title: 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 acceptorsubstrate specificity toward α1,6-fucosyl-GlcNAc-Asn or α1,6-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.
authors.affiliation: From the Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland 21201 and the Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742.
authors.forename: John P
authors.initials: JP
authors.lastname: Giddens
authors.affiliation: From the Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland 21201 and.
authors.forename: Joseph V
authors.initials: JV
authors.lastname: Lomino
authors.affiliation: From the Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland 21201 and the Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742.
authors.forename: Mohammed N
authors.initials: MN
authors.lastname: Amin
authors.affiliation: From the Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland 21201 and the Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742 wang518@umd.edu.
authors.forename: Lai-Xi
authors.initials: LX
authors.lastname: Wang
mesh_terms: D000907:Antibodies, Bacterial / Q000737:chemistry; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D041965:Flavobacteriaceae / Q000235:genetics / Q000378:metabolism; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism
chemical_list: D000907:Antibodies, Bacterial; D058846:Antibodies, Monoclonal, Murine-Derived; D001426:Bacterial Proteins; D006023:Glycoproteins; D016695:Glycosyltransferases
keywords: enzyme catalysis; glycoprotein; glycosidase; glycosylation; oligosaccharide