Role of N-glycosylation in the expression of human SLC26A2 and A3 anion transport membrane glycoproteins 1.

The human solute carrier 26 (SLC26) gene family of anion transporters consists of 10 members (SLC26A1-A11, A10 being a pseudogene) that encode membrane glycoproteins with 14 transmembrane segments and a C-terminal cytoplasmic sulfate transporter anti-sigma antagonist domain. Thus far, mutations in eight members of the SLC26 family (A1-A6, A8, and A9) have been linked to diseases in humans. Our goal is to characterize the role of N-glycosylation and the effect of mutations in SLC26A2 and A3 proteins on their functional expression in transfected HEK-293 cells. We found that certain mutants were retained in the endoplamic reticulum via an interaction with the lectin chaperone calnexin. Some could escape protein quality control and traffic to the cell surface upon removal of the N-glycosylation sites. Furthermore, we found that loss of N-glycosylation reduced expression of SLC26A2 at the cell surface. Loss of N-glycosylation had no effect on the stability of SLC26A3, yet resulted in a profound decrease in transport activity. Thus, N-glycosylation plays three roles in the functional expression of SLC26 proteins: (1) to retain misfolded proteins in the endoplamic reticulum, (2) to stabilize the protein at the cell surface, and (3) to maintain the transport protein in a functional state.

Molecular analysis of human solute carrier SLC26 anion transporter disease-causing mutations using 3-dimensional homology modeling.

The availability of the first crystal structure of a bacterial member (SLC26Dg) of the solute carrier SLC26 family of anion transporters has allowed us to create 3-dimensional models of all 10 human members (SLC26A1-A11, A10 being a pseudogene) of these membrane proteins using the Phyre2 bioinformatic tool. The homology modeling predicted that the SLC26 human proteins, like the SLC26Dg template, all consist of 14 transmembrane segments (TM) arranged in a 7+7 inverted topology with the amino-termini of two half-helices (TM3 and 10) facing each other in the centre of the protein to create the anion-binding site, linked to a C-terminal cytosolic sulfate transporter anti-sigma factor antagonist (STAS) domain. A plethora of human diseases are associated with mutations in the genes encoding human SLC26 transporters, including chondrodysplasias with varying severity in SLC26A2 (~50 mutations, 27 point mutations), congenital chloride-losing diarrhea in SLC26A3 (~70 mutations, 31 point mutations) and Pendred Syndrome or deafness autosomal recessive type 4 in SLC26A4 (~500 mutations, 203 point mutations). We have localized all of these point mutations in the 3-dimensional structures of the respective SLC26A2, A3 and A4 proteins and systematically analyzed their effect on protein structure. While most disease-causing mutations may cause folding defects resulting in impaired trafficking of these membrane glycoproteins from the endoplasmic reticulum to the cell surface - as demonstrated in a number of functional expression studies - the modeling also revealed that a number of pathogenic mutations are localized to the anion-binding site, which may directly affect transport function.

The ENU-3 protein family members function in the Wnt pathway parallel to UNC-6/Netrin to promote motor neuron axon outgrowth in C. elegans.

The axons of the DA and DB classes of motor neurons fail to reach the dorsal cord in the absence of the guidance cue UNC-6/Netrin or its receptor UNC-5 in C. elegans. However, the axonal processes usually exit their cell bodies in the ventral cord in the absence of both molecules. Strains lacking functional versions of UNC-6 or UNC-5 have a low level of DA and DB motor neuron axon outgrowth defects. We found that mutations in the genes for all six of the ENU-3 proteins function to enhance the outgrowth defects of the DA and DB axons in strains lacking either UNC-6 or UNC-5. A mutation in the gene for the MIG-14/Wntless protein also enhances defects in a strain lacking either UNC-5 or UNC-6, suggesting that the ENU-3 and Wnt pathways function parallel to the Netrin pathway in directing motor neuron axon outgrowth. Our evidence suggests that the ENU-3 proteins are novel members of the Wnt pathway in nematodes. Five of the six members of the ENU-3 family are predicted to be single-pass trans-membrane proteins. The expression pattern of ENU-3.1 was consistent with plasma membrane localization. One family member, ENU-3.6, lacks the predicted signal peptide and the membrane-spanning domain. In HeLa cells ENU-3.6 had a cytoplasmic localization and caused actin dependent processes to appear. We conclude that the ENU-3 family proteins function in a pathway parallel to the UNC-6/Netrin pathway for motor neuron axon outgrowth, most likely in the Wnt pathway.