N-Glycosylation of glucose transporter-1 (Glut-1) is associated with increased transporter affinity for glucose in human leukemic cells

Abstract

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 ( K m) without a change in V max. 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.