GLYCOSYLATION IN CAENORHABDITIS ELEGANS

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 研究中心，而研究中心不一定是研究者所在的机构。 秀丽隐杆线虫是发育、先天免疫和宿主-病原体相互作用的一个有吸引力的模型，所有这些过程都涉及碳水化合物识别。该生物体易于在培养物中维持，并且在发育和遗传上具有良好的特征，并且基因组被完全测序。几个研究小组已经报道了该生物体中的N-聚糖结构（1-3）。保守聚糖如高甘露糖、短甘露糖型杂合聚糖和复合聚糖已被发现，但一些由基因组序列预测的高级复合聚糖在最近的研究中很少或不存在。这一信息加上一些新的寡糖和其他似乎是保守的线虫的存在暗示，这种生物体的N-糖基化库的整体知识是不完整的。 寡糖的还原胺化可用于提高HPLC和MS分析的灵敏度。C.的2-氨基苯甲酰胺衍生物。已分析并比较了来自L1-4期、成虫期和Dauer期的线虫N-聚糖。应用了使用MALDI-TOF、PSD和QoTOF MS进行的经检测的C-18色谱和离线MS分析。PNGase F释放的聚糖包含五种一般类型，包括高甘露糖、磷酰胆碱取代的、C6岩藻糖基、复合类型和后两者的杂合形式。每个发育阶段的色谱图、检测到的糖型和离子丰度都是唯一的。磷酰胆碱取代和Ce-岩藻糖基寡糖是最丰富的和结构多样的幼虫1和Dauer阶段。其中隐含了一些新颖的结构。成虫线虫的聚糖不太复杂，与混合发育阶段的聚糖最相似，这一观察结果并不太令人惊讶，因为成虫在混合样品中的质量最丰富。幼虫1和Dauer幼虫在混合样品中的代表性不足，这可能解释了为什么我们观察到的一些高阶复合物和磷酰胆碱寡糖罕见或未被其他实验室检测到，因为迄今为止报告的所有其他研究都涉及混合阶段的聚糖。这些数据表明，2-氨基苯甲酰胺寡糖衍生物的荧光检测和离线MS分析可以用于分析和比较相关的生物样品。为进一步促进和加强这一方法，正在制定在线分析战略。

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.