Investigation of antigen processing using partially assembled MHC class I molecules
The processing of antigens for presentation to CD8+ T-cells in association with MHC class I molecules involves many chaperones and accessory proteins in the ER. Once processed into a trimeric complex of beta-2-microglobulin (p2m), heavy chain and antigenic peptide the MHC class I molecule is complete and can traffic to the cell surface where it interacts with the T cell receptor of CD8+ T-cells. The main focus of this work is the use of MHC class I fusion proteins, which by means of a covalent linker form partially assembled class I molecules. Using a fusion protein in which an antigenic HLA-A2 binding peptide is linked to p2m (PB), it has been possible to show by immunoprecipitation that class I molecules can continue to interact with the peptide-loading complex after high affinity peptide has bound. This data is further supported by peptide release assays in which class I molecules are not released from the peptide-loading complex upon provision of high affinity peptide. The expression of fusion proteins in which p2in is linked to the heavy chain alleles HLA-B44 (p2m-B44) or HLA-A2 (p2m-A2), has highlighted an allelic difference in the requirement of MHC class I molecules for chaperones in the antigen processing pathway. Specifically it has been possible to show that while P2m-A2 is able to express in murine fibroblast K41 cells, p2m-B44 is not. This inability to express is not as a result of the absence of human tapasin, as previously reported, but because of some other unknown factor that is present in human cells but not in murine K41 cells. Interestingly the expression of p2m-B44 in K41 cells also disrupts native MHC class I expression implying that the construct may be occupying chaperone molecules in the ER and delaying processing of mouse class I molecules. Furthermore, in the same system it has been possible to show that the major function of calreticulin is not the recruitment of P2m to heavy chain, since the p2m-A2 fusion protein, like normal class I expressed 8-10 fold less well in calreticulin deficient cells than in 'wild type' cells.