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Ferrata Storti Foundation
Haematologica 2020 Volume 105(8):2164-2173
Hodgkin Lymphoma
Vitamin K-dependent carboxylation of coagulation factors: insights from a cell-based functional study
Zhenyu Hao, Da-Yun Jin, Darrel W. Stafford, and Jian-Ke Tie
Department of Biology, the University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
ABSTRACT
Vitamin K-dependent carboxylation is a post-translational modifica- tion essential for the biological function of coagulation factors. Defects in carboxylation are mainly associated with bleeding disor- ders. With the discovery of new vitamin K-dependent proteins, the impor- tance of carboxylation now encompasses vascular calcification, bone metabolism, and other important physiological processes. Our current knowledge of carboxylation, however, comes mainly from in vitro studies carried out under artificial conditions, which have a limited usefulness in understanding the carboxylation of vitamin K-dependent proteins in native conditions. Using a recently established mammalian cell-based assay, we studied the carboxylation of coagulation factors in a cellular environment. Our results show that the coagulation factor’s propeptide controls substrate binding and product releasing during carboxylation, and the propeptide of factor IX appears to have the optimal affinity for efficient carboxylation. Additionally, non-conserved residues in the propeptide play an important role in carboxylation. A cell-based functional study of naturally occurring mutations in the propeptide successfully interpreted the clinical phenotype of warfarin’s hypersensitivity during anticoagulation therapy in patients with these mutations. Unlike results obtained from in vitro studies, results from our cell-based study indicate that although the propeptide of osteocal- cin cannot direct the carboxylation of the coagulation factor, it is required for the efficient carboxylation of osteocalcin. This suggests that the coagu- lation factors may have a different mechanism of carboxylation from osteo- calcin. Together, results from this study provide insight into efficiently con- trolling one physiological process, such as coagulation without affecting the other, like bone metabolism.
Introduction
Vitamin K-dependent (VKD) carboxylation is a post-translational modification that converts specific glutamate residues (Glu) to gamma-carboxyglutamate residues (Gla) in VKD proteins. It is essential for the biological function of proteins that control blood coagulation, vascular calcification, bone metabolism, and other important physiological processes.1 Carboxylation has mostly been associated with coagulation, since it was originally observed in the clotting factor, prothrombin (PT).2 Defects of VKD carboxylation have long been known to cause bleeding dis- orders.3 There are two types of coagulation factors, one is procoagulant proteins which include PT, FVII, FIX, and FX. The other is anticoagulant proteins which include PC, PS, and PZ. The biological functions of these clotting factors require 9-13 Glu residues at the N-terminus of the mature protein (referred to as the Gla domain) to be properly modified by VKD carboxylation.
Carboxylation is catalyzed by an integral membrane protein gamma-glutamyl carboxylase (GGCX), which utilizes the reduced form of vitamin K, carbon diox- ide, and oxygen as co-factors. This modification involves the subtraction of the gamma-hydrogen from the Glu residue followed by the addition of a carbon diox- ide (carboxyl group). Simultaneously, reduced vitamin K is oxidized to vitamin K epoxide to provide the energy required for the carboxylation reaction. The enzy-
Correspondence:
JIAN-KE TIE
jktie@email.unc.edu
Received: June 13, 2019. Accepted: October 11, 2019. Pre-published: October 17, 2019.
doi:10.3324/haematol.2019.229047
Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/105/8/2164
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