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

Abstract

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.

Characterizing human α-1,6-fucosyltransferase (FUT8) substrate specificity and structural similarities with related fucosyltransferases

“…These reductions in kcat/Km include Lys216 that H-bonds with N1 (160-fold reduction in kcat/Km), Gln470 that Hbonds with N2 (80-fold reduction), Asp494 adjacent to N3 (800-fold reduction), His535 that H-bonds with N3 (11-fold reduction), and Gln502 that H-bonds with N4 (16-fold reduction). A similar reduction in activity was previously seen for mutants in the SH3 domain including H535A as well as H535A/Lys541A and T550A/L552A double mutants in prior studies (93) and a collection of residues conserved between human FUT8 and chicken c-Src (87) demonstrating the critical role of the SH3 domain in providing acceptor substrate specificity and affinity. Thus, while the donor binding site is exceptionally sensitive to mutational perturbation, the acceptor binding site has fixed points of essential interaction and other regions that contribute incremental binding energy to catalytic efficiency.…”

Differential Labeling of Glycoproteins with Alkynyl Fucose Analogs

“…FUT1, 2, 4, and 9 were expressed as full-length forms in HEK293 cells and immunopurified as described [ 50 ], and FUT3 and 5 were similarly expressed in HEK293 cells and purified ( Supplementary Figure S6A ). FUT8 was expressed as a his-tagged soluble form in COS7 cells and purified from the media [ 51 ]. Lacto-N-neotetraose (LNnT) and a biantennary N-glycan (GnGnbiAsn-PNS) were used as an acceptor for FUT1-4 and FUT8, respectively [ 50 , 51 ].…”

“…FUT8 was expressed as a his-tagged soluble form in COS7 cells and purified from the media [ 51 ]. Lacto-N-neotetraose (LNnT) and a biantennary N-glycan (GnGnbiAsn-PNS) were used as an acceptor for FUT1-4 and FUT8, respectively [ 50 , 51 ]. A β4-galactosylated biantennary N-glycan (GGnGGnbi-PA) that was prepared as described previously [ 50 ] was used as an acceptor for FUT3 and FUT5 ( Supplementary Figure S6B ).…”

FUT8 Alpha-(1,6)-Fucosyltransferase in Cancer

“…The SH3 domain is typical of signal transduction molecules in the cytosol, but is unique to FUT8 compared to other glycosyltransferases (and likely plays are an important role in FUT8’s activity and core fucosylation) [ 44 ]. FUT8 is mainly localized to the Golgi with a type II topology, however, Tomida et al have reported that FUT8 can be partially localized to the cell surface in an SH3-dependent manner [ 44 , 45 ].…”

“…The SH3 domain is typical of signal transduction molecules in the cytosol, but is unique to FUT8 compared to other glycosyltransferases (and likely plays are an important role in FUT8’s activity and core fucosylation) [ 44 ]. FUT8 is mainly localized to the Golgi with a type II topology, however, Tomida et al have reported that FUT8 can be partially localized to the cell surface in an SH3-dependent manner [ 44 , 45 ]. Amino acid His535 in the SH3 domain is an essential residue for the enzymatic activity of FUT8, and ribophorin I (RPN1) has been identified as an SH3-dependent binding protein of FUT8, that can stimulate core fucosylation [ 44 ].…”