Modulation of kidney urea transporter UT-A3 activity by alpha2,6-sialylation.
Modulation of kidney urea transporter UT-A3 activity by alpha2,6-sialylation.
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通过 α2,6-唾液酸化调节肾脏尿素转运蛋白 UT-A3 活性
DOI：
10.1007/s00424-016-1802-0
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发表时间：
2016-07
期刊：
影响因子：
--
通讯作者：
Chen G
中科院分区：
文献类型：
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作者：
Qian X;Sands JM;Song X;Chen G
两种尿素转运蛋白UT-A1和UT-A3在肾末端内髓集管（IMCD）中表达，对浓缩尿的产生很重要。UT-A1作为所有UT-A尿素转运蛋白中最大的异构体，受到了广泛的关注和研究；然而，UT-A3的作用及其调控机制研究较少。在本研究中，我们研究了糖基化修饰对UT-A3的调节。定点诱变证实了UT-A3在Asn279处存在一个糖基化位点。糖基化的缺失降低了福斯克林刺激的UT-A3细胞膜表达和尿素运输活性。UT-A3有两种糖基化形式，45和65 kDa。使用糖特异性结合凝集素，检测UT-A3糖基化谱。45 kda的形式被凝集素cona （Con A）和Galant husnivalis凝集素（GNL）拉低，表明这是一种含有大量甘露糖的未成熟聚糖（Man），而65 kda的形式是一种由乙酰氨基葡萄糖（GlcNAc）和聚n -乙酰乳酸（poly-LacNAc）组成的成熟聚糖，分别被小麦胚芽凝集素（WGA）和番茄凝集素拉低。有趣的是，成熟的UT-A3聚糖含有大量的唾液酸。我们探索了负责指导UT-A3唾液化的酶。唾液基转移酶st6galii，而不是ST3GalIV，分解代谢UT-A3 α2,6-唾液基化。PDB处理激活蛋白激酶C (PKC)，促进UT-A3聚糖唾液化和膜表面表达。PKC抑制剂chelerythrine阻断st6gali诱导的UT-A3唾液化。ST6GalI唾液酰化的增加增加了UT-A3蛋白的稳定性和尿素运输活性。总之，我们的研究揭示了st6gali介导的唾液酰化修饰对UT-A3调节的新机制，该机制可能在肾脏尿素重吸收和尿浓缩机制中发挥重要作用。
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.