title: N-Glycosylation is required for Na+-dependent vitamin C transporter functionality.
abstract: The human sodium-dependent vitamin C transporters (hSVCT1 and hSVCT2) mediate cellular uptake of ascorbic acid. Both these transporters contain potential sites for N-glycosylation in their extracellular domains (Asn-138, Asn-144 [hSVCT1]; Asn-188, Asn-196 [hSVCT2]), however the role of N-glycosylation in transporter function is unexplored. On the basis of the result that tunicamycin decreased (14)C-ascorbic acid uptake in HepG2 cells, we systematically ablated all consensus N-glycosylation sites in hSVCT1 and hSVCT2 to resolve any effects on ascorbic acid uptake, transporter expression and targeting. We show that removal of individual N-glycosylation sites significantly impairs protein expression and consequently ascorbic acid uptake for hSVCT1 mutants (N138Q is retained intracellularly) and for hSVCT2 mutants (all of which reach the cell surface). N-Glycosylation is therefore essential for vitamin C transporter functionality.
authors.affiliation: Departments of Medicine, Physiology and Biophysics, University of California, Irvine, CA 92697, USA. vsubrama@uci.edu
authors.forename: Veedamali S
authors.initials: VS
authors.lastname: Subramanian
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Marchant
authors.forename: Jack C
authors.initials: JC
authors.lastname: Reidling
authors.forename: Hamid M
authors.initials: HM
authors.lastname: Said
mesh_terms: D001205:Ascorbic Acid / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D009154:Mutation; D029363:Organic Anion Transporters, Sodium-Dependent / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D060347:Sodium-Coupled Vitamin C Transporters; D027981:Symporters / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D029363:Organic Anion Transporters, Sodium-Dependent; D060347:Sodium-Coupled Vitamin C Transporters; D027981:Symporters; D014415:Tunicamycin; D001205:Ascorbic Acid