Functional effects of single amino acid substitutions in the region of Phe113 to Asp138 in the plasminogen activator inhibitor 1 molecule.
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 coli and 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 (t12 22-31 min) at physiological pH and temperature than the other variants. However, in the presence of vitronectin they were both almost equally stable (t12 2.3 h) as wtPAI-1 (t12 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.
Sui
G C
GC
Department of Clinical Chemistry and Blood Coagulation, Karolinska Hospital, Karolinska Institute, S-171 76 Stockholm, Sweden.
Wiman
B
B
D019943:Amino Acid Substitution; D001224:Aspartic Acid; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D019791:Guanidine / Q000494:pharmacology; D006493:Heparin / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D012967:Sodium Dodecyl Sulfate / Q000378:metabolism; D013329:Structure-Activity Relationship; D013696:Temperature; D010959:Tissue Plasminogen Activator / Q000378:metabolism; D019096:Vitronectin / Q000378:metabolism
D017395:Plasminogen Activator Inhibitor 1; D019096:Vitronectin; D001224:Aspartic Acid; D012967:Sodium Dodecyl Sulfate; D010649:Phenylalanine; D006493:Heparin; D010959:Tissue Plasminogen Activator; D019791:Guanidine