Functional Significance of Glycosylation of Cl‐/oxalate exchanger Slc26a6

Abstract

The brush border Cl‐/oxalate exchanger, Slc26a6, plays a crucial role in enteric oxalate secretion. Mice devoid of Slc26a6 develop profound hyperoxalemia, hyperoxaluria, and calcium oxalate urolithiasis. Modifications such as glycosylation that alter Slc26a6 trafficking or function may therefore be important determinants of oxalate homeostasis. The study aims are to confirm Slc26a6 glycosylation and to characterize its functional significance. By use of glycosidic enzyme digestions and site‐specific mutagenesis of transfected human Slc26a6 expressed in OKP cells, we demonstrate that Slc26a6 is glycosylated and that carbohydrate residues are added via N‐glycosidic linkage at two distinct sites in the putative second extracellular loop. Biotinylation studies suggest that glycosylation is not absolutely essential for surface delivery but does dramatically affect the efficiency by which Slc26a6 is processed and delivered to the cell surface. Moreover, cell‐surface biotinylation trafficking studies indicate that rates of endocytic retrieval of enzymatically deglycosylated Slc26a6 are 2‐3 fold higher than that observed with fully glycosylated Slc26a6, further supporting a role for glycosylation in Slc26a6 trafficking. Finally, investigation of the intrinsic transport function of Slc26a6, assayed as Cl‐ gradient‐dependent uptake of [14C]oxalate normalized to cell‐surface expression, demonstrate that deglycosylation achieved through either mutation or enzymatic digest consistently reduces transport rates by up to 75%. In conclusion, we confirm that Slc26a6 is glycosylated and demonstrate that the presence of glycan residues at two glycosylation sites is critically important for both protein trafficking and oxalate transport function. NIH DK33793.