Mutagenesis of the N-glycosylation site of hNaSi-1 reduces transport activity

Hongyan Li and Ana M. Pajor

Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555-0641

Submitted 24 April 2003 ; accepted in final form 10 July 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human Na+-sulfate cotransporter (hNaSi-1) belongs to the SLC13 gene family, which also includes the high-affinity Na+-sulfate cotransporter (hSUT-1) and the Na+-dicarboxylate cotransporters (NaDC). In this study, the location and functional role of the N-glycosylation site of hNaSi-1 were studied using antifusion protein antibodies. Polyclonal antibodies against a glutathione S-transferase fusion protein containing a 65-amino acid peptide of hNaSi-1 (GST-Si65) were raised in rabbits, purified, and then used in Western blotting and immunofluorescence experiments. The antibodies recognized native NaSi-1 proteins in pig and rat brush-border membrane vesicles as well as the recombinant proteins expressed in Xenopus oocytes. Wild-type hNaSi-1 and two N-glycosylation site mutant proteins, N591Y and N591A, were functionally expressed and studied in Xenopus oocytes. The apparent mass of N591Y was not affected by treatment with peptide-N-glycosylase F, in contrast to the mass of wild-type hNaSi-1, which was reduced by up to 15 kDa, indicating that Asn591 is the N-glycosylation site. Although the cell surface abundance of the two glycosylation site mutants, N591Y and N591A, was greater than that of wild-type hNaSi-1, both mutants had greatly reduced Vmax, with no change in Km. These results suggest that Asn591 and/or N-glycosylation is critical for transport activity in NaSi-1.

antifusion protein antibodies; Xenopus oocytes; sulfate; immunofluorescence


THE LOW-AFFINITY Na+-sulfate cotransporter NaSi-1 is located on the apical membrane of renal proximal tubule epithelial cells and plays an important role in maintaining sulfate homeostasis (5, 17, 19). NaSi-1 couples three Na+ ions to the transport of an oxyanion, such as sulfate, thiosulfate, and selenate (4). NaSi-1 belongs to the SLC13 (solute carrier 13) gene family, which also includes the high-affinity Na+-sulfate co-transporter (SUT-1) and the Na+-dicarboxylate cotransporters (NaDC) (19). The sequences of NaSi-1 and the NaDC transporters are ~40% identical, and they are likely to have similar protein structures.

The NaSi-1 transporters contain a single consensus sequence for N-glycosylation at the COOH terminus, in addition to two or three other sites in the proteins (3, 15, 20). All members of the SLC13 gene family contain one or two consensus N-glycosylation sequences at the COOH terminus, and there is experimental evidence that the COOH-terminal sequence is the N-glycosylation site in NaDC-1 (22). Although in vitro translation experiments suggest a single N-glycosylation site in rat NaSi-1 (20), the location of this site in NaSi-1 transporters has not been identified, and the functional role of N-glycosylation in NaSi-1 is not known. Mutagenesis studies of the rabbit NaDC-1 suggest that N-glycosylation is required for protein trafficking, rather than transport activity (22). However, there is evidence for a functional role of N-glycosylation in some transport proteins. For example, mutagenesis of the N-glycosylation site in the GLUT-1 glucose transporter resulted in increased Km (1). Mutagenesis of N-glycosylation sites in the neuronal GABA transporter GAT1 resulted in a reduction in substrate turnover and altered voltage sensitivity (16).

In this study, polyclonal antibodies against the human NaSi-1 (hNaSi-1) were produced using a glutathione S-transferase (GST) fusion protein containing a 65-amino acid peptide of hNaSi-1 (GST-Si65). The antibodies recognized native and recombinant proteins. The consensus sequence for N-glycosylation at the COOH terminus in hNaSi-1 was removed by mutating Asn591 to tyrosine or alanine. The mutant proteins were similar in mass to the enzymatically deglycosylated wild-type hNaSi-1, suggesting that Asn591 is the N-glycosylation site in hNaSi-1. Therefore, hNaSi-1 resembles the other members of the SLC13 gene family, in that it has a glycosylated COOH-terminal tail. However, unlike the NaDC transporters, mutagenesis of Asn591 did not impair protein trafficking but produced a large decrease in Vmax. Therefore, Asn591 and/or N-glycosylation in hNaSi-1 is required for transport activity.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of hNaSi-1 cDNA. The hNaSi-1 was amplified from human kidney cDNA by polymerase chain reaction (PCR) using the Advantage cDNA PCR kit (Clontech) and sequence-specific primers based on a human sequence of unknown function (accession no. AK026413 [GenBank] , GenBank) that had a high sequence identity with rat NaSi-1 (20). Later, this sequence was shown to be identical to the hNaSi-1 identified by functional expression (15). The PCR product was ligated into the pCR2.1 vector using the TOPO TA cloning kit according to manufacturer's directions (InVitrogen). The hNaSi-1 cDNA was then subcloned into the SalI/MscI sites of the pSPORT1 expression vector (GIBCO) containing the 3'-untranslated region from NaDC-2, which facilitates expression of transporter proteins in oocytes (2). The plasmid construct, pSPORT/hNaSi-1, was sequenced at the Sealy Center for Molecular Science [University of Texas Medical Branch at Galveston (UTMB)]. The secondary structure of hNaSi-1 was predicted using a combination of hydropathy analysis described by Kyte and Doolittle (14) and Rao and Argos (26).

