Erythroid Band Research Articles

Comodulation of NO-Dependent Vasodilation by Erythroid Band 3 and Hemoglobin: A GP.Mur Athlete Study.

GP.Mur, a red blood cell (RBC) hybrid protein encoded by glycophorin B-A-B, increases expression of erythroid band 3 (Anion Exchanger-1, SLC4A1). GP.Mur is extremely rare but has a prevalence of 1–10% in regions of Southeast Asia. We unexpectedly found slightly higher blood pressure (BP) among healthy Taiwanese adults with GP.Mur. Since band 3 has been suggested to interact with hemoglobin (Hb) to modulate nitric oxide (NO)-dependent hypoxic vasodilation during the respiratory cycle, we hypothesized that GP.Mur red cells could exert differentiable effects on vascular tone. Here we recruited GP.Mur-positive and GP.Mur-negative elite male college athletes, as well as age-matched, GP.Mur-negative non-athletes, for NO-dependent flow-mediated dilation (FMD) and NO-independent dilation (NID). The subjects were also tested for plasma nitrite and nitrate before and after arterial occlusion in FMD. GP.Mur+ and non-GP.Mur athletes exhibited similar heart rates and blood pressure, but GP.Mur+ athletes showed significantly lower FMD (4.8 ± 2.4%) than non-GP.Mur athletes (6.5 ± 2.1%). NO-independent vasodilation was not affected by GP.Mur. As Hb controls intravascular NO bioavailability, we examined the effect of Hb on limiting FMD and found it to be significantly stronger in GP.Mur+ subjects. Biochemically, plasma nitrite levels were directly proportional to individual band 3 expression on the red cell membrane. The increase of plasma nitrite triggered by arterial occlusion also showed small dependency on band 3 levels in non-GP.Mur subjects. By the GP.Mur comparative study, we unveiled comodulation of NO-dependent vasodilation by band 3 and Hb, and verified the long-pending role of erythroid band 3 in this process.

The Diego blood group system: a review

Abstract The Diego blood group system (DI) currently encompasses 22 antigens. Three of the antigens are of high prevalence and the other 19 are of low prevalence. The antigens of the Diego blood group system are carried on the erythroid band 3 protein anion exchanger 1 (AE1), the product of a single gene, SLC4A1(solute carrier family 4, anion exchanger, member 1). AE1 is a member of a family of three anion exchangers or transporters expressed in a variety of tissues. This protein is involved in carbon dioxide transport from tissues to lungs. It is also found in the kidney, where it is involved in acid secretion. Antibodies to Diego system antigens with the exception of anti-Dia, -Dib, -Wra, -ELO and -DISK do not seem to be of clinical significance for transfusion or of importance in hemolytic disease of the fetus and newborn. Immunohematology 2013;29:73–81.

Analysis of the Mobilities of Band 3 Populations Associated with Ankyrin Protein and Junctional Complexes in Intact Murine Erythrocytes

Current models of the erythrocyte membrane depict three populations of band 3: (i) a population tethered to spectrin via ankyrin, (ii) a fraction attached to the spectrin-actin junctional complex via adducin, and (iii) a freely diffusing population. Because many studies of band 3 diffusion also distinguish three populations of the polypeptide, it has been speculated that the three populations envisioned in membrane models correspond to the three fractions observed in diffusion analyses. To test this hypothesis, we characterized band 3 diffusion by single-particle tracking in wild-type and ankyrin- and adducin-deficient erythrocytes. We report that ∼40% of total band 3 in wild-type murine erythrocytes is attached to ankyrin, whereas ∼33% is immobilized by adducin, and ∼27% is not attached to any cytoskeletal anchor. More detailed analyses reveal that mobilities of individual ankyrin- and adducin-tethered band 3 molecules are heterogeneous, varying by nearly 2 orders of magnitude and that there is considerable overlap in diffusion coefficients for adducin and ankyrin-tethered populations. Taken together, the data suggest that although the ankyrin- and adducin-immobilized band 3 can be monitored separately, significant heterogeneity still exists within each population, suggesting that structural and compositional properties likely vary considerably within each band 3 complex.

