Killing by cytotoxic T cells and natural killer cells: multiple granule serine proteases as initiators of DNA fragmentation.
ABSTRACT The vectorial secretion of the contents of highly specialized cytoplasmic granules is of pivotal importance to the killing by cytotoxic T cells and natural killer cells. The purification and biochemical characterization of some of the granule constituents, in particular the pore-forming protein perforin, had engendered the notion that the killing of cellular targets was largely an osmotic phenomenon analogous to the insult delivered by complement attack. However, the apparent absence of membrane perforation in various examples of lymphocyte-mediated killing, and the observation that perforin alone could not account for apoptosis associated with programmed cell death, suggested that perforin activity represented, at best, only a part of the whole mechanism. More recently, the characterization of a large family of granule serine proteases (granzymes) has provided evidence that these molecules may collaborate in the killing process by inducing a 'suicide' pathway in target cells, resulting in DNA fragmentation. However, the serine proteases are inactive alone, their natural substrates have not been defined and they require access into the target cell cytoplasm via perforin-induced pores to exert their deleterious effects. Thus, we propose that the cytotoxic granule-mediated mechanism comprises at least two interdependent arms, perforin and serine proteases, that together are capable of inflicting cell death by osmotic shock and/or nuclear collapse.
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ABSTRACT: Granulysin is a newly described lytic molecule expressed by CTL and NK cells. Three mRNA (519, 520, and 522) and two protein products of 15 and 9 kDa are encoded by the granulysin gene. Stable transfectants overexpressing the predominate 520 mRNA were generated to determine the protein products originating from the translation of this message. A transfectant of the NK cell tumor YT overexpressed both 15 and 9 kDa proteins while a transfectant of the T cell tumor HuT78 produced mainly 15 kDa granulysin. Thus the 520 mRNA is sufficient for production of both 15 and 9 kDa granulysin. 9 kDa granulysin accumulated via post-translational processing of 15 kDa protein and was present intracellularly but not in the cell culture supernatant, indicating specific retention of the 9 kDa protein. An inhibitor of granule acidification, concanamycin A, blocked the processing of 15 kDa granulysin to the 9 kDa form. A deduced structural difference between the two forms of the protein and a decrease in lytic activity of 9 kDa granulysin at granule pH suggest two mechanisms by which a granulysin expressing cell is protected from autolysis during the biosynthesis of this potentially harmful molecule.Molecular Immunology 06/1999; 36(7):413-22. DOI:10.1016/S0161-5890(99)00063-2 · 3.00 Impact Factor