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Philip S. Low - One of the best experts on this subject based on the ideXlab platform.
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Oxygen regulates the Band 3-ankyrin bridge in the human erythrocyte membrane.
Biochemical Journal, 2013Co-Authors: Marko Stefanovic, Estela Puchulu-campanella, Gayani C. Kodippili, Philip S. LowAbstract:The oxygenation state of erythrocytes is known to impact several cellular processes. As the only known O2-binding protein in red blood cells, haemoglobin has been implicated in the oxygenation-mediated control of cell pathways and properties. Band 3, an integral membrane protein linked to the spectrin/actin cytoskeleton, preferentially binds deoxygenated haemoglobin at its N-terminus, and has been postulated to participate in the mechanism by which oxygenation controls cellular processes. Because the ankyrin-binding site on Band 3 is located near the deoxyHb (deoxygenated haemoglobin)-binding site, we hypothesized that deoxyHb might impact the association between Band 3 and the underlying erythrocyte cytoskeleton, a link that is primarily established through Band 3–ankyrin bridging. In the present paper we show that deoxygenation of human erythrocytes results in displacement of ankyrin from Band 3, leading to release of the spectrin/actin cytoskeleton from the membrane. This weakening of membrane–cytoskeletal interactions during brief periods of deoxygenation could prove beneficial to blood flow, but during episodes of prolonged deoxygenation, such as during sickle cell occlusive crises, could promote unwanted membrane vesiculation.
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interaction of deoxyhemoglobin with the cytoplasmic domain of murine erythrocyte Band 3
Biochemistry, 2012Co-Authors: Martiana F Sega, Haiyan Chu, John A Christian, Philip S. LowAbstract:The partial pressure of oxygen constitutes an important factor in the regulation of human erythrocyte physiology, including control of cell volume, membrane structure, and glucose metabolism. Because Band 3 is thought to be involved in all three processes and because binding of hemoglobin (Hb) to the cytoplasmic domain of Band 3 (cdb3) is strongly oxygen-dependent, the possibility that the reversible association of deoxyhemoglobin (deoxyHb) with cdb3 might constitute an O2-dependent sensor that mediates O2-regulated changes in erythrocyte properties arises. While several lines of evidence support this hypothesis, a major opposing argument lies in the fact that the deoxyHb binding sequence on human cdb3 is not conserved. Moreover, no effect of O2 pressure on Hb–Band 3 interactions has ever been demonstrated in another species. To explore whether Band 3–Hb interactions might be widely involved in O2-dependent regulation of erythrocyte physiology, we undertook characterization of the effect of O2 on Band 3–H...
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regulation of membrane cytoskeletal interactions by tyrosine phosphorylation of erythrocyte Band 3
Blood, 2011Co-Authors: Emanuela Ferru, Francesco Michelangelo Turrini, Katie Giger, Antonella Pantaleo, Estela Campanella, Jesse L Grey, Ken Ritchie, Rosa Vono, Philip S. LowAbstract:The cytoplasmic domain of Band 3 serves as a center of erythrocyte membrane organization and constitutes the major substrate of erythrocyte tyrosine kinases. Tyrosine phosphorylation of Band 3 is induced by several physiologic stimuli, including malaria parasite invasion, cell shrinkage, normal cell aging, and oxidant stress (thalassemias, sickle cell disease, glucose-6-phosphate dehydrogenase deficiency, etc). In an effort to characterize the biologic sequelae of Band 3 tyrosine phosphorylation, we looked for changes in the polypeptide's function that accompany its phosphorylation. We report that tyrosine phosphorylation promotes dissociation of Band 3 from the spectrin-actin skeleton as evidenced by: (1) a decrease in ankyrin affinity in direct binding studies, (2) an increase in detergent extractability of Band 3 from ghosts, (3) a rise in Band 3 cross-linkability by bis-sulfosuccinimidyl-suberate, (4) significant changes in erythrocyte morphology, and (5) elevation of the rate of Band 3 diffusion in intact cells. Because release of Band 3 from its ankyrin and adducin linkages to the cytoskeleton can facilitate changes in multiple membrane properties, tyrosine phosphorylation of Band 3 is argued to enable adaptive changes in erythrocyte biology that permit the cell to respond to the above stresses.
