Genetics - Theses
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ItemInvestigating the structure and function of lymphocyte perforin(University of Melbourne, 2003)Cytotoxic T Lymphocytes (CTL) and Natural Killer (NK) cells are involved in the elimination of virus-infected and malignant cells. Perforin, a key cytotoxin secreted by cytotoxic lymphocytes acts synergistically with the co-secreted serine proteases (granzymes) to kill the target cell. The devastating effects of perforin deficiency are mirrored in perforin-deficient mice and children diagnosed with familial haemophagocytic lymphohistiocytosis (FHL), a lethal immune deficiency requiring bone marrow transplantation as the only successful therapy. Perforin�s pivotal role in killer cell function makes it an attractive target for therapeutic intervention. Currently however, a large gap exists in our understanding of how perform operates at the molecular level, principally due to a lack of expression systems capable of synthesising this cytotoxic protein. This thesis describes a novel retroviral expression system that was successfully used to express wild type perforin, allowing the first ever mutagenic analysis of the molecule. Using this technology, perforin was expressed in Rat Basophilic Leukemia (RBL) cells, which can synthesis and store the protein in secretory granules. Degranulation and perforin release were achieved through the use of an anti-trinitrophenyl (TNP) IgE antibody to crosslink the Fee receptor on RBL cells with TNP-labelled EL-4 target cells. This resulted in death of the EL-4 cells, however RBL cells transduced with empty viral vector did not induce cell death. Using the same methodology, two mutations identified in FHL (P5 mutation: G429E and P6 mutation: P345L) were expressed in the RBL cells and shown to be associated with complete loss of cytotoxic function. Both mutated perforin molecules were correctly targeted to the secretory granules, and released upon Fee receptor crosslinking. This suggested that in each case, the defect in perforin function mapped downstream of release from cytotoxic lymphocytes. Retroviral transduction was also used to investigate the role of putative calcium-binding aspartate residues located in perforin�s carboxy terminus C2-like domain. The negatively charged aspartate side chains have been predicted to cluster and bind calcium ions, which is an obligate requirement for perform function. Single and joint mutation of two of the five aspartate residues conserved in rat, mouse and human perforins caused a complete loss of perforin-mediated cytotoxicity, suggesting that aspartate residues 484 and 486 are both indispensable for perforin function.