Post-Translational Modification of a Type 1 Diabetes Autoantigen

描述（由申请人提供）：本项目的目标是使用蛋白质组学研究与嗜铬粒蛋白A相关的翻译后修饰，嗜铬粒蛋白A是最近发现的致糖尿病CD 4 T细胞克隆BDC-2.5的自身抗原。初步数据显示，ChgA衍生肽的抗原性可在用酶组织转氨酶（TGase）处理后显著增加。额外的数据表明，从？细胞肿瘤含有醛或酮官能团。在具体目标1中，我们将研究TGase处理的肽的化学性质。为此，我们将测试 的TGase处理改变ChgA肽，并使用蛋白质组学纯化，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？细胞为此，我们将使用生物化学衍生和质谱方法纯化衍生抗原并鉴定修饰的化学性质。含有醛或酮基的经TGase处理的肽和蛋白质可与伯胺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-Ag 7含有几个赖氨酸残基，在具体的目的3中，我们将研究TGase处理的肽或？细胞抗原与I-Ag 7形成共价键。为此，我们将共价标记I-Ag 7使用？细胞抗原和用TGase处理的生物素标记的肽。蛋白质组学和质谱方法将用于纯化和鉴定共价连接。1型糖尿病（T1 D）中的免疫后修饰抗原迄今尚未被描述，并且可能在该疾病的起始中起关键作用。

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.