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 kDa and 65 kDa. Using sugar specific-binding lectins, the UT-A3 glycosylation profile was examined. The 45 kDa form was pulled down by lectin Con A and 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), poly-N-acetyllactosame (poly-LacNAc) that was pulled down by 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 PKC by PDB treatment promoted UT-A3 glycan sialylation and membrane surface expression. 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 in kidney urea reabsorption and the urinary concentrating mechanism.

Mammalian urine concentration: a review of renal medullary architecture and membrane transporters

“…UT-A3 has a single glycosylation site at N279 and mutation of this site reduces forskolin-stimulated membrane expression and urea transport (Qian et al 2016). Sialylation of the mature glycosylated UT-A3 by sialyltransferase increases protein stability, cell surface expression, and urea transport activity via a PKA pathway (Qian et al 2016).…”

Urea transport and clinical potential of urearetics

“…Since PKC inhibition blocked the induction of UT-A1 sialylation by high glucose, this pathway may be important in ameliorating the osmotic diuresis caused by glucosuria in patients with diabetes mellitus (26). PKC also enhances UT-A3 glycan sialylation, and this effect is mediated by ST6GalI (27). …”

Estudo da via de transporte de água e/ou amônia através dos transportadores de ureia (UTs) quando expressos em oócitos da rã >i<Lithobates catesbeianus>/i<.

“…Portanto, os resultados da Figura 16 demonstram que a mutação dos resíduos conservados de Thr para Val na proteína mUT-A2 WT , mUT-A3 WT ou mUT-B WT não prejudicou a expressão da proteína na membrana plasmática dos oócitos, uma vez que as colunas correspondentes às proteínas mutantes no resultado de "western blot" apresentaram bandas semelhantes às bandas da proteína WT descritas na literatura (YOU et al, 1993;TRINH-TRANG-TAN et al, 2002;BAGNASCO et al, 2006;MACIVER et al, 2008;SU et al, 2012;QIAN et al, 2016). O experimento para a medida das variações do pHS foi iniciado com a ponta do eletrodo pHS na solução de "ND96" extracelular ("banho") -~ 300 µm longe da superfície do oócitopara calibrar o eletrodo no pH 7,50 da solução controle.…”