Substrate Preference and Interplay of Fucosyltransferase 8 and <i>N</i>-Acetylglucosaminyltransferases

Tseng, Tzu-Hao; Lin, Ting‐Yi; Chen, Chien‐Yu; Chen, Chein‐Hung; Lin, Jung-Lee; Hsu, Tsui-Ling; Wong, Chi‐Huey

doi:10.1021/jacs.7b03729

Cited by 43 publications

(39 citation statements)

References 26 publications

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“…Two previous studies have explored the acceptor substrate specificity of FUT8 to ascertain what features promote or impair FUT8 activity 31,32 and these results are summarised in Figure 3D. It is clear through comparison to our structure that a bisecting GlcNAc would be sterically occluded by the SH3 domain of FUT8 ( Figure 3A), consistent with this modification's ability to block corefucosylation 31,32 . Modifications of the a6branch of the glycan are well-tolerated by FUT8, and it is clear from our structure that there is sufficient space to accommodate most types of truncation, elongation or branching at this position.…”

Section: Structural Insights Into N-glycan Acceptor Substrate Recognisupporting

confidence: 77%

Structural basis of substrate recognition and catalysis by fucosyltransferase 8

Järvå

Dramićanin

Lingford

et al. 2020

Journal of Biological Chemistry

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Fucosylation of the inner-most Nacetylglucosamine (GlcNAc) of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can dramatically affect the activities and half-lives of glycoproteins, effects that are relevant to understanding the invasiveness of some cancers, the development of monoclonal antibody therapeutics, and the etiology of a congenital glycosylation disorder. The acceptor substrate preferences of FUT8 are well characterized and provide a framework for understanding N-glycan maturation in the Golgi; however the structural basis for these substrate preferences and the mechanism through which catalysis is achieved remain unknown. Here, we describe several structures of mouse and human FUT8 in the apo state and in complex with guanosine diphosphate (GDP), a mimic of the donor substrate, and with a glycopeptide acceptor substrate at 1.80-2.50 Å resolutions. These structures provide insights into a unique conformational change associated with donor substrate binding, common strategies employed by fucosyltransferases to coordinate GDP, features that define acceptor substrate preferences, and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also revealed how FUT8 dimerization plays an important role in defining the acceptor substrate-binding site. Collectively, this information significantly builds on our understanding of the core fucosylation process. https://www.jbc.org/cgi/

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Section: Structural Insights Into N-glycan Acceptor Substrate Recognisupporting

confidence: 77%

Structural basis of substrate recognition and catalysis by fucosyltransferase 8

Järvå

Dramićanin

Lingford

et al. 2020

Journal of Biological Chemistry

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“…(C) A close up of the active site illustrating a potential role for E373 and K369 as a proton relay to facilitate electrophilic migration of fucose from GDP-Fuc to the 6-hydroxyl group of the innermost GlcNAc. (D) A selection of N-glycans known to be modified or not modified by FUT8 31, 32 , for comparison to the structure depicted in (A).…”

Section: Resultsmentioning

confidence: 99%

“…Two previous studies have explored the acceptor substrate specificity of FUT8 to ascertain what features promote or impair FUT8 activity 31, 32 and these results are summarised in Figure 3D . It is clear through comparison to our structure that a bisecting GlcNAc would be sterically occluded by the SH3 domain of FUT8 ( Figure 3A ), consistent with this modification’s ability to block core-fucosylation 31, 32 . Modifications of the α6-branch of the glycan are well-tolerated by FUT8, and it is clear from our structure that there is sufficient space to accommodate most types of truncation, elongation or branching at this position.…”

Section: Resultsmentioning

confidence: 99%

“…With core-fucosylation playing such an important role in the function of proteins and the maturation of N-glycans, a great deal of effort has been invested in profiling the acceptor substrate preferences of this enzyme. Our structures provide a basis for understanding the vagaries of FUT8 substrate preference 31, 32 . What is clear from this work is that the SH3 domain of FUT8 plays a defining role in recognising N-glycan acceptor substrates.…”

Section: Discussionmentioning

confidence: 99%

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Structural basis of substrate recognition and catalysis by fucosyltransferase 8

Järvå¹,

Dramićanin²,

Lingford³

et al. 2020

Preprint

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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, ...

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“…In animals and humans, core-fucosylation is catalyzed solely by the mammalian α1,6-fucosyltransferase, FUT8 27,28 . However, FUT8 has a very strict substrate specificity, requires the presence of a free GlcNAc at the α1,3-linked mannose arm in the N-glycan as the substrate and usually is unable to fucosylate full-size mature N-glycans 29–32 . Only until recently we have provided the first examples showing that FUT8 could catalyze in vitro fucosylation of some high-mannose N-glycans lacking a free GlcNAc at the α1,3-linked mannose arm when the glycan is present in an appropriate protein or other context 33 .…”

Section: Introductionmentioning

confidence: 99%

Designer α1,6-Fucosidase Mutants Enable Direct Core Fucosylation of Intact N-Glycopeptides and N-Glycoproteins

Zhu

Christopher

et al. 2017

J. Am. Chem. Soc.

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Core fucosylation of N-glycoproteins plays a crucial role in modulating the biological functions of glycoproteins. Yet, the synthesis of structurally well-defined, core-fucosylated glycoproteins remains a challenging task due to the complexity in multi-step chemical synthesis or the inability of the biosynthetic α1,6-fucosyltransferase (FUT8) to directly fucosylate full-size mature N-glycans in a chemoenzymatic approach. We report in this paper the design and generation of potential α1,6-fucosynthase and fucoligase for direct core-fucosylation of intact N-glycoproteins. We found that mutation at the nucleophilic residue (D200) did not provide a typical glycosynthase from this bacterial enzyme, but several mutants with mutation at the general acid/base residue E274 of the Lactobacillus casei α1,6-fucosidase, including E274A, E274S, and E274G, acted as efficient glycoligases that could fucosylate a wide variety of complex N-glycopeptides and intact glycoproteins by using α-fucosyl fluoride as a simple donor substrate. Studies on the substrate specificity revealed that the α1,6-fucosidase mutants could introduce an α1,6-fucose moiety specifically at the Asn-linked GlcNAc moiety not only to GlcNAc-peptide, but also to high-mannose and complex type N-glycans in the context of N-glycopeptides, N-glycoproteins, and intact antibodies. This discovery opens a new avenue to a wide variety of homogeneous, core-fucosylated N-glycopeptides and N-glycoproteins that are hitherto difficult to obtain for structural and functional studies.

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Substrate Preference and Interplay of Fucosyltransferase 8 and N-Acetylglucosaminyltransferases

Cited by 43 publications

References 26 publications

Structural basis of substrate recognition and catalysis by fucosyltransferase 8

Structural basis of substrate recognition and catalysis by fucosyltransferase 8

Structural basis of substrate recognition and catalysis by fucosyltransferase 8

Designer α1,6-Fucosidase Mutants Enable Direct Core Fucosylation of Intact N-Glycopeptides and N-Glycoproteins

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