One‐pot Multienzyme (OPME) Chemoenzymatic Synthesis of Brain Ganglioside Glycans with Human ST3GAL II Expressed in <i>E. coli</i>

Yang, Xiaoxiao; Yu, Hai; Yang, Xiaohong; Kooner, Anoopjit Singh; Yuan, Yue; Luu, Bryant; Chen, Xi

doi:10.1002/cctc.202101498

Cited by 8 publications

(10 citation statements)

References 60 publications

(91 reference statements)

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“…To facilitate the purification of enzymes that will be used for glycoprotein in vitro N-glycan processing, an His 6 -tag was introduced at the C-terminus of each recombinant enzyme to allow its easy purification by Ni 2+ -affinity columns. Furthermore, we found that fusing a maltose binding protein (MBP) at the N-terminus of the target recombinant protein and removing the N-terminal transmembrane domain of mammalian glycosyltransferases by truncation worked well to improve their soluble expression in E. coli [ 47 ]. These were the strategies that guided our general design to construct the plasmids for expressing target recombinant enzymes.…”

Section: Resultsmentioning

confidence: 99%

Glycoprotein In Vitro N-Glycan Processing Using Enzymes Expressed in E. coli

Zhang

et al. 2023

Molecules

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Protein N-glycosylation is a common post-translational modification that plays significant roles on the structure, property, and function of glycoproteins. Due to N-glycan heterogeneity of naturally occurring glycoproteins, the functions of specific N-glycans on a particular glycoprotein are not always clear. Glycoprotein in vitro N-glycan engineering using purified recombinant enzymes is an attractive strategy to produce glycoproteins with homogeneous N-glycoforms to elucidate the specific functions of N-glycans and develop better glycoprotein therapeutics. Toward this goal, we have successfully expressed in E. coli glycoside hydrolases and glycosyltransferases from bacterial and human origins and developed a robust enzymatic platform for in vitro processing glycoprotein N-glycans from high-mannose-type to α2–6- or α2–3-disialylated biantennary complex type. The recombinant enzymes are highly efficient in step-wise or one-pot reactions. The platform can find broad applications in N-glycan engineering of therapeutic glycoproteins.

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Section: Resultsmentioning

confidence: 99%

Glycoprotein In Vitro N-Glycan Processing Using Enzymes Expressed in E. coli

Zhang

et al. 2023

Molecules

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“…Haemophilus ducreyi vST3Gal-I 59 (GT29) myxoma virus-infected European rabbit kidney RK13 cells hST3GAL-II 42 (GT29) human CjCst-I 41,44 (GT42) Campylobacter jejuni Sialyltransferases and mutants for synthesizing α2−6-linked sialosides Pd2,6ST 11,60,61 Campylobacter jejuni Sialic acid aldolases EcAldolase 70 Escherichia coli PmAldolase 71 Pasteurella multocida Crystal structures 72 CMP-Nonulosonic acid synthetases NmCSS 70 Neisseria meningitidis NmCSS_S81R and NmCSS_Q163A 73 Crystal structures 74 EcCSS 70 Escherichia coli SaVCSS 70 Streptococcus agalactiae PmCSS 73 Pasteurella multocida HdCSS 73 Haemophilus ducreyi LpCLS 75 Legionella pneumophila 58 although it was capable of α2−3-sialylating the unbranched Core 1 T antigen (a β1−3-linked galactoside) on glycopeptides. 43 Human ST3GAL-II uses β1−3-linked galactosides as acceptor substrates efficiently for the high-yield synthesis of the glycans of gangliosides GT1b, GD1a, and GM1b.…”

Section: (Gt80)mentioning

confidence: 99%

Enabling Chemoenzymatic Strategies and Enzymes for Synthesizing Sialyl Glycans and Sialyl Glycoconjugates

Chen

2023

Acc. Chem. Res.

