Figure 1
From: Vitamin K epoxide reductase and its paralogous enzyme have different structures and functions
Effect of helix-breaking proline residue on the proposed membrane topologies of VKOR and VKORL. (A) TMHMM prediction of VKOR membrane topology. The top line shows the predicted TMDs, and the striped lines indicate the probability of the regions’ functioning as the TMD. (B,C) Schematic representation of the membrane topology of VKOR and the VKOR charge mutant (VKOR-CM). The location of I86 is indicated. (D,E) Cell-based activity assay of VKOR, VKORL and their mutants. Wild-type VKOR, VKOR-I86P, VKOR-CM and VKOR-CM-I86P mutants (D) were transiently expressed in HEK293-DGKO reporter cells, and the media was supplemented with 2.5 μM KO; 48 hours post-transfection, carboxylated reporter protein FIXgla-PC levels were measured by ELISA. Data is presented as mean ± SD of three independent experiments (n = 3), ***p < 0.001 (unpaired t-test) compared VKOR with VKOR-I86P and VKOR-CM with VKOR-CM-I86P. VKOR, VKOR-I86P, VKORL, and VKORL-I93P mutants (E) were transiently expressed in HEK293-DGKO reporter cells, and the cells were grown in media supplemented with 2.5 μM KO in the absence (black bars, KO) or presence (grey bars, KO + WF) of 2 μM warfarin 4 hours post-transfection; 48 hours later carboxylated FIXgla-PC concentration in the cell culture supernatant was measured by ELISA. Data is presented as mean ± SD of three independent experiments (n = 3), ***p < 0.001 (two-way ANOVA), compared to the respective VKOR or VKORL variant with KO and KO + warfarin (WF).
