Targeted inactivation of murine band 3 (AE1) gene produces a hypercoagulable state causing widespread thrombosis in vivo.

Only 5% to 10% of band 3 null mice survive the neonatal period. To determine the cause of death, 3 adult and 11 newborn band 3 null mice were submitted for histopathologic examination. All but 1 pup showed evidence of thrombosis including: (1) large thrombotic lesions in the heart, which were partially organized, calcified in some fields, and endothelialized, indicating a process that developed premortem (3 of 3 adults and 6 of 11 pups). (2) Subcapsular necrotic areas in the liver suggestive of premortem ischemic events caused by arteriolar occlusions (8 of 11 pups). (3) Large vein thrombi (4 of 11 pups). To investigate the etiology of this hypercoagulable state, we have used the Russell's viper venom test (RVV) to show that red blood cells (RBCs) from band 3 null mice significantly shorten the RVV clotting time of normal plasma in a dose-dependent fashion, whereas RBCs from normal mice have no effect, suggesting that the membrane of band 3 null RBCs provides a suitable surface for activation of the prothrombinase complex. Using flow cytometry, we have examined the phosphatidylserine (PS)-specific binding of fluorescein isothiocyanate (FITC)-annexin V to normal and band 3 null RBCs. A subpopulation of cells (3% to 5% of RBCs) with increased FITC-annexin V binding was detected in band 3 null RBCs as compared with normal RBCs. Furthermore, the entire cell population of band 3 null RBCs shows a measurable increase in the mean fluorescence intensity, suggesting that band 3 null RBCs may have increased PS exposure on the outer membrane leaflet. These findings are further supported by direct fluorescence microscopy of normal and band 3 null RBCs labeled with FITC-annexin V. Based on these observations, we postulate that the high mortality of band 3 null mice may be related to a hypercoagulable state, which appears to originate from changes in the phospholipid composition of the membrane leading to PS exposure on the outer leaflet.

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.

Complete deficiency of glycophorin A in red blood cells from mice with targeted inactivation of the band 3 (AE1) gene.

Glycophorin A is the major transmembrane sialoglycoprotein of red blood cells. It has been shown to contribute to the expression of the MN and Wright blood group antigens, to act as a receptor for the malaria parasite Plasmodium falciparum and Sendai virus, and along with the anion transporter, band 3, may contribute to the mechanical properties of the red blood cell membrane. Several lines of evidence suggest a close interaction between glycophorin A and band 3 during their biosynthesis. Recently, we have generated mice where the band 3 expression was completely eliminated by selective inactivation of the AE1 anion exchanger gene, thus allowing us to study the effect of band 3 on the expression of red blood cell membrane proteins. In this report, we show that the band 3 -/- red blood cells contain protein 4.1, adducin, dematin, p55, and glycophorin C. In contrast, the band 3 -/- red blood cells are completely devoid of glycophorin A (GPA), as assessed by Western blot and immunocytochemistry techniques, whereas the polymerase chain reaction (PCR) confirmed the presence of GPA mRNA. Pulse-label and pulse-chase experiments show that GPA is not incorporated in the membrane and is rapidly degraded in the cytoplasm. Based on these findings and other published evidence, we propose that band 3 plays a chaperone-like role, which is necessary for the recruitment of GPA to the red blood cell plasma membrane.

Red cell membranes of ankyrin-deficient nb/nb mice lack band 3 tetramers but contain normal membrane skeletons.

