Coexpression of band 3 mutants and Rh polypeptides: differential effects of band 3 on the expression of the Rh complex containing D polypeptide and the Rh complex containing CcEe polypeptide.

K562 cells were stably transfected with cDNAs encoding the band 3 found in Southeast Asian ovalocytosis (B3SAO, deletion of residues 400-408), band 3 with a transport-inactivating E681Q point mutation (B3EQ), or normal band 3 (B3). Flow cytometric analysis and quantitative immunoblotting revealed that B3SAO expressed alone was translocated to the plasma membrane, at levels similar to B3 or B3EQ. Nine monoclonal antibodies that reacted with extracellular loops of B3 also reacted with B3SAO, although the affinity of most antibodies for the mutant protein was reduced. Both known Wr(b) epitopes were expressed on K562/B3SAO cells, demonstrating that B3SAO interacts with glycophorin A. The growth rates of K562 clones expressing equivalent amounts of B3 and B3EQ were the same, suggesting that the potentially toxic transport function of band 3 may be regulated in K562 cells. The band 3-mediated enhancement of Rh antigen reactivity and the depression of Rh epitopes on SAO erythrocytes were investigated by comparing the coexpression of B3, B3SAO, or B3EQ in K562 clones expressing exogenous RhcE or RhD polypeptides. The results are consistent with an interaction between band 3 and the Rh polypeptide-Rh glycoprotein (RhAG) complex, which may enhance translocation of the complex or affect its conformation in the plasma membrane. The data suggest that the interaction between band 3 and the RhD-RhAG complex is weaker than it is between band 3 and the RhCcEe-RhAG complex.

Human BTR1, a new bicarbonate transporter superfamily member and human AE4 from kidney.

We report the cloning, characterization, and chromosomal localization of two novel human members of the bicarbonate transporter superfamily, BTR1 (Bicarbonate Transporter Related protein-1) and AE4 (Anion Exchange protein 4). BTR1 is a novel mammalian protein. The BTR1 gene maps to chromosome 20p12 and encodes a 100 kDa protein predominantly expressed in the kidney, salivary glands, testis, thyroid glands, and trachea. The AE4 gene maps to chromosome 5q23-31 and encodes a 104 kDa protein expressed mainly in the kidney. Human AE4 shares 84% identity with the recently reported rabbit AE4, a sodium independent, Cl(-)/HCO(-)(3) exchanger located on the apical membrane of beta-intercalated kidney cells.

Glycophorin A dimerization and band 3 interaction during erythroid membrane biogenesis: in vivo studies in human glycophorin A transgenic mice.

Band 3 and glycophorin A (GPA) are the 2 most abundant integral proteins in the human erythrocyte membrane. Earlier studies suggested that the 2 proteins may associate not only in the mature erythrocyte membrane, but also during their posttranslational processing and intracellular trafficking. The purpose of this study was to directly examine the GPA-band 3 interaction in vivo and determine the nature of this association during erythroid membrane biogenesis. Transgenic mice were generated expressing the human glycophorin A gene and were used to examine how the induction of human GPA expression affected the levels of murine GPA and band 3 expression in the red cell membrane. Murine GPA expression was reduced in erythrocytes expressing human GPA, whereas the level of band 3 expression remained constant, implying a tight coupling of band 3 and GPA expression in the membrane of mature red cells. In vivo GPA dimerization was not modulated solely by the GPA transmembrane motif, but the distance between this motif and the basic residues on the cytoplasmic side of the transmembrane domain may also be important. In addition, GPA monomers with varying degrees of glycosylation dimerized, providing clear evidence that carbohydrate structures on the extracellular domain do not affect dimerization. The association between the multiple transmembrane-spanning protein, band 3, and the single transmembrane-spanning sialoglycoprotein, GPA, may serve as a model for interactions of other multi-pass and single-pass polypeptides during membrane biogenesis.

Lutheran blood group glycoprotein and its newly characterized mouse homologue specifically bind alpha5 chain-containing human laminin with high affinity.