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Role of Band 3 in regulating metabolic flux of red blood cells
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Ian A. Lewis, M. Estela Campanella, John L Markley, Philip S. LowAbstract:Deoxygenation elevates glycolytic flux and lowers pentose phosphate pathway (PPP) activity in mammalian erythrocytes. The membrane anion transport protein (Band 3 or AE1) is thought to facilitate this process by binding glycolytic enzymes (GEs) and inhibiting their activity in an oxygen-dependent manner. However, this regulatory mechanism has not been demonstrated under physiological conditions. In this study, we introduce a 1H-13C NMR technique for measuring metabolic fluxes in intact cells. The role of Band 3 in mediating the oxygenated/deoxygenated metabolic transition was examined by treating cells with pervanadate, a reagent that prevents the GE–Band 3 complex from forming. We report that pervanadate suppresses oxygen-dependent changes in glycolytic and PPP fluxes. Moreover, these metabolic alterations were not attributable to modulation of bisphosphoglycerate mutase, direct inhibition of GEs by pervanadate, or oxidation, which are the major side effects of pervanadate treatment. These data provide direct evidence supporting the role of Band 3 in mediating oxygen-regulated metabolic transitions.
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characterization of the deoxyhemoglobin binding site on human erythrocyte Band 3 implications for o2 regulation of erythrocyte properties
Blood, 2008Co-Authors: Haiyan Chu, Andrew G Breite, Peter Ciraolo, Robert S Franco, Philip S. LowAbstract: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, oxygenation-dependent 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) the Hb binding site on Band 3 lies proximal to binding sites for glycolytic enzymes, Band 4.1 and ankyrin, suggesting possible mechanisms through which multifarious erythrocyte properties might be regulated by the oxygenation state of the cell.
Dieter Schubert - One of the best experts on this subject based on the ideXlab platform.
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the complex of Band 3 protein of the human erythrocyte membrane and glyceraldehyde 3 phosphate dehydrogenase stoichiometry and competition by aldolase
Biochimica et Biophysica Acta, 2002Co-Authors: Bogdan Von Ruckmann, Dieter SchubertAbstract:Abstract The cytoplasmic domain of Band 3, the main intrinsic protein of the erythrocyte membrane, possesses binding sites for a variety of other proteins of the membrane and the cytoplasm, including the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and aldolase. We have studied the stoichiometry of the complexes of human Band 3 protein and GAPDH and the competition by aldolase for the binding sites. In addition, we have tried to verify the existence of mixed Band 3/GAPDH/aldolase complexes, which could represent the nucleus of a putative glycolytic multienzyme complex on the erythrocyte membrane. The technique applied was analytical ultracentrifugation, in particular sedimentation equilibrium analysis, on mixtures of detergent-solubilized Band 3 and dye-labelled GAPDH, in part of the experiments supplemented by aldolase. The results obtained were analogous to those reported for the binding of hemoglobin, aldolase and Band 4.1 to Band 3: (1) the predominant or even sole Band 3 oligomer forming the binding site is the tetramer. (2) The Band 3 tetramer can bind up to four tetramers of GAPDH. (3) The Band 3/GAPDH complexes are unstable. (4) Artificially stabilized Band 3 dimers also represent GAPDH binding sites. In addition it was found that aldolase competes with GAPDH for binding to the Band 3 tetramer, and that ternary complexes of Band 3 tetramers, GAPDH and aldolase do exist.
