High-specificity affinity reagents for the detection of glycan sialylation

用于检测聚糖唾液酸化的高特异性亲和试剂

• 批准号: 8994739
• 负责人: Loretta Yang
• 金额: $ 34.93万
• 依托单位: GLYCOSENSORS AND DIAGNOSTICS, LLC
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-15 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8994739
• 关键词: Address; Affinity; Antibodies; Antigens; Binding; Biological Assay; Biological Markers; Carbohydrates; Cell surface; Complex; Computer Simulation; Detection; Development; Directed Molecular Evolution; Disease; Disease Marker; Engineering; Enzymes; Epitopes; Eukaryota; Evolution; Galactose; Glycopeptides; Glycoproteins; Glycoside Hydrolases; Health; Homologous Gene; Human; Kinetics; Lectin; Libraries; Ligands; Link; Lipids; Location; Malignant neoplasm of prostate; Mass Spectrum Analysis; Metabolism; Methods; Modification; Mutagenesis; Nature; Neuraminic Acids; Neuraminidase; Nucleic Acids; Phase; Plant Lectins; Polymers; Polysaccharides; Positioning Attribute; Preparation; Process; Proliferating; Property; Protein Subunits; Proteins; Randomized; Reagent; Rheumatoid Arthritis; Sampling; Sialic Acids; Specificity; Spectrometry; Staging; Streptococcus pneumoniae; Structure; Substrate Specificity; Surface; Techniques; Tissue Sample; Toxic effect; Variant; Western Blotting; Yeasts; adhesion receptor; base; carbohydrate binding protein; design; genetic linkage analysis; genetic manipulation; glycosylation; glycosyltransferase; histological stains; histological studies; improved; in vivo; mutant; pathogen; preference; response; sialylation

DESCRIPTION (provided by applicant): 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 will convert the NanB sialidase from S. pneumococcus into a high-specificity affinity reagent for the detection 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(r)". A NanB Lectenz(r) addresses a key need in disease glycomarker detection: namely, a robust and easy to produce reagent specific for �3-linked sialic acid. This reagent could be employed in an affinity matrix for sample enrichment, which in conjunction with existing MS based methods could provide linkage information. It could also be employed in histological studies, or Western blots, etc. Lectenz(r) offer numerous advantages over plant lectins: they are engineered to be high affinity and yet retain the exquisite substrate specificityof 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(r) development parallel those of antibody evolution, Lectenz(r) have the tremendous benefit of employing a protein naive template that has the desired specificity.

描述（由申请人提供）：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，因此它们对于正常代谢过程的控制至关重要，相反，它们充当病原体粘附受体。其次，在正常状态下不存在或含量较低的特定聚糖结构在疾病状态下会增殖或改变其序列。最后，糖基化的变化可能存在于许多蛋白质中，包括那些高度丰富的蛋白质。因此，聚糖结构（例如末端唾液酸）正常水平的变化可能是疾病状态的标志。为了克服当前在疾病相关聚糖的发现和利用方面的局限性，需要新的高度特异性的试剂。使用结构引导 通过基因操作，我们将肺炎球菌的 NanB 唾液酸酶转化为高特异性亲和试剂，用于检测糖肽和糖蛋白的唾液酸修饰。由于这种蛋白质具有凝集素样特性，但源自酶，因此被称为“Lectenz(r)”。 NanB Lectenz(r) 解决了疾病糖标记物检测的关键需求：即一种稳定且易于生产的 3-连接唾液酸特异性试剂。该试剂可用于亲和基质中进行样品富集，与现有的基于 MS 的方法相结合可以提供连锁信息。它还可用于组织学研究或蛋白质印迹等。与植物凝集素相比，Lectenz(r) 具有许多优势：它们被设计为具有高亲和力，但保留了内源酶的精致底物特异性，它们可以有效地生产，并且对于人类同系物有可能以低毒性在体内使用。尽管 Lectenz(r) 开发的某些方面与抗体进化相似，但 Lectenz(r) 具有采用具有所需特异性的蛋白质天然模板的巨大优势。