Mutant Band Research Articles

Elf5 is essential for early embryogenesis and mammary gland development during pregnancy and lactation

Elf5 is an epithelial-specific ETS factor. Embryos with a null mutation in the Elf5 gene died before embryonic day 7.5, indicating that Elf5 is essential during mouse embryogenesis. Elf5 is also required for proliferation and differentiation of mouse mammary alveolar epithelial cells during pregnancy and lactation. The loss of one functional allele led to complete developmental arrest of the mammary gland in pregnant Elf5 heterozygous mice. A quantitative mRNA expression study and Western blot analysis revealed that decreased expression of Elf5 correlated with the downregulation of milk proteins in Elf5(+/-) mammary glands. Mammary gland transplants into Rag(-/-) mice demonstrated that Elf5(+/-) mammary alveolar buds failed to develop in an Elf5(+/+) mammary fat pad during pregnancy, demonstrating an epithelial cell autonomous defect. Elf5 expression was reduced in Prolactin receptor (Prlr) heterozygous mammary glands, which phenocopy Elf5(+/-) glands, suggesting that Elf5 and Prlr are in the same pathway. Our data demonstrate that Elf5 is essential for developmental processes in the embryo and in the mammary gland during pregnancy.

New Insights into the Function of N-Terminal 11 Amino Acids of Band 3 from Structural and Functional Study of a Naturally Occuring Band 3 Variant.

The 911 amino acid human erythroid AE1 (eAE1) Cl-/HCO3- exchanger SLC4A1 (usually called band 3) is the major intrinsic membrane protein of red cells. The N-terminal cytoplasmic domain of AE1 represents the anchoring site for membrane-associated proteins such as ankyrin, protein 4.2, protein 4.1, glycolytic enzymes (including aldolase and glyceraldeyde-3-phosphate dehydrogenase (GAPDH) and hemoglobin. We identified marked band 3 deficiency in the second son of a consanguineous marriage with a life-threatening nonimmune hemolytic anemia. The patient was transfusion-dependent prior to splenectomy. SDS-PAGE and immunoblotting analysis of the proband red cell membrane proteins showed approximately 12±4% of band 3 and protein 4.2 compared to controls. Direct nucleotide sequence of SLC4A1 gene showed a single base substitution (T->C) at position +2 in the donor splice site of intron 2 (Band 3 Neapolis). Functionally, the mutation causes an altered splicing with the consequent formation of two different mature mRNAs, one including intron 2 and one skipping exon 2. While intron 2 retention leads to premature translation termination, exon 2 skipping causes the loss of the normal start site of eAE1 protein translation. The purification of mutant band 3 and its characterization by MALDI mass spectrometry demonstrated the lack of the first 11 amino acids due to the usage of second in frame start site. Real-time RT-PCR analyses of reticulocyte mRNA showed a marked decrement in band 3 transcription accounting for protein deficiency. The lack of the 11 N-terminal amino acids resulted in complete absence of membrane bound aldolase while other glycolitic enzymes (for example GAPDH) were membrane bound. Syk tyrosine kinase recognized the truncated band 3 as a substrate in vitro. In spite of this ability to be phosphorylated by Syk and to recruit Lyn tyrosine kinase in vitro, we were unable to demonstrate Tyr-phosphorylation of mutant band 3 in intact erythrocytes following stimulation by oxidative stress. This finding implies a requirement for the 11 N-terminal amino acids for the sequential Tyr-phosphorylation of band 3 in intact red cell membranes. The mutant band 3 was largely present in the high molecular weight aggregate fraction (about 5.2 fold higher than control), indicating its increased tendency to cluster in the membrane. The spontaneous clustering of truncated band 3 strongly suggests that the negatively charged N-terminal domain may regulate oligomeric state of band 3 in the membrane. Biophysical characterization showed that band 3 deficiency resulted in decreased cohesion between lipid bilyer and spectrin based membrane skeleton accounting for membrane loss. The structural and functional characterization of the naturally occuring mutant band 3 has enabled us to identify a significant role for the 11 N-terminal amino acids in band 3 function and in red cell membrane physiology.

