Fig 1.
Graphical presentation of GDP-fucose biosynthesis pathways in mammalian cells.
(A) scheme presenting the de novo biosynthesis pathway of GDP-fucose. (B) diagram illustrating the fucose-derived pathway of GDP-fucose synthesis.
Fig 2.
Analysis of intracellular concentration of GDP-fucose and the level of fucosylated structures in the control and fucose-fed knockouts generated in the HEK293T cell line.
(A) and (B) present intracellular concentration of GDP-fucose and the level of fucosylated structures in the TSTA3KO cell line, respectively. (C) and (D) present the intracellular concentration of GDP-fucose and the level of fucosylated structures in the GMDSKO cell line, respectively. (E) and (F) show the intracellular concentration of GDP-fucose and the level of fucosylated structures in the FCSKKO cell line, respectively. Data are presented as mean ± SEM. Each sample was run in three biological replicates, ns, not significant and ****, p < 0.0001, as determined using one-way ANOVA with the Tukey post hoc test.
Fig 3.
Analysis of the effect of supplementation of the TSTA3KO and the GMDSKO HEK293T cell lines with different fucose concentrations.
(A) quantification of intracellular GDP-fucose concentration. (B) quantification of the percentage of the core-fucosylated N-glycan structures; each sample was run in three biological replicates. Data are represented as mean ± SEM. Each sample was run in three biological replicates, ns, not significant; **p < 0.01; and ****p < 0.0001, as determined using one-way ANOVA with the Tukey post hoc test.
Fig 4.
Changes in levels of proteins involved in GDP-fucose synthesis.
(A) FPGT western blotting analysis of the wild-type, TSTA3KO and GMDSKO HEK293T cells without and after supplementation with fucose. As a loading control, the anti-GAPDH antibody was used. (B) FCSK western blotting analysis of the wild-type, TSTA3KO and GMDSKO HEK293T cells after supplementation with or without fucose. As a loading control, the anti-GAPDH antibody was used. The main change in FCSK protein level in TSTA3KO cell line was indicated with a green box. (C) GMDS western blotting analysis of the wild-type and FCSKKO HEK293T cells. As a loading control, the anti-GAPDH antibody was used. (D) TSTA3 western blotting analysis of the wild-type and FCSKKO HEK293T cells. As a loading control, the anti-GAPDH antibody was used. The main change in TSTA3 protein level in FCSKKO cell line was indicated with a green box. For (A) and (B), ns, not significant; *, p < 0.05 as determined using one-way ANOVA with the Tukey post-hoc test. P values that trend to be statistically significant are also shown. For (C) and (D), ns, not significant; *, p < 0.05 as determined using Welch’s t-test. Data are represented as mean ± SEM. Each sample was run at least in two biological replicates.
Fig 5.
Overexpression of FCSK and FPGT in TSTA3KO and GMDSKO HEK293T cell lines.
(A) intracellular concentration of GDP-fucose of wild-type, TSTA3KO and GMDSKO HEK293T cells, and in cells with overexpressed FPGT and FCSK proteins (tagged with HA and cmyc, respectively) in knockouts. (B) quantification of the percentage of fucosylated structures in wild-type, TSTA3KO and GMDSKO HEK293T cells and in cells with overexpressed FPGT or FCSK proteins in knockouts. (C) western blotting of HA in GMDSKO and TSTA3KO cells and overexpressed FPGT and FCSK in cell lines deficient in TSTA3 and GMDS enzymes. (D) western blotting of fucokinase in GMDSKO and TSTA3KO cells and overexpressed FPGT and FCSK in cell lines deficient in TSTA3 and GMDS enzymes. As a loading control, the anti-HSP60 antibody was used. For (A) and (B), ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 as determined using one-way ANOVA with the Tukey post-hoc test. Data are represented as mean ± SEM. Each sample was run in three biological replicates.
Table 1.
Fucose concentration (expressed in μM) in wild-type, TSTA3KO, GMDSKO and FCSKKO HEK293T cell lines.
Fig 6.
Influence of TSTA3, GMDS and FCSK protein depletion on the level of proteins engaged in fucose absorption.
(A) intracellular concentration of GDP-fucose measured in TSTA3KO and GMDSKO HEK293T cell lines after supplementation with 3H-fucose. (B) result of 3H-fucose labelled N-glycans isolated from the TSTA3KO and the GMDSKO HEK293T cells. For (A) and (B); ns, not significant; ****, p < 0.0001 as determined using one-way ANOVA with the Tukey post-hoc test. Data are presented as mean ± SEM. Each sample was run at least in three biological replicates. Without and after supplementation with fucose, the wild-type, TSTA3KO and GMDSKO HEK293T cells were subjected to western blot analysis of (C) GLUT1, (D) CaSR. Anti-calnexin and anti-GAPDH antibodies, respectively, were used as the loading control. (E) wild-type and FCSKKO cells were employed for western blot analysis of GLUT1. For the presentation of equal loading of samples, the anti-GAPDH antibody was used. For (C) and (D), ns, not significant; *, p < 0.05; **, p <0.01 as determined using one-way ANOVA with the Tukey post-hoc test. P values that trend to be statistically significant are also shown. For (E), ns, not significant as determined using Welch’s t-test. Data are represented as mean ± SEM. Each sample was run at least in two biological replicates.
Fig 7.
Influence of TSTA3, GMDS and FCSK protein inactivation on the level of proteins involved in fucose recycling in mammalian cells.
(A) and (B) wild-type, TSTA3KO and GMDSKO HEK293T cells fed and not fed with fucose as well as FCSKKO cells were analysed by western blotting to determine protein levels of FUOM; as a loading control, an anti-calnexin antibody and anti-HSP60 antibody were used. (C) and (D) western blotting of FUCA1 performed on wild-type, TSTA3KO, GMDSKO, supplemented and unsupplemented in fucose and FCSKKO cell lysates. Anti-HSP60 and anti-calnexin antibodies were used as a loading control. For (A) and (D), ns, not significant as determined using one-way ANOVA with the Tukey post-hoc test. For (B) and (C), ns, not significant as determined using Welch’s t-test. Data are represented as mean ± SEM. Each sample was run at least in two biological replicates.