Band 3 Missense Mutations and Stomatocytosis: Insight into the Molecular Mechanism Responsible for Monovalent Cation Leak

Missense mutations in the erythroid band 3 protein (Anion Exchanger 1) have been associated with hereditary stomatocytosis. Features of cation leaky red cells combined with functional expression of the mutated protein led to the conclusion that the AE1 point mutations were responsible for Na+ and K+ leak through a conductive mechanism. A molecular mechanism explaining mutated AE1-linked stomatocytosis involves changes in AE1 transport properties that become leaky to Na+ and K+. However, another explanation suggests that point-mutated AE1 could regulate a cation leak through other transporters. This short paper intends to discuss these two alternatives.

Hemolytic anemia and distal renal tubular acidosis in two Indian patients homozygous for SLC4A1/AE1 mutation A858D

Familial distal renal tubular acidosis (dRTA) can be caused by mutations in the Cl2/HCO32 exchanger of the renal Type A intercalated cell, kidney AE1/SLC4A1. dRTA-associated AE1 mutations have been reported in families from North America, Europe, Thailand, Malaysia, Papua-New Guinea, Taiwan, and the Philippines, but not India. The dRTA mutation AE1 A858D has been detected only in the context of compound heterozygosity. We report here two unrelated Indian patients with combined hemolytic anemia and dRTA who share homozygous A858D mutations of the AE1/SLC4A1 gene. The mutation creates a novel restriction site that is validated for diagnostic screening.

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.

Substitution Glu480Lys in erythroid band 3 corresponds to the Fr(a) blood group antigen and supports existence of the second ectoplasmic loop of band 3.

Polymorphisms in extracellular loops of RBC band 3 correspond to antigens of the Diego blood group system. Of the seven putative extracellular loops, no mutations have until recently been found in the second, fifth, and sixth loops. We detected a substitution Glu480Lys that would be located in its second ectoplasmic loop. We hypothesized the substitution may underlie a novel antigen of the Diego system. IAT was performed using two different multispecific sera containing anti-Fr(a) and a series of multispecific sera containing antibodies against other blood group antigens of the Diego blood group system but not agglutinating Fr(a+) RBCs. Biosynthesis of band 3 was studied by RT-PCR of reticulocyte RNA and electrophoresis of solubilized RBC membranes. Anion exchange function of band 3 was studied by measuring the influx of radiolabeled sulfate. RBCs from the Glu480Lys carrier were agglutinated with sera containing anti-Fr(a) and not by sera with specificities for other antigens of the Diego system. We detected identical quantities of mRNA corresponding to the two band-3 alleles and normal content of band 3 in the RBC membranes, as well as normal sulfate influx into RBCs from the Fr(a) heterozygote. We confirmed the previously reported molecular basis of the Fr(a) antigen, thus providing supportive evidence for the existence of the second extracellular loop of band 3. We also demonstrated that this substitution does not affect mRNA stability, surface expression, and anion exchange function of band 3.

Identification of quantitative trait loci that modify the severity of hereditary spherocytosis inwan, a new mouse model of band-3 deficiency

AbstractDefects in red blood cell (RBC) membrane skeleton components cause hereditary spherocytosis (HS). Clinically, HS varies significantly even among individuals with identical gene defects, illustrating the profound effects of genetic background on disease severity. We exploited a new spontaneous mouse model,wan, which arose on the inbred C3H/HeJ strain, to identify quantitative trait loci (QTL) that modify the HS phenotype. Homozygouswanmice have severe HS due to a complete deficiency of erythroid band 3. A QTL analysis of RBC count, hemoglobin, hematocrit, mean corpuscular volume (MCV), and mean corpuscular hemoglobin content (MCHC) was performed inwan/wanmice from an F2 intercross between C3H/HeJ+/wanand CAST/Ei+/+F1 hybrids. Hematologic and survival data from C3H, CAST/Ei F2wanhomozygotes support the hypothesis that genetic modifiers significantly influence the band-3 null HS phenotype. Significant QTL were identified for the MCV trait only, suggesting that RBC membrane characteristics are a target for modifier gene action. The most significant quantitative trait locus,Hsm1(hereditaryspherocytosismodifier 1), localizes to mouse Chromosome 12 and is dominant. The peak LOD score was obtained with a marker forSpnb1encoding erythroid β-spectrin, an obvious candidate gene. (Blood. 2004;103: 3233-3240)

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.

