Substrate behavior of plasminogen activator inhibitor-1 is not associated with a lack of insertion of the reactive site loop.
Plasminogen activator inhibitor-1 (PAI-1) is a unique member of the serpin superfamily. In the present study, we have evaluated the effect of substitution, with a proline, at positions P5, P7, P14, P15, or P16, on the conformational flexibility and functional properties of PAI-1. These mutants (PAI-1-P5, IIe-->Pro at P5; PAI-1-P7, Ala-->Pro at P7; PAI-1-P14, Thr-->Pro at P14; PAI-1-P15, Gly-->Pro at P15; PAI-1-P16, Ser-->Pro at P16) were purified and fully characterized. WtPAI-1 had a specific activity of 68 +/- 10% (mean +/- SD, n = 6) whereas PAI-1-P5, PAI-1-P7, and PAI-1-P16 had specific activities of 34 +/- 9.3%, 42 +/- 10%, and 36 +/- 11%, respectively. PAI-1-P14 and PAI-1-P15 did not exhibit significant inhibitory activity. Conformational analysis revealed that wtPAI-1 preparations contained 12 +/- 2.0% substrate, whereas PAI-1-P5, PAI-1-P7, and PAI-1-P16 were characterized with a significantly (p < 0.001) increased substrate behavior (i.e., 43 +/- 6.1%, 42 +/- 1.5% and 22 +/- 1.7%, respectively). The inactive variants PAI-1-P14 and PAI-1-P15 behaved exclusively as substrates toward various serine proteinases. Heat denaturation studies revealed that cleavage of any noninhibitory substrate form of PAI-1 resulted in an insertion of the NH2-terminal side of the reactive site loop. Incubation with plasmin showed the presence of a unique plasmin cleavage site (Lys191-Ser192) exclusively present in all latent forms studied. We conclude that (a) the entire P5 to P16 region in PAI-1 plays an important role in the functional and conformational properties of PAI-1; (b) the substrate behavior of serpins is not associated with a lack of insertion of the reactive site loop; (c) the identification of a plasmin cleavage site in latent PAI-1 may provide new insights in the mechanisms for the inactivation of storage pools of PAI-1.
Gils
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A
Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium.
Knockaert
I
I
Declerck
P J
PJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli; D005341:Fibrinolysin / Q000378:metabolism; D014644:Genetic Variation; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D017354:Point Mutation; D011392:Proline; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D015183:Restriction Mapping; D012697:Serine Endopeptidases / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000378:metabolism*; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D011392:Proline; D012697:Serine Endopeptidases; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator