Modulation of kidney urea transporter UT-A3 activity by alpha2,6-sialylation

Abstract

Two urea transporters, UT-A1 and UT-A3, are expressed in the kidney terminal inner medullary collecting duct (IMCD) and are important for the production of concentrated urine. UT-A1, as the largest isoform of all UT-A urea transporters, has gained much attention and been extensively studied; however, the role and the regulation of UT-A3 are less explored. In this study, we investigated UT-A3 regulation by glycosylation modification. A site-directed mutagenesis verified a single glycosylation site in UT-A3 at Asn279. Loss of the glycosylation reduced forskolin-stimulated UT-A3 cell membrane expression and urea transport activity. UT-A3 has two glycosylation forms, 45 and 65 kDa. Using sugar-specific binding lectins, the UT-A3 glycosylation profile was examined. The 45-kDa form was pulled down by lectin concanavalin A (Con A) and Galant husnivalis lectin (GNL), indicating an immature glycan with a high amount of mannose (Man), whereas the 65-kDa form is a mature glycan composed of acetylglucosamine (GlcNAc) and poly-N-acetyllactosame (poly-LacNAc) that was pulled down by wheat germ agglutinin (WGA) and tomato lectin, respectively. Interestingly, the mature form of UT-A3 glycan contains significant amounts of sialic acid. We explored the enzymes responsible for directing UT-A3 sialylation. Sialyltransferase ST6GalI, but not ST3GalIV, catabolizes UT-A3 α2,6-sialylation. Activation of protein kinase C (PKC) by PDB treatment promoted UT-A3 glycan sialylation and membrane surface expression. The PKC inhibitor chelerythrine blocks ST6GalI-induced UT-A3 sialylation. Increased sialylation by ST6GalI increased UT-A3 protein stability and urea transport activity. Collectively, our study reveals a novel mechanism of UT-A3 regulation by ST6GalI-mediated sialylation modification that may play an important role in kidney urea reabsorption and the urinary concentrating mechanism.

Keywords: Glycosylation; Protein kinase C; Sialyltransferase; Urea transporter.

Figure 1. A single glycosylation site at N279 in UT-A3

A: Structure diagram showing a glycosylation site at N279 located in the large extracellular loop. B: PNGase F digestion. FLAG-UT-A3 WT and FLAG-UT-A3 N279 were transiently transfected into HEK293 cells. 2 days later, the cell membrane lipid raft fractions were collected and de-glycosylated by PNGase F at 37°C for 2h. UT-A3 was analyzed by Western blot with FLAG antibody. C: UT-A3 membrane expression. HEK293 cells were transiently transfected with UT-A3 WT or N279Q for 48h. Cells were pre-treated with 10 μM forskolin (FSK) for 20 min and processed for cell surface biotinylation. Membrane UT-A3 proteins were precipitated by steptavidin beads followed by Western blot with FLAG antibody. The bar graphs show the percent increase in UT-A3 protein levels resulting from FSK treatment (** p<0.01, NS: no significance, n=3).

Figure 2. Un-glycosylated UT-A3 has reduced urea transport activity

Oocytes were injected with UT-A3 or UT-A3 N279Q cRNAs (A) or pre-injected with 10 ng tunicamycin (Tuni) then injected with UT-A3 cRNA (B). After 3 days. Urea transport activity was measured by ¹⁴C-labeled urea flux (n=6 oocyte/time point; mean ± SD). UT-A3 protein expression on the cell membrane was examined by biotinylation and followed by Western blot with a UT-A1 NH₂-terminal antibody. A representative blot was shown from 3 different experiments.

Figure 3. Lectin affinity analysis of UT-A3 glycans

HEK293 cells transfected with UT-A3 WT or N279Q (A) or rat kidney inner medulla (IM) tissues (B) were processed for lipid raft isolation by 5%-40% (v/v) sucrose gradient ultracentrifugation. Lipid raft fractions (2-5) were collected for lectin pull-down assay and followed by Western blot with antibodies to UT-A1 NH₂-terminus. A representative blot was shown from >3 different experiments.

Figure 4. Sialytransferase ST6GalI directs UT-A3 sialylation and cell membrane expression

UT-A3 membrane expression. HEK293 cells co-transfected UT-A3 without or with ST6GalI (ST6) or ST3GalIV (ST3) were processed for cell surface biotinylation. The total and the cell membrane UT-A3 proteins were examined by Western blot with FLAG antibody. UT-A3 protein sialylation was examined by SNA pull-down assay. Bar graph showed the cell membrane UT-A3 expression as a percent of control (** P<0.01, n=4).

Figure 5. Activation of PKC pathway promotes UT-A3 sialylation

A: UT-A3 sialylation by PDB. HEK 293 cells were transiently transfected with UT-A3 for 48h followed by PDBu treatment for 4h. Sialylated UT-A3 was pulled down by SNA and then examined by western blot. Con A lectin was used as a control. Bar graph showed the band densities of sialylated UT-A3 as a percent of control (0 PDBu) (** P<0.01, n=3). B: UT-A3 membrane expression. HEK293 cells were transiently transfected with UT-A3 alone or together with ST6GalI for 48h, and then treated with or without 2 μM chelerythrine (chel) for 2h. Cell surface proteins were biotinylated and analyzed by Western blot with FLAG antibody. C: Inhibition of ST6GalI-induced sialylation by PKC inhibitor. HEK293 cells transfected with UT-A3 alone or together with ST6GalI for 48h, then treated with chelerythrine for 2 h, sialylated UT-A3 was pulled down by SNA and then examined by western blot. B, C showed the representative western blots from three independent experiments.

Figure 6. Effect of ST6GalI on UT-A3 protein degradation

HEK293 cells transfected with UT-A3 alone or together with ST6GalI were treated with 100 μg/ml cycloheximide (CHX) for the indicated time. The cells were lysed with RIPA buffer and total UT-A3 protein levels were evaluated by Western blot with FLAG antibody. Bar graph showed the band densities of UT-A3 (including both 65- and 45-kDa) as a percent of control (0 h) (* P<0.05, ** P<0.01, n=3).

Figure 7. Effect of ST6GalI on UT-A3 activity

Oocytes were injected with cRNAs of UT-A3 (2 ng/cell) alone or plus ST6GalI (2 ng/cell). After 3 days, rea transport activity was measured by ¹⁴C-labeled urea flux (n=6 oocyte/time point; mean ± SD). UT-A3 protein expression on the cell membrane was examined by biotinylation and sialylated UT-A3 was pulled down by SNA, followed by Western blot with a UT-A1 NH₂-terminal antibody (experiments n=3).