Characterization of the deoxyhemoglobin binding site on human erythrocyte band 3: implications for O2 regulation of erythrocyte properties

Abstract

Band 3, the major protein of the human erythrocyte membrane, associates with multiple metabolic, ion transport, and structural proteins. Functional studies demonstrate that the oxygenation state of the erythrocyte regulates cellular properties performed by these and/or related proteins. Because deoxyhemoglobin, but not oxyhemoglobin, binds band 3 reversibly with high affinity, these observations raise the hypothesis that hemoglobin might regulate erythrocyte properties through its reversible, oxygenationdependent association with band 3. To explore this hypothesis, we have characterized the binding site of deoxyHb on human erythrocyte band 3. We report that (1) deoxyHb binds to residues 12-23 of band 3; (2) mutation of residues on either side of this sequence greatly enhances affinity of deoxyHb for band 3, suggesting that evolution of a higher affinity interaction would have been possible had it been beneficial for survival; (3) Hb does not bind to 2 other sequences in band 3 despite their high sequence homology to residues 12-23, and (4) IntroductionSeveral lines of evidence suggest that the oxygenation state of the erythrocyte (RBC) regulates multiple erythrocyte properties. First, the activities of many membrane solute transporters change with the oxygen content of the cell, including volume regulatory transporters (eg, Na ϩ /H ϩ exchanger), cation coupled Cl Ϫ cotransporters, amino acid transporters, and ion channels. [1][2][3][4][5][6][7][8][9][10] The K/Cl cotransporter, for example, is reported to be 20-fold more active in oxygenated than in deoxygenated RBCs. 9,10 Second, erythrocyte metabolism is modulated by the O 2 tension of the medium. Thus, glucose flux through the pentose phosphate pathway proceeds twice as rapidly in oxygenated as in deoxygenated cells, and glucose consumption in glycolysis is inhibited by oxygenation. 11 Third, erythrocyte deoxygenation may affect membrane structural properties. For example, deoxygenated hemoglobin (Hb) (but not oxygenated Hb [oxyHb]) competes with ankyrin for binding to band 3, and band 3 retention in detergent-extracted membrane skeletons is greatly reduced in skeletal pellets from deoxygenated cells (M. Stefanovic and P.S.L., unpublished data, December 2005). Furthermore, ankyrin epitopes are more accessible to antiankyrin antibodies in deoxygenated than in oxygenated RBCs. Taken together, these latter findings imply a looser association of ankyrin with the membrane in deoxygenated than in oxygenated erythrocytes.In searching for a mechanism to account for oxygenation effects on red cell properties, identification of both the O 2 sensor and downstream effector was considered important. A major candidate for the O 2 sensor was presumed to be Hb, because the O 2 dependence of the above properties generally follows the O 2 dissociation curve of Hb. 4,12,13 The downstream effector has been more uncertain, but band 3 has represented the prime candidate, because it constitutes the only established binding site of Hb on the membrane. Moreover, multiple observation...

Membrane association of peroxiredoxin-2 in red cells is mediated by the N-terminal cytoplasmic domain of band 3

“…Whereas, the cdb3 forms a sticky complex with hemichromes, with an affinity presumed to be much higher than that for hemoglobin, even if no structural basis for this interaction has been well established [28,31]. We also have been able to document small conformational changes in PRDX2 following its binding cdb3 using spectroscopic approaches.…”

“…Different stimuli such as the state of tyrosine phosphorylation state or deoxygenation can modulate the ability of these proteins to bind band 3 [2,3]. We therefore evaluated whether membrane association of PRDX2 in red cell is affected following treatment with Na-vanadate, a phosphatase(s) inhibitor [29,30] or by binding of deoxyhemoglobin to bind to band 3 following deoxygenation [31]. Two Tyr-residues (Tyr 8 and 21) present in N-terminal domain of band 3 are targets of Syk-Lyn tyrosine kinase pathway [30].…”

Regulation of membrane-cytoskeletal interactions by tyrosine phosphorylation of erythrocyte band 3

“…Band 3 (AE1) catalyzes the exchange of anions (primarily HCO 3 Ϫ for Cl Ϫ ) across the erythrocyte membrane, 6 anchors the spectrin/ actin cytoskeleton to the lipid bilayer, 7 organizes and regulates a complex of glycolytic enzymes, 8,9 participates in control of erythrocyte lifespan, 10,11 nucleates several important membrane-spanning proteins, 12 and serves as a docking site for multiple peripheral membrane proteins, including protein 4.1, protein 4.2, and several kinases and phosphatases. [13][14][15][16] Not surprisingly, mutations in band 3 are frequently associated with various hemolytic diseases. 17 Perhaps because of its many important functions, band 3 is also a prominent substrate of Ser/Thr kinases, 18,19 and is the major substrate of the cell's protein tyrosine kinases.…”

Physical and Functional Links between Anion Exchanger-1 and Sodium Pump

“…Multiple binding sites for other proteins including cytoskeletal proteins ankyrin-1 (ANK1), glycolytic enzymes, and hemoglobin have been identified in the N-terminal domain of eAE1. [7][8][9][10][11] However, none of these appears to interact with the N terminus of kAE1. 9,12,13 The crystal structure of the N-terminal domain of eAE1 has been solved.…”