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. 2011 Dec;25(12):4531–4539. doi: 10.1096/fj.11-185991

Mature N-linked glycans facilitate UT-A1 urea transporter lipid raft compartmentalization

Guangping Chen *,1, Ashley G Howe *, Gang Xu *, Otto Fröhlich †, Janet D Klein *, Jeff M Sands *,†
PMCID: PMC3236619  PMID: 21965602

Abstract

The UT-A1 urea transporter is a glycoprotein with two different glycosylated forms of 97 and 117 kDa. In this study, we found the 117-kDa UT-A1 preferentially resides in lipid rafts, suggesting that the glycosylation status may interfere with UT-A1 lipid raft trafficking. This was confirmed by a site-directed mutagenesis study in MDCK cells. The nonglycosylated UT-A1 showed reduced localization in lipid rafts. By using sugar-specific binding lectins, we further found that the UT-A1 in nonlipid rafts contained a high amount of mannose, as detected by concanavalin A, while the UT-A1 in lipid rafts was the mature N-acetylglucosamine-containing form, as detected by wheat germ agglutinin. In the inner medulla (IM) of diabetic rats, the more abundant 117-kDa UT-A1 in lipid rafts was the mature glycosylation form, with high amounts of N-acetylglucosamine and sialic acid. In contrast, in the IM of normal rats, the predominant 97-kDa UT-A1 was the form enriched in mannose. Functionally, inhibition of glycosylation by tunicamycin or elimination of the glycosylation sites by mutation significantly reduced UT-A1 activity in oocytes. Taken together, our study reveals a new role of N-linked glycosylation in regulating UT-A1 activity by promoting UT-A1 trafficking into membrane lipid raft subdomains.—Chen, G., Howe, A. G., Xu, G., Fröhlich, O., Klein, J. D., Sands, J. M. Mature N-linked glycans facilitate UT-A1 urea transporter lipid raft compartmentalization.

Keywords: glycosylation, apical membrane, trafficking, lectin, sucrose gradient centrifugation

The vasopressin-regulated urea transporter UT-A1, expressed in the inner medullary collecting duct (IMCD), plays a key role in the urine-concentrating mechanism (1–3). A UT-A1/UT-A3-knockout mouse has a seriously impaired urinary concentrating ability (2). In recent years, significant progress has been made in understanding the regulation of the urea transporter (4–9). However, as a membrane protein, the molecular mechanism of how UT-A1 traffics to the apical membrane in polarized epithelial cells is still not clear.

N-linked glycosylation is one of the most common post-translational modifications of membrane proteins. Glycosylation plays important roles in many aspects of glycoprotein function, such as modulating the protein's biological activity; directing protein folding; regulating membrane protein trafficking, surface expression, and membrane localization; or stabilizing the mature protein (10–12). Glycosylation dysfunction has been linked to a number of human diseases (10, 12).

Structurally, UT-A1 has 4 potential consensus N-linked glycosylation sites, at Asn13, Asn279, Asn544, and Asn742. Only Asn279 and Asn742 have been proven to be sites of UT-A1 glycosylation (4). Immunoblotting studies of normal rat renal inner medulla (IM) demonstrate that UT-A1 has 2 bands, a predominant protein band of 97 kDa and a less abundant protein band of 117 kDa (13, 14). These two bands are not caused by gene splicing but by different degrees of post-translational glycosylation. After deglycosylation by PNGase F treatment, both the 97- and 117-kDa bands disappear and yield a single 88-kDa deglycosylated UT-A1 (13, 14). In the IM tip, which contains the terminal IMCD, UT-A1 is detected as 97- and 117-kDa glycoproteins. However, in the IM base, which contains the initial IMCD, only the 97-kDa form is detected. Nevertheless, both the 97-kDa and the more mature 117-kDa glycoprotein forms of UT-A1 are phosphorylated in response to vasopressin (AVP) stimulation (15).

