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: 9883115
• 负责人: Sheng-Cheng Wu
• 金额: $ 74.06万
• 依托单位: GLYCOSENSORS AND DIAGNOSTICS, LLC
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9883115
• 关键词: Address; Advanced Development; Affinity; Affinity Chromatography; Animals; Antibodies; Attenuated; Binding; Biological; Biological Markers; Biological Models; Bovine Serum Albumin; Carbohydrates; Cell Extracts; Cell surface; Cells; Chromatography; Data; Detection; Development; Disease; Disease Marker; Disease model; Engineering; Enzymes; Evolution; Force of Gravity; Gene Library; Genomics; Glycopeptides; Glycoproteins; Homologous Gene; Human; Intervention; Kinetics; Lectin; Ligand Binding; Location; MCF7 cell; Mass Spectrum Analysis; Medicine; Metabolism; Methods; Modification; Mutagenesis; Neuraminidase; Peptides; Performance; Phase; Plant Lectins; Plant Resins; Polysaccharides; Preparation; Procedures; Process; Production; Program Development; Proliferating; Property; Proteins; Proteomics; Protocols documentation; Reagent; Reporting; Research; Research Infrastructure; Risk; Sampling; Sialic Acids; Signal Transduction; Site; Sodium Chloride; Specificity; Streptococcus pneumoniae; Structure; Substrate Specificity; Techniques; Technology; Toxic effect; Validation; Variant; adhesion receptor; alpha-Fetoproteins; animal tissue; asialofetuin; base; epigenomics; fast protein liquid chromatography; formative assessment; genetic manipulation; glycosylation; high throughput technology; human tissue; improved; in vivo; interest; ionization; metabolomics; model development; novel; pathogen; phenomics; scale up; sialylation; tool; transcriptomics

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.

项目总结 多糖具有几种独特的性质，使其成为疾病生物标记物具有吸引力。 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 它们在控制正常代谢过程中起着至关重要的作用，反过来，它们也起病原体的作用。 黏附受体。其次，特定的糖链结构不存在，或者在正常情况下含量很低。 在疾病状态下，它们的状态、增殖或改变它们的序列。最后，可以发现糖基化的变化。 在许多蛋白质中，包括那些高度丰富的蛋白质。因此，糖链的正常水平发生了变化 末端唾液酸等结构可能是疾病状态的标志。新的高特异性试剂是 以克服目前在发现和开发与疾病相关的多糖方面的限制。 使用结构引导的遗传操作，我们正在从肺炎链球菌转化纳米B唾液酸酶 成为一种高特异性亲和试剂，用于检测糖肽的所有类型的唾液酸修饰 和糖蛋白。因为这种蛋白质具有类似凝集素的性质，但它是从一种酶中提取的，所以它被称为 A“Lectenz®”。NanB Lectenz®解决了疾病糖标记物检测的关键需求：即，坚固和 易于生产的试剂，专用于检测所有类型的唾液酸聚糖。这种试剂可以用来 在用于样品浓缩的亲和力矩阵中，与现有的基于MS的方法相结合可以提供 链接信息。糖肽样品浓缩通过消除非糖基化来辅助血糖分析 多肽，否则将衰减来自低电离效率的糖肽的信号。 糖基化位点作图对于充分表征和利用糖链作为特异性标记是必不可少的。 疾病状态，但目前还没有试剂可以检测唾液酸聚糖，独立于类型 与唾液酸有关的键。因此，目前，一些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聚糖。 与植物凝集素相比，Lectenz®提供了许多优势：它们的设计具有高亲和力，但仍能保留 内源性酶的精致底物专一性，它们可以有效地产生，并且对人类来说 同系物有可能在低毒性的情况下用于体内。鉴于Lectenz®的某些方面 与抗体进化平行发展，Lectenz®具有使用蛋白质的巨大好处 天真的模板，具有所需的专一性。