The elliptocytoses, ovalocytosis and related disorders.
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Africa↗
Biomedical subjects
J Palek
Publications and source records attributed to J Palek.
A common type of the spectrin alpha I 46-50a-kD peptide abnormality in hereditary elliptocytosis and pyropoikilocytosis is associated with a mutation distant from the proteolytic cleavage site. Evidence for the functional importance of the triple helical model of spectrin.
We studied nine individuals from five unrelated families with alpha I/46-50a hereditary elliptocytosis (HE) or hereditary pyropoikilocytosis (HPP), including one of the original HHP probands first reported by Zarkowsky and colleagues (1975. Br. J. Haematol. 29:537-543). Biochemical analysis of erythrocyte membrane proteins from these patients revealed, as a common abnormality, the presence of the alpha I/46-50a peptide after limited tryptic digestion of spectrin. The polymerase chain reaction was utilized to study the structure of the DNA encoding the alpha I domain of spectrin in the affected individuals. The DNA sequence of the alpha-spectrin gene encoding the region of the alpha-spectrin chain surrounding the abnormal proteolytic cleavage site was normal. We identified a point mutation causing the replacement of a highly conserved leucine residue by proline at position 207 in the alpha-spectrin chain, a site 51 residues to the amino-terminal side of the abnormal proteolytic cleavage site. Analysis of the proposed triple helical model of spectrin repeats reveals that the mutation occurs in helix 2 at a position directly opposite the abnormal proteolytic cleavage site in helix 3, making this the first report of a mutation occurring in helix 2 of a repeat in the alpha I domain of spectrin. These results add to the molecular heterogeneity of mutations associated with HE/HPP and provide further support for the proposed triple helical model of spectrin. Disruption of this proposed alpha-helical structure by helix-breaking proline substitutions may result in a functionally defective spectrin chain.
Amino Acid Sequence↗
Protein immunolocalization in the spread erythrocyte membrane skeleton.
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Ankyrins↗
The expression and synthesis of the band 3 protein initiates the formation of a stable membrane skeleton in murine Rauscher-transformed erythroid cells.
While the temporal sequences of the synthesis and assembly of membrane skeletal proteins has been studied during erythroid maturation, relatively little is known about the events which initiate the assembly of membrane skeleton at the early stages of mammalian erythroid commitment. To investigate the early events that initiate the assembly of the membrane skeleton in mammalian erythroid cells, we have studied the synthesis and assembly of membrane skeletal proteins in murine Rauscher erythroleukemia virus-transformed cells. These cells are blocked in differentiation at around the early progenitor (burst forming unit-erythroid, BFUe) cell stage but can be induced to differentiate in vitro. Pulse-labeling studies reveal that Rauscher cells actively synthesize alpha spectrin, beta spectrin, ankyrin and band 4.1 proteins. However, the synthesis of the band 3 protein and its mRNA are barely detectable in these cells. The peripheral membrane skeletal components assemble only transiently in the membrane skeleton and turn over rapidly, resulting in about 20-fold lower steady state levels than are found in mature erythrocytes. Upon induction with erythropoietin and dimethyl sulfoxide, the mRNA level and synthesis of band 3 are increased about 50-fold. In contrast, the synthesis of spectrin, ankyrin and band 4.1 is increased only about 1.5 to 2.0-fold. However, after induction, the fraction of these proteins assembled on the membrane is increased, their half-lives on the membrane are nearly doubled with a concomitant 4 to 5-fold increase in their steady-state levels. These results suggest that the synthesis of peripheral membrane proteins is detected at the earliest stages of erythroid commitment and increases only slightly during further differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Biogenesis of normal and abnormal red blood cell membrane skeleton.
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Animals↗
Deletion in erythrocyte band 3 gene in malaria-resistant Southeast Asian ovalocytosis.
Southeast Asian ovalocytosis (SAO) is a hereditary condition that is widespread in parts of Southeast Asia. The ovalocytic erythrocytes are rigid and resistant to invasion by various malarial parasites. We have previously found that the underlying defect in SAO involves band 3 protein, the major transmembrane protein, which has abnormal structure and function. We now report two linked mutations in the erythrocyte band 3 gene in SAO: (i) a deletion of codons 400-408 and (ii) a substitution, A----G, in the first base of codon 56 leading to substitution of Lys-56 by Glu-56. The first defect leads to a deletion of nine amino acids in the boundary of cytoplasmic and membrane domains of band 3. This defect has been detected in all 30 ovalocytic subjects from Malaysia, the Philippines, and two unrelated coastal regions of Papua New Guinea, whereas it was absent in all 30 controls from Southeast Asia and 20 subjects of different ethnic origin from the United States. The Lys-56----Glu substitution has likewise been found in all SAO subjects. However, it has also been detected in 5 of the 50 control subjects, suggesting that it represents a linked polymorphism. We conclude that the deletion of codons 400-408 in the band 3 gene constitutes the underlying molecular defect in SAO.
