Carbohydrate Binding Module Family 94

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• Author: Takatsugu Miyazaki
• Responsible Curator: Takatsugu Miyazaki

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CAZy DB link

https://www.cazy.org/CBM94.html

Ligand specificities

CBM94 was established in 2022 after the structural and functional characterization of the C-terminal domains of human N-acetylglucosaminyltransferase IVa (GnT-IVa, MGAT4A; GT54; EC 2.4.1.145) and an ortholog from lepidopteran insect Bombyx mori [1]. The CBM94 proteins from human and B. mori showed affinity toward N-acetylglucosamine, N,N’-diacetylchitobiose, and p-nitrophenyl β-N-acetylglucosaminide with K_a values of 242–1,970 M⁻¹. No affinity was detected for other monosaccharides, including glucose, mannose, galactose, L-fucose, and N-acetylgalactosamine, some of which are components of matured N-glycans [1]. Nagae et al. demonstrated that the C-terminal domain of mouse GnT-IVa has binding ability for GlcNAc and GlcNAcβ1-2Man using NMR titration analysis [2]. Furthermore, comprehensive frontal affinity chromatography analysis using 157 glycans showed that mouse CBM94 has affinity for N-glycans with β-(1→2) and β-(1→4)-linked GlcNAc at the non-reducing ends. On the other hand, it showed low affinity for N-glycan with only β-(1→2)-linked GlcNAc, which is the substrate of GnT-IV [2]. Therefore, CBM94 prefers product N-glycans rather than substrate N-glycans.

Structural Features

CBM94 domains of GnT-IV enzymes comprise of around 150 amino acid residues. The crystal structures of the CBM94 domains in human and mouse GnT-IVa and B. mori ortholog were determined at 1.97, 1.95, and 1.47 Å resolution (PDB ID 7XTL, 7VMT, and 7XTM), respectively. The mammalian CBM94 adopt β-sandwich fold comprising nine β-strands and three short α-helices, while B. mori CBM94 has a similar fold but lacks one α-helix. They are structurally homologous to CBM32 proteins, such as GlcNAc-binding CBM32 domain of Clostridium perfringens GH84 β-N-acetylglucosaminidase NagH. The 1.15-Å resolution structure of B. mori CBM94 in complex with β-GlcNAc (PDB ID 7XTN) indicates that Tyr429, Trp445, Asp480, and Trp535 contribute to GlcNAc binding. These residues are completely conserved among CBM94 domains in mammalian GnT-IV isozymes (GnT-IVa, GnT-IVb, and GnT-IVc) except that Tyr429 is substituted to Phe in GnT-IVc.

Functionalities

Content in this section should include, in paragraph form, a description of:

• Functional role of CBM: Describe common functional roles such as targeting, disruptive, anchoring, proximity/position on substrate.
• Most Common Associated Modules: 1. Glycoside Hydrolase Activity; 2. Additional Associated Modules (other CBM, FNIII, cohesin, dockerins, expansins, etc.)
• Novel Applications: Include here if CBM has been used to modify another enzyme, or if a CBM was used to label plant/mammalian tissues? Etc.

Family Firsts

First Identified

Sugar-binding ability of the C-terminal domains of human and mouse GnT-IVa (MGAT4A) and Bombyx mori ortholog was identified independently by two groups [1, 2].

First Structural Characterization

Crystal structures of the C-terminal domains of human and mouse GnT-IVa (MGAT4A) and Bombyx mori ortholog were determined independently by two groups [1, 2]. β-GlcNAc-bound structure of B. mori CBM94 was also determined [1].

References

1. Oka N, Mori S, Ikegaya M, Park EY, and Miyazaki T. (2022). Crystal structure and sugar-binding ability of the C-terminal domain of N-acetylglucosaminyltransferase IV establish a new carbohydrate-binding module family. Glycobiology. 2022;32(12):1153-1163. DOI:10.1093/glycob/cwac058 |
2. Nagae M, Hirata T, Tateno H, Mishra SK, Manabe N, Osada N, Tokoro Y, Yamaguchi Y, Doerksen RJ, Shimizu T, and Kizuka Y. (2022). Discovery of a lectin domain that regulates enzyme activity in mouse N-acetylglucosaminyltransferase-IVa (MGAT4A). Commun Biol. 2022;5(1):695. DOI:10.1038/s42003-022-03661-w |
3. Osada N, Nagae M, Nakano M, Hirata T, and Kizuka Y. (2022). Examination of differential glycoprotein preferences of N-acetylglucosaminyltransferase-IV isozymes a and b. J Biol Chem. 2022;298(9):102400. DOI:10.1016/j.jbc.2022.102400 |