Functions of individual gamma-carboxyglutamic acid (Gla) residues of human protein c. Determination of functionally nonessential Gla residues and correlations with their mode of binding to calcium.
Previous studies from this laboratory have been directed toward elucidation of the roles of individual gamma-carboxyglutamic acid (Gla) residues in Gla domain-related Ca(2+)-directed properties of human protein C (PC) and activated protein C (APC). On the basis of results using recombinant variants of PC containing highly conservative (Asp) mutations of individual Gla residues, it was previously proposed that Gla6, Gla14, and Gla19 may not be essential for properties associated with the Ca(2+)-dependent conformation of the Gla domain of these proteins. In this study, we have demonstrated that radical mutations to Val of Gla residues 14 and 19 resulted in 94% and 82%, respectively, of the Gla domain-related, Ca(2+)- and phospholipid- (PL-) dependent anticoagulant (APTT) activity of wild-type recombinant (wtr) APC, while [Gla6-->Val]r-APC showed a complete loss of this same activity. The more conservative mutant [Gla6-->Gln]r-APC possessed 4% of the APTT activity of wtr-APC, whereas [Gla6-->Asp]r-APC was nearly fully active. As with wtr-PC, both [Gla6-->Val]r-PC and [Gla6-->Gln]r-PC displayed Ca(2+)-dependent intrinsic fluorescence quenching, suggesting that they adopted a Ca(2+)-induced conformation. However, Ca2+ titration data suggested that these conformations were not identical to that undergone by wtr-PC. In addition, the Ca(2+)-mediated binding parameters of [Gla6-->Val]r-PC and [Gla6-->Gln]r-PC to acidic PL vesicles were found to be defective. These data were interpreted at the molecular level using a model for the Gla domain of PC based on the X-ray crystal structure of the Ca2+/bovine prothrombin fragment 1 complex.(ABSTRACT TRUNCATED AT 250 WORDS)
Christiansen
W T
WT
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
Tulinsky
A
A
Castellino
F J
FJ
D015055:1-Carboxyglutamic Acid / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000925:Anticoagulants; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002460:Cell Line; D017077:Culture Media, Conditioned; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010743:Phospholipids / Q000494:pharmacology; D011486:Protein C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
D000925:Anticoagulants; D017077:Culture Media, Conditioned; D010743:Phospholipids; D011486:Protein C; D011994:Recombinant Proteins; D015055:1-Carboxyglutamic Acid; D002118:Calcium