Original Article
Molecular Defect of the Band 3 Protein in Southeast Asian Ovalocytosis
N Engl J Med 1990; 323:1530-1538November 29, 1990
- Abstract
- Abstract
Background.
Southeast Asian ovalocytosis is a form of hereditary elliptocytosis in which the red cells are rigid and resistant to malaria invasion. The underlying molecular defect is unknown.
Methods and Results.
We studied the red cells of 54 patients with ovalocytosis and 122 normal controls. We found that ovalocytes contain a structurally and functionally abnormal band 3 protein, the principal transmembrane protein of red cells. The structural lesion of ovalocyte band 3 was revealed by limited proteolytic cleavage of the protein, which produced fragments of abnormal size that were derived from the cytoplasmic domain of the protein. The structural lesion was present in all the subjects with ovalocytosis but none of the controls. This region of band 3 serves as the principal binding site for the membrane skeleton, a submembrane protein network composed of ankyrin, spectrin, actin, and protein 4.1. The structural defect is dominantly inherited, being tightly linked with the inheritance of ovalocytosis (the probability of linkage is in excess of 10 million to 1 ). Ovalocyte band 3 bound considerably more tightly than normal band 3 to ankyrin, which connects the membrane skeleton to the band 3 protein. This tight binding of ovalocyte band 3 to the underlying skeleton containing ankyrin was directly confirmed in intact cells by the finding that ovalocyte band 3 had markedly reduced lateral mobility in the membrane.
Conclusions.
The red cells in Southeast Asian ovalocytosis carry a structurally and functionally abnormal band 3 protein. This molecular defect may underlie the increased rigidity of the red cells and their resistance to invasion by malaria parasites. (N Engl J Med 1990; 323:1530–8.)
Article Activity
- Article
THE hereditary elliptocytosis syndrome encompasses a heterogeneous group of inherited disorders, the common feature of which is the presence of elliptical red cells on peripheral-blood film.1 Recently, molecular defects of spectrin and protein 4.1, the principal proteins of the red-cell-membrane skeleton, were identified in many people with hereditary elliptocytosis.1 Hereditary elliptocytosis is uncommon in Westerners. A related condition, designated Southeast Asian ovalocytosis or stomatocytic elliptocytosis,1 , 2 is widespread in parts of Southeast Asia, particularly in Malaysia, Papua New Guinea, the Philippines, and Indonesia,2 3 4 5 6 7 8 9 with a prevalence reaching 30 percent in certain ethnic groups.6 , 7 The condition has recently attracted considerable attention for several reasons. First, ovalocytic red cells were found to be very rigid,10 an abnormality thought to have a deleterious effect on their survival in vivo.11 Affected persons, however, are asymptomatic, having no clinically detectable hemolysis.2 Second, ovalocytes are resistant to invasion by various strains of malaria parasites in vitro,12 , 13 and in areas of endemic malaria they contain reduced numbers of intracellular parasites in vivo.5 , 10 , 14
The normal red-cell membrane is supported by a membrane skeleton consisting of a hexagonal lattice of complexes of oligomeric actin that are connected in a two-dimensional network by fiber-like spectrin tetramers, together with protein 4.1 and other accessory proteins.15 This membrane skeleton is attached to the membrane proper primarily by a linkage of the β subunit of spectrin to ankyrin, which in turn connects the skeleton to the chief transmembrane protein, band 3.16 In this article, we show that ovalocytes contain a structurally and functionally abnormal band 3 protein that binds tightly to ankyrin and that this dominantly inherited defect is linked with the inheritance of ovalocytosis.
