Effect of carbohydrate side chain of tissue-type plasminogen activator on its interaction with plasminogen activator inhibitor-1

Abstract

Summary Tissue-type plasminogen activator (t-PA) has a variable N-glycosylation processing in different cell lines. We studied an effect of the carbohydrate side chain of t-PA molecule on the interaction of t-PA with plasminogen activator inhibitor-1 (PAI-1). First, we analysed the carbohydrate composition of t-PAs derived from human diploid fibroblasts (fibroblast t-PA) and Bowes melanoma cells (melanoma t-PA). Per 1 mol of protein fibroblast t-PA possesses 2.2 mol of sialic acid and 7.7 mol of mannose, whereas melanoma t-PA had 0.9 mol of sialic acid and 9.7 mol of mannose. The second-order rate constants (pH 7.4, 37°C) for the interactions between type I or type II single-chain t-PA and PAI-1 were determined after fractionation of t-PA by lysine-Sepharose chromatography. The rate constants of type I and type II single-chain fibroblast t-PA were significantly different ( P 6 and type II-1.70 ± 0.05 × 10 6 M −1 s −1 , respectively, whereas those of melanoma t-PA were almost similar. Sialidase treatment of type I single-chain fibroblast t-PA apparently increased the rate constant, 1.68 ± 0.10 × 10 6 M −1 s −1 , in almost an equal way to that of type II t-PA. On the other hand, modification of high mannose type oligosaccharide with endoglycosidase H treatment did not alter the rate constants of all types of t-PAs. These results suggest that the sialic acid of N-glycosylation at the site 184 on single-chain t-PA modulates the interaction with PAI-1.