title: alpha-Fucose-mediated binding and degradation of tissue-type plasminogen activator by HepG2 cells.
abstract: The glycoprotein tissue-type plasminogen activator (t-PA) is subject to hepatic clearance in humans. Here, the interaction of t-PA with a well-differentiated hepatoma cell line (HepG2) was examined. Suspended HepG2 cells bound 125I-t-PA in a specific, saturable, and reversible fashion through a Ca(2+)-dependent, active site-independent mechanism. Binding isotherms indicated a high affinity system with a single class of saturable binding sites (Kd 39 nM; maximum binding capacity 493,000 sites per cell). Bound t-PA was rapidly degraded at 37 degrees C in a manner inhibited by lysosomotropic agents or metabolic inhibitors. Pretreatment of t-PA with monoclonal antibodies against the EGF/fibronectin finger domain, but not kringle 2 or kringle 1, reduced total binding by 86%. Binding of 125I-t-PA to HepG2 cells was inhibited by monosaccharides fucose and galactose and by the neoglycoprotein fucosyl-albumin. Enzymatic removal of alpha-fucose residues, but not alpha-galactose, high mannose, or complex oligosaccharide from 125I-t-PA, reduced specific binding by 60 +/- 5%. Binding was also inhibited by high, but not low, molecular weight urokinase, which contains an EGF-based threonine-linked alpha-fucose homologous to that of t-PA. These data suggest that EGF-associated O-linked alpha-fucose may mediate t-PA binding and degradation by HepG2 cells. This mechanism may be relevant to other proteins with analogous structures.
authors.affiliation: Department of Pediatrics, Cornell University Medical College, New York 10021.
authors.forename: K A
authors.initials: KA
authors.lastname: Hajjar
authors.forename: C M
authors.initials: CM
authors.lastname: Reynolds
mesh_terms: D000643:Ammonium Chloride / Q000494:pharmacology; D001386:Azides / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D002738:Chloroquine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005643:Fucose / Q000494:pharmacology*; D017127:Glycation End Products, Advanced; D006601:Hexoses / Q000494:pharmacology*; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D008113:Liver Neoplasms; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D012709:Serum Albumin / Q000494:pharmacology; D019810:Sodium Azide; D012969:Sodium Fluoride / Q000494:pharmacology; D013237:Stereoisomerism; D010959:Tissue Plasminogen Activator / Q000187:drug effects / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D000093423:Glycated Serum Albumin
chemical_list: D001386:Azides; D017127:Glycation End Products, Advanced; D006601:Hexoses; D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D012709:Serum Albumin; D000643:Ammonium Chloride; D005643:Fucose; D002738:Chloroquine; D012969:Sodium Fluoride; D019810:Sodium Azide; C067262:PLAT protein, human; D010959:Tissue Plasminogen Activator; D000093423:Glycated Serum Albumin