Mapping of the epitope of a monoclonal antibody protecting plasminogen activator inhibitor-1 against inactivating agents.
Plasminogen activator inhibitor-1 (PAI-1) belongs to the serpin family of serine proteinase inhibitors. Serpins inhibit their target proteinases by an ester bond being formed between the active site serine of the proteinase and the P1 residue of the reactive centre loop (RCL) of the serpin, followed by insertion of the RCL into beta-sheet A of the serpin. Concomitantly, there are conformational changes in the flexible joint region lateral to beta-sheet A. We have now, by site-directed mutagenesis, mapped the epitope for a monoclonal antibody, which protects the inhibitory activity of PAI-1 against inactivation by a variety of agents acting on beta-sheet A and the flexible joint region. Curiously, the epitope is localized in alpha-helix C and the loop connecting alpha-helix I and beta-strand 5A, on the side of PAI-1 opposite to beta-sheet A and distantly from the flexible joint region. By a combination of site-directed mutagenesis and antibody protection against an inactivating organochemical ligand, we were able to identify a residue involved in conferring the antibody-induced conformational change from the epitope to the rest of the molecule. We have thus provided evidence for communication between secondary structural elements not previously known to interact in serpins.
Bødker
Julie S
JS
Laboratory of Cellular Protein Science, Department of Molecular Biology, University of Aarhus, Denmark. jsb@mb.au.dk
Wind
Troels
T
Jensen
Jan K
JK
Hansen
Martin
M
Pedersen
Katrine E
KE
Andreasen
Peter A
PA
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000918:Antibody Specificity; D001665:Binding Sites; D003001:Cloning, Molecular; D000939:Epitopes / Q000737:chemistry; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016254:Mutagenesis, Insertional; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000276:immunology*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D012697:Serine Endopeptidases