The association between familial distal renal tubular acidosis and mutations in the red cell anion exchanger (band 3, AE1) gene.

In distal renal tubular acidosis (dRTA) the tubular secretion of hydrogen ion in the distal nephron is impaired, leading to the development of metabolic acidosis, frequently accompanied by hypokalemia, nephrocalcinosis, and metabolic bone disease. The condition can be familial, when it is usually inherited as an autosomal dominant, though there is a rarer autosomal recessive form associated with nerve deafness. It has been shown that the autosomal dominant form of dRTA is associated with a defect in the anion exchanger (AE1) of the renal collecting duct intercalated cell. This transporter is a product of the same gene (AE1) as the erythrocyte anion exchanger, band 3. In this review we will look at the evidence for this association. Studies of genomic DNA from families with this disorder have shown, both by genetic linkage studies and by DNA sequencing, that affected individuals are heterozygous for mutations in the AE1 gene whilst unaffected family members have a normal band 3 sequence. Mutations have been found in the region of proposed helices 6 and 7 of the membrane domain of band 3 and involve amino acids Arg-589 and Ser-613, and in the COOH-terminal domain of band 3. Studies of red cell band 3 from these families have provided information on the effect these mutations have on the structure and function of erythrocyte band 3. Expression studies of the erythroid and kidney isoforms of the mutant AE1 proteins, in Xenopus laevis oocytes, have shown that they retained chloride transport activity, suggesting that the disease in the dRTA families is not related simply to the anion transport activity of the mutated proteins. A possible explanation for the dominant effect of these mutant AE1 proteins in the kidney cell is that these mutations affect the targeting of AE1 from the basolateral to the apical membrane of the alpha-intercalated cell.     

Erythroid 5‐aminolevulinate synthase mediates the upregulation of membrane band 3 protein expression by iron

Iron deficiency leads to abnormal expression and function of band 3 protein in erythrocytes, but the underlying mechanisms remain elusive. The mRNA of erythroid‐specific 5‐aminolevulinate synthase (eALAS) contains an iron response element and the eALAS protein is an important mediator of iron utilization by erythrocytes. In this study, we investigated the effect of short hairpin RNA (shRNA) mediated silencing of eALAS on the expression of band 3 protein induced by iron. By real‐time RT‐PCR and Western blot we showed that at mRNA and protein level iron‐induced expression of band 3 protein was lower in eALAS‐shRNA transfected K562 cells than in control cells. Of note, the lowest expression was detected in K562 cells cultured in iron deficiency condition (p < 0.01). Thus either iron deficiency or depletion of eALAS could suppress the expression of erythroid band 3 protein. These results demonstrated for the first time that iron and the iron‐regulatory system regulate the expression of the erythrocyte membrane proteins. Copyright © 2010 John Wiley & Sons, Ltd.     

Expression, purification, and characterization of the functional dimeric cytoplasmic domain of human erythrocyte band 3 in Escherichia coli.

The cytoplasmic domain of the human erythrocyte membrane protein, band 3 (cdb3), contains binding sites for hemoglobin, several glycolytic enzymes, band 4.1, band 4.2, and ankyrin, and constitutes the major linkage between the membrane skeleton and the membrane. Although erythrocyte cdb3 has been partially purified from proteolyzed red blood cells, further separation of the water-soluble 43-kDa and 41-kDa proteolytic fragments has never been achieved. In order to obtain pure cdb3 for crystallization and site-directed mutagenesis studies, we constructed an expression plasmid that has a tandemly linked T7 promoter placed upstream of the N-terminal 379 amino acids of the erythrocyte band 3 gene. Comparison of several Escherichia coli strains led to the selection of the BL21 (DE3) strain containing the pLysS plasmid as the best host for efficient production of cdb3. About 10 mg of recombinant cdb3 can be easily purified from 4 L of E. coli culture in two simple steps. Comparison of cdb3 released from the red blood cell by proteolysis with recombinant cdb3 reveals that both have the same N-terminal sequence, secondary structure, and pH-dependent conformational change. The purified recombinant cdb3 is also a soluble stable dimer with the same Stokes radius as erythrocyte cdb3. The affinities of the two forms of cdb3 for ankyrin are essentially identical; however, recombinant cdb3 with its unblocked N-terminus exhibits a slightly lower affinity for aldolase.     

