Extraction of erythrocyte membrane proteins by sulfhydryl inhibitors.
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Human red cell membrane proteins were extracted by incubation of the ghost with hypotonic phosphate buffer (pH 7.4), N-ethylmaleimide and p-hydroxy-mercuribenzoate. In paroxysmal nocturnal hemoglobinuria (PNH), hereditary spherocytosis (HS) and hereditary elliptocytosis, the amount of proteins extracted by these procedures was significantly less than the amount extractable from the ghost of normal and aplastic anemia red cells. Polypeptide patterns of red cell membranes in these hematological disorders were essentially similar to those of normal ghosts. Analysis of the supernatant by SDS polyacrylamide gel electrophoresis revealed that this reduction was mainly due to the reduced amount of peripheral proteins extracted. The extraction of peripheral proteins by sulfhydryl reagents was accompanied by shape changes resulting in the formation of membrane vesicles, suggesting an important role of peripheral proteins in the maintenance of ghost shape. It is also suggested that qualitative abnormalities of peripheral proteins such as altered reactivity to sulfhydryl reagents and/or strong binding to the membrane are present in PNH, HS and hereditary elliptocytosis red cells. (+info)
Hereditary spherocytosis and elliptocytosis erythrocytes show a normal transbilayer phospholipid distribution.
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Phosphatidylserine (PS) asymmetry was determined in red blood cells from patients with hereditary spherocytosis and elliptocytosis. No PS-exposing subpopulations were detected using the very sensitive method with fluorescently labeled annexin V. Treatment with N-ethylmaleimide or adenosine triphosphate (ATP) depletion to inactivate the flipase did not lead to formation of PS-exposing subpopulations in these cells, but elevated intracellular calcium levels did lead to extensive scrambling of the PS asymmetry. Although interactions of the membrane skeleton with the phospholipid bilayer have been suggested to stabilize the asymmetric distribution of PS across the bilayer, our data show that red blood cells with a severely damaged membrane skeleton are able to preserve asymmetry, even under conditions in which restoration of the asymmetric distribution is excluded. Moreover, the loss of membrane asymmetry in these cells requires active scrambling involving high levels of intracellular calcium as in normal cells. Our data show that the severe disorder of the membrane skeleton found in these cells does not affect the activity of flipase or scramblase, indicating that these proteins are not regulated by, nor coupled to the membrane skeleton assembly, and that possible thrombotic events in spherocytosis patients are not likely associated with altered PS topology of the red blood cells. (+info)
Prevention of cerebral malaria in children in Papua New Guinea by southeast Asian ovalocytosis band 3.
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Southeast Asian ovalocytosis (SAO) occurs at high frequency in malarious regions of the western Pacific and may afford a survival advantage against malaria. It is caused by a deletion of the erythrocyte membrane band 3 gene and the band 3 protein mediates the cytoadherence of parasitized erythrocytes in vitro. The SAO band 3 variant may prevent cerebral malaria but it exacerbates malaria anemia and may also increase acidosis, a major determinant of mortality in malaria. We undertook a case-control study of children admitted to hospital in a malarious region of Papua New Guinea. The SAO band 3, detected by the polymerase chain reaction, was present in 0 of 68 children with cerebral malaria compared with six (8.8%) of 68 matched community controls (odds ratio = 0, 95% confidence interval = 0-0.85). Median hemoglobin levels were 1.2 g/dl lower in malaria cases with SAO than in controls (P = 0.035) but acidosis was not affected. The remarkable protection that SAO band 3 affords against cerebral malaria may offer a valuable approach to a better understanding of the mechanisms of adherence of parasitized erythrocytes to vascular endothelium, and thus of the pathogenesis of cerebral malaria. (+info)
Autosomal recessive distal renal tubular acidosis associated with Southeast Asian ovalocytosis.
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BACKGROUND: A defect in the anion exchanger 1 (AE1) of the basolateral membrane of type A intercalated cells in the renal collecting duct may result in a failure to maintain a cell-to-lumen H+ gradient, leading to distal renal tubular acidosis (dRTA). Thus, dRTA may occur in Southeast Asian ovalocytosis (SAO), a common AE1 gene abnormality observed in Southeast Asia and Melanesia. Our study investigated whether or not this renal acidification defect exists in individuals with SAO. METHODS: Short and three-day NH4Cl loading tests were performed in 20 individuals with SAO and in two subjects, including their families, with both SAO and dRTA. Mutations of AE1 gene in individuals with SAO and members of the two families were also studied. RESULTS: Renal acidification in the 20 individuals with SAO and in the parents of the two families was normal. However, the two clinically affected individuals with SAO and dRTA had compound heterozygosity of 27 bp deletion in exon 11 and missense mutation G701D resulting from a CGG-->CAG substitution in exon 17 of the AE1 gene. Red cells of the two subjects with dRTA and SAO and the family members with SAO showed an approximate 40% reduction in sulfate influx with normal 4,4'-di-isothiocyanato-stilbene-2,2'-disulfonic acid sensitivity and pH dependence. CONCLUSION: These findings suggest that compound heterozygosity of abnormal AE1 genes causes autosomal recessive dRTA in SAO. (+info)
Elliptocytosis in patients with C-terminal domain mutations of protein 4.1 correlates with encoded messenger RNA levels rather than with alterations in primary protein structure.
