Role of N-glycosylation in cell surface expression and protection against proteolysis of the intestinal anion exchanger SLC26A3
Role of N-glycosylation in cell surface expression and protection against proteolysis of the intestinal anion exchanger SLC26A3
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DOI：
10.1152/ajpcell.00165.2011
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发表时间：
2012-03-01
影响因子：
5.5
通讯作者：
Yamashita, Yukari
中科院分区：
文献类型：
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作者：
Hayashi, Hisayoshi;Yamashita, Yukari
Hayashi H，Yamashita Y。N-糖基化在细胞表面表达和防止肠道阴离子交换器 SLC26A3 蛋白水解中的作用。 Am J Physiol Cell Physiol 302：C781-C795，2012。首次发表于 2011 年 12 月 7 日； doi:10.1152/ajpcell.00165.2011.-SLC26A3 是一种 Cl-/HCO3- 交换剂，在肠道吸收 Cl- 中起主要作用。它的突变会导致先天性失氯性腹泻。研究表明，SLC26A3 是糖基化的，其附着的碳水化合物位于细胞外，可能具有调节功能。然而，糖基化的作用尚未明确确定。我们使用生化修饰和定点突变的方法来防止糖基化。使用糖苷酶的去糖基化实验表明，SLC26A3 的成熟糖基化形式存在于质膜上，并且假定的大第二细胞外环包含所有 N 连接碳水化合物。与野生型相比，SLC26A3 的去糖基化导致转运活性降低，尽管仍然观察到强烈的细胞内 pH 变化，这表明 N-糖基化对于转运活性并不是绝对必要的。为了定位糖基化位点，我们通过用谷氨酰胺替换天冬酰胺 (N) 来突变五个共有位点。免疫印迹表明 SLC26A3 在 N153、N161 和 N165 处被糖基化。 SLC26A3 的去糖基化会导致细胞表面加工缺陷，从而导致细胞表面表达减少。我们还评估了 SLC26A3 是否受到胰蛋白酶消化的保护。虽然成熟的糖基化 SLC26A3 在用胰蛋白酶处理后几乎没有分解，但去糖基化的 SLC26A3 对胰蛋白酶的敏感性增加，表明寡糖可以保护 SLC26A3 免受胰蛋白酶消化。总之，我们的数据表明，SLC26A3 的 N-糖基化对于细胞表面表达和防止蛋白水解降解非常重要，这可能有助于了解先天性糖基化疾病的发病机制。
Hayashi H, Yamashita Y. Role of N-glycosylation in cell surface expression and protection against proteolysis of the intestinal anion exchanger SLC26A3. Am J Physiol Cell Physiol 302: C781-C795, 2012. First published December 7, 2011; doi:10.1152/ajpcell.00165.2011.-SLC26A3 is a Cl-/HCO3- exchanger that plays a major role in Cl- absorption from the intestine. Its mutation causes congenital chloride-losing diarrhea. It has been shown that SLC26A3 are glycosylated, with the attached carbohydrate being extracellular and perhaps modulating function. However, the role of glycosylation has yet to be clearly determined. We used the approaches of biochemical modification and site-directed mutagenesis to prevent glycosylation. Deglycosylation experiments with glycosidases indicated that the mature glycosylated form of SLC26A3 exists at the plasma membrane, and a putative large second extracellular loop contains all of the N-linked carbohydrates. Deglycosylation of SLC26A3 causes depression of transport activity compared with wild-type, although robust intracellular pH changes were still observed, suggesting that N-glycosylation is not absolutely necessary for transport activity. To localize glycosylation sites, we mutated the five consensus sites by replacing asparagine (N) with glutamine. Immnoblotting suggests that SLC26A3 is glycosylated at N153, N161, and N165. Deglycosylation of SLC26A3 causes a defect in cell surface processing with decreased cell surface expression. We also assessed whether SLC26A3 is protected from tryptic digestion. While the mature glycosylated SLC26A3 showed little breakdown after treatment with trypsin, deglycosylated SLC26A3 exhibited increased susceptibility to trypsin, suggesting that the oligosaccharides protect SLC26A3 from tryptic digestion. In conclusion, our data indicate that N-glycosylation of SLC26A3 is important for cell surface expression and for protection from proteolytic degradation that may contribute to the understanding of pathogenesis of congenital disorders of glycosylation.