Figure 2.

The consequences of PLPHP deficiency on the intracellular vitamin B₆metabolism in cultured human and yeast cells.A, intracellular content of PLP, PNP, PMP, pyridoxal (PL), pyridoxine (PN), and pyridoxamine (PM) in control and PLPHP-deficient fibroblasts cultured in complete DMEM containing no vitamin B₆, 20 μM PN, 20 μM PL, or 20 μM PM for 96 h. The type of B₆ vitamer present in the culture medium is indicated on the x-axis. Data are means from n = 6 to 9 biological replicates per cell line and condition, ±SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared to control (only comparisons at the same culturing condition are shown). B, comparison of intracellular PLP, PNP, PMP, PL, PN, and PM content in control and PLPHP-deficient HEK293 cells cultured in complete DMEM containing no vitamin B₆, 20 μM PN, 20 μM PL, or 20 μM PM for 96 h. The type of B₆ vitamer present in the culture medium is indicated on the x-axis. Data are means from n = 6 to 12 biological replicates per cell line and condition, ±SD. ∗p < 0.05 compared to control (only comparisons at the same culturing condition are shown). C, PNPO enzyme activity with PNP as the substrate in control and PLPHP-deficient fibroblasts and HEK293 cells. PNPO activity was measured at subsaturating (0.4 μM) and saturating (2 μM) PNP concentrations. Data are means from n = 3 biological replicates per cell line and condition, ±SD. ∗p < 0.05 compared to control. D, B₆ vitamer profiles in mitochondrial and cytosolic fractions, and total cell lysates in control and PLPHP-deficient HEK293 cells cultured with 19.4 μM pyridoxine (standard DMEM). Data are means from n = 5 to 6 biological replicates per cell line, ±SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared to control. E, Western blot showing PLPHP protein expression in total cell extracts, and cytosolic and mitochondrial fractions in HEK293 cells cultured with pyridoxine (standard complete DMEM). F, intracellular B₆ vitamer concentrations in WT and YBL036C-deficient (ybl036cΔ) yeast grown on glucose, oleate, or ethanol as carbon source. Data are means from n = 3 independent experiments, ±SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared to WT yeast grown on the same carbon source. CS, citrate synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PLP, pyridoxal 5′-phosphate; PLPHP, pyridoxal 5′-phosphate homeostasis protein; PMP, pyridoxamine 5′-phosphate; PNP, pyridoxine 5′-phosphate; PNPO, pyridox(am)ine 5′-phosphate oxidase.