Chemical and Chemoenzymatic Synthesis of Glycoproteins for Deciphering Functions

Wang, Lai-Xi; Amin, Md. Ruhul

doi:10.1016/j.chembiol.2014.01.001

Cited by 150 publications

(150 citation statements)

References 153 publications

(184 reference statements)

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“…Thus, methods that can lead to the production of structurally well defined, homogeneous glycoforms of antibodies are highly desirable for both functional studies and for the development of better antibody-based therapeutics. In parallel to the attempt to control glycosylation through host glycosylation pathway engineering (14 -20), a chemoenzymatic glycosylation remodeling method, which involves endoglycosidase-catalyzed deglycosylation and subsequent transglycosylation of intact antibodies, has been emerging as a promising approach to obtain homogeneous antibody glycoforms (21). We have initially shown that the Fc glycans of recombinant IgG-Fc domain could be remodeled through the enzymatic deglycosylation-transglycosylation steps under the catalysis of an appropriate endoglycosidase, including Endo-A 2 and Endo-D, without the need of denaturing the proteins (22)(23)(24).…”

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confidence: 99%

Glycosynthase Mutants of Endoglycosidase S2 Show Potent Transglycosylation Activity and Remarkably Relaxed Substrate Specificity for Antibody Glycosylation Remodeling

Tong

Yang

et al. 2016

Journal of Biological Chemistry

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109

139

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Glycosylation can exert a profound impact on the structures and biological functions of antibodies. Glycosylation remodeling using the endoglycosidase-catalyzed deglycosylation and transglycosylation approach is emerging as a promising platform to produce homogeneous glycoforms of antibodies, but the broad application of this method will require the availability of highly efficient glycosynthase mutants. We describe in this paper a systematic site-directed mutagenesis of an endoglycosidase from Streptococcus pyogenes of serotype M49 (Endo-S2) and the evaluation of the resulting mutants for their hydrolysis and transglycosylation activities. We found that mutations at the Asp-184 residue gave mutants that demonstrated significantly different properties, some possessed potent transglycosylation activity with diminished hydrolysis activity but others did not, which would be otherwise difficult to predict without the comparative study. In contrast to the previously reported Endo-S mutants that are limited to action on complex type N-glycans, the Endo-S2 glycosynthases described here, including D184M and D184Q, were found to have remarkably relaxed substrate specificity and were capable of transferring three major types (complex, high-mannose, and hybrid type) of N-glycans for antibody glycosylation remodeling. In addition, the Endo-S2 glycosynthase mutants were found to be much more active in general than the Endo-S mutants for transglycosylation. The usefulness of these Endo-S2 glycosynthase mutants was exemplified by an efficient glycosylation remodeling of two therapeutic monoclonal antibodies, rituximab and trastuzumab (Herceptin).

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confidence: 99%

Glycosynthase Mutants of Endoglycosidase S2 Show Potent Transglycosylation Activity and Remarkably Relaxed Substrate Specificity for Antibody Glycosylation Remodeling

Tong

Yang

et al. 2016

Journal of Biological Chemistry

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109

139

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“…Recent reviews have highlighted advancements in the study of glycosyl transferases, 75 glycotherapies, 76 and methods for preparing glycoproteins for performing structure-function studies. [77][78][79] Wu and coworkers developed a technology for the evaluation of LacNAc levels in histology sections. Taking advantage of the promiscuity of a1,3-fucosyltransferase in binding modified monosaccharides, the researchers were able to transfer an alkyne-labeled fucose to LacNAc-decorated glycans within formalin-fixed paraffin-embedded tissue samples, which were subsequently detected with a fluorescent click partner.…”

Section: Glycan Remodelingmentioning

confidence: 99%

Recent Advances in Chemoenzymatic Bioconjugation Methods

Rabuka¹

2015

Organic Chemistry Insights

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Genetic fusions of either full enzymes or peptide tags with a protein of interest have enabled the synthesis of protein conjugates with precise control over the site of attachment and number of payloads incorporated. Engineering of protein glycans, depending on the application, can lead to similar control for nonengineered glycoproteins. Recent advances in the field of chemoenzymatic modifications of proteins for site-specific protein conjugation will be reviewed. These techniques have been used in an array of fields for the execution of innovative and valuable experiments. Specific industrial applications of these technologies will be highlighted.KEY WORDS: bioconjugation, antibody drug conjugate, protein tag, transferase, ligase, glycoconjugate, chemoenzymatic CITATION: mcFarland and rabuka. recent advances in chemoenzymatic Bioconjugation methods.

