Membrane Protein Disease Research Group, Department of Physiology, School of Molecular and Systems Medicine, University of Alberta, Edmonton, Canada T6G 2H7.
Biochemistry. 2011 Mar 29;50(12):2157-69. doi: 10.1021/bi101887z. Epub 2011 Feb 14.
Mutations in the SLC4A11 protein, reported as a sodium-coup-led borate transporter of the human plasma membrane, are responsible for three corneal dystrophies (CD): congenital hereditary endothelial dystrophy type 2, Harboyan syndrome, and late-onset Fuch's CD. To develop a rational basis to understand these diseases, whose point mutations are found throughout the SLC4A11 sequence, we analyzed the protein biochemically. Hydropathy analysis and an existing topology model for SLC4A1 (AE1), a bicarbonate transporter with the lowest evolutionary sequence divergence from SLC4A11, formed the basis to propose an SLC4A11 topology model. Immunofluorescence studies revealed the cytosolic orientation of N- and C-termini of SLC4A11. Limited trypsinolysis of SLC4A11 partially mapped the folding of the membrane and cytoplasmic domains of the protein. The binding of SLC4A11 to a stilbenedisulfonate inhibitor resin (SITS-Affi-Gel) was prevented by preincubation with H(2)DIDS, with a significantly higher half-maximal effective concentration than AE1. We conclude that stilbenedisulfonates interact with SLC4A11 but with a lower affinity than other SLC4 proteins. Disease-causing mutants divided into two classes on the basis of the half-maximal [H(2)DIDS] required for resin displacement and the fraction of protein binding H(2)DIDS, likely representing mildly misfolded and grossly misfolded proteins. Disease-causing SLC4A11 mutants are retained in the endoplasmic reticulum of HEK 293 cells. This phenotype could be partially rescued in some cases by growing the cells at 30 °C.
SLC4A11 蛋白中的突变被报道为人类质膜的钠耦合硼酸转运体，可导致三种角膜营养不良（CD）：先天性遗传性内皮营养不良 2 型、Harboyan 综合征和晚期 Fuch 的 CD。为了开发一种合理的基础来理解这些疾病，其点突变发现于 SLC4A11 序列的各个部位，我们对该蛋白进行了生化分析。疏水性分析和现有的 SLC4A1（AE1）拓扑模型，一种与 SLC4A11 进化序列差异最小的碳酸氢盐转运体，为提出 SLC4A11 拓扑模型奠定了基础。免疫荧光研究显示 SLC4A11 的 N 端和 C 端位于细胞质中。SLC4A11 的有限胰蛋白酶水解部分绘制了该蛋白的膜和细胞质结构域的折叠图。SLC4A11 与 stilbenedisulfonate 抑制剂树脂（SITS-Affi-Gel）的结合被 H(2)DIDS 的预孵育所阻止，其半最大有效浓度显著高于 AE1。我们得出结论，stibenedisulfonates 与 SLC4A11 相互作用，但亲和力低于其他 SLC4 蛋白。致病突变根据树脂置换所需的半最大 [H(2)DIDS] 和 H(2)DIDS 结合蛋白的分数分为两类，可能代表轻度错误折叠和严重错误折叠的蛋白质。致病的 SLC4A11 突变体在 HEK 293 细胞的内质网中被保留。在某些情况下，通过在 30°C 下培养细胞，可以部分挽救这种表型。