Site-directed mutagenesis. Site-directed mutagenesis was performed using the oligonucleotide-directed method of Kunkel (13). Escherichia coli CJ236 transformed with pSPORT/hNaSi-1 was grown in uridine-containing medium to produce double-stranded plasmids that contain uracil in place of thymine. Single-stranded DNA was rescued using M13K07 helper phage and then employed as a template for mutagenesis using the Muta-gene in vitro mutagenesis kit (Bio-Rad). The mutations of the putative N-glycosylation site at position 591 were introduced with the following antisense primers (with mutated nucleotides in boldface): 1) 5'-CATGGTCTCATAACTCATAGCAGG-3' (Asn591 to Tyr) and 2) 5'-CATGGTCTCAGCACTCATAGCAGG-3' (Asn591 to Ala). The sequences of the mutated plasmids were verified by the Sealy Center for Molecular Science.

Preparation of GST-Si65 fusion protein. Amino acid residues 173-237 of hNaSi-1, located in an intracellular loop between putative transmembrane domains 4 and 5, were amplified by PCR, with the pSPORT/hNaSi-1 construct used as a template. The reaction was performed with the following primers, which contained restriction sites for BamHI or EcoRI to facilitate subcloning: 5'-CGTGGATCCTTCAACGGATCAACCAAC-3' and 5'-ATGAATTCCGTGGCCCTTCTTTGT-3'. A touchdown PCR was performed using the Advantage cDNA PCR kit (Clontech) with the following conditions: denaturation at 95°C for 3 min followed by five cycles (94°C for 30 s, 72°C for 30 s), 5 cycles (94°C for 30 s, 70°C for 30 s), and 25 cycles (94°C for 30 s, 68°C for 30 s). The PCR product was first ligated into the pCR2.1 vector using the TOPO TA cloning kit (InVitrogen) and then subcloned into the BamHI and EcoRI sites of the pGEX-2T vector (Amersham). The pGEX-2T vector is designed to produce a fusion protein of the inserted cDNA sequence at the COOH-terminal end of GST. The construct is called pGEX/hNaSi-1, and the fusion protein is called GST-Si65. The sequence of the pGEX/hNaSi-1 recombinant plasmid was verified by the Sealy Center for Molecular Science.

The methods used for expression and purification of GSTSi65 fusion protein were as described previously (22). E. coli DH5{alpha} cells were transformed with the pGEX/hNaSi-1 recombinant plasmid or only pGEX-2T vector and then cultured in rich medium (28). Protein synthesis was induced by the addition of isopropyl-{beta}-D-galactopyranoside. After 2 h of growth, the cells were harvested, lysed, centrifuged, and then purified using affinity chromatography with a glutathione-Sepharose 4B column (Amersham). The bound proteins (GST-Si65 or GST) were eluted from the columns using buffer containing 10 mM glutathione. The eluted proteins were concentrated and desalted into phosphate-buffered saline (PBS) through a Centricon-10 column (Amicon) according to manufacturer's directions. The protein samples were separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and stained with Coomassie blue (22). The mass of the protein was estimated by comparison with BenchMark prestained protein ladder (InVitrogen).

Antibody preparation and purification. The purified GSTSi65 fusion protein was sent to Covance Research Products (Denver, PA) to raise polyclonal antibodies in two rabbits. Titers of the immune sera were tested by Covance with enzyme-linked immunosorbent assay. The final immune serum with the highest titer was purified through a GST affinity column to remove anti-GST antibodies. The GST affinity column was prepared using the Aminolink immobilization kit (Pierce) with 20 mg of purified GST protein. The purified immune serum containing the anti-hNaSi-1 antibodies was then used for subsequent Western blotting and immunofluorescence experiments.

Western blotting. Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 8% or 6% tricine gels and then transferred to nitrocellulose membranes (0.45 µm; Schleicher & Schuell) (22). For some experiments, a preparative gel with a single sample well was run, and the membrane was cut into strips. The membranes or strips were blocked with PBS-TM (PBS containing 0.5% Carnation instant nonfat dry milk and 0.05% Tween 20) at room temperature for 1 h or at 4°C overnight. The blots were incubated with 1:1,000 dilution of the antifusion protein antibodies in PBS-TM at room temperature for 2 h and then washed three times with PBS-TM. For fusion protein blocking experiments, the immune serum was preincubated with 2 mg/ml of GST-Si65 fusion protein or GST at 37°C for 1 h and then applied to the blots. The secondary antibody, horseradish peroxidase-linked anti-rabbit IgG from donkey (Amersham Biosciences), was diluted to 1:5,000 in PBS-TM and applied at room temperature for 1 h. The blots were then washed three times with PBS. Antibody binding was detected using the Supersignal West Pico chemiluminescent substrate kit (Pierce), and images were acquired using Image Station 440CF (Eastman Kodak) with six captures of 5 min to prevent saturation of the image. The mass of hNaSi-1 was estimated by comparison with chemiluminescent protein size standards (MagicMark Western Standard, InVitrogen).

Preparation of brush-border membrane vesicles. Brush-border membranes of pig and rat kidneys were prepared using an Mg2+ precipitation method as described previously (21). The pig kidneys were donated by the Cardiology Department, University of Arizona. Sprague-Dawley rats (male, aged 5-6 mo) were purchased from Harlan and housed for <1 wk at the UTMB Animal Care Facility under controlled lighting and temperature conditions. The rats were allowed free access to laboratory chow and water. All animal experiments were done using protocols approved by the Institutional Animal Care and Use Committee. The kidney cortex was dissected and homogenized with a Polytron in 300 mM mannitol, 1 mM EDTA, 20 mM MES-Tris (pH 6), and 0.1 mM phenylmethylsulfonyl fluoride (PMSF). Then 100 mM MgCl2 stock solution was added to a final concentration of 10 mM, and the sample was mixed in the cold for 20 min. The samples were then centrifuged four times at 6,000 g for 10 min, with the supernatant taken each time. The membranes were pelleted twice at 35,000 g for 30 min and resuspended each time in 300 mM mannitol, 1 mM EDTA, 20 mM HEPESTris (pH 7.4), and 0.1 mM PMSF. The final pellet was resuspended in 300 mM mannitol, 20 mM HEPES-Tris (pH 7.4), and 0.1 mM MgSO4 and then stored at -80°C until use. The alkaline phosphatase enrichment of the pig brush borders was 10.5-fold above that of the initial homogenate. The concentrations of the brush-border membrane vesicles (BBMV) were measured using the Bio-Rad protein assay kit.

