Analytical tools for the analysis of clustered O-glycans in clinical samples

用于分析临床样品中聚集的 O-聚糖的分析工具

• 批准号: 9338092
• 负责人: Matthew B Renfrow
• 金额: $ 3.5万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-15 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9338092
• 关键词: Academy; Acetylgalactosamine; Address; Amino Acid Sequence; Amino Acids; Antibodies; Antigen-Antibody Complex; Area; Autoimmune Diseases; Biological Process; Biomedical Research; Biopsy; Chronic; Chronic Kidney Failure; Clinical; Cluster Analysis; Communicable Diseases; Complex; Data; Databases; Detection; Development; Diagnostic; Disease; Disease Outcome; Disease Progression; Enzyme-Linked Immunosorbent Assay; Epitope Mapping; Epitopes; Exhibits; Fostering; Funding; Future; Galactose; Glomerulonephritis; Glycobiology; Glycopeptides; Glycoproteins; Health; Heterogeneity; Housing; IgA1; Immunoassay; Immunoglobulin A; Immunoglobulins; Individual; Inflammatory; Kidney; Kidney Diseases; Knowledge; Label; Laboratories; Lead; Lectin; Maps; Methods; Modification; Molecular; National Research Council; Outcome; Pathologic Processes; Patients; Peptides; Phenotype; Polymers; Polysaccharides; Population; Post-Translational Protein Processing; Progress Reports; Protein Analysis; Protein Glycosylation; Proteins; Publishing; Reagent; Reference Standards; Research; Role; Running; Sampling; Series; Serum; Site; Tandem Repeat Sequences; Technology; Testing; Time; Tn antigen; United States National Academy of Sciences; Viral Tumor Antigens; Work; analytical tool; base; biomarker discovery; clinical biomarkers; cohort; disorder control; glycosylated serum protein; glycosylation; insight; interest; ionization; monomer; multidisciplinary; novel; prognostic significance; public health relevance; research study; sialylation; specific biomarkers; technology development; tool

 DESCRIPTION (provided by applicant): Glycosylation is one of the most common post-translational modifications of proteins; over half of mammalian proteins are glycosylated. Patients with several autoimmune disorders, chronic inflammatory diseases, and some infectious diseases exhibit abnormal glycosylation of serum immunoglobulins and other glycoproteins. The biological functions of these modifications in health and disease continue to be a significant area of interest in biomedical research. Specifically, the task of defining site-specific glycoprotein heterogeneity is recognized as an area that still needs a considerable amount of effort to fully understand the role of glycan heterogeneity. We have developed robust workflows for the analysis of the IgA1 clustered O-glycan heterogeneity in clinical samples from patients with a chronic kidney disease, IgA nephropathy (IgAN), the most predominant form of glomerulonephritis in the world. Our outcome-based approach for profiling the entire range of IgA1 O-glycoforms from clinical samples has made significant contributions to the field of IgAN research, but also to the broader field of profiling protein glycosylation heterogeneity. Our findings have provided a means of detecting shifts in glycan heterogeneity through the application of relative quantitative, label-free analysis of the entire population of IgA1 glycofors in a single serum sample. Based on our novel workflows, we have identified subsets of IgA1 O-glycoforms in patients with IgAN that are significantly increased/decreased in their relative abundance compared to healthy controls. Going forward, absolute quantitation of individual O-glycoforms is needed. There are no established methods for the quantitative analysis of intact clustered O-glycopeptides or O-glycoproteins with Core 1 type O-glycans. Current quantitative immunoassays for detection of aberrant Core 1 glycans (Tn and sialyl-Tn antigens) are not well defined in terms of what protein, amino acid, or adjacent O-glycan context they recognize. In this proposal, we will adapt our existing methods for relative quantitative profiles into absolute quantitative strategies with O-glycopeptide reference standards. We hypothesize that this approach will provide a needed tool for heavily O-glycosylated proteins to become reliable clinical biomarkers. We have successfully synthesized in-house a series of complex clustered O-glycopeptides to serve as reference standards and novel reagents for the analysis of clustered O-glycan-specific epitopes. We will apply our quantitative strategies to the analysis of two IgAN patient cohorts to identify specific Tn antigen-presenting IgA1 O-glycoforms that lead to the formation of nephritogenic immune complexes. We will also make use of our accumulated knowledge of clustered O-glycan heterogeneity in tandem repeat amino acid sequences to characterize the accessible Tn antigen epitopes for recognition by naturally occurring and commercially available antibodies.

 描述（由应用程序提供）：糖基化是蛋白质最常见的翻译后修饰之一；超过一半的哺乳动物蛋白是糖基化的。患有多种自身免疫性疾病，慢性炎性疾病和一些传染病的患者表现出血清免疫球蛋白和其他糖蛋白的异常糖基化。这些修饰在健康和疾病中的生物学功能仍然是生物医学研究中的重要领域。具体而言，定义特定地点糖蛋白异质性的任务被认为是一个仍然需要考虑的努力来充分了解聚糖异质性的作用的领域。我们开发了强大的工作流程，用于分析来自慢性肾脏疾病，IGA肾病（IGAN）患者的临床样本中IGA1聚集的O-聚糖异质性，这是世界上最主要的肾小球肾炎形式。我们基于结果的方法，用于分析临床样本的整个IgA1 O-糖型范围，这对IGAN研究领域做出了重大贡献，但也对较广泛的分析蛋白糖基化异质性的领域做出了贡献。我们的发现提供了一种通过对单个血清样品中IgA1糖型整个种群进行相对定量的无标签分析来检测聚糖异质性转移的方法。基于我们的新工作流程，我们已经确定了与健康对照组相比，IGAN患者的IgA1 O-糖型子集的相对丰度显着增加/减少。展望未来，需要对单个O-糖型的绝对定量。没有建立的方法来定量分析完整的O-糖肽或具有核心1型O-聚糖的O-糖蛋白。当前用于检测异常核心1聚糖（TN和siAllyl-TN抗原）的定量免疫测定尚未很好地定义，这些蛋白质，氨基酸或邻近的O-散甘糖环境都无法识别。在此提案中，我们将使用现有的方法将相对定量概况的方法调整为具有O-糖肽参考标准的绝对定量策略。我们假设这种方法将为大量的O-糖基化蛋白提供可靠的临床生物标志物提供所需的工具。我们已经成功合成了内部一系列复杂的O-糖肽，以作为参考标准和新型试剂，用于分析簇的O-聚糖特异性表位。我们将应用定量策略对两个IGAN患者队列的分析，以鉴定特定的TN抗原呈递IgA1 O-糖型，从而导致形成肾脏源性免疫复合物。我们还将利用串联重复氨基酸序列中的O-GlyCan异质性的积累知识，以表征可访问的TN抗原表位，以通过自然发生和市售的抗体识别。