The functions of the first epidermal growth factor homology region of human protein C as revealed by a charge-to-alanine scanning mutagenesis investigation

Variant proteins containing charge-to-alanine mutations of single amino acid residues and clusters of such groups contained in the epidermal growth factor 1 (EGF1) homology unit of human protein C (PC) have been accomplished, resulting in the following recombinant (r) mutant proteins: r-[E⁵⁶A/H⁵⁷A]PC; r-[H⁶⁶A]PC; r-[D⁷¹A]PC; r-[D⁷⁹A/R⁸¹A]PC; r-[E⁸⁵A/R⁸⁷A]PC; and r-[R⁹¹A/E⁹²A]PC. Studies of the mutant proteins with a variety of Ca²⁺-dependent and Ca²⁺-independent monoclonal antibodies not only led to identification of the epitopes of these antibodies, but also confirmed the importance of D/β-hydroxyaspartic acid (Hya)⁷¹ as one probable coordination site for Ca²⁺. Employing these antibodies, it was also revealed that Ca²⁺ binding to its site in the EGF1 region of PC did not influence Ca²⁺ binding or adoption of the Ca²⁺-dependent conformation of the gamma-carboxyglutamic acid domain of this same protein. In addition, the Ca²⁺-induced inhibition of PC activation by thrombin, and the kinetic constants for activation of PC by the thrombin/thrombomodulin complex, were only modestly affected by any of the mutations. The mutants r-[E⁵⁶A/H⁵⁷A]APC and r-[H⁶⁶A]APC displayed at least 70% of wild type r-APC activity in a fVIII inactivation assay, while r-[D⁷⁹A/R⁸¹A]APC, r-[E⁸⁵A/R⁸⁷A]APC and r-[R⁹¹A/E⁹²A]APC possessed only approximately 40% activity in that same assay. The special role of D/Hya⁷¹ in this process was confirmed by showing that r-[D⁷¹A]APC was inactive in the fVIII-inactivation assay. These findings demonstrate that some of the charged residues of EGF1, most notably those in the carboxyterminal region of this domain, participate as partial determinants of the anticoagulant activity of APC. Overall, with the exceptions noted, the data generally suggest that the charged residues of the EGF1 domain of PC, and the Ca²⁺ binding site contained within this module, are likely more involved with maintenance of the overall structural integrity of this module rather than with its direct functional interactions with effectors, activators, or substrates of PC and APC. Lastly, functional Ca²⁺ binding to the Gla domain of PC is not significantly influenced by the binding of Ca²⁺ to the EGF1 module.