N-glycan Core Tri-fucosylation Requires Golgi ?-mannosidase III Activity that Impacts Nematode Growth and Behaviour

Jonatan Kendler; Florian Wöls; Saurabh Thapliyal; Elsa Arcalis; Hanna Gabriel; Sascha Kubitschek; Daniel Malzl; Maria R. Strobl; Dieter Palmberger; Thomas Luber; Carlo Unverzagt; Katharina Paschinger; Dominique A. Glauser; Iain B. H. Wilson; Shi Yan

doi:10.1016/j.jbc.2024.107944

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Title (eng)

N-glycan Core Tri-fucosylation Requires Golgi ?-mannosidase III Activity that Impacts Nematode Growth and Behaviour

Author

Jonatan Kendler

University of Veterinary Medicine Vienna

Florian Wöls

Saurabh Thapliyal

Elsa Arcalis

Hanna Gabriel

University of Veterinary Medicine Vienna

Sascha Kubitschek

Daniel Malzl

Maria R. Strobl

Dieter Palmberger

Thomas Luber

Carlo Unverzagt

Katharina Paschinger

Dominique A. Glauser

Iain B. H. Wilson

Shi Yan

University of Veterinary Medicine Vienna

Abstract (eng)

N-glycans with complex core chitobiose modifications are observed in various free-living and parasitic nematodes but are absent in mammals. Using Caenorhabditis elegans as a model, we demonstrated that the core N-acetylglucosamine (GlcNAc) residues are modified by three fucosyltransferases (FUTs), namely FUT-1, FUT-6, and FUT-8. Interestingly, FUT-6 can only fucosylate N-glycans lacking the ?1,6-mannose upper arm, indicating that a specific ?-mannosidase is required to generate substrates for subsequent FUT-6 activity. By analyzing the N-glycomes of aman-3 KOs using offline HPLC-MALDI-TOF MS/MS, we observed that the absence of aman-3 abolishes ?1,3-fucosylation of the distal GlcNAc of N-glycans, which suggests that AMAN-3 is the relevant mannosidase on whose action FUT-6 depends. Enzymatic characterization of recombinant AMAN-3 and confocal microscopy studies using a knock-in strain (aman-3::eGFP) demonstrated a Golgi localization. In contrast to the classical Golgi ?-mannosidase II (AMAN-2), AMAN-3 displayed a cobalt-dependent ?1,6-mannosidase activity toward N-glycans. Using AMAN-3 and other C. elegans glycoenzymes, we were able to mimic nematode N-glycan biosynthesis in vitro by remodeling a fluorescein conjugated-glycan and generate a tri-fucosylated structure. In addition, using a high-content computer-assisted C. elegans analysis platform, we observed that aman-3 deficient worms display significant developmental delays, morphological, and behavioral alterations in comparison to the WT. Our data demonstrated that AMAN-3 is a Golgi ?-mannosidase required for core fucosylation of the distal GlcNAc of N-glycans. This enzyme is essential for the formation of the unusual tri-fucosylated chitobiose modifications in nematodes, which may play important roles in nematode development and behavior.

Keywords (eng)

AnimalsPolysaccharides MetabolismGolgi Apparatus MetabolismGolgi Apparatus EnzymologyAlpha-Mannosidase MetabolismAlpha-Mannosidase GeneticsCaenorhabditis Elegans ProteinsGlycosylationFucose MetabolismFucosyl TransferasesFucosyl MetabolismFucosyl GeneticsCaenorhabditis Elegans MetabolismCaenorhabditis Elegans EnzymologyCaenorhabditis Elegans Genetics

Type (eng)

journal article

Language

[eng]

Persistent identifier

https://phaidra.vetmeduni.ac.at/o:3863

DOI

10.1016/j.jbc.2024.107944

Is in series

Title (eng)

Journal of Biological Chemistry

Volume