Abstract 53 Allergy and Immunology Platform, Monday, 5/3

Granulysin is a newly described peptide expressed by cytotoxic T lymphocytes (CTL) and Natural Killer (NK) cells which co-localizes with perforin in secretory lytic granules. Granulysin shares sequence homology with the saposin-like family of proteins. Granulysin was recently shown to selectively induce apoptosis of tumor cells (J. of Immunology, 1998, 161:1758-64) and lysis of parasites, fungi and bacteria (Science, 1998, 282:121-25). To determine possible mechanisms of granulysin's bactericidal actions, we studied the effects of recombinant granulysin on large unilamellar liposomes. The fluorescent probe ANTS and its collisional quencher DPX were encapsulated in 1-palmitoyl-2-oleoyl- phosphatidylcholine (POPC), phosphatidylglycerol (POPG), POPC/POPG (1/1), POPC/cardiolipin (1/1) and POPC/phosphatidylserine (1/1) liposomes prepared by freeze-thawing and extrusion of mutilamellar vesicles through 0.2 µm polycarbonate filters. Release of liposome contents was measured by the change in fluorescence recorded in a spectrofluorometer and was normalized to that obtained after detergent lysis. Granulysin caused dose-dependent lysis of the liposomes but required the presence of negatively charged phospholipids and was maximum with pure POPG and POPC/cardiolipin. Lowering the solvent pH to 5 markedly decreased granulysin's effects, but addition of DTT increased the final extents of lysis twofold. We conclude that granulysin's membrane lytic action requires the presence of negatively charged phospholipids. It is inhibited at the acidic pH of the vesicle granulysin is stored intracellularly and does not appear to depend on an intact tertiary structure of the peptide.