Expression system for structural and functional studies of human glycosylation enzymes

Moremen, Kelley W.; Ramiah, Annapoorani; Stuart, Melissa A.; Steel, Jason C.; Li, Meng; Forouhar, F.; Moniz, Heather; Gahlay, Gagandeep Kaur; Gao, Zhongwei; Chapla, Digantkumar; Wang, S.; Yang, Jeong‐Yeh; Prabhakar, Pradeep Kumar; Johnson, Roy W.; Rosa, Nelson Souto; Geisler, Christoph; Nairn, Alison V.; Seetharaman, J.; Wu, Shuangxiu; Tong, Liang; Gilbert, Harry J.; LaBaer, Joshua; Jarvis, Donald L.

doi:10.1038/nchembio.2539

Cited by 201 publications

(233 citation statements)

References 62 publications

(98 reference statements)

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“…The DNA sequence encoding amino acid residues 68-575 of the HsFUT8 was codon optimized and synthesized by Gen-Script (USA) for expression in HEK293 cells (see Supplementary Table 2 for the codon optimized sequence). The DNA, containing at the 5′-end a recognition sequence for KpnI, and at the 3′ end a stop codon and a recognition sequence for XhoI, was cloned into a modified pHLSec containing after the secretion signal sequence a 12xHis tag, a superfolder GFP 35 and a Tobacco Etch Virus (TEV) cleavage site, rendering the vector pHLSec-12His-GFP-TEV-HsFUT8. Both the synthesis of the HsFUT8 construct and the engineered pHLSec together with the cloning of HsFUT8 into pHLSec-12His-GFP-TEV were performed by GenScript.…”

Section: Discussionmentioning

confidence: 99%

Structural basis for substrate specificity and catalysis of α1,6-fucosyltransferase

et al. 2020

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Core-fucosylation is an essential biological modification by which a fucose is transferred from GDP-β-L-fucose to the innermost N-acetylglucosamine residue of N-linked glycans. A single human enzyme α1,6-fucosyltransferase (FUT8) is the only enzyme responsible for this modification via the addition of an α-1,6-linked fucose to N-glycans. To date, the details of substrate recognition and catalysis by FUT8 remain unknown. Here, we report the crystal structure of FUT8 complexed with GDP and a biantennary complex N-glycan (G0), which provides insight into both substrate recognition and catalysis. FUT8 follows an S N 2 mechanism and deploys a series of loops and an α-helix which all contribute in forming the binding site. An exosite, formed by one of these loops and an SH3 domain, is responsible for the recognition of branched sugars, making contacts specifically to the α1,3 arm GlcNAc, a feature required for catalysis. This information serves as a framework for inhibitor design, and helps to assess its potential as a therapeutic target.

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Section: Discussionmentioning

confidence: 99%

Structural basis for substrate specificity and catalysis of α1,6-fucosyltransferase

et al. 2020

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“…Soluble GalNAcT-1, T-2 and T-10 enzymes were expressed according to a published procedure (6). Soluble GalNAcT-7 was expressed as a fusion construct with superfolder GFP in pGEn2-DEST (a kind gift from Kelley Moremen, University of Goergia, Athens, USA) in HEK293-F cells according to the manufacturer’s instructions (Thermo) (7). Briefly, a 30 mL culture was transfected with 293Fectin (Thermo) according to the manufacturer’s instructions.…”

Section: Supporting Informationmentioning

confidence: 99%

Metabolic precision labeling enables selective probing of O-linkedN-acetylgalactosamine glycosylation

Debets¹,

Tastan

Wisnovsky³

et al. 2020

Preprint

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38Protein glycosylation events that happen early in the secretory pathway are often dysregulated during 39 tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that 40 would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other 41 monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based 42 design process to develop the monosaccharide probe GalNAzMe that is specific for cancer-relevant 43

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“…Cell-surface glycan structures are not template-derived. Instead, they are the products of complex metabolic pathways where biosynthetic enzymes encode the regiospecific branching and extension of mature glycan structures (2,3). Understanding the details of substrate recognition, catalytic mechanisms, and regulation of these enzymes is key to understanding glycan diversity and their roles in human disease.…”

mentioning

confidence: 99%

Human N -acetylglucosaminyltransferase II substrate recognition uses a modular architecture that includes a convergent exosite

Kadirvelraj

Yang

Sanders

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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Asn-linked oligosaccharides are extensively modified during transit through the secretory pathway, first by trimming of the nascent glycan chains and subsequently by initiating and extending multiple oligosaccharide branches from the trimannosyl glycan core. Trimming and branching pathway steps are highly ordered and hierarchal based on the precise substrate specificities of the individual biosynthetic enzymes. A key committed step in the synthesis of complex-type glycans is catalyzed by -acetylglucosaminyltransferase II (MGAT2), an enzyme that generates the second GlcNAcβ1,2- branch from the trimannosyl glycan core using UDP-GlcNAc as the sugar donor. We determined the structure of human MGAT2 as a Mn-UDP donor analog complex and as a GlcNAcManGlcNAc-Asn acceptor complex to reveal the structural basis for substrate recognition and catalysis. The enzyme exhibits a GT-A Rossmann-like fold that employs conserved divalent cation-dependent substrate interactions with the UDP-GlcNAc donor. MGAT2 interactions with the extended glycan acceptor are distinct from other related glycosyltransferases. These interactions are composed of a catalytic subsite that binds the Man-α1,6- monosaccharide acceptor and a distal exosite pocket that binds the GlcNAc-β1,2Man-α1,3Manβ- substrate "recognition arm." Recognition arm interactions are similar to the enzyme-substrate interactions for Golgi α-mannosidase II, a glycoside hydrolase that acts just before MGAT2 in the Asn-linked glycan biosynthetic pathway. These data suggest that substrate binding by MGAT2 employs both conserved and convergent catalytic subsite modules to provide substrate selectivity and catalysis. More broadly, the MGAT2 active-site architecture demonstrates how glycosyltransferases create complementary modular templates for regiospecific extension of glycan structures in mammalian cells.

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Expression system for structural and functional studies of human glycosylation enzymes

Cited by 201 publications

References 62 publications

Structural basis for substrate specificity and catalysis of α1,6-fucosyltransferase

Structural basis for substrate specificity and catalysis of α1,6-fucosyltransferase

Metabolic precision labeling enables selective probing of O-linkedN-acetylgalactosamine glycosylation

Human N -acetylglucosaminyltransferase II substrate recognition uses a modular architecture that includes a convergent exosite

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