FUNCTIONAL-EFFECTS OF SINGLE AMINO-ACID SUBSTITUTIONS IN THE REGION OF PHE(113) TO ASP(138) IN THE PLASMINOGEN-ACTIVATOR INHIBITOR-1 MOLECULE

Thirteen amino acid substitutions have been introduced within the stre tch phe(113) to Asp(138) in the plasminogen activator inhibitor 1 (PAI -1) molecule by site-directed mutagenesis. The different proteins and wild-type (wt) PAI-I have been overexpressed in Escherichia coil and p urified by chromatography on heparin-Sepharose and on anhydrotrypsin-a garose. The PAI-1 variants have been characterized by their reactivity with tissue plasminogen activator (tPA), interactions with vitronecti n or heparin, and stability. Most PAI-I variants, except for Asp(125) --> Lys, phe(126) --> Ser and Arg(133) --> Asp, displayed a high spont aneous inhibitory activity towards CPA, which did not change greatly o n reactivation with 4 M guanidinium chloride, followed by dialysis at pH 5.5. The variants Asp(125) --> Lys and Arg(133) --> Asp became much more active after reactivation and they were also more rapidly transf ormed to inactive forms (t(1/2)22-31 min) at physiological pH and temp erature than the other variants. However, in the presence of vitronect in they were both almost equally stable (t(1/2) 2.3 h) as wtPAI-1 (t(1 /2) 3.0 h). The mutant Glu(130) --> Lys showed an increased stability, both in the absence and in the presence of vitronectin compared with wtPAI-1. Nevertheless a similar affinity between all the active PAI-1 variants and vitronectin was observed. Further, all mutants, including the three mutants with low activity, were to a large extent adsorbed on anhydrotrypsin-agarose and were eluted in a similar fashion. In acc ordance with these data, the three variants with a low activity were a ll to a large extent cleaved as a result of their reaction with CPA, s uggesting that they occurred predominantly in the substrate conformati on. Our results do not support the presence of a binding site for vitr onectin in this part of the molecule, but rather that it might be invo lved in controlling the active PAI-1 to substrate transition. Partly, this region of the PAI-1 molecule (Arg(115) to Arg(118)) seems also to be involved in the binding of heparin to PAI-1.