Importance of the Hinge Region between α-Helix F and the Main Part of Serpins, Based upon Identification of the Epitope of Plasminogen Activator Inhibitor Type 1 Neutralizing Antibodies

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-33...

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Bibliographic Details
Published in:The Journal of biological chemistry 2000-03, Vol.275 (9), p.6375-6380
Main Authors: Bijnens, Ann-Pascale, Gils, Ann, Knockaert, Isabelle, Stassen, Jan M., Declerck, Paul J.
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Antibodies, Monoclonal - immunology
Binding Sites
Epitopes - immunology
Humans
Models, Molecular
Molecular Sequence Data
Mutation
Plasminogen Activator Inhibitor 1 - chemistry
Plasminogen Activator Inhibitor 1 - immunology
Protein Structure, Secondary
Sequence Alignment
Serine Proteinase Inhibitors - chemistry
Serine Proteinase Inhibitors - genetics
Serine Proteinase Inhibitors - immunology
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Summary: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 Glu128-Val129-Glu130-Arg131and Lys154 (at the hinge region between α-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-33H1F7 for PAI-1 were 2.7 ± 1.6 × 109m−1 and 5.4 ± 1.7 × 109m−1, respectively, but decreased between 13- and 270-fold upon mutation of Lys154 to Ala154 or Glu128-Val129-Glu130-Arg131to 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 (KA = 4–5 × 109m−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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.275.9.6375