Transcription of cRNA. The pSPORT/hNaSi-1 plasmid construct was linearized with XbaI purified using RNase-free Chroma-spin 1000 columns (Clontech) and employed as a template for in vitro cRNA transcription using the T7 mMessage mMachine kit (Ambion). The cRNA was resuspended in RNase-free water to a final concentration of 1 µg/µl and stored at -80°C.

Preparation and injection of Xenopus oocytes. Adult female Xenopus laevis (Xenopus I) were maintained at 18°C and a 12:12-h light-dark cycle. They were fed Xenopus chow (Xenopus I) supplemented with dried liver three times a week. For removal of oocytes, the frogs were anesthetized by submersion in 0.1% 3-aminobenzoic acid ethyl ester (MS-222) buffered to pH 7 with NaHCO3, and a portion of the ovary was surgically removed. The oocytes were placed in Barth's solution [in mM: 88 NaCl, 2.4 NaHCO3, 1 KCl, 0.33 Ca(NO3)2, 0.41 CaCl2, 0.82 MgSO4, and HEPES 10, pH 7.4] and separated using forceps. The oocytes were incubated for 1 h in collagenase solution [Ca2+-free Barth's solution containing 2 mg/ml collagenase A (Roche) and 1 mg/ml trypsin inhibitor type III-0 (Sigma)] and then for 45 min in 100 mM potassium phosphate and 1 mg/ml BSA. Stage V and VI oocytes were sorted and then incubated at 18°C in Barth's solution supplemented with 100 µg/ml gentamicin sulfate, 50 µg/ml tetracycline, 2.5 mM sodium pyruvate, and 5% heat-inactivated horse serum at 18°C. The oocytes were injected on the following day with 50 nl of hNaSi-1 cRNA (1 µg/µl). Preliminary experiments showed that maximal expression of hNaSi-1 was reached after injection of >=30 ng of cRNA, and injection of 50 ng reduces variability because of inaccuracies in injection volume. The medium and culture vials were changed daily.

Biotinylation of Xenopus oocytes. Groups of five oocytes were washed three times with PBS (pH 9) and then incubated in 0.5 ml of 0.5 mg/ml sulfo-NHS-LC-biotin (Pierce) for 10 min at room temperature, as described previously, with additional washes (8, 23). The oocytes were rinsed with quench buffer (100 mM glycine in PBS) at room temperature and then incubated in the same quench buffer for 20 min on ice. The oocytes were incubated on ice for 30 min with lysis buffer (1% Triton X-100, 150 mM NaCl, and 20 mM Tris, pH 7.6) supplemented with protease inhibitors (10 µg/ml pepstatin, 10 µg/ml leupeptin, and 0.5 mM PMSF). The solubilized oocytes were centrifuged for 15 min at 14,000 g, and the supernatants were incubated with 50 µl of Immunopure immobilized streptavidin (Pierce) at 4°C overnight. On the next day, the samples were washed and pelleted in the following buffers: lysis buffer (see above), high-salt buffer (0.1% Triton X-100, 20 mM Tris, and 50 mM NaCl, pH 7.5), and no-salt buffer (50 mM Tris, pH 7.5). The washed streptavidin-biotin complexes were then used in Western blots.

Preparation of Xenopus oocyte plasma membranes. Plasma membranes were prepared from Xenopus oocytes by the method of Geering and colleagues (7). Each group of 50 oocytes was washed twice and then homogenized by 20 passes with a glass-Teflon homogenizer in homogenization buffer (83 mM NaCl, 1 mM MgCl2, and 10 mM HEPES-Tris, pH 7.9). The samples were centrifuged twice at 1,000 g for 10 min, and the pellet was discarded each time. The supernatants were transferred to a microcentrifuge tube and spun down at maximum speed (model 5415C, Brinkmann Instruments) for 20 min. The pellets were resuspended in homogenization buffer containing protease inhibitors (0.5 mM PMSF, 50 µg/µl leupeptin, and 16.7 µg/µl pepstatin) and then stored at -20°C until use. The concentrations of the protein samples were measured using a Bio-Rad protein assay kit.

Deglycosylation of membranes with peptide-N-glycosidase F. For deglycosylation, 4 µg of the oocyte plasma membranes or 20 µg of pig or rat brush-border membranes were denatured in 0.5% SDS and 1% {beta}-mercaptoethanol, boiled for 10 min, and then incubated in 50 mM sodium phosphate buffer (pH 7.5), 1% NP-40, and 1 µl peptide-N-glycosidase F (PNGase F, 500 U/µl; New England BioLabs) at 37°C for 1 h (22). Controls were incubated with water instead of PNGase F. The samples were then used in Western blots.