Lutheran blood group glycoproteins (Lu gps) are receptors for the extracellular matrix protein, laminin. Studies suggest that Lu gps may contribute to vaso-occlusion in sickle cell disease and it has recently been shown that sickle cells adhere to laminin isoforms containing the alpha5 chain (laminin 10/11). Laminin alpha5 is present in the subendothelium and is also a constituent of bone marrow sinusoids, suggesting a role for the Lu/laminin interaction in erythropoiesis. The objectives of the current study were to define more precisely the molecular interactions of the extracellular and intracellular regions of human Lu and to clone and characterize a mouse homologue. To this end, complementary DNA and genomic clones for the mouse homologue were sequenced and the mouse Lu gene mapped to a region on chromosome 7 with conserved synteny with human 19q13.2. Mouse and human Lu gps are highly conserved (72% identity) at the amino acid sequence level and both mouse and human Lu gps specifically bind laminin 10/11 with high affinity. Furthermore, the first 3, N-terminal, immunoglobulin superfamily domains of human Lu are critical for this interaction. The results indicated that the cytoplasmic domain of BRIC 221-labeled human Lu gp is linked with the spectrin-based skeleton, affording the speculation that this interaction may be critical for signal transduction. These results further support a role for Lu gps in sickle cell disease and indicate the utility of mouse models to explore the function of Lu gp-laminin 10/11 interaction in normal erythropoiesis and in sickle cell disease.

Band 3 mutations, renal tubular acidosis and South-East Asian ovalocytosis in Malaysia and Papua New Guinea: loss of up to 95% band 3 transport in red cells.

We describe three mutations of the red-cell anion exchangerband 3 (AE1, SLC4A1) gene associated with distalrenal tubular acidosis (dRTA) in families from Malaysia and Papua NewGuinea: Gly(701)-->Asp (G701D), Ala(858)-->Asp(A858D) and deletion of Val(850) (DeltaV850). The mutationsA858D and DeltaV850 are novel; all three mutations seem to berestricted to South-East Asian populations. South-East Asianovalocytosis (SAO), resulting from the band 3 deletion of residues400-408, occurred in many of the families but did not itselfresult in dRTA. Compound heterozygotes of each of the dRTA mutationswith SAO all had dRTA, evidence of haemolytic anaemia and abnormal red-cell properties. The A858D mutation showed dominant inheritance and therecessive DeltaV850 and G701D mutations showed a pseudo-dominantphenotype when the transport-inactive SAO allele was also present. Red-cell and Xenopus oocyte expression studies showed that theDeltaV850 and A858D mutant proteins have greatly decreased aniontransport when present as compound heterozygotes (DeltaV850/A858D,DeltaV850/SAO or A858D/SAO). Red cells with A858D/SAO had only 3% ofthe SO(4)(2-) efflux of normal cells, thelowest anion transport activity so far reported for human red cells. The results suggest dRTA might arise by a different mechanism for eachmutation. We confirm that the G701D mutant protein has an absoluterequirement for glycophorin A for movement to the cell surface. Wesuggest that the dominant A858D mutant protein is possibly mis-targetedto an inappropriate plasma membrane domain in the renal tubular cell,and that the recessive DeltaV850 mutation might give dRTA because ofits decreased anion transport activity.

Red-cell glycophorin A-band 3 interactions associated with the movement of band 3 to the cell surface.

We have examined the mechanism by which glycophorin A (GPA) facilitates the movement of the human red-cell anion exchanger (band 3, AE1) to the cell surface. GPA itself forms stable dimers in membranes and detergent solution. Four mutants of human GPA with impaired dimerization were prepared (L75I, I76A, G79L and G83L). All four GPA mutants enhanced band 3 translocation to the Xenopus oocyte plasma membrane in the same way as wild-type GPA, showing that the GPA monomer is sufficient to mediate this process. Cell-surface expression of the natural band 3 mutant G701D has an absolute requirement for GPA. GPA monomers also rescued the cell-surface expression of this mutant band 3. Taking into account other evidence, we infer that the site of GPA interaction with band 3 is located outside the GPA dimerization interface but within the GPA transmembrane span. The results of examination of the cell-surface expression of GPA and band 3 in different K562 erythroleukaemia cell clones stably transfected with band 3 are consistent with the movement of GPA and band 3 to the cell surface together. We discuss the pathways by which band 3 moves to the cell surface in the presence and the absence of GPA, concluding that GPA has a role in enhancing the folding and maturation of band 3. We propose that GPA functions in erythroid cells to assist with the incorporation of large amounts of properly folded band 3 into the membrane within a limited time span during erythroid maturation.