Methods
Subjects
A total of 54 subjects with ovalocytosis, 55 controls matched for race, and 67 other controls (white and black Americans) were studied. Forty-five of the subjects with ovalocytosis were from Malaysia, eight from Papua New Guinea, and one from the Philippines. The diagnosis of Southeast Asian ovalocytosis was established by determining the percentage of ovalocytes on blood smears stained with Wright—Giemsa stain; 25 percent or more indicated ovalocytosis (Fig. 1Figure 1
Blood Film from a Subject with Southeast Asian Ovalocytosis.). In accordance with recently adopted diagnostic criteria for elliptocytosis,1 subjects were considered positive if they had the appearance of dominantly inherited ovalocytosis (25 percent or more ovalocytes) on peripheral-blood films.Studies of Salt-Induced Changes in Red-Cell Ghosts and Membrane Skeletons
Venous blood from the normal controls and the subjects with Southeast Asian ovalocytosis was collected in sterile tubes containing citrate—phosphate—dextrose and used within two weeks. Ghost membranes were prepared from washed erythrocytes by the method of Dodge et al.,17 with 5 mM sodium phosphate (pH 7.4). The spectrin—actin skeletons (or Triton shells) were produced by extracting lipids and the integral membrane proteins from ghosts with 1 percent Triton X-100 in phosphate buffer (pH 7.4) at 0°C for 30 minutes. Sodium chloride (final concentration, 150 mM) was added to freshly prepared ghosts or Triton shells at 0°C to initiate the salt-induced changes in shape characterized by the formation of surface spicules. After a 10-minute incubation period, the ghosts or Triton shells were fixed with glutaraldehyde and examined with a phase-contrast light microscope attached to a Newvicon camera and video monitor (MTI, Michigan City, Ind.).18
Detection of Structural Abnormalities of the Band 3 Protein by Limited Proteolysis
Erythrocyte ghosts were suspended in 10 mM phosphate buffer (pH 7.4) and digested with N-tosyl-L-phenylalanine chloromethyl ketone—trypsin (1:250 to 1:2000 wt/wt) for three, six, and nine hours at 0°C. The reaction was terminated by adding a protease inhibitor, diisopropyl fluorophosphate (1 mM), or by heating the samples to 100°C for one minute in the presence of 1 percent sodium dodecyl sulfate (SDS) and 20 mM dithiothreitol.
In a parallel experiment, membranes depleted of spectrin, actin (band 5), and glyceraldehyde-3-phosphate dehydrogenase (band 6) were prepared by extracting these proteins from normal and ovalocyte ghost membranes as previously described.19 These membranes were digested with papain ( 1 to 10 μg per milliliter) for one hour at 23°C,19 and the reaction was terminated with sodium iodoacetate (final concentration, 10 mM). Aliquots were electrophoresed in the presence of SDS as described below.
Protein Electrophoresis and Immunoblotting
Membrane-protein digests were electrophoresed on either SDS—polyacrylamide (12 percent) or gradient SDS—polyacrylamide (15 to 25 percent) slab gels by the procedure of Laemmli.20 The gels were stained for protein with Coomassie blue. For the immunochemical analysis by Western blotting, the gels were electrophoretically transferred to nitrocellulose paper; probed with polyclonal rabbit anti—band 3 antibodies (gift of Drs. Manjit Hanspal and Rajiv Kalraiya), anti—cytoplasmic domain of band 3 antibodies (gift of Ms. Catherine Korsgren), or antiserum to peptides corresponding to residues 142 through 154 of human erythroid band 321 (gift of Dr. Philip S. Low); and treated with secondary antibodies conjugated to peroxidase, followed by a color development after the addition of 3,3′-diaminobenzidine.22
Interaction of Ankyrin with the Cytoplasmic Domain of the Band 3 Protein
Two approaches were used to study the interaction of ankyrin with the cytoplasmic domain of the band 3 protein. The first involved investigating the binding of purified, radioactive-labeled ankyrin to inside-out membrane vesicles containing the band 3 protein but stripped of ankyrin.23 Alternatively, the binding of ankyrin to the cytoplasmic domain of band 3 was measured directly in solution. The water-soluble 41,000- and 43,000-dalton cytoplasmic domain of band 3 was prepared by the proteolysis of inside-out vesicles (previously stripped of ankyrin) with α-chymotrypsin and purified in a native form by column chromatography with use of DE-52 cellulose (Whatman, Maidstone, United Kingdom) and Ultrogel AcA-44 (LKB, Gaithersburg, Md.) as previously described.23 The binding of ankyrin to the cytoplasmic domain of band 3 was measured as reported,23 with a constant concentration of band 3 fragment and variable concentrations of ankyrin in the reaction mixtures.
Studies of the Lateral Mobility of Band 3 in the Red-Cell Membrane
Venous blood from one subject with ovalocytosis and one normal control was collected in acid—citrate—dextrose and shipped overnight to Boston for immediate processing. Red cells were washed and labeled as described.24 Briefly, band 3 was specifically labeled by incubating intact red cells with eosin-5-maleimide (Molecular Probes, Eugene, Oreg.). Glycophorin was specifically labeled by conjugating fluorescein-5-thiosemicarbazide (Molecular Probes) to glycophorin-linked sialic acid moieties. The phospholipid analogue fluorescein-phosphatidylethanolamine (Avanti Polar Lipids, Birmingham, Ala.) was incorporated directly into intact red-cell membranes.