Immunofluorescence of Xenopus oocytes. Oocytes were injected with cRNA of wild-type hNaSi-1 and two glycosylation mutants, N591Y and N591A. Four days after injection, the oocytes were washed three times with PBS, and the plasma membranes were labeled with 25 µg/ml Alexa Fluor 594 conjugated wheat germ agglutinin (WGA; Molecular Probes) at room temperature for 30 min. The 36-kDa dimeric WGA is a carbohydrate-binding lectin that binds to sialic acid and N-acetylglucosaminyl residues of glycoproteins and glycolipids (9). After incubation, the oocytes were washed three times with PBS and then frozen and cut into sections (5 µm) by the Histopathology Core Facility (UTMB). Frozen sections were treated with blocking buffer (1% goat serum in PBS) at room temperature for 1 h. The antifusion protein antibodies were diluted to 1:100 in the same blocking buffer and then applied at room temperature for 1 h. The slides were washed three times with PBS. The secondary antibodies (Alexa Fluor 488 goat anti-rabbit IgG, 2 mg/ml; Molecular Probes) were diluted to 1:500 and then applied to the samples at room temperature for 1 h. The slides were washed again three times with PBS. Vectashield mounting medium (Vector) was added to the slides, which were then covered with microscope cover glass, and the edges were sealed with nail polish. The immunofluorescence images were observed with x10/0.3 NA Plan Fluor objectives on a Nikon Eclipse E800 upright epifluorescence microscope. Standard band-pass emission filter sets for tetramethylrhodamine isothiocyanate and fluorescein isothiocyanate were used to observe the red (Alexa Fluor 594) and green (Alexa Fluor 488) fluorescent dyes, respectively. Images were acquired by a digital camera (Roper Scientific CoolSNAP FX cooled charge coupled device monochrome 12 bit), recorded with Metavue 4.67, and analyzed with MetaMorph 5.0 imaging software (Universal Imgaing, Downingtown, PA). The red fluorescent WGA-labeled membrane of each oocyte was used as a mask to outline the green immunofluorescence area of the same oocyte. The average intensity of the overlapped signals (yellow) was then calculated.

Radiotracer uptake assay. The transport of [35S]sulfate (Perkin Elmer Life Science) was measured in Xenopus oocytes 3 or 4 days after cRNA injection. Each group of five oocytes was rinsed with room temperature choline buffer (100 mM choline chloride, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 10 mM HEPES, pH 7.5). Transport buffer containing [35S]sulfate in Na+ buffer (containing 100 mM NaCl in place of choline chloride) was added to initiate the uptake. After a timed period of incubation, three additions of 4 ml of ice-cold choline buffer were used to stop the reaction and wash the oocytes. Individual oocytes were transferred to scintillation vials and dissolved in 0.25 ml of 10% SDS. Then 3 ml of scintillation cocktail were added to each vial, and the radioactivity was counted in a scintillation counter.

Transport data analysis. In kinetic experiments, sulfate uptakes in individual cRNA-injected oocytes have been corrected for background uptakes by subtracting the mean uptakes in control, uninjected oocytes. The uptake rates were fitted to the Michaelis-Menten equation as follows: v = Vmax * [S]/(Km + [S]), where [S] is the concentration of sulfate, Vmax is the maximum uptake rate, and Km is the concentration of sulfate that produces 0.5 Vmax. The nonlinear regression of the data to the equation was done using Sigma Plot 2000 (SPSS).


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of antifusion protein antibodies. The hNaSi-1 protein is predicted to have 11 transmembrane domains (Fig. 1). This model is based on hydropathy analysis of the hNaSi-1 sequence (14) and comparison with other members of the SLC13 gene family (22, 29). Amino acids 173-237 of hNaSi-1, located in an intracellular loop between putative transmembrane domains 4 and 5, have a high antigenic index (12). Also, the sequence alignments of the SLC13 gene family members have a very low amino acid sequence identity in this region (~50% between rat and human NaSi-1). Therefore, this part of the sequence was selected as an immunogenic peptide in a GST fusion protein to raise antibodies against hNaSi-1. The cDNA sequence coding for the 65-amino acid peptide was subcloned into the pGEX-2T vector, which contains the coding sequence of GST. The GST-Si65 fusion protein consists of 65 amino acid residues of hNaSi-1 fused to the COOH-terminal end of GST.



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Fig. 1. Secondary structure model of human Na+-sulfate cotransporter (hNaSi-1) containing 11 transmembrane domains. The NH2 terminus is located intracellularly and the COOH-terminal tail is located extracellularly. Asparagines from the 4 consensus N-glycosylation sequences are highlighted in black, and glycosylation site at Asn591 is shown by a Y. Peptide (amino acid residues 173-237) used for producing antibodies is highlighted in gray.

 

Figure 2 shows a Coomassie-stained gel of samples taken during the purification of GST and GST-Si65 fusion protein. The expression of the protein in E. coli cells reached a maximum level 2 h after isopropyl-{beta}-D-galactopyranoside induction. The soluble fraction containing the GST or GST-Si65 protein was purified through a GST affinity chromatography column. The two proteins, GST (28 kDa) and GST-Si65 (34 kDa), differ in mass by ~6 kDa, which is close to the predicted mass of 7 kDa for the 65-amino acid hNaSi-1 peptide. The purified GST-Si65 fusion protein was injected into two rabbits to raise antibodies. At 3 mo after the first injection, the immune sera from the two rabbits had ELISA titers of 56,000x and 36,000x. The final immune serum with the highest titer was purified by removing anti-GST antibodies and used in all subsequent experiments. The purified immune serum had reduced reactivity against the purified GST protein (Fig. 3A).



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Fig. 2. Expression and purification of glutathione S-transferase (GST) and GST-Si65 fusion protein. Expression of proteins in Escherichia coli cells was induced by isopropyl-{beta}-D-galactopyranoside (IPTG), and then proteins were extracted and purified through a glutathione-Sepharose 4B column. Protein samples taken at each step were separated on a 10% SDS-polyacrylamide gel and stained with Coomassie blue. A: expression and purification of GST-Si65 fusion protein. Lane 2, protein samples from cells before IPTG; lane 3, protein samples after induction with IPTG; lane 4, soluble extract; lane 5, column flow-through; lane 6, sample eluted with glutathione buffer. B: purified GST (lane 2) and GST-Si65 fusion (lane 3) proteins. Size standards are shown in lane 1 in A and B.

