N-linked glycosylation of the thyroid Na+/I- symporter (NIS) -: Implications for its secondary structure model
N-linked glycosylation of the thyroid Na+/I- symporter (NIS) -: Implications for its secondary structure model
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DOI：
10.1074/jbc.273.35.22657
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发表时间：
1998-08-28
影响因子：
4.8
通讯作者：
Carrasco, N
中科院分区：
文献类型：
--
作者：
Levy, O;De la Vieja, A;Carrasco, N
Na~+/I~-转运体(NIS)是本实验室(Dai，G.，Levy，O.和Carrasco，N.(1996)Natural 379,458-460)在分子水平上鉴定和鉴定的一种618个氨基酸的膜糖蛋白，它催化I-主动聚集到甲状腺细胞内。由于成熟的MS是高度糖基化的，所以它在SDS-聚丙烯酰胺凝胶电泳中作为一种宽的多肽迁移，其分子质量(类似于90-110 kDa)高于非糖基化的NIS(类似于50 kDa)。我们分别和同时用谷氨酰胺替换了NIS的三个N-连接糖基化共识序列中的天冬酰胺残基，并评估了突变对COS细胞中NIS功能和稳定性的影响。所有突变体都是活性的，显示出50%-90%的野生型NIS活性，包括完全非糖基化的三重突变体。这表明在相当大的程度上，NIS的功能和稳定性在部分甚至全部没有N-连接的糖基化的情况下保持不变。我们还发现ASN(225)是糖基化的，从而证明含有该氨基酸残基的亲水环面对的是细胞外环境，而不是先前提出的胞浆。我们证明了NH_1末端也面向细胞外，提出了与这些发现相一致的新的二级结构模型。
The Na+/I- symporter (NIS), a 618-amino acid membrane glycoprotein that catalyzes the active accumulation of I- into thyroid cells, was identified and characterized at the molecular level in our laboratory (Dai, G., Levy, O., and Carrasco, N. (1996) Nature 379, 458-460), Because mature MS is highly glycosylated, it migrates in SDS-polyacrylamide gel electrophoresis as a broad polypeptide of higher molecular mass (similar to 90-110 kDa) than nonglycosylated NIS (similar to 50 kDa), Using site-directed mutagenesis, we substituted both separately and simultaneously the asparagine residues in all three putative N-linked glycosylation consensus sequences of NIS with glutamine and assessed the effects of the mutations on function and stability of NIS in COS cells. All mutants were active and displayed 50-90% of wild-type NIS activity, including the completely nonglycosylated triple mutant. This demonstrates that to a considerable extent, function and stability of NIS are preserved in the partial or even total absence of N-linked glycosylation. We also found that Asn(225) is glycosylated, thus proving that the hydrophilic loop that contains this amino acid residue faces the extracellular milieu rather than the cytosol as previously suggested. We demonstrated that the NH, terminus faces extracellularly as well, A new secondary structure model consistent with these findings is proposed,