Calreticulin, a component of the endoplasmic reticulum and of cytotoxic lymphocyte granules, regulates perforin-mediated lysis in the hemolytic modelsystem

Cytotoxic lymphocytes kill virally infected cells with specialized cytotoxi c granules containing perforin, a protein that forms toxic pores in the tar get cell membrane. These specialized cytotoxic granules also contain calret iculin, an endoplasmic reticulum chaperone protein. The calcium-independent association of perforin and calreticulin prompted our evaluation of calret iculin's potential to function as a regulatory molecule that protects cytot oxic lymphocytes from their own perforin. We report here that 10(-7) M calr eticulin blocked perforin-mediated lysis in the hemolytic model system usin g erythrocytes as targets. Previously, we found that millimolar levels of c alcium in the hemolytic assays dissociate high-affinity perforin-calreticul in complexes, which makes it unlikely that perforin associates with calreti culin in solution when hemolysis is blocked. Calreticulin may affect perfor in at the erythrocyte membrane. We observed calcium-dependent binding of ca lreticulin to erythrocyte membranes with a K-d Of 2.7 x 10(-7) M and a satu ration average of 10(5) molecules calreticulin per erythrocyte. At concentr ations that blocked hemolysis, calreticulin occupied many of the calreticul in membrane-binding sites and was in molar excess of perforin. These observ ations open the possibilities that membrane-bound calreticulin prevents hyd rophobic entry of perforin into membranes and (or) prevents perforin from a ssembling into polyperforin pores.