The study of lipid rafts has become a high-emphasis field over the past decade. Advances in cell membrane structure reveal that the plasma membrane contains many specialized microdomains, referred to as lipid rafts, floating in the membrane. The lipid raft is a highly ordered membrane structure enriched in cholesterol and sphingolipids (16). By compartmentalizing proteins or recruiting other signaling proteins, lipid rafts have been shown to be key regulators of many membrane proteins (16–19). In polarized epithelial cells, lipid rafts are believed to be in the apical membrane (20). Differential partitioning in lipid raft microdomains is an important determinant of the apical vs. basolateral localization of the PMCA2w and 2z splice variants (21). Many transporters and channels are reported to be associated with lipid rafts, such as Na-K-2Cl cotransporter (NKCC1; refs. 17, 22), TRPM8 channel (18), Na/H exchanger (NHE3; ref. 23), epithelial (ENaC) Na channels (24), and CFTR (25). Being associated with lipid rafts is important for the physiological role of these proteins.

We previously reported that UT-A1 urea transporter is associated with lipid rafts, both in stably expressing UT-A1 HEK-293 cells (8) and in freshly isolated rat kidney IMCD suspensions (9). In this study, we have the interesting finding that the two glycosylation forms of UT-A1 display differential distribution in lipid rafts. The highly glycosylated 117-kDa UT-A1 preferentially partitions into the lower-density membrane fractions comprised of lipid rafts. We further found that the mature N-linked glycosylation with high amounts of N-acetylglucosamine (in MDCK cells) and sialic acid (in IMCD) is critically involved in the specific lipid raft membrane localization of UT-A1. We hypothesize that association with lipid rafts, mediated by N-linked glycosylation, could represent an important mechanism of UT-A1 apical membrane targeting.

MATERIALS AND METHODS

Glycan mutant expressing UT-A1 MDCK cell lines and cell culture

UT-A1 has 2 glycosylation sites, at Asn279 and Asn742 (4). The two single-glycosylation mutants (A1m1 and A1m2) and one double mutant (m1m2) of UT-A1 MDCK cell lines were generated using our previously described MDCK-FRT (Flp-In system) cell lines (26). All cells were grown in 10% FCS DMEM containing 400 μg/ml hygromycin (EMD Chemicals, Newark, NJ, USA).

Animal preparation

All animal protocols were approved by the Emory University Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (Charles River Laboratories, Franklin, CT, USA) weighing 125–200 g were used in this study. We used the well-established streptozotocin (STZ)-induced diabetic rat model (13). Rats were injected with STZ (62.5 mg/kg body weight prepared fresh in 0.1 M citrate buffer, pH 4.0) or vehicle into a tail vein. After STZ injection (24 h), diabetes was confirmed by measuring blood glucose (One Touch Profile Diabetes Tracking Kit; Lifescan, Milpitas, CA, USA). At 7 d after injection, rats were sacrificed by decapitation, and kidney tissues were collected for tubule suspension preparation.

Kidney IMCD tubule suspension preparation

Rat kidney IMCD suspensions were prepared by digestion with hyaluronidase and collagenase B (Sigma-Aldrich, St. Louis, MO, USA) as described previously (9). After assessing the quality of suspended tubules, they were used for lipid raft isolation.

Membrane lipid raft isolation by sucrose density gradient ultracentrifugation

Lipid rafts from freshly isolated IMCD tubules or cultured UT-A1 MDCK cells were prepared with a 5–40% sucrose discontinuous gradient according to our published protocol (8), following Fattakhova et al. (27) with some modifications. Briefly, rat IMCD suspensions or UT-A1 MDCK cells were homogenized in 0.5% Brij 96V (Sigma)/TNEV buffer (10 mM Tris-HCl, pH 7.5; 150 mM NaCl; 5 mM EDTA; 2 mM Na vanadate; and protease inhibitor cocktail) on ice for 30 min. Supernatant (500 μl) was mixed with an equal volume of 80% sucrose in TNEV and transferred into a polyallomer centrifuge tube (13×51 mm; Beckman Coulter, Palatine, IL, USA). Three milliliters of 35% sucrose in TNEV was carefully layered on top of the mixture, followed by another 1-ml layer of 5% sucrose. The sucrose gradient was then centrifuged in a SW 50.1 rotor (Beckman Coulter) at 34,000 rpm (∼110,000 g) for 20 h at 4°C. After centrifugation, fractions were collected starting from the top to bottom of the tube. Thirteen fractions (∼400 μl) were collected, and equal volumes of each fraction were analyzed by 4–15% gradient SDS-PAGE and immunoblotted with relevant antibodies. Band densities were quantitated with the ImageJ program (U.S. National Institutes of Health, Bethesda, MD, USA).

