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

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.