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.

项目概要 聚糖具有多种独特的特性，使其作为疾病生物标志物的开发具有吸引力。 首先，它们在细胞表面的位置使它们成为细胞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，它们充当病原体 其次，正常情况下不存在或含量较低的特定聚糖结构。 状态、增殖或改变它们在疾病状态下的序列，最后，可能会发现糖基化的变化。 许多蛋白质，包括那些高度丰富的蛋白质，因此聚糖的正常水平发生变化。 末端唾液酸等结构可能是疾病状态的标记。 为了克服目前在疾病相关聚糖的发现和利用方面的限制。 使用结构引导的基因操作，我们正在将肺炎链球菌中的 NanB 唾液酸酶转化为 成为高特异性亲和试剂，用于检测糖肽的所有类型的唾液酸修饰 由于这种蛋白质具有凝集素样特性，但源自酶，因此被称为糖蛋白。 “Lectenz®” NanB Lectenz® 解决了疾病糖标记物检测的关键需求：即强大且可靠的检测方法。 易于生产用于检测所有类型唾液酸化聚糖的试剂。 在用于样品富集的亲和矩阵中，与现有的基于 MS 的方法相结合可以提供 糖肽样品富集通过消除非糖基化来辅助糖组分析。 肽，否则会减弱来自电离效率低的糖肽的信号。 糖基化位点图谱对于充分表征和利用聚糖作为特定标记物至关重要 疾病状态，但目前不存在可以检测唾液酸化聚糖的试剂，无论其类型如何 因此，目前许多试剂具有不同的特异性和作用。 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：它们经过精心设计，具有高亲和力，同时保留 由于内源酶的精细底物特异性，它们可以被有效地生产，并且对于人类 除 Lectenz® 的某些方面外，同系物具有低毒性的体内应用潜力。 与抗体进化平行的发展，Lectenz® 具有使用蛋白质的巨大好处 具有所需特异性的朴素模板。