The role of ankyrin in the formation and stabilization of the spectrin-based skeletal meshwork and of band 3 oligomers was studied by characterizing, in nb/nb mouse red cells, the effect of ankyrin deficiency on skeletal ultrastructure, band 3-skeleton associations, and band 3 oligomeric states. Despite severe ankyrin deficiency, nb/nb mouse red cell skeletal components formed a relatively uniform two-dimensional hexagonal array of junctional complexes cross-linked by spectrin tetramers. Treatment of nb/nb ghosts with the nonionic detergent C12E8 (octaethylene glycol n-dodecyl monoether) resulted in nearly complete extraction of band 3. The extracted band 3 was present exclusively as band 3 dimers. Fluorescence photobleaching recovery and polarized fluorescence depletion measurements showed increases in the laterally (33% vs 10%) and rotationally (90% vs 76%) mobile fractions of band 3 in intact nb/nb compared to control red cells. The rotational correlation time of the major fraction of band 3 molecules was 10-fold shorter in nb/nb compared to control red cells, indicating a significant relaxation of rotational constraints in nb/nb cells. These data suggest that, although ankyrin plays a major role in strengthening the attachment of the skeleton to the membrane bilayer, ankyrin is not required for the formation of a stable two-dimensional spectrin-based skeleton. The absence of band 3 tetramers in the membrane of ankyrin-deficient red cells suggests that ankyrin is required for the formation of stable band 3 tetramers.

Characterization of the underlying molecular defect in hereditary spherocytosis associated with spectrin deficiency.

Several subsets of patients with hereditary spherocytosis (HS) have been defined based on the specific red blood cell membrane protein deficiencies involving spectrin, ankyrin, band 3, and protein 4.2. Mutations of the genes encoding these proteins are currently being uncovered. Regarding spectrin, only three isolated cases of beta-spectrin gene mutations were recently reported in association with HS and spectrin deficiency. We have screened the coding region of the beta-spectrin gene using the SSCP technique, in 40 families with HS associated with spectrin deficiency or combined spectrin and ankyrin deficiencies. In this report we describe six frameshift and nonsense mutations and four missense mutations of the beta-spectrin gene in 11 unrelated families. Taking advantage of modifications in the restriction enzyme recognition sequences introduced by the mutations, we show, in all cases of frameshift and nonsense mutations, the loss of heterozygosity at the cDNA level when compared to genomic DNA, reflecting the absence of the mutant mRNA transcripts. In one family with a large pedigree including six generations and 112 members, we firmly establish the autosomal dominant inheritance of one of the beta-spectrin null mutations. Most of the mutations described are responsible for a phenotype of mild to moderate autosomal dominant form of HS associated with a conspicuous spherocytosis with frequent spiculated cells (8% to 15% acanthocytes). One missense mutation appears to be associated with a recessive form of the disease. Five common restriction enzyme polymorphisms of the coding region of the beta-spectrin gene are also described. Overall, these findings underscore the importance of the beta-spectrin gene mutations in the pathogenesis of HS and reemphasizes the extreme heterogeneity of the underlying molecular basis of this condition.

Erythrocyte membrane alterations in Plasmodium falciparum malaria sequestration.

Plasmodium falciparum malaria, the most lethal form of human malaria, claims at least 2 million lives worldwide each year. Recently, there has been a significant advance in our understanding of the molecular basis of P. falciparum sequestration, a distinctive pathologic feature that often leads to fatal human cerebral malaria. Parasite-derived VAR proteins (Plasmodium falciparum-infected erythrocyte membrane protein 1) have been cloned and identified as antigenically diverse cytoadherent receptors localized to the knob protrusions that act as attachment points in parasite sequestration. Evidence now supports the hypothesis that cryptic regions of band 3 protein are parasite-induced, host-derived erythrocyte receptors mediating parasite sequestration. Knob structures have been localized to spectrin-actin-protein 4.1 junctions in intact spread membrane skeletons. A recombinant domain of knob-associated histidine-rich protein, a major protein found in both membrane-intact and isolated knobs, has been shown to associate with filamentous actin and spectrin. Parasite- and host-derived erythrocyte membrane proteins involved in P. falciparum sequestration are discussed in this review.

[Relation between types of erythrocyte membrane defects and the clinical picture in hereditary spherocytosis]. / Vztah mezi typem membránového defektu erytrocytu a klinickým obrazem u hereditární sférocytózy.

