Structure, biosynthesis, and function of the hexose transporter in Chinese hamster ovary cells deficient in N-acetylglucosaminyltransferase 1 activity.
We have used a Chinese hamster ovary cell line deficient in N-acetylglucosaminyltransferase 1 activity (Lec1) to study the effects of altered asparagine-linked oligosaccharides on the structure, biosynthesis, and function of glucose transporter protein. Immunoblots of membranes of Lec1 cells show a glucose transporter protein of Mr 40,000, whereas membranes of wild-type (WT) cells contain a broadly migrating Mr 55,000 form similar to that observed in several other mammalian tissues. The total content of immunoreactive glucose transporters in Lec1 cells is 3.5-fold greater than that of WT cells. Digestion with endoglycosidases, treatment with inhibitors of glycosylation, and interactions with agarose-bound lectins demonstrate that glucose transporters of both cell lines derive from a similar Mr 38,000 core polypeptide and that both contain asparagine-linked oligosaccharide. Transporters in Lec1 cells contain primarily "undecorated" but "trimmed" mannose-type asparagine-linked oligosaccharides, while the protein in WT cells contains a mixture of "decorated" and "trimmed" asparagine-linked oligosaccharides. Biosynthetic and turnover studies demonstrate that Lec1 cells, in contrast to WT cells, are unable fully to process the core asparagine-linked oligosaccharides of maturing glucose transporters. When radiolabeled in methionine-deficient medium both Lec1 and WT cells show similar rates of synthesis and turnover of glucose transporter proteins. It should be noted, however, that starvation for a critical amino acid may alter the ability of the cell to synthesize or degrade proteins. The abilities of Lec1 and WT cells to transport hexoses and to interact with the inhibitor cytochalasin B are very similar. The results indicate that, although altered asparagine-linked glycosylation can affect the content and biogenesis of glucose transporters, these changes do not greatly modify cellular hexose uptake. The possibility that alterations in asparagine-linked glycosylation may change the cell surface localization or acquisition of a "functional conformation" of the glucose transporter is also suggested.
Haspel
H C
HC
Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Revillame
J
J
Rosen
O M
OM
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D003571:Cytochalasin B / Q000494:pharmacology; D005260:Female; D005964:Glucosyltransferases / Q000172:deficiency*; D006031:Glycosylation; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis / Q000502:physiology*; D017351:N-Acetylglucosaminyltransferases; D010053:Ovary / Q000201:enzymology / Q000378:metabolism
D009004:Monosaccharide Transport Proteins; D003571:Cytochalasin B; D005964:Glucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I