Studies of the interaction between human protein S and human C4b-binding protein using deletion variants of recombinant human protein S

Human protein S interacts noncovalently with human C4b-binding protein (C4BP). We have studied this interaction using deletion variants of recombinant human protein S. Two deletion variants were constructed by restriction enzyme digestion and in vitro site-specific mutagenesis of the human protein S cDNA. The variants were stably expressed in C127 cells. Recombinant proteins were purified using Fast Flow Q anion-exchange chromatography. The activated protein C (APC) cofactor activity, C4BP binding properties and reactivity to different monoclonal antibodies against human protein S were examined. The first variant (E variant), which has a deletion of the third epidermal growth factor (EGF)-like domain (deletion of exon VII, corresponding to amino acid residues ASP-160 to Asp-202) expresses normal APC cofactor activity in a plasma system. This activity was inhibited by the addition of purified C4BP. The second variant (L variant), which has a deletion of the C-terminal loop of the sex hormone binding globulin (SHBG-)like domain (deletion of exon XV, corresponding to amino acid residues Asp-583 to Ser-635) also expresses normal APC cofactor activity in plasma. This activity could only be partially inhibited by the addition of purified C4BP. Binding of the recombinant proteins to C4BP was studied in a system using purified proteins. The E variant binds to C4BP with the same affinity similar as recombinant wild type protein S (apparent K(d) ~ 10¹⁰ M). The L variant, however, shows a markedly reduced affinity for binding to C4BP (apparent K(d) ~ 10^-7 M). Three different Ca²⁺-independent monoclonal antibodies (S5, S12, S17) against protein S, which do not interfere with the APC cofactor activity and C4BP binding of protein S, were used to screen the deletion variants for possible conformational changes. Two of these showed the same affinity for the E and L variant as for wild type recombinant protein S. The third, S12, which recognizes an epitope in the vicinity of ser-460, reacts normally with the E variant but has a strongly reduced affinity for the L variant, although the presence of the epitope could be clearly demonstrated by immunoblotting under denaturing conditions. This suggests that the deletion of the C-terminal loop induces a conformational change in protein S which affects the epitope for S12. Therefore although our results indicate that the C-terminal loop of the SHBG-like domain of human protein S is involved in the interaction with C4BP, we cannot exclude the possibility that the deletion of the C-terminal loop induces a conformational change that results in a loss of binding affinity for C4BP elsewhere in the protein S molecule.