A natural killer cell granule protein that induces DNA fragmentation and apoptosis.

Abstract

SummaryWe report the purification from a rat natural killer (RNK) large granular lymphocyte leukemia of a 32-kD granule protein that induces rapid DNA fragmentation and apoptosis. The protein, which we have called "fragmentin" was capable of causing DNA from intact YAC-1 cells to be cleaved into oligonucleosomal-sized fragments and producing severe chromatin condensation within 1 h. Amino acid sequence of tryptic peptides indicated that fragmentin was highly homologous to the NK and T cell granule serine proteases RNK protease 1 and mouse cytotoxic T cell protease I (CCPI)/granzyme B. Preincubation with the serine esterase inhibitor 3,4-dichloroisocoumarin blocked fragmentin-induced DNA damage, but had no effect on cytolysin. Fragmentin activity against four lymphoma target cells was completely dependent on the presence of cytolysin. Fragmentin produced rapid membrane damage as well as DNA fragmentation at nonlytic cytolysin doses, suggesting that ffagmentin activity was not limited to its effects on the nucleus. Fragmentin and cytolysin activity were completely inhibited by EGTA, indicating the process was Ca 2+ dependent. A role for cytolysin in endocytosis of ffagmentin was suggested by the observation that treatment of YAC-1 with cytochalasin B or sodium azide and 2-deoxyglucose blocked DNA fragmentation but not cytolysin activity. A 30-kD Not-CBZ-t-lysine thiobenzyl esterase, which copurified with ffagmentin, was inactive on its own but was able to synergistically amplify the DNA damage induced by fragmentin in the presence of cytolysin. Fragmentin activity was not dependent on protein synthesis, as cycloheximide treatment of YAC-1 cells did not prevent DNA damage. We postulate that fragmentin is the molecular mediator of NK cell-mediated DNA fragmentation and apoptosis.

Apoptosis in disease

“…Furthermore, there is intense interest in the possibility that proteases may trigger apoptosis by cleaving as yet unknown cytoplasmic substrates. This is because proteases may play a crucial role in the killing of targets by cytotoxic lymphocytes [82], and even more exciting data indicate that interleukin-1 P-converting enzyme (ICE) may act on an uncharacterized substrate to initiate apoptosis [83]. Indeed, the homology of ICE with a gene-driving programmed cell death in the C. elegans nematode illustrates that molecular characterization of cell death mutations in simple organisms will also have an important impact [4].…”

A quarter century of granzymes

“…Initial studies indicated that GrB-induced in vitro cell lysis ( 51 Cr-release), rapid DNA fragmentation, and chromatin condensation. [15][16][17] Indeed, identification of the DNA fragmenting activity (fragmentin) of GrB was an important piece of the puzzle in understanding the mechanism of CTL-induced death. Later experiments revealed that purified GrB and perforin or adenovirus treatment induced many classical features of apoptosis, such as membrane blebbing, phosphatidylserine exposure, release of cytochrome C, dissipation of Dc m , generation of ROS, and plasma membrane permeability to vital dyes at early timepoints.…”

Perforin-dependent nuclear entry of granzyme B precedes apoptosis, and is not a consequence of nuclear membrane dysfunction

“…The second involves exocytosis of the contents of cytoplasmic granules from the killer cell toward the target (Young and Cohn, 1986;Smyth and Trapani, 1995). The apoptotic effects of cytolytic granules can be mimicked by exposing cells to just two molecules, the pore-forming protein perforin, and the serine protease granzyme B (grB) (Shi et al, 1992). Neither perforin nor grB is able to induce apoptosis in the absence of the other agent (Shi et al, 1992;Duke et al, 1989).…”

“…The apoptotic effects of cytolytic granules can be mimicked by exposing cells to just two molecules, the pore-forming protein perforin, and the serine protease granzyme B (grB) (Shi et al, 1992). Neither perforin nor grB is able to induce apoptosis in the absence of the other agent (Shi et al, 1992;Duke et al, 1989). Accordingly, perforin-deficient mice exhibit defective cytolysis against virus-infected, allogeneic and tumor targets (Kagi et al, 1994;Lowin et al, 1994), and the killer lymphocytes of mice lacking grB induce delayed cell death (Heusel et al, 1994).…”