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cytoskeleton membrane connections in the human erythrocyte membrane Band 4 1 binds to tetrameric Band 3 protein
Biochimica et Biophysica Acta, 1997Co-Authors: Bogdan Von Ruckmann, Thomas Jons, Frank Dolle, Detlev Drenckhahn, Dieter SchubertAbstract:Abstract Band 4.1 provides, besides ankyrin, the main linkage between the erythrocyte membrane and its cytoskeleton. Its predominant binding sites in the membrane are located on the glycophorins. However, the cytoplasmic domain of Band 3 can also bind Band 4.1. We have studied which of the different Band 3 oligomers observed (monomers, dimers, tetramers) can act as Band 4.1 binding sites, by equilibrium sedimentation experiments on mixtures of purified Band 3 and dye-labelled Band 4.1 in solutions of a nonionic detergent. At low molar ratios of Band 4.1 and Band 3, the sedimentation equilibrium distributions obtained could all be perfectly fitted assuming that only two dye-labelled particles were present: uncomplexed Band 4.1 and a complex formed between one Band 4.1 molecule and one Band 3 tetramer. The presence of small amounts of complexes containing Band 3 monomers or dimers could not be completely ruled out but is unlikely. On the other hand, stabilized Band 3 dimers effectively bound Band 4.1. At higher molar Band 4.1/Band 3 ratio, the Band 3 tetramer apparently could bind up to at least four Band 4.1 molecules. The Band 4.1/Band 3 tetramer complex was found to be unstable. The results described, together with those reported previously, point at a prominent role of tetrameric Band 3 in ligand binding.
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associations between erythrocyte Band 3 protein and aldolase in detergent solution determining their stoichiometry by analytical ultracentrifugation
FEBS Journal, 1996Co-Authors: Elfriede Huber, Gerhard Spatzkumbel, Hans G Baumert, Dieter SchubertAbstract:The cytoplasmic domain of Band 3, the predominant polypeptide of the erythrocyte membrane, represents a binding site for certain glycolytic enzymes. We have studied the association between human Band 3 protein and aldolase, in order to clarify the role of the different Band 3 oligomers as ligand binding sites. The experiments were performed on mixtures of solubilized Band 3 and aldolase in solutions of a nonionic detergent, nonaethyleneglycol lauryl ether. The main technique applied was sedimentation equilibrium analysis in an analytical ultracentrifuge. In addition, nonequilibrium centrifugation techniques were used. To facilitate the evaluations, the aldolase was labelled with a dye. The following results were obtained. (1) With unmodified Band 3, aldolase is bound exclusively or at least predominantly to the Band 3 tetramer (but not to monomers or dimers). (2) The Band 3 tetramer can bind up to four aldolase tetramers. (3) The Band 3 tetramer/aldolase complex is unstable on the time scale of the techniques used. (4) Stable Band 3 dimers (stabilized either covalently or noncovalently) can also associate with aldolase and can bind up to two aldolase tetramers. The results described, together with those reported previously, point at a prominent role of the Band 3 tetramer in ligand binding.
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erythrocyte Band 3 protein strongly interacts with phosphoinositides
FEBS Letters, 1994Co-Authors: Andreas Hanicak, Dieter Maretzki, Barbara Reimann, Antonie J. W. G. Visser, Karel W.a. Wirtz, Dieter SchubertAbstract:85% of the phosphorus coisolated with Band 3 protein during separation of the intrinsic proteins of the human erythrocyte membrane by zonal electrophoresis in high concentrations of acetic acid was found to be derived from phosphoinositides, mainly phosphatidylinositol 4,5-bisphosphate. When native Band 3 protein and pyrene-labelled phospholipids were present in micelles of the nonionic detergent nonaethyleneglycol lauryl ether, strong resonance energy transfer was observed between the tryptophan residues and phosphatidylinositol 4,5-bisphosphate and, to a smaller degree, phosphatidylinositol 4-phosphate. We conclude that Band 3 protein strongly interacts with phosphoinositides, in particular with phosphatidylinositol 4,5-bisphosphate.
Jiri Palek - One of the best experts on this subject based on the ideXlab platform.
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targeted disruption of the murine erythroid Band 3 gene results in spherocytosis and severe haemolytic anaemia despite a normal membrane skeleton
Nature Genetics, 1996Co-Authors: Christopher D Southgate, Athar H Chishti, Betsy Mitchell, Jiri PalekAbstract:Band 3 is the most abundant integral protein of the red blood cell membrane. It performs two critical biological functions: maintaining ionic homeostasis, by transporting Cl- and HCO3-ions, and providing mechanical stability to the erythroid membrane. Erythroid Band 3 (AE1) is one of three anion exchangers that are encoded by separate genes. The AE1 gene is transcribed by two promoters: the upstream promoter produces erythroid Band 3, whereas the downstream promoter initiates transcription of the Band 3 isoform in kidney. To assess the biological consequences of Band 3 deficiency, we have selectively inactivated erythroid but not kidney Band 3 by gene targeting in mice. Although no death in utero occurred, the majority of homozygous mice die within two weeks after birth. The erythroid Band 3 null mice show retarded growth, spherocytic red blood cell morphology and severe haemolytic anaemia. Remarkably, the Band 3-/- red blood cells assembled normal membrane skeleton thus challenging the notion that the presence of Band 3 is required for the stable biogenesis of membrane skeleton. The availability of Band 3-/- mice offers a unique opportunity to investigate the role of erythroid Band 3 in the regulation of membrane-skeletal interactions, anion transport and the invasion and growth of malaria parasite into red blood cells.
