DRA (Downregulated in adenoma, SLC26A3) is a major apical intestinal Cl/HCO3 exchanger, which is expressed in complexand hybrid N-glycosylated forms. Although the importance of N-glycosylation is evident from the significantly reduced transportactivity 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 activityof DRA-N0 was reduced by 70% compared with the wild-type construct. Absolute expression of DRA-N0 was significantlyreduced 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 notsupport a difference in the relative expression of DRA and DRA-N0 in the plasma membrane and in plasma membrane-derivedlipid 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 waspredicted using AlphaFold3 with complex N-glycans covalently attached to N153, N161, and N164 in the transport domain. Thisrevealed multiple inward- and outward-facing conformations of the protein. The number of interdomain contacts of the transportdomain-bound glycans with the scaffold domain was higher in the inward-facing state. Because substrate release to the cytoplasmrepresents the rate-limiting step in many transport proteins, this suggests that in DRA, glycans stabilize the inward-facingstate facilitating anion transport.NEW & NOTEWORTHY Deficient N-glycosylation decreases DRA transport activity but does not significantly affect trafficking tothe 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. Favoringthe inward-facing state may facilitate more efficacious anion transport, as substrate release from this state into the cytoplasm isa rate limiting step for numerous transport proteins.
Achilles et al. (Wed,) studied this question.