Identification of a novel DI*02(2558T) allele associated with weakened expression of DI2 antigen.

BACKGROUND: The distribution of DI1/DI2 antigens of the Diego blood group system is polymorphic in Mongoloid populations and the corresponding alloantibodies are clinically significant. Here a novel DI variant was found by donor screening, and the effect of the novel and previously reported mutations on expression of DI1/DI2 antigens and Band 3 protein was explored. STUDY DESIGN AND METHODS: DNA samples of 1150 Chinese donors were collected. DI*01/DI*02 genotyping was determined by Sanger sequencing. For the carrier of novel allele, the expression of Band 3 and DI1/DI2 antigens on red blood cells (RBCs) was detected by Western blot and flow cytometry, respectively. in vitro expression studies were conducted by transfecting the mutant (including the novel and three reported DI*02(2534T), DI*02(2358_2359insCAC), and DI*02(2572T) alleles) or wild-type DI*02 constructs into HEK 293T cells, the expression of Band 3 and DI1/DI2 antigens was analyzed. RESULTS: A novel heterozygous mutation (c.2558C>T, p.Thr853Met), which is located near the DI1/DI2 polymorphism site (c.2561T>C), was identified in a donor with DI:-1,2 phenotype. Reduced expression of DI2 antigen was observed on the RBCs, while weakened expression of Band 3 and absence of DI2 antigen were detected in cells transfected with the mutant DI*02(2558T) construct. In addition, absent or decreased expression of Band 3 and DI2 antigen was also detected in cells transfected with three reported mutant constructs. CONCLUSION: The novel DI*02(2558T) allele and three previously described DI mutations can affect the expression of Band 3 protein and/or DI2 antigen and/or interfere with DI*01/DI*02 genotyping result.

Expression and role of anion exchanger 1 in esophageal squamous cell carcinoma.

Recent studies have described important roles for the anion exchanger (AE) in epithelial carcinogenesis and tumor behavior. The objectives of the present study were to investigate the role of AE1 in the regulation of genes involved in tumor progression and the clinicopathological significance of its expression in esophageal squamous cell carcinoma (ESCC). An immunohistochemical analysis was performed on 61 primary tumor samples obtained from ESCC patients who underwent esophagectomy. AE1 was primarily located in the cell membranes or cytoplasm of carcinoma cells, and its distribution pattern was related to the histological degree of the differentiation of SCC or the pT category. Among patients with pT2-3 ESCC, the 5-year survival rate of patients with diffuse AE1 expression (40.2%) was significantly lower than that of patients with focal expression (74.0%). AE1 was strongly expressed in KYSE150 and TE8 human ESCC cells. The depletion of AE1 using siRNA inhibited cell proliferation, migration, and invasion and induced apoptosis. The results of the microarray analysis revealed that MAPK and Hedgehog signaling pathway-related genes, such as DHH, and GLI1, were down-regulated in AE1-depleted KYSE150 cells. In conclusions, the results of the present study suggest that the diffuse expression of AE1 is related to a worse prognosis in patients with advanced ESCC, and that it regulates tumor progression by affecting MAPK and Hedgehog signaling pathways. These results provide an insight into the role of AE1 as a mediator of and/or a biomarker for ESCC.

El antígeno Diego alcanza los 60 años de edad: su descubrimiento y desarrollo / Diego antigen reach 60 years old: discovery and development

El antigeno Diego fue descubierto en junio de 1953 por el hematólogo estadounidense Philip Levine (1900-1987) en una muestra de sangre enviada desde Venezuela por el pediatra Miguel Raga Mendoza (1917-1986). El propósitus, de nombre Diego, había fallecido a los 3 días de edad por causa de una enfermedad hemolítica del recién nacido. Levine bautizó al nuevo antigeno con el nombre de diego y lo clasificó como un factor privado o familiar de baja prevalencia. En 1955, los hematólogos Miguel Layrisse (1919-2002) y Tulio Arents (1918-1990), y el obstetra Rafael Dominguez Sisco (1908-1980) llegaron a la conclusión de que el antígeno Diego tenía una mayor frecuencia que la reportada por Levine y que por tanto constituía un grupo sanguíneo de alta prevalencia en poblaciones indigenas venezolanas. Estos resultados fueron extendidos a otras poblaciones indígenas de América, demostrandose también su existencia en personas de origen asiático (mongoloides) y su ausencia en las razas caucasoide y negroide. El antígeno Diego se transformó así en el primer marcador mongoloide de gran valor antropológico, genético y clínico. En la década de 1990 se demostró que el antígeno Diego estaba asociado con la proteína eritrocitaria denominada banda 3; esta funciona como un intercambiador de aniones (AE-1) que se expresa también en células del túbulo renal. Actualmente, el grupo snguíneo Diego está formado por 22 antígenos o alelos.