The fluorescence-photobleaching-recovery technique25 was used to measure the lateral mobility of band 3, glycophorin, and fluorescein-phosphatidylethanolamine in the membranes of fluorescently labeled intact red cells. In this technique, a single red cell is observed under a fluorescence microscope, with a focused laser beam as the source of excitation. A small area of membrane is exposed to a brief, intense laser pulse, causing irreversible bleaching of the fluorophore. The recovery of fluorescence that results from the lateral diffusion of unbleached fluorophore into the bleached area is measured. The analysis of fluorescence-recovery curves yields the fraction of fluorescently labeled protein or lipid that is free to diffuse in the plane of the membrane (the mobile fraction), as well as the diffusion coefficient of the mobile fraction.
Our fluorescence-photobleaching-recovery apparatus and analytical methods were as previously described,24, with the following modifications. Two computer-linked acousto-optic modulators (N350850–3, Newport Electro-Optics Systems, Fountain Valley, Calif.) were used to produce the monitoring and photobleaching laser pulses. A multichannel sealer (model 370, Nicolet Instrument, Madison, Wis.) was used to collect fluorescence data from the amplifier—discriminator. The experiment was controlled by a computer (model 386i, Sun Microsystems, Mountain View, Calif.) and a custom-built timing board (provided by J.D. Corbett, V.G. Bose, and D.E. Golan). The radius of the Gaussian beam at the sample plane, as determined by a two-dimensional emission-scanning technique,26 was 0.55 p,m for measurements of protein mobility and 1.86 μm for lipids. The photobleaching power at the sample plane was approximately 2 mW. The bleaching time was 300 msec for measurements of protein mobility and 80 msec for lipids. The intensity of the measuring beam was approximately 3 μW. The sample temperature was kept at 37±0.1°C with use of a thermal microscope stage. Data were fitted by nonlinear least-squares analysis,27 as previously described.24
Statistical Analysis
The lod score indicating the logarithmic value of probability (log odds) was estimated separately for 14 families (two or three generations) according to the established method reviewed by Race and Sanger.28 It was calculated as follows: the probability of observing the coinheritance of ovalocytosis and a band 3 abnormality that was assumed to be genetically linked was divided by the probability of observing the coinheritance of ovalocytosis and a band 3 abnormality that was not genetically linked. Autosomal linkage is considered to be established if the sum of the lod scores in the families reaches 3 (odds in favor of linkage, 1000:1).
Other Methods
The following previously reported methods were also used: protein concentration29; binding of 125I-labeled spectrin to inside-out vesicles30; membrane-protein electrophoresis according to the methods of Fairbanks et al.31 and Laemmli,20 followed by the quantitation of protein bands by densitometric tracing; spectrin dimerdimer self-association in solution32; interaction of spectrin, actin, and band 4.133; heat-induced spectrin cross-linking after the incubation of washed red cells at 46 to 52°C for 30 minutes34; and tryptic digestion of spectrin.35
Results
Resistance to Salt-Induced Changes in Shape
Red cells from subjects with Southeast Asian ovalocytosis have previously been found to be rigid, resisting changes in shape after exposure to echinocytogenic (spicule-forming) agents.4 To establish whether the primary lesion underlying membrane rigidity resides in the red-cell membrane or the membrane skeleton (containing only the spectrin—actin network but devoid of lipids and integral proteins), we compared the changes in the shape of red-cell ghosts with those in the shape of membrane skeletons in normal controls and subjects with ovalocytosis. In a hypotonic buffer both the ovalocytic red-cell ghosts and their skeletons had the characteristic oval shape (Fig. 2Figure 2
Responses of Control and Ovalocyte Ghosts and Their Skeletons to an Increase in the Salt Concentration of the Suspending Solution.). On subsequent incubation in isotonic buffer (5 mM sodium phosphate [pH 7.4] and 150 mM sodium chloride) at 0°C, both the normal ghosts and their membrane skeletons underwent a marked spiculation and shrinkage. In striking contrast, ovalocyte ghosts resisted salt-induced condensation and echinocytic transformation of their shape, whereas their skeletons underwent the same degree of shrinkage as the skeletons of normal cells. On the basis of this finding, we concluded that the molecular lesion of ovalocytic red cells was unlikely to involve proteins of the skeleton. We suspected an abnormality of one of the integral membrane proteins, such as band 3 protein, which binds the skeleton to the membrane and which is present in ghosts but not in membrane skeletons.The possibility that a defect of an integral membrane protein, rather than a skeletal protein, is involved in ovalocytosis was further suggested by our failure to detect any abnormalities in the composition12 and function of skeletal protein, including the binding of spectrin dimers to spectrin-depleted inside-out membrane vesicles, spectrin dimer—dimer self-association, the binding of spectrin to F-actin in the presence of protein 4.1, heat-induced spectrin cross-linking in the membrane, and limited tryptic digestion of spectrin (data not shown). In ovalocyte ghosts the ratio of band 4.1b to band 4.1a was slightly increased (data not shown). Changes in this ratio characteristically reflect increased numbers of young red cells in the peripheral blood.36 Furthermore, the ultrastructure of ovalocyte-membrane skeletons appeared normal on negative-staining electron microscopy37 (data not shown).
