The inhibitory effect of calumenin on the vitamin K-dependent gamma-carboxylation system. Characterization of the system in normal and warfarin-resistant rats.
The vitamin K-dependent gamma-carboxylation system is responsible for post-translational modification of vitamin K-dependent proteins, converting them to Gla-containing proteins. The system consists of integral membrane proteins located in the endoplasmic reticulum membrane and includes the gamma-carboxylase and the warfarin-sensitive enzyme vitamin K(1) 2,3-epoxide reductase (VKOR), which provides gamma-carboxylase with reduced vitamin K(1) cofactor. In this work, an in vitro gamma-carboxylation system was designed and used to understand how VKOR and gamma-carboxylase work together as a system and to identify factors that can regulate the activity of the system. Results are presented that demonstrate that the endoplasmic reticulum chaperone protein calumenin is associated with gamma-carboxylase and inhibits its activity. Silencing of the calumenin gene with siRNA resulted in a 5-fold increase in gamma-carboxylase activity. The results provide the first identification of a protein that can regulate the activity of the gamma-carboxylation system. The propeptides of vitamin K-dependent proteins stimulate gamma-carboxylase activity. Here we show that the factor X and prothrombin propeptides do not increase reduced vitamin K(1) cofactor production by VKOR in the system where VKOR is the rate-limiting step for gamma-carboxylation. These findings put calumenin in a central position concerning regulation of gamma-carboxylation of vitamin K-dependent proteins. Reduced vitamin K(1) cofactor transfer between VKOR and gamma-carboxylase is shown to be significantly impaired in the in vitro gamma-carboxylation system prepared from warfarin-resistant rats. Furthermore, the sequence of the 18-kDa subunit 1 of the VKOR enzyme complex was found to be identical in the two rat strains. This finding supports the notion that different forms of genetic warfarin resistance exist.
Wajih
Nadeem
N
Departments of Internal Medicine and Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA.
Sane
David C
DC
Hutson
Susan M
SM
Wallin
Reidar
R
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D019735:Carbon-Carbon Ligases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D008297:Male; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D014812:Vitamin K / Q000494:pharmacology*; D064417:Vitamin K Epoxide Reductases; D014859:Warfarin / Q000494:pharmacology*
D002135:Calcium-Binding Proteins; C107182:Calu protein, mouse; D004791:Enzyme Inhibitors; D014812:Vitamin K; D014859:Warfarin; D006899:Mixed Function Oxygenases; D064417:Vitamin K Epoxide Reductases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase