Caspase 3-mediated Proteolysis of the N-terminal Cytoplasmic Domain of the Human Erythroid Anion Exchanger 1 (Band 3)

Abstract

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 p72 syk , 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, TATD 45 and EQGD 205 (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 Apoptosis is required to maintain the balance between cell proliferation and cell death. Nuclear collapse and DNA fragmentation are universal features of apoptosis in nucleated cells. However, the nucleus itself is not required for apoptosis, since apoptotic stimuli induce apoptotic morphological features in anucleate cells (1-3). The signaling events that culminate in some of the key plasma membrane changes associated with cell death in vivo, such as externalization of PS, 1 operate independently of the nucleus (3). While the process of apoptosis has been studied in-depth in nucleated cells, the role of apoptosis regulatory molecules in anucleate cells is poorly understood. The anucleate mature circulating human erythrocyte is cleared by macrophages after its 120-day life span. The senescent erythrocyte shares at least one feature in common with nucleated cells undergoing apoptosis. The externalization of PS on the outer leaflet of the red cells results in recognition by macrophages probably through a receptor that engages PS (4). We hypothesized that the circulating human erythrocyte potentially shares part of the apoptotic machinery of nucleated cells, and that this machinery may be triggered as a consequence of red cell senescence, pathology such as in diseases like sickle cell anemia, thalassemia, and oxidative insult to red cells. Proteases play a critical role in the expression of mammalian apoptosis. The caspases are a specialized family of cysteine-dependent aspartate-directed proteases (5-7). The caspases recognize a tetrapeptide sequence on their substrates with an aspartate residue at the fourth position. Caspase activation is a critical event in the onset of apoptosis (5). Human caspase 3 is perhaps the most universal apoptosis mediator. It is present in most mammalian cells (8). Its deletion by gene knockout blocks neuronal death during brain development with consequent lethality (9). Studies in our laboratory have demonstrated that caspase 3 is present in mature human erythrocytes and activated by oxidative stress (10). Berg et al. (11) have also demonstrated that caspases 3 and 8 are present in mature human erythrocytes.The cytoskeleton of an apoptotic cell undergoes profound changes. Underlying these changes is the cleavage of many cytoskeletal proteins by caspases. Thes...

Nitrite enhances RBC hypoxic ATP synthesis and the release of ATP into the vasculature: a new mechanism for nitrite-induced vasodilation

“…This same peptide activates caspase 3 in the RBC (7,27), which cleaves the NH 2 -terminal end of band 3 (27). The inhibition of ATP release does not involve a decrease in glycolysis or the inhibition of the signal transduction pathway responsible for the release of ATP.…”

PGE2 in the regulation of programmed erythrocyte death

“…Caspases comprise a family of cysteine endopeptidases expressed in erythrocytes. 47,48 However, the role of caspases during stress-induced programmed erythrocyte death seems to be more complex. Oxidative stress 47 and erythrocyte aging 48 obviously lead to stimulation of caspase-3 and degradation of target proteins, for example, erythrocyte anion exchanger 1 (band 3), while caspases are not activated after hyperosmotic shrinkage 7 or ionomycin treatment.…”

“…47,48 However, the role of caspases during stress-induced programmed erythrocyte death seems to be more complex. Oxidative stress 47 and erythrocyte aging 48 obviously lead to stimulation of caspase-3 and degradation of target proteins, for example, erythrocyte anion exchanger 1 (band 3), while caspases are not activated after hyperosmotic shrinkage 7 or ionomycin treatment. 2 Thus, to the extent that calcium activates the enzymes responsible for the breakdown of membrane phosphatidylserine asymmetry and degradation of the cytoskeleton, an increase of cytosolic Ca 2 þ activity is expected to trigger the clearance of the affected erythrocytes.…”

Apoptotic mechanisms in the control of erythropoiesis

“…113 First, it was observed that activated caspase-3 can be detected in old, but not in young red blood cells; 114 second, this activated caspase-3 is able to cleave cell membrane band 3, disrupting its interaction with the peripheral membrane protein 4.2. 115 According to these observations, it was suggested that some caspases activated during red cell aging could participate in the degradation of crucial erythrocyte membrane proteins involved in the maintenance of shape and function. In line with these findings, it was observed that a loss of band 3 is associated with premature erythroid cell death (dyserythropoiesis).…”