SP27
SLC35A2 PROVIDES A NOVEL ROLE FOR GLYCOSYLATION IN GLUCOSE
UPTAKE AND CELLULAR METABOLISM.
Annear, NMP1, Maxwell, PH2, Ashcroft, M1
1
Centre for Cell Signalling and Molecular Genetics, Division of Medicine,
The Rayne Building, University College London, London WC1E 6JF
2
School of Clinical Medicine, University of Cambridge, UK
INTRODUCTION:
Glucose transport is central to cellular metabolism1. The facilitative glucose transporter,
GLUT1 is a glycoprotein that is N-glycosylated at a single site2. GLUT1 is commonly
upregulated in human cancers, a phenotype that is at least in part mediated by the transcription
factor, hypoxia-inducible factor (HIF)3, including the commonest form of kidney cancer, clear
cell renal cell carcinoma (CCRCC), where mutations in the von Hippel-Lindau tumour
suppressor (VHL) are frequently observed, leading to HIF upregulation4. A number of cancers
of different tissue origin have been reported to harbour somatic missense, deletion and nonsense
mutations in the glycosylation gene, SLC35A2, which encodes the UDP-galactose transporter5.
The role of SLC35A2 in modulating glucose transport has not hitherto been explored.
METHODS:
The glycosylation mutant chinese hamster ovary (CHO) cell line Lec8, which harbours an
inactivating mutation in Slc35a2, and parental CHO cells were used. Lectin binding was
assessed by fluorescence microscopy, Glut1 expression was analysed by western blot, and
uptake of the fluorescent glucose analogue, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2deoxyglucose (2-NBDG) was assessed by flow cytometry. Human renal cancer cell lines (786O and RCC10), either expressing empty vector (EV) or re-expressing VHL, were used to assess
expression of SLC35A2 by western blot and by reverse transcription polymerase chain reaction.
RESULTS:
We found that Lec8 cells exhibit enhanced electrophoretic mobility of Glut1 protein, altered
lectin binding, and reduced uptake of 2-NBDG, which was time- and concentration-dependent,
and reduced whole cell ATP content. Importantly, we found that enhanced Glut1
electrophoretic mobility, altered lectin binding, decreased 2-NBDG uptake and decreased whole
cell ATP were all reversed by re-expression of Slc35a2 in Lec8 cells. Furthermore, treatment
with the hexokinase inhibitor, 2-deoxyglucose led to a more significant reduction in whole cell
ATP content in wild-type compared with Lec8 cells. Taken together, our data indicate that
Slc35a2 modulates the rate of glucose transport in CHO cells, and this may be mediated by
Glut1 glycosylation state. Interestingly, we found that in two independent human CCRCC cell
lines, SLC35A2 protein and mRNA expression was increased following re-expression of VHL.
CONCLUSION:
Our study links glycosylation with glucose uptake and cellular metabolism, and may provide
new insight into targeting glucose uptake in renal carcinoma.
REFERENCES:
1.
Augustin R. The Protein Family of Glucose Transport Facilitators: It's Not Only About
Glucose After All. IUBMB Life 2010; 62(5): 315-33.
2.
Asano T, Katagiri H, Takata K, et al. The role of N-glycosylation of GLUT1 for
glucose transport activity. Journal of Biological Chemistry 1991; 266(36): 24632-6.
3.
Ouiddir A, Planes C, Fernandes I, VanHesse A, Clerici C. Hypoxia upregulates activity
and expression of the glucose transporter GLUT1 in alveolar epithelial cells. American journal
of respiratory cell and molecular biology 1999; 21(6): 710-8.
4.
Maxwell PH, Wiesener MS, Chang GW, et al. The tumour suppressor protein VHL
targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 1999;
399(10353251): 271-5.
5.
Bamford S, Dawson E, Forbes S, et al. The COSMIC (Catalogue of Somatic Mutations
in Cancer) database and website. . Br J Cancer 2004; 91: 355-8.