Anion Exchange Protein 1, Erythrocyte↗
Erythropoietin enhances the assembly of alpha,beta spectrin heterodimers on the murine erythroblast membranes by increasing beta spectrin synthesis.
Previous biosynthetic studies have revealed that in both mammalian and chicken erythroid cells, alpha spectrin is synthesized in 2-3-fold excess over beta spectrin. However, in the membrane skeleton, the two polypeptides are assembled in equimolar amounts, suggesting that the association of alpha spectrin with the membrane skeleton is rate-limited by the amount of beta spectrin synthesized. Here we have analyzed the synthesis and transcription of alpha and beta spectrin in Friend virus-infected murine erythroblasts (FVA cells) in vitro during the erythropoietin-dependent period of maturation. Erythropoietin (Epo) increases the synthesis of beta spectrin but not alpha spectrin, thereby altering the alpha/beta spectrin synthetic ratio. When immature FVA cells are exposed for 24 h to a standard dose of 0.2 unit/ml Epo for maximum effect, the mRNA content and the synthesis of beta spectrin are increased resulting in about 1.3-1.5-fold excess of beta spectrin over alpha spectrin in the cytosol. On the membrane, the incorporation of both alpha and beta spectrins is increased equally by about 30-35%. Furthermore, nuclear run-off transcription measurements indicate that the increased beta spectrin gene expression is regulated at the level of transcription. We conclude that in cells with strong Epo stimulus, the expression and synthesis of beta spectrin are increased resulting in an increase in the amount of alpha,beta spectrin heterodimers available for membrane assembly.
Animals↗
Uncoupling of the spectrin-based skeleton from the lipid bilayer in sickled red cells.
The distribution of spectrin and band 3 in deoxygenated reversibly sickled cells was visualized by immunofluorescence and immunoelectron microscopy. Antibodies against band 3, the major lipid-associated transmembrane protein, labeled the entire cell body, including the entire length of the long protruding spicule, whereas antibodies against spectrin labeled only the cell body and the base region of the spicules. The results suggest that the formation of long spicules during sickling is associated with a continuous polymerization of hemoglobin S polymers, presumably through gaps in the spectrin-actin meshwork, and a subsequent uncoupling of the lipid bilayer from the submembrane skeleton.
Anemia, Sickle Cell↗
Molecular basis of spectrin and ankyrin deficiencies in severe hereditary spherocytosis: evidence implicating a primary defect of ankyrin.
While varying degrees of spectrin deficiency have been found in the majority of patients with hereditary spherocytosis (HS), a combined severe deficiency of both spectrin and the spectrin-binding protein, ankyrin, has been reported only in two patients with severe HS. To elucidate the molecular basis of these protein deficiencies, we have studied the synthesis, assembly, and the mRNA levels of spectrin and ankyrin in peripheral blood reticulocytes in one of the previously reported probands. Pulse-labeling studies showed that in HS reticulocytes, the synthesis of alpha-spectrin was comparable with control reticulocytes while that of beta-spectrin was increased about fourfold, presumably reflecting increased erythropoietic drive. On the HS reticulocyte membrane, the amount of newly assembled spectrin was reduced to about half of the control values, presumably reflecting a decrease in the synthesis of the spectrin binding protein, ankyrin: the ankyrin synthesis was nearly absent in the cytosol and the amounts of membrane-associated ankyrin were reduced to about half of the normal values. The changes in the amounts of spectrin and ankyrin mRNAs quantitated by slot blot and Northern blot analyses were comparable with changes in the synthesis of these proteins: The alpha spectrin mRNA was within a control range and the beta-spectrin mRNA was slightly increased, while the amounts of ankyrin mRNA were reduced to about 50% of control values. We conclude that the primary defect underlying the combined spectrin and ankyrin deficiency is a deficiency of ankyrin mRNA leading to a reduced synthesis of ankyrin which, in turn, underlies the decreased assembly of spectrin on the membrane.
Adult↗
Spinal cord compression secondary to extramedullary hematopoiesis: a noninvasive management based on MRI.
We report the first case of spinal cord compression secondary to extramedullary hematopoiesis in which the diagnosis was made by magnetic resonance imaging (MRI). Based on the great advantage of this imaging test that visualizes the entire spine, the noninvasive approach to such patients, using low-dose radiation therapy and guided by MRI is discussed. This management appears to be both efficacious and safe.
Adult↗
Four different mutations in codon 28 of alpha spectrin are associated with structurally and functionally abnormal spectrin alpha I/74 in hereditary elliptocytosis.
