CHINESE-HAMSTER OVARY CELLS WITH REDUCED HEXOKINASE-ACTIVITY MAINTAINNORMAL GDP-MANNOSE LEVELS

Parental Chinese hamster ovary (CHO) cells were mutagenized and subjec ted first to a mannose suicide selection technique and second to a scr een of individual colonies grown on polyester discs for reduced mannos e incorporation into protein. The incorporation of radioactivity for t he selection and the screen was conducted at 41.5 degrees C instead of die normal growth temperature of 34 degrees C in order to allow for t he isolation of temperature-sensitive lesions. This selection/screenin g procedure resulted in the isolation of MI5-4 cells, which had three- to five-fold lower incorporation of [2-H-3]mannose into mannose 6-pho sphate mannose 1-phosphate, GDP-mannose, oligosaccharide-lipid, and gl ycoprotein at 41.5 degrees C. We detected no difference in the qualita tive pattern of mannose-labeled lipid-linked oligosaccharides compared to parental cells. MI5-4 cells synthesized dolichol. The defect of MI 5-4 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, incor poration of labeled mannose from the medium was significantly decrease d. 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-m annose, not a substantial reduction in the level of GDP-mannose. Consi stent with these results, no alterations in the glycosylation of a mod el glycoprotein, G protein of vesicular stomatitis virus, were observe d. These cells grew slower than parental cells, especially in low-gluc ose medium. J. Cell. Biochem. 72:56-66, 1999. (C) 1999 Wiley-Liss, Inc .