On June 1953, the American hematologist Philip Levine (1900-1987) discovered a new erythrocyte antigen in the blood of a sick child collected in Venezuela by the pediatrician Miguel Raga Mendoza (1917-1986). The propositus, named Diego, was affected by a hemolytic disease of the newborn and died 3 days after delivery. Levine named the antigen Diego (Diª) and classified it as a private or familial factor of low prevalence. In 1955, the hematologists Miguel Layrisse (1919-2002) and Tulio Arends (1918-1990), and the obstetrician Rafael Dominguez Sisco (1908-1980) concluded that the Diego antigen had a greater frecuency than that reported by Levine, constituting a blood group of high prevalence in Venezuelan aboriginal populations. Similar results were obtained in other aboriginal populations of the American continent. The Diego antigen was also present in high frequency in people of asiatic origin (mongoloids), and absent in caucasoid and negroid people. Thus, the Diego antigen became the first mongoloid marker of great anthropological, genetic and clinical importance. In 1992, the Diego antigen was found associated with the erythrocyte protein named band 3, later known to function as an anion exchanger (AE-1). Band 3 is also expressed on cells of the renal tubules. Presenthy, the Diego blood group is formed by 22 antigens or allelles.

Functional rescue of a kidney anion exchanger 1 trafficking mutant in renal epithelial cells.

Mutations in the SLC4A1 gene encoding the anion exchanger 1 (AE1) can cause distal renal tubular acidosis (dRTA), a disease often due to mis-trafficking of the mutant protein. In this study, we investigated whether trafficking of a Golgi-retained dRTA mutant, G701D kAE1, or two dRTA mutants retained in the endoplasmic reticulum, C479W and R589H kAE1, could be functionally rescued to the plasma membrane of Madin-Darby Canine Kidney (MDCK) cells. Treatments with DMSO, glycerol, the corrector VX-809, or low temperature incubations restored the basolateral trafficking of G701D kAE1 mutant. These treatments had no significant rescuing effect on trafficking of the mis-folded C479W or R589H kAE1 mutants. DMSO was the only treatment that partially restored G701D kAE1 function in the plasma membrane of MDCK cells. Our experiments show that trafficking of intracellularly retained dRTA kAE1 mutants can be partially restored, and that one chemical treatment rescued both trafficking and function of a dRTA mutant. These studies provide an opportunity to develop alternative therapeutic solutions for dRTA patients.

Ductal plate malformation-like arrays in early explants after a Kasai procedure are independent of splenic malformation complex (heterotaxy).

Biliary atresia has at least 2 proposed forms, the common perinatal and the less common embryonic subtype with earlier onset and/or extrahepatic developmental anomalies. Histologic evidence of ductal plate malformation (DPM)-like change in liver has been proposed both as a marker for the embryonic type and as a predictor of poor outcome after Kasai portoenterostomy. We investigated the prevalence of DPM-like change in liver explants in usual biliary atresia (BA) and in BA with splenic malformation syndrome (BASM). Liver sections from 8 patients with BA and 6 with BASM, all of whom had a Kasai procedure followed by explant before age 2 years, were analyzed using hematoxylin and eosin, trichrome, CK7, and AE1/AE3 stains. Each block was scored for inflammation and fibrosis. We estimated the number of portal areas per block and counted the number of definite and possible examples of DPM-like change, defined as a circumferential duct complex arranged around a fibrovascular core. We assessed whether the frequency per portal area was related to low and high scores for either inflammation or fibrosis. Definite and possible examples of DPM-like arrays were present in about 10% of portal areas in both patient groups, but these were unevenly distributed. There was no statistical difference between BA and BASM in terms of the number of examples per portal area. No correlation existed between degree of fibrosis and the intensity of portal inflammation and the number of DPM-like arrays. Ductal plate malformation-like arrays do not distinguish perinatal BA from BA associated with heterotaxy in liver explants after a failed Kasai procedure.