[35S]-methionine labeling and autoradiography

MDCK cells stably expressing either wild-type (WT) UT-A1 or its double-glycan mutant (UT-A1-m1m2) were grown in 10-cm tissue culture dishes. After reaching confluence, cells were washed with phosphate-buffered saline (PBS) and starved of methionine by incubating with methionine-free minimum Eagle's medium (Sigma) for 45 min at 37°C. The cells were labeled for 1 h at 37°C with 0.1 mCi/ml [35S]-methionine (Perkin Elmer Life Sciences, Shelton, CT, USA). After washing with PBS, the cells were processed for lipid raft isolation as described above. Thirteen sucrose fractions (∼400 μl) were collected. Fifty microliters was used for Western blot as the total fraction proteins. The remaining fractions were diluted with 800 μl of RIPA lysis buffer and immunoprecipitated by adding 5 μl of UT-A1 antibody for 2 h at 4°C, followed by incubation overnight with protein A agarose beads (Thermo Scientific, Rockford, IL, USA). Immunoprecipitates were washed with lysis buffer, resuspended in Laemmli sample buffer, and then resolved by SDS-PAGE. The gel was dried and analyzed by autoradiography.

Lectin pulldown assay

The selected sucrose gradient fractions, fractions 2–4 as lipid rafts and fractions 10–12 as nonrafts, were pooled and diluted with 2 vol of RIPA buffer. Equal amounts of pooled fractions were incubated with 30 μl of a serial agarose-bound lectin suspension at 4°C overnight. After washing, the precipitated samples were used for Western blot with UT-A1 antibody. Agarose-bound concanavalin A (Con A), Galanthus nivalis lectin (GNL), wheat germ agglutinin (WGA), Sambucus nigra lectin (SNA), and tomato lectin all were purchased from Vector Laboratories Inc. (Burlingame, CA, USA).

Oocyte experiment

Xenopus oocytes were harvested, defolliculated, and maintained as detailed by Romero et al. (28). Capped rat UT-A1 cRNA was synthesized with T7 polymerase using the mMessage mMachine T7 Ultra Kit (Ambion, Austin, TX, USA). UT-A1 cRNAs (2 ng) in total volume of 23 nl water were injected into each oocyte. For tunicamycin treatment, at 2 h prior to cRNA injection, oocytes were preinjected with 10 ng tunicamycin (Sigma). After 3 d, healthy oocytes were selected for functional study and protein expression, as described previously (9).

Western blot analysis

Western blotting was performed as described previously (9). Blots were probed with primary antibody, followed by anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, Piscataway, NJ, USA) and developed by ECL (GE Healthcare). A new N-terminal UT-A1 antibody was raised by immunizing a rabbit with a 19-aa peptide of LPEPLSSRYKLYESELSSP. Since UT-A1 and UT-A3 share the same N-terminal sequence, this antibody can detect both UT-A1 and UT-A3 of the IM. Anti-caveolin-1 was purchased from BD Bioscience (Pasadena, CA, USA) and anti-Epac was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Statistical analysis

Urea flux data are expressed as means ± sd. A paired Student's t test was used to assess statistically significant differences between two groups. One-way analysis of variance (ANOVA) followed by Tukey HSD test was used for multiple group analysis.

RESULTS

The 117-kDa form of UT-A1 is preferentially associated with lipid raft microdomains

Due to differential glycosylation, UT-A1 is expressed in the kidney terminal IMCD as two glycosylated forms of 97 and 117 kDa (14). When performing fractionation using a discontinuous sucrose gradient to prepare lipid rafts from kidney IMCD suspensions, we found that although both are detected in low-density fractions of the lipid raft subdomains, the two forms of UT-A1 are associated with different lipid rafts (Fig. 1A). The highly glycosylated form of 117-kDa UT-A1 preferred to reside in less buoyant lipid rafts (fractions 1–3). Under normal conditions, UT-A1 from renal IM shows a stronger protein band of 97 kDa than that of 117 kDa. However, the ratio of 117 to 97 kDa is higher in fractions 1–3. This ratio decreased in higher-density fractions. Figure 1B is the quantitative analysis of 97- and 117-kDa band densities from 3 independent experiments. In Fig. 1A, caveolin was used as the marker of membrane lipid raft, and Epac was used as the marker of cytosolic proteins.

