N-glycosylation of glucose transporter-1 (Glut-1) is associated with increased transporter affinity for glucose in human leukemic cells.
To elucidate the role of N-glycosylation in the functional activity of the universal glucose transporter, Glut-1, we investigated effects of the N-glycosylation inhibitor, tunicamycin, on glucose transport by human leukemic cell lines K562, U937 and HL60. Treatment with tunicamycin produced a 40-50% inhibition of 2-deoxyglucose uptake and this was associated with a 2-2.5-fold decrease in transporter affinity for glucose (Km) without a change in Vmax. Leukemic K562, U937 and HL60 cells expressed Glut-1 transporter protein. With K562 cells Glut-1 appeared as a broad band of 50-60 kDa, whereas with U937 and HL60 cells a diffuse band was observed at approximately 55 kDa. Treatment of K562 cells with tunicamycin for 18 h, resulted in extensive loss of the 50-60 kDa glycoprotein, appearance of a 30-40 kDa band and increased staining of a 45 kDa band. With U937 cells, tunicamycin treatment resulted in the appearance of a 30-40 kDa band and increased staining of a 45 kDa band. With HL60 cells loss of the 55 kDa Glut-1 band was observed and a band of 45 kDa appeared. Tunicamycin-treatment resulted in 75-90% inhibition in [3H]mannose incorporation but only 20-25% inhibition in [3H]thymidine and [3H]leucine incorporation. In contrast, tunicamycin had little effect on the viability and MTT responses of the cells used. These results suggest that in leukemic cells N-glycosylation of Glut-1 plays an important role in maintaining its structure and functional integration.
Ahmed
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N
Malaghan Institute of Medical Research, Wellington School of Medicine, Wellington South, New Zealand. nahmed@mail.newcastle.edu.au
Berridge
M V
MV
D019325:3-O-Methylglucose / Q000378:metabolism / Q000493:pharmacokinetics; D002455:Cell Division / Q000187:drug effects; D003847:Deoxyglucose / Q000378:metabolism / Q000493:pharmacokinetics; D005947:Glucose / Q000378:metabolism* / Q000493:pharmacokinetics; D051272:Glucose Transporter Type 1; D006031:Glycosylation / Q000187:drug effects; D018922:HL-60 Cells; D006801:Humans; D020014:K562 Cells; D007938:Leukemia / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis / Q000378:metabolism*; D013778:Tetrazolium Salts; D013844:Thiazoles; D014415:Tunicamycin / Q000494:pharmacology; D020298:U937 Cells
D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; C495677:SLC2A1 protein, human; D013778:Tetrazolium Salts; D013844:Thiazoles; D014415:Tunicamycin; D019325:3-O-Methylglucose; D003847:Deoxyglucose; C022616:thiazolyl blue; D005947:Glucose