Trafficking and folding defects in hereditary spherocytosis mutants of the human red cell anion exchanger

Hereditary spherocytosis (HS) is a common inherited hemolytic anemia caused by mutations in erythrocyte proteins including the anion exchanger, AE1 (band 3). This study examined seven missense mutations (L707P, R760Q, R760W, R808C, H834P, T837M, and R870W) located in the membrane domain of the human AE1 that are associated with this disease. The HS mutants, constructed in full-length AE1 cDNA, could be transiently expressed to similar levels in HEK 293 cells. Immunofluorescence, cell surface biotinylation, and pulse chase labeling showed that the HS mutants all exhibited defective cellular trafficking from the endoplasmic reticulum to the plasma membrane. Impaired binding to an inhibitor affinity matrix indicated that the mutant proteins had non-native structures and may be misfolded. Further characterization of the HS R760Q mutant showed no change in its oligomeric structure or turnover (half-life=15 h) compared to wild-type AE1, suggesting the mutant was not aggregated or targeted for rapid degradation via the proteasome. Intracellular retention of HS mutant AE1 would lead to destruction of the protein during erythroid development and would account for the lack of HS mutant AE1 in the plasma membrane of the mature red cell.     

Identification of immunoreactive forms of human erythrocyte band 3 in nonerythroid cells

Antibodies directed to the cytoplasmic domain of human erythrocyte band 3, the major integral protein of the erythrocyte membrane which is thought to be the main anchoring site of the membrane cytoskeleton, were demonstrated in the present study to react with the membrane of various nonerythroid cells, such as human leucocytes, fibroblasts or human umbilical mesenchyme cells, amniotic epithelium and vascular smooth muscle. In cultured fibroblasts staining was confined to small dots and streaks associated with both the dorsal and ventral cell membrane. In human lymphocytes band 3 antigen accompanied capping of concanavalin A binding surface receptors. The immunoreactive form of band 3 in fibroblasts was shown by immunoblotting studies to be a polypeptide of approximately 60 000 dalton. This polypeptide is immunologically and electrophoretically related to a major immunoreactive form of band 3 naturally occurring in the red blood cell membrane. Considering the recent identification in nonerythroid cells of immunoreactive forms of other major components of the erythrocyte membrane cytoskeleton, the present observation in nucleated cells of a polypeptide related to erythrocyte band 3 may indicate some of the features of erythrocyte membrane architecture are also present in nonerythroid cells.     

Identification of two novel mutations in POU4F3 gene associated with autosomal dominant hearing loss in Chinese families

Autosomal dominant non‐syndromic hearing loss is genetically heterogeneous with 47 genes identified to date, including POU4F3. In this study, by using a next‐generation sequencing panel targeting 127 deafness genes, we identified a pathogenic frameshift mutation c.704_705del and a missense mutation c.593G>A in two three‐generation Chinese families with late‐onset progressive ADNSHL, respectively. The novel mutations of POU4F3 co‐segregated with the deafness phenotype in these two families. c.704_705del caused a frameshift p.T235fs and c.593G>A caused an amino acid substitution of p.R198H. Both mutations led to an abnormal and incomplete protein structure. POU4F3 with either of the two mutations was transiently transfected into HEI‐OC1 and HEK 293 cell lines and immunofluorescence assay was performed to investigate the subcellular localization of mutated protein. The results indicated that both c.704_705del (p.T235fs) and c.593G>A (p.R198H) could impair the nuclear localization function of POU4F3. The p.R198H POU4F3 protein was detected as a weak band of the correct molecular weight, indicating that the stability of p.R198H POU4F3 differed from that of the wild‐type protein. While, the p.T235fs POU4F3 protein was expressed with a smaller molecular weight, implying this mutation result in a frameshift and premature termination of the POU4F3 protein. In summary, we report two novel mutations of POU4F3 associated with progressive ADNSHL and explored their effects on POU4F3 nuclear localization. These findings expanded the mutation spectrum of POU4F3 and provided new knowledge for the pathogenesis of POU4F3 in hearing loss.     