1280. Insulator Elements Confer High Level, Position Independent, Copy Number Dependent, Uniform Expression of a Linked Transgene Directed by the Erythroid Band 3 (AE1) Promoter in Mice

1280. Insulator Elements Confer High Level, Position Independent, Copy Number Dependent, Uniform Expression of a Linked Transgene Directed by the Erythroid Band 3 (AE1) Promoter in Mice

Amino acid substitutions in human erythroid protein band 3 account for the low-incidence antigens NFLD and BOW.

The low-incidence red cell antigens NFLD (700.37) and BOW (700.46) were first described in 1984 and 1988, respectively. Recent investigations showed that antigens of the Diego blood group system (including a number of low-incidence antigens) are coded by SLC4A1 (solute carrier family 4, anion exchanger member 1 gene). Among these newly characterized Diego system antigens is Wu (designated DI9). Because a serologic relationship among Wu, NFLD, and BOW has been established, a series of genetic and molecular investigations of SLC4A1 in relation to NFLD and BOW were undertaken. By the use of exon-specific primers, single-strand conformational polymorphism (SSCP) analysis of SLC4A1 was performed on DNA isolated from an NFLD+ person from Japan, from the members of a Canadian kindred segregating for NFLD, and from two unrelated BOW+ persons. Exons displaying SSCPs were subjected to genetic linkage analysis (for NFLD only) and DNA sequencing. SSCPs in DNA amplified from exons 12 and 14 of SLC4A1 were observed for all NFLD+ subjects. Linkage between each of these polymorphisms and NFLD was established with peak lods = 4.82 at theta = 0.00 for combined paternal and maternal meiosis. DNA sequencing of exons 12 and 14 of SLC4A1 from NFLD+ persons identified A-->T and C-->G mutations that underlie Glu429Asp and Pro561Ala substitutions in human erythroid band 3 protein (band 3). DNA from the two unrelated BOW+ persons only exhibited an SSCP in exon 14 of SLC4A1. Subsequent DNA sequencing revealed a C-->T mutation that accounts for a Pro561Ser substitution in band 3. SLC4A1 codes for the low-incidence red cell antigens NFLD and BOW. In light of these findings, both antigens have been assigned to the Diego blood group system.

Erythroid band 3 variants and disease

This review describes some of the naturally occurring band 3 (AE1) variants and their association with disease. Southeast Asian Ovalocytic (SAO) band 3, an inactive and misfolded protein, is probably only maintained in certain populations because it provides protection against the cerebral form of malaria. Many mutations that cause instability of band 3, either at the mRNA or protein level, result in hereditary spherocytosis (HS). Some polymorphisms alter amino acid residues in the extracellular loops of band 3 and are associated with blood group antigens. A truncated form of AE1 is expressed in kidney cells and certain AE1 mutations are associated with distal renal tubular acidosis (dRTA). The molecular basis of these variants and their effect on the structure and function of band 3 are discussed. The association between band 3 and glycophorin A (GPA) and the structure/function changes of band 3 in the absence of GPA are also described.

A Gly565-->Ala substitution in human erythroid band 3 accounts for the Wu blood group polymorphism.

