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

用于富集唾液酸聚糖的泛特异性工具和试剂

• 批准号: 10662901
• 负责人: Sheng-Cheng Wu
• 金额: $ 3.72万
• 依托单位: GLYCOSENSORS AND DIAGNOSTICS, LLC
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-11 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10662901
• 关键词: Address; Affinity; Antibodies; Attenuated; Binding; Biological Markers; Carbohydrates; Cell surface; Detection; Development; Disease; Disease Marker; Engineering; Enzymes; Evolution; Glycopeptides; Glycoproteins; Homologous Gene; Human; Kinetics; Lectin; Location; Metabolism; Methods; Modification; Neuraminidase; Peptides; Plant Lectins; Polysaccharides; Proliferating; Property; Proteins; Reagent; Sampling; Sialic Acids; Signal Transduction; Site; Specificity; Streptococcus pneumoniae; Structure; Substrate Specificity; Toxic effect; adhesion receptor; base; genetic manipulation; glycosylation; in vivo; ionization; pathogen; tool

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.

项目摘要 Glycans具有几种不同的特性，使其发展为疾病生物标志物的出现。 首先，它们在细胞表面上的位置使它们成为蜂窝相互作用的第一个接触点，因此 它们对于控制正常代谢过程至关重要，相反，它们起病原体的作用 粘附受体。其次，不存在的特定聚糖结构，或者在正常状态下含量较低 状态，在疾病状态下增殖或改变其顺序。最后，可能会发现糖基化的变化 在许多蛋白质中，包括高度丰富的蛋白质。这种聚糖正常水平的变化 诸如唾液酸末端的结构可能是疾病状态的标志。新的高度特异性试剂是 为了克服与疾病相关的聚糖的发现和剥削中的当前限制所必需的。 使用结构引导的遗传操纵，我们正在转化肺炎链球菌的Nanb唾液酸酶 进入高特异性亲和力试剂，以检测所有类型的糖酸修饰 和糖蛋白。因为这样的蛋白质具有类似讲座的特性，但源自酶，因此称为 “Lectenz®”。 NanbLectenz®涉及疾病糖标记检测的关键需求：即健壮和 易于产生的试剂，特异性用于检测所有类型的溶解的糖基。该试剂可以使用 在样品酶的亲和力矩阵中，与现有的基于MS的方法结合使用可以提供 链接信息。通过消除非糖基化的糖肽样品富集糖分析 肽，否则会减弱离子化效率较低的糖肽的信号。 糖基化位点映射对于充分表征和利用聚糖作为特定标记至关重要 疾病状态，但目前尚无可以检测到脱糖的聚糖的试剂，与独立于类型的类型 与唾液酸相关的连锁。目前，这是许多规格不同的试剂， 必须采用亲和力来捕获或检测所有形式的脱糖聚糖。 Lectenz®提供了与植物讲座相比的许多优势：它们的设计为高亲和力，但保留 内源性酶的独家底物特异性，它们可能是有效产生的，对于人类 同源物具有低毒性的体内携带的潜力。而Lectenz®的某些方面 发育平行于抗体进化的发展，Lectenz®具有使用蛋白质的巨大好处 具有期望特异性的天真模板。