Topology Structure Of Neutral Amino Acid Transporter SNAT2

SNAT2is the second member of the sodium-coupled neutral amino acidtransporters (SNATs), which belong to the SLC38(Solute carrier family38) gene family.It plays an important role in glutamate-glutamine cycle in the brain andgluconeogenesis in the liver. On the other hand, SNAT2appears to be the majorregulated isoform in response to endocrine and nutrient stimuli. It has been reportedthat alterations in glutamate transporters is responsible for severalneurodegenerative disorders. Except the physiological importance, little is knownabout its functional and structural properties. Hydropathy analysis predicts thatSNAT2contains11transmembrane helices (TM) with an intracelluar N terminus andan extracelluar C terminus, The transport process mediated by SNAT2is sensitive tothe external pH, coupled the anion leak conductance. Since the crystal structure ofSNAT2is unavailable so far, sequence homology was used to investigate the functionand structure of SNAT2.In order to detect expression and localization of SNAT2on the membraneconveniently, the C terminus of enhanced green fluorescence protein (EGFP) waslinked to the N terminus of SNAT2in the mammalian expression vector pBK-CMV(Δ[1098-1300]) by PCR, single restriction endonuclease digestion and ligation techniques.Since there was a terminator between gene EGFP and SNAT2，site-directedmutagenesis was used to mutate the terminator into Leucine. Enzyme digestion andsequence were used to confirm the plasmid pBK-CMV⊿-EGFP-SNAT2. After theplasmid pBK-CMV-EGFP-SNAT2was transiently transfected into HEK293T cells for36hours, the expression and localization of EGFP-SNAT2fusion protein were detectedby western blot and laser scanning confocal microscope (LSCM). The result showedthat EGFP-SNAT2expressed and localized on the cell membrane normally. Theconstruction of the expression vector of pBK-CMV-EGFP-SNAT2will be of greatbenefit to the investigation of structure and function of SNAT2in the future.In order to investigate the topology structure of neutral amino acid transporterSNAT2, the location of N terminus and C terminus of SNAT2, the site of N-glycosylation and the number of transmemberane domains should be investigated.Firstly, two plasmids，namely pBK-CMV⊿-HA--SNAT2and pBK-CMV⊿-SNAT2-HA，were used to determine the location of N terminus and C terminus of SNAT2.pBK-CMV⊿-SNAT2-HA was constructed previously in our laboratory. pBK-CMV⊿-HA--SNAT2was constructed by using enzyme digestion and ligation. Then theplasmid was verified by double digestion and sequencing. The result of Western Blotshowed that HA-SNAT2could express normally in HEK293T cells. After two plasmidswere obtained, Alexa Fluor488labeled HA antibody was used to detect the directionof N terminus and C terminus of SNAT2. It has been shown that N terminus wasextracellular while C terminus was inside of the membrane.Secondly, two N-glycosylation sites of SNAT2were predicted previously, N258and N272. After these two sites were mutated into glutamine, Western Blot was usedto identify whether these two sites were glycosylated. According to the change ofmobility, it could be concluded that these two sites were the N-glycosylation sites.But the mobility shift on SDS-PAGE wasn’t consistent with the SNAT2protein digestedby PNGase F, so we deduced there was another N-glycosylation site in SNAT2. N254was identified to be another N-glycosylation site by using the same method.We will utilize chemical surface labeling approach with a membraneimpermeable Cys-directed reagent, MTSEA-biotin, to get the number of thetransmemberane domains in the future.