Engineered probes for sialoglycan detection

用于唾液酸聚糖检测的工程探针

• 批准号: 10653008
• 负责人: T M Iverson
• 金额: $ 45.02万
• 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 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10653008
• 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; Detection; Development; Diagnostic; Diagnostic Reagent Kits; Disaccharides; Disease; Disease Outbreaks; Engineering; Enzymes; Glycols; Glycoproteins; Harvest; Human; Laboratories; Lectin; Letters; Libraries; Ligands; Link; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Measures; Mediating; Medical; Methods; Modification; Outcome; Plasma; Polysaccharides; Positioning Attribute; Process; Property; Protein Engineering; Protein Glycosylation; 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; cancer biomarkers; cancer type; coronavirus receptor; detection limit; diagnostic tool; experimental study; glycosylation; instrumentation; preference; protein purification; receptor; scaffold; sialic acid binding Ig-like lectin

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.

项目概要/摘要 唾液酸聚糖可能是较差的抗原，这意味着开发有效的抗体可能具有挑战性 相反，唾液酸聚糖图谱很大程度上依赖于质谱分析，这需要专业知识。 很少有实验室可以使用仪器和仪器，该领域最近的一项努力是扩大范围。 在天然存在的唾液酸聚糖结合蛋白中发现选择性，以开发聚糖检测 该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) 的亲和力和窄选择性然而，在谱中仍然存在重大差距。 被识别的唾液酸聚糖。 在这里，我们重点关注细胞中可能丰富的唾液酸聚糖或疾病中普遍存在但缺乏的唾液酸聚糖。 我们建议创建用于检测的实用探针，特别是 α2,3 连接和 α2,6 连接唾液聚糖。 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的探针 基于结构的蛋白质工程。 在目标 1 中，我们设计了细菌 Siglec 样粘附素，以创建用于三和四的探针库。 糖类，每一种都以高亲和力和窄选择性结合一个 α2,3 连接的唾液酸聚糖。 在目标 2 中，我们应用工程原理来开发 α2,3 连接唾液酸聚糖和 重点关注 α2,6 连接唾液酸 Tn 抗原二糖，这是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的效用，并进行了交叉验证 这些通过亲和捕获和质谱分析来区分聚糖丰度和谱系。 作为这一目标的一部分，将分发健康捐赠者与癌症患者。 用于凝集素阵列和低通量测定。