[Show abstract][Hide abstract] ABSTRACT: We have examined the N-glycans present during the developmental stages of Caenorhabditis elegans using two approaches, 1) a combination of permethylation followed by MALDI-TOF mass spectrometry (MS) and 2) derivatization
with 2-aminobenzamide followed by separation by high-performance liquid chromatography and analyses by MALDI-TOF MS, post
source decay (PSD) MS, and MALDI-QoTOF MS/MS. The N-glycan profile of each developmental stage (Larva 1, Larva 2, Larva 3, Larva 4, and Dauer and adult) appears to be unique.
The pattern of complex N-glycans was stage-specific with the general trend of number and abundance of glycans being Dauer ≈ L1 > adult ≈ L4 > L3 ≈
L2. Dauer larvae contained complex N-glycans with higher molecular masses than those seen in other stages. MALDI-QoTOF MS/MS of Hex4HexNAc4 showed an N-acetyllac-tosamine substitution not previously observed in C. elegans. Phosphorylcholine (Pc)-substituted glycans were also found to be stage-specific. Higher molecular weight Pc-containing glycans,
including fucose-containing ones such as difucosyl Pc-glycan (Pc1dHex2Hex5HexNAc6) seen in Dauer larvae, have not been observed in any organism. Pc2Hex4HexNAc3, from Dauer larvae, when subjected to PSD MS analyses, showed Pc may substitute both core and terminally linked GlcNAc; no
such structure has previously been reported in any organism. C. elegans-specific fucosyl and native methylated glycans were found in all developmental stages. Taken together, the above results
demonstrate that in-depth investigation of the role of the above N-glycans during C. elegans development should lead to a better understanding of their significance and the ways that they may govern interactions, both
within the organism during development and between the mobile nematode and its pathogens.
[Show abstract][Hide abstract] ABSTRACT: srf-3 is a mutant of C. elegans that is resistant to infection by Microbacterium nematophilum and to binding of the biofilm produced by Yersinia pseudotuberculosis and Yersinia pestis. Recently, SRF-3 was characterized as a nucleotide sugar transporter of the Golgi apparatus occurring exclusively in hypodermal seam cells, pharyngeal cells, and spermatheca. Based on the above observations, we hypothesized that srf-3 may have altered glyconjugates that may enable the mutant nematode to grow unaffected in the presence of the above pathogenic bacteria. Following analyses of N- and O-linked glycoconjugates of srf-3 and wild type nematodes using a combination of enzymatic degradation, permethylation, and mass spectrometry, we found in srf-3 a 65% reduction of acidic O-linked glycoconjugates containing glucuronic acid and galactose as well as a reduction of N-linked glycoconjugates containing galactose and fucose. These results are consistent with the specificity of SRF-3 for UDP-galactose and strongly suggest that the above glycoconjugates play an important role in allowing adhesion of M. nematophilum or Y. pseudotuberculosis biofilm to wild type C. elegans. Furthermore, because seam cells as well as pharyngeal cells secrete their glycoconjugates to the cuticle and surrounding surfaces, the results also demonstrate the critical role of these cells and their secreted glycoproteins in nematode-bacteria interactions and offer a mechanistic basis for strategies to block such recognition processes.
[Show abstract][Hide abstract] ABSTRACT: The biosynthesis in vitro of phosphorylcholine oligosaccharides in Caenorhabditis elegans has been investigated. Here we show that extracts of C. elegans' microsomes transfer phosphorylcholine from L-alpha-dipalmitoyl phosphatidylcholine to hybrid and complex type N-linked oligosaccharides containing mannose residues disubstituted with N-acetylglucosamine. The reaction products are consistent with structures reported for C. elegans as well those found in the filarial nematodes Acanthocheilonema viteae, Onchocerca volvulus, and Brugia malayi, strongly supporting the concept that the phosphorylcholine oligosaccharide biosynthetic enzymes are conserved in this group of organisms. Because it is thought that phosphorylcholine substitution of oligosaccharides modulates host immune response in filarial infections, this in vitro system may help in gaining an understanding of the basis for this response.
Proceedings of the National Academy of Sciences 04/2004; 101(10):3404-8. DOI:10.1073/pnas.0400384101 · 9.67 Impact Factor