Epitope mapping for four monoclonal antibodies against human plasminogen activator inhibitor type-1: implications for antibody-mediated PAI-1-neutralization and vitronectin-binding.
The inhibitory mechanism of serine proteinase inhibitors of the serpin family is based on their unique conformational flexibility. The formation of a stable proteinase-serpin complex implies insertion of the reactive centre loop of the serpin into the large central beta-sheet A and a shift in the relative positions of two groups of secondary structure elements, the smaller one including alpha-helix F. In order to elucidate this mechanism, we have used phage-display and alanine scanning mutagenesis to map the epitopes for four monoclonal antibodies against alpha-helix F and its flanking region in the serpin plasminogen activator inhibitor-1 (PAI-1). One of these is known to inhibit the reaction between PAI-1 and its target proteinases, an effect that is potentiated by vitronectin, a physiological carrier protein for PAI-1. When combined with the effects these antibodies have on PAI-1 activity, our epitope mapping points to the mobility of amino-acid residues in alpha-helix F and the loop connecting alpha-helix F and beta-strand 3A as being important for the inhibitory function of PAI-1. Although all antibodies reduced the affinity of PAI-1 for vitronectin, the potentiating effect of vitronectin on antibody-induced PAI-1 neutralization is based on formation of a ternary complex between antibody, PAI-1 and vitronectin, in which PAI-1 is maintained in a state behaving as a substrate for plasminogen activators. These results thus provide new details about serpin conformational changes and the regulation of PAI-1 by vitronectin and contribute to the necessary basis for rational design of drugs neutralizing PAI-1 in cancer and cardiovascular diseases.
Wind
T
T
Laboratory of Cellular Protein Science, Department of Molecular and Structural Biology, University of Aarhus, Denmark. wind@biobase.dk
Jensen
M A
MA
Andreasen
P A
PA
D000409:Alanine / Q000235:genetics; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000494:pharmacology; D001666:Binding Sites, Antibody; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011487:Protein Conformation; D013045:Species Specificity; D019096:Vitronectin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D019151:Peptide Library; D017395:Plasminogen Activator Inhibitor 1; D019096:Vitronectin; D000409:Alanine