Influence of Six Mutations of the Protein C Gene on the Gla Domain Conformation and Calcium Affinity

Summary

The protein C Gla domain was studied in six families presenting a type II hereditary deficiency characterized by low activity in a coagulation assay and normal activity in an amidolytic assay. Five of these mutations, previously described by our group, affected Arg-5, Arg-1, Arg 229 and Ser 252. We report here the first natural Glu 7 to Asp mutation in a sixth family.

We evaluated the binding of the mutated protein C to H_n, a monoclonal antibody (mAb) known to recognize the sequence Phe 4 to Arg 9 of the Gla domain; the presence of calcium ions suppresses the recognition of this epitope by H_n. Mutation of Arg 229 to Gin and Ser 252 to Asn did not modify the inhibition of protein C binding, whereas the Arg-1 to His mutation resulted in a loss of inhibition in the presence of CaCl₂. This suggests that the protein C of this patient shows impaired carboxylation.

The protein C from patients bearing the mutations Arg-5 to Trp, Arg-1 to Cys and Glu 7 to Asp bound poorly to H_n mAb, even in the absence of calcium ions. The calcium affinity of the Gla domain was studied by pseudo-affinity chromatography, in which protein C was successively eluted from a Mono Q column by CaCl₂10 mM and NaCl 0.6 M. Protein C from the patient bearing the Arg-5 to Asp mutation had a normal elution profile, suggesting that a modification of the propeptide cleavage site impairs the conformation of the Gla domain but not carboxylation. Conversely, the patient bearing the mutation Arg-1 to Cys had an abnormal elution profile, showing that calcium binding affinity was impaired probably due to a defective carboxylation (the behaviour of the Glu 7 to Asp mutant lacking one Gla residue validated our experimental approach).

These results confirm that mutations in the serine protease domain do not modify the conformation of the N-terminal part of the Gla domain. Arg-1 to His, Arg-1 to Cys and Glu 7 to Asp mutations strongly modified affinity for calcium ions, whereas the Arg-5 to Trp mutation probably modifies the conformation of the Gla domain.

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