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

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Abstract
Tissue-type plasminogen activator (t-PA), when isolated from human colon firboblast (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 is 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 than that of type I t-PA from the same cell line, while the Km values for activation of Gly-plasminogen were similar (0.13-0.18 .mu.M). The stimulated activity of glycosyl t-PA was similar to that of type II t-PA. No effect of glycosylation was seen when each t-PA was assayed directly with H-D-Val-Gly-Arg-p-nitroanilide. In a clot lysis assay, type II t-PA was 23-26% more active than the type I t-PA from the same cell line, while type I and type II m-t-PA were about 30% more active, respectively, than type I and type II hcf-t-PA. The fibrin binding ability of each t-PA correlated very well with clot lysis activity but not with the stimulated indirect amidolytic activity. Together, these results suggest that sequon occupancy of Asn-184 (in addition to Asn-117 and -448) significantly decreases the fibrin-dependent stimulation of t-PA activity. Occupancy of sequons Asn-117 and -448 decreases the unstimulated activity of t-PA, and the nature of the oligosaccharide at Asn-448 influences both fibrin and clot lysis activity.

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