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Abstract: FR-PO624

UT-A2-Mediated Water Transport Is Regulated by N-Glycosylation

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic

Authors

  • Kabutomori, Jessica, Sao Paulo Univ., Sao Paulo, SÃO PAULO, Brazil
  • Pina-Lopes, Neydiana, Sao Paulo Univ., Sao Paulo, SÃO PAULO, Brazil
  • Geyer, R. Ryan, Sao Paulo Univ., Sao Paulo, SÃO PAULO, Brazil
  • Musa-Aziz, Raif, Sao Paulo Univ., Sao Paulo, SÃO PAULO, Brazil
Background

Urea transporters (UTs) are transmembrane glycoproteins that facilitate the diffusion of urea across cell membranes, and play a crucial role in the urinary concentrating mechanisms, important for maintaining nearly constant blood plasma osmolality. Renal UTs include: UT-A1 and UT-A3, which are expressed on the apical and basolateral membranes of the inner medullary CD, respectively; UT-A2, localized in the thin descending limb of the inner and outer renal medulla; and UT-B, expressed in the endothelial cells of the descending vasa recta and red blood cells. The X-tal structure of bovine UT-B shows that three monomers assemble to form a homotrimer. Each monomer contains 10 transmembrane helices folded into independent urea channels. Helices 5 and 6 are connected by an extracellular loop that can be glycosylated. Indeed, protein glycosylation is known to modulate protein structure/activity. UT-A2 is glycosylated at Asparagine-210, yielding 45 kDa and 55 kDa glycoforms. Previously, we showed that wild-type mouse UT-A2 (mUT-A2WT) enhances water transport when expressed in Lithobates catesbeianus oocytes, and that two conserved Threonine residues (T176 and T338) are required for this transport. Here, we investigated the role of glycosylation on UT-A2-mediated water transport.

Methods

First, the N210 residue of mUT-A2 was mutated to glutamine (Q), generating UT-A2N210Q. Next, mUT-A2WT and UT-A2N210Q (c-Myc tagged) cRNA, or H2O was injected into Lithobates oocytes. UT-A2 cell surface expression was assessed by biotinylation and western blot analyses, with and without PNGase F. Pf was assessed by placing the oocytes in a hypotonic solution and monitoring the rate of cell swelling with video microscopy.

Results

We observed an immunoreactive band at 34 kDa after treating mUT-A2WT with PNGase F, a molecular weight consistent with unglycosylated monomer. No bands were detected in the 45-55 kDa range with mUT-A2N210Q, indicating lack of glycosylation. The Pf value for mUT-A2WT (0.0013±0.0001, n=27) was significantly greater than UT-A2N210Q (0.0005±0.00004, n=25) and H2O oocytes (0.0006±0.00004, n=30).

Conclusion

These results indicate that the oocytes were not only capable of adding N-linked glycans to membrane proteins, but also that this post-translational modification affects activity. This observation is relevant to urine concentration mechanisms.

Funding

  • Government Support - Non-U.S.