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

 描述(申请人提供)：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质是糖基化的。一些自身免疫性疾病、慢性炎症性疾病和一些感染性疾病的患者表现出血清免疫球蛋白和其他糖蛋白的糖基化异常。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一个重要领域。具体地说，确定特定部位糖蛋白异质性的任务被认为是一个仍然需要大量努力才能充分理解多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--世界上最主要的肾小球肾炎--IgA肾病(IgAN)患者的临床样本中的IgA1聚集型O-葡聚糖异质性。我们基于结果的方法从临床样本中分析整个范围的IgA1O-糖型，为IgAN研究领域做出了重大贡献，也为分析蛋白质糖基化异质性的更广泛领域做出了贡献。我们的发现提供了一种方法，通过应用相对定量的、无标记的分析单个血清样本中的整个IgA1糖基来检测糖链异质性的变化。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患者中IgA1 O-糖型的相对丰度显著增加/减少的亚群。展望未来，需要对单个O-糖型进行绝对定量。对于完整的簇状O-糖肽或带有核心1型O-糖链的O-糖蛋白的定量分析，目前还没有成熟的方法。目前用于检测异常核心1多聚糖(TN和sialyl-Tn抗原)的定量免疫分析方法不能很好地根据它们识别的蛋白质、氨基酸或邻近的O-糖链环境进行定义。在这项提案中，我们将把现有的相对定量图谱方法调整为具有O-糖肽参考标准的绝对定量策略。我们假设，这种方法将为重度O-糖基化蛋白成为可靠的临床生物标记物提供必要的工具。我们已经成功地合成了一系列复杂的簇状O-糖肽，作为分析簇状O-糖特异性表位的参考标准和新型试剂。我们将应用我们的定量策略对两个IgAN患者队列进行分析，以确定导致肾炎免疫复合体形成的特定TN抗原递呈的IgA1O-糖体。我们还将利用我们积累的在串联重复氨基酸序列中聚集的O-葡聚糖异质性的知识来表征可获得的TN抗原表位，以供自然产生的和商业上可用的抗体识别。

 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.