Structural Abnormality of Band 3 Revealed by Limited Proteolytic Digestion
To explore a possible structural abnormality of band 3 protein, the most abundant integral protein of the membrane, we subjected both the red-cell ghosts and inside-out membrane vesicles to limited tryptic digestion and separated the fragments by electrophoresis. As revealed on gels stained with Coomassie blue, a new protein band (molecular weight, 25,000) was present in whole-membrane digests derived from ovalocyte ghosts but not in those from ghosts of normal red cells (Fig. 3Figure 3
Limited Tryptic Digestion and Immunoblotting of Control Erythrocyte (C) and Ovalocyte (Ov) Ghosts.A). The presence of this new 25,000-dalton band in the ovalocyte-membrane digests was associated with a concomitant reduction (13 to 50 percent) in a 22,000-dalton band that was present in digests of both normal and ovalocyte membranes. The subsequent electrophoretic transfer to nitrocellulose paper, followed by immunoblotting with affinity-purified antibodies against the isolated cytoplasmic domain of band 3 (Fig. 3B), unequivocally identified both the 22,000-dalton and 25,000-dalton bands as polypeptide fragments of the cytoplasmic domain of band 3. The presence of the additional 25,000-dalton fragment was also detected in the ovalocyte-derived inside-out vesicles by limited tryptic digestion (data not shown). Part of the 25,000-dalton fragment was released from the vesicles and could be immunostained by antiserum to peptides corresponding to residues 142 through 154 of human erythroid band 3 (Fig. 3C).Earlier studies have suggested that the 22,000-dalton fragment released from normal inside-out vesicles by trypsin represents the N terminus of band 3 corresponding to amino acid residues 1 through 180.38 , 39 In an attempt to identify the subdomain of the N-terminal 22,000- and 25,000-dalton fragments responsible for the abnormal pattern of cleavage of the band 3 protein in ovalocytes, we subjected the fragments to a further limited digestion with papain to remove the 9000-dalton fragment from the N terminus.19 The results showed that the normal 22,000-dalton fragment produced a fragment of 13,000 dal tons, whereas the ovalocyte 22,000- and 25,000-dalton fragments produced an additional 16,000-dalton band (Fig. 3D). Both the 13,000-dalton and the 16,000-dalton fragments could be immunostained by antipeptide residues 142 through 154 of band 3. Furthermore, when the entire cytoplasmic domain, namely the 40,000- and 43,000-dalton fragments of ovalocyte band 3, was digested with papain to remove the 9000-dalton fragment from the N-terminal end, a 34,000-dalton fragment was detected in addition to the normal 31,000-dalton fragment (Fig. 3E and 3F). Taken together, the data from trypsin and papain digestion suggest that the peptide alteration in ovalocytes is located within the 16,000-dalton segment of the C-terminal end of the abnormal 25,000-dalton trypsin fragment of band 3 (Fig. 4Figure 4
Comparison of the Digestion Products of Red-Cell Band 3 Protein in Normal Controls (N) and Subjects with Ovalocytosis (Ov).).Linkage of the Structural Abnormality of the Band 3 Protein to the Inheritance of Ovalocytosis
The abnormality of band 3 was present in all the samples from subjects with ovalocytosis, including 45 Malaysians, 8 Melanesians, and 1 Filipino, whereas erythrocyte membranes from 106 controls, including American whites and blacks, Southeast Asians, 11 stored blood samples, and 5 patients with other types of hemolytic anemia, were all normal (Table 1Table 1
Association of the Structural Defect of the Band 3 Protein with Southeast Asian Ovalocytosis.). The presence of both the normal 22,000-dalton peptide and the abnormal 25,000-dalton peptide in ovalocyte membranes suggested a simple heterozygous state for a structurally abnormal band 3 protein. Both the structural alteration of band 3 and ovalocytosis were inherited as dominant traits (Fig. 5Figure 5
Pedigrees of Three Malaysian Kindreds with Southeast Asian Ovalocytosis.) and were closely linked: a combined lod score of 7.0 (equivalent to a probability of 10 million to 1 in favor of linkage) was obtained from 14 families with ovalocytosis, thus strongly supporting linkage between the inheritance of the structural abnormality of band 3 and ovalocytosis.Abnormal Function of the Ovalocyte Band 3 Protein
