Propeptide recognition by the vitamin K-dependent carboxylase in early processing of prothrombin and factor X.
Precursors of vitamin K-dependent proteins are synthesized with a propeptide that is believed to target these proteins for gamma-carboxylation by the vitamin K-dependent carboxylase. In this study synthetic propeptides were used to investigate gamma-carboxylation of the prothrombin and factor X precursors in rat liver microsomes. The extent of prothrombin processing by the carboxylase was also investigated. Antisera raised against the human prothrombin and factor X propeptides only recognized precursors with the respective propeptide regions. The data demonstrate structural differences in the propeptide region of the prothrombin and the factor X carboxylase substrates which raises questions about the hypothesis of a common propeptide binding site on the carboxylase for all precursors of vitamin K-dependent proteins. The hypothesis of separate binding sites is supported by data which demonstrate differences in binding of the prothrombin and factor X precursors to membrane fragments from rough and smooth microsomes. gamma-Carboxylation of the prothrombin precursors in vitro was investigated with conformational specific antibodies raised against a portion of the Gla (gamma-carboxyglutamic acid) region extending from residue 15 to 24. The synthetic peptide used as antigen contains three of the ten potential Gla sites in prothrombin. It is shown that these antibodies do not recognize mature prothrombin but recognize the decarboxylated protein. It is also demonstrated that the epitope is Ca2(+)-dependent. The antibodies were used to assess gamma-carboxylation of the prothrombin precursor in membrane fragments from microsomal membranes. The results suggest that microsomal gamma-carboxylation does not involve Glu residues 16, 19 and 20 of the Gla region.
Wallin
R
R
Rheumatology/Department of Medicine, Wake Forest University, Bowman Gray School of Medicine, Winston-Salem, NC 27103.
Turner
R
R
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019735:Carbon-Carbon Ligases; D004792:Enzyme Precursors / Q000235:genetics*; D005170:Factor X / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis; D011499:Protein Processing, Post-Translational; D011516:Prothrombin / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D004792:Enzyme Precursors; D010455:Peptides; D011516:Prothrombin; D005170:Factor X; D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase