Figure 3. Markedly reduced levels of γ-carboxylated MGP are
crucial but not exclusive for causing facial dysmorphologies
(a-c) γ-Carboxylation dose-response curves of GRP, MGP, and BGLAP
measured by ELISA. The y-axis represents normalized γ-carboxylation (%)
to wt. The x-axis represents K1 concentrations
[0.1-100 µM]. In each figure dose-response curve for GGCX wt is
depicted in black. Compound heterozygous pathogenic variants in one
particular patient are represented by the same color with each allele
variant in solid and dotted line. Error bars depict SD of triplicate
measurements of n = 3 experiments. (a) γ-Carboxylation
dose-response curves with respect to variant found in patients having
facial dysmorphologies. (b) γ-Carboxylation dose-responses for a variant
reported in a patient having facial dysmorphologies, whose mother had
severe hyperemesis gravidarum. The second allele harbors a nonsense
variant. (c) Patients with low γ-carboxylated MGP values that did not
developed facial dysmorphologies.
Figure 4. Identification of the Vitamin K Hydroquinone
binding site in GGCX
(a) A snapshot of the GGCX structural model (grey) embedded in membrane
(green) with bound KH2 (brown). The model is depicted in
ribbon format, whereas the membrane is depicted by its molecular
surface. KH2 is depicted in stick format. (b) Close up
view of the equilibrated docking pose 6 (brown) with ten interacting
residues (blue) and critical interacting residue F299 (purple).
Interactions by Pi-Pi and hydrophobic binding with the head group of
KH2 are depicted by red and green lines, respectively.
The GGCX structural model is depicted in ribbon format while
KH2 and interacting residues are depicted in stick
format. (c) Receptor and ligand movement RMSD (c-α backbone) for GGCX wt
(black), M174R (blue), and S300F (red). The y-axis represents RMSD and
x-axis represents simulation time. (d) Colocalization images of HEK293TGGCX -/- cells expressing GGCX wt, F299S, M174R,
S300F. First panel represents Dapi counter stain followed by PDI stained
with Alexa Fluor 488 (green), GGCX stained with Alexa Fluor 594 (red)
and a merged picture. The Pearson´s correlation co-efficient is given as
Rcoloc in the merged picture. (e) The Pearson´s correlation coefficients
of GGCX loss-of-function variants with PDI. Error bars depict SD ofn = 10 analyzed cells. **** p < 0.001 (f)
γ-Carboxylation dose-response curves of VKD proteins with respect to
pathogenic variant showing loss-of-function (M174R) and variants
involved in KH2 binding (F299S and S300F). The x-axis
represents K1 concentrations [0.1-100 µM]. Error
bars represent SD of n = 3 measurements of n = 3
experiments.