Glycosylation dependent conformational transitions in plasminogen activator inhibitor-1: evidence for the presence of two active conformations

Abstract Plasminogen activator inhibitor-1 (PAI-1) is a unique member of the serpin superfamily because of its conformational and functional flexibility. Different systems have been used to express PAI-1, e.g. Chinese hamster overy (CHO) cells, Escherichia coli cells and HT 1080 cells. Although glycosylation may influence the biochemical properties of proteins, to date minor differences have been observed between glycosylated and non-glycosylated PAI-1. In the present study, we have investigated the effect of glycosylation on the inactivation of PAI-1 by Triton X-100 and the associated pathways of conformational transitions. Whereas in the absence of Triton X-100, the observed PAI-1 stability was independent on the source of PAI-1, the addition of Triton X-100 revealed major glycosylation-dependent differences in the inactivation process of PAI-1. Incubation at 0°C in the presence of Triton X-100 resulted in a conversion of the active conformation to a substrate-like conformation for all PAI-1 molecules examined. The rate (k) of this conversion was 0.00016 s –1 and 0.00121 s –1 for non-glycosylated PAI-1 and CHO-PAI-1, respectively. When incubating non-glycosylated PAI-1 with 0.2% Triton X-100 at 37°C, two consecutive conformational transitions occur ultimately resulting in a complete conversion to the latent conformation. However, incubation of recombinant CHO-PAI-1 under these conditions yielded significantly different pathways of conformational transitions, i.e. a rapid conversion (k 1 >0.035 s –1 ) of part (39%) of the active conformation into a stable substrate conformation and a slower conversion (k 1 =0.0004 s –1 ) of the remaining part (61%) of the active conformation into the latent conformation, revealing the existence of two distinct active conformations. In conclusion, this is the first report describing significant differences between glycosylated and non-glycosylated PAI-1. Both the rate and the pathways of induced conformational transitions and concomitant inactivation of PAI-1 depend strongly on the glycosylation.