Chemical and Biomolecular Engineering Research and Publications


Conformational Changes in an Epitope Localized to the NH2-terminal Region of Protein C

Date of this Version

November 1989


Originally Published in The ournal of biological chemistry ,by American Society of Biochemistry and molecular biology Inc. in Vol 264,Issue of November 5 1989. pp- 18781-18788.


Murine monoclonal antibodies, developed following immunization with human protein C, were characterized for their ability tboin d antigen in thpe resence of either CaClz or excess EDTA. Three stablec lones were obtained which produced antiboditehsa t bound to protein C only itnh e presence of EDTA. Allt hree antibodies bound to the light choafi np rotein C on immunoblots and also bound to the homologous proteins factor X and prothrombin in solid-phase radioimmunoassays. One antibody, 7D7B10 was purified and studied further. The binding of 7D7B10 to human protein C was characterized bya KOo f 1.4 nM. In competition studies, it was found that the relative affinityo f the antibody for protein C was 20-40-fold higher than for prothrombin, fragment 1 of prothrombin, or factor X. In contrast, 7D7B10 was unable to bind to factor 1X or bovine protein C. The effect of varying Ca2+ concentration on the interaction of the antibody with protein C was complex. Low concentrations of Ca2+ enhanced the formation of the protein C-antibody complex with half-maximal effect occurring at approximately6 0 PM metal ion. However, higher concentrations of Ca2+ completely inhibited 7D7B10 binding to protein C with a K o .o~f 1.1 mM. Furthermore, millimolar concentrations of Mn2+, Ba2+, or Mg2+ also completely abolished antibody binding to protein C. The location of the epitope was delineatedby immunoblotting and peptide studies andf ound to be present in the NHz-termin1a5l residues of protein C. Although residues corresponding to positions 10-13 of human protein C wernee cessary for maximal binding of the antibody, they were not sufficient. No evidence could be found for involvement of the epitope in metal binding per se. Therefore, the effect of Ca2+ on antibody binding is thought to be due to metal-dependent conformational changes in protein C. It seems likelyth at Ca2+ occupatioonf a high affinity site, shown by others to be located in the epidermal growth factor-like domain, causes a conformational change in the NHz-terminarel gion of protein C which is favorable for antibody interaction, whereas Ca2+ binding to thleo w affinity site($, known to be present in the y-carboxyglutamic acid domain, causes an unfavorable conformational change.

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