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Band 3 memphis a widespread polymorphism with abnormal electrophoretic mobility of erythrocyte Band 3 protein caused by substitution aag gag lys glu in codon 56
Blood, 1992Co-Authors: Petr Jarolim, H L Rubin, Sen Zhai, Kenneth E. Sahr, T J Mueller, Jiri PalekAbstract:Band 3 Memphis (b3M) is a variant of the erythrocyte Band 3 protein detected in individuals of virtually all ethnic groups and characterized by a reduced mobility of proteolytic fragments derived from the N-terminus of the cytoplasmic domain of Band 3 (cdb3). We have sequenced Band 3 cDNA corresponding to cdb3 in 12 heterozygotes for the b3M polymorphism including one white, one black, one Chinese, one Philippino, one Malay, and seven Melanesian subjects. In all individuals, we found a single-base substitution in codon 56 of one Band 3 allele changing lysine to glutamic acid (AAG----GAG) which, in some of them, was linked with an additional mutation in cdb3. Since the change of codon 56 from AAG to GAG was the only mutation in the studied individuals found within the cDNA segment coding for the abnormally migrating fragment of cdb3, we conclude that it represents the underlying molecular basis of the b3M polymorphism. We further support this conclusion by showing that electrophoresis in the presence of 4 mol/L urea abolished the difference in migration between proteolytic products of b3M and normal Band 3, and that a fusion protein prepared from cDNA coding for the b3M allele again exhibits reduced electrophoretic mobility compared with the normal fusion protein. Finally, since most of the previously cloned mouse, rat, and chicken Band 3 and Band 3-related proteins contain glutamic acid in the position corresponding to amino acid 56 in the human Band 3, we propose that the Memphis variant is the evolutionarily older form of Band 3.
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Band 3 tuscaloosa pro327 arg327 substitution in the cytoplasmic domain of erythrocyte Band 3 protein associated with spherocytic hemolytic anemia and partial deficiency of protein 4 2
Blood, 1992Co-Authors: Petr Jarolim, H L Rubin, Cathy Korsgren, Jiri Palek, Josef T Prchal, Carl M. CohenAbstract:Protein 4.2 is a major red blood cell (RBC) protein that interacts with the Band 3 protein and with ankyrin. Inherited deficiencies of this protein are associated with spherocytic hemolytic anemia, but the molecular basis of this defect is unknown. We have studied the underlying defect in a patient with spherocytic hemolytic anemia whose RBCs had a partial (29% +/- 5%) deficiency of protein 4.2. We have first studied the binding of normal ankyrin and protein 4.2 to patient inside-out vesicles (IOVs) stripped of peripheral proteins. While the binding of ankyrin was normal, the predicted maximal binding capacity of patient IOVs for Band 4.2 was 20% to 33% lower than that of control IOVs, suggesting a defect in the cytoplasmic domain of Band 3 (cdb3). An additional line of evidence pointing to a possible abnormality of Band 3 was an abnormal proteolytic digest of cdb3. To elucidate the underlying molecular defect, we have cloned and sequenced the cDNA coding for cdb3 from the patient. One Band 3 allele was found to be normal, while clones corresponding to the other allele contained two mutations: substitution A\---|-G in nucleotide 166, changing codon 56 from AAG to GAG (Lys\---|-Glu), and substitution C\---|-G in nucleotide 980, changing codon 327 from CCC to CGC (Pro\---|-Arg). Since the Lys56\---| -Glu56 substitution is found in a common asymptomatic variant of the Band 3 protein designated Band 3 Memphis, we conclude that either the Pro327\---|-Arg327 substitution itself, or in combination with the Band 3 Memphis polymorphism, underlies the abnormal binding of protein 4.2 to cdb3 and results in the spherocytic.
