Perforin lytic activity is controlled by calreticulin

The components within cytotoxic lymphocyte granules are responsible for a s ignificant fraction of T and NK cell-mediated death. Perforin is stored in these granules together with calreticulin, Calreticulin has long been recog nized as a chaperone protein of the endoplasmic reticulum (ER) and is the o nly resident ER protein to be found in the cytotoxic granules. Here we impl icate a role for calreticulin in killing and report that it controls osmoti c lysis mediated by purified perforin, Calreticulin, at a concentration of 2.2 x 10(-7) M, completely blocked perforin-mediated lysis, Inhibition was stable and held over 5 h. Recombinant calreticulin, at a concentration of 8 .8 x 10-7 M, also blocked lysis, indicating the inhibition was due to calre ticulin and not a copurifying protein in the native calreticulin preparatio ns. Using calreticulin domain fragments (expressed as GST fusion proteins), we found inhibitory activity in the high capacity calcium-binding C-domain , which does not bind perforin, The N- or P domains, which can bind perfori n, were unable to block lysis, The inhibition of lysis was independent of g ranzyme inactivation or the ability of calreticulin to sequester calcium. O ur data indicate that calreticulin regulation of perforin-mediated lysis pr obably occurs without direct interaction with perforin. We propose a novel model in which calreticulin stabilizes membranes to prevent polyperforin po re formation.