Expression of anion exchanger 1 sequestrates p16 in the cytoplasm in gastric and colonic adenocarcinoma.

p16(INK4A) (p16) binds to cyclin-dependent kinase 4/6 and negatively regulates cell growth. Recent studies have led to an understanding of additional biologic functions for p16; however, the detailed mechanisms involved are still elusive. In this article, we show an unexpected expression of anion exchanger 1 (AE1) in the cytoplasm in poorly and moderately differentiated gastric and colonic adenocarcinoma cells and in its interaction with p16, thereby sequestrating the protein in the cytoplasm. Genetic alterations of p16 and AE1 were not detectable. Forced expression of AE1 in these cells sequestrated more p16 in the cytoplasm, whereas small interfering RNA-mediated silencing of AE1 in the cells induced the release of p16 from the cytoplasm to the nucleus, leading to cell death and growth inhibition of tumor cells. By analyzing tissue samples obtained from patients with gastric and colonic cancers, we found that 83.33% of gastric cancers and 56.52% of colonic cancers coexpressed AE1 and p16 in the cytoplasm. We conclude that AE1 plays a crucial role in the pathogenesis of gastric and colonic adenocarcinoma and that p16 dysfunction is a novel pathway of carcinogenesis.

[Expression of band 3 protein on erythrocytes of malignant tumor patients and its impact on proliferation of K562 cells].

BACKGROUND & OBJECTIVE: Membrane domain of band 3 protein (mdb3) mediates transmembrane exchange of chloride and bicarbonate, and regulates intracellular pH. It has been found recently that abnormality of CI(-)/HCO3(-) exchange, which mainly leads to change of intracellular pH, may be involved in cell proliferation and apoptosis. This study was to explore expression of band 3 protein on erythrocytes, and its impact on proliferation of K562 cells. METHODS: Anion transport activity of band 3 protein on erythrocytes of 8 malignant tumor patients was measured using SPQ fluorescent probe. Expression of band 3 protein was detected by Western blot. Plasmid pYD1-mdb3 was constructed, and transfected into K562 cells. Cl- transport activity and proliferation of K562 cells were detected after transfection. RESULTS: Of the 8 patients, 7 showed increase of anion transport activity on erythrocytes, 5 showed increase of band 3 protein expression. To some extent, expression of mdb3 enhanced proliferation of K562 cells. CONCLUSION: Expression of band 3 protein is enhanced on erythrocytes of some malignant tumors, and might be a candidate marker of malignant tumors.

Defects in processing and trafficking of the AE1 Cl-/HCO3- exchanger associated with inherited distal renal tubular acidosis.

Distal renal tubular acidosis (dRTA) results from impaired urinary acidification by the renal collecting duct. Acquired dRTA can be secondary to diverse pathological processes, including diabetic, ischemic, fibrosing, or immunological processes; less frequently it presents as a familial disorder with either an autosomal recessive or dominant pattern of transmission. Mutations in the SLC4A1/AE1/band 3 Cl(-)/HCO(3)(-) exchanger gene have been identified as causes for both dominant and recessive forms of dRTA. These mutations comprise a group almost entirely distinct from the SLC4A1 mutations that underlie the familial hemolytic anemia of hereditary spherocytosis. Why does one group of mutations express almost exclusively an isolated erythroid phenotype, whereas the second group of mutations expresses almost exclusively a phenotype explicable entirely by defective function of renal collecting duct type A intercalated cells? This review summarizes current research addressing this central question in the pathobiology of inherited dRTA associated with mutations in the SLC4A1 gene. Studying dRTA-associated mutant AE1 polypeptides can provide novel insights into the biology of the intercalated cell and the collecting duct as well as more generally into mechanisms by which epithelial cells generate and maintain functional polarity.

Band 3 as a marker of erythrocyte changes in pregnancy.

Modifications in the erythrocyte membrane protein band 3 seem to mark the cell for death. A decrease in band 3 high molecular weight aggregates (HMWAg) and a rise in its proteolytic fragments (Pfrag) were described for younger erythrocytes. The aim of this work was to study the band 3 profile as a marker of erythrocyte changes in pregnancy and postpartum. We performed a cross-sectional study in non-pregnant controls (n = 24), in women in the first (n = 64), second (n = 48) and third (n = 67) trimesters of gestation, and also in the puerperium (24-48 h after delivery; n = 32); we also carried out a longitudinal study (n = 23) during the three trimesters of normal pregnancy. We evaluated the band 3 profile (% of band 3 monomer, HMWAg, and Pfrag) and the membrane-bound haemoglobin. Total serum bilirrubin, glutathione peroxidase activity, red blood cell (RBC) count, haematocrit (Ht), haemoglobin (Hb) concentration, the haematimetric indices, and red cell distribution width were also evaluated. Similar results were found in pregnancy in both the cross-sectional and longitudinal studies. We found that the RBC count, Hb, and Ht decreased significantly in pregnancy and in puerperium. Band 3 profile in the first trimester of pregnancy, when compared with controls, presented significantly reduced HMWAg and increased Pfrag. Comparing the first with the third trimester, we found a significant reduction in band 3 and a significant rise in Pfrag. However, between these same periods, HMWAg did not decrease. Our data suggest band 3 profile as a marker of erythrocyte changes in pregnancy, which are independent of the 'physiological anaemia' of pregnancy. These changes suggest an increase in damaged RBCs, but also an increase in younger RBCs in the maternal circulation.

