PERFORIN-ENHANCING PROTEIN, A LOW-MOLECULAR-WEIGHT PROTEIN OF CYTOTOXIC LYMPHOCYTE GRANULES, ENHANCES PERFORIN LYSIS

Perforin is a 68 kD protein found in the granules of cytotoxic lymphoc ytes and is used by lymphocytes to form lethal pores in the membranes of the cells they kill, We and others have found that when perforin is purified, its lytic activity is markedly reduced, ELISAs indicated th at our final recovery of perforin protein was excellent,We decided to determine if depletion of other granule proteins contributed to the lo ss of lytic activity. We isolated perforin to the point where lytic ac tivity was diminished and added back granule proteins that had no lyti c activity or detectable (anti-genic) perforin. Perforin teas isolated by Cu2+-immobilized metal affinity chromatography (IMAC) followed by phenyl-Superose hydrophobic interaction chromatography (HIC), Its lyti c activity was enhanced by a low molecular weight (<15 kD) protein, pe rforin enhancing protein (PEPr). We have isolated PEPr by two methods, HIC and MonoQ. Nonlytic PEPr restored perforin to close to its origin al lytic activity. A protein similar if not identical to PEPr was also detectable as I-125-labeled protein associated with lytic perforin, W e propose that PEPr acts in conjunction with perforin to form lethal p ores and suggest that PEPr may be the rat equivalent of the human cyto toxic lymphocyte protein, granulysin. (C) 1997 Academic Press.