Fig. 1
Expression and post-translational processing of NHE6 ΔES mutant protein is impaired in transfected AP-1 and SH-SY5Y cells. a Schematic drawing of the predicted membrane topology of mammalian NHE6v1 (based on sequence alignment and transmembrane organization of NHE1 proposed by Wakabayashi et al. [127] and Nygaard et al. [128]) and locations of mutations (red shading) identified by Gilfillan et al. [8] in patients with Christianson syndrome. The blue shading in the second extracellular loop (EL2) represents the additional 32 amino acids (residues 145–176) present in the NHE6v1 splice-variant. Two predicted N-glycosylation sites within EL2 are also illustrated. b AP-1 and c, SH-SY5Y cells were transiently transfected (24 h) with NHE6HA WT or ΔES mutant. Total cell lysates of WT and ΔES-transfectants were examined by SDS-PAGE. The immunoblots were probed with a mouse monoclonal anti-HA antibody (α-HAm) to detect NHE6v1. NHE6v1 migrates as multiple bands: slower migrating high molecular weight bands representing the fully-glycosylated (fg) and core-glycosylated (cg) dimeric forms of the exchanger (~200 and 175 kDa, respectively) that do not fully dissociate under SDS-PAGE conditions and faster migrating fully-glycosylated (fg, ~100 kDa) and core-glycosylated (cg, ~70 kDa) and unglycosylated (ug, ~65 kDa) forms of the monomeric protein. To control for protein loading, the blots were reprobed with a mouse monoclonal anti-GAPDH antibody (α-GAPDHm). d, e To confirm the nature of the oligosaccharide modifications of the NHE6 bands, AP-1 cells transiently transfected (24 h) with WT or ΔES constructs were lysed in non-detergent buffers and post-nuclear supernatants were left untreated or incubated with either endoglycosidase H (EndoH), which cleaves only asparagine-linked mannose-rich oligosaccharides (i.e., core-glycosylated) but not more highly processed complex oligosaccharides (i.e., fully-glycosylated), or peptide-N-glycosidase F (PNGaseF) which cleaves between the innermost N-acetylglucosamine and asparagine residues of all oligosaccharide structures (i.e., high mannose, hybrid, and complex). The lysates were then subjected to SDS-PAGE and immunoblotting with an α-HAm antibody. The data are representative of three independent experiments