Physical and functional links between anion exchanger-1 and sodium pump.

Anion exchanger-1 (AE1) mediates chloride-bicarbonate exchange across the plasma membranes of erythrocytes and, via a slightly shorter transcript, kidney epithelial cells. On an omnivorous human diet, kidney AE1 is mainly active basolaterally in α-intercalated cells of the collecting duct, where it is functionally coupled with apical proton pumps to maintain normal acid-base homeostasis. The C-terminal tail of AE1 has an important role in its polarized membrane residency. We have identified the ß1 subunit of Na(+),K(+)-ATPase (sodium pump) as a binding partner for AE1 in the human kidney. Kidney AE1 and ß1 colocalized in renal α-intercalated cells and coimmunoprecipitated (together with the catalytic α1 subunit of the sodium pump) from human kidney membrane fractions. ELISA and fluorescence titration assays confirmed that AE1 and ß1 interact directly, with a Kd value of 0.81 µM. GST-AE1 pull-down assays using human kidney membrane proteins showed that the last 11 residues of AE1 are important for ß1 binding. siRNA-induced knockdown of ß1 in cell culture resulted in a significant reduction in kidney AE1 levels at the cell membrane, whereas overexpression of kidney AE1 increased cell surface sodium pump levels. Notably, membrane staining of ß1 was reduced throughout collecting ducts of AE1-null mouse kidney, where increased fractional excretion of sodium has been reported. These data suggest a requirement of ß1 for proper kidney AE1 membrane residency, and that activities of AE1 and the sodium pump are coregulated in kidney.

Partial pyruvate kinase deficiency aggravates the phenotypic expression of band 3 deficiency in a family with hereditary spherocytosis.

In a family with mild dominant spherocytosis, affected members showed partial band 3 deficiency. The index patient showed more severe clinical symptoms than his relatives, and his red blood cells displayed concomitant low pyruvate kinase activity. We investigated the contribution of partial PK deficiency to the phenotypic expression of mutant band 3 in this family. Pyruvate kinase deficiency and band 3 deficiency were characterized by DNA analysis. Results of red cell osmotic fragility testing, the results of cell deformability obtained by the Automated Rheoscope and Cell Analyzer and the results obtained by Osmotic Gradient Ektacytometry, which is a combination of these tests, were related to the red cell ATP content. Spherocytosis in this family was due to a novel heterozygous mutation in SLC4A1, the gene for band 3. Reduced PK activity of the index patient was attributed to a novel mutation in PKLR inherited from his mother, who was without clinical symptoms. Partial PK deficiency was associated with decreased red cell ATP content and markedly increased osmotic fragility. This suggests an aggravating effect of low ATP levels on the phenotypic expression of band 3 deficiency.

Genetic diagnosis of band 3 deficiency using a quenching probe (QProbe)-PCR assay in bovine embryos.

The present study was conducted to develop a simple and rapid procedure to determine the genotype of band 3 deficiency in bovine embryos by a novel real-time PCR method using a fluorescent quenching-based probe (QProbe-PCR). QProbe-PCR successfully distinguished wild type and R664X mutant alleles by melting curve analysis. Minimal amounts of DNA template were required for the detection of wild type/wild type alleles, mutant/mutant alleles, and wild type/mutant alleles; their amounts were 10 pg, 25 pg, and 50 pg, respectively. When 10 blastomeres were used as a DNA sample, accuracies of genotyping by QProbe-PCR were 100% and 89% in embryos homozygous for the wild type allele and heterozygous for the wild type and mutant alleles, respectively. QProbe-PCR takes approximately 2 h for genotyping and requires lesser time than the conventional method using PCR-RFLP, which requires digestion with a restriction enzyme and electrophoresis. Our data showed that QProbe-PCR is a useful method for rapid analysis of the genetic deficiency in preimplantation embryos. Reduction in the time required for genotyping enabled the transfer of genetically selected embryos to recipient cows on the day of embryo collection. These results suggest that determination of the genotype for the genetic deficiency in embryos is useful to select animals free from the genetic disease, and it also makes it possible to produce an animal model homozygous for the mutation.

[Band 3 deficiency as a cause of hereditary spherocytosis].

