Distinctive domains and activity regulation of core fucosylation enzyme FUT8.

BACKGROUND: Core fucose, a structure added to the reducing end N-acetylglucosamine of N-glycans, has been shown to regulate various physiological and pathological processes, including melanoma metastasis, exacerbation of chronic obstructive pulmonary disease, and severe outcomes in COVID-19. SCOPE OF REVIEW: Recent research has shed light on regulation of the activity and subcellular localization of a1,6-fucosyltransferase (FUT8), the glycosyltransferase responsible for core fucose biosynthesis, unraveling the mechanisms for controlling core fucosylation in vivo. MAJOR CONCLUSIONS: This review summarizes the various features of FUT8, including its domains, structures, and substrate specificity. Additionally, we discuss the potential involvement of FUT8-binding proteins, such as oligosaccharyltransferase subunits, in the regulation of FUT8 activity, substrate specificity, and the secretion of FUT8. GENERAL SIGNIFICANCE: We anticipate that this review will contribute to a deeper understanding of the control of core fucose levels in vivo and involvement of core fucosylation in FUT8-relevant functions and diseases.

The stem region of α1,6-fucosyltransferase FUT8 is required for multimer formation but not catalytic activity.

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.

ER entry pathway and glycosylation of GPI-anchored proteins are determined by N-terminal signal sequence and C-terminal GPI-attachment sequence.

Newly synthesized proteins in the secretory pathway, including glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs), need to be correctly targeted and imported into the endoplasmic reticulum (ER) lumen. GPI-APs are synthesized in the cytosol as preproproteins, which contain an N-terminal signal sequence (SS), mature protein part, and C-terminal GPI-attachment sequence (GPI-AS), and translocated into the ER lumen where SS and GPI-AS are removed, generating mature GPI-APs. However, how various GPI-APs are translocated into the ER lumen in mammalian cells is unclear. Here, we investigated the ER entry pathways of GPI-APs using a panel of KO cells defective in each signal recognition particle-independent ER entry pathway-namely, Sec62, GET, or SND pathway. We found GPI-AP CD59 largely depends on the SND pathway for ER entry, whereas prion protein (Prion) and LY6K depend on both Sec62 and GET pathways. Using chimeric Prion and LY6K constructs in which the N-terminal SS or C-terminal GPI-AS was replaced with that of CD59, we revealed that the hydrophobicity of the SSs and GPI-ASs contributes to the dependence on Sec62 and GET pathways, respectively. Moreover, the ER entry route of chimeric Prion constructs with the C-terminal GPI-ASs replaced with that of CD59 was changed to the SND pathway. Simultaneously, their GPI structures and which oligosaccharyltransferase isoforms modify the constructs were altered without any amino acid change in the mature protein part. Taking these findings together, this study revealed N- and C-terminal sequences of GPI-APs determine the selective ER entry route, which in turn regulates subsequent maturation processes of GPI-APs.

The SH3 domain in the fucosyltransferase FUT8 controls FUT8 activity and localization and is essential for core fucosylation.

Core fucose is an N-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 in vitro 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.