Structural basis of substrate recognition and catalysis by fucosyltransferase 8

Abstract

18Fucosylation of the inner-most N-acetyl-glucosamine (GlcNAc) of N-glycans by fucosyltransferase 8 19 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple 20 modification can have a dramatic impact on the activity and half-life of glycoproteins. These effects 21 are relevant to understanding the invasiveness of some cancers, the development of monoclonal 22 antibody therapeutics, and to a congenital disorder of glycosylation. The acceptor substrate preferences 23 of FUT8 are well characterised and provide a framework for understanding N-glycan maturation in 24 the Golgi, however the structural basis for these substrate preferences and the mechanism through 25 which catalysis is achieved remains unknown. Here, we describe several structures of mouse and 26 human FUT8 in the apo state and in complex with guanosine diphosphate (GDP), a mimic of the donor 27 substrate, and a glycopeptide acceptor substrate. These structures provide insights into: a unique 28 conformational change associated with donor substrate binding; common strategies employed by 29 fucosyltransferases to coordinate GDP; features that define acceptor substrate preferences; and a likely 30 mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also 31 reveal how FUT8 dimerisation plays an important role in defining the acceptor substrate binding site. 32Collectively, this information significantly builds on our understanding of the core-fucosylation 33 process. 34 35 EGFR signalling 1, 14 . These animals also exhibited behavioural abnormalities 15 . Many of these 51 phenotypes are also observed in patients with the recently described FUT8 congenital disorder of 52 glycosylation (CDG-FUT8) 16 . In contrast to CDG-FUT8, which features the ablation of FUT8 activity, 53 many cancers upregulate FUT8 expression and this correlates with a poor 54 prognosis 17 . In melanomas, increased FUT8 activity stabilises L1CAM to promote metastasis 18 . 55Metastasis is also promoted by FUT8 in breast cancers, where increased core-fucosylation of TGFb1R 56 promotes strong constitutive signalling through this receptor and tumour cell migration 19 . The 57 increased core fucosylation of a-fetoprotein is also a well-established biomarker of hepatocellular 58 carcinoma (HCC) 20 . 59Some have speculated that FUT8 antagonists may have therapeutic potential for the treatment 60 of cancer 9,18 , though questions remain around how a hypothetic FUT8 antagonist might impact host 61 immune responses to tumour cells. Regardless, no drug-like small molecule inhibitors have yet been 62 reported for FUT8, or any other human fucosyltransferase (FUT). To some degree, drug discovery 63 efforts are impeded by a limited structural understanding of this enzyme and the mechanism it employs 64to perform core fucosylation. The only reported FUT8 structure possesses no bound ligands, 21 and our 65 only insights into donor and acceptor substrate binding come from STD-NMR, molecular dynamics 66 and docking studies 22, ...

Promiscuity and specificity of eukaryotic glycosyltransferases

“…This is a large recognition motif which occurs on a restricted set of oligosaccharides. The requirement for this recognition motif may be dependent on the dimerization of FUT8, which allows its SH3 domain to form a specific binding pocket [64,65]. FUT8's activity is inhibited by the addition of a bisecting GlcNAc to its recognition motif [53,56], likely due to steric hindrance [64,65] (Figure 3A, bottom).…”

“…The requirement for this recognition motif may be dependent on the dimerization of FUT8, which allows its SH3 domain to form a specific binding pocket [64,65]. FUT8's activity is inhibited by the addition of a bisecting GlcNAc to its recognition motif [53,56], likely due to steric hindrance [64,65] (Figure 3A, bottom). However, FUT8 can fucosylate certain oligosaccharides lacking the α1,3-Man GlcNAc, if they are attached to specific peptides [52,54] (Figure 3A, top).…”