Immunological detection of conformational neoepitopes associated with the serpin activity of plasminogen activator inhibitor type-2

Darren N. Saunders, Kathy M L Buttigieg, Alison Gould, Virginia McPhun, Mark S. Baker*

*Corresponding author for this work

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

The physiological roles of plasminogen activator inhibitor-2 (PAI-2) are not yet well understood. Kinetic studies suggest a role in the regulation of plasminogen activator-driven proteolysis in many cell types. This study describes a monoclonal antibody (2H5), which uniquely recognizes neoepitope determinants on PAI-2 appearing after thermodynamic relaxation of the molecule. Enzyme-linked immunosorbent assays and native polyacrylamide gel electrophoresis immunoblotting confirmed the specificity of 2H5 for urokinase type plasminogen activator-PAI-2 complexes. Examination of the affinity of 2H5 for complexes formed between PAI-2 and a synthetic 14-mer reactive site loop peptide, PAI-2 treated with tissue plasminogen activator, or thrombin suggests that the 2H5 epitope is determined exclusively by sequences found only on PAI-2 following proteolytic cleavage of the Arg380-Thr381 bond and insertion of the reactive site loop into β-sheet A. Peptides lacking both the P13 (Glu368) and P14 (Thr367) residues did not induce a conformational change or affect the inhibitory activity of PAI-2, indicating that one or both of these residues are critical for PAI-2 function. To our knowledge, this is the first description of a monoclonal antibody that can distinguish conformational changes in PAI-2 related specifically to its potential biological function(s).

Original languageEnglish
Pages (from-to)10965-10971
Number of pages7
JournalJournal of Biological Chemistry
Volume273
Issue number18
DOIs
Publication statusPublished - 1 May 1998
Externally publishedYes

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