Biochemical characterization and molecular cloning of a novel endothelial-specific sialomucin.
We have generated rat monoclonal antibodies (MoAbs) against cell surface antigens of the mouse endothelioma cell line bEND.3. Three antibodies (V.1A7, V.5C7, and V.7C7) were selected, all of which recognize a 75-kD antigen on bEND.3 cells and bind selectively to endothelial cells in cryostat sections of mouse tissues. A cDNA for the antigen was isolated from a bEND.3 pCDM8 expression library by using transient expression in COS-7 cells and immunoselection with the three MoAbs. This cDNA coded for a novel, type I membrane protein of 248 amino acids with an extracellular domain rich in threonine and serine residues (35%). The protein is sensitive to O-sialoglycoprotein endopeptidase, indicating that it belongs to the class of sialomucin-like proteins. Therefore, we suggest the name endomucin. Treatment of isolated endomucin by sialidase and O-glycosidase reduced the apparent molecular weight to 45 kD and abolished binding of all three antibodies, indicating that carbohydrates are directly or indirectly involved in the formation of the antibody epitopes. Immunohistological analysis of all examined mouse tissues showed that endomucin is an endothelial antigen found in venous endothelium as well as in capillaries, but not on arterial endothelium. Interestingly, high endothelial venules of peripheral and mesenteric lymph nodes as well as of Peyers's patches were negative for staining with the three MoAbs.
Morgan
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SM
Cell Adhesion Group, Institute of Molecular Medicine, John Radcliffe Hospital, and the Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.
Samulowitz
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Darley
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Simmons
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Vestweber
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D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D000954:Antigens, Surface / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D004730:Endothelium, Vascular / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D004789:Enzyme Activation; D006026:Glycoside Hydrolases / Q000378:metabolism; D008666:Metalloendopeptidases / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009077:Mucins / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D009928:Organ Specificity; D051381:Rats; D052116:Sialomucins; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D018076:DNA, Complementary; D009077:Mucins; D052116:Sialomucins; D006026:Glycoside Hydrolases; D008666:Metalloendopeptidases; C070738:O-sialoglycoprotein endopeptidase