Interaction between a Ca²⁺-Binding Protein Calreticulin and Perforin, a Component of the Cytotoxic T-Cell Granules

Abstract

Calreticulin is a component of cytotoxic T-lymphocyte and NK lymphocyte granules. We report here that granule-associated calreticulin terminates with the KDEL endoplasmic reticulum retrieval amino acid sequence and somehow escapes the KDEL retrieval system. In perforin knock-out mice calreticulin is still targeted into the granules. Thus, calreticulin will traffic without perforin to cytotoxic granules. In the granules, calreticulin and perforin are associated as documented by (i) copurification of calreticulin with perforin but not with granzymes and (ii) immunoprecipitation of a calreticulin-perforin complex using specific antibodies. By using calreticulin affinity chromatography and protein ligand blotting we show that perforin binds to calreticulin in the absence of Ca2+ and the two proteins dissociate upon exposure to 0.1 mM or higher Ca2+ concentration. Perforin interacts strongly with the P-domain of calreticulin (the domain which has high Ca2+-binding affinity and chaperone function) as revealed by direct protein-protein interaction, ligand blotting, and the yeast two-hybrid techniques. Our results suggest that calreticulin may act as Ca2+-regulated chaperone for perforin. This action will serve to protect the CTL during biogenesis of granules and may also serve to regulate perforin lytic action after release.

Perforin Lytic Activity Is Controlled by Calreticulin

“…The lytic granules contain perforin, serine-dependent proteases, called granzymes (reviewed in Ref. 3), and calreticulin (4,5). Granule-initiated cell death occurs via both necrosis and apoptosis (6).…”

“…Calreticulin is a calcium-binding resident protein of the endoplasmic reticulum (ER), 3 belonging to the KDEL protein family (15). Calreticulin is the only KDEL protein found in cytotoxic lymphocyte granules, which suggests that it has a novel function in granulemediated cell death (5). In the lumen of the ER, calreticulin functions as a protein chaperone.…”

“…Calreticulin binds perforin with affinities great enough to support coprecipitation. However, precipitable complexes fail to form in the presence of calcium (5). The P-domain of calreticulin (see Fig.…”

“…Perforin has sites for N-glycosylation, a process dependent on calcium. Calreticulin may chaperone N-glycosylated perforin; however, calreticulin appears in the granules of lymphocytes from perforin "knockout" mice (5). Calreticulin binds calcium with both high affinity (K d ϳ1 M)/low capacity (1 mol of calcium/mol of protein), localized in the P-domain, and low affinity (K d ϳ1 mM)/high capacity (25 mol of calcium/mol of protein), localized in the C-domain (17).…”

Characterization of DNA vaccines encoding the domains of calreticulin for their ability to elicit tumor-specific immunity and antiangiogenesis

“…The P domain is thought to be critical for the lectin-like chaperone activity of calreticulin [19]. The P domain of calreticulin also interacts with PDI [17] and perforin [20,21]. The Cterminal region of the protein is highly acidic and terminates with the KDEL ER retrieval sequence [22].…”