Hereditary elliptocytosis (HE) Sp alpha I/74 is a disorder associated with defective spectrin (Sp) heterodimer self-association and an abnormal tryptic cleavage of the 80-kD alpha I domain of Sp resulting in increased amounts of a 74-kD peptide. The molecular basis of this disorder is heterogeneous and mutations in codons 28, 46, 48, and 49 (codons 22, 40, 42, and 43 in the previous nomenclature which did not include the six NH2-terminal amino acids) have been reported. In this study we present data on seven unrelated HE Sp alpha I/74 kindred from diverse racial backgrounds in whom we identified four different mutations all occurring in exon 2 of alpha Sp at codon 28. Utilizing the polymerase chain reaction we established a CGT----CTT; Arg----Leu 28 mutation in one kindred of Arab/Druze origin. In two unrelated white kindred of English/European origin the substitution is CGT----AGT; Arg----Ser 28 and in two apparently unrelated white kindred from New Zealand, the mutation is CGT----TGT; Arg----Cys 28. Finally, in one American black kindred and in a black kindred from Ghana the mutation involves CGT----CAT; Arg----His 28. Allele specific oligonucleotide hybridization confirmed that the probands are heterozygous for the respective mutant alleles. All four point mutations abolished an Aha II restriction enzyme site which allowed verification of linkage of the mutation with HE Sp alpha I/74. Our results imply that codon 28 of alpha Sp is a "hot spot" for mutations and also indicate that Arg 28 is critical for the conformational stability and functional self association of Sp heterodimers.
Base Sequence↗
Molecular defect of the band 3 protein in southeast Asian ovalocytosis.
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.
Anion Exchange Protein 1, Erythrocyte↗
Effect of hemoglobin oxidation products on the stability of red cell membrane skeletons and the associations of skeletal proteins: correlation with a release of hemin.
Oxidative injury to hemoglobin (Hb) leads to formation of methemoglobin (MetHb), reversible hemichromes (rHCRs), and irreversible hemichromes (iHCRs). iHCRs precipitate and form Heinz bodies that attach to the red cell membrane causing injury that leads to hemolysis. The molecular mechanisms of this membrane damage have not been fully elucidated. We have studied the effect of Hb oxidation products on the mechanical stability of red cell membrane skeletons and the associations of membrane skeletal proteins. Hb and MetHb stabilized the isolated membrane skeletons, whereas further oxidation to rHCRs abolished this stabilizing effect. Crude iHCRs prepared by phenylhydrazine oxidation of Hb destabilized membrane skeletons by decreasing formation of the spectrin-protein 4.1-actin complex, the effect similar to that of pure hemin. Whereas virtually no hemin was released from Hb and MetHb, high concentrations of hemin were released from crude iHCR preparations. After removal of this hemin fraction by Dowex resin, the iHCRs lost their destabilizing effect. We conclude that as the oxidation of Hb proceeds, the stabilizing effect of Hb on the membrane skeleton is gradually lost and the deleterious effect increases. The destabilization of the red cell membrane skeleton in the presence of crude iHCR is caused by release of hemin, which lowers the stability of membrane skeleton by weakening the spectrin-protein 4.1-actin interaction.
Actins↗
Alteration of the erythrocyte membrane skeletal ultrastructure in hereditary spherocytosis, hereditary elliptocytosis, and pyropoikilocytosis.
The membrane skeleton of normal erythrocytes is largely organized into a hexagonal lattice of junctional complexes (JC) crosslinked by spectrin tetramers, and occasional double tetramers and hexamers. To explore possible skeletal alterations in hereditary spherocytosis (HS), elliptocytosis (HE), and pyropoikilocytosis (HPP), we have studied the ultrastructure of the spread membrane skeletons from a subpopulation of HS patients with a partial spectrin deficiency ranging from 43% to 86% of normal levels, and in patients with HPP who, in addition to a mild spectrin deficiency, also carried a mutant spectrin that was dysfunctional, thus reducing the ability of spectrin dimers to assemble into tetramers. Membrane skeletons derived from Triton-treated erythrocyte ghosts were examined by negative staining electron microscopy. HS membrane skeletons contained structural elements, consisting of JC and spectrin filaments similar to the normal skeleton. However, less spectrin filaments interconnected the JC, and the decrease of spectrin filaments attached to JC appeared to correlate with the severity of spectrin deficiency. Only in severe HS associated with severe spectrin deficiency was the loss of spectrin sufficient enough to disrupt the overall skeletal architecture. In contrast, membrane skeletons prepared from red blood cells (RBCs) of subjects with HPP were strikingly different from HS RBCs with a comparable degree of spectrin deficiency. Although HPP RBCs were only mildly deficient in spectrin, their skeletal lattice was grossly disrupted, in contrast to only mild ultrastructural abnormalities of HS membrane skeletons with a nearly identical degree of spectrin deficiency. Skeletons from patients with common mild HE or asymptomatic carriers, carrying the mutant spectrin but having normal spectrin content, exhibited a moderate disruption of the skeletal lattice. We propose that the above differences in skeletal ultrastructure may underlie differences in the biomechanical properties and morphology of HS, HE, and HPP RBCs.
