GLYCOSYLATION IN CAENORHABDITIS ELEGANS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 秀丽线虫是发育、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的识别。该生物体很容易在培养中维护，在发育和遗传上具有良好的特征，并且基因组完全测序。几个小组已经报道了这种生物体中的N-糖链结构(1-3)。保守的多聚糖，如高甘露糖和短的哺乳动物类型的杂交糖和复合糖已经被发现。然而，由基因组序列预测的一些高阶复杂糖链在最近的研究中很少或没有。这些信息，再加上一些新的寡糖和其他似乎在线虫中保守的存在，表明对这种生物的N-糖基化谱系的整体知识是不完整的。 低聚糖的还原胺化在高效液相色谱和质谱仪分析中都有助于提高灵敏度。对线虫L1-4期、成虫期和Dauer期N-糖链的2-氨基苯甲酰胺衍生物进行了分析和比较。采用荧光检测C-18色谱柱和离线质谱仪进行分析。PNGase F释放的多糖包括高甘露糖、磷酰胆碱取代、Ce岩藻糖基、复合型和后两者的杂合型五大类。每个发育阶段的层析图谱、检测到的糖形式和离子丰度都是独一无二的。磷酰胆碱取代和Ce-岩藻糖基低聚糖在幼虫1期和Dauer期含量最丰富，结构也最多样化。隐含了一些新的结构。成虫的多糖不那么复杂，最接近于混合发育阶段的多糖，这一观察结果并不太令人惊讶，因为在混合样本中，成虫的质量最丰富。幼虫1和Dauer幼虫在混合样本中的表达不足，这可能解释了为什么我们观察到的一些高阶复合体和磷酰胆碱低聚糖很少或没有被其他实验室检测到，因为到目前为止报道的所有其他研究都涉及混合阶段的多糖。这些数据表明，2-氨基苯甲酰胺低聚糖衍生物的荧光检测和离线MS分析可以作为相关生物样品分析和比较的有用手段。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.