NATURALLY-OCCURRING ARG(-1) TO HIS MUTATION IN HUMAN PROTEIN-C LEADS TO ABERRANT PROPEPTIDE PROCESSING AND SECRETION OF DYSFUNCTIONAL PROTEIN-C

The dysfunctional protein C from a thrombophilic patient heterozygote for a G(1388) to A converting the codon for Arg(-1) to His was purifie d from plasma and characterized. N-terminal amino acid sequence analys is of the light chain of the protein C demonstrated that the dysfuncti onal protein C is elongated with one amino acid, namely the mutated Hi s. This finding is compatible with disruption by the mutated His of th e original basic propeptidase recognition sequence (Arg(-5)-Ile-Arg-Ly s-Arg(-1)), resulting in a shift of the cleavage site to a new positio n, Lys(-2)-His(-1), which follows an alternative basic amino acid prop eptidase recognition sequence (Arg(-5)-Ile-Arg-Lys(-2)). Because the m utation affects the propeptide that directs the gamma-carboxylation co nverting Glu to Gla residues in the Gla domain, it was investigated wh ether the mutation impaired this reaction. Gla fragment obtained by cl eavage of the dysfunctional protein C light chain with endoproteinase Asp-N was isolated by reverse-phase highperformance liquid chromatogra phy, methylated, and subjected to N-terminal sequence analysis, The me thylation step enabled the positive identification of Gla residues as well as the determination of the relative amount of Gla and Glu residu es at each of the nine gamma-carboxylation sites of the Gla domain, Th e analysis showed that all nine potential gamma-carboxylation sites of the dysfunctional protein C were normally carboxylated. This result i s compatible with the notion that position -1 is not a part of the rec ognition element for the gamma-carboxylase, In conclusion, evidence is provided showing that the mutation leads to aberrant propeptide proce ssing and secretion of dysfunctional normally carboxylated protein C e xtended with the mutated His. (C) 1997 by The American Society of Hema tology.