Characterization of recombinant epidermal growth factor (EGF)-like modules from vitamin-K-dependent protein S expressed in Spodoptera cells -: The cofactor activity depends on the N-terminal EGF module in human protein S

Epidermal growth factor (EGF)-like modules in protein S, a physiological anticoagulant protein that functions as a cofactor to activated protein C, have been expressed in Spodoptera tells using baculovirus. EGF modules 1-3, 1-4, 2-3 and 2-4, were produced on a preparative scale. The isolated modules were mole than 95% homogenous, as judged by sequence determination. Ca-45(2+)-ligand blotting experiments indicated that recombinant proteins that contained the fourth EGF module, i.e. EGF 1-3 and 2-4, bound Ca2+ with high affinity. The Ca-45(2+)-ligand blotting results, together with results of competitive binding experiments using monoclonal antibodies as structural probes, indicated that the recombinant proteins had been folded to a native conformation, EGF modules 1-3 and 1-4 inhibited the interaction between activated protein C and protein S, whereas modules 2-3 and 2-4 had no effect on this interaction. It is thus apparent that EGF module 1 is crucial Tor the interaction between protein S and activated protein C, Moreover, EGF modules 1-4 were approximately 10-fold more effective in inhibiting the interaction than modules 1-3, suggesting a very weak interaction between module 4 and activated protein C or that this module is important to keep module 1 in a conformation that is optimal for interaction with activated protein C.