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 HUMANPROTEIN-S

Epidermal growth factor (EGF)-like modules in protein S, a physiologic al anticoagulant protein that functions as a cofactor to activated pro tein 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 seque nce determination. Ca-45(2+)-ligand blotting experiments indicated tha t 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 blott ing results, together with results of competitive binding experiments using monoclonal antibodies as structural probes, indicated that the r ecombinant proteins had been folded to a native conformation, EGF modu les 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 inter action. It is thus apparent that EGF module 1 is crucial Tor the inter action between protein S and activated protein C, Moreover, EGF module s 1-4 were approximately 10-fold more effective in inhibiting the inte raction than modules 1-3, suggesting a very weak interaction between m odule 4 and activated protein C or that this module is important to ke ep module 1 in a conformation that is optimal for interaction with act ivated protein C.