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

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.

Lovastatin-Induced Phosphatidylinositol-4-Phosphate 5-Kinase Diffusion from Microvilli Stimulates ROMK Channels

“…44 Briefly, mpkCCD c14 cell suspensions were homogenized in 0.5% Brij 96V (Sigma-Aldrich) and 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 2 mM Na vanadate, and protease inhibitor cocktail buffer (TNEV) on ice for 30 minutes. Supernatant (500 ml) was mixed with an equal volume of 80% sucrose in TNEV and transferred into a centrifuge tube (13351 mm; Beckman Coulter, Palatine, IL); 3 ml 35% sucrose in TNEV was carefully layered on top of the mixture followed by another 1-ml layer of 5% sucrose.…”

Activation of Protein Kinase C-α and Src Kinase Increases Urea Transporter A1 α-2, 6 Sialylation

“…8 In addition, diabetes increases DAG by glycolysis, which results in a high level of PKC pathway activation. 17 To evaluate whether UT-A1 sialylation is regulated by PKC, UT-A1-MDCK cells were treated by the PKC activator phorbol dibutyrate (PDBu), and total UT-A1 and sialylated UT-A1 were examined.…”

“…IMCD suspensions were prepared and treated with PDBu for 6 hours. Because the cell PM UT-A1 is concentrated in lipid raft microdomains, 8 we particularly examined cell membrane UT-A1 in lipid raft domains. As shown in Figure 1B, PDBu treatment did not change UT-A1 distribution in lipid rafts.…”

“…7 The 117-kD glycoform is fully glycosylated and contains poly-N-acetyllactosamine, whereas the 97-kD glycoform is a hybrid form with a high level of mannose. 8 A functional study using tubule perfusion showed that the increased 117-kD glycoform in the IM is associated with increased urea transport activity. 9 This finding suggests that changes in the relative abundances of the 97-and 117-kD forms of UT-A1 may have important regulatory roles for UT-A1 function.…”

“…19 We previously reported that diabetes not only causes an increase of UT-A1 protein abundance but also, results in UT-A1 glycan changes. 8 UT-A1 glycan undergoes increased sialylation 8 ; however, the mechanism of how UT-A1 is sialylated and the significance of increased UT-A1 sialylation remain unclear. In this study, we show that activation of PKC promotes UT-A1 sialylation.…”