Having found structural alterations of the cytoplasmic domain of the band 3 protein in ovalocytosis, we next examined the function of this protein in terms of its binding to ankyrin. For these experiments, we prepared inside-out membrane vesicles that were stripped of ankyrin. Because of their inside-out orientation, the ankyrin-binding region of the band 3 protein was exposed on the outer surface of the vesicles. Figure 6Figure 6
Binding of 125I-Labeled Ankyrin in Cells from Normal Controls (●) and Ovalocytes (○).A shows that the vesicles prepared from ovalocytes bound substantially larger amounts of 125I-labeled ankyrin than the corresponding controls. The binding of ankyrin was destroyed almost completely by preincubation at temperatures higher than 60°C (data not shown).The finding that more ankyrin bound to ovalocyte inside-out vesicles than to normal vesicles was further supported by the finding that the water-soluble 40,000- and 43,000-dalton fragments (representing the cytoplasmic domain of band 3) prepared from ovalocyte inside-out vesicles inhibited the binding of 125I-labeled ankyrin to normal ankyrin-stripped inside-out vesicles considerably more than did the normal 40,000- and 43,000-dalton band 3 fragments (Fig. 6B). We also directly assessed the specific binding of ankyrin to the isolated cytoplasmic domain of band 3 prepared from subjects with ovalocytosis or controls (Fig. 6C). The stoichiometric ratio of binding was higher in subjects with ovalocytosis than in controls (moles of ankyrin per mole of cytoplasmic domain of band 3, 0.54 vs. 0.39 of normal), confirming the results with inside-out membrane vesicles.
Specific Immobilization of Band 3 in Membranes of Intact Ovalocytes
In order to assess whether increased binding of band 3 to ankyrin also occurs in intact ovalocytes, we used the fluorescence-photobleaching-recovery technique to measure the lateral mobility of fluorescently labeled band 3 in the membranes of intact ovalocytes. A mean (±SD) of only 16±14 percent of labeled band 3 molecules were free to diffuse in ovalocyte membranes, as compared with 73±9 percent in normal red-cell membranes. The diffusion coefficient of the mobile band 3 molecules was not significantly different in ovalocytic and normal red-cell membranes. Furthermore, neither the fractional mobilities nor the diffusion coefficients of glycophorin and the phospholipid analogue fluorescein-phosphatidylethanolamine were significantly different in ovalocyte and normal membranes (Table 2Table 2
Lateral Mobility of Band 3, Glycophorin, and Phospholipid in Membranes of Intact Control and Ovalocytic Red Cells.*). These data demonstrate that band 3 is specifically immobilized in the intact ovalocyte membrane. Since the fractional mobility of band 3 in membranes of both normal and abnormal red cells appears to be regulated primarily by high-affinity binding between band 3 and ankyrin,40 this finding supports the hypothesis that interaction between band 3 and ankyrin is abnormally increased in intact ovalocytes.Discussion
We present data indicating that red cells from subjects with Southeast Asian ovalocytosis contain both structurally and functionally abnormal band 3 protein. Furthermore, we have shown that these subjects are heterozygotes carrying both the normal and abnormal band 3 proteins, as evidenced by structural analysis of this protein with limited proteolysis; they have both the normal 22,000-dalton fragment produced by trypsin cleavage of the N-terminal cytoplasmic domain of the normal band 3 protein and a 25,000-dalton fragment cleaved from the abnormal band 3 protein. We further found that this defect is inherited in an autosomal dominant mode and that the inheritance of the structural defect is tightly linked with the inheritance of ovalocytosis, with a probability of linkage of 10 million to 1, as indicated by a lod score of 7. We thus conclude that the molecular defect of the band 3 protein constitutes the primary molecular lesion in Southeast Asian ovalocytosis.
