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 cells using baculovirus. EGF modules 1-3, 1-4, 2-3 and 2-4 were produced on a preparative scale. The isolated modules were more than 95% homogeneous, as judged by sequence determination. 45Ca2+-ligand blotting experiments indicated that recombinant proteins that contained the fourth EGF module, i.e. EGF 1-4 and 2-4, bound Ca2+ with high affinity. The 45Ca2+-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 for 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.
Stenberg
Y
Y
Department of Clinical Chemistry, University of Lund, University Hospital, Malmö, Sweden.
Drakenberg
T
T
Dahlbäck
B
B
Stenflo
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses; D011487:Protein Conformation; D017293:Protein S / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
D017293:Protein S; D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; D002118:Calcium