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Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry
Published on: July 8, 2010
Perforin lytic activity is controlled by calreticulin
S A Fraser1, R Karimi, M Michalak
1Cell and Molecular Biology Program, School of Medicine and College of Agriculture, University of Nevada, Reno, NV 89557, USA. saf@scs.unr.edu
Journal of Immunology (Baltimore, Md. : 1950)
|February 7, 2001
Summary
Calreticulin, an endoplasmic reticulum (ER) protein, inhibits perforin-mediated cell lysis. This protein regulates osmotic lysis by stabilizing cell membranes, preventing pore formation by polyperforin.
Area of Science:
- Immunology
- Cell Biology
- Protein Biochemistry
Background:
- Cytotoxic lymphocytes utilize granule components for cell-mediated killing.
- Perforin and calreticulin are co-stored in cytotoxic granules.
- Calreticulin, an endoplasmic reticulum chaperone, is uniquely found in these granules.
Purpose of the Study:
- To investigate the role of calreticulin in cytotoxic lymphocyte-mediated cell death.
- To determine if calreticulin regulates perforin-dependent osmotic lysis.
- To elucidate the mechanism by which calreticulin affects perforin activity.
Main Methods:
- Perforin-mediated osmotic lysis assays using purified perforin.
- Inhibition studies with native and recombinant calreticulin.
- Analysis of calreticulin domain fragments (GST fusion proteins) for inhibitory activity.
- Assessment of inhibition independence from granzyme inactivation or calcium sequestration.
Main Results:
- Calreticulin significantly inhibited perforin-mediated osmotic lysis in a dose-dependent manner.
- Inhibitory activity was localized to the C-domain of calreticulin, which does not bind perforin.
- Inhibition was stable over 5 hours and independent of granzyme inactivation or calcium binding.
- Recombinant calreticulin confirmed the inhibitory effect.
Conclusions:
- Calreticulin plays a regulatory role in perforin-mediated cell lysis.
- The inhibitory mechanism likely involves membrane stabilization, preventing polyperforin pore formation.
- Calreticulin regulates lysis independently of direct interaction with perforin or calcium sequestration.
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