Interaction of deoxyhemoglobin with the cytoplasmic domain of murine erythrocyte band 3

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 O(2)-dependent sensor that mediates O(2)-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 O(2) 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 O(2)-dependent regulation of erythrocyte physiology, we undertook characterization of the effect of O(2) on band 3-Hb interactions in the mouse. We report here that murine band 3 binds deoxyHb with significantly greater affinity than oxyHb, despite the lack of significant homology within the deoxyHb binding sequence. We further map the deoxyHb binding site on murine band 3 and show that deletion of the site eliminates deoxyHb binding. Finally, we identify mutations in murine cdb3 that either enhance or eliminate its affinity for murine deoxyHb. These data demonstrate that despite a lack of homology in the sequences of both murine band 3 and murine Hb, a strong oxygen-dependent association of the two proteins has been conserved.     

红细胞膜带4.2蛋白的结构与功能

带4.2蛋白是一种重要的红细胞膜蛋白,与红细胞的形态、可变形性及携氧功能有至关重要的联系。它通过与带3蛋白(阴离子通道蛋白)、锚蛋白结合,稳定的连接在细胞膜的内表面,连接着膜骨架网架结构与细胞膜,是膜骨架与脂质双分子层连接的重要纽带。带4.2蛋白的缺失会引起球形或椭圆形红细胞增多症及不同程度的溶血性贫血,严重的情况需要摘除脾脏来进行治疗。近年来研究认为,带4.2蛋白在维持细胞膜骨架的完整性和稳定性方面扮演了重要角色。现对带4.2蛋白结构及功能的研究状况进行综述。     

Disruption of C-terminal cytoplasmic domain of betaPS integrin subunit has dominant negative properties in developing Drosophila

We have analyzed a set of new and existing strong mutations in the myospheroid gene, which encodes the betaPS integrin subunit of Drosophila. In addition to missense and other null mutations, three mutants behave as antimorphic alleles, indicative of dominant negative properties. Unlike null alleles, the three antimorphic mutants are synthetically lethal in double heterozygotes with an inflated (alphaPS2) null allele, and they fail to complement very weak, otherwise viable alleles of myospheroid. Two of the antimorphs result from identical splice site lesions, which create a frameshift in the C-terminal half of the cytoplasmic domain of betaPS. The third antimorphic mutation is caused by a stop codon just before the cytoplasmic splice site. These mutant betaPS proteins can support cell spreading in culture, especially under conditions that appear to promote integrin activation. Analyses of developing animals indicate that the dominant negative properties are not a result of inefficient surface expression, or simple competition between functional and nonfunctional proteins. These data indicate that mutations disrupting the C-terminal cytoplasmic domain of integrin beta subunits can have dominant negative effects in situ, at normal levels of expression, and that this property does not necessarily depend on a specific new protein sequence or structure. The results are discussed with respect to similar vertebrate beta subunit cytoplasmic mutations.     

The structure and function of band 3 (AE1): Recent developments (Review)

