N-glycan core tri-fucosylation requires Golgi a-mannosidase III activity that impacts nematode growth and behavior

Publikationen als Autor:in / Herausgeber:in der Vetmeduni

Yan, Shi; Kendler, Jonatan

Abstrakt

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 a1,6-mannose upper arm, indicating that a specific a-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 a1,3-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 a-mannosidase II (AMAN-2), AMAN-3 displayed a cobalt-dependent a1,6-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 a-mannosidase required for core fucosylation of the distal GlcNAc of N-glycans. This enzyme is essential for the formation of the unusual trifucosylated chitobiose modifications in nematodes, which may play important roles in nematode development and behavior.

Dokumententyp

Originalarbeit

Open Access Type

Gold

WoS ID

WOS:001383279400001

Repository Phaidra

o:3863