The role of N-glycosylation of GLUT1 for glucose transport activity.
To elucidate a functional role of N-glycosylation in glucose transporters, we introduced oligonucleotide-directed mutagenesis in GLUT1 cDNA to remove the possible site for N-linked glycosylation. The wild-type and the mutated GLUT1 cDNAs which induced a mutation of Asn at residue 45 to Asp, Tyr, or Gln were transfected and stably expressed into Chinese hamster ovary cells. The expressed wild-type and the mutated GLUT1 was demonstrated to be a broad band of a 45-60-kDa form and a sharp band of a 38-kDa form on Western blot analysis, respectively, indicating no glycosylation in the mutated GLUT1. Although the cell surface labeling of the glucose transporters demonstrated the presence of the glycosylation-defective glucose transporters on the cells surface, photoaffinity labeling of glycosylation-defective GLUT1 with [3H] cytochalasin B and a photoreactive mannose derivative, [3H]2-N-4-(1-azi-2,2,2,trifluoroethyl)benzoyl-1,3-bis(D-mannos+ ++-4-yloxy)-2- propylamine in the membranes was observed to be 40-70 and 15-30% of that of the wild-type GLUT1, respectively. The kinetic study of 2-deoxyglucose uptake revealed that the glycosylation-defective GLUT1 had a 2-2.5-fold greater Km value for 2-deoxyglucose uptake compared with the wild-type GLUT1. These observations strongly suggest that 1) N-glycosylation of GLUT1 glucose transporter is only on Asn 45 and 2) N-glycosylation plays an important role in maintaining a structure of glucose transporter with high affinity for glucose, thus, with high transport activity.
Asano
T
T
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Katagiri
H
H
Takata
K
K
Lin
J L
JL
Ishihara
H
H
Inukai
K
K
Tsukuda
K
K
Kikuchi
M
M
Hirano
H
H
Yazaki
Y
Y
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D015153:Blotting, Western; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D003571:Cytochalasin B / Q000378:metabolism; D004247:DNA / Q000235:genetics; D003847:Deoxyglucose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D006031:Glycosylation; D006601:Hexoses / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010777:Photochemistry; D011817:Rabbits; D014162:Transfection
D000345:Affinity Labels; D006601:Hexoses; D009004:Monosaccharide Transport Proteins; D003571:Cytochalasin B; D004247:DNA; D003847:Deoxyglucose; D005947:Glucose