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.

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.

Characterization of seven low incidence blood group antigens carried by erythrocyte band 3 protein.

Recent studies have demonstrated that band 3 carries antigens of the Diego blood group system and have elucidated the molecular basis of several previously unassigned low incidence and high incidence antigens. Because the available serological data suggested that band 3 may carry additional low incidence blood group antigens, we screened band 3 genomic DNA encoding the membrane domain of band 3 for single-strand conformational polymorphisms. We found that the putative first ectoplasmic loop of band 3 carries blood group antigen ELO, 432 Arg-->Trp; the third putative loop harbors antigens Vga (Van Vugt), 555 Tyr-->His, BOW 561 Pro-->Ser, Wu (Wulfsberg), 565 Gly-->Ala, and Bpa (Bishop), 569 Asn-->Lys; and the putative fourth ectoplasmic loop carries antigens Hga (Hughes), 656 Arg-->Cys, and Moa (Moen), 656 Arg-->His. We studied erythrocytes from carriers of five of these blood group antigens. We found similar levels of reticulocyte mRNA corresponding to the two band 3 gene alleles, normal content and glycosylation of band 3 in the red blood cell membrane, and normal band 3-mediated sulfate influx into red blood cells, suggesting that the mutations do not have major effect on band 3 structure and function. In addition to elucidating the molecular basis of seven low incidence blood group antigens, these results help to create a more accurate structural model of band 3.

Assessment of topogenic functions of anticipated transmembrane segments of human band 3.

Band 3 protein is a typical multispanning membrane protein whose membrane topology has been extensively studied from various protein chemical approaches. To clarify the membrane topogenesis of this multispanning protein on the endoplasmic reticulum, the topogenic functions of the anticipated transmembrane segments were individually assessed in an in vitro system using two series of model proteins in which each segment was placed in either a "stop-transfer" context or a "translocation initiation" context. They were expressed in a cell-free system containing rough microsomal membranes, and their topologies were evaluated by taking advantage of either sensitivity to protease or accessibility to N-glycosylation. We found that some segments seem to possess insufficient topogenic functions for membrane integration: the second transmembrane segment (TM2) is insufficient for the stop-transfer sequence, and TM3, TM5, and TM7 are not sufficient for the translocation initiation. In contrast to these phenomena, we herein demonstrate that TM2 shows an efficient stop-transfer function when it is near the preceding TM1 and suggest that TM3, TM5, and TM7 are followed by TM segments with a strong topogenic function to form Nexo/Ccyt topology, via which the preceding segments are integrated into the membrane. From these results, we propose that the interactions between the TMs should be operative during membrane integration, and that the segments with a weak topogenic function are given a transmembrane orientation by their following TMs.

Temporal synthesis of band 3 oligomers during terminal maturation of mouse erythroblasts. Dimers and tetramers exist in the membrane as preformed stable species.

