Activated protein C-protein C inhibitor complex formation: characterization of a neoepitope provides evidence for extensive insertion of the reactive center loop.
Protein C inhibitor, a serine proteinase inhibitor (serpin), is the physiologically most important inhibitor of activated protein C. We have made a monoclonal antibody (M36) that binds with equally high affinity to an epitope present in activated protein C-protein C inhibitor complexes and cleaved loop-inserted protein C inhibitor. Insertion of a synthetic N-acetylated tetradecapeptide (corresponding to residues P1-P14 of the reactive center loop) into beta-sheet A of the uncleaved inhibitor also exposed the epitope. The antibody had no apparent affinity for native uncleaved inhibitor or for the free peptide. Synthetic P1-P14 analogues, with Arg P13 or Ala P9 substituted to the residues found in mouse protein C inhibitor (Thr and Ile, respectively), were also inserted in beta-sheet A. The Arg P13/Thr substitution led to a greatly impaired reactivity with the antibody, whereas the Ala P9/Ile mutation resulted in a modest loss of reactivity with the antibody. These results indicate that complex formation leads to insertion of the reactive center loop in beta-sheet A from Arg P14 and presumably beyond Ala P9. Moreover, to the best of our knowledge, this is the first instance where the neoepitope of a complexation-specific monoclonal antibody has been localized to the loop-inserted part of beta-sheet A, the part of the serpin where the complexation-induced conformational change is most conspicuous.
Strandberg
K
K
Department of Clinical Chemistry, University Hospital, Malmö, Lund University, S-205 Malmö, Sweden.
Kjellberg
M
M
Erb
E M
EM
Persson
U
U
Mosher
D F
DF
Villoutreix
B O
BO
Stenflo
J
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D000918:Antibody Specificity; D001665:Binding Sites; D003198:Computer Simulation; D004789:Enzyme Activation; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011486:Protein C / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D018046:Protein C Inhibitor / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D011486:Protein C; D018046:Protein C Inhibitor