Post-Translational Modification of a Type 1 Diabetes Autoantigen

1 型糖尿病自身抗原的翻译后修饰

• 批准号: 8279897
• 负责人: THOMAS DELONG
• 金额: $ 12.24万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8279897
• 关键词: Aldehydes; Amines; Amino Acid Motifs; Amino Acids; Antigen Targeting; Antigen-Presenting Cells; Antigens; Autoantigens; Binding; Biochemical; Biotin; CD4 Positive T Lymphocytes; Cell Extracts; Cells; Chemicals; Chromogranin A; Data; Disease; Enzymes; Epitopes; Fractionation; Generations; Goals; Histocompatibility Antigens Class II; In Vitro; Incubated; Insulin-Dependent Diabetes Mellitus; Label; Ligands; Location; Lysine; MHC Class II Genes; Major Histocompatibility Complex; Mediating; Methods; Modification; Nature; Pancreas; Pathogenesis; Peptides; Play; Post-Translational Protein Processing; Proteins; Proteomics; Reagent; Research; Research Personnel; Role; T cell response; T-Lymphocyte; T-Lymphocyte Epitopes; Testing; Tissues; Transglutaminases; antigen binding; autoreactive T cell; cell type; covalent bond; functional group; neoplastic cell; research study; transglutaminase 2

DESCRIPTION (provided by applicant): The goal of this project is to use proteomics to investigate a post-translational modification that is associated with Chromogranin A, the recently discovered autoantigen for the diabetogenic CD4 T cell clone BDC-2.5. Preliminary data shows that the antigenicity of ChgA-derived peptides can be increased significantly upon treatment with the enzyme tissue transglutaminase (TGase). Additional data indicates that natural antigen obtained from ?-cell tumors contains an aldehyde or keto functional group. In specific aim 1 we will investigate the chemical nature of the TGase-treated peptide. For this we will test the effect of TGase treatment on altered ChgA peptides, and use proteomic purification, mass spectrometric and biochemical derivatization methods to investigate the role of peptide TGase interactions. In specific aim 2 we will investigate the presence of an aldehyde or keto group in antigen obtained from pancreatic ?-cells. For this we will use biochemical derivatization and mass spectrometric methods to purify derivatized antigen and to identify the chemical nature of the modification. TGase-treated peptides and proteins containing an aldehyde or keto group can form covalent bonds with primary amines, such as lysines. The major histocompatibility complex class II (MHC II) molecule I-Ag7 contains several lysine residues and in specific aim 3 we will investigate whether TGase-treated peptides or ?-cell antigens form a covalent bond with I-Ag7. For this we will covalently label I-Ag7 using ?-cell antigen and biotin-labeled peptides that were treated with TGase. Proteomic and mass spectrometric methods will be used to purify and identify the covalent attachment. Post-translationally modified antigens in type 1 diabetes (T1D) have so far not been described and could play be a key role in the initiation of this disease. PUBLIC HEALTH RELEVANCE: In this project the investigator proposes to study the role of post-translational modifications in Chromogranin A (ChgA), the recently identified autoantigen for the diabetogenic T cell clone BDC-2.5. The modifications to be investigated are (1) the enzymatic modification of ChgA-derived peptides with the enzyme tissue transglutaminase, and (2) the naturally occurring presence of a carbonyl functional group in ChgA isolated from pancreatic ?-cells. Additionally, the interactions of the class major histocompatibility complex (MHC I) with modified antigens will be examined.

描述（由申请人提供）：该项目的目标是利用蛋白质组学来研究与 Chromogranin A 相关的翻译后修饰，Chromogranin A 是最近发现的致糖尿病 CD4 T 细胞克隆 BDC-2.5 的自身抗原。初步数据表明，经组织转谷氨酰胺酶 (TGase) 处理后，ChgA 衍生肽的抗原性可显着增加。附加数据表明从α细胞肿瘤获得的天然抗原含有醛或酮官能团。在具体目标 1 中，我们将研究 TGase 处理的肽的化学性质。为此我们来测试一下效果 TGase 处理改变的 ChgA 肽，并使用蛋白质组纯化、质谱和生化衍生化方法研究肽 TGase 相互作用的作用。在具体目标 2 中，我们将研究从胰腺 β 细胞获得的抗原中是否存在醛基或酮基。为此，我们将使用生化衍生化和质谱方法来纯化衍生化抗原并鉴定修饰的化学性质。经过 TGase 处理的含有醛基或酮基的肽和蛋白质可以与伯胺（例如赖氨酸）形成共价键。主要组织相容性复合体 II 类 (MHC II) 分子 I-Ag7 含有几个赖氨酸残基，在具体目标 3 中，我们将研究 TGase 处理的肽或 β-细胞抗原是否与 I-Ag7 形成共价键。为此，我们将使用 β-细胞抗原和经 TGase 处理的生物素标记肽共价标记 I-Ag7。将使用蛋白质组学和质谱方法来纯化和鉴定共价连接。迄今为止，1 型糖尿病 (T1D) 中的翻译后修饰抗原尚未被描述，但可能在这种疾病的发生中发挥关键作用。 公共健康相关性：在该项目中，研究者建议研究嗜铬蛋白 A (ChgA) 翻译后修饰的作用，ChgA 是最近发现的致糖尿病 T 细胞克隆 BDC-2.5 的自身抗原。要研究的修饰是(1)用组织转谷氨酰胺酶对ChgA衍生肽进行酶促修饰，以及(2)从胰腺β细胞分离的ChgA中天然存在的羰基官能团。此外，还将检查主要组织相容性复合物 (MHC I) 类与修饰抗原的相互作用。