Characterization of plasminogen activator inhibitor 1 mutants containing the P13 to P10 region of ovalbumin or antithrombin III: evidence that the P13 residue contributes significantly to the active to substrate transition

• Published in: Biochimica et biophysica acta Vol. 1387; no. 1; pp. 291 - 297
• Main Authors: Gils, Ann, Vleugels, Nele, Tobback, Kristof, Knockaert, Isabelle, Declerck, Paul J.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 08.09.1998
• Subjects: Antithrombin III - chemistry; Chimeric; Conformational transition; Enzyme Stability - genetics; Kinetics; Mutant; Mutation - genetics; Ovalbumin - chemistry; Plasminogen activator inhibitor 1; Plasminogen Activator Inhibitor 1 - chemistry; Protein Conformation; Recombinant Fusion Proteins - chemistry; Serine Endopeptidases - metabolism; Serine Proteinase Inhibitors - chemistry; Serpin; Tissue Plasminogen Activator - metabolism; Urokinase-Type Plasminogen Activator - metabolism; PAI-1-ovalbumin, PAI-1 in which the P13 to P10 region has been replaced by the corresponding region of ovalbumin; serpin, serine proteinase inhibitor; Chimeric; PAI-1-antithrombin III, PAI-1 in which the P13 to P10 region has been replaced by the corresponding region of antithrombin III; wt, wild-type; t-PA, tissue-type plasminogen activator; PAI-1, plasminogen activator inhibitor-1; Plasminogen activator inhibitor 1; Serpin; PAI-1-P13 (Val→Glu), PAI-1 in which Val at position P13 has been replaced by Glu; PBS, phosphate-buffered saline; u-PA, urokinase-type plasminogen activator; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; Mutant; Conformational transition
• DOI: 10.1016/S0167-4838(98)00139-3
• ISSN: 0167-4838; 0006-3002

The serpin plasminogen activator inhibitor 1 (PAI-1) can occur, in vitro, in both an inhibitory and a non-inhibitory but cleavable substrate form. In the present study, we have evaluated the effect of replacing the P13 to P10 region of PAI-1 (Val-Ala-Ser-Ser), with the P13 to P10 region of either the non-inhibitory serpin ovalbumin (Glu-Val-Val-Gly; PAI-1-ovalbumin) or the inhibitory serpin antithrombin III (Glu-Ala-Ala-Ala; PAI-1-antithrombin III). In addition, we have replaced Val at position P13 with Glu (PAI-1-P13 (Val→Glu)). Wild-type (wt) PAI-1 revealed specific activities of 80±9% (mean±S.D., n=4) of the theoretical maximum value towards t-PA. PAI-1-ovalbumin, PAI-1-antithrombin III and PAI-1-P13 (Val→Glu) revealed specific activities of 86±15%, 77±11%, and 100±30% respectively, towards t-PA and similar inhibitory properties towards u-PA. Surprisingly, upon inactivation at 37°C, the active conformation of the PAI-1 mutants converted partly into a substrate conformation (i.e. 52±5.2%, 55±8.2% and 46±4.6% for PAI-1-ovalbumin, PAI-1-antithrombin III and PAI-1-P13 (Val→Glu), respectively) and partly into a latent conformation. This is in contrast to active wtPAI-1 which, as expected, is converted to the latent conformation (i.e. 86±1.0%). In conclusion, even though replacement of the P13 to P10 region of PAI-1 by the corresponding region of a non-inhibitory serpin or of an inhibitory serpin, does not directly affect its inhibitory properties, the nature of the amino acids in this region and of P13 in particular, contributes to its conformational transitions.