From total number of 130 patients with hereditary spherocytosis from 75 families in 119 patients from 69 families a defect of membrane proteins was detected. In 23 families (33.3%) a spectrin defect was involved, in 32 families (46.3%) a combined defect of spectrin and ancyrine and in 14 families (20.3%) a defect of band 3 proteins. Investigation of the membrane defect and the clinical and laboratory picture revealed that the band 3 protein defect of spectrin and ancyrine. There are significant differences in the clinical picture of the two latter defects.

Does a patient with hereditary spherocytosis qualify for preoperative autologous blood donation?

BACKGROUND: Hereditary spherocytosis (HS) is characterized by osmotically fragile spherocytic red cells (RBCs), mild to moderate hemolysis, and splenomegaly. Little is known about the safety of blood bank storage of HS RBCs. CASE REPORT: A 50-year-old man with mild autosomally dominant HS asked to make an autologous RBC deposit before his scheduled surgery. His RBCs were serially tested for osmotic fragility during 4 weeks of blood bank storage in CPD with adenine-saline. Aliquots of his fresh and 4-week-old RBCs were also labeled with 51Cr for measurement of in vivo survival. The osmotic fragility of the patient's fresh RBCs was normal, with 50-percent hemolysis at approximately 0.43-percent NaCl and a sigmoid lysis curve. Incubated osmotic fragility (24 hours at 37 degrees C) was abnormal, with 50-percent hemolysis at 0.64-percent NaCl. Median survival of his fresh 51Cr-labeled RBCs was moderately shortened at 17.5 days. Osmotic fragility increased during storage, with 50-percent lysis occurring at 0.58-percent and 0.62-percent NaCl after 2 and 4 weeks, respectively. Volunteer normal donor RBCs exhibited 50-percent lysis in 0.48-percent NaCl after 4 weeks. Median survival of the patient's 4-week-old 51Cr-labeled RBCs was severely shortened, at 3.5 days. At surgery, intraoperatively salvaged RBCs demonstrated osmotic fragility identical to that of the patient's RBCs freshly obtained by venipuncture. CONCLUSION: HS RBCs may lose membrane under optimum storage conditions, becoming unsuitable for transfusion at surgery. Intraoperative autologous transfusion may be preferable to preoperative deposit for avoidance of allogeneic RBC transfusion in patients with HS.

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.

Combination of two mutant alpha spectrin alleles underlies a severe spherocytic hemolytic anemia.

We studied a patient with a severe spherocytic hemolytic anemia without family history of spherocytosis. Analysis of patient's erythrocyte membrane proteins revealed spectrin deficiency and a truncated alpha spectrin protein. We determined that the patient is a compound heterozygote with two mutations in alpha spectrin gene. Mutation in the paternal allele, designated alpha spectrin(PRAGUE), is a transition A to G in the penultimate position of intron 36 that leads to skipping of exon 37, frameshift, and production of the truncated alpha spectrin protein. The maternal allele, designated alpha spectrin(LEPRA), contains transition C-->T in position -99 of intron 30. This mutation enhances an alternative acceptor splice site 70 nucleotides upstream from the regular site. The alternative splicing causes a frameshift and premature termination of translation leading to a significant decrease in alpha spectrin production. The alpha(LEPRA) mutation is linked to a spectrin alphaIIa marker that was found to be associated with recessive or nondominant spectrin-deficient hereditary spherocytosis in approximately 50% of studied families. We conclude that the alpha(LEPRA) mutation combined in trans with the alpha(PRAGUE) mutation underlie the severe hemolytic anemia in the proband. We suggest that allele alpha spectrin(LEPRA) may be frequently involved in pathogenesis of recessive or nondominant spectrin-deficient hereditary spherocytosis.

