N-linked glycans are required on epithelial Na+ channel subunits for maturation and surface expression
N-linked glycans are required on epithelial Na+ channel subunits for maturation and surface expression
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DOI：
10.1152/ajprenal.00195.2017
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发表时间：
2018-03-01
影响因子：
4.2
通讯作者：
Kleyman, Thomas R.
中科院分区：
文献类型：
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作者：
Kashlan, Ossama B.;Kinlough, Carol L.;Kleyman, Thomas R.
上皮Na+通道（ENaC）亚基在内质网中进行n -链糖基化，并在内质网中组装成α - β - γ复合物。n-糖基化的6、13和5个共识位点（Asn-X-Ser/Thr）分别位于小鼠α -、β -和γ -亚基的细胞外结构域。由于ENaC n -连接聚糖的重要性尚未得到充分解决，我们研究了阻止特定亚基n -糖基化对通道功能、表达、成熟和折叠的影响。在非洲爪蟾卵母细胞或Fischer大鼠甲状腺细胞中异种表达缺乏单个亚基上n -连接聚糖的α - β -ENaC可降低ENaC活性以及对细胞外Na+的抑制反应。β亚基上缺乏n -连接聚糖也阻碍了胰蛋白酶激活通道。然而，无论哪个亚基被修饰，剪切应力对通道的激活是与n链聚糖无关的。我们还发现，在任何一个亚基上缺乏n -链聚糖都会降低同源亚基的总水平和表面水平。β亚基上缺乏n -连接的聚糖对总水平的影响最大，γ亚基和α亚基上缺乏n -连接的聚糖分别具有中等和适度的影响。最后，具有野生型β亚基的通道对有限胰蛋白酶蛋白水解的敏感性高于β亚基上缺乏n -连接聚糖的通道。我们的研究结果表明，每个亚基上的n链聚糖对于通道的适当折叠、成熟、表面表达和功能是必需的。
Epithelial Na+ channel (ENaC) subunits undergo N-linked glycosylation in the endoplasmic reticulum where they assemble into an alpha beta gamma complex. Six, 13, and 5 consensus sites (Asn-X-Ser/Thr) for N-glycosylation reside in the extracellular domains of the mouse alpha-, beta-, and gamma-subunits, respectively. Because the importance of ENaC N-linked glycans has not been fully addressed, we examined the effect of preventing N-glycosylation of specific subunits on channel function, expression, maturation, and folding. Heterologous expression in Xenopus oocytes or Fischer rat thyroid cells with alpha beta gamma-ENaC lacking N-linked glycans on a single subunit reduced ENaC activity as well as the inhibitory response to extracellular Na+. The lack of N-linked glycans on the beta-subunit also precluded channel activation by trypsin. However, channel activation by shear stress was N-linked glycan independent, regardless of which subunit was modified. We also discovered that the lack of N-linked glycans on any one subunit reduced the total and surface levels of cognate subunits. The lack of N-linked glycans on the beta-subunit had the largest effect on total levels, with the lack of N-linked glycans on the gamma- and alpha-subunits having intermediate and modest effects, respectively. Finally, channels with wild-type beta-subunits were more sensitive to limited trypsin proteolysis than channels lacking N-linked glycans on the beta-subunit. Our results indicate that N-linked glycans on each subunit are required for proper folding, maturation, surface expression, and function of the channel.