Reports published in 1976 and 1980 described the low-incidence red cell antigen Wu. Distinction of Wu from all other known low-incidence antigens and from the ABO, Rh, Lutheran, Duffy, Kidd, P, and X-linked blood group systems allowed Wu to be placed in the International Society of Blood Transfusion's 700 series, designated as 700013. Recently, a blood donor apparently homozygous for Wu has been identified. This report documents the serologic and molecular findings of samples from this individual and the members of his family. Blood samples from 26 members of a kindred of Dutch descent segregating for Wu were collected and analyzed. Red cells were subjected to titration and enzymatic tests, while DNA was analyzed by polyacrylamide gel electrophoresis for single-strand conformational polymorphism (SSCP) and nucleotide differences by DNA sequencing. Serologic investigations conducted on red cells of the propositus and two of his siblings consistently revealed higher titers with various sera containing anti-Wu than did cells from their parents or children. Treatment of intact red cells with alpha-chymotrypsin completely abolished Wu recognition. Because erythroid band 3 is cleaved by alpha-chymotrypsin, the possible relationship between Wu and AE1 (the gene controlling erythroid band 3 expression) was investigated by molecular methods. SSCP analysis of DNA revealed that all Wu+ family members exhibited a mobility shift in exon 14 of AE1. The nature of the SSCP was defined by DNA sequencing as a G-->C mutation that resulted in a Gly565-->Ala substitution in human erythroid band 3. Three members of the kindred are homozygous for the low-incidence red cell antigen Wu. A G-->C mutation in AE1 gives rise to a Gly565-->Ala substitution in band 3, thereby accounting for the Wu red cell polymorphism. In light of these findings, the International Society of Blood Transfusion Working Party has provisionally assigned Wu to the Diego blood group system (designated 010009 or D19).

Blood group antigens Rb(a), Tr(a), and Wd(a) are located in the third ectoplasmic loop of erythroid band 3.

Rb(a), Tr(a), and Wd(a) are three low-incidence blood group antigens that have not been assigned to a particular structure of the red cell membrane. Recent genetic and serologic data suggested erythroid band 3 as a possible carrier of these three antigens. Ten band 3 gene exons that encode the membrane domain of band 3 were screened for single strand conformation polymorphism (SSCP). Exons displaying SSCP were cloned and sequenced, and the presence of the mutations was verified by restriction digestion. Substitutions 548 Pro-->Leu, 551 Lys-->Asn, and 557 Val-->Met, all located in the third ectoplasmic loop of band 3, were detected in the subjects with Rb(a+), Tr(a+), and Wd(a+) red cells, respectively. The presence of the Rb(a) and Wd(a) mutations was confirmed in additional carriers of these blood group antigens. Chymotryptic cleavage at Tyr 553 and Tyr 555 abolished the agglutinability of Tr(a+) and Wd(a+) cells with the corresponding antisera, further demonstrating that the epitopes are located in the third ectoplasmic loop of band 3. Similar quantities of mRNA corresponding of the two band 3 alleles, a normal pattern of red cell membrane proteins, and normal DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid, disodium salt)-inhibitable sulfate flux were detected, which suggests that the mutations do not affect band 3 mRNA stability or band 3 protein expression and transport function. Wd(a) and Rb(a), and tentatively Tr(a), can be assigned to the Diego blood group system.

A Thr552—&gt;Ile substitution in erythroid band 3 gives rise to the Warrior blood group antigen

Recent family studies established that the low-incidence red cell antigen WARR is not part of the MNS, Lutheran, Lewis, Duffy, Kidd, Xg, Chido/ Rodgers, Kx, or Gerbich blood group systems. Continued serologic and genetic studies of WARR suggest that it is carried on erythroid band 3. To test the hypothesis that expression of WARR is controlled by the anion exchanger 1 gene (AE1), AE1 intronic primers that flank the exons encoding the membrane domain of band 3 were prepared. Polymerase chain reaction-amplified products corresponding to exons 11-20 of AE1 were analyzed for single-strand conformational polymorphism (SSCP) in DNA from WARR-positive and WARR-negative individuals. An SSCP was detected in exon 14. Subsequent sequencing revealed a C-->T mutation in codon 552 that leads to a Thr-->Ile substitution. Because the C-->T mutation eliminates a Bbs I restriction site, it was possible to confirm the phenotypes of all family members. To study the possible effect of the Thr552-->Ile substitution on the expression and function of band 3, polymerase chain reaction-amplified reverse-transcribed reticulocyte mRNA was digested with Bbs I. Both alleles of band 3 mRNA were detected in similar quantities, which suggests that the substitution underlying WARR did not interfere with mRNA stability. Comparison of sodium dodecyl sulfate-polyacrylamide gel electrophoretic mobility and size patterns revealed no difference between proteins isolated from WARR-positive and WARR-negative red cells. Further, the presence of WARR did not alter the di-isothiocyano-dihydrostilbene disulfonate (DIDS)-inhibitable influx of radiolabeled sulfate. Although it appears inconsequential to the function of band 3, the red cell polymorphism known as WARR is controlled by AE1. WARR should be therefore included in the Diego blood group system.

