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

Citation
Rm. Camire et al., Enhanced gamma-carboxylation of recombinant factor X using a chimeric construct containing the prothrombin propeptide, BIOCHEM, 39(46), 2000, pp. 14322-14329
Citations number
46
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
00062960 → ACNP
Volume
39
Issue
46
Year of publication
2000
Pages
14322 - 14329
Database
ISI
SICI code
0006-2960(20001121)39:46<14322:EGORFX>2.0.ZU;2-O
Abstract
Factor Xa is the serine protease component of prothrombinase, the enzymatic complex responsible for thrombin generation. Production of recombinant fac tor X/Xa has proven to be difficult because of inefficient gamma -carboxyla tion, a critical post-translational modification. The affinities of the vit amin K-dependent propeptides for the gamma -carboxylase vary over 2 logs, w ith the propeptide of factor X having the highest affinity followed by the propeptides of factor VII, protein S, factor LX, protein C, and prothrombin [Stanley, T. B. (1999) J. Biol. Chem. 274, 16940-16944], On the basis of t his observation, it was hypothesized that exchanging the propeptide of fact or X with one that binds the gamma -carboxylase with a reduced affinity wou ld enhance gamma -carboxylation by allowing greater substrate turnover, A c himeric cDNA consisting of the human prothrombin signal sequence and propep tide followed by mature human factor:X was generated and stably transfected into HEK 293 cells, and modified factor X was purified from conditioned me dium, The results indicate that on average 85% of the total factor X produc ed with the prothrombin propeptide was fully gamma -carboxylated, represent ing 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 -carbo xylase for the propeptide greatly influences the extent of gamma -carboxyla tion. It was also observed that regardless of which propeptide sequence is directing gamma -carboxylation (factor X or prothrombin), two pools of fact or X are secreted; one is uncarboxylated and a second is fully gamma -carbo xylated, supporting the notion that the gamma -carboxylase is a processive enzyme.