Although the bulk of the band 3 protein is embedded in the lipid bilayer and transverses it many times,41 both the N terminus and the C terminus of the protein are located in the cytoplasm. The protein has at least two main functions in the red-cell membrane. In addition to transporting anions across the membrane, a function assigned to the transmembrane segment of the protein, it also provides the principal binding site for ankyrin, a protein that links the spectrin-based submembrane skeleton to the red-cell membrane. The critical role of the band 3 protein in the assembly of the red-cell membrane is well illustrated by studies of the biogenesis of the membrane during erythroid development: although spectrin, ankyrin, and protein 4.1 are synthesized in early erythroid precursors, their assembly into a stable membrane skeleton takes place only after the synthesis of band 3 has been initiated.42 The binding site for ankyrin is located in the N-terminal cytoplasmic segment of the protein and is likely to contain several noncontinuous regions, including amino acids 118 through 162, 174 through 186, and sequences near Cys-201 and Cys-317.21 , 43 The N terminus is unique in that it contains a very negative stretch of amino acids, the most negatively charged stretch known in nature, and it serves as an attachment site for hemoglobin, Heinz bodies, and several glycolytic enzymes.44 Our studies assigned the structural abnormality of the ovalocyte band 3 to the cytoplasmic domain because of the presence of abnormally migrating peptides that reacted specifically with antibodies against the cytoplasmic domain of the band 3 protein (Fig. 3B). The abnormally migrating peptides were produced both by cleavage with trypsin, which cuts the cytoplasmic domain of the band 3 protein at amino acid 180, as well as with papain, with the site of papain cleavage being about 9000 daltons from the N terminus.19 The normal 13,000-dalton and abnormal 16,000-dalton fragments stained specifically with antibodies raised against residues 142 through 154. We thus concluded that the structural lesion resides between amino acids 60 (the possible site of papain cleavage) and 180 (site of tryptic cleavage) of the cytoplasmic domain of the band 3 protein.
We have also detected functional alterations in the band 3 protein characterized by a tight binding of the cytoplasmic domain of band 3 to ankyrin. Such tight binding was demonstrated by a variety of approaches, including the binding of ankyrin to inside-out vesicles containing intact band 3 protein and the binding of ankyrin to the soluble cytoplasmic domain of the band 3 protein. Furthermore, the fragment derived from the abnormal band 3 was more efficient than its normal counterpart in displacing ankyrin from the inside-out vesicles in a study of competitive inhibition. Even more important, we provide direct evidence for an immobilization of the band 3 protein in the membranes of intact ovalocytes, as evidenced by a marked restriction in the lateral mobility of band 3 protein in the fluorescence-photobleaching study.
Thus, in contrast to red cells in other membrane disorders, such as elliptocytosis or spherocytosis, which have either dysfunctional or deficient membrane skeletal proteins,1 red cells in Southeast Asian ovalocytosis are unique in that the underlying defect involves a hyperstable rather than an unstable protein linkage, producing a rigid red-cell membrane. Two variant forms of band 3 have previously been reported with an elongated N terminus.45 , 46 Both variants are hematologically normal, however, with normal red-cell morphologic features, and the red cells do not appear to be resistant to invasion by malaria parasites in vitro.47 , 48 The exact location of the amino acid mutations in these two variants remains to be established and compared with the presumed mutation in the ovalocyte band 3.
The mechanism whereby an excessively tight binding of band 3 to ankyrin accounts for an overall increase in membrane rigidity and possibly for a marked resistance of ovalocytes to invasion by various strains of malaria parasites is open to further study. Since the contact site between the cytoplasmic domain of band 3 and ankyrin is regulated by phosphorylation21 and since the entry of malaria parasites into cells appears to require the phosphorylation of as yet undefined membrane proteins,49 further elucidation of the role of the abnormal band 3 in malaria resistance may provide important clues about the molecular mechanisms by which malaria parasites enter cells, and may serve as a focus for future therapeutic interventions.