Characterization of 13 Novel Band 3 Gene Defects in Hereditary Spherocytosis With Band 3 Deficiency

AbstractHereditary 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.

Characterization of 13 novel band 3 gene defects in hereditary spherocytosis with band 3 deficiency

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.

Targeted disruption of the murine erythroid band 3 gene results in spherocytosis and severe haemolytic anaemia despite a normal membrane skeleton.

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.

Volume-associated osmolyte fluxes in cell lines with or without the anion exchanger.

To investigate the involvement of a red cell-type anion exchanger in the volume-sensitive amino acid release, the hyposmolarity-evoked release of D-[3H]aspartate and [3H]taurine was examined in three cell lines: 1) wild-type Chinese hamster ovary (CHO-K1) cells, expressing an anion exchanger activity (Cl-/SO4(2-)) functionally similar to the erythroid band 3; 2) a mutant CHO cell type (CHO 605) lacking this anion exchanger activity; and 3) 293 cells in which the Cl-/HCO3(-) anion exchanger is absent. All cell types accumulated D-[3H]aspartate and [3H]taurine under isosmotic conditions, and, similarly, in the three cell lines, cell swelling evoked by hyposmolarity induced a rapid and transient increase in the amino acid efflux. Blockers of the anion exchanger and/or Cl- channels [niflumic acid, dipyridamole, diphenylamine-2-carboxylate,5-nitro-2-(3-phenylpropylamino)-benzoi c acid, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid] were potent inhibitors of amino acid efflux in the three cell lines. 125I- efflux, used as a marker for Cl- fluxes, was also markedly increased in response to cell swelling in all cell lines, and this efflux was inhibited by the anion exchanger/Cl- channel blockers. These results do not support a role for an anion exchanger in the hyposmolarity-induced amino acid efflux and suggest that amino acids and Cl- may be transported by the same or a similar mechanism, presumably an anion channel-like structure.

Cl- -HCO3- exchange in developing neonatal rat cardiac cells. Biochemical identification and immunolocalization of band 3-like proteins.

The Cl- -HCO3- exchanger is the main anionic exchanger (AE) that alleviates alkaline loads in cardiac cells. We recently identified in adult ventricular cells two membrane proteins (80 and 120 kD) immunologically related to the erythroid band 3 and likely to mediate the anion exchange. In the present study, we further investigated the Cl- -HCO3- exchanger activity concomitantly with the expression and intracellular localization of the band 3-like proteins during the development of neonatal rat cardiac cells maintained in culture for 17 days. Microspectrofluorometric measurements of pHi in single cells show that neonatal rat cardiomyocytes display a fully functional DIDS-sensitive Cl- -HCO3- exchanger at early stages of development. Neither basal pHi nor the anion exchange activity changes with different stages of the culture. In Western blotting with an anti-whole erythroid band 3 antibody, we found both the 80- and the 120-kD band 3-like proteins in whole heart and cultured neonatal cardiac cells. The 80-kD protein was also recognized by an anti-AE1 antiserum, whereas the 120-kD protein was specifically detected by an anti-cardiac AE3 antibody. Thus, we propose that the proteins are encoded by two different genes, AE1 and AE3, respectively. Subcellular fractionation of isolated and cultured cardiomyocytes revealed the presence of both proteins in the membrane, nuclear, and myofibril fractions. The results obtained in biochemical experiments corroborate the confocal images of immunostained neonatal cells, which demonstrate perinuclear location of band 3-like proteins at an early stage of development and their appearance within myofilaments after cell maturation.(ABSTRACT TRUNCATED AT 250 WORDS)