Figure 6.

Accumulation of phospho- and phosphohexose-polyprenol alongside truncated N-linked oligosaccharide species in DHRSX/SRD5A3-deficient cells

(A–D) Dolichol-phosphate or polyprenol-phosphate (A and C), and dolichol-phospho-hexose or polyprenol-phospho-hexose (B and D) were measured in wild-type, DHRSX KO, and SRD5A3 KO HAP1 cells and their respective complementations (A and B), as well as EBV-immortalized lymphoblasts from controls, patients, and parents (C and D). Data are TIC-normalized AUC (means ± SEM, n = 4; ^∗p < 0.05; ^∗∗∗∗p < 0.0001; §p < 0.05 compared to every control; #p < 0.05 compared to one of the controls). See also Table S2, and Figures S6A and S6B. Dolichol- and polyprenol-phospho-hexose represent mixtures of mannose and glucose derivatives.

(E) Mechanisms underlying the glycosylation defect in DHRSX and SRD5A3 deficient cells integrating published data and our present paper. The inset table presents the ratio of polyprenol to dolichol (see Figure 2B), as well as the ratios of the corresponding phospho and phosphohexose derivatives (A and B) (means ± SEM, n = 4). The percentages alongside red dashed lines indicate the relative activity of the indicated enzyme when using a polyprenol-instead of dolichol-derived substrate (DOLK, dolichol kinase⁴⁰; DPM1/2/3, Dolichol-phosphate mannosyltransferase⁴¹; ALG3, Alpha-1,3-Mannosyltransferase⁴²; DPAGT1, UDP-N-acetylglucosamine—dolichyl-phosphate N-acetylglucosamine-phosphotransferase^43,44. Erroneous transfer of an immature glycan (Man₅GlcNAc₂) to nascent glycoproteins leads to linear Man-5 NLOs (see “abnormal glycosylation” box), that are subsequently trimmed to Man-4 NLOs by the enzyme EDEM3.⁴⁵ Alternatively, branched Man-5 can be formed during normal N-glycosylation as a consequence of successive glycan trimming (arrows on the right side). Castanospermine allows us to determine the origin of Man-5 glycans, since it inhibits $\alpha$-glucosidases I/II required for this trimming, thereby preventing the formation of branched Man-5.

(F–H) N-linked oligosaccharide (NLO) HPLC profiles obtained from wild-type (F), DHRSX KO (G), and SRD5A3 KO (H) HAP1 cells labeled with 100 μCi [2-³H]-Mannose, showing an accumulation of truncated NLOs, primarily Man-4, Man-5, and Glc₁Man_5/M₆ species upon inactivation of DHRSX or SRD5A3. See also Figures S6D, S6E, and S6F.

(I–K) N-linked oligosaccharide (NLO) HPLC profiles obtained from wild-type (I), DHRSX KO (J), and SRD5A3 KO (K) HAP1 cells labeled with 100 μCi [2-³H]-Mannose and treated with 50 μmol/L castanospermine prior to metabolic labeling. The castanospermine-resistant accumulation of Man₄, Man₅, and Glc₁Man_5/M₆ species indicates that these are due to the transfer of an incomplete lipid-linked olichosaccharide, rather than trimming of mature NLOs.

(L) Ratio of the abundance of Man₅ N-linked to Man₉ N-linked oligosaccharides (NLO) detected in metabolic labeling experiments in wild-type (WT), DHRSX KO, and SRD5A3 KO HAP1 cells and their respective complementations (shown in E–G). Data are presented normalized to WT in a log 2 scale (means ± SEM, n = 2–4; ^∗p < 0.05; ^∗∗p < 0.01).

See also Figure S6.