Inverted Race Tube Assay for Circadian Clock Studies of the Neurospora Accessions

Although the Neurospora crassa circadian clock has been studied for forty years, population studies of natural accessions have been limited by technical difficulties associated with the conventional race tube assay (CRTA) that is used to measure asexual development (conidiation). Due to the buildup of CO2 in the CRTA that represses banding, a mutant strain band (bd) has been utilized for increased visualization of the banding phenotype. In order to study the circadian clock in natural accessions of Neurospora multiple techniques have been explored. One such technique, the rubidium chloride-supplemented race tube assay (RRTA) has been used successfully. Here we present a new technique, the Inverted Race Tube Assay (IRTA) that is a simple modification of the CRTA. We analyzed 5 natural accessions of Neurospora using CRTA, IRTA and RRTA and discuss the advantages of the IRTA in natural variation studies in Neurospora.

Band 3 Walton, a C-terminal deletion associated with distal renal tubular acidosis, is expressed in the red cell membrane but retained internally in kidney cells

Human band 3 Walton is an AE1 mutation that results in the deletion of the 11 COOH-terminal amino acids of the protein and is associated with dominant distal renal tubular acidosis. The properties of band 3 Walton expressed with normal band 3 in the heterozygous mutant erythrocytes and the kidney isoform expressed in Xenopus oocytes and in the Madin-Darby canine kidney cell line were examined. The mutant erythrocytes have normal hematology but have reduced band 3 Walton content. Transport studies showed that erythrocyte band 3 Walton has normal sulfate transport activity, and kidney band 3 Walton has normal chloride transport activity when expressed in Xenopus oocytes. The mutant protein is clearly able to reach the cell surface of erythrocytes and oocytes. In contrast, while normal kidney band 3 was expressed at the cell surface in the kidney cell line, the Walton mutant protein was retained intracellularly within the kidney cells. The results demonstrate that band 3 Walton is targeted differently in erythrocytes and kidney cells and indicate that the COOH-terminal tail of band 3 is required to allow movement to the cell surface in kidney cells. It is proposed here that the mutant band 3 gives rise to dominant distal renal tubular acidosis by inhibiting the movement of normal band 3 to the cell surface. It is suggested that this results from the association of the normal and mutant proteins in band 3 hetero-oligomers, which causes the intracellular retention of normal band 3 with the mutant protein.

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.

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.

Red blood cell membrane disorders.

The recent discovery of the specific molecular defects in many patients with hereditary spherocytosis and hereditary elliptocytosis/pyropoikilocytosis partially clarifies the molecular pathology of these diseases. HE and HPP are caused by defects in the horizontal interactions that hold the membrane skeleton together, particularly the critical spectrin self-association reaction. Single gene defects cause red cells to elongate as they circulate, by a unknown mechanism, and are clinically harmless. The combination of two defective genes or one severe alpha spectrin defect and a thalassaemia-like defect in the opposite allele (alphaLELY) results in fragile cells that fragment into bizarre shapes in the circulation, with haemolysis and sometimes life-threatening anaemia. A few of the alpha spectrin defects are common, suggesting they provide an advantage against malaria or some other threat. HS, in contrast, is nearly always caused by family-specific private mutations. These involve the five proteins that link the membrane skeleton to the overlying lipid bilayer: alpha and beta spectrin, ankyrin, band 3 and protein 4.2. Somehow, perhaps through loss of the anchorage band 3 provides its lipid neighbours (Peters et al, 1996), microvesiculation of the membrane surface ensues, leading to spherocytosis, splenic sequestration and haemolysis. Future research will need to focus on how each type of defect causes its associated disease, how the spleen aggravates membrane skeleton defects (a process termed 'conditioning'), how defective red, cells are recognized and removed in the spleen, and why patients with similar or even identical defects can have different clinical severity. Emphasis also needs to be given to improving diagnostic tests, particularly for HS, and exploring new options for therapy, like partial splenectomy, which can ameliorate symptoms while better protecting patients from bacterial sepsis and red cell parasites, and perhaps from atherosclerosis (Robinette & Franmeni, 1977) and venous thrombosis (Stewart et al, 1996).

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.

Genetic studies into inherited and sporadic hemolytic uremic syndrome

Genetic studies into inherited and sporadic hemolytic uremic syndrome

Genetic studies into inherited and sporadic hemolytic uremic syndrome

Hemolytic uremic syndrome (HUS) in adults carries a high morbidity and mortality, and its cause remains unknown despite many theories. Although familial HUS is rare, it affords a unique opportunity to elucidate underlying mechanisms that may have relevance to acquired HUS. We have undertaken a genetic linkage study based on a candidate gene approach. A common area bounded by the markers D1S212 and D1S306, a distance of 26 cM located at 1q32 segregated with the disease (Z max 3.94). We demonstrate that the gene for factor H lies within the region. Subsequent mutation analysis of the factor H gene has revealed two mutations in patients with HUS. In an individual with the sporadic/relapsing form of the disease we have found a mutation comprising a deletion, subsequent frame shift and premature stop codon leading to half normal levels of serum factor H. In one of the three families there is a point mutation in exon 20 causing an arginine to glycine change, which is likely to alter structure and hence function of the factor H protein. Factor H is a major plasma protein that plays a critical regulatory role in the alternative pathway of complement activation. In light of these findings and previous reports of HUS in patients with factor H deficiency, we postulate that abnormalities of factor H may be involved in the etiology of HUS.

