Engineered probes for sialoglycan detection

用于唾液酸聚糖检测的工程探针

• 批准号: 10266164
• 负责人: T M Iverson
• 金额: $ 45.19万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-20 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10266164
• 关键词: Adhesives; Affinity; Antibodies; Antigens; Arthritis; Autoimmune Diseases; Autoimmunity; Bacterial Adhesins; Bacterial Attachment Site; Bacterial Infections; Binding; Binding Proteins; Biological; Biological Assay; Blood specimen; Cancer Patient; Cells; Crystallization; Detection; Development; Diagnostic; Diagnostic Reagent Kits; Disaccharides; Disease; Disease Outbreaks; Engineering; Enzymes; Glycols; Glycoproteins; Harvest; Human; Laboratories; Lectin; Letters; Libraries; Ligands; Link; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Mediating; Medical; Methods; Modification; Outcome; Plasma; Polysaccharides; Positioning Attribute; Process; Property; Protein Engineering; Proteins; Reagent; Resolution; Role; SARS coronavirus; Sampling; Severe Acute Respiratory Syndrome; Sialic Acids; Sialyltransferases; Signal Transduction; Specificity; Stains; Streptococcus adhesin; Structure; Techniques; Time; Tn antigen; Trisaccharides; Virus Diseases; Work; base; cancer biomarkers; cancer type; coronavirus receptor; experimental study; glycosylation; instrumentation; preference; receptor; scaffold; sialic acid binding Ig-like lectin; tool

PROJECT SUMMARY/ABSTRACT Sialoglycans can be poor antigens, meaning that it can be challenging to develop effective antibodies for their detection. Instead, sialoglycan mapping heavily relies upon mass spectrometry, which requires expertise and instrumentation available to few laboratories. One recent push in the field has been to harvest the breadth of selectivity found within naturally-occurring sialoglycan-binding proteins in order to develop glycan-detecting proteins. This strategy identified a number of sialoglycan-binding proteins, particularly those that bind with high affinity and narrow selectivity to sialyl-T antigen (sTa). However, major gaps remain in the spectrum of the sialoglycans that are recognized. Here, we focus on sialoglycans that are likely abundant on cells or that are prevalent in disease but that lack practical probes for their detection, specifically α2,3 linked and α2,6 linked sialoglycans. We propose to create probes that recognize these glycans by tailoring the specificity of existing sialoglycan binding proteins using structure-based protein engineering. In Aim 1, we engineer the bacterial Siglec-like adhesins to create a library of probes for tri- and tetra- saccharides, each of which binds one α2,3 linked sialoglycan with high affinity and narrow selectivity. In Aim 2, we apply engineering principles to the development of probes for α2,3 linked sialoglycans and focus on the α2,6 linked sialyl Tn antigen disaccharide, a biomarker for cancer. In Aim 3, we evaluate the utility of these probes in measuring glycans in human plasma, and cross-validated these by affinity capture and mass spectrometry. The ability to distinguish glycan abundance and repertoire in healthy donors versus cancer patients will be included as a part of this aim. Successful probes will be distributed for use both in lectin arrays and in low-throughput assays.

项目总结/摘要 唾液酸聚糖可能是较差的抗原，这意味着开发针对唾液酸聚糖的有效抗体可能是具有挑战性的。 他们的检测。相反，唾液酸聚糖图谱在很大程度上依赖于质谱法，这需要专业知识 和仪器设备，只有少数几个实验室可以使用。最近在该领域的一项努力是收获 在天然存在的唾液酸聚糖结合蛋白中发现的选择性，以开发聚糖检测 proteins.这种策略鉴定了许多唾液酸聚糖结合蛋白，特别是那些与高唾液酸聚糖结合的蛋白。 对唾液酸-T抗原（sTa）的亲和力和窄的选择性。然而，在这一领域仍然存在重大差距， 唾液酸聚糖被识别。 在这里，我们关注的唾液酸聚糖可能是丰富的细胞或普遍存在的疾病，但缺乏 用于其检测的实用探针，特别是α 2，3连接和α 2，6连接的唾液酸聚糖。我们建议开设 探针通过调整现有唾液酸聚糖结合蛋白的特异性来识别这些聚糖， 结构蛋白质工程 在目标1中，我们设计了细菌Siglec样粘附素，以创建用于三-和四- α 2，3-连接的唾液酸聚糖，每一个都以高亲和力和窄选择性结合一个α 2，3-连接的唾液酸聚糖。 在目标2中，我们将工程原理应用于α 2，3连接唾液酸聚糖的探针开发， 关注α 2，6连接唾液酸Tn抗原二糖，一种癌症生物标志物。 在目标3中，我们评估了这些探针在测量人血浆中聚糖中的实用性，并交叉验证了 通过亲和捕获和质谱分析。区分聚糖丰度和库的能力， 健康捐赠者与癌症患者的对比将作为这一目标的一部分。成功的探针将被分发 用于凝集素阵列和低通量测定。