The interaction of a Ca2+-dependent monoclonal antibody with the protein C activation peptide region. Evidence for obligatory Ca2+ binding to both antigen and antibody.
Protein C undergoes Ca2+-induced conformational changes required for activation by the thrombin-thrombomodulin complex. A Ca2+-dependent monoclonal antibody (HPC4) that blocks protein C activation was used to study conformational changes near the activation site in protein C. The half-maximal Ca2+ dependence was similar for protein C and gamma-carboxy-glutamic acid-domainless protein C for binding to HPC4 (205 +/- 23 and 110 +/- 29 microM Ca2+, respectively), activation rates (214 +/- 22 and 210 +/- 37 microM), and intrinsic fluorescence of gamma-carboxyglutamic acid-domainless protein C (176 +/- 34 microM). Protein C heavy chain binding to HPC4 was half-maximal at 36 microM Ca2+, although neither the heavy chain nor HPC4 separately bound Ca2+ with high affinity. The epitope was lost when the activation peptide was released. A synthetic peptide, P (6-17), which spans the activation site, exhibited Ca2+-dependent binding to HPC4 (half-maximal binding = 6 microM Ca2+). Thus, each decrease in antigen structure resulted in a reduced Ca2+ requirement for binding to HPC4. Tb3+ and Ca2+ binding studies demonstrated a Ca2+-binding site in HPC4 required for high affinity antigen binding. These studies provide the first direct evidence for a Ca2+-induced conformational change in the activation region of a vitamin K-dependent zymogen. Furthermore, Ca2+ binding to HPC4 is required for antigen binding. The multiple roles of Ca2+ described may be useful in interpretation of other metal-dependent antibody/antigen interactions.
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000936:Antigen-Antibody Complex / Q000032:analysis; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D004789:Enzyme Activation; D000939:Epitopes / Q000032:analysis; D006801:Humans; D011486:Protein C / Q000378:metabolism*; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000378:metabolism; D018179:Receptors, Thrombin; D013725:Terbium / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D011486:Protein C; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D013725:Terbium; D013917:Thrombin; D002118:Calcium