Figure 1.

Figure 1.

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Endogenous UT-A1 distribution in membrane lipid rafts after fractionation. A) Rat IMCD suspension was prepared and lysed in 0.5% Brij 96V/TNEV, then applied to 5–40% discontinuous sucrose gradient ultracentrifugation. Equal sizes (∼400 μl) of fractions were collected from the top to bottom. Each fraction was subjected to immunoblotting for UT-A1 and for lipid raft marker caveolin and cytosolic protein marker Epac. B) Quantitative densitometric analysis of 97- and 117-kDa UT-A1 bands in the membrane fractions from 3 independent experiments. Sum of the intensities in fractions 1–12 was set to 100%, and the band intensity in each fraction is expressed as a percentage of the sum total.

Glycosylation affects UT-A1 lipid raft association

As shown above, the higher level of glycosylation enables UT-A1 to float with the lower-density lipid rafts. This suggests that glycosylation may affect the partitioning of UT-A1 in membrane lipid rafts. To gain further insight into the role of glycosylation in raft segregation, we used the MDCK cells stably expressing WT UT-A1 and the UT-A1 glycosylation site mutants (A1m1, A1m2, and m1m2; ref. 4). Unlike UT-A1 in IMCD suspensions (Fig. 1A) and transfected HEK-293 cells (8), which is mainly distributed in lipid rafts, UT-A1 in transfected MDCK cells has a much wider distribution in the lipid raft fractions as well as in the nonraft fractions. We took advantage of these cells, which enabled us to compare UT-A1 trafficking to lipid raft vs. nonraft subdomains. As seen in Fig. 2, the single glycosylation site mutation severely impaired UT-A1's association with lipid raft subdomains. The double-mutant UT-A1 is almost absent in lipid raft fractions. But clearly, UT-A1 protein was still seen in the nonlipid raft fractions. The same membranes were stripped and reprobed for caveolin.

Figure 2.

Figure 2.

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Lipid raft analysis of UT-A1 glycosylation site mutants by sucrose density gradient fractionation. MDCK cells stably expressing UT-A1 WT, two single glycosylation site mutants, A1m1 (N279Q) and A1m2 (N742Q), and the double mutant m1m2 (N279Q/N742Q) were lysed in 0.5% Brij 96V/TNEV and subjected to 5–40% sucrose gradient ultracentrifugation. Fractions were immunoblotted using UT-A1 antibody. The same membrane was stripped and reprobed with caveolin antibody. Western blot results are representative of >3 independent experiments.

Loss of glycosylation impairs UT-A1 trafficking to lipid rafts

The decreased UT-A1 expression in lipid rafts could be due to either impaired apical membrane delivery and/or protein retrieval from lipid rafts. To investigate whether glycosylation affects UT-A1 trafficking to the lipid raft compartments, the newly synthesized UT-A1 delivery to the rafts was examined by 35S-methionine incorporation and autoradiography. As seen in Fig. 2, the general expression of glycan mutant UT-A1 is less in all sucrose fractions. The newly synthesized UT-A1 labeled by 35S-methionine is also less (Fig. 3A). This is consistent with our previous publication that loss of glycans reduces UT-A1 cell membrane expression (4). However, when comparing the UT-A1 in lipid raft (fractions 2–4) and nonlipid raft compartments (fractions 10–12), the newly synthesized UT-A1 trafficking to rafts is markedly reduced (Fig. 3A). The total 35S-methionine incorporated into protein in the sucrose fractions is comparable between the two types of MDCK cells (Fig. 3B). This suggests that UT-A1 lacking glycans reduces its trafficking to lipid raft compartments. Figure 3C is the densitometry quantification of Fig. 3A from 3 independent experiments.

Figure 3.

Figure 3.