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

Band 3 is the most abundant integral protein of the red blood cell membrane. It performs two critical biological functions: maintaining ionic homeostasis, by transporting Cl- and HCO3-ions, and providing mechanical stability to the erythroid membrane. Erythroid band 3 (AE1) is one of three anion exchangers that are encoded by separate genes. The AE1 gene is transcribed by two promoters: the upstream promoter produces erythroid band 3, whereas the downstream promoter initiates transcription of the band 3 isoform in kidney. To assess the biological consequences of band 3 deficiency, we have selectively inactivated erythroid but not kidney band 3 by gene targeting in mice. Although no death in utero occurred, the majority of homozygous mice die within two weeks after birth. The erythroid band 3 null mice show retarded growth, spherocytic red blood cell morphology and severe haemolytic anaemia. Remarkably, the band 3-/- red blood cells assembled normal membrane skeleton thus challenging the notion that the presence of band 3 is required for the stable biogenesis of membrane skeleton. The availability of band 3-/- mice offers a unique opportunity to investigate the role of erythroid band 3 in the regulation of membrane-skeletal interactions, anion transport and the invasion and growth of malaria parasite into red blood cells.

Hereditary spherocytosis: a review of the clinical and molecular aspects of the disease.

Hereditary spherocytosis is a common and very heterogeneous hemolytic anemia caused by defects of the red cell membrane proteins. In recent years, major advances in our understanding of the red cell membrane skeleton and a better characterization of its individual components have allowed a brighter insight into the pathogenesis of the disease. In this article, we present an overview of the erythrocyte skeleton and its individual constituents. We also review the clinical aspects of the disease and describe the currently known molecular defects involving the membrane proteins which have been shown to play an essential role in the underlying mechanism of hereditary spherocytosis. Finally we examine several models that have been proposed in an attempt to clarify the mechanism leading from the initial molecular insult to the clinical phenotype.

Reduced invasion and growth of Plasmodium falciparum into elliptocytic red blood cells with a combined deficiency of protein 4.1, glycophorin C, and p55.

In this investigation, we have measured the invasion and growth of the malaria parasite Plasmodium falciparum into elliptocytic red blood cells (RBCs) obtained from subjects with homozygous hereditary elliptocytosis. These elliptocytic RBCs have been previously characterized to possess molecular defects in protein 4.1 and glycophorin C. Our results show that the invasion of Plasmodium falciparum into these protein 4.1 (-) RBCs is significantly reduced. Glycophorin C (-) Leach RBCs were similarly resistant to parasite invasion in vitro. The intracellular development of parasites that invaded protein 4.1 (-) RBCs was also dramatically reduced. In contrast, no such reduction of intracellular parasite growth was observed in the glycophorin C (-) Leach RBCs. In conjunction with our recent finding that a third protein termed p55 is also deficient in protein 4.1 (-) and glycophorin C (-) RBCs, the present data underscore the importance of the membrane-associated ternary complex between protein 4.1, glycophorin C, and p55 during the invasion and growth of malaria parasites into human RBCs.

Hereditary spherocytosis with spectrin deficiency due to an unstable truncated beta spectrin.

Red cell membrane protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and direct quantitation by radioimmunoassay or cytofluorometry defines four distinct subsets of patients with hereditary spherocytosis: Patients with isolated spectrin deficiency, combined spectrin and ankyrin deficiency, band 3 deficiency, and protein 4.2 deficiency. In regard to the first group, only one mutation of beta spectrin has been reported in the literature. We describe a spectrin variant characterized by a truncated beta chain, and associated with hereditary spherocytosis and isolated spectrin deficiency. The clinical phenotype consists of a moderate hemolytic anemia with spherocytosis and frequent spiculation of the red cells. We present the biochemical characteristics of this mutant protein and show that it constitutes only 12% of the total spectrin on the membrane. We show that the truncation of the protein is the result of a single point mutation at position +1 (G-->A) of the donor consensus splice site of intron 17 leading to an aberrant beta spectrin transcriptional message lacking exons 16 and 17. To elucidate the basis for the decreased amount of the truncated protein on the membrane and the overall spectrin deficiency, we provide evidence that the mutated gene is transcribed but its mRNA is less abundant than its normal counterpart in reticulocytes; we also show that the mutant protein is synthesized in decreased amounts in the cytoplasm of erythroid progenitor cells, and appears to be susceptible to proteolytic degradation. This mutant spectrin underscores the importance of the regulatory role played by the beta spectrin molecule in the assembly of alphabeta spectrin heterodimers on the membrane.

