ROLE OF Ii CLEAVAGE AND RELEASE IN ANTIGEN PRESENTATION

该项目的长期目标是确定 抗原处理和提呈过程中的不变链(I-I)。至 为实现这一目标，提出了以下目标：(1)界定 导致I-I从II类MHC裂解和释放的条件 分子；(2)表征在过程中产生的切割片段 导致I-I释放的蛋白降解事件；(3)鉴定 I-I区(S)，与II类MHC或抗原相关(S) 结合部位；以及，(4)确定多肽是否与II类结合 MHC分子依赖于I-I的去除或通过I-I的去除而增强。至 解决目标1，拟议的实验旨在模仿 内体环境(低pH和/或存在特定的蛋白酶) 以诱导II类MHC释放I-I 免疫沉淀和SDS-PAGE分析。作为目标2的一部分， 在I-I的切割和释放过程中产生的片段将是 用N端抗体进行免疫印迹鉴定 I-I和兔抗血清中的(VicY1)和C末端(E1)表位 与I-I内不同区域相对应的合成肽。部分 片段的N-末端测序将对片段进行 通过反相高效液相色谱或2D电泳法分离 电印迹。在目标3中，分离的I-I片段导致以下结果 将检查I-I从II类MHC分子中的裂解和释放 因为它们有能力阻止肽的呈现，这可能是由于 结合到第II类MHC分子上的抗原结合部位；因此， 识别阻止脱位的I-I序列。拥有 阐明了导致I-I去除的条件，在目标4中 条件将在流感多肽存在的情况下重现 用交联剂衍生并加碘。它们的绑定 在紫外光诱导的交联之后，将进行评估 暴露，通过抗II类抗体的免疫沉淀， 电泳法和放射自显影技术。或者，绑定可以是 通过凝胶过滤将结合肽从游离肽中分离出来。 这些研究得出的答案将起到双重作用：(A) 提高我们对免疫学中一个基本问题的认识和(B) 确定II中的一个区域，该区域可能在合成中起骨干作用 基于多肽的疫苗。

The long-term objective of this project is to define the role of the invariant chain (I-i) in antigen processing and presentation. To approach this goal the following aims are proposed: (1) To define the conditions that lead to cleavage and release of I-i from class II MHC molecules; (2) To characterize the cleavage fragments generated during the proteolytic events which lead to I-i release; (3) To identify the region(s) within I-i which associate(s) with class II MHC or the antigen binding site; and, (4) To determine whether peptide binding to class II MHC molecules depends upon or is enhanced by the removal of I-i. To address Aim 1, the proposed experiments are aimed at mimicking the endosomal environment (low pH and/or the presence of specific proteases) to induce I-i release from class II MHC as detected by immunoprecipitation and SDS-PAGE analysis. As part of Aim 2, the fragments generated during cleavage and release of I-i will be characterized through Western blotting with antibodies to N-terminal (VicY1) and C-terminal (E1) epitopes in I-i as well as rabbit antisera to synthetic peptides corresponding to various regions within I-i. Partial N-terminal sequencing of the fragments will be performed on fragments isolated by either reverse phase HPLC or 2D electrophoresis and electroblotting. In Aim 3, isolated I-i fragments which result following cleavage and release of I-i from class II MHC molecules will be examined for their ability to block peptide presentation presumably as a result of binding to the antigen binding site on class II MHC molecules;. thus, identifying the I-i sequence which blocks the desetope. Having elucidated the conditions which lead to I-i removal, in Aim 4 those conditions will be reproduced in the presence of influenza peptides derivatized with a crosslinking reagent and iodinated. Their binding will then be assessed, subsequent to crosslinking induced by U.V. light exposure, through immunoprecipitation with anti-class II antibodies, electrophoresis, and autoradiography. Alternatively, binding can be examined through gel filtration to separate bound from free peptides. The answers derived from these studies will serve a dual role: (a) enhance our understanding of a fundamental question in immunology and (b) identify a region in Ii which might serve as a backbone in the synthesis of peptide-based vaccines.