ROLE OF Ii CLEAVAGE AND RELEASE IN ANTIGEN PRESENTATION

该项目的长期目标是确定 不变链（I-i）在抗原加工和呈递中的作用。 到 为了实现这一目标，提出了以下目标：（1）定义 导致I-i从II类MHC裂解和释放的条件 分子;（2）为了表征切割过程中产生的切割片段 导致I-i释放的蛋白水解事件;（3）为了鉴定I-i释放的蛋白水解事件， I-i内与II类MHC或抗原相关的区域 结合位点;以及（4）确定肽是否结合II类 MHC分子依赖于I-i的去除或通过I-i的去除而增强。 到 地址目标1，拟议的实验旨在模仿 内体环境（低pH和/或存在特定蛋白酶） 诱导I-i从II类MHC释放，如通过 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被 通过蛋白质印迹法用N-末端 I-i中的VicY 1和C-末端（E1）表位以及针对 对应于I-i内不同区域的合成肽。部分 将对片段进行片段的N-末端测序 通过反相HPLC或2D电泳分离， 电印迹。 在目标3中，分离的I-i片段，其产生如下： 将检测I-i从II类MHC分子的切割和释放 因为它们阻断肽呈递的能力可能是由于 与II类MHC分子上的抗原结合位点结合;因此，在本发明中， 鉴定阻断desetope的I-i序列。 具有 阐明了导致I-i去除的条件，在目标4中， 条件将在流感肽存在下重现 用交联剂衍生化并碘化。 它们的结合 然后在紫外光诱导交联后进行评估 暴露，通过与抗II类抗体的免疫沉淀， 电泳和放射自显影。 或者，结合可以是 通过凝胶过滤进行检查，以将结合的肽与游离的肽分离。 从这些研究中得出的答案将发挥双重作用： 增强我们对免疫学中一个基本问题的理解，以及（B） 确定Ii中可能作为合成骨架的区域 基于肽的疫苗。

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.