Red cell membrane remodeling in sickle cell anemia. Sequestration of membrane lipids and proteins in Heinz bodies.

In red cells from patients with sickle cell anemia, hemoglobin S denatures and forms Heinz bodies. Binding of Heinz bodies to the inner surface of the sickle cell membrane promotes clustering and colocalization of the membrane protein band 3, outer surface-bound autologous IgG and, to some extent, the membrane proteins glycophorin and ankyrin. Loss of transbilayer lipid asymmetry is also found in certain populations of sickle red cells. The lateral distribution of sickle cell membrane lipids has not been examined, however. In this report, we examine by fluorescence microscopy the incorporation and distribution of the fluorescent phospholipid analogues 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-phosphatidylserine and NBD-phosphatidylcholine in sickle red cells. Both phospholipid analogues are observed to accumulate prominently at sites of Heinz bodies. Accumulation at sites of Heinz bodies is also shown by 1,'1-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, a fluorescent lipid analogue that readily crosses membranes, but not by fluorescein-phosphatidylethanolamine, an analogue that is localized to the outer leaflet of the membrane. Double labeling and confocal microscopy techniques show that NBD-lipids, band 3 protein, protein 4.1, ankyrin, and spectrin are all sequestered within sickle red cells and colocalized at sites of Heinz bodies. We propose that Heinz bodies provide a hydrophobic surface on which sickle red cell membrane lipids and proteins are sequestered.

Molecular basis of spectrin deficiency in beta spectrin Durham. A deletion within beta spectrin adjacent to the ankyrin-binding site precludes spectrin attachment to the membrane in hereditary spherocytosis.

We describe a spectrin variant characterized by a truncated beta chain and associated with hereditary spherocytosis. The clinical phenotype consists of a moderate hemolytic anemia with striking spherocytosis and mild spiculation of the red cells. We describe the biochemical characteristics of this truncated protein which constitutes only 10% of the total beta spectrin present on the membrane, resulting in spectrin deficiency. Analysis of reticulocyte cDNA revealed the deletion of exons 22 and 23. We show, using Southern blot analysis, that this truncation results from a 4.6-kb genomic deletion. To elucidate the basis for the decreased amount of the truncated protein on the membrane and the overall spectrin deficiency, we show that (a) the mutated gene is efficiently transcribed and its mRNA abundant in reticulocytes, (b) the mutant protein is normally synthesized in erythroid progenitor cells, (c) the stability of the mutant protein in the cytoplasm of erythroblasts parallels that of the normal beta spectrin, and (d) the abnormal protein is inefficiently incorporated into the membrane of erythroblasts. We conclude that the truncation within the beta spectrin leads to inefficient incorporation of the mutant protein into the skeleton despite its normal synthesis and stability. We postulate that this misincorporation results from conformational changes of the beta spectrin subunit affecting the binding of the abnormal heterodimer to ankyrin, and we provide evidence based on binding assays of recombinant synthetic peptides to inside-out-vesicles to support this model.

Overexpression of AE1 Prague, but not of AE1 SAO, inhibits wild-type AE1 trafficking in Xenopus oocytes.

