Carboxylative efficacy of trans and cis MK7 and comparison with other vitamin K isomers

Abstract

Carboxylative enzymes are involved in many pathways and their regulation plays a crucial role in many of these pathways. In particular, γ-glutamylcarboxylase (GGCX) converts glutamate residues (Glu) into γ-carboxyglutamate (Gla) of the vitamin K-dependent proteins (VKDPs) activating them. VKDPs include at least 17 proteins involved in processes such as blood coagulation, blood vessels calcification, and bone mineralization. VKDPs are activated by the reduced form of vitamin K, naturally occurring as vitamin K1 (phylloquinone) and K2 (menaquinones, MKs). Among these, MK7 is the most efficient in terms of bioavailability and biological effect. Similarly to other trans isomers, it is produced by natural fermentation or chemically in both trans and cis. However, the efficacy of the biological effect of the different isomers and the impact on humans are unknown. Our study assessed carboxylative efficacy of trans and cis MK7 and compared it with other vitamin K isomers, evaluating both the expression of residues of carboxylated Gla-protein by western blot analysis and using a cell-free system to determine the GGCX activity by HPLC. Trans MK7H₂ showed a higher ability to carboxylate the 70 KDa GLA-protein, previously inhibited in vitro by warfarin treatment. However, cis MK7 also induced a carboxylation activity albeit of a small extent. The data were confirmed chromatographically, in which a slight carboxylative activity of cis MK7H₂ was demonstrated, comparable with both K1H₂ and oxidized trans MK7 but less than trans MK7H₂ . For the first time, a difference of biological activity between cis and trans configuration of menaquinone-7 has been reported.

Keywords: GGCX; carboxylation; cis/trans isomers; phylloquinone; vitamin MK7.

FIGURE 1

Chemical structure of trans and cis conformations of menaquinone‐7 (MK7)

FIGURE 2

Qualitative western blot of Gla‐protein in nHDF cells. (A) Pattern of Gla‐protein expression after 24 h treatment with 200 μM of warfarin (Warf) compared with untreated cells (Ctrl). (B) 70 kDa Gla‐protein variation after co‐incubation with warfarin (200 μM) and a range of trans MK7 concentration

FIGURE 3

Western blot and densitometric analysis of about 70 kDa Gla‐protein in nHDFs treated for 24 h with warfarin (200 μM) and/or MK7 in trans or cis conformation (1 and 10 μM). Protein levels are reported as Gla‐protein fold change in cells treated with the same concentration of cis vs trans MK7. Data were normalized to β‐actin. Data are represented as mean ± SD. T test: *p ≤ 0.05; **p ≤ 0.01

FIGURE 4

GGCX activity of reduced MK4 (MK4H2), oxidized trans MK7 (trans MK7), reduced trans MK7 (trans MK7H2), and reduced cis MK7 (cis MK7H2) expressed as percentage of reduced vitamin K1 (KH2) activity. *p ≤ 0.05