Optimal Synthetic Glycosylation of a Therapeutic Antibody

Parsons, Thomas B.; Struwe, Weston B.; Gault, Joseph; Yamamoto, Keisuke; Taylor, Thomas A.; Raj, Ritu; Wals, Kim; Mohammed, Shabaz; Robinson, Carol V.; Benesch, Justin L. P.; Davis, Benjamin G.

doi:10.1002/ange.201508723

Cited by 28 publications

(20 citation statements)

References 57 publications

(44 reference statements)

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“…Recently, Endo-F3 was converted to a glycosynthase, and the mutant enzyme was capable of transferring bi-and triantennary complex type N-glycans using sugar oxazoline donor substrates to synthesize core-fucosylated complex glycopeptides (42). Endo-S and its glycosynthase, which specifically and efficiently acts on the IgG-Fc domain of N-glycans, have been used for chemoenzymatic synthesis of IgGs with structurally defined glycoforms for functional studies (43)(44)(45). More recently, Endo-S2 was shown to have a more flexible substrate specificity and high efficiency in transferring complex, hybrid, and high mannose-type N-glycans onto corefucosylated or non-fucosylated IgG molecules (46).…”

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confidence: 99%

Generation of a Mutant Mucor hiemalis Endoglycosidase That Acts on Core-fucosylated N-Glycans

Katoh

Katayama

Tomabechi

et al. 2016

Journal of Biological Chemistry

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Endo-␤-N-acetylglucosaminidase M (Endo-M), an endoglycosidase from the fungus Mucor hiemalis, is a useful tool for chemoenzymatic synthesis of glycoconjugates, including glycoprotein-based therapeutics having a precisely defined glycoform, by virtue of its transglycosylation activity. Although Endo-M has been known to act on various N-glycans, it does not act on core-fucosylated N-glycans, which exist widely in mammalian glycoproteins, thus limiting its application. Therefore, we performed site-directed mutagenesis on Endo-M to isolate mutant enzymes that are able to act on mammalian-type core-␣1,6-fucosylated glycans. Among the Endo-M mutant enzymes generated, those in which the tryptophan at position 251 was substituted with alanine or asparagine showed altered substrate specificities. Such mutant enzymes exhibited increased hydrolysis of a synthetic ␣1,6-fucosylated trimannosyl core structure, whereas their activity on the afucosylated form decreased. In addition, among the Trp-251 mutants, the W251N mutant was most efficient in hydrolyzing the core-fucosylated substrate. W251N mutants could act on the immunoglobulin G-derived core-fucosylated glycopeptides and human lactoferrin glycoproteins. This mutant was also capable of transferring the sialyl glycan from an activated substrate intermediate (sialyl glycooxazoline) onto an ␣1,6-fucosyl-N-acetylglucosaminyl biotin. Furthermore, the W251N mutant gained a glycosynthase-like activity when a N175Q substitution was introduced and it caused accumulation of the transglycosylation products. These findings not only give insights into the substrate recognition mechanism of glycoside hydrolase family 85 enzymes but also widen their scope of application in preparing homogeneous glycoforms of core-fucosylated glycoproteins for the production of potent glycoprotein-based therapeutics.Endo-␤-N-acetylglucosaminidases (ENGases, 3 EC 3.2.1.96) are enzymes that hydrolyze ␤-1,4 glycosidic linkages within the N,NЈ-diacetylchitobiose moiety in the N-glycan core structure and liberate a large part of the glycan, thus leaving the innermost N-acetylglucosamine residue (often attached to the core fucose) on the aglycon. These enzymes are distributed in a wide range of organisms, from bacteria living in various ecological niches to higher eukaryotes, including humans. In eukaryotes, the endoglycosidase activity of this enzyme is thought to be involved in the processing of free oligosaccharides in the cytosol (1-5), and in bacterial species, they may play roles in the acquisition of carbohydrates as energy sources from the environment (6, 7) or compromise the host defense system, contributing to the virulence of pathogenic bacteria (7-9).There are two main classes of these endoglycosidases based on their amino acid sequences, within the carbohydrate-active enzymes (CAZy) database: glycoside hydrolase (GH) families GH18 and GH85. GH18 endoglycosidases, including the enzymes used frequently in glycoprotein analysis, such as Endo-H from Streptomyces plicatus (10), Endo-F1, -F2, and -F3 ...

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confidence: 99%

Generation of a Mutant Mucor hiemalis Endoglycosidase That Acts on Core-fucosylated N-Glycans

Katoh

Katayama

Tomabechi

et al. 2016

Journal of Biological Chemistry

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“…This platform has been especially efficient in synthesis of various glycopeptides and for remodeling of simple glycoproteins such as ribonuclease B. In addition, it has also been successfully applied to the glycan remodeling of therapeutic IgG antibodies due to the discovery of the antibody-specific glycosynthases derived from Endo-S and Endo-S2 20,21,23-26 . Nevertheless, the application of this method for producing homogeneous glycoproteins with more complex glycosylation patterns remained to be tested.…”

Section: Introductionmentioning

confidence: 99%

Glycan Remodeling of Human Erythropoietin (EPO) Through Combined Mammalian Cell Engineering and Chemoenzymatic Transglycosylation

Yang

Zhu

et al. 2017

ACS Chem. Biol.