The low-frequency MNS blood group antigens Ny(a) (MNS18) and Os(a) (MNS38) are associated with GPA amino acid substitutions.

BACKGROUND: Antigens of the MNS blood group system are located on two sialoglycoproteins, GPA and GPB, encoded by GYPA and GYPB. The molecular backgrounds of the low-frequency antigens Ny(a) and Os(a) are not known. STUDY DESIGN AND METHODS: Immunoblotting and a monoclonal antibody-specific immobilization of erythrocyte antigens (MAIEA) assay were used to analyze Os(a). PCR-amplified products of the coding exons of GYPA were studied by single-strand conformation polymorphism analysis, and exon 3 was sequenced. Synthetic peptides were used in hemagglutination-inhibition tests. RESULTS: Sequencing of GYPA exon 3 of two unrelated Ny(a+) persons revealed heterozygosity for a T194A base change encoding an Asp27Glu substitution. Immunoblotting with anti-Os(a) and an MAIEA assay with MoAbs to GPA showed that Os(a) is on GPA. Sequencing exon 3 of an Os(a+) person from the only family with Os(a) revealed heterozygosity for a C273T base change encoding a Pro54Ser substitution. A synthetic peptide representing part of GPA with the Os(a) mutation (VRTVYPSEEETGE) completely inhibited anti-Os(a), whereas the control peptide (VRTVYPPEEETGE) did not inhibit anti-Os(a). CONCLUSION: Ny(a) and Os(a) are low-frequency antigens of the MNS blood group system that represent Asp27Glu and Pro54Ser substitutions in GPA, respectively.

Topology studies with biosynthetic fragments identify interacting transmembrane regions of the human red-cell anion exchanger (band 3; AE1).

The red-cell anion exchanger (band 3; AE1) is a multispanning membrane protein that traverses the bilayer up to 14 times and is N-glycosylated at Asn-642. We have shown that the integrity of six different loops are not essential for stilbene disulphonate-sensitive chloride uptake in Xenopus oocytes. We used an N-glycosylation mutagenesis approach to examine the orientation of the N-terminus and the endogenous glycosylation site of each C-terminal fragment by cell-free translation. The fragments initiating in the loops preceding spans 2, 9 and 11 did not insert into the membrane with the expected orientation. Furthermore, N-glycosylation of Asn-642 might facilitate the membrane integration of span 7. The correct integration of spans 2-3 required the presence of the region containing span 4 and that the luminal exposure of the C-terminus of span 7 is increased in the presence of the region including span 6 or span 8. The results suggest the span 8 region is required for the correct folding of spans 9-10, at least in the presence of the span 11-12 region. Our results suggest that there are intramolecular interactions between the regions of transmembrane spans 1 and 2, 2 and 4, 4 and 5, 7 and 8, 8 and 9-10, and 9-10 and 11-12. Spans 1, 4, 5, 6 and 8 might act as a scaffold for the assembly of spans 2-3, 7 and 9-10. This approach might provide a general method for dissecting the interactions between membrane-spanning regions of polytopic membrane proteins.

Structural model for the organization of the transmembrane spans of the human red-cell anion exchanger (band 3; AE1).