赤血球膜蛋白質の構造・機能と疾患病態

The investigation of red cell anion exchange protein, band 3 has been at the center of two fundamental questions on functions of membrane proteins : how they regulate cell morphology, and how they regulate homeostasis in the cell and throughout the whole organism. Our recent studies on bovine hereditary band 3 deficiency, a novel disorder inherited in an autosomal dominant mode in which homozygous animals totally lack band 3 due to a nonsense mutation Arg664 → stop, have addressed the importance of band 3 in red cell morphology and homeostasis. The animals with total lack of band 3 had red cells that were also deficient in other major membrane skeletal proteins, resulting in a distorted and disrupted membrane skeletal network. Their red cell membranes, therefore, were extremely unstable and showed a continuous loss of surface area, leading to spherocyte formation and anemia. The decreased 4.1a/4.1b ratio implicated a high rate of hematopoiesis compensatory to survive. Rapid anion exchange sensitive to stilbene disulfonate was completely absent in band 3-deficient cells. The animals had slight acidosis and their blood bicarbonate concentration and total CO2 were lower than control levels but within a normal range, suggesting that there are alternate or multiple strategies for CO2 and pH homeostasis. These observations demonstrate that band 3 indeed contributes to red cell membrane organization, CO2 transport, and acid-base homeostasis, but it is not always essential. A putative dominant negative effect of the mutant band 3 protein on expression of functional band 3 is also discussed.

Familial distal renal tubular acidosis is associated with mutations in the red cell anion exchanger (Band 3, AE1) gene.

All affected patients in four families with autosomal dominant familial renal tubular acidosis (dRTA) were heterozygous for mutations in their red cell HCO3-/Cl- exchanger, band 3 (AE1, SLC4A1) genes, and these mutations were not found in any of the nine normal family members studied. The mutation Arg589--> His was present in two families, while Arg589--> Cys and Ser613--> Phe changes were found in the other families. Linkage studies confirmed the co-segregation of the disease with a genetic marker close to AE1. The affected individuals with the Arg589 mutations had reduced red cell sulfate transport and altered glycosylation of the red cell band 3 N-glycan chain. The red cells of individuals with the Ser613--> Phe mutation had markedly increased red cell sulfate transport but almost normal red cell iodide transport. The erythroid and kidney isoforms of the mutant band 3 proteins were expressed in Xenopus oocytes and all showed significant chloride transport activity. We conclude that dominantly inherited dRTA is associated with mutations in band 3; but both the disease and its autosomal dominant inheritance are not related simply to the anion transport activity of the mutant proteins.

The structure and function of band 3 (AE1): recent developments (review).

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

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.

Detection of ras gene mutations in perioperative peripheral blood with pancreatic adenocarcinoma.

Surgeons wish to know of any correlation between an operation and the incidence of metastasis. In perioperative periods, pancreatic cancer cells were identified by detecting mutant K‐ras gene by two‐step PCR and RFLP analysis in blood samples taken from peripheral blood. In no case was K‐ras point mutation detected in blood before operation, although the mutant band was observed in all cases at the time the lesion was resected. Surprisingly, in five of ten cases, positive bands were identified just after laparotomy, before we had reached the primary lesion. In almost all cases, mutant K‐ras was detected until the fourteenth postoperative day. These findings suggest that cancer cells exist in the circulation, and have a potential for hematogenous metastasis during the perioperative period. In conclusion, surgical stress causes hematogenous dissemination of pancreatic cancer cells, and surgeons should employ the appropriate anti‐metastasis therapy in the perioperative period.

