Department of Medicine, University of California, Irvine, CA 92697, United States of America.
Department of Medicine, University of California, Irvine, CA 92697, United States of America.
Int J Biol Macromol. 2021 Dec 1;192:1178-1184. doi: 10.1016/j.ijbiomac.2021.10.058. Epub 2021 Oct 18.
Ascorbic acid (AA) uptake in neurons occurs via a Na-dependent carrier-mediated process mediated by the sodium-dependent vitamin C transporter-2 (SVCT2). Relatively little information is available concerning the network of interacting proteins that support human (h)SVCT2 trafficking and cell surface expression in neuronal cells. Here we identified the synaptogenic adhesion protein, calsyntenin-3 (CLSTN3) as an hSVCT2 interacting protein from yeast two-hybrid (Y2H) screening of a human adult brain cDNA library. This interaction was confirmed by co-immunoprecipitation, mammalian two-hybrid (M2H), and co-localization in human cell lines. Co-expression of hCLSTN3 with hSVCT2 in SH-SY5Y cells led to a marked increase in AA uptake. Reciprocally, siRNA targeting hCLSTN3 inhibited AA uptake. In the J20 mouse model of Alzheimer's disease (AD), mouse (m)SVCT2 and mCLSTN3 expression levels in hippocampus were decreased. Similarly, expression levels of hSVCT2 and hCLSTN3 were markedly decreased in hippocampal samples from AD patients. These findings establish CLSTN3 as a novel hSVCT2 interactor in neuronal cells with potential pathophysiological significance.
抗坏血酸（AA）通过钠离子依赖的载体介导的过程被神经元摄取，该过程由钠离子依赖的维生素 C 转运蛋白-2（SVCT2）介导。关于支持人类（h）SVCT2在神经元细胞中的运输和细胞表面表达的相互作用蛋白网络，目前所知甚少。在这里，我们通过对人成人大脑 cDNA 文库进行酵母双杂交（Y2H）筛选，鉴定出突触发生黏附蛋白 calsyntenin-3（CLSTN3）是 hSVCT2 的相互作用蛋白。这种相互作用通过共免疫沉淀、哺乳动物双杂交（M2H）和在人细胞系中的共定位得到了证实。在 SH-SY5Y 细胞中与 hSVCT2 共表达 hCLSTN3 导致 AA 摄取显著增加。相反，靶向 hCLSTN3 的 siRNA 抑制 AA 摄取。在阿尔茨海默病（AD）的 J20 小鼠模型中，海马体中的 mSVCT2 和 mCLSTN3 表达水平降低。同样，AD 患者海马体样本中的 hSVCT2 和 hCLSTN3 表达水平也显著降低。这些发现确立了 CLSTN3 作为神经元细胞中 hSVCT2 的新型相互作用蛋白，具有潜在的病理生理意义。