Band 3, the anion transport protein of the erythrocyte membrane, exists in the membrane as a mixture of dimers (B3D) and tetramers (B3T). The dimers are not linked to the skeleton and constitute the free mobile band 3 fraction. The tetramers are linked to the skeleton by their interaction with ankyrin. In this report we have examined the temporal synthesis and assembly of band 3 oligomers into the plasma membrane during red cell maturation. The oligomeric state of newly synthesized band 3 in early and late erythroblasts was analyzed by size-exclusion high-pressure liquid chromatography of band 3 extracts derived by mild extraction of plasma membranes with the nonionic detergent C12E8 (octaethylene glycol n-dodecyl monoether). This analysis revealed that at the early erythroblast stage, the newly synthesized band 3 is present predominantly as tetramers, whereas at the late stages of erythroid maturation, it is present exclusively as dimers. To examine whether the dimers and tetramers exist in the membrane as preformed stable species or whether they are interconvertible, the fate of band 3 species synthesized during erythroblast maturation was examined by pulse-chase analysis. We showed that the newly synthesized band 3 dimers and tetramers are stable and that there is no interconversion between these species in erythroblast membranes. Pulse-chase analysis followed by cellular fractionation showed that, in early erythroblasts, the newly synthesized band 3 tetramers are initially present in the microsomal fraction and later incorporated stably into the plasma membrane fraction. In contrast, in late erythroblasts the newly synthesized band 3 dimers move rapidly to the plasma membrane fraction but then recycle between the plasma membrane and microsomal fractions. Fluorescence photobleaching recovery studies showed that significant fractions of B3T and B3D are laterally mobile in early and late erythroblast plasma membranes, respectively, suggesting that many B3T-ankyrin complexes are unattached to the membrane skeleton in early erythroblasts and that the membrane skeleton has yet to become tightly organized in late erythroblasts. We postulate that in early erythroblasts, band 3 tetramers are transported through microsomes and stably incorporated into the plasma membrane. However, when ankyrin synthesis is downregulated in late erythroblasts, it appears that B3D are rapidly transported to the plasma membrane but then recycled between the plasma membrane and microsomal compartments. These observations may suggest novel roles for membrane skeletal proteins in stabilizing integral membrane protein oligomers at the plasma membrane and in regulating the endocytosis of such proteins.

Effect of site-directed mutagenesis of the arginine residues 509 and 748 on mouse band 3 protein-mediated anion transport.

Using site-directed mutagenesis, the arginine residues 509 and 748 in mouse band 3 protein were substituted by Lys, Thr, and Cys, or by Lys and Gln, respectively. After expression in Xenopus oocytes of the cRNAs encoding wild type band 3 or any one of the band 3 mutants, chloride equilibrium exchange was measured. When the flux measurements were performed two to three days after microinjection of the cRNAs, in contrast to the wild type, neither one of the mutants was able to accomplish transport, with the possible exception of the mutants R509K and R748K both of which showed some transport activity of doubtful significance. Immunoprecipitates revealed that the Arg 748 mutants were expressed similar to the wild type band 3 while no expression of the Arg 509 mutants could be detected. When the flux measurements were performed only 3 h after microinjection of the cRNAs, transport activity was observed in the oocytes that had received cRNAs encoding wild type band 3. In some oocytes of a population, a very slight transport activity was brought about by cRNA encoding Arg 509 mutants. No transport activity could be detected after injection of the Arg 748 mutant. Immunoprecipitation demonstrated the successful biosynthesis of wild type band 3 and of both the Arg 509 and the Arg 748 mutants. The experiments suggest that mutation of Arg 748 leads to biosynthesis of an inactive form of the band 3 protein, while that of Arg 509 results in expression of an abnormally folded, possibly functionally more or less intact form, which is proteolytically degraded within less than one day.

Mapping the ends of transmembrane segments in a polytopic membrane protein. Scanning N-glycosylation mutagenesis of extracytosolic loops in the anion exchanger, band 3.

Band 3, the anion exchanger of human erythrocytes, contains up to 14 transmembrane (TM) segments and has a single endogenous site of N-glycosylation at Asn642 in extracellular (EC) loop 4. The requirements for N-glycosylation of EC loops and the topology of this polytopic membrane protein were determined by scanning N-glycosylation mutagenesis and cell-free translation in a reticulocyte lysate supplemented with microsomal membranes. The endogenous and novel acceptor sites located near the middle of the 35 residue EC loop 4 were efficiently N-glycosylated; however, no N-glycosylation occurred at sites located within sharply defined regions close to the adjacent TM segments. Acceptor sites located in the center of EC loop 3, which contains 25 residues, were poorly N-glycosylated. Expansion of this loop with a 4-residue insert containing an acceptor site increased N-glycosylation. Acceptor sites located in short (<10 residues) loops (putative EC loops 1, 2, 6, and 7) were not N-glycosylated; however, insertion of EC loop 4 into EC loops 1, 2, or 7, but not 6, resulted in efficient N-glycosylation. Acceptor sites in putative intracellular (IC) loop 5 exhibited a similar pattern of N-glycosylation as EC loop 4, indicating a lumenal disposition during biosynthesis. To be efficiently N-glycosylated, EC loops in polytopic membrane proteins must be larger than 25 residues in size, with acceptor sites located greater than 12 residues away from the preceding TM segment and greater than 14 residues away from the following TM segment. Application of this requirement allowed a significant refinement of the topology of Band 3 including a more accurate mapping of the ends of TM segments. The strict distance dependence for N-glycosylation of loops suggests that TM segments in polytopic membrane proteins are held quite precisely within the translocation machinery during the N-glycosylation process.