 


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Fig. 3. A: slot blots of purified GST protein (1 pg-10 ng/well). Blots were probed with the crude anti-NaSi-1 antibodies (unpurified) and with the antibodies after they were passed over a GST-affinity column to remove anti-GST antibodies. B: Western blots of pig renal brush-border membrane vesicles separated on an 8% tricine-SDS gel. Nitrocellulose strips containing 10 µg of protein were blocked in PBS + nonfat dry milk and Tween 20, and antifusion protein antibodies (diluted to 1:1,000) and secondary antibodies (diluted to 1:5,000) were added. Lane 1, chemiluminescent size standards (MagicMark); lane 2, strip probed with preimmune serum; lane 3, crude immune serum; lane 4, strip probed with serum purified with the GST-affinity column. Blocked immune serum was prepared by incubating purified immune serum with GST (GST block, lane 6) or GST-Si65 fusion protein (GST-Si65 block, lane 5) at 37°C for 1 h. Immunoreactive protein at ~63 kDa was not seen in strips incubated with fusion protein-blocked immune serum (lane 5).

 

Western blots of renal brush-border membranes. BBMV prepared from pig renal cortex were tested by Western blotting (Fig. 3B). No signal was obtained with the preimmune serum. An immunoreactive protein signal centered at 63 kDa was detected with the crude (unpurified) or GST column-purified immuneserum. This immunoreactive signal disappeared after the immune serum was blocked with the GST-Si65 fusion protein but did not show any change with GST-blocked immune serum. Although no sequence is available for the pig NaSi-1, the predicted mass of rat and human NaSi-1 is ~66 kDa (15, 20). Therefore, the protein signal at 63 kDa most likely represents glycosylated NaSi-1 transporter in pig renal brush-border membranes.

Deglycosylation of native NaSi-1 in pig and rat renal membranes. Brush-border membranes prepared from pig and rat kidney were deglycosylated using PNGase F. The native NaSi-1 in pig and rat membranes has an apparent mass of ~62-64 kDa (Fig. 4). The results with rat renal membranes are similar to previous reports of rat NaSi-1 (17, 27). Treatment of the membranes with PNGase F increased the mobility of the proteins from pig and rat to an apparent mass of 45 kDa. Therefore, the antifusion protein antibodies recognize native NaSi-1 in pig and rat renal brush-border membranes. The native proteins are N-glycosylated in renal membranes, and the mature N-glycosylation contributes ~17-20 kDa to the mass of the protein.



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Fig. 4. Western blot of brush-border membranes from pig and rat kidney. Each lane contains 20 µg of protein. Brush-border membranes were incubated with water (-) or peptide-N-glycosidase F (PNGase F, +). Samples were separated on 8% tricine gels and transferred to nitrocellulose. Blot was incubated with 1:1,000 dilution of antifusion protein antibodies and then with 1:5,000 dilution of secondary antibodies. Size standards are shown in leftmost lane.

 

N-glycosylation of hNaSi-1 expressed in Xenopus oocytes. On the basis of the secondary structure model of hNaSi-1 shown in Fig. 1, Asn591 in the COOH-terminal tail is the most likely candidate for N-glycosylation. The rabbit NaDC-1 is N-glycosylated at a single site, Asn578, also located at the COOH-terminal tail, and all members of the SLC13 gene family contain one or two consensus sequences for N-glycosylation at the COOH terminus (29). Therefore, Asn591 in hNaSi-1 was mutated to tyrosine or alanine (N591Y or N591A) to determine whether it is the N-glycosylation site. Plasma membranes prepared from control uninjected oocytes or oocytes expressing wild-type hNaSi-1 or N591Y were incubated with PNGase F to remove N-linked oligosaccharides (18). Control samples were incubated with water. The samples were then analyzed in Western blots (Fig. 5). The antibodies recognized two protein bands at 53 and 64 kDa in control wild-type hNaSi-1. After treatment with PNGase F, a single 49-kDa protein was detected. Therefore, the 53- and 64-kDa proteins are likely to represent the core and mature glycosylated forms of hNaSi-1 protein, respectively. The N591Y mutant had a smaller apparent mass than wild-type hNaSi-1, ~52 kDa, which was not affected by PNGase F treatment. Therefore, Asn591 is likely to be the residue used as the single N-glycosylation site in hNaSi-1.



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Fig. 5. Western blot of plasma membranes from Xenopus oocytes expressing wild-type hNaSi-1 and the glycosylation site mutant N591Y. Plasma membranes were deglycosylated using PNGase F (+) or incubated with water (-) as control. Samples were separated on a 6% tricine-SDS gel and then transferred to a nitrocellulose membrane. Each lane contains 4 µg of protein. Blot was incubated with 1:1,000 dilution of antifusion protein antibodies and then with 1:5,000 dilution of secondary antibodies. Size standards are shown in leftmost lane.

 

It was somewhat surprising that the apparent mass of N591Y (~52 kDa) was not identical to that of the deglycosylated wild-type hNaSi-1 (49 kDa). Therefore, to determine whether the 3-kDa difference might be due to a difference in migration rate of the protein in the gel between wild-type hNaSi-1 and the tyrosine-containing mutant, a second mutant in which alanine was at position 591 (N591A) was tested and compared with N591Y in Western blots of biotinylated oocytes. The N591A mutant had the same apparent mass as N591Y (49 kDa, which was reduced by 6 kDa compared with wild-type hNaSi-1; Fig. 6), suggesting that the replacement residue does not alter the protein's migration rate.



