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 journalArticlepeer-review

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

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Chinese hamster ovary cells with reduced hexokinase activity maintain normal GDP-mannose levels'. Together they form a unique fingerprint.

Cite this