Accurate profiles of IgA1 O-glycan heterogeneity in IgA nephropathy

IgA 肾病中 IgA1 O-聚糖异质性的准确分析

• 批准号: 7468774
• 负责人: Matthew B Renfrow
• 金额: $ 20.63万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-15 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7468774
• 关键词: Acetylgalactosamine; Address; Antibodies; Antigen-Antibody Complex; Binding; Biological Assay; Biopsy; Classification; Complex; Cyclotrons; Deposition; Diagnosis; Digestion; Disaccharides; Disease; Disease Marker; Dissociation; Electrons; End stage renal failure; Endopeptidases; Enzyme-Linked Immunosorbent Assay; Fourier Transform; Future; Galactose; Glomerulonephritis; Glycopeptides; Heterogeneity; IgA1; Immunoglobulin A; Individual; Inpatients; Invasive; Investigation; Ions; Kidney; Kidney Diseases; Laboratories; Lead; Lectin; Link; Localized; Lupus Nephritis; Mass Spectrum Analysis; Methodology; Methods; Molecular; Myeloma Proteins; N-terminal; Pathogenesis; Pathology; Patients; Pattern; Peptide Hydrolases; Polysaccharides; Population; Protein Glycosylation; Protocols documentation; Public Health; Range; Recruitment Activity; Renal Circulation; Reporting; Research; Role; Sampling; Series; Serum; Site; Techniques; Testing; Time; Trypsin; design; disorder control; glycoprotein structure; glycosylation; insight; mesangial cell; novel; research study; tandem mass spectrometry

DESCRIPTION (provided by applicant): IgA nephropathy (IgAN) is the most common form of glomerulonephritis in the world and is a leading cause of end-stage renal disease. Although the mechanisms of its pathogenesis remain unclear, reports from several laboratories indicate a key role for an aberrantly glycosylated IgA1 present in the circulation and renal deposits. This IgA1 is galactose (Gal)-deficient in some of its 3-5 O-linked glycans in the hinge region (HR) of the heavy chain. Thus, in patients with IgAN, the IgA1 O-linked glycans are truncated, with terminal N acetylgalactosamine (GalNAc) or sialylated GalNAc. A variety of methods (glycan-specific lectin binding assays, MALDI-TOF MS methods, and chromatographic analysis of glycan composition) have detected clear differences between normal healthy controls' and IgAN patients' O-glycan populations in IgA1. However, the sites of attachment of normal and Gal-deficient glycans remain to be defined. Thus, a method is needed that can provide detailed structural information about the population of O-glycopeptides within each sample. Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) provides unmatched mass accuracy in the identification of biomolecules, including the population of IgA1 O-glycopeptides enzymatically released from serum IgA1. We have recently localized (for the first time by a direct method) the multiple O-glycan chains in three different IgA1 HR glycopeptides by electron capture dissociation (ECD) FT-ICR tandem MS. This methodology enables us to test our hypothesis that the populations of serum IgA1 O-glycans differ in composition and sites of attachment in patients with IgAN when compared to normal healthy and disease controls and, furthermore, that these differences can serve as markers of the disease and define the pathological features. To establish accurate profiles of IgA1 glycoforms in IgAN patients we propose the following: 1. Provide an FT-ICR accurate mass profile of serum IgA1 glycoforms found inpatients with IgAN as well as a baseline profile from normal healthy and disease controls. and 2. Localize sites of O-glycan attachment in individual serum IgA1 glycoforms from patients with IgAN, normal healthy controls, and patients with other forms of glomerulonephritis by ECD FT-ICR tandem mass spectrometry. We perform this analysis on a total of 115 samples (45 IgAN, 25 lupus nephritis, and 45 healthy controls). These studies will provide detailed structural information about the aberrant glycosylation patterns found in IgAN, give insights into the pathogenesis of this disease, and may provide a novel noninvasive method for diagnosis of IgAN. PUBLIC HEALTH RELEVANCE: This two year project seeks to use novel mass spectrometry techniques to define the sites of aberrant O-glycosylation of serum IgA1 in patients with IgA nephropathy (IgAN). This can only be done in context of understanding the sites of IgA1 O-glycosylation in normal healthy controls and disease controls. The results will provide insights into the pathology of the IgAN and may provide a non-invasive method of diagnosing th3disease.

描述(申请人提供)：免疫球蛋白肾病(IgAN)是世界上最常见的肾小球肾炎形式，是终末期肾脏疾病的主要原因。尽管其发病机制尚不清楚，但几个实验室的报告表明，糖基化的IgA1在循环和肾脏沉积中起着关键作用。该IgA1在重链的铰链区(HR)的一些3-5 O连接的糖链中缺乏半乳糖(Gal)。因此，在IgAN患者中，IgA1 O连接的糖链被截断，末端为N-乙酰半乳糖胺(GalNAc)或唾液酸半乳糖。多种方法(糖链特异性凝集素结合分析、MALDI-TOF MS方法和糖链组成的层析分析)已经检测到正常健康对照组和IgAN患者的O-糖群在IgA1上的明显差异。然而，正常和半乳糖缺乏的多糖的附着位置仍有待确定。因此，需要一种方法来提供关于每个样品中O-糖肽种群的详细结构信息。傅立叶变换离子回旋共振质谱仪(FT-ICR MS)在鉴定生物分子方面提供了无与伦比的质量准确性，包括从血清中酶解释放的IgA1 O-糖肽。我们最近通过电子捕获解离(ECD)FT-ICR串联MS定位了(首次通过直接方法)三种不同的IgA1 HR糖肽中的多个O-糖链。这种方法使我们能够检验我们的假设，即与正常健康和疾病对照组相比，IgAN患者血清IgA1O-糖链的组成和附着位置不同，而且，这些差异可以作为疾病的标志并确定病理特征。为了建立IgAN患者血清IgA1糖型的准确图谱，我们提出如下建议：1.提供在IgAN患者中发现的血清IgA1糖型的FT-ICR准确质量图谱以及正常健康和疾病对照的基线图谱。2.用ECD FT-ICR串联质谱仪对IgAN患者、正常对照组和其他类型肾小球肾炎患者血清IgA1糖体中O-葡聚糖的附着部位进行定位。我们对115例样本(45例IgAN、25例狼疮性肾炎和45例健康对照)进行了分析。这些研究将提供有关在IgAN中发现的异常糖基化模式的详细结构信息，为该疾病的发病机制提供见解，并可能为IgAN的诊断提供一种新的非侵入性方法。公共卫生相关性：这个为期两年的项目寻求使用新的质谱学技术来确定IgA肾病(IgAN)患者血清IgA1异常O-糖基化的位置。这只能在了解正常健康对照和疾病对照中的IgA1O-糖基化位点的情况下进行。这一结果将为了解IgAN的病理机制提供洞察力，并可能提供一种诊断这种疾病的非侵入性方法。