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

Haruka Seki, Yibo Huang, Takatoshi Arakawa, Chihaya Yamada, Takashi Kinoshita, Shogo Iwamoto, Yujiro Higuchi, Kaoru Takegawa, Shinya Fushinobu

Research output: Contribution to journalArticlepeer-review

Abstract

N-Linked glycans play important roles in various cellular and immunological events. Endo-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 fucosespecific 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.

Original languageEnglish
Pages (from-to)17143-17154
Number of pages12
JournalJournal of Biological Chemistry
Volume294
Issue number45
DOIs
Publication statusPublished - Nov 8 2019

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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