Band 3 protein accounts for the largest percentage of whole erythrocyte membrane proteins. Abnormalities in this protein are closely associated with pathologies including hereditary spherocytosis (HS), Southeast Asian ovalocytosis and distant renal tubular acidosis. Currently, EMA binding capacity measurement in erythrocytes is the most useful screening test for diagnosing HS. We have also demonstrated reduced EMA binding capacity in patients with HS who have deficiencies of membrane proteins such as ankyrin not directly binding to EMA and who have as yet undetectable abnormalities of membrane proteins. However, even patients with hereditary elliptocytosis, who have a partial spectrin deficiency, were found to show reduced EMA binding capacity. Six of 7 had spherocytic elliptocytosis. Therefore, it is necessary to meticulously diagnose HS by ruling out all other possibilities.

Cell-specific mitotic defect and dyserythropoiesis associated with erythroid band 3 deficiency.

Most eukaryotic cell types use a common program to regulate the process of cell division. During mitosis, successful partitioning of the genetic material depends on spatially coordinated chromosome movement and cell cleavage. Here we characterize a zebrafish mutant, retsina (ret), that exhibits an erythroid-specific defect in cell division with marked dyserythropoiesis similar to human congenital dyserythropoietic anemia. Erythroblasts from ret fish show binuclearity and undergo apoptosis due to a failure in the completion of chromosome segregation and cytokinesis. Through positional cloning, we show that the ret mutation is in a gene (slc4a1) encoding the anion exchanger 1 (also called band 3 and AE1), an erythroid-specific cytoskeletal protein. We further show an association between deficiency in Slc4a1 and mitotic defects in the mouse. Rescue experiments in ret zebrafish embryos expressing transgenic slc4a1 with a variety of mutations show that the requirement for band 3 in normal erythroid mitosis is mediated through its protein 4.1R-binding domains. Our report establishes an evolutionarily conserved role for band 3 in erythroid-specific cell division and illustrates the concept of cell-specific adaptation for mitosis.

Analysis of novel sph (spherocytosis) alleles in mice reveals allele-specific loss of band 3 and adducin in alpha-spectrin-deficient red cells.

Five spontaneous, allelic mutations in the alpha-spectrin gene, Spna1, have been identified in mice (spherocytosis [sph], sph(1J), sph(2J), sph(2BC), sph(Dem)). All cause severe hemolytic anemia. Here, analysis of 3 new alleles reveals previously unknown consequences of red blood cell (RBC) spectrin deficiency. In sph(3J), a missense mutation (H2012Y) in repeat 19 introduces a cryptic splice site resulting in premature termination of translation. In sph(Ihj), a premature stop codon occurs (Q1853Stop) in repeat 18. Both mutations result in markedly reduced RBC membrane spectrin content, decreased band 3, and absent beta-adducin. Reevaluation of available, previously described sph alleles reveals band 3 and adducin deficiency as well. In sph(4J), a missense mutation occurs in the C-terminal EF hand domain (C2384Y). Notably, an equally severe hemolytic anemia occurs despite minimally decreased membrane spectrin with normal band 3 levels and present, although reduced, beta-adducin. The severity of anemia in sph(4J) indicates that the highly conserved cysteine residue at the C-terminus of alpha-spectrin participates in interactions critical to membrane stability. The data reinforce the notion that a membrane bridge in addition to the classic protein 4.1-p55-glycophorin C linkage exists at the RBC junctional complex that involves interactions between spectrin, adducin, and band 3.

Erythrocyte membrane protein destabilization versus clinical outcome in 160 Portuguese Hereditary Spherocytosis patients.

Hereditary Spherocytosis (HS) is a haemolytic anaemia caused by erythrocyte protein membrane defects - spectrin, ankyrin, band 3 or protein 4.2 - that lead to membrane destabilization. This study aimed to evaluate the prevalence of protein deficiencies and the role of membrane proteins or membrane-linked proteins in membrane disturbance and in HS clinical outcome. A total of 215 Portuguese individuals were studied - 203 from 71 families plus 12 individual unrelated subjects; 160 of them were diagnosed with HS. They were classified as presenting mild, moderate or severe forms of HS according to the degree of haemolytic anaemia. Standardized electrophoretic erythrocyte membrane protein analysis was used to identify and quantify protein deficiencies. Band 3 and ankyrin were found to account for the majority of the erythrocyte protein defects underlying HS. Increasing isolated protein deficiency or increasing imbalance between combined protein deficiencies seemed to underlie HS severity, by increasing membrane destabilization. There was an increased membrane linkage of the cytosolic proteins, glyceraldehyde-3-phosphate dehydrogenase and peroxiredoxin 2, and of denatured haemoglobin, suggesting that this linkage could interfere with membrane structure. Our data suggest that the quantification and the analysis of RBC membrane proteins may be helpful in predicting the clinical outcome of HS.

