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.

项目摘要/摘要 唾液酸可能是较差的抗原，这意味着开发有效的抗体可能是挑战 他们的检测。相反，Sialoglycan映射在很大程度上依赖于质谱，这需要专业知识 和仪器可用于少数实验室。该领域最近的一个推动是收获宽度 在天然存在的唾液聚糖结合蛋白中发现的选择性，以开发聚糖检测 蛋白质。该策略确定了许多唾液酸结合蛋白，尤其是那些与高结合的蛋白 亲和力和对SiAllyl-T抗原（STA）的选择性狭窄。但是，主要差距仍然存在于 被认可的唾液。 在这里，我们专注于可能富含细胞或在疾病中流行但缺乏细胞的唾液聚糖。 它们检测的实际问题，特别是α2,3连接的α2,6链接的唾液聚糖。我们建议创建 通过使用使用现有的唾液聚糖结合蛋白的特异性来识别这些聚糖的问题 基于结构的蛋白质工程。 在AIM 1中，我们设计了细菌Siglec样的粘附剂，以创建三库和四方的问题库。 糖，每种糖结合了一个α2,3与高亲和力和狭窄的选择性相关的唾液酸。 在AIM 2中，我们将工程原理应用于α2,3链接的唾液和的问题的发展 专注于α2,6连接的siAllyl TN抗原二糖，这是一种癌症的生物标志物。 在AIM 3中，我们评估了这些问题在测量人血浆中的聚糖中的效用，并进行了交叉验证 这些通过亲和力捕获和质谱法。区分聚糖丰度和曲目的能力 健康的捐助者与癌症患者将被包括在内。成功的问题将分发 用于在讲座阵列和低通量测定中使用。