Structural basis for the specific cleavage of core-fucosylated N-glycans by endo-β-N-acetylglucosaminidase from the fungus Cordyceps militaris

• Published in: The Journal of biological chemistry 2019-11, Vol.294 (45), p.17143-17154
• Main Authors: Seki, Haruka, Huang, Yibo, Arakawa, Takatoshi, Yamada, Chihaya, Kinoshita, Takashi, Iwamoto, Shogo, Higuchi, Yujiro, Takegawa, Kaoru, Fushinobu, Shinya
• Format: Article
• Language: English
• Subjects: Acetylglucosaminidase - chemistry; Acetylglucosaminidase - metabolism; Amino Acid Sequence; Binding Sites; Catalytic Domain; Cordyceps - enzymology; Cordyceps militaris; core-fucosylated N-glycan; endo-β-N-acetylglucosaminidase (ENGase); Enzymology; Fucose - metabolism; GH18; glycoprotein; glycoside hydrolase; Glycosylation; Models, Molecular; monoclonal antibody; N-linked glycosylation; Polysaccharides - metabolism; Substrate Specificity; substrate-assisted mechanism; X-ray crystallography
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA119.010842

N-Linked glycans play important roles in various cellular and immunological events. Endo-β-N-acetylglucosaminidase (ENGase) can release or transglycosylate N-glycans and is a promising tool for the chemoenzymatic synthesis of glycoproteins with homogeneously modified glycans. The ability of ENGases to act on core-fucosylated glycans is a key factor determining their therapeutic utility because mammalian N-glycans are frequently α-1,6-fucosylated. Although the biochemistries and structures of various ENGases have been studied extensively, the structural basis for the recognition of the core fucose and the asparagine-linked GlcNAc is unclear. Herein, we determined the crystal structures of a core fucose-specific ENGase from the caterpillar fungus Cordyceps militaris (Endo-CoM), which belongs to glycoside hydrolase family 18. Structures complexed with fucose-containing ligands were determined at 1.75–2.35 Å resolutions. The fucose moiety linked to GlcNAc is extensively recognized by protein residues in a round-shaped pocket, whereas the asparagine moiety linked to the GlcNAc is exposed to the solvent. The N-glycan–binding cleft of Endo-CoM is Y-shaped, and several lysine and arginine residues are present at its terminal regions. These structural features were consistent with the activity of Endo-CoM on fucose-containing glycans on rituximab (IgG) and its preference for a sialobiantennary substrate. Comparisons with other ENGases provided structural insights into their core fucose tolerance and specificity. In particular, Endo-F3, a known core fucose-specific ENGase, has a similar fucose-binding pocket, but the surrounding residues are not shared with Endo-CoM. Our study provides a foothold for protein engineering to develop enzymatic tools for the preparation of more effective therapeutic antibodies.