The Fine Structure of Caenorhabditis elegans N-Glycans

Abstract

We report the fine structure of a nearly contiguous series of N-glycans from the soil nematode Caenorhabditis elegans. Five major classes are revealed including high mannose, mammalian-type complex, hybrid, fucopausimannosidic (five mannose residues or fewer substituted with fucose), and phosphocholine oligosaccharides. The high mannose, complex, and hybrid N-glycan series show a high degree of conservation with the mammalian biosynthetic pathways. The fuco-pausimannosidic glycans contain a novel terminal fucose substitution of mannose. The phosphocholine oligosaccharides are high mannose type and are multiply substituted with phosphocholine. Although phosphocholine oligosaccharides are known immunomodulators in human nematode and trematode infections, C. elegans is unique as a non-parasitic nematode containing phosphocholine N-glycans. Therefore, studies in C. elegans should aid in the elucidation of the biosynthetic pathway(s) of this class of biomedically relevant compounds. Results presented here show that C. elegans has a functional orthologue for nearly every known enzyme found to be deficient in congenital disorders of glycosylation types I and II. This nematode is well characterized genetically and developmentally. Therefore, elucidation of its N-glycome, as shown in this report, may place it among the useful systems used to investigate human disorders of glycoconjugate synthesis such as the congenital disorders of glycosylation syndromes.

Molecular Basis of Anti-horseradish Peroxidase Staining in Caenorhabditis elegans

“…These substrates are utilized by core ␣1,3-fucosyltransferases from plants and insects (26,44), as well as by the core ␣1,6-fucosyltransferase of C. elegans. 4 It was only when we wished to rule out that the native core ␣1,6-fucosyltransferase could utilize MM that we detected a core fucosyltransferase in wild-type C. elegans extracts capable of transferring to this structure. As shown in Fig.…”

“…Over the years, it has become obvious that the glycosylation of invertebrate glycoproteins is significantly different from that of mammals, but although organisms such as Caenorhabditis elegans have been studied to a great extent in terms of molecular and developmental biology, an exact understanding of their glycosylation has lagged behind. Recently, however, a number of studies have indicated that the glycoconjugates of C. elegans have a number of unusual features (1)(2)(3)(4)(5)(6). None of the studies published so far agree absolutely as to the types of N-linked oligosaccharides, but the consensus would appear to be that this organism has a range of N-glycans carrying one or many fucose residues, with or without methyl or phosphorylcholine groups, as well as the standard oligomannose structures (7).…”

“…There is also only fragmented data on the fucosylation of other nematodes: up to three fucose residues on the core of N-glycans (45,46) and the presence of a Lewis-type fucosyltransferase (47) in Haemonchus, fucosylation of artho-series glycolipids from Ascaris suum (48), and the presence of Lewis epitopes in Dictyocaulus viviparus (49). In C. elegans, the single obvious ␣1,6-fucosyltransferase homologue (FUT-8) has been expressed in an active recombinant form, 4 and the determination of the enzymatic activity of only one ␣1,2-fucosyltransferase (FUT-2 or CE2FT1) has been published (16) but left its biological substrate unrevealed. However, the activity of most of the ␣1,2-fucosyltransferase homologues is still unknown, whereas published and preliminary data suggested that the ␣1,3-fucosyltransferase homologues were Lewis-type enzymes (42,50,51).…”

“…However, preliminary data indicated the presence of a fucosyltransferase activity capable of modifying a trimannosyl N-glycan core (MM). 4 We followed up this latter result by using MALDI-TOF MS and RP-HPLC assays of wild-type and mutant C. elegans. Wild-type extracts displayed activity toward dabsyl-MM as shown by an increase, in the presence of GDP-Fuc, in the m/z corresponding to the mass of one fucose.…”

A glycomics platform for the analysis of permethylated oligosaccharide alditols

“…Oligosaccharides were permethylated using the method of Ciucanu and Kerek [35] with minor modifications as described previously [36]. Sample recovery was 70% to 95% based on phenol sulfuric acid assay for neutral hexose and/or peak intensity on MALDI-TOF MS analysis using permethylated oligosaccharide standards.…”