Eduardo Lissi - One of the best experts on this subject based on the ideXlab platform.
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peroxynitrite oxidizes erythrocyte membrane Band 3 protein and diminishes its anion transport capacity
Free Radical Research, 2007Co-Authors: Gloria Celedon, Gustavo González, José Pino, Eduardo LissiAbstract:We describe an altered membrane Band 3 protein-mediated anion transport in erythrocytes exposed to peroxynitrite, and relate the loss of anion transport to cell damage and to Band 3 oxidative modifications. We found that peroxynitrite down-regulate anion transport in a dose dependent relation (100–300 μmoles/l). Hemoglobin oxidation was found at all peroxynitrite concentrations studied. A dose-dependent Band 3 protein crosslinking and tyrosine nitration were also observed. Band 3 protein modifications were concomitant with a decrease in transport activity. ( − )-Epicatechin avoids Band 3 protein nitration but barely affects its transport capacity, suggesting that both processes are unrelated. N-acetyl cysteine partially reverted the loss of Band 3 transport capacity. It is concluded that peroxynitrite promotes a decrease in anion transport that is partially due to the reversible oxidation of Band 3 cysteine residues. Additionally, Band 3 tyrosine nitration seems not to be relevant for the loss of its anio...
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peroxyl oxidized erythrocyte membrane Band 3 protein with anion transport capacity is degraded by membrane bound proteinase
Free Radical Research, 2004Co-Authors: Gloria Celedon, Gustavo González, Veronica Ferrer, Eduardo LissiAbstract:Human red blood cells anion exchange protein (Band 3) exposed to peroxyl radicals produced by thermolysis of 2,2'-azo-bis(2-amidinopropane) (AAPH) is degraded by proteinases that prevent accumulation of oxidatively damaged proteins. To assess whether this degradation affects anion transport capacity we used the anionic fluorescent probe 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-y) amino] ethanosulfonate (NBD-taurine). A decrease of Band 3 function was observed after exposure to peroxyl radicals. In the presence of proteinase inhibitors the decrement of anion transport through Band 3 was smaller indicating that removal achieved by proteinases includes oxidized Band 3 which still retain transport ability. Proteinases recognize Band 3 aggregates produced by peroxyl radicals as was evaluated by immunoblotting. It is concluded that decrease of Band 3 transport capacity may result from a direct protein oxidation and from its degradation by proteinases and that Band 3 aggregates removal may prevent macrophage recognition of the senescent condition which would lead to cell disposal.
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peroxyl oxidized erythrocyte membrane Band 3 protein with anion transport capacity is degraded by membrane bound proteinase
Free Radical Research, 2004Co-Authors: Gloria Celedon, Gustavo González, Veronica Ferrer, Eduardo LissiAbstract:Human red blood cells anion exchange protein (Band 3) exposed to peroxyl radicals produced by thermolysis of 2,2′-azo-bis(2-amidinopropane) (AAPH) is degraded by proteinases that prevent accumulation of oxidatively damaged proteins. To assess whether this degradation affects anion transport capacity we used the anionic fluorescent probe 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-y) amino] ethanosulfonate (NBD-taurine). A decrease of Band 3 function was observed after exposure to peroxyl radicals. In the presence of proteinase inhibitors the decrement of anion transport through Band 3 was smaller indicating that removal achieved by proteinases includes oxidized Band 3 which still retain transport ability. Proteinases recognize Band 3 aggregates produced by peroxyl radicals as was evaluated by immunoblotting. It is concluded that decrease of Band 3 transport capacity may result from a direct protein oxidation and from its degradation by proteinases and that Band 3 aggregates removal may prevent macrophage recogni...
Petr Jarolim - One of the best experts on this subject based on the ideXlab platform.
