Core Tri-fucosylation of Nematode N-glycans Requires Golgi α-mannosidase III Activity that Impacts Animal Growth and Behaviours
Authors: Kendler Jonatan , Wöls Florian , Thapliyal Saurabh , Arcalis Elsa , Gabriel Hanna , Kubitschek Sascha , Malzl Daniel , Strobl Maria R. , Palmberger Dieter , Luber Thomas , Unverzagt Carlo , Paschinger Katharina , Glauser Dominique A. , Wilson Iain B. H. , Yan Shi 
Published: July 2024

AbstractMany nematodes possess N-glycans with complex core chitobiose modifications, which is a feature observed in various free-living and parasitic nematodes but is absent in mammals. UsingCaenorhabditis elegansas a model to study N-glycan biosynthesis, we demonstrated that the coreN-acetylglucosamine (GlcNAc) residues can be modified by three fucosyltransferases in the Golgi, namely FUT-1, FUT-6 and FUT-8. While the asparagine-linked GlcNAc is modified with a α1,3- and α1,6-linked fucose by FUT-1 and FUT-8 respectively, the distal GlcNAc residue is α1,3-fucosylated solely by FUT-6. Interestingly, FUT-6 can only fucosylate N-glycan structures lacking the α1,6-mannose upper arm, indicating that a specific α-mannosidase is required to generate substrates for subsequent FUT-6 activity. By analysing the N-glycomes ofaman-3mutants (tm5400 and a CRISPR/Cas9 knockout,hex-2;hex-3;aman-3) using offline HPLC-MALDI-TOF MS/MS, we observed that the absence of theaman-3gene abolishes α1,3-fucosylation of the distal GlcNAc of N-glycans, which suggests that AMAN-3 is the relevant mannosidase on whose action FUT-6 depends. To further investigate it, we recombinantly expressed AMAN-3 in insect cells and characterised its enzymatic activityin vitro. In contrast to the classical Golgi α-mannosidase II (AMAN-2), AMAN-3 displayed a cobalt-dependent α1,6-mannosidase activity towards N-glycans. Using AMAN-3 and other recombinantC. elegansglycoenzymes, we remodelled a fluorescein conjugated-Man5GlcNAc2structure; we were able to mimic N-glycan biosynthesis in the Golgi and generate a tri-fucosylated glycanin vitro. We performed confocal microscopy studies using a knock-in strain (aman-3::eGFP) and could show the Golgi localisation of AMAN-3. In addition, using a high-content computer-assistedC. elegansanalysis platform, we observed that AMAN-3 deficient worms display significant developmental delays, morphological and behavioural alterations in comparison to the wild type. Therefore, our data suggested that AMAN-3 participates in nematode N-glycan biosynthesis in the Golgi and generates substrates for FUT-6; thereby, this enzyme is essential for the formation of the unusual tri-fucosylated chitobiose cores of nematode N-glycans, which may play important roles in nematode development and behaviour.BackgroundTri-fucosylation of N-glycan core is a conserved feature seen in the N-glycomes of several nematode species. However, beyond the three core fucosyltransferases, we know very little about the biosynthesis and biological function of these core modifications.ResultsComparative glycomics data revealed thataman-3mutants possess underfucosylated N-glycomes. Biochemical characterisation of AMAN-3 clarified its optimal reaction conditions and substrate specificity and, we demonstrated a Golgi localisation. Thereafter in vitro reconstruction of biosynthesis of a core tri-fucosylated N-glycan was achieved using 8 recombinantC. elegansglycoenzymes. Notably,aman-3deficient worms exhibited significant developmental and behavioural changes.ConclusionAMAN-3 is a Golgi α-mannosidase required for core fucosylation of the distalN-acetylglucosamine of N-glycoproteins.SignificanceThis study elucidates the key role of a novel Golgi α-mannosidase in the biosynthesis of the unusual N-glycans ofC. elegansand related nematodes, thereby setting the stage for new approaches to study the roles of glycan in the biology and immunology of nematode glycoproteins.