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

被引:28
作者
Saunders, DN
Buttigieg, KML
Gould, A
McPhun, V
Baker, MS [1 ]
机构
[1] Univ Wollongong, Dept Biol Sci, Wollongong, NSW 2522, Australia
[2] Biotech Australia Pty Ltd, Roseville, NSW 2069, Australia
[3] Australian Natl Univ, John Curtin Sch Med Res, Div Cell Biol, Canberra, ACT 2601, Australia
关键词
D O I
10.1074/jbc.273.18.10965
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The physiological roles of plasminogen activator inhibitor-a (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-P treated with tissue plasminogen activator, or thrombin suggests that the 2H5 epitope is determined exclusively by sequences found only on PAI-S following proteolytic cleavage of the Arg(380)-Thr(381) bond and insertion of the reactive site loop into beta-sheet A. Peptides lacking both the P13 (Glu(368)) and P14 (Thr(367)) 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).
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页码:10965 / 10971
页数:7
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