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35S-methionine incorporation and autoradiography. WT and glycan double mutant (m1m2) UT-A1 MDCK cells were pulsed with 0.1 mCi/ml 35S methionine for 1 h at 37°C as detailed in Materials and Methods and subsequently subjected to lipid raft isolation. A) Newly synthesized UT-A1 was immunoprecipitated by UT-A1 antibody and analyzed by autoradiography. B) Equal amounts of ultracentrifugation fractions were loaded directly to SDS-PAGE gel, and the total 35S-methionine-containing proteins were evaluated by autoradiography. C) Quantitative densitometric analysis of the newly synthesized UT-A1 in panel A from 3 independent experiments. Sum of the intensities in fractions 1–12 was set to 100%, and the band intensity in each fraction is expressed as a percentage of the sum total.

UT-A1 in lipid rafts displayed a mature glycosylation pattern

Interestingly, a significant amount of UT-A1 was identified in nonlipid raft compartments in MDCK cells (Fig. 2). It raised the question of why some UT-A1 goes to lipid rafts and other moves to nonlipid rafts. To figure out whether there is a difference of the glycosylation status of UT-A1 in rafts vs. in nonrafts, pulldown experiments using the sugar-specific binding lectins were performed. Lipid raft (fractions 2–4) and nonraft fractions (fractions 10–12) from sucrose density gradients of UT-A1 MDCK cells were incubated with different agarose-bound lectins. Surprisingly, UT-A1 in nonlipid rafts was only pulled down by α-linked mannose binding lectin Con A. In contrast, mature glycosylation of N-acetylglucosamine precipitated by WGA was only detected in UT-A1 from lipid raft (Fig. 4). UT-A1 glycans in MDCK cells lack SNA-binding sialic acid. In Fig. 4, with the extended exposure, we observed the 117-kDa high-glycosylation form enriched by WGA. Only the 97-kDa, but not the 117-kDa form, was previously reported in transfected cells (4, 5, 7, 26) and oocytes (8, 9, 29). The 117-kDa glycosylated UT-A1 was only found in lipid raft compartments. Our data show that differential N-glycosylation regulates UT-A1 distribution in lipid rafts.

Figure 4.

Figure 4.

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Lectin pulldown assays with UT-A1 MDCK cell samples. Lipid raft (fractions 2–4) and nonraft fractions (fractions 10–12) of UT-A1 MDCK cells were incubated with 30 μl of indicated agarose-bound lectins at 4°C overnight. After washing, the precipitated samples were used for Western blot with UT-A1 antibody. Bottom panel with deliberately extended exposure time shows the 117-kDa glycosylation band (arrowhead). Asterisk indicates nonspecific band. Images are representative of 3 independent experiments.

Increased 117-kDa UT-A1 in lipid rafts in STZ-diabetic rat IM

UT-A1 protein in the IM is increased in diabetes and the two glycosylated UT-A1 forms increase differentially (13). Diabetes causes an increase in the 117-kDa rather than 97-kDa glycoprotein in both the IM tip and base (13). We investigated whether any changes occurred in the abundance of the two glycosylated UT-A1 forms in membrane lipid rafts in STZ-diabetic rat IM. Figure 5A shows that the amount of the 117-kDa form is significantly increased in membrane lipid rafts and shifted to lower buoyant density fractions 1–3. In normal control rats, only the 97-kDa form is detected in IM base (the initial IMCD). In contrast, in IM base of diabetic rats, the increased UT-A1 is visible only in the 117-kDa form and is located in lower-density lipid raft pools (Fig. 5B). It is hard to see any 97-kDa band in either tip or base of STZ-diabetic rat IM.

Figure 5.

Figure 5.

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Lipid raft analysis of the two UT-A1 glycosylation forms in STZ-diabetic rat IM. Kidney IM tip (A) and base (B) dissected from normal or STZ-injected rats were lysed in 0.5% Brij 96V/TNEV and subjected to 5–40% sucrose gradient ultracentrifugation as described in Materials and Methods. Fractions were analyzed by immunoblotting for UT-A1. Images are representative of >3 independent experiments.

