Center for Highly Advanced Integration of Nano and Life Sciences (G-CHAIN), Gifu University, Gifu, Japan.
Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan.
J Biol Chem. 2020 Jun 5;295(23):7992-8004. doi: 10.1074/jbc.RA120.013079. Epub 2020 Apr 29.
Core fucose is an -glycan structure synthesized by α1,6-fucosyltransferase 8 (FUT8) localized to the Golgi apparatus and critically regulates the functions of various glycoproteins. However, how FUT8 activity is regulated in cells remains largely unclear. At the luminal side and uncommon for Golgi proteins, FUT8 has an Src homology 3 (SH3) domain, which is usually found in cytosolic signal transduction molecules and generally mediates protein-protein interactions in the cytosol. However, the SH3 domain has not been identified in other glycosyltransferases, suggesting that FUT8's functions are selectively regulated by this domain. In this study, using truncated FUT8 constructs, immunofluorescence staining, FACS analysis, cell-surface biotinylation, proteomics, and LC-electrospray ionization MS analyses, we reveal that the SH3 domain is essential for FUT8 activity both in cells and and identified His-535 in the SH3 domain as the critical residue for enzymatic activity of FUT8. Furthermore, we found that although FUT8 is mainly localized to the Golgi, it also partially localizes to the cell surface in an SH3-dependent manner, indicating that the SH3 domain is also involved in FUT8 trafficking. Finally, we identified ribophorin I (RPN1), a subunit of the oligosaccharyltransferase complex, as an SH3-dependent binding protein of FUT8. RPN1 knockdown decreased both FUT8 activity and core fucose levels, indicating that RPN1 stimulates FUT8 activity. Our findings indicate that the SH3 domain critically controls FUT8 catalytic activity and localization and is required for binding by RPN1, which promotes FUT8 activity and core fucosylation.
核心岩藻糖是一种由α1,6-岩藻糖基转移酶 8（FUT8）在高尔基器中合成的聚糖结构，对各种糖蛋白的功能具有关键调节作用。然而，细胞内 FUT8 活性是如何被调节的在很大程度上仍不清楚。在腔侧，FUT8 具有Src 同源 3（SH3）结构域，这在高尔基蛋白中不常见，通常存在于细胞质信号转导分子中，通常介导细胞质中的蛋白质-蛋白质相互作用。然而，其他糖基转移酶中并未发现 SH3 结构域，这表明 FUT8 的功能是由该结构域选择性调节的。在这项研究中，我们使用截断的 FUT8 构建体、免疫荧光染色、FACS 分析、细胞表面生物素化、蛋白质组学和 LC-电喷雾电离 MS 分析，揭示了 SH3 结构域对于细胞内和细胞表面的 FUT8 活性都是必需的，并确定了 SH3 结构域中的 His-535 是 FUT8 酶活性的关键残基。此外，我们发现尽管 FUT8 主要定位于高尔基器，但它也部分地以 SH3 依赖的方式定位于细胞表面，表明 SH3 结构域也参与 FUT8 的运输。最后，我们鉴定了核糖体蛋白 I（RPN1），一种寡糖基转移酶复合物的亚基，作为 FUT8 的 SH3 依赖性结合蛋白。RPN1 的敲低降低了 FUT8 的活性和核心岩藻糖水平，表明 RPN1 刺激了 FUT8 的活性。我们的研究结果表明，SH3 结构域对 FUT8 的催化活性和定位具有关键控制作用，并且是与 RPN1 结合所必需的，这促进了 FUT8 的活性和核心岩藻糖基化。
