Construction and characterization of plasminogen activator inhibitor-1 mutants in which part of the active site loop is deleted

Abstract

Plasminogen activator inhibitor-1 (PAI-1) can occur in a labile active inhibitory conformation, a non-inhibitory but cleavable substrate conformation and a non-reactive latent conformation. The active conformation is not stable and converts spontaneously into the latent conformation. To prevent this conversion, we have constructed, purified and characterized two mutants in which part of the reactive site loop is deleted: PAI-1-d(P6-P4) lacking residues P6 to P4 (Val-Ile-Val), and PAI-1-d(P9-P4) lacking residues P9 to P4 (Ser-Thr-Ala-Val-Ile-Val). Wild-type PAI-1 (wtPAI-1) revealed a specific activity of 54 ± 8% (mean ± SD, n = 4) of the theoretical maximum value towards tissue-type plasminogen activator (t-PA). Both deletion mutants revealed no inhibitory activity towards t-PA or towards urokinase-type plasminogen activator (u-PA). Conformational analysis of PAI-1-d(P6-P4) and PAI-1-d(P9-P4) revealed the formation of a cleaved derivative originating from the substrate form (55 ± 3% and 84 ± 8%, respectively) and non-reactive material (45 ± 3% and 16 ± 8%, respectively). Incubation at 37°C resulted in the conversion of the substrate conformation of both PAI-1 mutants into a non-reactive conformation. Reactivation experiments with guanidinium chloride revealed that for both mutants this non-reactive conformation represented a latent form from which the substrate properties could be restored up to 70–90%. In contrast to the latent form of wtPAI-1 and PAI-1-d(P6-P4), the non-reactive conformation of PAI-1-d(P9-P4) is not susceptible to plasmin cleavage outside the reactive site loop. Heat denaturation experiments revealed that the substrate conformation, as well as the latent conformation and the cleaved substrate derivative of PAI-1-d(P6-P4) and PAI-1-d(P9-P4), are less resistant to denaturation compared to wtPAI-1. In conclusion, shortening the reactive site loop of PAI-1 results in non-inhibitory mutants with an increased substrate behaviour. In contrast to the stable substrate conformation of wtPAI-1, the substrate conformation of these deletion mutants reveals an unexpected transition to the latent form.