GLYCOSYLATION IN CAENORHABDITIS ELEGANS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。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。低聚糖的还原胺化有助于在高效液相色谱和质谱学分析中提高灵敏度(4，5)。本文对线虫幼虫L1-4、成虫和Dauer期N-糖链的2-氨基苯甲酰胺衍生物进行了分析和比较。应用荧光检测的C-18层析和离线质谱分析，使用MALDI-TOF、PSD和QOTOF MS。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。在幼虫1期和Dauer期，磷酰胆碱取代的低聚糖和岩藻糖基的低聚糖含量最丰富，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。此外，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本研究中观察到的一些高阶复合体和磷胆碱寡糖在其他研究中很少或没有检测到，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。图1显示了一些高阶磷胆碱取代的N-葡聚糖。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分析可以作为分析和比较相关生物样品的有用方法。为了进一步促进和加强这一方法，有必要制定在线分析战略，并正在进行中。

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 mass spectrometric analyses (4,5). Here 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 mass spectrometric analysis using MALDI-TOF, PSD, and QoTOF MS were applied. The 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. Phosphorylcholine-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. Moreover, Larva 1 and Dauer larva are underrepresented in mixed samples, and this may explain why some higher order complex and phosphorylcholine oligosaccharides observed in the present study are rare or not detected in other studies since all other investigations reported to date have addressed glycans from mixed stages. Some higher order phosphocholine substituted N-glycans are shown in Figure 1. 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 warranted and ongoing.