Proteasome genes in the MHC: Implications for antigen processing

The aim of the work described in this thesis was to clone new genes from the class II region of the human major histocompatibility complex (MHC) and to investigate their function. The first section describes the cloning of LMP2 and LMP7, two genes with a proposed role in antigen processing. Both genes have homology to components of the proteasome, a 700 kD cytoplasmic complex, containing 10-20 subunits. The proteasome has well defined protease activities, suggesting that LMP2 and LMP7 may function in the degradation of cytoplasmically synthesised proteins to form antigenic peptides. This hypothesis is strengthened by the fact that the LMPs are closely linked to, and coordinately regulated with, another pair of genes, called TAP1 and TAP2. Studies with mutant cell lines have shown that the TAP gene products function to mediate the transport of antigenic peptides into the endoplasmic reticulum. Using antisera raised against LMP2 and LMP7, I showed that both proteins are synthesised as precursors. These precursors are cleaved at the N terminus to form mature proteins which are incorporated into the proteasome. The expression of both LMP2 and LMP7 proteins was up- regulated by interferon-y, a cytokine which also up-regulates MHC proteins at the cell surface. Further examination of the LMP7 locus revealed an unusual genomic structure. Two LMP7 transcripts with alternative first exons were isolated from cDNA libraries. In addition, three splice variants transcribed from the opposite strand to TAP1 and LMP7 were cloned. These did not contain an obvious open reading frame. A comparison of this locus with that of the mouse showed that only one of the alternative LMP7 exons was conserved and that the opposite strand transcripts were poorly conserved