Complementation studies with Co-expressed fragments of the human red cell anion transporter (Band 3; AE1). The role of some exofacial loops in anion transport.

We constructed cDNA clones encoding fragments of band 3 in which the membrane domain was truncated from either the N or the C terminus within each of the first four exofacial loops. The truncations containing the C terminus of the protein were fused with the cleavable N-terminal signal sequence of glycophorin A to facilitate the correct orientation of the most N-terminal band 3 membrane span. Cleavage of the glycophorin A signal sequence was observed, except when the truncation was in the first exofacial loop where the signal peptidase cleavage site was probably too close to the membrane. The anion transport activity of co-expressed complementary pairs of truncations which together contained the entire band 3 membrane domain was examined. The pairs of fragments divided in the third and fourth exofacial loops yielded transport activity, but the pair separated within the second exofacial loop was not active. We conclude that the integrity of the second exofacial loop, but not the third and fourth exofacial loops, is necessary for transport activity. The unusually stable association between the fragments divided in the second exofacial loop suggests that interactions may occur between polar surfaces on amphiphilic portions of the third and fifth transmembrane spans.

Human erythroid band 3 "anion exchanger 1" is expressed in transformed lymphocytes.

The anion transporter, band 3, is a ubiquitous protein. It is present in brain and all other tissues examined. Not only is band 3 present in cell membranes, but also in nuclear, Golgi and mitochondria membranes. There are four isoforms of band 3, the anion exchanger (AE) proteins, thus far discovered. They are products of different genes. Lymphocytes are reported to contain AE2, but not AE1. We hypothesized that induction or up-regulation of AE1 occurs when lymphocytes are transformed as an initial event in the path to malignancy. We transformed lymphocytes containing a single base mutation with Epstein Barr Virus (EBV). The mutation of band 3, high transport band 3 (HTbd3), exhibits anion transport that is 2-3 times normal in erythrocytes which contain AE1. This facilitated our identification of AE1 since the probability that 2 different gene products would have the same mutation approaches zero. Thus, we have a base mutation in addition to linear sequence to identify AE1. A 133 base pair (bp) fragment including the affected region was amplified from the mRNA of lymphocytes from the HTbd3 mutant. AE1 primers were used to amplify regions of interest. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to generate cDNA which was sequenced. The sequence of the crucial 133 base pair segment from high transport lymphocytes was 100% identical to the sequence published for the red blood cell band 3 of the same mutant. As reported previously for erythrocytes, this mutation is a C-->T base change which changes a proline to leucine in the protein sequence. Restriction enzyme digests of AE1 cDNA from normal and HTBD3 lymphocytes confirmed that the proposita was homozygous for the mutation, and showed the father to be heterozygous. Anion transport was increased in HTbd3 EBV transformed lymphocytes, as was the case with HTbd3 erythrocytes. AE2 was identified in lymphocytes by sequence. Thus, EBV transformed lymphocytes express the erythroid band 3 (AE1) in addition to AE2.

Glycosylation of multiple extracytosolic loops in Band 3, a model polytopic membrane protein.

N-glycosylated sites in polytopic membrane proteins are usually localized to single extracytosolic (EC) loops containing more than 30 residues [Landolt-Marticorena and Reithmeier (1994) Biochem. J. 302, 253-260]. This may be due to a biosynthetic restriction whereby only a single loop of nascent polypeptide is available to the oligosaccharyl transferase in the lumen of the endoplasmic reticulum. To test this hypothesis, two types of N-glycosylation mutants were constructed using Band 3, a polytopic membrane protein that contains up to 14 transmembrane segments and a single endogenous site of N-glycosylation at Asn-642 in EC loop 4. In the first set of mutants, an additional N-glycosylation acceptor site (Asn-Xaa-Ser/Thr) was constructed by site-directed mutagenesis in EC loop 3, with or without retention of the endogenous site. In the second set of mutants, EC loop 4 was duplicated and inserted into EC loop 2, again with or without retention of the endogenous site. Cell-free translation experiments using reticulocyte lysates showed that microsomes were able to N-glycosylate multiple EC loops in these Band 3 mutants. The acceptor site in EC loop 3 was poorly N-glycosylated, probably due to the suboptimal size (25 residues) of this EC loop. The localization of N-glycosylation sites to single EC loops in multi-span membrane proteins is probably due to the absence of suitably positioned acceptor sites on multiple loops.