Mutant of Chinese hamster ovary cells with altered mannose 6-phosphate receptor activity is unable to synthesize mannosylphosphoryldolichol.

Abstract

B4-2-1 is a Chinese hamster ovary cell mutant previously isolated and characterized as deficient in mannose 6-phosphate receptor activity. We show here that B4-2-1 is a pleiotropic mutant, defective in biosynthesis of asparagine-linked oligosaccharides. B4-2-1 is unable to synthesize mannosylphosphoryldolichol; the consequences of this defect on glycosylation are (i) biosynthesis of one major lipid-linked oligosaccharide, characterized by its resistance to endoglycosidase H and decreased size; this oligosaccharide is similar to a minor species of lipid-linked oligosaccharide found in parental cells; (ii) transfer of this oligosaccharide to newly synthesized proteins; and (iii) absence of normal "high-mannose" oligosaccharides on mature glycoproteins isolated from B4-2-1; glycoproteins from the mutant contain complex oligosaccharides as well as endoglycosidase H-resistant, amannosidase-sensitive species. While the glycosylation defect may alter adversely the function ofseveral glycoproteins in the mutant, including that of the mannose 6-phosphate receptor, it appears to have no effect on the formation or function ofthe mannose 6-phosphate recognition marker on acid hydrolases of B4-2-1.The uptake of acid hydrolases into human fibroblasts and Chinese hamster ovary (CHO) cells involves binding of a mannose 6-phosphate recognition marker on the enzymes to a receptor for mannose 6-phosphate on the cell surface (1)(2)(3). This receptor appears to be similar in the two cell types with respect to both structure (3) and function (4).The mannose 6-phosphate recognition marker and mannose 6-phosphate receptor appear to be involved not only in enzyme uptake but also in translocation of newly synthesized endogenous acid hydrolases to the lysosomes (2, 5). Most of the receptor has been shown to exist intracellularly (2). Human fibroblasts defective in mannose phosphorylation (6-9) fail to compartmentalize acid hydrolases but instead secrete those enzymes. Aberrant compartmentalization was also observed in CHO cell mutants defective in mannose 6-phosphate receptor activity (10).One ofthe receptor-defective CHO cell mutants, B4-2-1, was found to synthesize acid hydrolases ofincreased electrophoretic mobility (10). Since the receptor and the hydrolases are all glycoproteins, we speculated that this mutant might have a pleiotropic defect in glycosylation. We show here that B4-2-1 cells synthesize altered asparagine-linked oligosaccharides due to a defect in the biosynthesis of mannosylphosphoryldolichol. MATERIALS AND METHODSCells. Isolation and culture conditions for the CHO cell lines N211-1-8 and B4-2-1 (4) and WTB (11) have been described. All media contained 5% fetal bovine serum (Armour, Kankakee, IL); (North American Biologicals, Miami, FL). The cultures were checked routinely for mycoplasma (12) and were found to be negative. (-100 mCi/mmol) was prepared as described (13). Reagents. endo-p-N-acetylglucosaminidase H (endo H)Incubation of Cells with [2-3H]Mannose. Cells were labeled with 1 ml of a-modified minima...

Membrane mutants of animal cells: rapid identification of those with a primary defect in glycosylation.

“…In two crosses, the mass population of hybrids was tested for RIC sensitivity immeliately after fusion. The freshly formed hybrids exhibited essentially parental sensitivity to RIC, indicating that the B4-2-1 mutation behaves recessively as would be predicted by the fact that these cells lack dolichol-P-mannose synthetase activity (33 The complementation data in Table 4 and the LecR phenotypic data in Table 1 show that the B4-2-1 mutation is not similar to any of the previously described RIC-resistant CHO mutants Lecl, LeclA, Lec5, Lecl.Lec6, Lec8, Lec9, or LEC10 (26). It (Table 1).…”

“…The first three groups of mutants were selected for lectin resistance in single-step protocols, B4-2-1 survived a selection for a mannose-6-P receptor deficiency, and the remaining mutants were survivors of 3H-labeled sugar suicide protocols. All of the mutants had been previously tested for their lectin resistance or lectin binding properties or both with one or more lectins and, in many cases, exhibited phenotypes similar to those of mutants from our repertoire: 15B cells (isolated about the same time as Lecl CHO cells; 29) possessed a typical Lecl phenotype (1, 9); clone 13 (a novel phenotype when isolated; 2) and the abrin-resistant CHO-Ki mutants (18) appeared similar to each other and to Lec8 cells; the Jl LecR mutants isolated recently (14) exhibited phenotypes typical of Lecl (17B), Lec2 or Lec3 (19A), and Lec8 (21B) CHO cells; 1B4-2-1 was initially shown to be ricin resistant (21) and, subsequently, to be deficient in dolichol-P-mannose synthetase, resulting in the synthesis of altered N-linked carbohydrates (33); and the LecR properties of mutants 687 and 699 were consistent (for five different lectins) with those of a Lecl rtutant (699) and a related phenotype (10). The Cl mutant is a recent isolate which has not been extensively characterized.…”

Carbohydrate-deficient glycoprotein syndrome: not an N-linked oligosaccharide processing defect, but an abnormality in lipid-linked oligosaccharide biosynthesis?

“…The enzymatic defects in these cell lines include reduced levels of polyprenol reductase activity (required for the biosynthesis of dolichol), and of mannosylphosphoryldolichol synthase (required for the biosynthesis of Man-P-Dol; see 37,39,45,46). The abnormal LLO structure in the last case has been determined to be Glc3Man5GlcNAc2-P-P-Dol (36,37), again in contrast to the heterogeneous sizes of the LLO seen here. Saccharomyces cerevisiae sec59 cells, which are deficient in dolichol kinase activity (47), show reduced levels of Man incorporation into nascent proteins, as seen with the CDGS samples.…”

“…For another perspective, we also examined skin fibroblasts metabolically labeled with [2- (37)(38)(39)(40). The enzymatic defects in these cell lines include reduced levels of polyprenol reductase activity (required for the biosynthesis of dolichol), and of mannosylphosphoryldolichol synthase (required for the biosynthesis of Man-P-Dol; see 37,39,45,46). The abnormal LLO structure in the last case has been determined to be Glc3Man5GlcNAc2-P-P-Dol (36,37), again in contrast to the heterogeneous sizes of the LLO seen here.…”

Control of carbohydrate processing: increased beta-1,6 branching in N-linked carbohydrates of Lec9 CHO mutants appears to arise from a defect in oligosaccharide-dolichol biosynthesis.

“…radioactivity incorporated into water-soluble and chloroform-soluble fractions was determined ( Table 3). The aqueous fraction (containing water-soluble intermediates such as mannose, mannose-phosphate and GDP-mannose [17]) contained similar amounts of label in each cell line to those in the lipid fraction (containing monoglycosylated lipids such as mannosylphosphoryldolichol [32,35]). However, the chloroform-methanol-water fraction (containing oligosaccharide-lipid intermediates [32]) possessed 11 times more label when obtained from parental cells than from Lec9 cells, and the tryptic peptides were ca.…”