The gamma-carboxylation recognition site is sufficient to direct vitamin K-dependent carboxylation on an adjacent glutamate-rich region of thrombin in a propeptide-thrombin chimera.
The propeptides of the vitamin K-dependent proteins contain a gamma-carboxylation recognition site that is required for gamma-glutamyl carboxylation. To determine whether the propeptide is sufficient to direct carboxylation, two mutant prothrombin species were expressed and characterized with regard to posttranslational gamma-carboxylation. A double point mutant, in which serine substituted for cysteines 17 and 22 disrupted a conserved loop formed by a disulfide bond, was fully carboxylated when expressed in Chinese hamster ovary cells. A propeptide/thrombin chimeric protein, constructed by deleting the Gla, aromatic amino acid stack, and kringle domains of prothrombin, has the signal peptide and propeptide juxtaposed to a glutamate-rich COOH-terminal region of prothrombin, residues 249-530. Of the 8 glutamic acid residues contained within the first 40 residues of the NH2 terminus adjacent to the propeptide, at least seven were fully carboxylated as demonstrated by direct gamma-carboxyglutamic acid analysis of the alkaline hydrolysate and by NH2-terminal sequence analysis. These results indicate that the gamma-carboxylation recognition site within the prothrombin propeptide in a prothrombin propeptide-thrombin chimeric protein is sufficient to direct gamma-carboxylase-catalyzed carboxylation of adjacent glutamic acid residues in a glutamate-rich region of thrombin that is not normally gamma-carboxylated. Furthermore, the disulfide loop in the Gla domain of prothrombin is not required for complete carboxylation.
Furie
B C
BC
Center for Hemostasis and Thrombosis Research, Division of Hematology-Oncology, New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Ratcliffe
J V
JV
Tward
J
J
Jorgensen
M J
MJ
Blaszkowsky
L S
LS
DiMichele
D
D
Furie
B
B
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D019735:Carbon-Carbon Ligases / Q000378:metabolism*; D006224:Cricetinae; D004220:Disulfides / Q000737:chemistry; D018698:Glutamic Acid / Q000378:metabolism*; D018082:Kringles; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011516:Prothrombin / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013917:Thrombin / Q000235:genetics / Q000378:metabolism*; D014812:Vitamin K / Q000378:metabolism*
D004220:Disulfides; D010446:Peptide Fragments; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C050961:prothrombin precursor (13-29); D014812:Vitamin K; D018698:Glutamic Acid; D011516:Prothrombin; D013917:Thrombin; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase