title: Identification of a target site in plasminogen activator inhibitor-1 that allows neutralization of its inhibitor properties concomitant with an allosteric up-regulation of its antiadhesive properties.
abstract: The serpin plasminogen activator inhibitor-1 (PAI-1) has a dual function: 1) it plays an important role as a direct inhibitor of the plasminogen activation system, and 2) its interaction with the adhesive glycoprotein vitronectin suggests a role in tissue remodeling and metastasis, independent from its proteinase inhibitory properties. Unique to this serpin is the close association between its conformational and functional properties. Indeed, PAI-1 can occur in an active and a latent conformation, but both functions are exclusively present in the active conformation. We report here the epitope localization and functional effects of a monoclonal antibody (MA-124K1) that inhibits rat PAI-1 activity and simultaneously increases the binding of inactive PAI-1 to vitronectin (the affinity constant of PAI-1 for vitronectin is 2 x 10(7) m(-1) in the absence of MA-124K1 and 160 x 10(7) m(-1) in the presence of MA-124K1). To the best of our knowledge, this is the first monoclonal antibody dissociating the proteinase inhibitory properties from the vitronectin binding properties in PAI-1. Mutation of Glu(212) and/or Glu(220) in rat PAI-1 to Ala results in a strongly reduced affinity or absence of binding to MA-124K1. The three-dimensional structure of PAI-1 reveals that these residues constitute a conformational epitope close to the reactive-site loop and compatible with the effect of MA-124K1 on the inhibitory properties of PAI-1. However, the vitronectin binding site is localized at the opposite site of the molecule, indicating that the effect of MA-124K1 involves an allosteric modulation of the vitronectin binding site. Cell culture experiments revealed a significant reduction of cell attachment and migration in the presence of MA-124K1, providing evidence for the functional relevance of this antibody-mediated up-regulation of the vitronectin binding properties of PAI-1. In conclusion, a novel mechanism for interference with PAI-1 functions has been identified and is of importance in the modulation of cell migration and related events (e.g. tumor metastasis).
authors.affiliation: Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium.
authors.forename: T H
authors.initials: TH
authors.lastname: Ngo
authors.forename: M F
authors.initials: MF
authors.lastname: Hoylaerts
authors.forename: I
authors.initials: I
authors.lastname: Knockaert
authors.forename: E
authors.initials: E
authors.lastname: Brouwers
authors.forename: P J
authors.initials: PJ
authors.lastname: Declerck
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D000906:Antibodies; D018604:Epitope Mapping; D006801:Humans; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D015854:Up-Regulation; D019096:Vitronectin / Q000737:chemistry / Q000378:metabolism
chemical_list: D000906:Antibodies; D017395:Plasminogen Activator Inhibitor 1; D019096:Vitronectin