Supported by grants (HL-27215, HL-37462, HL-32854, and HL-15157) from the National Institutes of Health. Address reprint requests to Dr. Palek at the Division of Hematology/Oncology, St. Elizabeth's Hospital, 736 Cambridge St., Boston, MA 02135.
We are indebted to Drs. Manjit Hanspal, Rajiv Kalraiya, and Philip S. Low, Mr. Donald Howard, and Ms. Catherine Korsgren for supplying antibodies against band 3; to Ms. Pilarin Nichols and Mr. Patrick Yacono for technical assistance in conducting the experiments; to Dr. James Corbett for assistance in operating the fluorescence-photobleaching-recovery apparatus; to Ms. Gabriella Maitino for preparation of the manuscript; and to Ms. Joan Joos for preparation of the figures.
Source Information
From the Division of Hematology/Oncology and Department of Biomedical Research, St. Elizabeth's Hospital, Tufts University School of Medicine (S.-C.L., S.Z., J.P., T.C., P.J.); the Departments of Biological Chemistry and Molecular Pharmacology and Medicine, Harvard Medical School; and the Department of Medicine (Hematology Division), Brigham and Women's Hospital (D.E.G.), all in Boston; Mt. Sinai Hospital, Toronto (D.A., S.B.); the University of Toronto, Institute for Medical Research, Kuala Lumpur, Malaysia (K.H., M.Z.); the Papua New Guinea Red Cross, Boroko (D.B.); and the University of Papua New Guinea, Boroko (G.T.N.).
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Citing Articles
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Rossen Mirchev, Alexander Lam, David E. Golan. (2011) Membrane compartmentalization in Southeast Asian ovalocytosis red blood cells. British Journal of Haematology 155:1, 111-121
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Edward C Gordon-Smith, Narla Mohandas. 2010. Hereditary Disorders of the Red Cell Membrane. , 126-139.
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Anthony J. Baines. (2010) The spectrin–ankyrin–4.1–adducin membrane skeleton: adapting eukaryotic cells to the demands of animal life. Protoplasma 244:1-4, 99-131
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Somkiat Vasuvattakul. (2010) Molecular Approach for Distal Renal Tubular Acidosis Associated AE1 Mutations. Electrolytes & Blood Pressure 8:1, 25
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Emmanuelle Cordat. (2006) Unraveling trafficking of the kidney anion exchanger 1 in polarized MDCK epithelial cellsThis paper is one of a selection of papers published in this Special Issue, entitled CSBMCB — Membrane Proteins in Health and Disease.. Biochemistry and Cell Biology 84:6, 949-959
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Narla Mohandas, Anupama Narla. (2005) Blood group antigens in health and disease. Current Opinion in Hematology 12:2, 135-140
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Marion E Reid, Narla Mohandas. (2004) Red blood cell blood group antigens: structure and function. Seminars in Hematology 41:2, 93-117
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Patrick G Gallagher. (2004) Hereditary elliptocytosis: spectrin and protein 4.1R. Seminars in Hematology 41:2, 142-164
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Somkiat Vasuvattakul, Pa-Thai Yenchitsomanus, Prayong Vachuanichsanong, Peti Thuwajit, Charoen Kaitwatcharachai, Vichai Laosombat, Prida Malasit, Prapon Wilairat, Sumalee Nimmannit. (1999) Autosomal recessive distal renal tubular acidosis associated with Southeast Asian ovalocytosis. Kidney International 56:5, 1674-1682
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Vichai Laosombat, Supaporn Dissaneevate, Chayanon Peerapittayamongkol, Masafumi Matsuo. (1999) Neonatal hyperbilirubinemia associated with Southeast Asian ovalocytosis. American Journal of Hematology 60:2, 136-139
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Lesley J. Bruce, Susan M. Ring, Kay Ridgwell, David M. Reardon, Carol A. Seymour, Heidi M. Van Dort, Philip S. Low, Michael J.A. Tanner. (1999) South-east Asian ovalocytic (SAO) erythrocytes have a cold sensitive cation leak: implications for in vitro studies on stored SAO red cells. Biochimica et Biophysica Acta (BBA) - Biomembranes 1416:1-2, 258-270
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