Structural basis for the specific cleavage of core-fucosylated N-glycans by endo-β-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

doi:10.1074/jbc.ra119.010842

Abstract

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.

Highlights

N-Linked glycans play important roles in various cellular and immunological events
We previously discovered and characterized GH18 ENGases that are specific for core fucose-containing N-glycans from two fungal species (Beauveria bassiana and Cordyceps militaris) and the bacterium Sphingobacterium sp. strain HMA12 (Endo-SB– ORF1188) [30]
We provided a structural basis for the strict specificity of a GH18 ENGase for core-fucosylated N-glycans that are frequently present in mammal glycoproteins

Summary

To whom correspondence should be addressed

N-Linked glycans are oligosaccharides attached to Asn residues of proteins and have key functionalities in various cellular and immunological systems [1, 2]. Endo-␤-N-acetylglucosaminidases (ENGases, EC 3.2.1.96) hydrolytically cleave the ␤-1,4-glycosidic bonds within the core N,NЈ-diacetylchitobiose unit to release N-glycan, leaving a GlcNAc, with or without the core fucose, linked to the Asn residue of proteins (Fig. 1A) [10]. Structures of Endo-F3, Endo-S, and Endo-S2 only contain the octasaccharide of the biantennary complex glycan or high-mannose (Man7GlcNAc) glycan as a cleavage product complex [18, 20, 21]. The ENGase from C. militaris (hereafter called Endo-CoM) as well as the ENGase from B. bassiana (Endo-BB) and EndoSB–ORF1188 exhibit activity exclusively on core ␣-1,6-fucosylated biantennary complex-type oligosaccharides [30]. We report the crystal structures of Endo-CoM in the apo-form and complex forms with L-fucose and fucosecontaining ligands occupying the plus subsites. A mutational study and structural comparison with other ENGases provide structural insights into the core fucose-specific or nonspecific cleavage of N-glycans

Results and discussion

Concluding remarks

Experimental procedures