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Fig. 6. Western blots of biotinylated Xenopus oocytes expressing wild-type hNaSi-1 and glycosylation site mutants N591Y and N591A. Oocytes were incubated with sulfo-NHS-LC-biotin and then with streptavidin-agarose beads. Biotinylated samples were separated on a 6% tricine-SDS gel and transferred to a nitrocellulose membrane. Western blotting was performed using antifusion protein antibodies. Position of size standards is shown at right.

 

In control experiments, we found that it is not possible to measure the expression of hNaSi-1 by cell surface biotinylation using sulfo-NHS-LC-biotin. The streptavidin-agarose beads appear to bind nonspecifically to hNaSi-1, because there is a protein signal, even in control oocytes incubated without sulfo-NHS-LC-biotin (results not shown). Streptavidin and neutravidin immobilized on Ultralink support (Pierce) were also tested with similar results. This result is in contrast to oocytes expressing NaDC-1, which have no protein signals after streptavidin treatment when incubated without sulfo-NHSLC-biotin (results not shown). Therefore, immunofluorescence experiments were performed to compare the cell surface expression level of wild-type and mutant hNaSi-1 proteins.

Immunofluorescence of Xenopus oocytes. The carbohydrate-binding lectin WGA does not cross the plasma membrane, and it binds to extracellular sialic acid and N-acetylglucoaminyl residues (9). In this study, we used WGA conjugated to the red fluorescent dye Alexa Fluor 594 to label the plasma membrane of oocytes. Preliminary experiments showed that the dye did not penetrate the oocyte membrane and also survived the freezing-and-sectioning process (data not shown). The frozen oocyte sections were incubated with anti-NaSi-1 antibodies and green fluorescent secondary antibodies. The immunofluorescent signals of wild-type hNaSi-1 and the two N-glycosylation mutants were predominantly distributed on the plasma membrane, but some signals were also observed intracellularly (Fig. 7). The average intensity of the green immunofluorescence signal that overlapped with the red plasma membrane marker (yellow overlap signal) in oocytes injected with wild-type hNaSi-1 cRNA was considered to be the 100% control value. This average intensity (n = 11 oocytes) was used for calculating the relative fluorescence intensities of the experimental groups. The standard error among the control samples was also calculated and then expressed as a percentage of the mean (control 100 ± 11%, n = 11). The two Asn591 mutants had significantly stronger average immunofluorescent intensity than wild-type hNaSi-1: 137 ± 11% (n = 16) and 206 ± 14% (n = 16) of control for N591Y and N591A, respectively. There was no significant immunofluorescence in uninjected oocytes (results not shown). These results indicate that the two nonglycosylated mutants, N591Y and N591A, are more abundant on the cell surface than is wild-type hNaSi-1.



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Fig. 7. Immunofluorescence of Xenopus oocytes expressing wild-type hNaSi-1 and glycosylation site mutants N591Y and N591A. Oocytes were stained with the red fluorescent dye Alexa Fluor 594 conjugate of wheat germ agglutinin (WGA), frozen, and cut into sections. Frozen sections were probed with antifusion protein antibodies and then with green fluorescent secondary antibodies Alexa Fluor 488 conjugate of goat anti-rabbit IgG. WGA, red fluorescent WGA-stained membranes of oocytes expressing wild-type hNaSi-1, N591Y, and N591A; immune serum, green fluorescent signal from the same oocytes probed with antifusion protein antibodies and green fluorescent secondary antibodies; combination, color combinations of the first WGA and immune serum images. Images were acquired using standard filter sets (band-pass emission) for tetramethylrhodamine isothiocyanate (Alexa Fluor 594) and FITC (Alexa Fluor 488) at the same magnification using a x10/0.3 NA Plan Fluor objective.

 

Kinetics of sulfate uptakes by N591Y and N591A expressed in oocytes. The kinetic properties of the two Asn591 mutants were assessed in Xenopus oocytes. Preliminary studies showed that uptakes of 1 mM sulfate by oocytes expressing wild-type hNaSi-1 were linear up to 60 min. Therefore, 10-min uptakes were used to estimate initial rates in the kinetic experiments. The two Asn591 mutants had similar Km for sulfate compared with the wild-type hNaSi-1, but the maximum uptake rates were greatly reduced. The results for a single experiment are shown in Fig. 8. In three separate experiments, the mean Km was 376 ± 122, 466 ± 295, and 258 ± 66 µM and the maximum uptake rate (Vmax) was 1,135 ± 282, 298 ± 113, and 391 ± 88 (SE) pmol · oocyte-1 · h-1 for wild-type hNaSi-1, N591Y, and N591A, respectively. N591Y and N591A were expressed on the plasma membrane of oocytes (Fig. 7); therefore, the mutations affect the transport activity, rather than target hNaSi-1 proteins. Because Vmax is the product of the number of transporters and the turnover number, the results suggest that removal of Asn591 or lack of N-glycosylation results in a decrease in hNaSi-1 turnover number.



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Fig. 8. Kinetics of sulfate uptake by oocytes expressing wild-type hNaSi-1 and the glycosylation site mutants N591Y and N591A. Uptake of increasing concentrations of sulfate was measured over 10 min in the presence of 100 mM Na+. Values are means ± SE of 5 oocytes from a single frog. Km for sulfate is 215, 151, and 164 µM and Vmax is 1,604, 227, and 360 pmol · oocyte-1 · h-1 for wild-type hNaSi-1, N591Y, and N591A, respectively.

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The results of this study show that Asn591, located in the COOH terminus, is used as the N-glycosylation site in hNaSi-1. This finding verifies that the COOH terminus is extracellular in the Na+-sulfate cotransporters, similar to the other members of the SLC13 superfamily. The Na+-dicarboxylate co-transporter NaDC-1 is N-glycosylated at a single site at the COOH-terminal tail, and all members of the family have one or two consensus glycosylation sequences in the COOH terminus (22). Interestingly, the mutation of Asn591 to tyrosine or alanine in hNaSi-1 results in a large decrease in transport activity related to a decrease in turnover number with no effect on substrate affinity. Therefore, the COOH terminus in hNaSi-1 or the interaction of the COOH terminus with other domains or other proteins has an important functional role.