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“…Many elegant chemical and biochemical strategies have been explored for making homogeneous glycoproteins and mimics, including total chemical synthesis with native chemical ligation (13-17), chemoselective ligation (18, 19), chemoenzymatic synthesis (20 -24), and glycosylation pathway engineering in host expression systems (25-28). As part of these efforts, we have attempted to develop a chemoenzymatic method for construction of complex N-glycopeptides and glycoproteins that is based on the transglycosylation activity of a class of endo-␤-N-acetylglucosaminidases (the endoglycosidases that hydrolyze N-glycans of glycoproteins) for convergent native ligation of preassembled glycans and GlcNAc-peptide/ protein (20,23,24). The success of this approach relies on the availability of novel endoglycosidase-derived glycosynthase mutants that are devoid of hydrolysis activity but can use the highly activated glycan oxazolines as substrates for transglycosylation.…”

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confidence: 99%

Endo-F3 Glycosynthase Mutants Enable Chemoenzymatic Synthesis of Core-fucosylated Triantennary Complex Type Glycopeptides and Glycoproteins

Giddens

Lomino

Amin

et al. 2016

Journal of Biological Chemistry

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115

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Chemoenzymatic synthesis is emerging as a promising approach to the synthesis of homogeneous glycopeptides and glycoproteins highly demanded for functional glycomics studies, but its generality relies on the availability of a range of enzymes with high catalytic efficiency and well defined substrate specificity. We describe in this paper the discovery of glycosynthase mutants derived from Elizabethkingia meningoseptica endoglycosidase F3 (Endo-F3) of the GH18 family, which are devoid of the inherent hydrolytic activity but are able to take glycan oxazolines for transglycosylation. Notably, the Endo-F3 D165A and D165Q mutants demonstrated high acceptor substrate specificity toward ␣1,6-fucosyl-GlcNAc-Asn or ␣1, 6-fucosyl-GlcNAc-polypeptide in transglycosylation, enabling a highly convergent synthesis of core-fucosylated, complex CD52 glycopeptide antigen. The Endo-F3 mutants were able to use both bi-and triantennary glycan oxazolines as substrates for transglycosylation, in contrast to previously reported endoglycosidases derived from Endo-S, Endo-M, Endo-D, and Endo-A mutants that could not recognize triantennary N-glycans. Using rituximab as a model system, we have further demonstrated that the Endo-F3 mutants are highly efficient for glycosylation remodeling of monoclonal antibodies to produce homogeneous intact antibody glycoforms. Interestingly, the new triantennary glycan glycoform of antibody showed much higher affinity for galectin-3 than that of the commercial antibody. The Endo-F3 mutants represent the first endoglycosidase-based glycosynthases capable of transferring triantennary complex N-glycans, which would be very useful for glycoprotein synthesis and glycosylation remodeling of antibodies.Protein glycosylation is one of the most prevalent posttranslational modifications, found in almost all living organisms ranging from bacteria to eukaryotes (1). Glycosylation can profoundly affect a protein's intrinsic properties, such as folding, stability, intracellular trafficking, and immunogenicity. In addition, the oligosaccharide components of glycoproteins can participate directly in a number of important biological recognition processes, including cell adhesion, signaling, hostpathogen interactions, and immune responses (2-8). It is well documented that subtle changes in glycosylation can lead to a significant impact on the biological functions and, in the case of therapeutic glycoproteins, such as monoclonal antibodies, the in vivo stability and therapeutic efficacy (7, 9 -12). Natural and recombinant glycoproteins are usually produced as mixtures of glycoforms that differ only in the structures of pendant glycans, from which pure glycoforms are extremely difficult to isolate by current chromatographic techniques. As a result, synthetic homogeneous glycopeptides and glycoproteins emerge as indispensable tools for functional studies and for drug/vaccine discoveries. Many elegant chemical and biochemical strategies have been explored for making homogeneous glycoproteins and mimics, including total chemic...

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Chemical and Chemoenzymatic Synthesis of Glycoproteins for Deciphering Functions

Cited by 150 publications

References 153 publications

Glycosynthase Mutants of Endoglycosidase S2 Show Potent Transglycosylation Activity and Remarkably Relaxed Substrate Specificity for Antibody Glycosylation Remodeling

Glycosynthase Mutants of Endoglycosidase S2 Show Potent Transglycosylation Activity and Remarkably Relaxed Substrate Specificity for Antibody Glycosylation Remodeling

Recent Advances in Chemoenzymatic Bioconjugation Methods

Endo-F3 Glycosynthase Mutants Enable Chemoenzymatic Synthesis of Core-fucosylated Triantennary Complex Type Glycopeptides and Glycoproteins

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