Double Mutants as Tools for Elucidating N-glycosylation Pathways in Caenorhabditis elegans

Recent years, C. elegans began to be more popular in the study of glycomics. Interestingly, its glycome has been shown to have more complicated patterns than those of some higher organisms, e.g. Drosophila. The basis for this must be the presence of multiple glycosylation pathways involved in forming highly variable glycan structures. Here we report two novel double mutants as tools for elucidating N-glycosylation pathways in vivo: the H2H3 double mutant, lacking β-N-acetylhexosaminidase-2 (hex-2) and 3 (hex-3), and the F1F6 double mutant, lacking α-1, 3 fucosyltransferase-1 (fut-1) and 6 (fut-6). N-glycans of the double mutants were released by PNGase F, labeled with 2-aminopyridine and subsequently fractionated by HPLC, then analyzed by MALDI-TOF MS/MS. Preliminary data showed that the double mutants possess novel features. Both of the double mutants have a rather conspicuous difference in terms of reversed phase HPLC profiles, in comparison to their own parent mutants and the N2 wild type. A loss of anti-HRP epitope (core α-1,3-fucose) in an extract of the H2H3 double mutant, but not in the single hex-2 and hex-3 mutant extracts provides new evidence in biosynthesis of truncated N-glycans: HEX-2 and HEX-3 are main contributors in trimming the MGn to MM, together with Golgi mannosidase II, in the conversion of Man5Gn to MM. The abundance of a Gal-Fuc modification on the inner GlcNAc of N-glycan core increased the sensitivity to a toxic galectin CGL2. In comparison to the N2 wild type and fut-1 or fut-6 single mutant, the N-glycosylation pattern of the F1F6 double mutant has a general reduction in N-glycan fucosylation as judged by MALDI-TOF MS. Clues may be revealed when the glycan structures in each double mutant are carefully studied. It has to be noted that unexpected N-glycan structures were also observed in the double mutants, which increase the difficulties of any analyses.