Induction of target cell DNA release by the cytotoxic T lymphocyte granule protease granzyme A.

Abstract

The rapid breakdown of target cell DNA during CTL-mediated lysis has been difficult to explain by the granule exocytosis model of cytotoxicity. The involvement of CTL granule proteases in this process was strongly suggested by experiments in which CTL were pretreated with the serine protease inhibitor PMSF, in combination with agents that raise the pH of acidic intracellular compartments. While PMSF pretreatment alone had little effect on target lysis or DNA breakdown, the combination of PMSF and NH4Cl or monensin profoundly reduced target cell DNA release, while little effect was observed on target lysis, as measured by 51Cr release. CTL granule extracts cause release of 125I-DNA from detergent-permeabilized cells. This nuclear DNA-releasing (NDR) activity is inhibited by serine esterase inhibitors that also inhibit the granule BLT-esterase activity, and is specifically immunoabsorbed by antibodies to the CTL granule protease granzyme A. The NDR activity comigrates with BLT-esterase activity during subcellular fractionation, solubilization, gel filtration, and aprotinin-Sepharose affinity chromatography. SDS-PAGE analysis of the affinity-purified product indicates a molecular mass of 60,000 daltons under non-reducing conditions, which moves to 30,000 daltons upon reduction, consistent with previously reported behavior of granzyme A. When the purified material was reduced and alkylated, both esterase and NDR activities comigrated at 30,000 daltons upon gel filtration. Although fully lytic concentrations of purified LGL granule cytolysin alone failed to induce target cell DNA release, a combination of purified granzyme A and the cytolysin induces substantial DNA release.

Killing by cytotoxic T cells and natural killer cells: Multiple granule serine proteases as initiators of DNA fragmentation

“…The dependence of fragmentin-induced injury on the presence of PFP may be an inherent, effective cellular mechanism for restricting the range of activity of fragmentin. Hayes et al (1989) and Shiver et al (1992) suggest that by a similar mechanism to that proposed for fragmentin, a combination of PFP and granzyme-A can also induce DNA fragmentation in target cells. *^"^'^' These important observations have supported the original concept that proteolytic enzymes are involved in the lytic event, "" and have suggested something of the mode of action of the CL granule constituents, PFP and granzymes.…”

The Involvement of Macrophages and Lymphocytes in the Apoptosis of Enterocytes.

“…On the other hand, granzymes and fragmentin have been shown to rapidly induce the fragmentation of DNA. Noteworthily, these two proteins are active only in the presence of perforin (HAYES et al, 1989;SHI et al, 1992). Moreover, the intensity of their cytotoxic activity is shown to be dependent on perforin-dosage, with DNA damage increasing with perforin concentration.…”

“…LGLs correspond to a heterogenous cell population including NK cells, cytotoxic T lymphocytes (CTLs) and lymphokine-activated killer (LAK) cells (KANEDA, 1989). Cytoplasmic granules of the lymphocytes contain cytotoxic substances including perforin (PODACK et al, 1985), a family of serine esterases (MUNGER et al, 1988;HAYES et al, 1989;ZUNINO et al, 1990), and TIA-1 (TIAN et al, 1991) as effector molecules which kill target cells.…”

Mouse granzyme K has pro-inflammatory potential

“…1,2 On the basis of their transfer into target cells and secretion extracellularly, gzms were originally anticipated to act both intracellularly and mediate extracellular proteolytic events. 3 However, the discovery that isolated gzms induced apoptosis in the presence of perf 4,5 encouraged the notion that proteases acted primarily as death effectors.…”