Enhanced gamma-carboxylation of recombinant factor X using a chimeric construct containing the prothrombin propeptide

Biochemistry. 2000 Nov 21;39(46):14322-9. doi: 10.1021/bi001074q.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 1-Carboxyglutamic Acid / analysis
  • 1-Carboxyglutamic Acid / metabolism*
  • Amino Acid Sequence
  • Animals
  • CHO Cells
  • Carbon-Carbon Ligases / metabolism
  • Cell Line
  • Cricetinae
  • Factor X / genetics
  • Factor X / metabolism*
  • Genetic Vectors / chemical synthesis
  • Genetic Vectors / metabolism
  • Humans
  • Molecular Sequence Data
  • Protein Precursors / chemical synthesis
  • Protein Precursors / genetics
  • Protein Precursors / isolation & purification
  • Protein Precursors / metabolism*
  • Protein Sorting Signals* / genetics
  • Prothrombin / biosynthesis
  • Prothrombin / genetics
  • Prothrombin / isolation & purification
  • Prothrombin / metabolism*
  • Recombinant Fusion Proteins / chemical synthesis
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism*
  • Sequence Analysis, Protein

Substances

  • Protein Precursors
  • Protein Sorting Signals
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • 1-Carboxyglutamic Acid
  • Prothrombin
  • Factor X
  • Carbon-Carbon Ligases
  • glutamyl carboxylase