Zehnpfennig Britta, Wiriyasermkul Pattama, Carlson David A, Quick Matthias
From the Center for Molecular Recognition and.
Department of Psychiatry, Columbia University College of Physicians and Surgeons, New York, New York 10032.
J Biol Chem. 2015 Jun 26;290(26):16372-82. doi: 10.1074/jbc.M114.622555. Epub 2015 May 13.
The human Na(+)/multivitamin transporter (hSMVT) has been suggested to transport α-lipoic acid (LA), a potent antioxidant and anti-inflammatory agent used in therapeutic applications, e.g. in the treatment of diabetic neuropathy and Alzheimer disease. However, the molecular basis of the cellular delivery of LA and in particular the stereospecificity of the transport process are not well understood. Here, we expressed recombinant hSMVT in Pichia pastoris and used affinity chromatography to purify the detergent-solubilized protein followed by reconstitution of hSMVT in lipid bilayers. Using a combined approach encompassing radiolabeled LA transport and equilibrium binding studies in conjunction with the stabilized R-(+)- and S-(-)-enantiomers and the R,S-(+/-) racemic mixture of LA or lipoamide, we identified the biologically active form of LA, R-LA, to be the physiological substrate of hSMVT. Interaction of R-LA with hSMVT is strictly dependent on Na(+). Under equilibrium conditions, hSMVT can simultaneously bind ~2 molecules of R-LA in a biphasic binding isotherm with dissociation constants (Kd) of 0.9 and 7.4 μm. Transport of R-LA in the oocyte and reconstituted system is exclusively dependent on Na(+) and exhibits an affinity of ~3 μm. Measuring transport with known amounts of protein in proteoliposomes containing hSMVT in outside-out orientation yielded a catalytic turnover number (kcat) of about 1 s(-1), a value that is well in agreement with other Na(+)-coupled transporters. Our data suggest that hSMVT-mediated transport is highly specific for R-LA at our tested concentration range, a finding with wide ramifications for the use of LA in therapeutic applications.
人类钠/多种维生素转运体（hSMVT）已被认为可转运α-硫辛酸（LA），α-硫辛酸是一种有效的抗氧化剂和抗炎剂，用于治疗应用，如治疗糖尿病性神经病变和阿尔茨海默病。然而，LA细胞递送的分子基础，特别是转运过程的立体特异性尚不清楚。在这里，我们在毕赤酵母中表达重组hSMVT，并使用亲和色谱法纯化去污剂溶解的蛋白，随后将hSMVT重组到脂质双层中。我们采用了一种综合方法，包括放射性标记的LA转运和平衡结合研究，结合LA或硫辛酰胺的稳定化R-（+）-和S-（-）-对映体以及R，S-（+/-）外消旋混合物，我们确定LA的生物活性形式R-LA是hSMVT的生理底物。R-LA与hSMVT的相互作用严格依赖于钠离子。在平衡条件下，hSMVT可以在双相结合等温线中同时结合约2个R-LA分子，解离常数（Kd）分别为0.9和7.4μm。R-LA在卵母细胞和重组系统中的转运完全依赖于钠离子，亲和力约为3μm。在含有外向型hSMVT的蛋白脂质体中用已知量的蛋白质测量转运，得到的催化转换数（kcat）约为1 s-1，这一数值与其他钠偶联转运体非常一致。我们的数据表明，在我们测试的浓度范围内，hSMVT介导的转运对R-LA具有高度特异性，这一发现对LA在治疗应用中的使用具有广泛影响。
