DRA (Downregulated in adenoma, SLC26A3) is a major apical intestinal Cl-/HCO3- exchanger, which is expressed in complex and hybrid N-glycosylated forms. Although the importance of N-glycosylation is evident from the significantly reduced transport activity of non-N-glycosylated DRA constructs (DRA-N0), the underlying molecular mechanisms are controversial. Therefore, plasma membrane expression and lipid raft localization of glycosylation-deficient DRA-N0 were analyzed in HEK cells. The activity of DRA-N0 was reduced by 70% compared with the wild-type construct. Absolute expression of DRA-N0 was significantly reduced by ∼57% in the cell lysate and by 34 and 45% in the plasma membrane and in plasma membrane-derived lipid rafts, respectively. These amounts are insufficient to account for the reduction in activity. Furthermore, the statistical analysis did not support a difference in the relative expression of DRA and DRA-N0 in the plasma membrane and in plasma membrane-derived lipid rafts, indicating that N-glycosylation does not affect transport activity through trafficking and localization in these cell compartments. To gain insight into potential intramolecular effects of N-glycosylation on DRA, its three-dimensional structure was predicted using AlphaFold3 with complex N-glycans covalently attached to N153, N161, and N164 in the transport domain. This revealed multiple inward- and outward-facing conformations of the protein. The number of interdomain contacts of the transport domain-bound glycans with the scaffold domain was higher in the inward-facing state. Because substrate release to the cytoplasm represents the rate-limiting step in many transport proteins, this suggests that in DRA, glycans stabilize the inward-facing state facilitating anion transport.NEW & NOTEWORTHY Deficient N-glycosylation decreases DRA transport activity but does not significantly affect trafficking to the plasma membrane or to lipid rafts. Meanwhile, molecular modeling predicts stabilizing interdomain contacts of the glycans, covalently attached to the transport domain, with the scaffold domain having more contacts in the inward-facing state. Favoring the inward-facing state may facilitate more efficacious anion transport, as substrate release from this state into the cytoplasm is a rate limiting step for numerous transport proteins.
Keywords: SLC26A3; glycosylation; lipid rafts; plasma membrane trafficking; structure prediction.