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/文摘