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Title: Importance of the hinge region between alpha-helix F and the main part of serpins, based upon identification of the epitope of plasminogen activator inhibitor type 1 neutralizing antibodies
Authors: Bijnens, AP
Gils, Ann
Knockaert, I
Stassen, JM
Declerck, Paul # ×
Issue Date: Mar-2000
Publisher: Amer soc biochemistry molecular biology inc
Series Title: Journal of Biological Chemistry vol:275 issue:9 pages:6375-6380
Abstract: The serpin plasminogen activator inhibitor type 1 (PAI-1) is an important protein in the regulation of fibrinolysis and inhibits its target proteinases through formation of a covalent complex. In the present study, we have identified the epitope of two PAI-1 neutralizing monoclonal antibodies (MA-33H1F7 and MA-55F4C12). Based upon differential cross-reactivity data of these monoclonals with PAI-1 from different species and on a sequence alignment between these PAI-1s, combined with the three-dimensional structure, we predicted that the residues Glu(128)-Val(129)-Glu(130)-Arg(131) and Lys(154) (at the hinge region between alpha-helix F and the main part of the PAI-1-molecule) might form the major site of interaction. Therefore a variety of alanine mutants were generated and evaluated for their affinity toward both monoclonal antibodies. The affinity constants of MA-55F4C12 and MA-33HIF7 for PAI-1 were 2.1 +/- 1.6 x 10(9) M-1 and 5.4 +/- 1.7 x 10(9) M-1, respectively, but decreased between 13- and 270-fold upon mutation of Lys(154) to Ala(154) or Glu(128)-Val(129)-Glu(130)-Arg(131) to Ala-Ala-Ala-Ala. The combined mutations (PAI-1-EVER/K), however, resulted in an absence of binding to either of the antibodies. Both antibodies bound to PAI-1-wt/t-PA complexes with a similar affinity as to PAI-1-wt (K-A = 4-5 x 10(9) M-1). The epitope localization reveals the molecular basis for the neutralizing properties of both monoclonal antibodies. In addition, it provides new insights into the validity of various models that have been proposed for the serpin/proteinase complex, excluding full insertion of the reactive-site loop.
URI: 
ISSN: 0021-9258
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Laboratory for Pharmaceutical Biology (-)
× corresponding author
# (joint) last author

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