Molecular Basis of Altered Red Blood Cell Membrane Properties in Southeast Asian Ovalocytosis: Role of the Mutant Band 3 Protein in Band 3 Oligomerization and Retention by the Membrane Skeleton

Southeast Asian ovalocytosis (SAO) is an asymptomatic trait characterized by rigid, poorly deformable red cells that resist invasion by several strains of malaria parasites. The underlying molecular genetic defect involves simple heterozygous state for a mutant band 3 protein, which contains a deletion of amino acids 400 through 408, linked with a Lys 56-to-Glu substitution (band 3-Memphis polymorphism). To elucidate the contribution of the mutant SAO band 3 protein to increased SAO red blood cell (RBC) rigidity, we examined the participation of the mutant SAO band 3 protein in increased band 3 attachment to the skeleton and band 3 oligomerization. We found first that SAO RBC skeletons retained more band 3 than normal cells and that this increased retention preferentially involved the mutant SAO band 3 protein. Second, SAO RBCs contained a higher percentage of band 3 oligomer-ankyrin complexes than normal cells, and these oligomers were preferentially enriched by the mutant SAO protein. At the ultrastructural level, the increased oligomer formation of SAO RBCs was reflected by stacking of band 3-containing intramembrane particles (IMP) into longitudinal strands. The IMP stacking was not reversed by treating SAO RBCs in alkaline pH (pH 11), which is known to weaken ankyrin-band 3 interactions, or by removing the cytoplasmic domain of band 3 from SAO membranes with trypsin. Finally, we found that band 3 protein in intact SAO RBCs exhibited a markedly decreased rotational mobility, presumably reflecting the increased oligomerization and the membrane skeletal association of the SAO band 3 protein. We propose that the mutant SAO band 3 has an increased propensity to form oligomers, which appear as longitudinal strands of IMP and exhibit increased association with membrane skeleton. This band 3 oligomerization underlies the increase in membrane rigidity by precluding membrane skeletal extension, which is necessary for membrane deformation.

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.

Molecular characterization of the band 3 protein from Southeast Asian ovalocytes

Southeast Asian ovalocytosis (SAO) is a hereditary form of elliptocytosis resulting in rigid, oval-shaped erythrocytes resistant to invasion by malaria parasites. The molecular defect is due to deletion of codons 400-408, encoding a 9-amino-acid sequence located at the boundary between the cytosol and the first transmembrane segment in Band 3, the erythrocyte anion transport protein. We have carried out an extensive characterization of Band 3 isolated from SAO erythrocytes which contain about 50% mutant Band 3. A slightly higher proportion of Band 3 in SAO erythrocytes was left associated with the cytoskeleton after extraction of ghost membranes with non-ionic detergents. Size exclusion high performance liquid chromatography analysis showed that SAO Band 3 contained a higher proportion of tetramers relative to dimers (50% tetramer) than normal Band 3 (33% tetramer). The circular dichroism spectrum of Band 3 from SAO erythrocytes was very similar to the spectrum for normal Band 3. Enzymatic deglycosylation and tomato lectin binding showed that SAO Band 3 lacked the polylactosaminyl oligosaccharide found on normal Band 3. SAO Band 3 was unable to bind the anion transport inhibitor 4-benzamido-4'-aminostilbene-2,2'-disulfonate, suggesting a dramatic alteration in the inhibitor binding site. In conclusion, deletion of 9 amino acids from Band 3 on the cytosolic side of the membrane affects the properties (glycosylation and inhibitor binding) of Band 3 on the opposite side of the membrane without dramatic changes in the secondary and quaternary structure of the protein.

Basis of unique red cell membrane properties in hereditary ovalocytosis

Hereditary ovalocytes from a Mauritian subject are extremely rigid, with a shear elastic modulus about three times that of normal cells, and have increased resistance to invasion by the malaria parasite Plasmodium falciparum in vitro. The genetic anomaly resides in band 3; the protein gives rise to chymotryptic fragments with reduced mobility in SDS/polyacrylamide gel electrophoresis, but this is a result of anomalous binding of SDS and not a higher molecular weight. Analysis of the band 3 gene reveals (1) a point mutation (Lys56 → Glu), which also occurs in a common asymptomatic band 3 (Memphis) variant and governs the electrophoretic properties, and (2) a deletion of nine amino acid residues, including a proline residue, encompassing the interface between the membrane-associated and the N-terminal cytoplasmic domains. The interaction of the mutant band 3 with ankyrin appears unperturbed. The fraction of band 3 capable of undergoing translation diffusion in the membrane is greatly reduced in the ovalocytes. Cells containing the asymptomatic band 3 variant were normal with respect to all the properties that we have studied. Possible mechanisms by which a structural change in band 3 at the membrane interface could regulate rigidity are examined.