Functional effects of single amino acid substitutions in the region of Phe113 to Asp138 in the plasminogen activator inhibitor 1 molecule

Abstract

Thirteen amino acid substitutions have been introduced within the stretch Phe¹¹³ to Asp¹³⁸ in the plasminogen activator inhibitor 1 (PAI-1) molecule by site-directed mutagenesis. The different proteins and wild-type (wt) PAI-1 have been overexpressed in Escherichia coliand purified by chromatography on heparin–Sepharose and on anhydrotrypsin–agarose. The PAI-1 variants have been characterized by their reactivity with tissue plasminogen activator (tPA), interactions with vitronectin or heparin, and stability. Most PAI-1 variants, except for Asp¹²⁵ → Lys, Phe¹²⁶ → Ser and Arg¹³³ → Asp, displayed a high spontaneous inhibitory activity towards tPA, which did not change greatly on reactivation with 4 M guanidinium chloride, followed by dialysis at pH 5.5. The variants Asp¹²⁵ → Lys and Arg¹³³ → Asp became much more active after reactivation and they were also more rapidly transformed to inactive forms (t_½ 22–31 min) at physiological pH and temperature than the other variants. However, in the presence of vitronectin they were both almost equally stable (t_½ 2.3 h) as wtPAI-1 (t_½ 3.0 h). The mutant Glu¹³⁰ → 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 accordance with these data, the three variants with a low activity were all to a large extent cleaved as a result of their reaction with tPA, suggesting that they occurred predominantly in the substrate conformation. Our results do not support the presence of a binding site for vitronectin in this part of the molecule, but rather that it might be involved in controlling the active PAI-1 to substrate transition. Partly, this region of the PAI-1 molecule (Arg¹¹⁵ to Arg¹¹⁸) seems also to be involved in the binding of heparin to PAI-1.