We have examined the functional co-assembly of non-complementary pairs of N- and C-terminal polypeptide fragments of the anion transport domain (b3mem) of human red-cell band 3. cDNA clones encoding non-contiguous pairs of fragments with one transmembrane (TM) region omitted, or overlapping pairs of fragments with between one and ten TM regions duplicated, were co-expressed in Xenopus oocytes and a cell-free translation system. Stilbene disulphonate-sensitive chloride uptake assays in oocytes revealed that the omission of any single TM region of b3mem except spans 6 and 7 caused a complete loss of functional expression. In contrast, co-expressed pairs of fragments overlapping a single TM region 5, 6, 7, 8, 9-10 or 11-12 retained a high level of functionality, whereas fragments overlapping the clusters of TM regions 2-5, 4-5, 5-8 and 8-10 also mediated some stilbene disulphonate-sensitive uptake. The co-assembly of N- or C-terminal fragments with intact band 3, b3mem or other fragments was examined by co-immunoprecipitation in non-denaturing detergent solutions by using monoclonal antibodies against the termini of b3mem. All the fragments, except for TM spans 13-14, co-immunoprecipitated with b3mem. The medium-sized N-terminal fragments comprising spans 1-6, 1-7 or 1-8 co-immunoprecipitated particularly strongly with the C-terminal fragments containing spans 8-14 or 9-14. The fragments comprising spans 1-4 or 1-12 co-immunoprecipitated less extensively than the other N-terminal fragments with either b3mem or C-terminal fragments. There is sufficient flexibility in the structure of b3mem to allow the inclusion of at least one duplicated TM span without a loss of function. We propose a working model for the organization of TM spans of dimeric band 3 based on current evidence.

The expression of human blood group antigens during erythropoiesis in a cell culture system.

Phenotypic analysis of hematopoietic stem and progenitor cells has been an invaluable tool in defining the biology of stem cell populations. We use here flow cytometry to examine the expression of human erythroid-specific surface markers during the maturation of early committed erythroid cells derived from cord blood in vitro. The temporal order of the expression of erythroid specific markers was as follows: Kell glycoprotein (gp), Rh gp, Landsteiner Wiener (LW) gp, glycophorin A (GPA), Band 3, Lutheran (Lu) gp, and Duffy (Fy) gp. The time at which some of these markers appeared suggests possible roles for some of these erythroid-specific polypeptides during the differentiation of these committed progenitors. The early appearance of Kell gp raises the possibility that it may have an important role in the early stages of hematopoiesis or cell lineage determination. Kell gp may also be a useful marker for the diagnosis of erythroleukemia. The late expression of Lu gp suggests it may be involved in the migration of erythroid precursors from the marrow. Fy gp is also expressed late consistent with a role as a scavenger receptor for cytokines in the bone marrow and circulation. Rh c antigen appeared before Rh D antigen, and it is suggested that this may reflect a reorganization of the developing erythroid cell membrane involving the Rh polypeptides and other components, including GPA and Band 3.

Glycophorin A mutation Ala65 --> Pro gives rise to a novel pair of MNS alleles ENEP (MNS39) and HAG (MNS41) and altered Wrb expression: direct evidence for GPA/band 3 interaction necessary for normal Wrb expression.

We report here a novel Glycophorin A (GPA) mutation Ala65 --> Pro which gives rise to a low-incidence antigen HAG, lack of a high-incidence antigen ENEP and aberrant expression of the high-incidence Wrb antigen. Anti-ENEP was identified in the serum of a transfused male patient (E.H.) who was homozygous for a GPA Ala65 --> Pro mutation and possessed a novel low-incidence antigen which we have called HAG. An unrelated HAG-positive individual, heterozygous for the Ala65 --> Pro mutation, has also been identified. Anti-HAG was present in several multispecific antisera to low-incidence antigens and in one monospecific serum. Normal expression of the Wrb antigen depends on the presence of amino acid Glu658 of band 3 and on the presence of GPA. However, a specific epitope on GPA has not previously been implicated. DNA sequence analysis of band 3 from patient E.H. was normal in the region of Wra/Wrb polymorphism with homozygous presence of Glu658 and therefore the abnormal Wrb expression results from the Ala65 --> Pro mutation in GPA. The ENEP and HAG antigens have been assigned the MNS blood group system numbers 002.039 and 002.041, respectively, by the ISBT Working Party on Terminology for Red Cell Surface Antigens.

