Importance of N-terminal residues in plasminogen activator inhibitor 1 on its antibody induced latency transition.
The serpin plasminogen activator inhibitor-1 (PAI-1) is a well-known risk factor for thromboembolic and cardiovascular diseases. Many efforts have been made to reveal structure-function relationship in PAI-1, including the understanding of its unique latency transition. In this study, we describe the molecular characterization of PAI-1 neutralization by MA-159M12, a monoclonal antibody against rat PAI-1. Time-dependent inactivation of PAI-1, exposure of a trypsin cleavage site typically for the latent conformation and disappearance of elastase susceptibility revealed that MA-159M12 accelerated the active to latent, conformational transition (t1/2 120 +/- 12 min and 18 +/- 3.6 min in the absence and presence of MA-159M12, p < 0.0001). Cross-reactivity analysis of the antibody with various rat/human PAI-1 chimeras revealed that the epitope resides in alpha hA of rat PAI-1. Subsequent alanine-scanning mutagenesis and binding studies demonstrated that Pro2-Leu3-Pro4-Glu5 constitute the major residues of the epitope for MA-159M12. In conclusion, these findings demonstrate that, even though unexpected based on current knowledge on PAI-1 stability and function, interference with alpha hA results in a destabilisation of its active, inhibitory conformation. Therefore, alpha hA forms a putative target for the rational development of PAI-1 neutralizing components.
Ngo
Thu-Hoa
TH
Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Van Evenstraat 4, B-3000 Leuven, Belgium.
Zhou
Yan
Y
Stassen
Jean-Marie
JM
Declerck
Paul J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D018604:Epitope Mapping; D000939:Epitopes; D006801:Humans; D016297:Mutagenesis, Site-Directed; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D051381:Rats; D011993:Recombinant Fusion Proteins; D010959:Tissue Plasminogen Activator / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D017395:Plasminogen Activator Inhibitor 1; D011993:Recombinant Fusion Proteins; D010959:Tissue Plasminogen Activator