Characterization of glycolytic enzyme interactions with murine erythrocyte membranes in wild-type and membrane protein knockout mice.

Previous research has shown that glycolytic enzymes (GEs) exist as multienzyme complexes on the inner surface of human erythrocyte membranes. Because GE binding sites have been mapped to sequences on the membrane protein, band 3, that are not conserved in other mammalian homologs, the question arose whether GEs can organize into complexes on other mammalian erythrocyte membranes. To address this, murine erythrocytes were stained with antibodies to glyceraldehyde-3-phosphate dehydrogenase, aldolase, phosphofructokinase, lactate dehydrogenase, and pyruvate kinase and analyzed by confocal microscopy. GEs were found to localize to the membrane in oxygenated erythrocytes but redistributed to the cytoplasm upon deoxygenation, as seen in human erythrocytes. To identify membrane proteins involved in GE assembly, erythrocytes from mice lacking each of the major erythrocyte membrane proteins were examined for GE localization. GEs from band 3 knockout mice were not membrane associated but distributed throughout the cytoplasm, regardless of erythrocyte oxygenation state. In contrast, erythrocytes from mice lacking alpha-spectrin, ankyrin, protein 4.2, protein 4.1, beta-adducin, or dematin headpiece exhibited GEs bound to the membrane. These data suggest that oxygenation-dependent assembly of GEs on the membrane could be a general phenomenon of mammalian erythrocytes and that stability of these interactions depends primarily on band 3.

Hereditary stomatocytosis and cation-leaky red cells--recent developments.

The hereditary stomatocytoses (HSt) are a diverse group of conditions. Common features include hemolytic anemia, a red cell cation leak and morphological changes, but the severity of the condition can vary enormously. We have previously shown that one form of HSt (cryohydrocytosis), where the monovalent cation leak is increased at low temperature, results from amino acid substitutions in the membrane domain of band 3 (anion exchanger 1, SLC4A1). These substitutions appear to convert band 3 from an anion exchanger into a cation channel. More recently we found that over-hydrated hereditary stomatocytosis (OHSt) results from amino acid substitutions in Rh-associated glycoprotein (RhAG), a putative gas channel protein. Both band 3 and RhAG associate in the red cell membrane to form a macrocomplex that is thought to be involved in red cell gas exchange. In this paper I will review the data that has been published so far on the molecular basis of HSt. I will mention other similar conditions that cause either a cation leak or stomatocytosis or both, and consider the mechanisms of red cell shape change and permeability.

Increased acid load and deletion of AE1 increase Slc26a7 expression.

BACKGROUND/AIMS: Slc26a7 is a member of a family of anion transport proteins, Solute-Linked Carrier 26 (Slc26). Slc26a7, which can mediate Cl-/HCO3- exchange, is expressed in the acid-secreting, A-intercalated cells of the kidney collecting duct. On the basolateral side of the A-intercalated cells, Slc26a7 co-localizes with the anion exchanger 1 (AE1), a Cl-/HCO3- exchanger that mediates bicarbonate reabsorption in the collecting duct. METHODS: To test if Slc26a7 is involved in acid-base regulation, as its localization and function suggest, we examined the effect of acid loading and deletion of AE1 on Slc26a7 expression with quantitative real-time RT-PCR and Western blotting. RESULTS: Four days of acid loading increased Slc26a7 mRNA expression in the kidney inner medulla by 57% (n = 6 acid loaded vs. n = 6 control rats; p < 0.001), whereas mRNA expression in the outer medulla and the cortex did not change. Western blotting analysis demonstrated increased Slc26a7 protein expression in both outer (140%) and inner medulla (50%) in acid-loaded animals (n = 3) compared to controls (n = 3; p < 0.05). The expression of Slc26a7 mRNA was increased by 66% in the kidneys of AE1 knockout mice (n = 5) compared to the wild types (n = 5, p < 0.001). The increase in Slc26a7 mRNA correlated with a twofold increase in protein expression (p < 0.05). CONCLUSION: We suggest that the increase in Slc26a7 expression caused by acid challenge and deletion of AE1 represents an adaptive response, indicating that Slc26a7 contributes to the regulation of acid-base balance by the kidney.

Duplication of 10 nucleotides in the erythroid band 3 (AE1) gene in a kindred with hereditary spherocytosis and band 3 protein deficiency (band 3PRAGUE).

