Reduced utilization of Man5GlcNAc2-P-P-lipid in a Lec9 mutant of Chinese hamster ovary cells: Analysis of the steps in oligosaccharide-lipid assembly

Abstract

Recently we reported that CHB11-1-3, a Chinese hamster ovary cell mutant defective in glycosylation of asparagine-linked proteins, is defective in the synthesis of dolichol [Quellhorst et al., 343:19-26, 1997: Arch Biochem Biophys]. CHB11-1-3 was found to be in the Lec9 complementation group, which synthesizes polyprenol rather than dolichol. In this paper, levels of various polyprenyl derivatives in CHB11-1-3 are compared to levels of the corresponding dolichyl derivatives in parental cells. CHB11-1-3 was found to maintain near normal levels of Man5GlcNAc2-P-P-polyprenol and mannosylphosphorylpolyprenol, despite reduced rates of synthesis, by utilizing those intermediates at a reduced rate. The Man5GlcNAc2 oligosaccharide attached to prenol in CHB11-1-3 cells and to dolichol in parental cells is the same structure, as determined by acetolysis. Man5GlcNAc2-P-P-polyprenol and Man5GlcNAc5-P-P-dolichol both appeared to be translocated efficiently in an in vitro reaction. Glycosylation of G protein was compared in vesicular stomatitus virus (VSV)-infected parent and mutant; although a portion of G protein was compared in vesicular stomatitus virus (VSV)-infected parent and mutant; although a portion of G protein was normally glycosylated in CHB11-1-3 cells, a large portion of G was underglycosylated, resulting in the addition of either one or no oligosaccharide to G. Addition of a single oligosaccharide occurred randomly rather than preferentially at one of the two sites.

A pseudoautosomal glycosylation disorder prompts the revision of dolichol biosynthesis

“…When the conversion of polyprenol to polyprenal is defective, dolichol can be replaced by polyprenol as intermediate oligosaccharide acceptor, with polyprenol being much poorer in this respect than dolichol for several of the reactions necessary for N-glycosylation. 35 , 36 , 37 , 38 , 39 We wondered whether an increase of polyprenol-phosphate (Pol-P) or polyprenol-phospho-hexoses (Pol-P-Hex) could cause the glycosylation defect observed in DHRSX-deficient cells ( Figure 1 L).…”

“…These findings indicated that polyprenol can be phosphorylated in cells and that the imbalance between polyprenol-P-hexose and dolichol-P-hexose might be the cause of the glycosylation defect in DHRSX and SRD5A3-deficient cells. 35 , 38

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).

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“…Based on our observations and previous work in Chinese hamster ovary (CHO) cells, we hypothesized that the accumulation of polyprenol-P-hexoses could interfere with the activity of the required glycosyltransferases and that glycan assembly on polyprenol-PP might be less efficient. 35 , 36 , 37 , 38 , 39 Consequently, truncated high-mannose lipid linked oligosaccharides might accumulate or even be transferred onto glycoproteins ( Figure 6 E).…”

The N-glycosylation defect in Lec5 and Lec9 CHO cells is caused by absence of the DHRSX gene

“…Studies on human cell lines show that DHRSX deficiency leads to a decrease in dolichol and an increase in polyprenol, and similar changes in glycosylation to those previously observed with Lec5 and Lec9 cells (18). In particular, previous work on the glycosylation defect in CHO Lec5 and Lec9 cells showed that the conversion of polyprenol to dolichol is deficient (16, 19). As a result of this, N-glycan synthesis in Lec5 and Lec9 cells utilizes polyprenol-phosphate rather than dolichol-phosphate species, leading to perturbation in the synthesis and transfer of the LLO ( Fig.…”