The antifusion protein antibodies identified two protein bands at 53 and 64 kDa in plasma membranes of oocytes expressing wild-type hNaSi-1. These two protein bands probably represent different glycosylation states of the protein. NaDC-1 expressed in oocytes also is seen as two differently glycosylated proteins (22). After deglycosylation with PNGase F, the mass of hNaSi-1 was reduced to ~49 kDa, a difference of 4-15 kDa. Because the N591Y mutant had the same apparent mass before and after PNGase F treatment, it is likely that Asn591, at the COOH-terminal tail, is the only N-glycosylation site in hNaSi-1 protein.

Previous studies of the NaDC-1 transporter found that the succinate uptake and protein expression of a nonglycosylated mutant decreased 50% compared with the wild type, suggesting that N-glycosylation is required for protein trafficking, rather than function (22). In this study, the abundance of the nonglycosylated hNaSi-1 mutants on the plasma membrane was greater than that of wild-type hNaSi-1, indicating that trafficking is not affected by removal of Asn591. Therefore, the effects of glycosylation appear to be quite different in members of the same gene family. The mutations at Asn591 changed the Vmax of hNaSi-1 without changing Km for sulfate, suggesting that Asn591 or N-glycosylation at that site is involved in determining the substrate turnover rate. Previous studies of the mannose 6-phosphate-specific receptor suggested that N-glycosylation is required for the ligand-binding conformation of the receptor (10). Structural studies of N-glycopeptides suggest that N-glycosylation can affect their conformation by changing the specific secondary structure, such as {beta}-turns, and may also change covalent bonds and hydrogen bonds formed between the peptide backbone and side-chain atoms (25). Therefore, it is possible that the COOH terminus in hNaSi-1 interacts with other regions of the protein or with accessory proteins.

Generally, N-linked oligosaccharides may affect the biosynthesis, folding, sorting, or trafficking of proteins to the membrane and may also play a role in maintaining conformational stability and charge and in resisting proteolysis (24). Although many studies have shown changes in protein trafficking with removal of N-glycosylation sites (11, 22), there are examples of transport proteins that have functional effects of N-glycosylation. For example, studies of the GLUT-1 glucose transporter and the GABA transporter suggest that N-glycosylation may play a functional role (1, 16). Mutations of Asn45 in GLUT-1 resulted in an increase in the Km value, suggesting that N-glycosylation is involved in determining the structure of GLUT-1 that allows high substrate affinity (1). In the GABA transporter, mutations of N-glycosylation sites reduced turnover number and altered voltage sensitivity (16).

The specificity of the antifusion antibodies used in this study was tested using BBMV prepared from pig and rat kidney. The results showed immunoreactive proteins of 63 kDa in pig and 62 kDa in rat, both of which were deglycosylated with PNGase F to proteins of ~45 kDa. The predicted mass of rat and human NaSi-1 is ~66 kDa (15, 20), and there is no information on the mass of pig NaSi-1. The reported apparent mass of rat NaSi-1 varies from 59 to 62 kDa after in vitro translation, to 68 kDa in rat renal BBMV, and to 53 kDa in Sf9 cells expressing recombinant rat NaSi-1 (6, 17, 20, 27). The difference in the apparent mass of the protein between studies is probably related to differences in the amount of posttranslational modifications in the cells, the efficiency of denaturation of proteins from different sources, and the percent acrylamide used in the gels and the type of gel used (11).

In summary, polyclonal antibodies were prepared against a fusion protein of a 65-amino acid peptide of hNaSi-1 and GST. The antibodies recognized the purified fusion protein, the recombinant hNaSi-1 expressed in Xenopus oocytes, and the native transporters found in mammalian BBMV. Mutagenesis of the putative N-glycosylation site located at the COOH terminus showed that Asn591 is the N-glycosylation site in hNaSi-1. The other members of the SLC13 gene family also glycosylate one or two asparagines at the COOH-terminal tail. The plasma membrane abundance of the N591Y and N591A mutants was greater than that of the wild-type hNaSi-1. However, these mutants had greatly reduced transport activity, characterized by a decrease in Vmax with no change in Km. Therefore, it is likely that N-glycosylation plays an important role in substrate turnover in hNaSi-1.


    DISCLOSURES
 
This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-46269.


    ACKNOWLEDGMENTS
 
We thank Dr. Leoncio Vergara (Optical Imaging Laboratory) for advice on labeling plasma membranes and assistance with the immunofluorescence images and Drs. Steven Weinman and Stephen King for scientific discussions.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. M. Pajor, Dept. of Physiology and Biophysics, University of Texas Medical Branch, Galveston, TX 77555-0641 (E-mail: ampajor{at}utmb.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Asano T, Katagiri H, Takata K, Lin JL, Ishihara H, Inukai K, Tsukuda K, Kikuchi M, Hirano H, and Yazaki Y. The role of N-glycosylation of GLUT1 for glucose transport activity. J Biol Chem 266: 24632-24636, 1991.[Abstract/Free Full Text]

2. Bai L and Pajor AM. Expression cloning of NaDC-2, an intestinal Na+- or Li+-dependent dicarboxylate transporter. Am J Physiol Gastrointest Liver Physiol 273: G267-G274, 1997.[Abstract/Free Full Text]

3. Beck L and Markovich D. The mouse Na+-sulfate cotransporter gene Nas1. Cloning, tissue distribution, gene structure, chromosomal assignment, and transcriptional regulation by vitamin D. J Biol Chem 275: 11880-11890, 2000.[Abstract/Free Full Text]

