title: The distal hinge of the reactive site loop and its proximity: a target to modulate plasminogen activator inhibitor-1 activity.
abstract: The serpin plasminogen activator inhibitor type 1 (PAI-1) plays a regulatory role in various physiological processes (e.g. fibrinolysis and pericellular proteolysis) and forms a potential target for therapeutic interventions. In this study we identified the epitopes of three PAI-1 inhibitory monoclonal antibodies (MA-44E4, MA-42A2F6, and MA-56A7C10). Differential cross-reactivities of these monoclonals with PAI-1 from different species and sequence alignments between these PAI-1s, combined with the three-dimensional structure, revealed several charged residues as possible candidates to contribute to the respective epitopes. The production, characterization, and subsequent evaluation of a variety of alanine mutants using surface plasmon resonance revealed that the residues His(185), Arg(186), and Arg(187) formed the major sites of interaction for MA-44E4. In contrast, the epitopes of MA-42A2F6 and MA-56A7C10 were found to be conformational. The epitope of MA-42A2F6 comprises residues Lys(243) and Glu(350), whereas the epitope of MA-56A7C10 comprises residues Glu(242), Lys(243), Glu(244), Glu(350), Asp(355), and Arg(356). The participation of Glu(350), Asp(355), and Arg(356) provides a molecular explanation for the differential exposure of this epitope in the different conformations of PAI-1 and for the effect of these antibodies on the kinetics of the formation of the initial PAI-1-proteinase complexes. The localization of the epitopes of MA-44E4, MA42A2F6, and MA-56A7C10 elucidates two previously unidentified molecular mechanisms to modulate PAI-1 activity and opens new perspectives for the rational development of PAI-1 neutralizing compounds.
authors.affiliation: Laboratory for Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmaceutical Sciences, Katholieke Universiteit Leuven, Belgium.
authors.forename: A P
authors.initials: AP
authors.lastname: Bijnens
authors.forename: A
authors.initials: A
authors.lastname: Gils
authors.forename: J M
authors.initials: JM
authors.lastname: Stassen
authors.forename: A A
authors.initials: AA
authors.lastname: Komissarov
authors.forename: I
authors.initials: I
authors.lastname: Knockaert
authors.forename: E
authors.initials: E
authors.lastname: Brouwers
authors.forename: J D
authors.initials: JD
authors.lastname: Shore
authors.forename: P J
authors.initials: PJ
authors.lastname: Declerck
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001120:Arginine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D000939:Epitopes; D018698:Glutamic Acid / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D013552:Swine; D013997:Time Factors
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D017395:Plasminogen Activator Inhibitor 1; D001224:Aspartic Acid; D018698:Glutamic Acid; D006639:Histidine; D004247:DNA; D001120:Arginine; D008239:Lysine; D000409:Alanine