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

Abstract

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...

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

“…We have recently reported that a high-level, soluble expression of Endo-F3 and its mutants could be achieved using this vector (32). Following a similar method, the Endo-S2 was successfully expressed in Escherichia coli and was readily purified using immobilized metal ion affinity chromatography to obtain the soluble enzyme with a yield of more than 20 mg/liters.…”

“…These include a key asparagine residue for endoglycosidases Endo-A (Asn-171) (36), Endo-M (Asn-175) (37,38), and Endo-D (Asn-322) (24) of the GH85 family, or a key aspartic acid residue for the GH18 family endoglycosidases Endo-S (Asp-233) (25) and Endo-F3 (Asp-165) (32). Sequence alignment of Endo-S2 and Endo-S revealed that the Asp-184 of Endo-S2 was the residue equivalent to Asp-233 of Endo-S essential for promoting oxazolinium ion formation in hydrolysis (Fig.…”

“…These findings significantly expand the scope of the glycosylation remodeling strategy. For example, the previously reported Endo-S and Endo-F3 are specific for complex type N-glycans and are unable to efficiently transfer high-mannose and hybrid type N-glycans, and the Endo-F3 is efficient only for core-fucosylated GlcNAc acceptor (32).…”

“…Although the Endo-S glycosynthases were able to transfer biantennary complex type and modified Man3GlcNAc core, these mutants showed only marginal activity in transferring high-mannose type N-glycans. More recently, we have generated glycosynthase mutants from Endo-F3, another GH18 family endoglycosidase (32). It was found that the Endo-F3 glycosynthases, such as the D165A mutant, was able to transfer triantennary N-glycan to Fc domain of intact antibody, but they required ␣1,6-fucosylated GlcNAc moiety as the acceptor for transglycosylation and were unable to transfer to non-fucosylated GlcNAc acceptors.…”

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

“…Transglycosylation onto ␣1,6-fucosyl GlcNAc moieties by endoglycosidase was first reported in Endo-F2 and Endo-F3 (41). 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).…”