phaidra.vetmeduni.ac.at o:3863 DOI 10.1016/j.jbc.2024.107944 N-glycan Core Tri-fucosylation Requires Golgi ?-mannosidase III Activity that Impacts Nematode Growth and Behaviour 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. eng Animals Polysaccharides Metabolism Golgi Apparatus Metabolism Golgi Apparatus Enzymology Alpha-Mannosidase Metabolism Alpha-Mannosidase Genetics Caenorhabditis Elegans Proteins Glycosylation Fucose Metabolism Fucosyl Transferases Fucosyl Metabolism Fucosyl Genetics Caenorhabditis Elegans Metabolism Caenorhabditis Elegans Enzymology Caenorhabditis Elegans Genetics 2025-02-14T10:24:31.838Z LOMv1.0 Author BEGIN:VCARD VERSION:3.0 N:Kendler;Jonatan; FN:Jonatan Kendler END:VCARD BEGIN:VCARD VERSION:3.0 N:Wöls;Florian; FN:Florian Wöls END:VCARD BEGIN:VCARD VERSION:3.0 N:Thapliyal;Saurabh; FN:Saurabh Thapliyal END:VCARD BEGIN:VCARD VERSION:3.0 N:Arcalis;Elsa; FN:Elsa Arcalis END:VCARD BEGIN:VCARD VERSION:3.0 N:Gabriel;Hanna; FN:Hanna Gabriel END:VCARD BEGIN:VCARD VERSION:3.0 N:Kubitschek;Sascha; FN:Sascha Kubitschek END:VCARD BEGIN:VCARD VERSION:3.0 N:Malzl;Daniel; FN:Daniel Malzl END:VCARD BEGIN:VCARD VERSION:3.0 N:Strobl;Maria R.; FN:Maria R. Strobl END:VCARD BEGIN:VCARD VERSION:3.0 N:Palmberger;Dieter; FN:Dieter Palmberger END:VCARD BEGIN:VCARD VERSION:3.0 N:Luber;Thomas; FN:Thomas Luber END:VCARD BEGIN:VCARD VERSION:3.0 N:Unverzagt;Carlo; FN:Carlo Unverzagt END:VCARD BEGIN:VCARD VERSION:3.0 N:Paschinger;Katharina; FN:Katharina Paschinger END:VCARD BEGIN:VCARD VERSION:3.0 N:Glauser;Dominique A.; FN:Dominique A. Glauser END:VCARD BEGIN:VCARD VERSION:3.0 N:Wilson;Iain B. H.; FN:Iain B. H. Wilson END:VCARD BEGIN:VCARD VERSION:3.0 N:Yan;Shi; FN:Shi Yan X-ORCID:https://orcid.org/0000-0003-4949-9140 END:VCARD https://w3id.org/kim/hcrt/scheme https://w3id.org/kim/hcrt/text Textdokument Text LOMv1.0 yes https://creativecommons.org/licenses/by/4.0 application/pdf 6009977 https://phaidra.vetmeduni.ac.at/o:3863 https://phaidra.vetmeduni.ac.at/api/object/o:3863/thumbnail LOMv1.0 discipline