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Early biochemical studies defined 4 functional domains of the erythroid protein 4.1 (4.1R). From amino-terminal to carboxy-terminal, these are 30 kd, 16 kd, 10 kd, and 22/24 kd in size. Although the functional properties of both the 30-kd and the 10-kd domain have been demonstrated in red cells, no functional activities have been assigned to either the 16-kd or the 22/24-kd domain in these cells. We here describe new mutations in the sequence encoding the C-terminal 22/24-kd domain that are associated with hereditary elliptocytosis. An unusually mild phenotype observed in heterozygous and homozygous members of 1 family suggested heterogeneity in the pattern of expression of 4.1R deficiency. Using a variety of protein and messenger RNA (mRNA) quantification strategies, we showed that, regardless of the alteration in the C-terminal primary sequence, when the protein is produced, it assembles at the cell membrane. In addition, we found that alterations in red cell morphologic features and membrane function correlate with the amount of membrane-associated protein-and therefore with the amount of mRNA accumulated-rather than with the primary structure of the variant proteins. These data suggest that an intact sequence at exons 19 through 21 encoding part of the C-terminal 22/24-kd region is not required for proper protein 4.1R assembly in mature red cells. (Blood. 2000;95:1834-1841) (+info)
Mild spherocytic hereditary elliptocytosis and altered levels of alpha- and gamma-adducins in beta-adducin-deficient mice.
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The membrane skeleton, a dynamic network of proteins associated with the plasma membrane, determines the shape and mechanical properties of erythrocytes. Deficiencies or defects in membrane skeletal proteins are associated with inherited disorders of erythrocyte morphology and function. Adducin is one of the proteins localized at the spectrin-actin junction of the membrane skeleton. In this work we show that deficiency of beta-adducin produces an 80% decrease of alpha-adducin and a fourfold up-regulation of gamma-adducin in erythrocytes. beta-Adducin or any other isoform generated by translation of abnormally spliced messenger RNAs could not be detected by our antibodies either in ghosts or in cytoplasm of -/- erythrocytes. Actin levels were diminished in mutant mice, suggesting alterations in the actin-spectrin junctional complexes due to the absence of adducin. Elliptocytes, ovalocytes, and occasionally spherocytes were found in the blood film of -/- mice. Hematological values showed an increase in reticulocyte counts and mean corpuscular hemoglobin concentration, decreased mean corpuscular volume and hematocrit, and normal erythrocyte counts that, associated to splenomegaly, indicate that the mice suffer from mild anemia with compensated hemolysis. These modifications are due to a loss of membrane surface and dehydration that result in an increase in the osmotic fragility of red blood cells. The marked alteration in osmotic fragility together with the predominant presence of elliptocytes is reminiscent of the human disorder called spherocytic hereditary elliptocytosis. Our results suggest that the amount of adducin remaining in the mutant animals (presumably alphagamma adducin) could be functional and might account for the mild phenotype. (Blood. 2000;95:3978-3985) (+info)
Band 3 mutations, renal tubular acidosis and South-East Asian ovalocytosis in Malaysia and Papua New Guinea: loss of up to 95% band 3 transport in red cells.
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We describe three mutations of the red-cell anion exchangerband 3 (AE1, SLC4A1) gene associated with distalrenal tubular acidosis (dRTA) in families from Malaysia and Papua NewGuinea: Gly(701)-->Asp (G701D), Ala(858)-->Asp(A858D) and deletion of Val(850) (DeltaV850). The mutationsA858D and DeltaV850 are novel; all three mutations seem to berestricted to South-East Asian populations. South-East Asianovalocytosis (SAO), resulting from the band 3 deletion of residues400-408, occurred in many of the families but did not itselfresult in dRTA. Compound heterozygotes of each of the dRTA mutationswith SAO all had dRTA, evidence of haemolytic anaemia and abnormal red-cell properties. The A858D mutation showed dominant inheritance and therecessive DeltaV850 and G701D mutations showed a pseudo-dominantphenotype when the transport-inactive SAO allele was also present. Red-cell and Xenopus oocyte expression studies showed that theDeltaV850 and A858D mutant proteins have greatly decreased aniontransport when present as compound heterozygotes (DeltaV850/A858D,DeltaV850/SAO or A858D/SAO). Red cells with A858D/SAO had only 3% ofthe SO(4)(2-) efflux of normal cells, thelowest anion transport activity so far reported for human red cells. The results suggest dRTA might arise by a different mechanism for eachmutation. We confirm that the G701D mutant protein has an absoluterequirement for glycophorin A for movement to the cell surface. Wesuggest that the dominant A858D mutant protein is possibly mis-targetedto an inappropriate plasma membrane domain in the renal tubular cell,and that the recessive DeltaV850 mutation might give dRTA because ofits decreased anion transport activity. (+info)
Defective spectrin integrity and neonatal thrombosis in the first mouse model for severe hereditary elliptocytosis.
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Mutations affecting the conversion of spectrin dimers to tetramers result in hereditary elliptocytosis (HE), whereas a deficiency of human erythroid alpha- or beta-spectrin results in hereditary spherocytosis (HS). All spontaneous mutant mice with cytoskeletal deficiencies of spectrin reported to date have HS. Here, the first spontaneous mouse mutant, sph(Dem)/ sph(Dem), with severe HE is described. The sph(Dem) mutation is the insertion of an intracisternal A particle element in intron 10 of the erythroid alpha-spectrin gene. This causes exon skipping, the in-frame deletion of 46 amino acids from repeat 5 of alpha-spectrin and alters spectrin dimer/tetramer stability and osmotic fragility. The disease is more severe in sph(Dem)/sph(Dem) neonates than in alpha-spectrin-deficient mice with HS. Thrombosis and infarction are not, as in the HS mice, limited to adults but occur soon after birth. Genetic background differences that exist between HE and HS mice are suspect, along with red blood cell morphology differences, as modifiers of thrombosis timing. sph(Dem)/sph(Dem) mice provide a unique model for analyzing spectrin dimer- to-tetramer conversion and identifying factors that influence thrombosis. (+info)