Si Yunlong, Zhu Jiahui, Sayed Hend, Mayo Kevin H, Zhou Yifa, Tai Guihua, Su Jiyong
Jilin Province Key Laboratory for Chemistry and Biology of Natural Drugs in Changbai Mountain, School of Life Sciences, Northeast Normal University, Changchun 130024, China.
Jiangsu Key Laboratory of Brain Disease Bioinformation, Research Center for Biochemistry and Molecular Biology, Xuzhou Medical University, Xuzhou 221004, China.
Acta Biochim Biophys Sin (Shanghai). 2025 Jan 7;57(5):749-757. doi: 10.3724/abbs.2024182.
Glycan-mediated recognition plays a critical role in facilitating cell-cell and cell-matrix interactions. Galectin-8 (Gal-8), classified as a 'tandem-repeat' type of galectin, binds to cell surface glycans to modulate various cellular functions, including cell adhesion, migration, apoptosis, pathogen recognition, autophagy, and immunomodulation. Despite the known function of Gal-8 in binding to various glycosylated proteins, only a few interactions have been reported to date. In this study, mass spectrometry is used to identify CD98hc as a novel binding partner for Gal-8. Both the N-terminal and C-terminal carbohydrate recognition domains (CRDs) of Gal-8 (Gal-8N and Gal-8C) bind to CD98hc, an interaction that is specifically inhibited by lactose but not sucrose, as confirmed by pull-down assays. The binding affinity between CD98hc and Gal-8 measured by microscale thermophoresis (MST) is 1.51 ± 0.17 μM. In addition, Gal-8N and Gal-8C have the binding affinities of 0.22 ± 0.03 μM and 10.68 ± 1.69 μM, respectively. Gal-8N and Gal-8C are both involved in the recognition and binding process of CD98hc. Furthermore, both full-length Gal-8 and its individual CRDs bind specifically to -glycosylated glycans on CD98hc, as demonstrated by the use of tunicamycin to inhibit -glycosylation in cells. In addition, Gal-8 and its individual CRDs can pull down glycosylated CD98hc-ED but not free CD98hc-ED , indicating that the binding of Gal-8 to glycosylated CD98hc-ED is -glycosylation-dependent. Overall, our findings establish CD98hc as a novel binding partner for Gal-8 and provide insights for further exploration of the diverse biological functions of Gal-8.
聚糖介导的识别在促进细胞间和细胞与基质的相互作用中起着关键作用。半乳糖凝集素-8（Gal-8）被归类为“串联重复”型半乳糖凝集素，它与细胞表面聚糖结合以调节各种细胞功能，包括细胞黏附、迁移、凋亡、病原体识别、自噬和免疫调节。尽管已知Gal-8在与各种糖基化蛋白结合方面的功能，但迄今为止报道的相互作用却很少。在本研究中，采用质谱法鉴定出CD98hc是Gal-8的一种新的结合伴侣。Gal-8的N端和C端碳水化合物识别结构域（CRD）（Gal-8N和Gal-8C）均与CD98hc结合，下拉实验证实这种相互作用被乳糖特异性抑制，而蔗糖则无此作用。通过微量热泳动（MST）测定，CD98hc与Gal-8之间的结合亲和力为1.51±0.17μM。此外，Gal-8N和Gal-8C的结合亲和力分别为0.22±0.03μM和10.68±1.69μM。Gal-8N和Gal-8C均参与CD98hc的识别和结合过程。此外，全长Gal-8及其单个CRD均特异性结合CD98hc上的N-糖基化聚糖，这通过使用衣霉素抑制细胞中的N-糖基化得以证明。此外，Gal-8及其单个CRD可以下拉糖基化的CD98hc-ED，但不能下拉游离的CD98hc-ED，这表明Gal-8与糖基化的CD98hc-ED的结合是N-糖基化依赖性的。总体而言，我们的研究结果确定CD98hc是Gal-8的一种新的结合伴侣，并为进一步探索Gal-8的多种生物学功能提供了见解。
