Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Via P. Bucci 4C, 87036 Arcavacata di Rende, Italy.
Department of Molecular and Clinical Medicine, Wallenberg Laboratory, Sahlgrenska Academy, University of Gothenburg, 413 45 Gothenburg, Sweden.
Int J Mol Sci. 2018 Feb 25;19(3):648. doi: 10.3390/ijms19030648.
The human plasma membrane transporter ASCT2 is responsible for mediating Na- dependent antiport of neutral amino acids. New insights into structure/function relationships were unveiled by a combined approach of recombinant over-expression, site-directed mutagenesis, transport assays in proteoliposomes and bioinformatics. WT and Cys mutants of hASCT2 were produced in and purified for functional assay. The reactivity towards SH reducing and oxidizing agents of WT protein was investigated and opposite effects were revealed; transport activity increased upon treatment with the Cys reducing agent DTE, i.e., when Cys residues were in thiol (reduced) state. Methyl-Hg, which binds to SH groups, was able to inhibit WT and seven out of eight Cys to Ala mutants. On the contrary, C467A loses the sensitivity to both DTE activation and Methyl-Hg inhibition. The C467A mutant showed a Km for Gln one order of magnitude higher than that of WT. Moreover, the C467 residue is localized in the substrate binding region of the protein, as suggested by bioinformatics on the basis of the EAAT1 structure comparison. Taken together, the experimental data allowed identifying C467 residue as crucial for substrate binding and for transport activity modulation of hASCT2.
人血浆膜转运体 ASCT2 负责介导中性氨基酸的 Na+依赖性反向转运。通过重组过度表达、定点突变、在质体脂质体中的转运测定和生物信息学的综合方法，揭示了结构/功能关系的新见解。在 中产生了 WT 和 Cys 突变体 hASCT2 并进行了功能测定纯化。研究了 WT 蛋白对 SH 还原和氧化试剂的反应性，结果显示出相反的效果；当半胱氨酸残基处于巯基（还原）状态时，用 Cys 还原剂 DTE 处理会增加转运活性。与 SH 基团结合的甲基汞能够抑制 WT 和 8 个 Cys 到 Ala 突变体中的 7 个。相反，C467A 对 DTE 激活和甲基汞抑制均失去敏感性。与 WT 相比，C467A 突变体对 Gln 的 Km 高一个数量级。此外，根据 EAAT1 结构比较的生物信息学，C467 残基定位于蛋白质的底物结合区域。综上所述，实验数据表明 C467 残基对于 hASCT2 的底物结合和转运活性调节至关重要。
