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.
