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

Li, Tiezheng; Tong, Xin; Yang, Qiang; Giddens, John P.; Wang, Lai-Xi

doi:10.1074/jbc.m116.738765

Cited by 108 publications

(159 citation statements)

References 49 publications

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“…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).…”

mentioning

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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“…Similar phenomenon with this fucosidase was observed in the glycosylated and deglycosylated Fc domain of IgG antibodies including rituximab (unpublished data). A recent report also showed that the α-fucosidase from Bacteroides fragilis had similar selectivity in defucosylation 3721 . Finally, Endo-A catalyzed transglycosylation of glycoform 9 with the azide-Man3GlcNAc oxazoline gave the novel glycoform 10 .…”

Section: Resultsmentioning

confidence: 90%

“…Utilizing different substrate selectivity of respective endoglycosidases, we and other labs have developed a tool box of endoglycosidases and related glycosynthases for glycoprotein synthesis and glycosylation remodeling 12-15 . These include the glycosynthases derived from Endo-A (from Arthrobacter protophormiae ) 16 , Endo-M (from fungus Mucor hiemalis ) 17,18 , and Endo-D (from bacterium Streptococcus pneumoniae ) 19 of the glycoside hydrolase (GH) family 85 (GH85), as well as those derived from Endo-S (from Streptococcus pyogenes ) 20 , Endo-S2 21 (from Streptococcus pyogenes ), and Endo-F3 (from Elizabethkingia miricola ) 22 of the GH family 18 (GH18). This platform has been especially efficient in synthesis of various glycopeptides and for remodeling of simple glycoproteins such as ribonuclease B.…”

Section: Introductionmentioning

confidence: 99%

“…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%

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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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“…Core fucosylation of the Fc domain N-glycans plays a pivotal role in modulating the functions of antibodies and their therapeutic efficacy 8,74 . We have previously developed a chemoenzymatic Fc glycan remodeling method that consists of deglycosylation and glycosynthase-catalyzed glycan transfer to produce homogeneous Fc glycoforms 65,66,75 . If the fucoligase mutant could act on intact antibody, then it would further expand the repertoire of the toolbox for antibody Fc glycan engineering.…”

Section: Resultsmentioning

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

Cited by 108 publications

References 49 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

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

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