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.

Defective anion transport and marked spherocytosis with membrane instability caused by hereditary total deficiency of red cell band 3 in cattle due to a nonsense mutation.

We studied bovine subjects that exhibited a moderate uncompensated anemia with hereditary spherocytosis inherited in an autosomal incompletely dominant mode and retarded growth. Based on the results of SDS-PAGE, immunoblotting, and electron microscopic analysis by the freeze fracture method, we show here that the proband red cells lacked the band 3 protein completely. Sequence analysis of the proband band 3 cDNA and genomic DNA showed a C --> T substitution resulting in a nonsense mutation (CGA --> TGA; Arg --> Stop) at the position corresponding to codon 646 in human red cell band 3 cDNA. The proband red cells were deficient in spectrin, ankyrin, actin, and protein 4.2, resulting in a distorted and disrupted membrane skeletal network with decreased density. Therefore, the proband red cell membranes were extremely unstable and showed the loss of surface area in several distinct ways such as invagination, vesiculation, and extrusion of microvesicles, leading to the formation of spherocytes. Total deficiency of band 3 also resulted in defective Cl-/HCO3- exchange, causing mild acidosis with decreases in the HCO3- concentration and total CO2 in the proband blood. Our results demonstrate that band 3 indeed contributes to red cell membrane stability, CO2 transport, and acid-base homeostasis, but is not always essential to the survival of this mammal.

Characterization of red cell membrane proteins as a function of red cell density: annexin VII in different forms of hereditary spherocytosis.

Fresh human blood samples were collected from healthy controls and splenectomized and unsplenectomized patients with hereditary spherocytosis due to band 3 or ankyrin and spectrin deficiency. The erythrocytes were separated into age-related fractions using self-forming Percoll density gradients. Membrane proteins were analysed by 2D electrophoresis and identified by mass spectrometry. Annexin VII was present in reticulocytes but was then lost as the cells matured. A different pattern was found in band 3-deficient samples: annexin VII was in fact present in both mature and immature red cell membranes. Cytoskeletal anomalies may then influence the turn-over of annexin VII during erythrocyte maturation.

Genetic diagnosis of band 3 deficiency and sexing in bovine preimplantation embryos.

Band 3 deficiency with hereditary spherocytosis and hemolytic anemia in Japanese black cattle, band 3(Bov.Yamagata), is caused by a total lack of band 3 protein with an autosomal dominant inheritance. Genotyping for band 3 deficiency and sexing were successfully achieved in biopsied embryo cells with efficiencies of 98.4% and 97.4%, respectively. Transfer of the embryo that was determined as homozygous for the mutant allele into a recipient cow resulted in the production of a fetus exhibiting the genotype and red cell phenotypes characteristic of band 3(Bov.Yamagata). These results demonstrate that our procedure is reliable and applicable to produce animals free from or homozygous for the mutant allele by breeding carrier animals.

A Stem Cell Strategy Identifies Glycophorin C as a Major Erythrocyte Receptor for the Rodent Malaria Parasite Plasmodium berghei.

The clinical complications of malaria are caused by the parasite expansion in the blood. Invasion of erythrocytes is a complex process that depends on multiple receptor-ligand interactions. Identification of host receptors is paramount for fighting the disease as it could reveal new intervention targets, but the enucleated nature of erythrocytes makes genetic approaches impossible and many receptors remain unknown. Host-parasite interactions evolve rapidly and are therefore likely to be species-specific. As a results, understanding of invasion receptors outside the major human pathogen Plasmodium falciparum is very limited. Here we use mouse embryonic stem cells (mESCs) that can be genetically engineered and differentiated into erythrocytes to identify receptors for the rodent malaria parasite Plasmodium berghei. Two proteins previously implicated in human malaria infection: glycophorin C (GYPC) and Band-3 (Slc4a1) were deleted in mESCs to generate stable cell lines, which were differentiated towards erythropoiesis. In vitro infection assays revealed that while deletion of Band-3 has no effect, absence of GYPC results in a dramatic decrease in invasion, demonstrating the crucial role of this protein for P. berghei infection. This stem cell approach offers the possibility of targeting genes that may be essential and therefore difficult to disrupt in whole organisms and has the potential to be applied to a variety of parasites in diverse host cell types.

Uniquely higher incidence of isolated or combined deficiency of band 3 and/or band 4.2 as the pathogenesis of autosomal dominantly inherited hereditary spherocytosis in the Japanese population.

