Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Thyroid. 2021 Aug;31(8):1272-1281. doi: 10.1089/thy.2020.0931. Epub 2021 Jun 4.
The sodium/iodide (Na/I) symporter (NIS) mediates active transport of I into the thyroid gland. Mutations in the gene, which encodes NIS, cause I transport defects (ITDs)-which, if left untreated, lead to congenital hypothyroidism and consequent cognitive and developmental deficiencies. The ITD-causing NIS mutation Y348D, located in transmembrane segment (TMS) 9, was reported in three Sudanese patients. We generated cDNAs coding for Y348D NIS and mutants with other hydrophilic and hydrophobic amino acid substitutions at position 348 and transfected them into cells. The activity of the resulting mutants was quantitated by radioiodide transport assays. NIS glycosylation was investigated by Western blotting after endoglycosidase H (Endo H) and PNGase-F glycosidase treatment. Subcellular localization of the mutant proteins was ascertained by flow cytometry analysis, cell surface biotinylation, and immunofluorescence. The intrinsic activity of Y348D was studied by measuring radioiodide transport in membrane vesicles prepared from Y348D-NIS-expressing cells. Our NIS homology models and molecular dynamics simulations were used to identify residues that interact with Y348 and investigate possible interactions between Y348 and the membrane. The sequences of several Slc5 family transporters were aligned, and a phylogenetic tree was generated in ClustalX. Cells expressing Y348D NIS transport no I. Furthermore, Y348D NIS is only partially glycosylated, is retained intracellularly, and is intrinsically inactive. Hydrophilic residues other than Asp at position 348 also yield NIS proteins that fail to be targeted to the plasma membrane (PM), whereas hydrophobic residues at this position, which we show do not interact with the membrane, rescue PM targeting and function. Y348D NIS does not reach the PM and is intrinsically inactive. Hydrophobic amino acid substitutions at position 348, however, preserve NIS activity. Our findings are consistent with our homology model's prediction that Y348 should face the side opposite the TMS9 residues that coordinate Na and participate in Na transport, and with the notion that Y348 interacts only with hydrophobic residues. Hydrophilic or charged residues at position 348 have deleterious effects on NIS PM targeting and activity, whereas a hydrophobic residue at this position rescues NIS activity.
钠/碘（Na/I）转运体（NIS）介导碘向甲状腺的主动转运。编码 NIS 的 基因突变导致碘转运缺陷（ITD）-如果不治疗，会导致先天性甲状腺功能减退症以及随之而来的认知和发育缺陷。位于跨膜片段（TMS）9 中的 Y348D NIS 突变已在 3 名苏丹患者中报道。我们生成编码 Y348D NIS 和其他位于 348 位具有亲水和疏水性氨基酸取代的突变体的 cDNA，并将其转染到细胞中。通过放射性碘转运测定定量测定所得突变体的活性。用内切糖苷酶 H（Endo H）和 PNGase-F 糖苷酶处理后，通过 Western blot 研究 NIS 的糖基化。通过流式细胞术分析、细胞表面生物素化和免疫荧光确定突变蛋白的亚细胞定位。通过测量 Y348D-NIS 表达细胞制备的膜泡中的放射性碘转运来研究 Y348D 的固有活性。我们的 NIS 同源模型和分子动力学模拟用于鉴定与 Y348 相互作用的残基，并研究 Y348 与膜之间的可能相互作用。对几种 Slc5 家族转运体的序列进行了比对，并在 ClustalX 中生成了系统发育树。表达 Y348D NIS 的细胞不转运碘。此外，Y348D NIS 仅部分糖基化，滞留于细胞内，固有失活。位于 348 位的除天冬氨酸以外的亲水残基也产生不能靶向质膜（PM）的 NIS 蛋白，而位于该位置的疏水性残基不与膜相互作用，可挽救 PM 靶向和功能。Y348D NIS 无法到达 PM 且固有失活。然而，位于 348 位的疏水性氨基酸取代保留了 NIS 的活性。我们的研究结果与我们的同源模型的预测一致，即 Y348 应朝向与协调 Na 并参与 Na 转运的 TMS9 残基相对的一侧，并且 Y348 仅与疏水性残基相互作用。位于 348 位的亲水或带电残基对 NIS PM 靶向和活性具有有害影响，而该位置的疏水性残基可挽救 NIS 活性。
