GLYCOSYLATION IN CAENORHABDITIS ELEGANS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Caenorhabditis elegans is an attractive model for development, innate immunity and host-pathogen interactions, all processes where carbohydrate recognition is involved. The organism is easily maintained in culture and well characterized developmentally and genetically and the genome is completely sequenced. Several groups have reported the N-glycan structures in this organism (1-3). Conserved glycans such as high mannose and short mammalian-type hybrid and complex glycans have been found. However, some higher order complex glycans predicted by the genome sequence are rare or absent in recent studies. This information coupled with the presence of some novel oligosaccharides and others that appear to be conserved in nematodes hint that the overall knowledge of the N-glycosylation repertoire of this organism is incomplete. Reductive amination of oligosaccharides is useful for sensitivity enhancement in both HPLC and MS analyses. The 2-aminobenzamide derivatives of C. elegans N-glycans from larval stages L1-4, Adult, and Dauer have been analyzed and compared. Fluorescence-detected C-18 chromatography and off-line MS analysis using MALDI-TOF, PSD, and QoTOF MS were applied. PNGase F released glycans contained five general classes including high mannose, phosphoryl choline-substituted, Ce fucosyl, complex types, and hybrid forms of the last two. The chromatographic profiles, glycoforms detected and ion abundances were unique for each developmental stage. Phosphoryl choline-substituted and Ce-fucosyl oligosaccharides were most abundant and structurally diverse in Larva 1 and Dauer stages. Some novel structures were implied. The glycans of Adult nematodes were less complex and most closely resembled those of mixed developmental stages, a not too surprising observation since Adults are the most abundant by mass in mixed samples. Larva 1 and Dauer larva are underrepresented in mixed samples, and this may explain why some higher order complex and phosphoryl choline oligosaccharides we have observed are rare or not detected by other laboratories, since all other investigations reported to date have addressed glycans from mixed stages. These data show that fluorescence detection and off-line MS analysis of 2-aminobenzamide oligosaccharide derivatives can be a useful approach for analysis and comparison of related biological samples. To further facilitate and enhance the method, development of strategies for online analysis is ongoing.