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

Abstract

FUT8 is an essential α-1,6-fucosyltransferase that fucosylates the innermost GlcNAc of N-glycans, a process called core fucosylation. In vitro , FUT8 exhibits substrate preference for the biantennary complex N-glycan oligosaccharide (G0), but the role of the underlying protein/peptide to which N-glycans are attached remains unclear. Here, we explored the FUT8 enzyme with a series of N-glycan oligosaccharides, N-glycopeptides, and an Asn-linked oligosaccharide. We found that the underlying peptide plays a role in fucosylation of paucimannose (low mannose) and high-mannose N-glycans but not for complex-type N-glycans. Using saturation transfer difference (STD) NMR spectroscopy, we demonstrate that FUT8 recognizes all sugar units of the G0 N-glycan and most of the amino acid residues (Asn-X-Thr) that serve as a recognition sequon for the oligosaccharyltransferase (OST). The largest STD signals were observed in the presence of GDP, suggesting that prior FUT8 binding to GDP-β- l -fucose (GDP-Fuc) is required for an optimal recognition of N-glycans. We applied genetic engineering of glycosylation capacities in CHO cells to evaluate FUT8 core fucosylation of high-mannose and complex-type N-glycans in cells with a panel of well-characterized therapeutic N-glycoproteins. This confirmed that core fucosylation mainly occurs on complex-type N-glycans, although clearly only at selected glycosites. Eliminating the capacity for complex-type glycosylation in cells (KO mgat1 ) revealed that glycosites with complex-type N-glycans when converted to high mannose lost the core Fuc. Interestingly, however, for erythropoietin that is uncommon among the tested glycoproteins in efficiently acquiring tetra-antennary N-glycans, two out of three N-glycosites obtained Fuc on the high-mannose N-glycans. An examination of the N-glycosylation sites of several protein crystal structures indicates that core fucosylation is mostly affected by the accessibility and nature of the N-glycan and not by the nature of the underlying peptide sequence. These data have further elucidated the different FUT8 acceptor substrate specificities both in vitro and in vivo in cells, revealing different mechanisms for promoting core fucosylation.

Egg yolk sialylglycopeptide: purification, isolation and characterization of N-glycans from minor glycopeptide species

“…21 Normally during SGP purification the monoantennary glycopeptide is removed by size exclusion chromatography 22 or digested enzymatically to afford a homogenous albeit degalactosylated glycan. 23,24 Despite previous evidence for the presence of the monosialylated asymmetric glycan in egg yolk, recent publications identify it as a mixture. 25 Having an alternative source of asymmetric glycans would be of interest since they are difficult to synthesize and there is growing awareness of the biological implications of N-glycan asymmetry such as the complement-dependent cytotoxicity of monoclonal antibodies, 26 and influenza haemagglutinin binding.…”

“…21 Normally during SGP purification the monoantennary glycopeptide is removed by size exclusion chromatography 22 or digested enzymatically to afford a homogenous albeit de-galactosylated glycan. 23,24…”

Structural basis for the synthesis of the core 1 structure by C1GalT1

“…Interestingly, in the case of GalNAc-T2, the active conformation of GalNAc-T2, characterized by the shifting of a flexible loop from an open to a closed conformation, was completely achieved in the presence of UDP-GalNAc and less in the presence of UDP 20 . We also found that the GT-B fold FUT8 showed similar properties to the other two GTs, though FUT8 bound better to an N-glycan in the presence of GDP, and the nucleotide was not essential for binding to the N-glycan 37 , 38 . In addition, NleB/SseK and FUT8 also contained flexible loops that were ordered in the presence of the nucleotide as we found for GalNAc-T2, implying that the active site adopted an active conformation once that these flexible loops bound to the sugar nucleotide 20 , 35 , 36 , 38 .…”

Sequential in vitro enzymatic N-glycoprotein modification reveals site-specific rates of glycoenzyme processing

“…Core fucosylation is the attachment of an ⍺1,6-linked fucose to the GlcNAc that is directly attached to the asparagine at the core of N-linked glycans, a reaction which is catalyzed by the fucosyltransferase FUT8. This reaction is generally specific to complex N-glycans ( 44 ), and thus, we generated GlcNAc 2 Man 3 GlcNAc 2 glycans on our collection of reporter proteins by reacting with an excess of MGAT1, MAN2A1, and MGAT2 in the presence of the UDP-GlcNAc sugar donor. We then examined the rates of modification of the respective glycans with FUT8 ( Fig.…”