N-glycosylation and topology of the human SLC26 family of anion transport membrane proteins

Abstract

Li J, Xia F, Reithmeier RA. N-glycosylation and topology of the human SLC26 family of anion transport membrane proteins. Am J Physiol Cell Physiol 306: C943-C960, 2014. First published March 19, 2014 doi:10.1152/ajpcell.00030.2014.-The human solute carrier (SLC26) family of anion transporters consists of 10 members (SLCA1-11, SLCA10 being a pseudogene) that encode membrane proteins containing ϳ12 transmembrane (TM) segments with putative N-glycosylation sites (-NXS/T-) in extracellular loops and a COOHterminal cytosolic STAS domain. All 10 members of the human SLC26 family, FLAG-tagged at the NH2 terminus, were transiently expressed in HEK-293 cells. While most proteins were observed to contain both high-mannose and complex oligosaccharides, SLC26A2 was mainly in the complex form, SLC26A4 in the high-mannose form, and SLC26A8 was not N-glycosylated. Mutation of the putative N-glycosylation sites showed that most members contain multiple N-glycosylation sites in the second extracytosolic (EC) loop, except SLC26A11, which was N-glycosylated in EC loop 4. Immunofluorescence staining of permeabilized cells localized the proteins to the plasma membrane and the endoplasmic reticulum, with SLC26A2 highly localized to the plasma membrane. N-glycosylation was not a necessary requirement for cell surface expression as the localization of nonglycosylated proteins was similar to their wild-type counterparts, although a lower level of cell-surface biotinylation was observed. No immunostaining of intact cells was observed for any SLC26 members, demonstrating that the NH2-terminal FLAG tag was located in the cytosol. Topological models of the SLC26 proteins that contain an even number of transmembrane segments with both the NH2 and COOH termini located in the cytosol and utilized N-glycosylation sites defining the positions of two EC loops are presented. anion transport; calnexin; congenital chloride-losing diarrhea; CLD; membrane glycoproteins; membrane protein topology; membrane protein trafficking; D-glycosylation; SLC26; solute carrier THE SLC26 FAMILY OF ANION transporters is found in all kingdoms of life indicating a fundamental importance of these membrane proteins in biology. The human SLC26 family (Table 1) of anion transporters consists of 10 members (SLC26A1-11, A10 being a pseudo gene) (18,36,62,63). The human SLC26 proteins contain ϳ750 residues but range in size from 606 residues (SLC26A11) to 970 residues (SLC26A8). The proteins all consist of two major domains: a NH 2 -terminal membrane domain with ϳ12 putative transmembrane (TM) segments and a COOH-terminal STAS (sulfate transporter antagonist of anti-sigma factor) domain located in the cytosol (53). Structures of the STAS domain of several bacterial members of the SLC26 family have been obtained (4, 54), as well as the structure of a modified version of the STAS domain of rat SLC26A5 protein (40). All of the core structures resemble the Bacillus anti-sigma factor antagonist SpoIIAA, a 110 amino acid phospho-protein with an alpha/beta topology consisting of ...

Plasma Membrane Targeting of Endogenous NKCC2 in COS7 Cells Bypasses Functional Golgi Cisternae and Complex N-Glycosylation

“…However, most of NKCC2 from COS7 cells (densitometrically estimated 80–90%) appeared as core/high mannose (~120 kDa) and hybrid-type N-glycans (~135 kDa), whereas the complex N-glycosylated version of the transporter (~160 kDa) was barely detected. These results are consistent with the relatively low expression levels of the transporter in these cells and with the concept that complex N-glycosylation depends on the level of cargo reaching the complex N-glycosylation machinery of the Golgi, as has been suggested for many proteins (Stanley, 2011), including NKCC1 in COS7 cells (Singh et al, 2015) and other ion transporters (Li et al, 2014). The low levels, or even absence, of complex N-glycosylated NKCC2 is consistent with the cellular distribution of the co-transporter in COS7 cells, where most of the endogenous NKCC2 appeared localized in intracellular compartments but not in the plasma membrane.…”

Bicarbonate transport in health and disease

“…Recently, the structure of SLC26 proteins has been reviewed . SLC26 proteins have three distinct regions: a variable cytoplasmic N‐terminal region (51–106 amino acids), an integral membrane domain of 12 transmembrane segments, and a cytoplasmic C‐terminal STAS (sulfate transporter and anti‐Sigma factor antagonist) domain related to those found in some bacterial proteins. SLC26 proteins are dimeric , as also seen for SLC4 proteins.…”

N-linked glycosylation of N48 is required for equilibrative nucleoside transporter 1 (ENT1) function

“…Coe, unpublished). However, since we suspect that ENTs form dimers and we know that glycosylation of other transporters has been correlated with the formation of oligomers [20,21], we investigated the role of glycosylation of ENT1 in the formation of ENT dimers. As predicted, we observed that wt ENT1 co-immunoprecipitated with itself (HA-hENT1 with 3xFLAG-ENT1) but did not co-immunoprecipitate with 3xFLAG-N48Q-ENT1, or 3xFLAG-hLa (Figure 4) suggesting that N48Q mutant ENT1 is unable to form a complex with wt ENT1.…”

“…HEK293 (human embryonic kidney cell line), commonly used in membrane protein glycosylation studies [20–22], were grown in Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% (v/v) FBS in 10 cm 2 plates [ S -(4-nitrobenzyl)-6-thioinosine (NBTI) binding and Western blotting] or six-well plates (transport assays) at 37°C with 5% (v/v) CO 2 . Cells were transfected using the standard Polyjet protocol (SignaGen Laboratories) and incubated post transfection for ∼36 h. Equivalent transfection efficiency in wild type (wt) and mutant-transfected cells was confirmed by microscopy.…”