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The tremendous structural heterogeneity of N-glycosylation of glycoproteins poses a great challenge for deciphering the biological functions of specific glycoforms and for developing protein-based therapeutics. We have previously reported a chemoenzymatic glycan remodeling method for producing homogeneous glycoforms of N-glycoproteins including intact antibodies, which consist of endoglycosidase-catalyzed deglycosylation and novel glycosynthase-catalyzed transglycosylation, but its application to complex glycoproteins carrying multiple N-glycans remains to be examined. We report here site-selective chemoenzymatic glycosylation remodeling of recombinant human erythropoietin (EPO) that contains three N-glycans. We found that the generation of a HEK293S GnT I knockout FUT8 overexpressing cell line enabled the production of an unusual Man5GlcNAc2Fuc glycoform, which could be converted to the core-fucosylated GlcNAc-EPO intermediate acceptor for enzymatic transglycosylation. With this acceptor, homogeneous sialylated glycoform or azide-tagged glycoform were produced using the glycosynthase (EndoF3-D165A) catalyzed transglycosylation. Interestingly, a remarkable site-selectivity was observed in the transglycosylation reactions, leading to the introduction of two N-glycans selectively at the Asn-38 and Asn-83 sites, which was confirmed by a detailed MS/MS analysis of the transglycosylation product. Finally, a different N-glycan was attached at the third (Asn-24) site by pushing the enzymatic transglycosylation with a distinct glycan oxazoline, achieving the site-selective glycosylation modification of the protein. This study represents the first example of site-selective chemoenzymatic glycan engineering of complex glycoproteins carrying multiple N-glycans.

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“…Preparation of homogenous glycoproteins still poses a great challenge for the functional study of glycoproteins. To address this issue, our group and others have established an efficient chemoenzymatic approach to glycan remodeling of N-glycoproteins (Parsons et al, 2016; Wang & Amin, 2014). The approach exploits a glycosynthase to transfer a sugar oxazoline that mimics the transition state of glycan hydrolysis of the GlcNAc residue of a peptide or protein acceptor.…”

Section: Introductionmentioning

confidence: 99%

“… 7 Occasionally non-specific transfer of glycan oxazoline to a position other than GlcNAc may occur (Parsons et al, 2016) (Huang, Yang, Umekawa, Yamamoto, & Wang, 2010). To exclude this undesired possibility, the final product is examined after treatment with PNGase F. As illustrated in Fig.…”

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confidence: 99%

Chemoenzymatic Glycan Remodeling of Natural and Recombinant Glycoproteins

Yang

Wang

2017

Methods in Enzymology

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N-glycosylation plays important roles in modulating the biological functions of glycoproteins, such as protein folding, stability, and immunogenicity. However, acquiring homogeneous glycoforms of glycoproteins has been a challenging task for functional studies and therapeutic applications. In this chapter, we describe an efficient chemoenzymatic glycan remodeling protocol for making homogeneous glycoproteins that involves enzymatic deglycosylation and subsequent reglycosylation procedures. Two therapeutic glycoproteins, Herceptin (trastuzumab, a therapeutic monoclonal antibody) and erythropoietin (EPO, a glycoprotein hormone) were chosen as the model systems. The detailed protocol includes the deglycosylation of the Herceptin or EPO with a wild type endo-β-N-acetylglucosaminidase, to remove the heterogeneous N-glycans, leading to the GlcNAc-protein or Fucα1,6GlcNAc-protein intermediate. Then desired homogeneous N-glycans are attached to the acceptor by using an activated sugar oxazoline as the donor substrate and a specific glycosynthase (mutant of endoglycosidase) as the catalyst to reconstitute a homogeneous glycoform. Using this approach, Herceptin was remodeled to an afucosylated complex glycoform and a Man9GlcNAc2 glycoform, with the former showing significantly enhanced antibody-dependent cellular cytotoxicity. EPO was engineered to carry azide-tagged Man3GlcNAc2 glycans that could be further modified via click chemistry to introduce other functional groups.

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Optimal Synthetic Glycosylation of a Therapeutic Antibody

Cited by 28 publications

References 57 publications

Generation of a Mutant Mucor hiemalis Endoglycosidase That Acts on Core-fucosylated N-Glycans

Generation of a Mutant Mucor hiemalis Endoglycosidase That Acts on Core-fucosylated N-Glycans

Glycan Remodeling of Human Erythropoietin (EPO) Through Combined Mammalian Cell Engineering and Chemoenzymatic Transglycosylation

Chemoenzymatic Glycan Remodeling of Natural and Recombinant Glycoproteins

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