This review discusses recent advances in our understanding of the structure, function and molecular genetics of the membrane domain of red cell anion exchanger, band 3 (AE1), and its role in red cell and kidney disease. A new model for the topology of band 3 has been proposed, which suggests the membrane domain has 12 membrane spans, rather than the 14 membrane spans of earlier models. The major difference between the models is in the topology of the region on the C-terminal side of membrane spans 1-7. Two dimensional crystals of the deglycosylated membrane domain of band 3 have yielded two and three dimensional projection maps of the membrane domain dimer at low resolution. The human band 3 gene has been completely sequenced and this has facilitated the study of natural band 3 mutations and their involvement in disease. About 20% of hereditary spherocytosis cases arise from heterozygosity for band 3 mutations, and result in the absence or decrease of the mutant protein in the red cell membrane. Several other natural band 3 mutations are known that appear to be clinically benign, but alter red cell phenotype or are associated with altered red cell blood group antigens. These include the mutant band 3 present in Southeast Asian ovalocytosis, a condition which provides protection against cerebral malaria in children. Familial distal renal tubular acidosis, a condition associated with kidney stones, has been shown to result from a novel group of band 3 mutations. The total absence of band 3 has been described in animals-occurring naturally in cattle and after targeted disruption in mice. Some of these severely anaemic animals survive, so band 3 is not strictly essential for life. Although the band 3-negative red cells were very unstable, they contained a normally-assembled red cell skeleton, suggesting that the bilayer of the normal red cell membrane is stabilized by band 3 interactions with membrane lipids, rather than by interactions with the spectrin skeleton.     

Molecular basis and functional consequences of the dominant effects of the mutant band 3 on the structure of normal band 3 in Southeast Asian ovalocytosis

Southeast Asian ovalocytosis (SAO) human red cell membranes contain similar proportions of normal band 3 and a mutant band 3 with a nine amino acid deletion (band 3 SAO). We employed specific chemical modification and proteolytic cleavage to probe the structures of band 3 in normal and SAO membranes. When the membranes were modified specifically at lysine residues with N-hydroxysulfosuccinimide-SS-biotin, band 3 Lys-851 was not modified in normal membranes but quantitatively modified in SAO membranes. Normal and SAO membranes showed different patterns of band 3 proteolytic cleavage. Notably, many sites cleaved in normal membranes were not cleaved in SAO membranes, despite the presence of normal band 3 in these membranes. The mutant band 3 changes the structure of essentially all the normal band 3 present in the SAO membranes, and these changes extend throughout the normal band 3 molecules. The results also imply that band 3 in SAO membranes is present as hetero-tetramers or higher hetero-oligomers. The dominant structural effects of band 3 SAO on the other band 3 allele have important consequences on the functional and hematological properties of human red cells heterozygous for band 3 SAO. Analysis of the altered profile of biotinylation and protease cleavage sites suggests the location of exposed surfaces in the band 3 membrane domain and identifies likely interacting regions within the molecule. Our approach provides a sensitive method for studying structural changes in polytopic membrane proteins.     

Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) Deficiencies: HPRT1 Mutations in New Japanese Families and PRPP Concentration

Mutation of hypoxanthine guanine phosphoribosyltransferase (HPRT) gives rise to Lesch–Nyhan syndrome, which is characterized by hyperuricemia, severe motor disability, and self-injurious behavior, or HPRT-related gout with hyperuricemia. Four mutations were detected in two Lesch–Nyhan families and two families with partial deficiency since our last report. A new mutation of G to TT (c.456delGinsTT) resulting in a frameshift (p.Q152Hfs*3) in exon 3 has been identified in one Lesch–Nyhan family. In the other Lesch–Nyhan family, a new point mutation in intron 7 (c.532 + 5G > T) causing splicing error (exon 7 excluded, p.L163Cfs*4) was detected. In the two partial deficiency cases with hyperuricemia, two missense mutations of p.D20V (c.59A > T) and p.H60R (c.179A >G) were found. An increase of erythrocyte PRPP concentration was observed in the respective phenotypes and seems to be correlated with disease severity.     

