The United Graduate School of Agricultural Science, Gifu University, Gifu, Japan.
Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, Suita, Japan; Laboratory of Molecular Immunology, Immunology Frontier Research Center (IFReC), Osaka University, Suita, Japan.
J Biol Chem. 2022 Dec;298(12):102676. doi: 10.1016/j.jbc.2022.102676. Epub 2022 Nov 3.
Alpha-1,6-fucosyltransferase (FUT8) synthesizes core fucose in N-glycans, which plays critical roles in various physiological processes. FUT8, as with many other glycosyltransferases, is a type-II membrane protein, and its large C-terminal catalytic domain is linked to the FUT8 stem region, which comprises two α-helices. Although the stem regions of several glycosyltransferases are involved in the regulation of Golgi localization, the functions of the FUT8 stem region have not been clarified as yet. Here, we found that the FUT8 stem region is essential for enzyme oligomerization. We expressed FUT8Δstem mutants, in which the stem region was replaced with glycine/serine linkers, in FUT8-KO HEK293 cells. Our immunoprecipitation and native-PAGE analysis showed that FUT8 WT formed a multimer but FUT8Δstem impaired multimer formation in the cells, although the mutants retained specific activity. In addition, the mutant protein had lower steady-state levels, increased endoplasmic reticulum localization, and a shorter half-life than FUT8 WT, suggesting that loss of the stem region destabilized the FUT8 protein. Furthermore, immunoprecipitation analysis of another mutant lacking a part of the stem region revealed that the first helix in the FUT8 stem region is critical for multimer formation. Our findings demonstrated that the FUT8 stem region is essential for multimer formation but not for catalytic activity, providing insights into how the FUT8 protein matures and functions in mammalian cells.
α-1,6-岩藻糖基转移酶（FUT8）在 N-聚糖中合成核心岩藻糖，在各种生理过程中发挥着关键作用。与许多其他糖基转移酶一样，FUT8 是一种 II 型膜蛋白，其较大的 C 端催化结构域与 FUT8 茎区相连，该茎区由两个α-螺旋组成。尽管几个糖基转移酶的茎区参与了高尔基体定位的调节，但 FUT8 茎区的功能尚未阐明。在这里，我们发现 FUT8 茎区对于酶寡聚化是必不可少的。我们在 FUT8-KO HEK293 细胞中表达了 FUT8Δstem 突变体，其中茎区被甘氨酸/丝氨酸接头取代。我们的免疫沉淀和天然-PAGE 分析表明，FUT8 WT 形成多聚体，但 FUT8Δstem 会损害细胞中的多聚体形成，尽管突变体保留了特异性活性。此外，突变蛋白的稳态水平较低，内质网定位增加，半衰期比 FUT8 WT 短，这表明茎区的缺失使 FUT8 蛋白不稳定。此外，对缺失部分茎区的另一个突变体的免疫沉淀分析表明，FUT8 茎区的第一个螺旋对于多聚体形成至关重要。我们的研究结果表明，FUT8 茎区对于多聚体形成是必不可少的，但对于催化活性不是必需的，这为了解 FUT8 蛋白在哺乳动物细胞中的成熟和功能提供了新的视角。
