Calreticulin, a component of the endoplasmic reticulum and of cytotoxic lymphocyte granules, regulates perforin-mediated lysis in the hemolytic model system.
Cytotoxic lymphocytes kill virally infected cells with specialized cytotoxic granules containing perforin, a protein that forms toxic pores in the target cell membrane. These specialized cytotoxic granules also contain calreticulin, an endoplasmic reticulum chaperone protein. The calcium-independent association of perforin and calreticulin prompted our evaluation of calreticulin's potential to function as a regulatory molecule that protects cytotoxic lymphocytes from their own perforin. We report here that 10(-7) M calreticulin blocked perforin-mediated lysis in the hemolytic model system using erythrocytes as targets. Previously, we found that millimolar levels of calcium in the hemolytic assays dissociate high-affinity perforin-calreticulin complexes, which makes it unlikely that perforin associates with calreticulin in solution when hemolysis is blocked. Calreticulin may affect perforin at the erythrocyte membrane. We observed calcium-dependent binding of calreticulin to erythrocyte membranes with a Kd of 2.7 x 10(-7) M and a saturation average of 10(5) molecules calreticulin per erythrocyte. At concentrations that blocked hemolysis, calreticulin occupied many of the calreticulin membrane-binding sites and was in molar excess of perforin. These observations open the possibilities that membrane-bound calreticulin prevents hydrophobic entry of perforin into membranes and (or) prevents perforin from assembling into polyperforin pores.
Fraser
S A
SA
Cell and Molecular Biology Program, University of Nevada, Reno 89557-0046, USA. saf@scs.unr.edu
Michalak
M
M
Welch
W H
WH
Hudig
D
D
D002135:Calcium-Binding Proteins / Q000502:physiology*; D037282:Calreticulin; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000502:physiology*; D006461:Hemolysis / Q000502:physiology*; D006801:Humans; D007938:Leukemia / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D012261:Ribonucleoproteins / Q000502:physiology*; D014407:Tumor Cells, Cultured
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D012261:Ribonucleoproteins; D054353:Perforin