Increased 117-kDa UT-A1 in diabetic rat IM is a mature glycosylation form with high sialic acids

The native UT-A1 from kidney IM has two glycosylation forms. We probed the glycosylation profile of these two forms with lectin pulldown assays by using freshly prepared normal rat kidney IM tissue lysates (Fig. 6A). Tomato lectin, which is specific for poly-N-acetyllactosamine (poly-LacNAc) on the terminal ends of glycans, only binds 117-kDa UT-A1. GNL, on the other hand, which binds to mannose, only pulled down 97-kDa UT-A1. This result indicates that the two forms are due to different extent of glycosylation. The higher-molecular-mass form of 117 kDa is a complex N-glycan with heavier glycosylation.

Figure 6.

Figure 6.

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Lectin pulldown assays with kidney IM tissue lysates and lipid raft fractions. Normal rat kidney IM lysates (A) and lipid raft fractions (B) of 1–4 (both IM tip and base) from control or STZ-diabetic rats were pooled and incubated with 30 μl of indicated agarose-bound lectins at 4°C overnight. Lectin precipitated samples were analyzed by immunoblotting with UT-A1 antibody. Results are representative of 3 separate experiments.

We then explored whether there is any change in glycosylation profile of the increased UT-A1 in diabetic rat IM. Sucrose fractions 1–4, representing the lipid rafts, were pooled and used for lectin binding assay (Fig. 6B). In normal rat IM tip, both 117-kDa and 97-kDa bands are present in lipid rafts. Although a very small amount of 97-kDa UT-A1 was pulled down by WGA and SNA, the 97-kDa UT-A1 was shown to mainly contain Con A and GNL binding mannose. In contrast, the 117-kDa UT-A1 has high amounts of N-acetylglucosamine and sialic acid, which were precipitated by WGA and SNA, respectively. In IM tip of STZ diabetic rat, the increased 117-kDa UT-A1 in lipid rafts demonstrated the mature glycosylation with high amounts of N-acetylglucosamine and sialic acid, particularly the increased sialic acid. The same glycosylation profile of the increased 117-kDa UT-A1 was seen in rat IM base.

Inhibition of glycosylation affects UT-A1 urea transport activity

To investigate whether glycosylation is important for UT-A1 urea transport activity, we injected Xenopus oocytes with UT-A1 cRNA together with 10 ng/oocyte tunicamycin, an inhibitor of protein N-glycosylation. Western blot analysis (Fig. 7A) showed that UT-A1 cRNA injection into oocytes produced a single band of 97-kDa glycoprotein. Tunicamycin blocked UT-A1 glycosylation and brought its molecular mass from 97 down to 88 kDa, confirming that tunicamycin blocked UT-A1 glycosylation. Concurrently, treatment with tunicamycin decreased UT-A1 urea transport activity (Fig. 7B).

Figure 7.

Figure 7.

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Effect of tunicamycin treatment on UT-A1 activity in oocytes. A) At 2 h prior to injection of rat UT-A1 cRNA, Xenopus oocytes were preinjected with 10 ng tunicamycin; 3 d later, healthy oocytes were used for Western blot. Ten oocytes from each group were lysed in RIPA buffer, and equal amounts of protein were applied for immunoblotting using UT-A1 antibody. First 2 lanes are rat IM samples (normal and STZ-induced diabetic rats) showing the native UT-A1 with 97- and 117-kDa glycosylation bands. B) Urea uptake assay. Oocytes (5–6/time point) were incubated with 1 μCi 14C-urea/ml and 1 mM cold urea in uptake solution at different time points. After washing, each individual cell was dissolved in 10% SDS, followed by scintillation counting. Results are representative of 3 separate experiments. **P < 0.01.

Mutation of glycosylation sites results in reduced UT-A1 urea transport activity

Tunicamycin inhibits all N-glycoslyation, and therefore may alter proteins that directly or indirectly affect UT-A1 trafficking and stability. To exclude the possible nonspecific effects raised by tunicamycin, we performed the oocyte expression studies using our glycan-deficient UT-A1 mutants. Figure 8 shows that loss of the N-glycans results in reduced transporter activity when expressed in oocytes.

Figure 8.

Figure 8.

