; D006801:Humans; D008198:Lymph Nodes / Q000098:blood supply*; D008970:Molecular Weight; D019158:N-Acetylneuraminic Acid; D009439:Neuraminidase / Q000494:pharmacology; D014066:Palatine Tonsil / Q000098:blood supply*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012794:Sialic Acids / Q000032:analysis / Q000502:physiology; D014415:Tunicamycin / Q000494:pharmacology; D014471:Umbilical Veins / Q000737:chemistry*; D014699:Venules / Q000737:chemistry*
D001593:Benzyl Compounds; D015815:Cell Adhesion Molecules; D016207:Cytokines; D012794:Sialic Acids; D014415:Tunicamycin; C080739:benzyl-alpha-N-acetylgalactosamine; C076668:AOC3 protein, human; D006631:Amine Oxidase (Copper-Containing); D006596:Hexosaminidases; D009439:Neuraminidase; C060638:glycopeptide alpha-N-acetylgalactosaminidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D019158:N-Acetylneuraminic Acid; D000116:Acetylgalactosamine