Chen Junling, Wang Jiaqi, Sui Binglin, Jiang Pengwei, Wang Xumin, Wang Hongda, Liang Feng
School of Chemistry & Chemical Engineering, Wuhan University of Science and Technology, 947 Heping Street, Wuhan, Hubei 430081, China.
College of Chemical Engineering and Environmental Chemistry, Weifang University, Weifang 261061, China.
JACS Au. 2025 May 8;5(5):2246-2256. doi: 10.1021/jacsau.5c00193. eCollection 2025 May 26.
The asialoglycoprotein receptor (ASGPR) is a promising therapeutic target for drug delivery systems in hepatocellular carcinoma (HCC), exhibiting high affinity for specific carbohydrate residues and overexpression on malignant hepatic cells. However, their functional mechanisms remain poorly resolved at the single molecule level, hindering the rational optimization of ASGPR-targeted drug delivery systems. Here, we developed a trivalent -acetylgalactosamine (TriGalNAc)-functionalized ligand probe leveraging high affinity to enable the nanoscale visualization of ASGPR organization and trafficking via super-resolution imaging. Fixed cell imaging revealed pronounced clustering patterns of the ASGPR on HCC membranes. In live cell experiments, we observed the distribution changes of residual ASGPR and returned ASGPR on the membrane during endocytosis, identifying protein clusters as key functional platforms for mediated ligand uptake. Additionally, comparisons with ligand probe binding under varying cell states confirmed that ASGPR aggregation degree correlates with its ligand-binding capacity. Strikingly, disruption of membrane carbohydrate cross-linking dispersed ASGPR clusters and attenuated ligand binding. These findings resolve the nanoscale assembly of ASGPR in HCC and unveil clustering-dependent ligand-binding regulation, advancing a fundamental understanding of ASGPR biology while providing new insights to refine receptor-targeted therapeutics.
去唾液酸糖蛋白受体（ASGPR）是肝细胞癌（HCC）药物递送系统中一个很有前景的治疗靶点，它对特定碳水化合物残基具有高亲和力，且在恶性肝细胞上过表达。然而，其功能机制在单分子水平上仍未得到很好的解析，这阻碍了ASGPR靶向药物递送系统的合理优化。在此，我们开发了一种三价N - 乙酰半乳糖胺（TriGalNAc）功能化的配体探针，利用其高亲和力通过超分辨率成像实现ASGPR组织和运输的纳米级可视化。固定细胞成像显示ASGPR在HCC细胞膜上有明显的聚集模式。在活细胞实验中，我们观察到内吞过程中膜上残留ASGPR和回收ASGPR的分布变化，确定蛋白簇是介导配体摄取的关键功能平台。此外，在不同细胞状态下与配体探针结合的比较证实，ASGPR聚集程度与其配体结合能力相关。引人注目的是，膜碳水化合物交联的破坏分散了ASGPR簇并减弱了配体结合。这些发现解析了HCC中ASGPR的纳米级组装，并揭示了依赖于聚集的配体结合调节，推动了对ASGPR生物学的基本理解，同时为优化受体靶向治疗提供了新的见解。
