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

Abstract

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 ...

Generation of efficient mutants of endoglycosidase from Streptococcus pyogenes and their application in a novel one-pot transglycosylation reaction for antibody modification

“…Unexpectedly, the Endo-M N175Q mutant, without the help of Endo-S, showed approximately 5% transglycosylation efficiency towards core-fucosylated IgG ( Fig 4A ). This may be in conflict with a previous report by Katoh et al They reported that the Endo-M N175Q mutant scarcely transglycosylated SG-oxazoline to the core-fucosylated acceptor at a donor-to-acceptor ratio of 1:1, whereas their engineered Endo-M N175Q/W251 double mutant showed approximately 5% transglycosylation efficiency [ 32 ]. The apparent conflict may be related to differences in conditions such as the type of donor glycan and its ratio to acceptor.…”

Characterization of novel endo-β-N-acetylglucosaminidases from Sphingobacterium species, Beauveria bassiana and Cordyceps militaris that specifically hydrolyze fucose-containing oligosaccharides and human IgG

“…In summary, ENGases identified in this report could be useful as tools not only for analyzing the oligosaccharide contents of antibodies (basic research), but they could also be useful as tools for developing therapeutic antibody pharmaceuticals (biomedical application), such as an immunomodulatory therapeutic agent for the treatment of autoimmune disorders. In a recent study, it was reported that W251N mutant of Endo-M can catalyze the hydrolysis of fucosylated N -glycans, a feature is not seen in wild-type Endo-M 41 . At present, we do not know which amino acid residues in Endo-SBs are specifically involved in recognizing fucosylated oligosaccharides, because Endo-SBs belong to the GH18 family, whereas the Endo-M belongs to the GH85 family.…”

β‐Mannosylation through O‐Alkylation of Anomeric Cesium Alkoxides: Mechanistic Studies and Synthesis of the Hexasaccharide Core of Complex Fucosylated N‐Linked Glycans

“…This β ‐mannosylation via anomeric O ‐alkylation was next utilized in the synthesis of the hexasaccharide core of complex fucosylated N ‐linked glycans . For instance, bi‐antennary α ‐(2,3)‐sialylated core‐fucosylated N ‐glycan dodecasaccharide was found on alpha fetoprotein (AFP) isolated from patients with hepatocellular carcinoma.…”