Mutations in the AE1 (band 3) anion exchanger of human erythrocytes have been associated with altered red cell shape and heritable disease. The Southeast Asian Ovalocytosis (SAO) AE1 mutation, a 27 nt deletion producing the delta 400-408 form of AE1, and the AE1 Prague mutation, a 10 nt insertion producing a frameshift after AE1 aa 821 leading to premature termination, are found only in the heterozygous state. We therefore examined accumulation and function of wt AE1 polypeptide in Xenopus oocytes when coexpressed with AE1 SAO and with AE1 Prague. Our SAO construct lacked the K56E (AE1 Memphis) polymorphism present in the endogenous AE1 SAO protein. Neither mutant AE1 mediated Cl- uptake into cRNA-injected Xenopus oocytes. Coinjection of mutant and wt cRNAs led to dose-dependent inhibition of wt function by AE1 Prague, but not by SAO. Though in vitro translation of the two mutants revealed little difference in their insertion into microsomal membranes, AE1 Prague accumulated in Xenopus oocytes to lower levels than did AE1 SAO or wt. Unlike AE1 SAO polypeptide, AE1 Prague polypeptide was not detectable at the oocyte surface. Moreover, overexpression of AE1 Prague, in contrast to AE1 SAO, reduced the accumulation of wt AE1, at the oocyte surface. This inhibition occurred in the absence of detectable heteromer formation between the AE1 Prague and wt AE1 polypeptides.

Beta spectrin PRAGUE: a truncated beta spectrin producing spectrin deficiency, defective spectrin heterodimer self-association and a phenotype of spherocytic elliptocytosis.

Spherocytic elliptocytosis is a phenotypic hybrid between hereditary spherocytosis (HS) and hereditary elliptocytosis (HE) characterized by the presence of spheroovalocytes and spherocytes which exhibit increased osmotic fragility, indicating a deficiency of surface area. Both the spherocytic red cell morphology and the increased osmotic fragility distinguish this clinical entity from common HE. In contrast to common HE, the molecular basis of spherocytic elliptocytosis is unknown. Here we describe two members of a family who both have the characteristic features of spherocytic HE. We show that the underlying defect involves a G to C transversion at the -1 position of the acceptor splice site upstream of exon X of beta spectrin leading to skipping of exon X from the mutant beta spectrin mRNA allele. The mutant mRNA is present in reticulocytes in similar amounts as the normal mRNA. Pulse-labelling of erythroblasts prepared from peripheral blood in a two-phase liquid-culture system reveals a decreased synthesis of the truncated beta spectrin, a finding which is likely to underlie the moderately severe spectrin deficiency in the two patients. In addition, this mutant spectrin, similar to the previously reported spectrins, is defective in spectrin heterodimer self-association. The spectrin deficiency, which represents a common finding in the majority of patients with HS, together with weakened spectrin heterodimer self-association, as found in the majority of patients with common HE, provides a molecular explanation for the phenotype of spherocytic elliptocytosis in this kindred and, most likely, in other patients carrying similar beta spectrin mutations.

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.

Comparison of the ankyrin (AC)n microsatellites in genomic DNA and mRNA reveals absence of one ankyrin mRNA allele in 20% of patients with hereditary spherocytosis.

Combined deficiency of ankyrin and spectrin represents the most common biochemical abnormality in hereditary spherocytosis (HS). To examine whether a decrease in ankyrin mRNA represents a frequent cause of this type of HS, we took advantage of the reported (AC)n microsatellite polymorphism in the 3' untranslated region of ankyrin cDNA. We first measured the number of AC repeats in genomic DNA encoding erythrocyte ankyrin in 36 unrelated Czech HS patients with combined ankyrin and spectrin deficiency and found 21 of these subjects (58%) to be heterozygotes for the (AC)n microsatellite size. Further analysis of reticulocyte RNA showed that ankyrin cDNA from 7 of these 21 heterozygotes (33%) contained only one of the two ankyrin alleles. We conclude that approximately 1/3 of ankyrin-deficient autosomal dominant HS is caused by reduced expression of one ankyrin allele which, in turn, is caused by either a reduced transcription of one allele of the mutated ankyrin gene or abnormal processing or decreased stability of the mutant ankyrin mRNA. Because ankyrin deficiency is detected in approximately 60% of HS subjects, this result suggests that approximately 20% of all HS is caused by a decreased expression of one ankyrin mRNA allele.