Pan-specific tools and reagents for the enrichment of sialo-glycans

项目摘要 聚糖具有几种独特的性质，使其作为疾病生物标志物的发展具有吸引力。 首先，它们在细胞表面上的位置使它们成为细胞相互作用的第一个接触点， 它们在控制正常代谢过程中至关重要，相反，它们作为病原体发挥作用。 粘附受体第二，在正常细胞中不存在或含量较低的特定聚糖结构， 在疾病状态下表达、增殖或改变其序列。最后，糖基化的变化可能会被发现， 在许多蛋白质中，包括那些高度丰富的蛋白质。因此聚糖的正常水平的变化 结构，如末端唾液酸，可以是疾病状态的标志物。新型高特异性试剂 为了克服目前在发现和开发疾病相关聚糖方面的限制，需要进行这些研究。 使用结构指导的遗传操作，我们正在将NanB唾液酸酶从S.肺炎球菌 用于检测糖肽的所有类型的唾液酸修饰的高特异性亲和试剂 和糖蛋白。由于这种蛋白质具有凝集素样性质，但来源于一种酶，因此被称为 “Lectenz®"。NanB Lectenz®解决了疾病糖标志物检测的关键需求：即， 易于生产特异性检测所有类型唾液酸化聚糖的试剂。该试剂可用于 在用于样品富集的亲和基质中，其与现有的基于MS的方法结合可以提供 链接信息。糖肽样品富集通过消除非糖基化来辅助糖组学分析 肽，否则会减弱来自具有低电离效率的糖肽的信号。 糖基化位点作图在充分表征和利用聚糖作为特异性抗体的标记物中是必不可少的。 疾病状态，但目前，没有试剂存在，可以检测唾液酸化聚糖，独立的类型， 连接与唾液酸。因此，目前，许多具有不同特异性的试剂和 必须利用亲和力来捕获或检测所有形式的唾液酸化聚糖。 与植物凝集素相比，Lectenz®具有许多优势：它们被设计成具有高亲和力， 由于内源性酶的精细底物特异性，它们可以被有效地产生，并且对于人类来说， 同系物具有以低毒性在体内使用的潜力。而Lectenz®的某些方面 Lectenz®的发展与抗体进化的发展平行，Lectenz®具有使用蛋白质的巨大好处， 具有所需特异性的初始模板。

PROJECT SUMMARY Glycans have several distinct properties that make their development as disease biomarkers appealing. Firstly, their location on cell surfaces makes them the first point of contact for cellular interactions, and thus they are crucial in the control of normal metabolic processes, and conversely, they function as pathogen adhesion receptors. Secondly, specific glycan structures that are not present, or are in low amounts in normal state, proliferate or alter their sequence in disease states. And, lastly, changes in glycosylation may be found in many proteins, including those that are highly abundant. Thus changes in the normal levels of glycan structures, such as terminal sialic acid, may be markers of disease states. New highly-specific reagents are required in order to overcome current limitations in the discovery and exploitation of disease-related glycans. Using structurally-guided genetic manipulations, we are converting the NanB sialidase from S. pneumococcus into a high-specificity affinity reagent for the detection of all types of sialic acid modifications of glycopeptides and glycoproteins. Because such a protein has lectin-like properties, but is derived from an enzyme, it is called a "Lectenz®". A NanB Lectenz® addresses a key need in disease glycomarker detection: namely, a robust and easy to produce reagent specific for detecting all types of sialylated glycans. This reagent could be employed in an affinity matrix for sample enrichment, which in conjunction with existing MS based methods could provide linkage information. Glycopeptide sample enrichment aids glycomic analyses by eliminating non-glycosylated peptides, which would otherwise attenuate the signals from glycopeptides that have low ionization efficiency. Glycosylation site mapping is essential in fully characterizing and exploiting glycans as markers of specific disease states, but at present, no reagent exists that can detect sialylated glycans, independent of the type of linkage associated with the sialic acid. Thus, at present, a number of reagents with varying specificities and affinities must be employed to capture or detect all forms of sialylated glycans. Lectenz® offer numerous advantages over plant lectins: they are engineered to be high affinity and yet retain the exquisite substrate specificity of the endogenous enzyme, they may be efficiently produced, and for human homologues have the potential to be employed in vivo with low toxicity. Whereas some aspects of Lectenz® development parallel those of antibody evolution, Lectenz® have the tremendous benefit of employing a protein naïve template that has the desired specificity.