Abstract
The N-glycosylation of the model nematode Caenorhabditis elegans has proven to be highly variable and rather complex; it is an example to contradict the existing impression that "simple" organisms possess also a rather simple glycomic capacity. In previous studies in a number of laboratories, N-glycans with up to four fucose residues have been detected. However, although the linkage of three fucose residues to the N,N'-diacetylchitobiosyl core has been proven by structural and enzymatic analyses, the nature of the fourth fucose has remained uncertain. By constructing a triple mutant with deletions in the three genes responsible for core fucosylation (fut-1, fut-6 and fut-8), we have produced a nematode strain lacking products of these enzymes, but still retaining maximally one fucose residue on its N-glycans. Using mass spectrometry and HPLC in conjunction with chemical and enzymatic treatments as well as NMR, we examined a set of α-mannosidase-resistant N-glycans. Within this glycomic subpool, we can reveal that the core β-mannose can be trisubstituted and so carries not only the ubiquitous α1,3- and α1,6-mannose residues, but also a "bisecting" β-galactose, which is substoichiometrically modified with fucose or methylfucose. In addition, the α1,3-mannose can also be α-galactosylated. Our data, showing the presence of novel N-glycan modifications, will enable more targeted studies to understand the biological functions and interactions of nematode glycans.
Highlights
From the ‡Department fur Chemie, Universitat fur Bodenkultur, 1190 Wien, Austria; §Institut fur Organische Chemie, Universitat Wien, 1090 Wien, Austria; ¶Institutionen for Biomedicin, Goteborgs universitet, 405 30 Goteborg, Sweden; ʈDepartment fur Chromosomenbiologie, Max F
Bisecting Galactose on Nematode N-glycans that the core chitobiosyl region of nematode N-glycans is subject to a range of modifications, with up to three core fucose residues being present (␣1,3- and ␣1,6-linked on the reducing-terminal “proximal” GlcNAc and ␣1,3-linked on the second “distal” GlcNAc)
Thereby a pool of unusual mannosidase-resistant N-glycans was identified and, using mass spectrometry (MS) and NMR, we reveal their modification with bisecting galactose frequently capped with fucose or methylfucose
Summary
Preparation of the C. elegans Triple Mutant—Wild-type C. elegans (N2) and single mutants fut-1(ok892), fut-6(ok475) and fut-8(ok2558) were obtained from the Caenorhabditis Genetics Centre (CGC), University of Minnesota, USA. Hermaphrodites from the F2 generation were isolated and allowed to produce eggs prior to examination of the maternal genotypes by PCR. To generate the triple mutant, progeny of the ok2558 single mutant and N2 were crossed to fut-1;fut-6 double mutants prior to genotype screening. In total ϳ1500 MS and MS/MS spectra were manually interpreted on the basis of the mass, fragmentation pattern, and results of chemical and enzymatic treatments; isobaric structures present in different RP-HPLC fractions were defined on the basis of comparisons in the aforementioned parameters. Structural Elucidation Using Exoglycosidases and Chemical Treatment—In general, a 1 l aliquot of a HPLC fraction was mixed with 0.2 l exoglycosidase and 0.8 l 50 mM ammonium acetate solution, pH 5.0; after an overnight incubation at 37 °C, 0.5 l aliquot of the mixture was analyzed by MALDI-TOF MS. Glycans were identified from their MS/MS spectra by manual annotation; the nomenclature of Domon and Costello for fragment annotation was employed [25]
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