Increased Production of Functional Recombinant Human Clotting Factor IX by Baby Hamster Kidney Cells Engineered to Overexpress VKORC1, the Vitamin K 2,3-Epoxide-reducing Enzyme of the Vitamin K Cycle

Abstract

Some recombinant vitamin K-dependent blood coagulation factors (factors VII, IX, and protein C) have become valuable pharmaceuticals in the treatment of bleeding complications and sepsis. Because of their vitamin K-dependent post-translational modification, their synthesis by eukaryotic cells is essential. The eukaryotic cell harbors a vitamin K-dependent ␥-carboxylation system that converts the proteins to ␥-carboxyglutamic acid-containing proteins. However, the system in eukaryotic cells has limited capacity, and cell lines overexpressing vitamin K-dependent clotting factors produce only a fraction of the recombinant proteins as fully ␥-carboxylated, physiologically competent proteins. In this work we have used recombinant human factor IX (r-hFIX)-producing baby hamster kidney (BHK) cells, engineered to stably overexpress various components of the ␥-carboxylation system of the cell, to determine whether increased production of functional r-hFIX can be accomplished. All BHK cell lines secreted r-hFIX into serum-free medium. Overexpression of ␥-carboxylase is shown to inhibit production of functional r-hFIX. On the other hand, cells overexpressing VKORC1, the reduced vitamin K cofactor-producing enzyme of the vitamin Kdependent ␥-carboxylation system, produced 2.9-fold more functional r-hFIX than control BHK cells. The data are consistent with the notion that VKORC1 is the ratelimiting step in the system and is a key regulatory protein in synthesis of active vitamin K-dependent proteins. The data suggest that overexpression of VKORC1 can be utilized for increased cellular production of recombinant vitamin K-dependent proteins.

Vitamin K epoxide reductase significantly improves carboxylation in a cell line overexpressing factor X

“…While our paper was under review, Wajih et al reported that factor IX expression in baby hamster kidney (BHK) cells could also be helped by coexpressing VKOR. 21 Both this work and that of Hallgren et al 22 and Rehemtulla et al 13 report that GGCX expression actually decreases the amount of functional, carboxylated, vitamin Kdependent protein secreted from the cells. In fact, we recovered slightly more factor X from the cell line overexpressing GGCX than from any of the other cell lines.…”

r-VKORC1 Expression in Factor IX BHK Cells Increases the Extent of Factor IX Carboxylation but Is Limited by Saturation of Another Carboxylation Component or by a Shift in the Rate-Limiting Step

“…Also unknown is whether other cell lines contain similar amounts of this protein, which could impact the ability of r-VKORC1 to increase carboxylation in these lines. Two recent papers (35,36), which examined the effect of r-VKORC1 on VKD proteins secreted from BHK or 293 cells, also reported only 2-3-fold increases in the extent of carboxylated, secreted protein. Our determinations of the rates of in vitro and in vivo carboxylation suggest the possibility that the redox protein is critical to efficiency of carboxylation and that larger increases in carboxylation may not be possible until it is identified and coexpressed with VKORC1.…”

Compound heterozygosity of novel missense mutations in the gamma-glutamyl-carboxylase gene causes hereditary combined vitamin K–dependent coagulation factor deficiency

“…22 Consequently, VKD factor association with carboxylase results in more efficient vitamin K utilization, which is significant because the availability of KH 2 appears to regulate carboxylation. [23][24][25][26] In the absence of VKD substrate binding, KH 2 epoxidation to KO does not occur, 27 and this regulation prevents the unfettered formation of highly reactive and undesirable vitamin K intermediates. At present, the residues that make up the carboxylase active site to facilitate the reaction are largely unknown, due in part to the lack of a crystal structure or homology with other proteins that might indicate functional residues.…”

“…22 Consequently, VKD factor association with carboxylase results in more efficient vitamin K utilization, which is significant because the availability of KH 2 appears to regulate carboxylation. [23][24][25][26] In the absence of VKD substrate binding, KH 2 epoxidation to KO does not occur, 27 and this regulation prevents the unfettered formation of highly reactive and undesirable An Inside Blood analysis of this article appears at the front of this issue.Reprints: Jean-Philippe Rosa, Laboratory of Hemostasis and Thrombosis, U689 Institut National de la Santé Et de la Recherche Mé dicale, Hô pital Lariboisiè re, 41 boulevard de la Chapelle, 75475 Paris Cedex 10, France; e-mail: rosa@larib.inserm.fr.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 U.S.C.…”