4. Busch AE, Waldegger S, Herzer T, Biber J, Markovich D, Murer H, and Lang F. Electrogenic cotransport of Na+ and sulfate in Xenopus oocytes expressing the cloned Na+/SO42- transport protein NaSi-1. J Biol Chem 269: 12407-12409, 1994.[Abstract/Free Full Text]

5. Custer M, Murer H, and Biber J. Nephron localization of Na/SO42--cotransport-related mRNA and protein. Pflügers Arch 429: 165-168, 1994.[ISI][Medline]

6. Fucentese M, Winterhalter KH, Murer H, and Biber J. Functional expression and purification of histidine-tagged rat renal Na/phosphate (NaPi-2) and Na/sulfate (NaSi-1) cotransporters. J Membr Biol 160: 111-117, 1997.[ISI][Medline]

7. Geering K, Theulaz I, Verrey F, Hauptle MT, and Rossier BC. A role for the {beta}-subunit in the expression of functional Na+-K+-ATPase in Xenopus oocytes. Am J Physiol Cell Physiol 257: C851-C858, 1989.[Abstract/Free Full Text]

8. Gottardi CJ, Dunbar LA, and Caplan MJ. Biotinylation and assessment of membrane polarity: caveats and methodological concerns. Am J Physiol 37: F285-F295, 1995.[ISI]

9. Haugland RP. Antibodies, avidin, lectins and related products. In: Handbook of Fluorescent Probes and Research Products, edited by Gregory J. Eugene, OR: Molecular Probes, 2002, p. 185-264.

10. Hille A, Waheed A, and von Figura K. The ligand-binding conformation of Mr 46,000 mannose 6-phosphate-specific receptor. Acquisition of binding activity during in vitro synthesis. J Biol Chem 264: 13460-13467, 1989.[Abstract/Free Full Text]

11. Hirayama BA and Wright EM. Glycosylation of the rabbit intestinal brush border Na+/glucose cotransporter. Biochim Biophys Acta 1103: 37-44, 1992.[ISI][Medline]

12. Jameson BA and Wolf H. The antigenic index: a novel algorithm for predicting antigenic determinants. Comput Appl Biosci 4: 181-186, 1988.[Abstract]

13. Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci USA 82: 488-492, 1985.[Abstract]

14. Kyte J and Doolittle RF. A simple method for displaying the hydropathic character of a protein. J Mol Biol 157: 105-132, 1982.[ISI][Medline]

15. Lee A, Beck L, and Markovich D. The human renal sodium sulfate cotransporter (SLC13A1; hNaSi-1) cDNA and gene: organization, chromosomal localization, and functional characterization. Genomics 70: 354-363, 2000.[ISI][Medline]

16. Liu Y, Eckstein-Ludwig U, Fei J, and Schwarz W. Effect of mutation of glycosylation sites on the Na+ dependence of steady-state and transient currents generated by the neuronal GABA transporter. Biochim Biophys Acta 1415: 246-254, 1998.[ISI][Medline]

17. Lotscher M, Custer M, Quabius ES, Kaissling B, Murer H, and Biber J. Immunolocalization of Na/SO42- cotransport (NaSi-1) in rat kidney. Pflügers Arch 432: 373-378, 1996.[ISI][Medline]

18. Maley F, Trimble RB, Tarentino AL, and Plummer TH Jr. Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases. Anal Biochem 180: 195-204, 1989.[ISI][Medline]

19. Markovich D. Physiological roles and regulation of mammalian sulfate transporters. Physiol Rev 81: 1499-1533, 2001.[Abstract/Free Full Text]

20. Markovich D, Forgo J, Stange G, Biber J, and Murer H. Expression cloning of rat renal Na+/SO42- cotransport. Proc Natl Acad Sci USA 90: 8073-8077, 1993.[Abstract/Free Full Text]

21. Pajor AM, Hirayama BA, and Wright EM. Molecular evidence for two renal Na+/glucose cotransporters. Biochim Biophys Acta 1106: 216-220, 1992.[ISI][Medline]

22. Pajor AM and Sun N. Characterization of the rabbit renal Na+/dicarboxylate cotransporter using anti-fusion protein antibodies. Am J Physiol Cell Physiol 271: C1808-C1816, 1996.[Abstract/Free Full Text]

23. Pajor AM, Sun N, and Valmonte HG. Mutational analysis of histidine residues in the rabbit Na+/dicarboxylate co-transporter NaDC-1. Biochem J 331: 257-264, 1998.[ISI][Medline]

24. Paulson JC. Glycoproteins: what are the sugar chains for? Trends Biochem Sci 14: 272-276, 1989.[ISI][Medline]

25. Perczel A, Kollat E, Hollosi M, and Fasman GD. Synthesis and conformational analysis of N-glycopeptides. II. CD, molecular dynamics, and NMR spectroscopic studies on linear N-glycopeptides. Biopolymers 33: 665-685, 1993.[ISI][Medline]

26. Rao JKM and Argos P. A conformational preference parameter to predict helices in integral membrane proteins. Biochim Biophys Acta 869: 197-214, 1986.[ISI][Medline]

27. Sagawa K, DuBois DC, Han B, Almon RR, Biber J, Murer H, and Morris ME. Detection and quantitation of a sodium-dependent sulfate cotransporter (NaSi-1) by sandwich-type enzyme-linked immunosorbent assay. Pflügers Arch 437: 123-129, 1998.[ISI][Medline]

28. Sambrook J, Fritsch EF, and Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989.

29. Zhang FF and Pajor AM. Topology of the Na+/dicarboxylate cotransporter: the N-terminus and hydrophilic loop 4 are located intracellularly. Biochim Biophys Acta 1511: 80-89, 2001.[ISI][Medline]