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characterization of seven low incidence blood group antigens carried by erythrocyte Band 3 protein
Blood, 1998Co-Authors: Petr Jarolim, H L Rubin, J Storry, Dana Žáková, Marion E. ReidAbstract:Recent studies have demonstrated that Band 3 carries antigens of the Diego blood group system and have elucidated the molecular basis of several previously unassigned low incidence and high incidence antigens. Because the available serological data suggested that Band 3 may carry additional low incidence blood group antigens, we screened Band 3 genomic DNA encoding the membrane domain of Band 3 for single-strand conformational polymorphisms. We found that the putative first ectoplasmic loop of Band 3 carries blood group antigen ELO, 432 Arg→Trp; the third putative loop harbors antigens Vg a (Van Vugt), 555 Tyr→His, BOW 561 Pro→Ser, Wu (Wulfsberg), 565 Gly→Ala, and Bp a (Bishop), 569 Asn→Lys; and the putative fourth ectoplasmic loop carries antigens Hg a (Hughes), 656 Arg→Cys, and Mo a (Moen), 656 Arg→His. We studied erythrocytes from carriers of five of these blood group antigens. We found similar levels of reticulocyte mRNA corresponding to the two Band 3 gene alleles, normal content and glycosylation of Band 3 in the red blood cell membrane, and normal Band 3-mediated sulfate influx into red blood cells, suggesting that the mutations do not have major effect on Band 3 structure and function. In addition to elucidating the molecular basis of seven low incidence blood group antigens, these results help to create a more accurate structural model of Band 3.
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characterization of 13 novel Band 3 gene defects in hereditary spherocytosis with Band 3 deficiency
Blood, 1996Co-Authors: Petr Jarolim, H L Rubin, Josef T Prchal, J Murray, W M Taylor, S K Ballas, L M Snyder, L Chrobak, W D Melrose, V BrabecAbstract:Hereditary spherocytosis (HS) is a common hemolytic anemia of variable clinical expression. Pathogenesis of HS has been associated with defects of several red cell membrane proteins including erythroid Band 3. We have studied erythrocyte membrane proteins in 166 families with autosomal dominant HS. We have detected relative deficiency of Band 3 in 38 kindred (23%). Band 3 deficiency was invariably associated with mild autosomal dominant spherocytosis and with the presence of pincered red cells in the peripheral blood smears of unsplenectomized patients. We hypothesized that this phenotype is caused by Band 3 gene defects. Therefore, we screened Band 3 DNA from these 38 kindred for single strand conformational polymorphisms (SSCP). In addition to five mutations detected previously by SSCP screening of cDNA, we detected 13 new Band 3 gene mutations in 14 kindred coinherited with HS. These novel mutations consisted of two distinct subsets. The first subset included seven nonsense and frameshift mutations that were all associated with the absence of the mutant mRNA allele from reticulocyte RNA, implicating decreased production and/or stability of mutant mRNA as the cause of decreased Band 3 synthesis. The second group included five substitutions of highly conserved amino acids and one in-frame deletion. These six mutations were associated with the presence of comparable levels of normal and mutant Band 3 mRNA. We suggest that these mutations interfere with Band 3 biosynthesis leading thus to the decreased accumulation of the mutant Band 3 allele in the plasma membrane.
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duplication of 10 nucleotides in the erythroid Band 3 ae1 gene in a kindred with hereditary spherocytosis and Band 3 protein deficiency Band 3prague
Journal of Clinical Investigation, 1994Co-Authors: Petr Jarolim, H L Rubin, V Brabec, Shihchun Liu, M R Cho, L H Derick, Sara T O Saad, Seth L Alper, Carlo BrugnaraAbstract:We describe a duplication of 10 nucleotides (2,455-2,464) in the Band 3 gene in a kindred with autosomal dominant hereditary spherocytosis and a partial deficiency of the Band 3 protein that is reflected by decreased rate of transmembrane sulfate flux and decreased density of intramembrane particles. The mutant allele potentially encodes an abnormal Band 3 protein with a 3.5-kD COOH-terminal truncation; however, we did not detect the mutant protein in the membrane of mature red blood cells. Since the mRNA levels for the mutant and normal alleles are similar and since the Band 3 content is the same in the light and dense red cell fractions, we conclude that the mutant Band 3 is either not inserted into the plasma membrane or lost from the membrane prior to the release of red blood cells into circulation. We further show that the decrease in Band 3 content principally involves the dimeric laterally and rotationally mobile fraction of the Band 3 protein, while the laterally immobile and rotationally restricted Band 3 fraction is left essentially intact. We propose that the decreased density of intramembrane particles decreases the stability of the membrane lipid bilayer and causes release of lipid microvesicles that leads to surface area deficiency and spherocytosis.
