N-glycans on SLC26A3 do not significantly alter plasma membrane or lipid raft trafficking, but appear to stabilize interdomain contacts to stimulate transport.

Abstract

DRA (SLC26A3) is a major apical intestinal Cl^-/HCO₃^- exchanger, which is expressed in complex and hybrid N-glycosylated forms. While 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 to the wildtype construct. Absolute expression of DRA-N0 was significantly reduced by approximately 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 3D-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.