N-glycosylation critically regulates function of oxalate transporter SLC26A6.
The brush border Cl--oxalate exchanger SLC26A6 plays an essential role in mediating intestinal secretion of oxalate and is crucial for the maintenance of oxalate homeostasis and the prevention of hyperoxaluria and calcium oxalate nephrolithiasis. Previous in vitro studies have suggested that SLC26A6 is heavily N-glycosylated. N-linked glycosylation is known to critically affect folding, trafficking, and function in a wide variety of integral membrane proteins and could therefore potentially have a critical impact on SLC26A6 function and subsequent oxalate homeostasis. Through a series of enzymatic deglycosylation studies we confirmed that endogenously expressed mouse and human SLC26A6 are indeed glycosylated, that the oligosaccharides are principally attached via N-glycosidic linkage, and that there are tissue-specific differences in glycosylation. In vitro cell culture experiments were then used to elucidate the functional significance of the addition of the carbohydrate moieties. Biotinylation studies of SLC26A6 glycosylation mutants indicated that glycosylation is not essential for cell surface delivery of SLC26A6 but suggested that it may affect the efficacy with which it is trafficked and maintained in the plasma membrane. Functional studies of transfected SLC26A6 demonstrated that glycosylation at two sites in the putative second extracellular loop of SLC26A6 is critically important for chloride-dependent oxalate transport and that enzymatic deglycosylation of SLC26A6 expressed on the plasma membrane of intact cells strongly reduced oxalate transport activity. Taken together, these studies indicated that oxalate transport function of SLC26A6 is critically dependent on glycosylation and that exoglycosidase-mediated deglycosylation of SLC26A6 has the capacity to profoundly modulate SLC26A6 function.
Section of Nephrology, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut.
R Brent
RB
Thomson
Section of Nephrology, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut.
Claire L
CL
Thomson
Section of Nephrology, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut peter.aronson@yale.edu.
Peter S
PS
Aronson
D000818:Animals; D017920:Antiporters / Q000378:metabolism*; D018938:Caco-2 Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology; D002712:Chlorides / Q000378:metabolism; D006031:Glycosylation; D006706:Homeostasis / Q000502:physiology; D006801:Humans; D017136:Ion Transport / Q000502:physiology; D008297:Male; D026901:Membrane Transport Proteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D053040:Nephrolithiasis / Q000378:metabolism; D009893:Opossums; D010070:Oxalates / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D000076988:Sulfate Transporters
D017920:Antiporters; D002712:Chlorides; D026901:Membrane Transport Proteins; D010070:Oxalates; C466900:Slc26a6 protein, mouse; D000076988:Sulfate Transporters
SLC26A6; anion exchange; glycosylation; oxalate
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