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Metrics & MoreArticle Recommendations CONSPECTUS: Sialic acids are fascinating negatively charged nine-carbon monosaccharides. Sialic acid-containing glycans and glycoconjugates are structurally diverse, functionally important, and synthetically challenging molecules. We have developed highly efficient chemoenzymatic strategies that combine the power of chemical synthesis and enzyme catalysis to make sialic acids, sialyl glycans, sialyl glycoconjugates, and their derivatives more accessible, enabling the efforts to explore their functions and applications. The Account starts with a brief description of the structural diversity and the functional importance of naturally occurring sialic acids and sialosides. The development of one-pot multienzyme (OPME) chemoenzymatic sialylation strategies is then introduced, highlighting its advantages in synthesizing structurally diverse sialosides with a sialyltransferase donor substrate engineering tactic. With the strategy, systematic access to sialosides containing different sialic acid forms with modifications at C3/4/ 5/7/8/9, various internal glycans, and diverse sialyl linkages is now possible. Also briefly described is the combination of the OPME sialylation strategy with bacterial sialidases for synthesizing sialidase inhibitors. With the goal of simplifying the product purification process for enzymatic glycosylation reactions, glycosphingolipids that contain a naturally existing hydrophobic tag are attractive targets for chemoenzymatic total synthesis. A user-friendly highly efficient chemoenzymatic strategy is developed which involves three main processes, including chemical synthesis of lactosyl sphingosine as a water-soluble hydrophobic tag-containing intermediate, OPME enzymatic extension of its glycan component with a single C18-cartridge purification of the product, followed by a facile chemical acylation reaction. The strategy allows the introduction of different sialic acid forms and diverse fatty acyl chains into the products. Gram-scale synthesis has been demonstrated. OPME sialylation has also been demonstrated for the chemoenzymatic synthesis of sialyl glycopeptides and in vitro enzymatic N-glycan processing for the formation of glycoproteins with disialylated biantennary complex-type N-glycans. For synthesizing human milk oligosaccharides (HMOs) which are glycans with a free reducing end, acceptor substrate engineering and process engineering strategies are developed, which involve the design of a hydrophobic tag that can be easily installed into the acceptor substrate to allow facile purification of the product from enzymatic reactions and can be conveniently removed in the final step to produce target molecules. The process engineering involves heatinactivation of enzymes in the intermediate steps in multistep OPME reactions for the production of long-chain sialoside targets in a single reaction pot and with a single C18-cartridge purification process. In addition, a chemoenzymatic synthon strategy has been developed. It involves the design of a deriv...

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“…Depending on the buffer, pH, and temperature, sugar phosphates and NDP-sugars can decompose quickly within the time span relevant for enzymatic synthesis. Therefore, their formation in situ from the corresponding reducing sugar has become increasingly popular. − Partially driven by their high commercial value, the advent of one-pot multienzyme approaches in the early 1990s has led to simplified access to both natural and a limited range of unnatural NDP-sugars, alongside enabling improved characterization of glycosyltransferases. This section therefore focuses only on recent advancements in one-pot, biosynthetic routes to NDP-sugars, particularly around the regeneration of expensive cofactors, which are often required stoichiometrically.…”

Section: Using Biocatalysis To Provide the Building Blocks For Bioche...mentioning

confidence: 99%

Biocatalytic Approaches to Building Blocks for Enzymatic and Chemical Glycan Synthesis

Dolan

Cosgrove

Miller

2022

JACS Au

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While the field of biocatalysis has bloomed over the past 20−30 years, advances in the understanding and improvement of carbohydrate-active enzymes, in particular, the sugar nucleotides involved in glycan building block biosynthesis, have progressed relatively more slowly. This perspective highlights the need for further insight into substrate promiscuity and the use of biocatalysis fundamentals (rational design, directed evolution, immobilization) to expand substrate scopes toward such carbohydrate building block syntheses and/or to improve enzyme stability, kinetics, or turnover. Further, it explores the growing premise of using biocatalysis to provide simple, cost-effective access to stereochemically defined carbohydrate materials, which can undergo late-stage chemical functionalization or automated glycan synthesis/polymerization.

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One‐pot Multienzyme (OPME) Chemoenzymatic Synthesis of Brain Ganglioside Glycans with Human ST3GAL II Expressed in E. coli

Cited by 8 publications

References 60 publications

Glycoprotein In Vitro N-Glycan Processing Using Enzymes Expressed in E. coli

Glycoprotein In Vitro N-Glycan Processing Using Enzymes Expressed in E. coli

Enabling Chemoenzymatic Strategies and Enzymes for Synthesizing Sialyl Glycans and Sialyl Glycoconjugates

Biocatalytic Approaches to Building Blocks for Enzymatic and Chemical Glycan Synthesis

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