Immunological detection of conformational neoepitopes associated with the serpin activity of plasminogen activator inhibitor type-2.
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 beta-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).
Saunders
D N
DN
Department of Biological Sciences, University of Wollongong, Wollongong 2522, Australia.
Buttigieg
K M
KM
Gould
A
A
McPhun
V
V
Baker
M S
MS
D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D015153:Blotting, Western; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D017396:Plasminogen Activator Inhibitor 2 / Q000737:chemistry / Q000276:immunology*; D011487:Protein Conformation; D015843:Serpins / Q000737:chemistry / Q000276:immunology*; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D000939:Epitopes; D017396:Plasminogen Activator Inhibitor 2; D015843:Serpins