South-east asian ovalocytic (SAO) erythrocytes have a cold sensitive cation leak: implications for in vitro studies on stored SAO red cells.

South-east Asian ovalocytosis (SAO) results from the heterozygous presence of an abnormal band 3, which causes several alterations in the properties of the erythrocytes. Although earlier studies suggested that SAO erythrocytes are refractory to invasion in vitro by the malarial parasite Plasmodium falciparum, a more recent study showed that fresh SAO cells were invaded by the parasites, but became resistant to invasion on storage because intracellular ATP was depleted more rapidly than normal. Here we show that SAO red cells are much more leaky to sodium and potassium than normal red cells when stored in the cold. This leak was much less marked when the cells were stored at 25 or 37 degreesC. Incubation for 3.5 h at 37 degreesC of cold-stored SAO red cells did not restore sodium and potassium to normal levels, probably because the depleted ATP level in cold-stored SAO red cells is further reduced with incubation at 37 degreesC. The increased leakiness of SAO red cells is non-specific and extends to calcium ions, taurine, mannitol and sucrose. These results suggest that SAO red cells undergo a structural change on cooling. Since many of the reports describing altered properties of SAO red cells have used cells which have been stored in the cold, these results need re-evaluation using never-chilled SAO red cells to assess whether the cells have the same abnormal properties under in vivo conditions.

Structural studies on the effects of the deletion in the red cell anion exchanger (band 3, AE1) associated with South East Asian ovalocytosis.

We have carried out a solution-state NMR study of synthetic peptides patterned on the first membrane span of normal human band 3, and the same region of the mutant band 3 present in Southeast Asian ovalocytosis (SAO) which has a nine amino acid deletion. In 1:1 (v/v) chloroform/methanol, the 42 residue normal peptide (R389-K430) consisted of three helical regions. The slow solvent exchange of backbone amide protons revealed the helix from P403 to A416 was more stable than the "cytoplasmic" N-terminal helix from P391 to A400. These helices were separated by a sharp bend at P403, which is probably located at the boundary between the cytoplasmic domain and the first transmembrane span. The SAO deletion (A400-A408) removed the bend at P403, to leave a stable helix from P391 to A416 containing the residuum of the normal first transmembrane helix and with a hydrophobic turn replaced by a polar turn in the SAO peptide. Insertion of fragments of normal band 3 and band 3 SAO into microsomal membranes was investigated using a cell free translation system. A fragment composed of the cytoplasmic domain and the putative first membrane domain of normal band 3 (B3(1)) inserted stably into the membrane. However, the corresponding fragment of band 3 SAO [SAO(1)] did not integrate stably into membranes. Our results suggest that in SAO band 3, the region of the first membrane span of normal band 3 does not integrate properly into the membrane because it lacks a sufficiently long hydrophobic segment, and the deletion also disrupts a conserved structural subdomain at the membrane surface.

Erythroid band 3 variants and disease.

This review describes some of the naturally occurring band 3 (AEI) 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 AEI is expressed in kidney cells and certain AEI 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.

Functional cell surface expression of band 3, the human red blood cell anion exchange protein (AE1), in K562 erythroleukemia cells: band 3 enhances the cell surface reactivity of Rh antigens.

Human K562 erythroleukemia cells were transfected with human band 3 (anion exchanger 1 [AE1]) cDNA, using the pBabe retroviral vector. Stable K562 clones expressing band 3 were isolated by flow cytometry, and surface expression was quantified by immunoblotting. The function of band 3 expressed at the cell surface was demonstrated in chloride transport assays. K562 cells expressing band 3 also displayed high levels of the Wrb blood group antigen, confirming the role of band 3 in Wrb expression, and an increase in the low levels of endogenous Rh antigen activity. We also performed coexpression experiments with K562 clones that had previously been transduced with cDNAs encoding RhD or RhcE polypeptides. The transfection and expression of band 3 in these clones substantially increased the levels of RhD and cE antigen activity expressed on the cells and also increased the reactivity of the cells with antibody to the endogenous Rh glycoprotein (RhGP, Rh50). The increased reactivity of Rh antigens may result from cell surface or intracellular interactions of band 3 with the protein complex which contains the Rh polypeptides and RhGP, or from indirect effects of band 3 on the membrane environment. This work establishes a system for cell surface expression of band 3 in a mammalian cell line, which will enable further studies of the protein and its interactions with other membrane components.

