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.

Rosenwald, Anne G.; Stanley, Pamela; Krag, Sharon S.

doi:10.1128/mcb.9.3.914

Cited by 22 publications

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“…Lec8, Lec9, LEC10, LEC11, and LEC12. structural and enzymic studies, which identified the basis for the glycosylation change in each mutant (1,5,6,9,12,(26)(27)(28)(32)(33)(34)(35)(36)(37)(38); Krieger et al, in press). The N-linked carbohydrates in parental CHO cells would be expected to contain up to three or more lactosamine units (Gal 0-1,4-GlcNAc) (14).…”

Section: Resultsmentioning

confidence: 99%

“…Interestingly, amebic adherence was not apparently affected by the increased branching of N-linked carbohydrates that is characteristic of Lec9 and LEC10 mutants ( Table 1). Lec9 mutants synthesize an increased proportion of ,8-1,6-branched, N-linked carbohydrates (26,28), and LEC10 mutants add the bisecting GlcNAc to N-linked carbohydrates (1). It is of note that amebic adherence to Lec9 CHO cells was extremely sensitive to inhibition by Gal (98.8% inhibition).…”

Section: Resultsmentioning

confidence: 99%

“…CHO cells cultivated included a Gat-2 parent and Lecl, Lec2, Lec3, Lec4, Lec8, Lec9, LEC10, LEC11, LEC12, and ldlD.Lecl glycosylation mutants (28,(34)(35)(36)(37)(38). The specific clones used were as follows: Gat-Lecl.lN, Pro-Lec2.6A, Pro-Lec3.4B, Pro-Lec4.7B, Gat-Lec8.1C, Pro-Lec9.12A, Pro-LEC10.3C, Pro-LEC11.E7, Pro-LEC12.1B, and Pro-ldlD.Lecl.1.5.…”

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Characterization of cell surface carbohydrate receptors for Entamoeba histolytica adherence lectin

et al. 1989

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Binding and cytolysis of Chinese hamster ovary (CHO) cells by Entamoeba histolytica trophozoites is inhibitable by galactose (Gal) or N-acetyl-D-galactosamine (GalNAc). To better define the carbohydrate receptor for E. histolytica, we compared the binding and cytolytic target properties of 10 CHO glycosylation mutants. Each mutant expresses a uniquely altered array of N- and/or O-linked cell surface carbohydrates. Amebic adherence was reduced when lactosamine-containing N-linked carbohydrates were essentially absent (Lec1 mutant), almost undetectable when Gal and GalNAc residues were absent on both N- and O-linked carbohydrates (ldlD.Lec1 mutant), and enhanced for mutants with increased terminal Gal residues (Lec2 and Lec3). Parental CHO cells treated with neuraminidase to expose Gal residues behaved like Lec2 mutants. Binding of purified Gal or GalNAc lectin to parental, Lec1, ldlD.Lec1, and Lec2 mutant CHO cells corroborated the adherence results. The suitability of CHO cell mutants as targets for amebic cytolysis correlated with their glycosylation phenotype: the Lec1 mutants were less susceptible than parental CHO cells, the ldlD.Lec1 mutants were highly resistant, and the Lec2 mutants required higher concentrations of Gal for inhibition. The E. histolytica Gal or GalNAc adherence lectin bound preferentially to beta 1-6-branched, N-linked carbohydrates lacking terminal sialic acid or fucose residues. However, amebic lectin binding to either N- or O-linked cell surface carbohydrates was sufficient to initiate parasite cytolytic activity.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Characterization of cell surface carbohydrate receptors for Entamoeba histolytica adherence lectin

et al. 1989

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“…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).…”

Section: Discussionmentioning

confidence: 99%

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

Powell¹,

Paneerselvam²,

Vij³

et al. 1994

J. Clin. Invest.

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The carbohydrate-deficient glycoprotein syndrome (CDGS) is a developmental disease associated with an abnormally high isoelectric point of serum transferrin. Carbohydrate analyses of this glycoprotein initially suggested a defect in N-linked oligosaccharide processing, although more recent studies indicate a defect in the attachment of these sugar chains to the protein. We studied both serum glycoproteins and fibroblast-derived [2-3H]mannose-labeled oligosaccharides from CDGS patients and normal controls. While there was a decrease in the glycosylation of serum glycoproteins of affected individuals, differences were not seen in either monosaccharide composition or oligosaccharide structures. The lectin-binding profiles of glycopeptides from [2-3H]-mannose-labeled fibroblasts were likewise indistinguishable. However, the incorporation of [2-3H]mannose into both glycoproteins and the dolichol-linked oligosaccharide precursor was significantly reduced. Thus, at least in some patients, CDGS is not due to a defect in processing of N-linked oligosaccharides, but rather to defective synthesis and transfer of nascent dolichol-linked oligosaccharide precursors. This abnormality could result in both a failure to glycosylate some sites on some proteins, as well as secondary abnormalities in overall glycoprotein processing and/or function. (J.

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“…tein [Morrison and Lodish, 1975]. Normally in CHO cells, G protein is N-glycosylated at two sites [Stanley, 1982] and di-, mono-, and nonglycosylated forms can easily be separated by SDS-PAGE [Rosenwald et al, 1989].…”

Section: N-linked Glycosylation Of G Protein Of Vsv-infected Mi5-4 Cellsmentioning

confidence: 99%

Chinese hamster ovary cells with reduced hexokinase activity maintain normal GDP-mannose levels

et al. 1999

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Parental Chinese hamster ovary (CHO) cells were mutagenized and subjected first to a mannose suicide selection technique and second to a screen of individual colonies grown on polyester discs for reduced mannose incorporation into protein. The incorporation of radioactivity for the selection and the screen was conducted at 41.5 degrees C instead of the normal growth temperature of 34 degrees C in order to allow for the isolation of temperature-sensitive lesions. This selection/screening procedure resulted in the isolation of M15-4 cells, which had three- to five-fold lower incorporation of [2-3H]mannose into mannose 6-phosphate, mannose 1-phosphate, GDP-mannose, oligosaccharide-lipid, and glycoprotein at 41.5 degrees C. We detected no difference in the qualitative pattern of mannose-labeled lipid-linked oligosaccharides compared to parental cells. M15-4 cells synthesized dolichol. The defect of M15-4 cells was determined to be in hexokinase activity; crude cytosolic extracts were eight- to nine-fold lower in hexokinase activity in M15-4 cells compared to parental cells. As a result of this defect, incorporation of labeled mannose from the medium was significantly decreased. However, the level of GDP-mannose in M15-4 cells was 70% of normal. The phenotype of M15-4 was a lower specific activity of labeled GDP-mannose, not a substantial reduction in the level of GDP-mannose. Consistent with these results, no alterations in the glycosylation of a model glycoprotein, G protein of vesicular stomatitis virus, were observed. These cells grew slower than parental cells, especially in low-glucose medium.

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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.

Cited by 22 publications

References 21 publications

Characterization of cell surface carbohydrate receptors for Entamoeba histolytica adherence lectin

Characterization of cell surface carbohydrate receptors for Entamoeba histolytica adherence lectin

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

Chinese hamster ovary cells with reduced hexokinase activity maintain normal GDP-mannose levels

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