Caspase 3-mediated proteolysis of the N-terminal cytoplasmic domain of the human erythroid anion exchanger 1 (band 3).
The N-terminal cytoplasmic domain of the anion exchanger 1 (AE1 or band 3) of the human erythrocyte associates with peripheral membrane proteins to regulate membrane-cytoskeleton interactions, with glycolytic enzymes such as glyceraldehyde-3-phosphate dehydrogenase and aldolase, with the protein-tyrosine kinase p72syk, with hemoglobin and with hemichromes. We have demonstrated that the N-terminal cytoplasmic domain of band 3 (CDB3) is a substrate of the apoptosis executioner caspase 3 (1). CDB3 has two non-conventional caspase 3 cleavage sites, TATD45 and EQGD205 (2). In vitro treatment of recombinant CDB3 with caspase 3 generated two fragments, which could be blocked by pretreatment with the caspase 3 inhibitor Z-DEVD-fmk (3). Recombinant CDB3 in which the caspase 3 cleavage sites Asp45 and Asp205 were mutated, was resistant to proteolysis (4). Proteolytically derived fragments crossreactive with polyclonal anti-band 3 antibody appeared with simultaneous cleavage of poly (ADP-ribose) polymerase and procaspase 3 in staurosporine (STS)-treated HEK293 cells transiently transfected with CDB3 (5). In vivo cleavage of CDB3 could be blocked by pretreatment of cells with Z-DEVD-fmk or in cells transfected with mutant CDB3 (D45A, D205A) (6). Co-transfection experiments showed that STS-mediated cleavage of CDB3 diminished its interaction with the N-terminal domain of protein 4.2, confirming that such cleavage interferes with the interaction of CDB3 with cytoskeletal proteins (7). Active caspase 3 was observed in aged red cells but not in young cells. This red cell caspase 3 could cleave band 3 present in inside-out vesicles prepared from young erythrocytes arguing in favor of a physiological role of caspase 3 in aged erythrocytes.
Department of Chemistry, Bose Institute, 93/1 Acharya, Prafalla Chandra Road, Kolkata 700009, India.
Debabrata
D
Mandal
Veronique
V
Baudin-Creuza
Asima
A
Bhattacharyya
Shresh
S
Pathak
Jean
J
Delaunay
Manikuntala
M
Kundu
Joyoti
J
Basu
D000375:Aging; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry* / Q000378:metabolism; D017209:Apoptosis; D001665:Binding Sites; D015153:Blotting, Western; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D003599:Cytoskeleton / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009842:Oligopeptides / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014162:Transfection
D001457:Anion Exchange Protein 1, Erythrocyte; D004791:Enzyme Inhibitors; D009842:Oligopeptides; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; C505407:CASP3 protein, human; C505530:CASP8 protein, human; D053148:Caspase 3; D053181:Caspase 8; D020169:Caspases