Improve-WUST Joint Laboratory of Advanced Technology for Point-of-Care Testing and Precision Medicine, School of Chemistry & Chemical Engineering, Wuhan University of Science and Technology, 947 Heping Street, Wuhan, Hubei 430081, China.
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Research Center of Biomembranomics, 5625 Renmin Street, Changchun, Jilin 130022, China.
Anal Chem. 2023 Jun 20;95(24):9207-9218. doi: 10.1021/acs.analchem.3c00596. Epub 2023 Jun 5.
Nucleoside transporters (NTs) play an important role in the metabolism of nucleoside substances and the efficacy of nucleoside drugs. Its spatial information related to biofunctions at the single-molecule level remains unclear, owing to the limitation of the existing labeling methods and traditional imaging methods. Therefore, we synthesize the inhibitor-based fluorescent probe SAENTA-Cy5 and apply direct stochastic optical reconstruction microscopy (dSTORM) to conduct refined observation of human equilibrative nucleoside transporter 1 (hENT1), the most important and famous member of NTs. We first demonstrate the labeling specificity and superiority of SAENTA-Cy5 to the antibody probe. Then, we found different assembly patterns of hENT1 on the apical and basal membranes, which are further investigated to be caused by varying associations of membrane carbohydrates, membrane classical functional domains (lipid rafts), and associated membrane proteins (EpCAM). Our work provides an efficient method for labeling hENT1, which contributes to realize fine observation of NTs. The findings on the assembly features and potential assembly mechanism of hENT1 promote a better understanding of its biofunction, which facilitates further investigations on how NTs work in the metabolism of nucleoside and nucleoside analogues.
核苷转运体（NTs）在核苷物质代谢和核苷类药物疗效中发挥着重要作用。由于现有标记方法和传统成像方法的限制，其与生物功能相关的空间信息在单分子水平上仍不清楚。因此，我们合成了基于抑制剂的荧光探针 SAENTA-Cy5，并应用直接随机光学重建显微镜（dSTORM）对 NTs 中最重要和最著名的成员——人腔肠苷酸转运蛋白 1（hENT1）进行了精细观察。我们首先证明了 SAENTA-Cy5 对抗体探针的标记特异性和优越性。然后，我们发现 hENT1 在顶膜和基底膜上有不同的组装模式，进一步研究表明这是由膜碳水化合物、膜经典功能域（脂筏）和相关膜蛋白（EpCAM）的不同关联引起的。我们的工作为 hENT1 的标记提供了一种有效方法，有助于实现对 NTs 的精细观察。hENT1 的组装特征和潜在组装机制的发现促进了对其生物功能的更好理解，这有助于进一步研究 NTs 在核苷和核苷类似物代谢中的作用方式。