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Functional study of glycosylation mutant UT-A1 in oocyte. WT or glycan-deficient UT-A1 mutant cRNAs were injected into oocytes. After 3 d, oocyte UT-A1 protein expression was examined by Western blot (A), and urea transport activity was measured by 14C-urea flux (B), as described in the text. Results are representative of 3 separate experiments. *P < 0.05; **P < 0.01.

DISCUSSION

Recent advances in the study of cell membrane structure reveal an important role of lipid rafts in cell function. Numerous proteins are associated with and regulated by lipid rafts in the cell membrane. Lipid rafts participate in diverse cellular processes, such as intracellular protein sorting, membrane trafficking, endocytosis, signal transduction, and activity (19, 22, 30, 31). Urea transporter UT-A1 associates with lipid rafts and codistributes with the lipid raft protein caveolin-1 (9). In the current study, we found that the mature N-linked glycosylation promotes UT-A1 partitioning into membrane lipid raft compartments. This may have an important physiological significance for UT-A1 regulation. In response to stimulus, epithelial cells may increase UT-A1 apical membrane expression by changing glycosylation status, which facilitates UT-A1 lipid raft targeting.

It is well known that both N- and O-linked glycans attached to the extracellular aspects of some membrane proteins are important for protein apical location (32, 33). N-glycosylation has been proven to function as a sorting signal for apical secretory proteins. Growth hormone, which is nonglycosylated, is secreted from both sides, but it is secreted only from the apical side when glycosylated in MDCK cells (33). Indeed, most membrane proteins targeted to the plasma membrane possess N-linked glycosylation (34–36). AQP2 mutant (T125M), which causes hereditary recessive nephrogenic diabetes insipidus (NDI), is not glycosylated because the N-linked glycosylation motif is disrupted (12). UT-A1 urea transporter is glycosylated at residues Asn279 and Asn742. We have shown before that loss of N-glycosylation significantly reduces vasopressin-stimulated UT-A1 membrane trafficking and transport activity (4). But the mechanism of how glycosylation influences UT-A1 membrane apical trafficking is unknown.

Protein partitioning to membrane rafts occurs either via protein–protein interaction or by a variety of post-translational modifications, such as palmitoylation, myristoylation, acylation, and glycosylphosphatidylinositol modification (18, 22, 30, 37). The possible role for glycosylation as a lipid raft sorting signal has been appreciated in several reports (18, 31, 38). Huang et al. (31) reported that the rat mu opioid receptors (MOR) with different N-glycans have different raft associations. The 75-kDa MOR in caudate putamen was found mainly associated with lipid rafts, while the 66-kDa form in the thalamus was present in rafts and nonrafts without preference (31). When highly glycosylated, TRPM8 channels are associated to lipid rafts. Elimination of the N-glycan moieties of the TRPM8 channel produced a great reduction in the association of TRPM8 to the lipid raft fractions (18). In this study, we demonstrate that the nonglycosylated UT-A1 failed to traffic into lipid raft microdomains (Figs. 2 and 3). Therefore, association with lipid rafts mediated by glycosylation may regulate UT-A1 targeting to the apical membrane in polarized epithelial cells.

Unlike native UT-A1 in IMCD and UT-A1 in HEK-293 transfected cells, the heterologously expressed UT-A1 in MDCK cells was found in both lipid raft and nonraft subdomain pools of the cell membrane. By using a lectin binding assay, we were able to fingerprint the carbohydrate structures of the UT-A1 from the two different pools. We found that the mature, fully glycosylated UT-A1 containing N-acetylglucosame was only in lipid rafts. In contrast, the UT-A1 found in the nonlipid rafts possessed mostly mannose glycans. Another unexpected finding was that, compared to native UT-A1 from IMCD, UT-A1 from MDCK cells did not have sialic acid in its glycan chains (Fig. 4). Sialylation plays an important role in the regulation of glycoproteins. Differentially sialylated β1 integrins exhibit altered adhesion to collagen I (39). Increased levels of sialic acid are linked to cancer cell metastasis (40). The fully mature native UT-A1 from IMCD is sialylated, and its sialylation is markedly increased in a diabetic condition (Fig. 6). Most likely, the fact that UT-A1 lacked complete sialylation in MDCK cells was one of the reasons why some UT-A1 localized in nonraft domains.