To clarify the pathogenesis of hereditary spherocytosis (HS), red cell membrane protein components were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a 3.5-17% exponential gradient according to the method of Fairbanks et al. and of Laemmli in 47 HS cases from 32 unrelated Japanese families. The relative contents of each membrane protein fraction, which was stained by Coomassie blue, were expressed as their ratios to those of total membrane proteins. The density of each band of red cell membrane proteins in 47 HS patients was compared to that in 10 normal controls or in 4 high-reticulocyte controls. Various isolated or combined deficiencies of membrane proteins in these HS patients were detected by identifying the amounts of membrane proteins, which were > 1 S.D. (91%) or 2 S.D. (53%) of the mean values of normal controls, and > 1 S.D. (100%) or 2 S.D. (98%) of those of high-reticulocyte controls. Contrary to the commonly held belief that most of the autosomal dominantly-inherited HS demonstrate isolated or combined deficiency of ankyrin (ANK) and/or spectrin (SP), a much lower incidence of isolated or combined deficiency of SP and/or ANK was observed in these Japanese HS patients; 19% (> 1 S.D.) or 12% (2 S.D.) compared to normal controls, or 2% (1 S.D.) or 4% (2 S.D.) compared to high-reticulocyte controls. Instead, the incidence of isolated or combined deficiency of band 3 (B3) and/or band 4.2 (B4.2) was markedly elevated in these Japanese HS patients; 50% (1 S.D.) or 39% (2 S.D.) compared to normal controls, or 78% (1 S.D.) or 88% (2 S.D.) compared to high-reticulocyte controls. Other combined deficiencies were also observed, but the incidence was much lower. Therefore, distinct characteristics, i.e., higher incidence of isolated or combined deficiency of B4.2 and/or B3 with much lower incidence of ANK and/or SP deficiency, were observed in Japanese HS patients.

Flow cytometric analysis of band 3 protein of human erythrocytes.

Flow cytometry was used to quantify the transmembrane anion exchanger (band 3 protein) of human erythrocytes by covalently bound eosin-5-maleimide. In vitro and in vivo vesiculated red blood cells were investigated. The fluorescence and light scatter signals of cells after heat induced vesiculation, in vivo ageing, and in patients with hereditary spherocytosis were decreased. These results reflect a deficiency of band 3 protein which is presumably caused by membrane surface area loss. It was possible to distinguish control erythrocytes, erythrocytes from patients with hereditary spherocytosis, and from other forms of haemolytic anaemias on the basis of their light scatter and fluorescence signals characteristics.

[Hereditary spherocytosis -- prevalence of erythrocyte membrane protein deficiency]. / Esferocitose hereditária--prevalência dos défices proteicos da membrana do eritrócito.

The authors studied the relative prevalence of erythroid cytoskeletal protein defects and their relationship with the clinical course of Hereditary Spherocytosis (HS) in 39 Portuguese patients of North of Portugal (25 families). This study showed that, in the North of Portugal, HS is primarily due to anquirin deficiency (72%), followed by band 3 (20%). These findings are similar to the published data in other Caucasian populations. Anquirin primary defects have been difficult to diagnose before splenectomy, due to high reticulocytes counts.

[Hereditary spherocytosis: one year study of erythrocyte membrane proteins]. / La spherocytose hereditaire: bilan d'une annee d'experience de l'etude des proteines de la membrane erythrocytaire.

Hereditary spherocytosis (HS) is a congenital hemolytic anemia which affects one person out of 5000 in Northern Europe. HS is caused by defects of the red cell membrane proteins involving mainly ankyrin or band 3, and less frequently, protein 4.2 or spectrin. The reduction of red cell osmotic resistance and the recognition of spherocytes on the peripheral blood smear are the primary laboratory tests necessary to evocate a diagnosis of hereditary spherocytosis. Analysis of the red cell membrane proteins using polyacrylamide gel electrophoresis is used to quantify individual proteins and to identify the protein defect related to a diagnosis of HS. Samples from 47 patients and 25 controls were studied by electrophoresis of the red cell membrane proteins. Protein deficiencies related to HS were demonstrated for 21 patients. In 4 other cases, abnormalities of membrane proteins unrelated to HS were also demonstrated. Electrophoresis of the red cell membrane proteins allows the identification of the protein deficiency related to HS and thus confirms the diagnosis of HS, but also points to the underlying molecular defect, the inheritance pattern and the clinical aspects of the disease.