The Effect of Changes in the Leader Sequence of Human Protein C on Biosynthetic Processing and Gamma-Carboxylation

Abstract

Protein C is the precursor to a serine protease in plasma which contains gamma-carboxy glutamic acid and functions as a potent anticoagulant. Protein C shows considerable structural homology with the other vitamin K-dependent coagulation factors including prothrombin, factor VII, factor IX and factor X. Sequence analysis ofthe cDNAs for these proteins has revealedthe presence of a prepro leader sequence that contains a pre sequence or hydrophobic signal sequence and a propeptide containing a number of highly conserved amino acids. The pre region is removed from thegrowing polypeptide chain by signal peptidase, while the pro region is subsequently removed from the protein prior to secretion. Deletion mutants have been constructed in the propeptide portion of the cDNAfor human protein C in order to test the possibility that the propeptide portion of the 42 amino acid leader sequence serves as a molecular signal for gamma-carboxylation.Accordingly, these mutants containthe pre-peptide (hydrophobic leader) plusportions of the pro-peptide at the amino terminus of the light chain. These deletions include the removal of 4, 9, 12, 15, 16 or 17 amino acids from the carboxyl end of the leader sequence of 42 amino acids. The mutant proteins were expressed in carboxylation-competent mammalian cells and were then examined by Western blotting, barium citrate adsorption and precipitation, amino acid sequence analysis, and biological activity and compared with the native protein present in normal plasma. These studies have shown that deletions inthe pro-peptide region interfere with gamma-carboxylation and removal of the pro-peptide. Deletion of residues -1 through -12 had little effect on the carboxylationor secretion. Deletion of -1 through -17 completely abolished gamma-carboxylation, but had no measurable effect on secretion.Amino terminal sequence analysis of thelatter mutant showed that the light chainbegan with Thr-Pro-Ala-Pro... This corresponds to a sequence in the prepro leader starting at -24. This indicates that the signal peptidase cleavage site for human protein C is between residues -25 and -24 and removal of the pro-peptide had been blocked by the deletion.Furthermore, during biosynthesis and secretion, the amino-terminal region of the propeptide (residues from about -12 through -17) are important for carboxylation of protein C, while the carboxyl-terminal portion of the peptide (residues -1 through -4) are important for the removal of the proleader sequence by proteolytic processing.