Interaction between a Ca2+-binding protein calreticulin and perforin, a component of the cytotoxic T-cell granules.
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.
Molecular Biology of Membranes Research Group, University of Alberta, Edmonton, Canada.
C
C
Andrin
M J
MJ
Pinkoski
K
K
Burns
E A
EA
Atkinson
O
O
Krahenbuhl
D
D
Hudig
S A
SA
Fraser
U
U
Winkler
J
J
Tschopp
M
M
Opas
R C
RC
Bleackley
M
M
Michalak
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000502:physiology; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D037282:Calreticulin; D002460:Cell Line; D003594:Cytoplasmic Granules / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D017930:Genes, Reporter; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D011392:Proline / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D052899:Pore Forming Cytotoxic Proteins; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D054353:Perforin; D011392:Proline; D002118:Calcium