Functional effects of asparagine-linked oligosaccharide on natural and variant human tissue-type plasminogen activator.
The role of Asn-linked oligosaccharide in the functional properties of both human tissue-type plasminogen activator (t-PA) and a genetic variant of t-PA was studied. Nonglycosylated and glycosylated wild-type t-PA were produced in mammalian cells which express recombinant t-PA. These proteins were compared in fibrin binding and 125I-labeled fibrin clot lysis assays, using purified components. The nonglycosylated form showed higher fibrin binding, as well as higher fibrinolytic potency than the glycosylated form. Subsequently, prevention of glycosylation of a t-PA variant which lacked the finger and epidermal growth factor domains (delta FE), was carried out in an attempt to enhance its fibrinolytic activity. Glycosylation was prevented by changing Asn to Gln; at Asn-117 to produce delta FE1X t-PA, and at Asn-117, -184, and -448 to produce delta FE3X t-PA. All variants were similar to wild-type t-PA in their catalytic dependence on fibrinogen fragments, fibrinolytic activity in fibrin autography analysis, and plasminogen activator activity. In a clot lysis assay, using citrated human plasma, the fibrinolytic potency of the variants were comparable to that of wild-type t-PA at activator concentrations of 17-51 nM (approximately 1-3 micrograms/ml). At 0.5-5.1 nM (approximately 0.03-0.3 micrograms/ml), however, the variant proteins had lower fibrinolytic potency than wild-type t-PA. Fifty percent lysis in 1.5 h for wild-type, delta FE, delta FE1X, and delta FE3X t-PA, required 2.5, 10, 7.5, and 5.5 nM t-PA, respectively. The fibrinogenolytic activity in human plasma was measured for wild-type, delta FE, delta FE1X, and delta FE3X t-PA, and showed significant fibrinogen depletion after 3 h of incubation at 51 nM, decreasing to 11, 11, 50, and 72% of basal levels, respectively. These data indicate that partial or total nonglycosylated t-PA variants have a higher fibrinolytic versus fibrinogenolytic ratio than their fully glycosylated counterparts.
D001216:Asparagine / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000378:metabolism; D005340:Fibrinogen / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D009844:Oligosaccharides / Q000494:pharmacology*; D010959:Tissue Plasminogen Activator / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D009844:Oligosaccharides; D014415:Tunicamycin; D001216:Asparagine; D005337:Fibrin; D005340:Fibrinogen; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator