Mutants of Mucor hiemalis Endo-β-N-acetylglucosaminidase Show Enhanced Transglycosylation and Glycosynthase-like Activities

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100

137

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).

Section: Resultsmentioning

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

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100

137

“…However, based on sequence alignments of known GH85 enzymes, there is no obvious carboxylate residue that might serve to enhance the nucleophilicity of the acetamido group. Very recently, Yamamoto and co-workers (29) have proposed that a conserved asparagine fulfills the same function because they observed that deletion of the side chain by mutagenesis resulted in a dramatic loss of activity. Interestingly, endohexosaminidases, in which either of these residues are mutated in a fairly conservative manner, are still able to catalyze the second step of the reaction, using oxazoline substrates as donor sugars and transferring the saccharide to a suitable acceptor (29,30).…”

mentioning

confidence: 99%

“…Very recently, Yamamoto and co-workers (29) have proposed that a conserved asparagine fulfills the same function because they observed that deletion of the side chain by mutagenesis resulted in a dramatic loss of activity. Interestingly, endohexosaminidases, in which either of these residues are mutated in a fairly conservative manner, are still able to catalyze the second step of the reaction, using oxazoline substrates as donor sugars and transferring the saccharide to a suitable acceptor (29,30). On the basis of these results, Yamamoto and co-workers (11,29) have proposed that these enzymes use substrate-assisted catalysis.…”

mentioning

confidence: 99%

Streptococcus pneumoniae Endohexosaminidase D, Structural and Mechanistic Insight into Substrate-assisted Catalysis in Family 85 Glycoside Hydrolases

Abbott

Macauley

Vocadlo

et al. 2009

Endo-␤-D-glucosaminidases from family 85 of glycoside hydrolases (GH85 endohexosaminidases) act to cleave the glycosidic linkage between the two N-acetylglucosamine units that make up the chitobiose core of N-glycans. Endohexosaminidase D (Endo-D), produced by Streptococcus pneumoniae, is believed to contribute to the virulence of this organism by playing a role in the deglycosylation of IgG antibodies. Endohexosaminidases have received significant attention for this reason and, moreover, because they are powerful tools for chemoenzymatic synthesis of proteins having defined glycoforms. Here we describe mechanistic and structural studies of the catalytic domain (SpGH85) of Endo-D that provide compelling support for GH85 enzymes using a catalytic mechanism involving substrate-assisted catalysis. Furthermore, the structure of SpGH85 in complex with the mechanism-based competitive inhibitor NAG-thiazoline (K d ‫؍‬ 28 M) provides a coherent rationale for previous mutagenesis studies of Endo-D and other related GH85 enzymes. We also find GH85, GH56, and GH18 enzymes have a similar configuration of catalytic residues. Notably, GH85 enzymes have an asparagine in place of the aspartate residue found in these other families of glycosidases. We propose that this residue, as the imidic acid tautomer, acts analogously to the key catalytic aspartate of GH56 and GH18 enzymes. This topographically conserved arrangement of the asparagine residue and a conserved glutamic acid, coupled with previous kinetic studies, suggests these enzymes may use an unusual proton shuttle to coordinate effective general acid and base catalysis to aid cleavage of the glycosidic bond. These results collectively provide a blueprint that may be used to facilitate protein engineering of these enzymes to improve their function as biocatalysts for synthesizing glycoproteins having defined glycoforms and also may serve as a guide for generating inhibitors of GH85 enzymes.

“…We found that a quantitative conversion of the Fuc␣1,6GlcNAc-CD52 (8) to the glycopeptide (9) could be achieved within 2 h when a 3-fold excess of the glycan oxazoline (7) was used, demonstrating the efficiency of the Endo-F3 D165A-catalyzed transglycosylation. We have also tested several glycosynthases described previously, including the D233A mutant of Endo-S, the N322A and N322Q mutants of Endo-D, the N175Q mutant of Endo-M, and the N171A mutant of Endo-A (29,30,(32)(33)(34). We found that none of those glycosynthases were able to transfer triantennary complex glycans from the glycan oxazoline.…”

Section: Endo-f3 Mutant D165a Is Able To Transfer Triantennary N-glycmentioning

confidence: 99%

“…We have previously generated glycosynthases from endoglycosidases of both the GH85 and GH18 family by site-directed mutation at a key residue that is responsible for promoting the formation of the oxazolinium ion intermediate during hydrolysis, which proceeds in a substrate-assisted mechanism (29,30,(32)(33)(34). These include a key asparagine residue for the GH85 endoglycosidases Endo-A (Asn-171), Endo-M (Asn-275), and Endo-D (Asn-322) or a key aspartic acid residue for the GH18 family endoglycosidase Endo-S (Asp-233).…”

Section: Generation and Characterization Of Glycosynthase Mutants Of mentioning

confidence: 99%

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

Giddens

Lomino

Amin

et al. 2016

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114

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...