UT-A1 abundance is significantly increased in IMCD in STZ-induced diabetes. The increases in UT-A1, together with AQP2 and NKCC2/BSC1 proteins, are believed to be compensatory changes to the ongoing osmotic diuresis caused by glucosuria (13). A tubular perfusion study of isolated initial IMCDs showed that the increased urea transport activity correlates with an increase in UT-A1 protein abundance, particularly the appearance of the 117-kDa glycoprotein (41). Our lipid raft fractionation experiments showed that in diabetes, almost all up-regulated UT-A1 in cell membranes was in the 117-kDa form. The increased membrane 117-kDa UT-A1 was located in lipid rafts and shifted to lower-density fractions 1–3. The expression of various glycosyltransferases can change during cell or tissue development, resulting in alterations of glycosylation of a particular protein (32). It is not clear from the current study whether and how in diabetes the 97-kDa form was further glycosylated into the highly glycosylated 117-kDa form in lipid rafts. Membrane protein activity can be modulated by its specific localization within different microdomains at the plasma membrane (18, 19, 22, 37). Some membrane protein (channel) activity is higher outside of lipid raft microdomains (18), as seen in TRPM8. When confined into lipid raft domains, TRPM8 is less active. However, NKCC1 is strongly activated when moved into lipid rafts (22). The increased 117-kDa form of UT-A1 in lipid rafts may contribute to the enhanced urea permeability in IMCDs from diabetic rats (41). At this point, the 117-kDa UT-A1 could be the active form with a high activity in lipid raft subdomains.

Our current study clarified for the first time the difference in glycosylation structure between 97- and 117-kDa UT-A1. It was believed that both 97 and 117 kDa are mature glycosylation forms since they are insensitive to Endo H digestion (14). It is true that both forms are detected in the cell plasma membrane and respond to vasopressin treatment (42). However, our present study revealed that the 97-kDa UT-A1 is a hybrid glycan form with low content of terminal N-acetylglucosamine (GlcNAc) residues but high content of mannose glycans. The 117-kDa is a mature complex glycosylation form composed of GlcNAc, N-acetyllactosamine (LacNAc), and sialic acid carbohydrates. The sialylated UT-A1 was particularly increased in diabetes. Our study demonstrated that diabetes not only causes an increase in total UT-A1 protein abundance, but also causes changes in the sugar chain carbohydrate structure.

In addition, we identified the 117-kDa glycosylation form of UT-A1 in transfected cells. Native UT-A1 from IMCD demonstrated both the 97- and 117-kDa glycosylation forms. However, only one 97-kDa glycosylation form of UT-A1 was observed in heterologous expression systems, such as HEK-293 cells (5, 8, 9), MDCK cells (4, 26), CHO cells (29), mIMCD cells (7), LLC-PK1 cells (6), and oocytes (8, 9, 29). Our study clearly shows the existence of the 117-kDa form in transfected MDCK cells after enrichment for terminal N-acetylglucosamine glycans with WGA. It was undetectable before due to its low abundance compared with the 97-kDa form. Not very surprisingly, the 117-kDa glycosylated UT-A1 was found in lipid raft compartments in MDCK cells (Fig. 4).

In summary, the major finding in this study is that glycosylation affects UT-A1 trafficking to lipid raft compartments. In MDCK cells, mature glycosylated UT-A1 (containing N-acetylglucosamine) is localized in lipid rafts. In vivo, the 117-kDa UT-A1 is the mature glycosylation form, and the highly sialylated UT-A1 specifically increased in lipid raft subdomains in diabetes. The mature glycosylation acts as a targeting signal, determining UT-A1 trafficking into membrane lipid raft subdomains. Further studies will be needed to address whether the lipid raft-associated highly glycosylated 117-kDa UT-A1 represents the functional urea transporter and how the lipid rafts modulate UT-A1 activity. Also, it will be necessary to investigate how UT-A1 is sialylated by carbohydrate modifying enzymes (like sialyltransferase) and how sialylation regulates UT-A1 activity, particularly under diabetic conditions.

Acknowledgments

This work was supported by U.S. National Institutes of Health grants R01-DK087838 and R21-DK080431 (to G.C.), and R01-DK41707 (to J.M.S.).

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