A conserved epitope on several human vitamin K-dependent proteins. Location of the antigenic site and influence of metal ions on antibody binding.
A murine monoclonal antibody (designated H-11) produced by injecting mice with purified human protein C was found to bind several human vitamin K-dependent proteins. Using a solid-phase competitive radioimmunoassay with antibody immobilized onto microtiter plates, binding of 125I-labeled protein C to the antibody was inhibited by increasing amounts of protein C, prothrombin, and Factors X and VII over a concentration range of 1 X 10(-8) to 1 X 10(-6) M. Other vitamin K-dependent proteins including Factor IX and protein S did not inhibit or inhibited only at the highest concentration binding of radiolabeled protein C to the immobilized antibody. Chemical treatment of prothrombin with a variety of agents including denaturation by sodium dodecyl sulfate, reduction with mercaptoethanol followed by carboxymethylation with iodoacetic acid, citraconylation of lysine residues, removal of metal ion with EDTA, or heat decarboxylation did not destroy the antigenic site recognized by the antibody as measured by immunoblotting of prothrombin or prothrombin derivative immobilized onto nitrocellulose. Immunoblotting of purified vitamin K-dependent polypeptides with the monoclonal antibody following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose indicated that the antigenic site was found on the light chains of protein C and Factor X. Chymotrypsin digestion of prothrombin and isolation on QAE-Sephadex of the peptide representing amino-terminal residues 1-44 of prothrombin further localized the antigenic site recognized by the monoclonal antibody to the highly conserved gamma-carboxyglutamic acid-containing domain. The exact location of the antigenic determinant for antibody H-11 was established using synthetic peptides. Antibody H-11 bound specifically to synthetic peptides corresponding to residues 1-12 of Factor VII and 1-22 of protein C. Comparison of protein sequences of bovine and human vitamin K-dependent proteins suggests that the sequence Phe-Leu-Glu-Glu-Xaa-Arg/Lys is required for antibody binding. The glutamic acid residues in this peptide segment are the first 2 gamma-carboxyglutamic acid residues near the amino-terminal end in the native proteins. Increasing concentrations of Ca2+, Mg2+, or Mn2+ partially inhibited binding of 125I-protein C to the antibody in a solid-phase assay system with half-maximal binding observed at divalent metal ion concentrations of 2, 4, and 0.6 mM, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001798:Blood Proteins / Q000032:analysis*; D002352:Carrier Proteins / Q000032:analysis*; D000939:Epitopes / Q000032:analysis*; D005164:Factor IX / Q000032:analysis; D005167:Factor VII / Q000032:analysis; D005170:Factor X / Q000032:analysis; D006023:Glycoproteins / Q000032:analysis; D006801:Humans; D008670:Metals / Q000378:metabolism*; D051379:Mice; D011486:Protein C / Q000032:analysis / Q000276:immunology; D017293:Protein S; D011516:Prothrombin / Q000032:analysis; D011863:Radioimmunoassay; D014812:Vitamin K / Q000032:analysis*
D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D002352:Carrier Proteins; D000939:Epitopes; D006023:Glycoproteins; D008670:Metals; D011486:Protein C; D017293:Protein S; D014812:Vitamin K; D005167:Factor VII; D011516:Prothrombin; D005164:Factor IX; D005170:Factor X