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. http://creativecommons.org/licenses/by/4.0/ Text Wissenschaftlicher Artikel Elsevier doi:10.1016/j.jbc.2024.107944 Copyright © 2024 The Authors open access Journal of Biological Chemistry 2024 N-glycan Core Tri-fucosylation Requires Golgi ?-mannosidase III Activity that Impacts Nematode Growth and Behaviour eng application/pdf Text journal article 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 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 https://phaidra.vetmeduni.ac.at/o:3863