Enhanced gamma-carboxylation of recombinant factor X using a chimeric construct containing the prothrombin propeptide.
Factor Xa is the serine protease component of prothrombinase, the enzymatic complex responsible for thrombin generation. Production of recombinant factor X/Xa has proven to be difficult because of inefficient gamma-carboxylation, a critical post-translational modification. The affinities of the vitamin K-dependent propeptides for the gamma-carboxylase vary over 2 logs, with the propeptide of factor X having the highest affinity followed by the propeptides of factor VII, protein S, factor IX, protein C, and prothrombin [Stanley, T. B. (1999) J. Biol. Chem. 274, 16940-16944]. On the basis of this observation, it was hypothesized that exchanging the propeptide of factor X with one that binds the gamma-carboxylase with a reduced affinity would enhance gamma-carboxylation by allowing greater substrate turnover. A chimeric cDNA consisting of the human prothrombin signal sequence and propeptide followed by mature human factor X was generated and stably transfected into HEK 293 cells, and modified factor X was purified from conditioned medium. The results indicate that on average 85% of the total factor X produced with the prothrombin propeptide was fully gamma-carboxylated, representing a substantial improvement over a system that employs the native factor X propeptide, with which on average only 32% of the protein is fully gamma-carboxylated. These results indicate that the affinity of the gamma-carboxylase for the propeptide greatly influences the extent of gamma-carboxylation. It was also observed that regardless of which propeptide sequence is directing gamma-carboxylation (factor X or prothrombin), two pools of factor X are secreted; one is uncarboxylated and a second is fully gamma-carboxylated, supporting the notion that the gamma-carboxylase is a processive enzyme.
Camire
R M
RM
Department of Pediatrics and Pathology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Larson
P J
PJ
Stafford
D W
DW
High
K A
KA
D015055:1-Carboxyglutamic Acid / Q000032:analysis / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D019735:Carbon-Carbon Ligases / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D005170:Factor X / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000138:chemical synthesis / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000138:chemical synthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics; D011516:Prothrombin / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020539:Sequence Analysis, Protein
D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D015055:1-Carboxyglutamic Acid; D011516:Prothrombin; D005170:Factor X; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase