A novel glycosylation phenotype expressed by Lec23, a Chinese hamster ovary mutant deficient in α-glucosidase I

Manas K. Ray, Jing Yang, Subha Sundaram, Pamela Stanley

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Abstract

Lec23 Chinese hamster ovary (CHO) cells have been shown to possess a unique lectin resistance phenotype and genotype compared with previously isolated CHO glycosylation mutants (Stanley, P., Sallustio, S., Krag, S. S., and Dunn, B. (1990) Somatic Cell Mol. Genet. 16, 211-223). In this paper, a biochemical basis for the lec23 mutation is identified. The carbohydrates associated with the G glycoprotein of vesicular stomatitis virus (VSV) grown in Lec23 cells (Lec23/VSV) were found to possess predominantly oligomannosyl carbohydrates that bound strongly to concanavalin A-Sepharose, eluted 3 sugar eq beyond a Man9GlcNAc marker oligosaccharide on ion suppression high pressure liquid chromatography, and were susceptible to digestion with jack bean α-mannosidase. Monosaccharide analyses revealed that the oligomannosyl carbohydrates contained glucose, indicating a defect in α-glucosidase activity. This was confirmed by further structural characterization of the Lec23/VSV oligomannosyl carbohydrates using purified rat mammary gland α-glucosidase I, jack bean α-mannosidase, and 1H NMR spectroscopy at 500 MHz. [3H]Glucose-labeled Glc3Man9GlcNAc was prepared from CHO/VSV labeled with [3H]galactose in the presence of the processsing inhibitors castanospermine and deoxymannojirimycin. Subsequently, [3H]Glc2Man9GlcNAc was prepared by purified α-glucosidase I digestion of [3H] Glc3Man9GlcNAc. When these oligosaccharides were used as α-glucosidase substrates it was revealed that Lec23 cells are specifically defective in α-glucosidase I, a deficiency not previously identified among mammalian cell glycosylation mutants.

Original languageEnglish (US)
Pages (from-to)22818-22825
Number of pages8
JournalJournal of Biological Chemistry
Volume266
Issue number34
StatePublished - Dec 5 1991

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Glycosylation
Cricetulus
Viruses
Vesicular Stomatitis
Ovary
Mannosidases
Carbohydrates
Glucosidases
Phenotype
Jacks
Oligosaccharides
Cells
High pressure liquid chromatography
Digestion
Glucose
Monosaccharides
Viverridae
Galactose
Lectins
Sugars

ASJC Scopus subject areas

  • Biochemistry

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A novel glycosylation phenotype expressed by Lec23, a Chinese hamster ovary mutant deficient in α-glucosidase I. / Ray, Manas K.; Yang, Jing; Sundaram, Subha; Stanley, Pamela.

In: Journal of Biological Chemistry, Vol. 266, No. 34, 05.12.1991, p. 22818-22825.

Research output: Contribution to journalArticle

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