Granzyme B-induced and Caspase 3-dependent Cleavage of Gelsolin by Mouse Cytotoxic T Cells Modifies Cytoskeleton Dynamics

Praxedis Martin,Julián Pardo,Natalie Schill,Lars Jöckel,Matthias Berg,Christopher J Froelich,Reinhard Wallich,Markus M Simon

doi:10.1074/jbc.m109.056028

Abstract

Granule-associated perforin and granzymes (gzms) are key effector molecules of cytotoxic T lymphocytes (Tc cells) and natural killer cells and play a critical role in the control of intracellular pathogens and cancer. Based on the notion that many gzms, including A, B, C, K, H, and M exhibit cytotoxic activity in vitro, all gzms are believed to serve a similar function in vivo. However, more recent evidence supports the concept that gzms are not unidimensional but, rather, possess non-cytotoxic potential, including stimulation of pro-inflammatory cytokines and anti-viral activities. The present study shows that isolated mouse gzmB cleaves the actin-severing mouse protein, cytoplasmic gelsolin (c-gelsolin) in vitro. However, when delivered to intact target cells by ex vivo immune Tc cells, gzmB mediates c-gelsolin proteolysis via activation of caspases 3/7. The NH(2)-terminal c-gelsolin fragment generated by either gzmB or caspase 3 in vitro constitutively severs actin filaments without destroying the target cells. The observation that gzmB secreted by Tc cells initiates a caspase cascade that disintegrates the actin cytoskeleton in target cells suggests that this intracellular process may contribute to anti-viral host defense.

Highlights

The present study shows that gzmB cleaves both the isolated and cell lysate-associated forms of c-gelsolin in vitro but that proteolysis mediated by gzmB secreted by ex vivo immune Tc cells requires caspases 3 and/or 7
In light of the fact that certain viruses need an intact cytoskeleton for infection of cells, duplication, and/or egress [35,36,37], one could speculate that Tc cells interfere with viral life cycle through this novel gzmB-induced intracellular proteolytic pathway
The requirements include 1) achieving a minimum catalytic efficiency for the observed proteolytic event that is further documented by mutating amino acid (AA) at the cleavage site and 2) showing that the protein is cleaved after gzmB is delivered into intact cells by such agents as perf, SLO, and adenoviral particles

Summary

EXPERIMENTAL PROCEDURES

Mouse Strains—The following strains were maintained at the Max-Planck-Institute of Immunobiology, Freiburg. 5 mg/ml anti-FasL monoclonal antibody (mAb) (clone Jo2; BD Pharmingen) or the appropriate control antibody (hamster IgG; BD Pharmingen) or sublytic concentrations of streptolysin (SLO) [26] together with rec.gzmB were added to cell cultures and incubated at 37 °C and 7% CO2 for the indicated time points. The cells were stained with anti-rec.c-gelsolin IS diluted in saponin buffer/1ϫ Roti-Block (Roth) at room temperature for 45 min followed by incubation with an anti-rabbit IgG Alexa 488 (Invitrogen) or phalloidin Alexa Fluor 546 (Invitrogen) diluted in saponin buffer/1ϫ Roti-Block at room temperature for 45 min. For staining with the in situ cell death detection kit TMR red, MC.GelsolinϪ/Ϫ cells were treated and stained with anti-rec.c-gelsolin IS and anti-rabbit IgG Alexa488 as described above followed by staining with the in situ cell death detection kit TMR red according to manufacturer’s instructions.

RESULTS