Appropriate aglycone modification significantly expands the glycan substrate acceptability of α1,6-fucosyltransferase (FUT8)

Abstract

The α1,6-fucosyltransferase, FUT8, is the sole enzyme catalyzing the core-fucosylation of N-glycoproteins in mammalian systems. Previous studies using free N-glycans as acceptor substrates indicated that a terminal β1,2-GlcNAc moiety on the Man-α1,3-Man arm of N-glycan substrates is required for efficient FUT8-catalyzed core-fucosylation. In contrast, we recently demonstrated that, in a proper protein context, FUT8 could also fucosylate Man5GlcNAc2 without a GlcNAc at the non-reducing end. We describe here a further study of the substrate specificity of FUT8 using a range of N-glycans containing different aglycones. We found that FUT8 could fucosylate most of high-mannose and complex-type N-glycans, including highly branched N-glycans from chicken ovalbumin, when the aglycone moiety is modified with a 9-fluorenylmethyloxycarbonyl (Fmoc) moiety or in a suitable peptide/protein context, even if they lack the terminal GlcNAc moiety on the Man-α1,3-Man arm. FUT8 could also fucosylate paucimannose structures when they are on glycoprotein substrates. Such core-fucosylated paucimannosylation is a prominent feature of lysosomal proteins of human neutrophils and several types of cancers. We also found that sialylation of N-glycans significantly reduced their activity as substrate of FUT8. Kinetic analysis demonstrated that Fmoc aglycone modification could either improve the turnover rate or decrease the Km value depending on the nature of the substrates, thus significantly enhancing the overall efficiency of FUT8 catalyzed fucosylation. Our results indicate that an appropriate aglycone context of N-glycans could significantly broaden the acceptor substrate specificity of FUT8 beyond what has previously been thought.

FUT8-Directed Core Fucosylation of N-glycans Is Regulated by the Glycan Structure and Protein Environment

“…10 In addition, a recent report also supports our findings that the kinetic parameters are very similar to those of G0 and the G0-peptide, implying that the peptide does not influence the kinetic parameters of the G0peptide versus G0. 21 In short, while the presence of the peptide makes no difference in the kinetic parameters of FUT8 against G0 and the G0-peptide, it is clear that the peptide plays a critical role in the core fucosylation of the M3N2-peptide.…”

“… 10 In addition, a recent report also supports our findings that the kinetic parameters are very similar to those of G0 and the G0-peptide, implying that the peptide does not influence the kinetic parameters of the G0-peptide versus G0. 21 …”

“… 26 This also implies that these two very distant GTs with different structures and donor/acceptor substrates likely share the same sequon and that FUT8 also recognizes additional amino acids beyond the previously proposed result, suggesting that FUT8 might only recognize the Asn residue. 21 …”

“…It was previously suggested that the more the N-glycans are exposed to the solvent, regardless of the type of N-glycan, the more likely they will be core-fucosylated. 21 However, no analysis of the N-glycans in the context of their protein acceptor substrates or MD simulations was performed. The latter calculations allowed us to obtain a more realistic scenario (exemplified by the SASA values) that reflect a more dynamic situation characterized by several conformations of the protein and the glycans, rather than only the conformation found in the crystal structure.…”

Fucosyltransferase 2 inhibitors: Identification via docking and STD-NMR studies

“…The development of FUTs inhibitors is an appropriate strategy to treat various types of cancers. Despite the rigorous efforts for the development of FUT inhibitors, only a limited outcome has been reported [ 13 ]. There are many reasons for this marginal success; the key reasons include the lack of some FUTs crystal structures, complex transition state of FUTs reaction, and low binding affinity for acceptor ligands [ 14 ].…”