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Band 3 memphis a widespread polymorphism with abnormal electrophoretic mobility of erythrocyte Band 3 protein caused by substitution aag gag lys glu in codon 56
Blood, 1992Co-Authors: Petr Jarolim, H L Rubin, Sen Zhai, Kenneth E. Sahr, T J Mueller, Jiri PalekAbstract:Band 3 Memphis (b3M) is a variant of the erythrocyte Band 3 protein detected in individuals of virtually all ethnic groups and characterized by a reduced mobility of proteolytic fragments derived from the N-terminus of the cytoplasmic domain of Band 3 (cdb3). We have sequenced Band 3 cDNA corresponding to cdb3 in 12 heterozygotes for the b3M polymorphism including one white, one black, one Chinese, one Philippino, one Malay, and seven Melanesian subjects. In all individuals, we found a single-base substitution in codon 56 of one Band 3 allele changing lysine to glutamic acid (AAG----GAG) which, in some of them, was linked with an additional mutation in cdb3. Since the change of codon 56 from AAG to GAG was the only mutation in the studied individuals found within the cDNA segment coding for the abnormally migrating fragment of cdb3, we conclude that it represents the underlying molecular basis of the b3M polymorphism. We further support this conclusion by showing that electrophoresis in the presence of 4 mol/L urea abolished the difference in migration between proteolytic products of b3M and normal Band 3, and that a fusion protein prepared from cDNA coding for the b3M allele again exhibits reduced electrophoretic mobility compared with the normal fusion protein. Finally, since most of the previously cloned mouse, rat, and chicken Band 3 and Band 3-related proteins contain glutamic acid in the position corresponding to amino acid 56 in the human Band 3, we propose that the Memphis variant is the evolutionarily older form of Band 3.
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Band 3 tuscaloosa pro327 arg327 substitution in the cytoplasmic domain of erythrocyte Band 3 protein associated with spherocytic hemolytic anemia and partial deficiency of protein 4 2
Blood, 1992Co-Authors: Petr Jarolim, H L Rubin, Cathy Korsgren, Jiri Palek, Josef T Prchal, Carl M. CohenAbstract:Protein 4.2 is a major red blood cell (RBC) protein that interacts with the Band 3 protein and with ankyrin. Inherited deficiencies of this protein are associated with spherocytic hemolytic anemia, but the molecular basis of this defect is unknown. We have studied the underlying defect in a patient with spherocytic hemolytic anemia whose RBCs had a partial (29% +/- 5%) deficiency of protein 4.2. We have first studied the binding of normal ankyrin and protein 4.2 to patient inside-out vesicles (IOVs) stripped of peripheral proteins. While the binding of ankyrin was normal, the predicted maximal binding capacity of patient IOVs for Band 4.2 was 20% to 33% lower than that of control IOVs, suggesting a defect in the cytoplasmic domain of Band 3 (cdb3). An additional line of evidence pointing to a possible abnormality of Band 3 was an abnormal proteolytic digest of cdb3. To elucidate the underlying molecular defect, we have cloned and sequenced the cDNA coding for cdb3 from the patient. One Band 3 allele was found to be normal, while clones corresponding to the other allele contained two mutations: substitution A\---|-G in nucleotide 166, changing codon 56 from AAG to GAG (Lys\---|-Glu), and substitution C\---|-G in nucleotide 980, changing codon 327 from CCC to CGC (Pro\---|-Arg). Since the Lys56\---| -Glu56 substitution is found in a common asymptomatic variant of the Band 3 protein designated Band 3 Memphis, we conclude that either the Pro327\---|-Arg327 substitution itself, or in combination with the Band 3 Memphis polymorphism, underlies the abnormal binding of protein 4.2 to cdb3 and results in the spherocytic.
