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

Jessica L. O'Rear, Jane R. Scocca, Brian K. Walker, Adina Kaiden, Sharon S. Krag

Research output: Contribution to journalArticle

Abstract

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°C instead of the normal growth temperature of 34°C in order to allow for the isolation of temperature-sensitive lesions. This selection/screening procedure resulted in the isolation of MI5-4 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°C. We detected no difference in the qualitative pattern of mannose-labeled lipid-linked oligosaccharides compared to parental cells. MI5-4 cells synthesized dolichol. The defect of MI5-4 cells was determined to be in hexokinase activity, crude cytosolic extracts were eight- to nine-fold lower in hexokinase activity in MI5-4 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 MI5-4 cells was 70% of normal. The phenotype of MI5-4 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.

Original languageEnglish (US)
Pages (from-to)56-66
Number of pages11
JournalJournal of Cellular Biochemistry
Volume72
Issue number1
DOIs
StatePublished - Jan 1 1999

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Guanosine Diphosphate Mannose
Hexokinase
Mannose
Cricetulus
Ovary
Cells
Glycoproteins
Dolichol
Glycosylation
Defects
Polyesters
Radioactivity
Growth temperature
Oligosaccharides
Screening
Lipids
Glucose
Temperature
Complex Mixtures
Suicide

Keywords

  • G protein of vesicular stomatitis virus
  • N-linked glycosylation
  • Screen/selection mutant isolation procedure

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology

Cite this

Chinese hamster ovary cells with reduced hexokinase activity maintain normal GDP-mannose levels. / O'Rear, Jessica L.; Scocca, Jane R.; Walker, Brian K.; Kaiden, Adina; Krag, Sharon S.

In: Journal of Cellular Biochemistry, Vol. 72, No. 1, 01.01.1999, p. 56-66.

Research output: Contribution to journalArticle

O'Rear, Jessica L. ; Scocca, Jane R. ; Walker, Brian K. ; Kaiden, Adina ; Krag, Sharon S. / Chinese hamster ovary cells with reduced hexokinase activity maintain normal GDP-mannose levels. In: Journal of Cellular Biochemistry. 1999 ; Vol. 72, No. 1. pp. 56-66.
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