Analysis of band 3 cytoplasmic domain phosphorylation and association with ankyrin

The phosphorylation of the cytoplasmic domain of band 3 by the human erythrocyte membrane kinase and casein kinase A has been investigated. The cytoplasmic domain of band 3 was released from erythrocyte vesicles by treatment with alpha-chymotrypsin and isolated as a 43,000-Da peptide. Both the membrane kinase and casein kinase A catalyzed the incorporation of about 1 mol of phosphate per mole of the band 3 fragment. The phosphorylation of the band 3 fragment by both kinases was not additive, suggesting that the two enzymes might recognize the same phosphorylation sites. Also in support of this notion was the observation that the phosphopeptide maps of the band 3 fragment phosphorylated by the two kinases were identical. Phosphoamino acid analysis of the band 3 fragment phosphorylated by casein kinase A revealed the presence of approximately equal amounts of phosphoserine and phosphothreonine and, to a lesser extent, phosphotyrosine. The interaction between the 43,000-Da peptide with ankyrin and the effect of phosphorylation on this interaction have been examined. The band 3 fragment was found to form two different types of complexes, termed C1 and C2, with ankyrin in a saturable manner. The C1 and C2 complexes contained about 1.7 and 0.43 mol of band 3 fragment per mole of ankyrin, respectively. Interestingly, these binding stoichiometries were found to be reduced by half by the phosphorylation of ankyrin but not by the phosphorylation of the band 3 fragment. The results suggest that the structure and dynamics of the erythrocyte membrane cytoskeletal network may be regulated by phosphorylation.     

The orientation of eosin-5-maleimide on human erythrocyte band 3 measured by fluorescence polarization microscopy.

The dominant motional mode for membrane proteins is uniaxial rotational diffusion about the membrane normal axis, and investigations of their rotational dynamics can yield insight into both the oligomeric state of the protein and its interactions with other proteins such as the cytoskeleton. However, results from the spectroscopic methods used to study these dynamics are dependent on the orientation of the probe relative to the axis of motion. We have employed polarized fluorescence confocal microscopy to measure the orientation of eosin-5-maleimide covalently reacted with Lys-430 of human erythrocyte band 3. Steady-state polarized fluorescence images showed distinct intensity patterns, which were fit to an orientation distribution of the eosin absorption and emission dipoles relative to the membrane normal axis. This orientation was found to be unchanged by trypsin treatment, which cleaves band 3 between the integral membrane domain and the cytoskeleton-attached domain. this result suggests that phosphorescence anisotropy changes observed after trypsin treatment are due to a rotational constraint change rather than a reorientation of eosin. By coupling time-resolved prompt fluorescence anisotropy with confocal microscopy, we calculated the expected amplitudes of the e-Dt and e-4Dt terms from the uniaxial rotational diffusion model and found that the e-4Dt term should dominate the anisotropy decay. Delayed fluorescence and phosphorescence anisotropy decays of control and trypsin-treated band 3 in ghosts, analyzed as multiple uniaxially rotating populations using the amplitudes predicted by confocal microscopy, were consistent with three motional species with uniaxial correlation times ranging from 7 microseconds to 1.4 ms.     

Structural and functional characterization of band 3 from Southeast Asian ovalocytes.

To determine why deletion of the nine amino acids joining the membrane and cytoplasmic domains of band 3 from Southeast Asian ovalocytes (SAO) renders the erythrocytes rigid, we compared the structural and functional properties of SAO and normal band 3. Calorimetric data, inhibitor binding studies, and anion transport assays all reveal that the membrane-spanning domain of SAO band 3 is denatured, while proteolysis studies and circular dichroism spectroscopy suggest the mutant domain retains much secondary structure. It is concluded that the transmembrane helices of SAO band 3 are dissociated and randomized but not unfolded. The cytoplasmic domain of SAO band 3 was shown to be structurally and functionally normal based on (i) calorimetric properties, (ii) native conformational change, (iii) ability to form an intersubunit disulfide bond, (iv) affinity and capacity for binding ankyrin and protein 4.1, and (v) kinetics of association with ankyrin. However, both normal and mutant isoforms of band 3 in SAO cells were found to adhere nonspecifically to the spectrin skeleton. Further, when SAO cells were osmotically swollen, the detergent extractability of band 3 became normal. We propose that much of band 3 is nonspecifically entrapped in the spectrin network in SAO cells and that this nonspecific adhesion may be responsible for the rigidity of the SAO erythrocyte.     

