Epitope mapping of monoclonal antibodies directed to PAI-1 using PAI-1/PAI-2 chimera and PAI-1-derived synthetic peptides.
Plasminogen activator inhibitor type-1 is a key regulatory protein of the fibrinolytic system that is involved in a variety of physiological and pathophysiological processes. A panel of 14 monoclonal antibodies directed against plasminogen activator inhibitor type-1 was analyzed regarding epitope specificity on plasminogen activator inhibitor type-1. For this purpose, chimera consisting of plasminogen activator inhibitor type-1 and another plasminogen activator inhibitor, plasminogen activator inhibitor type-2, with different portions of the respective wild-type proteins, were generated and plasminogen activator inhibitor type-1-derived 20-mer and 10-mer linear peptides were synthesized. Nine of the 14 monoclonal antibodies recognized an epitope located in the region between amino acid 76-188 of plasminogen activator inhibitor type-1, which encompasses the binding sites for vitronectin, heparin, and part of the fibrin binding region. Of these nine monoclonal antibodies, six reacted with a quadruple plasminogen activator inhibitor type-1 mutant (N152H, K156T, Q321L, M356I), and seven detected a plasminogen activator inhibitor type-1 deletion mutant (DeltaF111-H114). Two of the remaining five monoclonal antibodies recognized epitopes located between amino acid 209-227 and amino acid 352-371, respectively, while the other three antibodies reacted with wild-type plasminogen activator inhibitor type-1, only. Additional experiments revealed that two of the 14 mAbs neutralized and one monoclonal antibodies increased plasminogen activator inhibitor type-1 activity toward urokinase-type plasminogen activator, one of its target proteases.
Muehlenweg
B
B
Frauenklinik, München, Germany.
Guthaus
E
E
de Prada
N A
NA
Schmitt
M
M
Schmiedeberg
N
N
Kotzsch
M
M
Creutzburg
S
S
Kramer
M D
MD
Kessler
H
H
Wilhelm
O G
OG
Magdolen
V
V
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D018604:Epitope Mapping; D006801:Humans; D016056:Immunodominant Epitopes; D010446:Peptide Fragments / Q000276:immunology; D017395:Plasminogen Activator Inhibitor 1 / Q000276:immunology*; D017396:Plasminogen Activator Inhibitor 2 / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D016056:Immunodominant Epitopes; D010446:Peptide Fragments; D017395:Plasminogen Activator Inhibitor 1; D017396:Plasminogen Activator Inhibitor 2; D011993:Recombinant Fusion Proteins