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

Abstract

N-glycans with complex core chitobiose modifications (CCMs) 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, namely FUT-1, FUT-6 and FUT-8. Interestingly, FUT-6 can only fucosylate N-glycans 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 of aman-3 knockouts using offline HPLC-MALDI-TOF MS/MS, we observed that the absence of aman-3 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. Enzymatic characterisation of recombinant AMAN-3 and confocal microscopy studies using a knock-in strain (aman-3::eGFP) demonstrated a Golgi localisation. 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 C. elegans glycoenzymes, we were able to mimic nematode N-glycan biosynthesis in vitro by remodelling 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 behavioural alterations in comparison to the wild type. Our data demonstrated that AMAN-3 is a Golgi α-mannosidase required for core fucosylation of the distal GlcNAc of N-glycans. This enzyme is essential for the formation of the unusual tri-fucosylated CCMs in nematodes, which may play important roles in nematode development and behaviour.

Keywords: N-glycan; fucosylation; glycosylation; mannosidase; nematode.