Control of band 3 lateral and rotational mobility by band 4.2 in intact erythrocytes: release of band 3 oligomers from low-affinity binding sites.

Band 4.2 is a human erythrocyte membrane protein of incompletely characterized structure and function. Erythrocytes deficient in band 4.2 protein were used to examine the functional role of band 4.2 in intact erythrocyte membranes. Both the lateral and the rotational mobilities of band 3 were increased in band 4.2-deficient erythrocytes compared to control cells. In contrast, the lateral mobility of neither glycophorins nor a fluorescent phospholipid analog was altered in band 4.2-deficient cells. Compared to controls, band 4.2-deficient erythrocytes manifested a decreased ratio of band 3 to spectrin, and band 4.2-deficient membrane skeletons had decreased extractability of band 3 under low-salt conditions. Normal band 4.2 was found to bind to spectrin in solution and to promote the binding of spectrin to ankyrin-stripped inside-out vesicles. We conclude that band 4.2 provides low-affinity binding sites for both band 3 oligomers and spectrin dimers on the human erythrocyte membrane. Band 4.2 may serve as an accessory linking protein between the membrane skeleton and the overlying lipid bilayer.     

Flexibility of the cytoplasmic domain of the anion exchange protein, band 3, in human erythrocytes.

The rotational flexibility of the cytoplasmic domain of band 3, in the region that is proximal to the inner membrane surface, has been investigated using a combination of time-resolved optical anisotropy (TOA) and saturation-transfer electron paramagnetic resonance (ST-EPR) spectroscopies. TOA studies of rotational diffusion of the transmembrane domain of band 3 show a dramatic decrease in residual anisotropy following cleavage of the link with the cytoplasmic domain by trypsin (E. A. Nigg and R. J. Cherry, 1980, Proc. Natl. Acad. Sci. U.S.A. 77:4702-4706). This result is compatible with two independent hypotheses: 1) trypsin cleavage leads to dissociation of large clusters of band 3 that are immobile on the millisecond time scale, or 2) trypsin cleavage leads to release of a constraint to uniaxial rotational diffusion of the transmembrane domain. ST-EPR studies at X- and Q-band microwave frequencies detect rotational diffusion of the transmembrane domain of band 3 about the membrane normal axis of reasonably large amplitude that does not change upon cleavage with trypsin. These ST-EPR results are not consistent with dissociation of clusters of band 3 as a result of cleavage with trypsin. Global analyses of the ST-EPR data using a newly developed algorithm indicate that any constraint to rotational diffusion of the transmembrane domain of band 3 via interactions of the cytoplasmic domain with the membrane skeleton must be sufficiently weak to allow rotational excursions in excess of 32 degrees full-width for a square-well potential. In support of this result, analyses of the TOA data in terms of restricted amplitude uniaxial rotational diffusion models suggest that the membrane-spanning domain of that population of band 3 that is linked to the membrane skeleton is constrained to diffuse in a square-well of approximately 73 degrees full-width. This degree of flexibility may be necessary for providing the unique mechanical properties of the erythrocyte membrane.     

Two chicken erythrocyte band 3 mRNAs are generated by alternative transcriptional initiation and differential RNA splicing.

The erythrocyte anion transport protein (band 3) mediates two distinct cellular functions: it provides plasma membrane attachment sites for the erythroid cytoskeletal network, and it also functions as the anion transporter between the erythrocyte cytoplasm and extracellular milieu. We previously showed that two chicken band 3 polypeptides are encoded by two different mRNAs with different translation initiation sites. Here we show that these two band 3 mRNAs are transcribed from two separate promoters within a single gene. In addition, the two pre-mRNAs are differentially spliced, leading to fusion with coding exons used in common in the two mRNAs. The chicken erythrocyte band 3 gene is therefore the first example of a gene that has two promoters within a single locus which function equally efficiently in one cell type at the same developmental stage.