Functional reassembly of the anion transport domain of human red cell band 3 (AE1) from multiple and non-complementary fragments.

We constructed cDNA clones encoding N-terminal, C-terminal and internal polypeptide fragments of the human red cell anion exchanger (band 3; AE1). The internal fragments comprised between one and seven putative transmembrane spans with two or more spans deleted from both termini of the membrane domain of band 3. Sets of three, four or five complementary fragments, which together represented the complete amino acid sequence of the membrane domain, were co-expressed in Xenopus oocytes. Stilbene disulphonate-sensitive chloride uptake assays revealed that all six of the three-fragment combinations and two of the four-fragment combinations reassembled functionally in vivo. Unexpectedly, co-expression of a non-complementary pair of fragments comprising the first five and last seven putative transmembrane spans (i.e. entirely lacking spans six and seven) was also found to be sufficient to generate stilbene disulphonate-sensitive chloride uptake.

NMR solution structure of a cytoplasmic surface loop of the human red cell anion transporter, band 3.

The membrane domain of the human red cell anion transport protein, band 3, is too large to be studied by solution nuclear magnetic resonance spectroscopy (NMR), and its amphiphilic nature requires the use of detergents for solubilization. An alternative approach is to divide the protein into smaller (trans-membrane or surface loop) domains for NMR study. We report the structure of a 46-residue synthetic peptide that corresponds to the cytoplasmic surface loop connecting the putative 12th and 13th trans-membrane spans (residues 796-841) in the 14 span model of band 3. This peptide was shown by circular dichroism (CD) to be 38% helical in 30% trifluoroacetic acid. Two regions of helix (one close to the N-terminus of the peptide and one close to the C-terminus of the peptide) were identified by NMR. Long-range nuclear Overhauser effect (NOE) cross-peaks showed the two helices to be in near proximity. The helices were separated by a proline-rich loop that exhibited local order but was mobile with respect to the rest of the peptide. We discuss how the NMR structure of this loop fits the current models of band 3 structure and topology and the results of recent mutagenesis experiments. A cyclic version of this peptide was synthesized and studied by CD, but NMR studies were not possible due to the low solubility of this peptide.

Mutations in the chloride-bicarbonate exchanger gene AE1 cause autosomal dominant but not autosomal recessive distal renal tubular acidosis.

Primary distal renal tubular acidosis (dRTA) is characterized by reduced ability to acidify urine, variable hyperchloremic hypokalemic metabolic acidosis, nephrocalcinosis, and nephrolithiasis. Kindreds showing either autosomal dominant or recessive transmission are described. Mutations in the chloride-bicarbonate exchanger AE1 have recently been reported in four autosomal dominant dRTA kindreds, three of these altering codon Arg589. We have screened 26 kindreds with primary dRTA for mutations in AE1. Inheritance was autosomal recessive in seventeen kindreds, autosomal dominant in one, and uncertain due to unknown parental phenotype or sporadic disease in eight kindreds. No mutations in AE1 were detected in any of the autosomal recessive kindreds, and analysis of linkage showed no evidence of linkage of recessive dRTA to AE1. In contrast, heterozygous mutations in AE1 were identified in the one known dominant dRTA kindred, in one sporadic case, and one kindred with two affected brothers. In the dominant kindred, the mutation Arg-589/Ser cosegregated with dRTA in the extended pedigree. An Arg-589/His mutation in the sporadic case proved to be a de novo mutation. In the third kindred, affected brothers both have an intragenic 13-bp duplication resulting in deletion of the last 11 amino acids of AE1. These mutations were not detected in 80 alleles from unrelated normal individuals. These findings underscore the key role of Arg-589 and the C terminus in normal AE1 function, and indicate that while mutations in AE1 cause autosomal dominant dRTA, defects in this gene are not responsible for recessive disease.