Effects of N-glycosylation on in vitro activity of Bowes melanoma and human colon fibroblast-derived tissue plasminogen activator

• Published in: Biochemistry (Easton) 1989-09, Vol.28 (19), p.7662-7669
• Main Authors: Wittwer, Arthur J, Howard, Susan C, Carr, Linda S, Harakas, Nikos K, Feder, Joseph, Parekh, Raj B, Rudd, Pauline M, Dwek, Raymond A, Rademacher, Thomas W
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Blood coagulation. Blood cells; cell lines; Cells, Cultured; Colon - cytology; Enzyme Activation - drug effects; Fibrin; Fibroblasts; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods, hemostasis, fibrinolysis; Glycosylation; Humans; man; Melanoma - pathology; Molecular and cellular biology; Plasminogen - metabolism; Tissue Plasminogen Activator - metabolism; Tumor Cells, Cultured; Tunicamycin - pharmacology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00445a022

Tissue-type plasminogen activator (t-PA), when isolated from human colon fibroblast (hcf) cells, is N-glycosylated differently than when isolated from the Bowes melanoma (m) cell line (Parekh et al., 1988). Both hcf- and m-t-PA can be separated into type I t-PA (with three occupied N-glycosylation sequons, at Asn-117, -184, and -448) and type II t-PA (with two occupied sequons, at Asn-117 and -448). Oligosaccharide analysis of each of these types of t-PA indicates that hcf-t-PA and m-t-PA have no glycoforms in common, despite having the same primary amino acid sequence. We have therefore compared in vitro the enzymatic activities and fibrin binding of type I and type II hcf- and m-t-PA with those of aglycosyl t-PA isolated from tunicamycin-treated cells. Plasminogen activation kinetics were determined by using an indirect amidolytic assay with Glu-plasminogen and a chromogenic plasmin substrate. In the absence of stimulator, there was little difference in activity between type I and type II t-PA, but the activity of aglycosyl t-PA was 2-4-fold higher than that of the corresponding glycosylated t-PA. In the presence of a fibrinogen fragment stimulator, the Kcat value of type II t-PA was approximately 5-fold that of type I t-PA from the same cell line, while the Km values for activation of Glu-plasminogen were similar (0.13-0.18 microM). The stimulated activity of glycosyl t-PA was similar to that of type II t-PA.