Cytoskeleton↗
Structural and functional heterogeneity of alpha spectrin mutations involving the spectrin heterodimer self-association site: relationships to hematologic expression of homozygous hereditary elliptocytosis and hereditary pyropoikilocytosis.
Defects involving alpha spectrin (Sp) are found in patients with hereditary elliptocytosis and a related disorder, hereditary pyropoikilocytosis (HPP). We have previously found that the severity of hemolysis was related to the total spectrin content of the cells and the percentage of unassembled dimeric Sp (SpD) in the membranes, which, in turn, reflected the amount of mutant Sp in the cell. However, no data are available comparing differences in the function of various alpha Sp mutations to clinical severity. We now report studies of nine homozygotes or double heterozygotes for four alpha Sp mutations: alpha 1/74, alpha 1/46, alpha 1/65, and alpha 1/61, whose red blood cells (RBCs) contained only the mutant Sp and no normal Sp. Sp alpha 1/74, Sp alpha 1/46, and alpha 1/65 homozygotes differed strikingly in the severity of hemolysis that correlated with the severity of mutant Sp dysfunction, as reflected by the fraction of unassembled SpD in the membranes and the self-association of mutant Sp on inside-out vesicles. Homozygotes for Sp alpha 1/74 had a very severe hemolytic anemia and their SpD were virtually incapable of self-association, whereas SpD alpha 1/46 were not as severely affected. The Sp alpha 1/65 homozygotes had a relatively mild hemolytic anemia and their SpD showed the least impairment of function. Ultrastructural examination of membrane skeletons from subjects whose SpD self-association was severely impaired showed gross skeletal disruption and loss of hexagonal structure. In striking contrast, the homozygote for the mildly dysfunctional Sp alpha 1/65 had only a moderate disruption of the skeleton. Some of the homozygous or doubly heterozygous subjects also exhibited a partial deficiency of Sp that correlated with a RBC morphology characteristic of HPP, namely, marked microspherocytosis with virtual absence of elliptocytes. These data demonstrate striking differences in the function and structure of various alpha Sp mutants that underlie differences in clinical expression.
Adolescent↗
cDNA sequence for human erythrocyte ankyrin.
The cDNA for human erythrocyte ankyrin has been isolated from a series of overlapping clones obtained from a reticulocyte cDNA library. The composite cDNA sequence has a large open reading frame of 5636 base pairs (bp) with the complete coding sequence for a polypeptide of 1879 amino acids with a predicted molecular mass of 206 kDa. The derived amino acid sequence contained 194 residues that were identical to those obtained by direct amino acid sequencing of 11 ankyrin proteolytic peptides. The primary sequence contained 23 highly homologous repeat units of 33 amino acids within the 90-kDa band 3 binding domain. Two cDNA clones showed evidence of apparent mRNA processing, resulting in the deletions of 486 bp and 135 bp, respectively. The 486-bp deletion resulted in the removal of a 16-kDa highly acidic peptide, and the smaller deletion had the effect of altering the COOH terminus of the molecule. Radiolabeled ankyrin cDNAs recognized two erythroid message sizes by RNA blot analysis, one of which was predominantly associated with early erythroid cell types. An ankyrin message was also observed in RNA from the human cerebellum by the same method. The ankyrin gene is assigned to chromosome 8 using genomic DNA from a panel of sorted human chromosomes.
Amino Acid Sequence↗
Genetics of the red cell membrane skeleton.
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Animals↗
Severe hemolysis and red cell fragmentation caused by the combination of a spectrin mutation with a thrombotic microangiopathy.
Two patients are described who presented with severe hemolysis and erythrocyte fragmentation. One patient had renal allograft rejection and disseminated intravascular coagulation, and the other had thrombotic thrombocytopenia purpura. The severity of hemolysis and the red cell abnormalities were considerably more profound than usually seen in patients with thrombotic microangiopathies. After evaluation of blood smears prepared before the onset of the disease and biochemical characterization of proteins of the red blood cell skeleton, a mutation of the skeletal protein spectrin, designated Sp alpha l/65, was identified. In the heterozygous form, this mutation manifests as mild, often asymptomatic, hereditary elliptocytosis. We conclude that in these two patients with thrombotic microangiopathy, the intrinsic red cell membrane instability resulting from the underlying skeletal defect aggravated the mechanical red cell fragmentation, producing morphological features similar to the severe hemolytic form of hereditary elliptocytosis or hereditary pyropoikilocytosis.
Adolescent↗