Post-Translational Modification of a Type 1 Diabetes Autoantigen

描述(申请人提供)：这个项目的目标是利用蛋白质组学来研究与嗜铬粒蛋白A相关的翻译后修饰，嗜铬粒蛋白A是最近发现的糖尿病致CD4T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衍生肽的抗原性显著增强。更多的数据表明，从？细胞肿瘤中获得的天然抗原含有一个醛或酮官能团。在特定的目标1中，我们将研究TGase处理的多肽的化学性质。为此，我们将测试其效果 TGase处理改变的ChgA多肽，并利用蛋白质组学纯化、质谱学和生化衍生化方法研究多肽TGase相互作用的作用。在特定目标2中，我们将调查从胰腺细胞获得的抗原中是否存在乙醛或酮基。为此，我们将使用生化衍生化和质谱学方法来纯化衍生化抗原，并鉴定修饰的化学性质。经TGase处理的含有醛或酮基的多肽和蛋白质可以与伯胺(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-Ag7含有几个赖氨酸残基，在特定的目标3中，我们将研究TGase处理的多肽或？细胞抗原是否与I-Ag7形成共价键。为此，我们将使用TGase处理过的？细胞抗原和生物素标记的多肽来共价标记I-Ag7。蛋白质组学和质谱学方法将被用来纯化和鉴定共价连接。到目前为止，1型糖尿病(T1D)的翻译后修饰抗原还没有被描述，可能在这种疾病的发生中发挥关键作用。 公共卫生相关性：在这个项目中，研究人员建议研究嗜铬粒蛋白A(ChgA)翻译后修饰的作用，ChgA是最近发现的糖尿病T细胞克隆BDC-2.5的自身抗原。将要研究的修饰包括(1)用酶组织转谷氨酰胺酶对ChgA衍生的多肽进行酶修饰，以及(2)从胰腺细胞中分离的ChgA中自然存在的羰基官能团。此外，还将检查主要组织相容性复合体(MHC I)类与修饰抗原的相互作用。

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. 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.