Topological Structures and Membrane Nanostructures of Erythrocytes after Splenectomy in Hereditary Spherocytosis Patients via Atomic Force Microscopy

Hereditary spherocytosis is an inherited red blood cell membrane disorder resulting from mutations of genes encoding erythrocyte membrane and cytoskeletal proteins. Few equipments can observe the structural characteristics of hereditary spherocytosis directly expect for atomic force microscopy In our study, we proved atomic force microscopy is a powerful and sensitive instrument to describe the characteristics of hereditary spherocytosis. Erythrocytes from hereditary spherocytosis patients were small spheroidal, lacking a well-organized lattice on the cell membrane, with smaller cell surface particles and had reduced valley to peak distance and average cell membrane roughness vs. those from healthy individuals. These observations indicated defects in the certain cell membrane structural proteins such as α- and β-spectrin, ankyrin, etc. Until now, splenectomy is still the most effective treatment for symptoms relief for hereditary spherocytosis. In this study, we further solved the mysteries of membrane nanostructure changes of erythrocytes before and after splenectomy in hereditary spherocytosis by atomic force microscopy. After splenectomy, the cells were larger, but still spheroidal-shaped. The membrane ultrastructure was disorganized and characterized by a reduced surface particle size and lower than normal Ra values. These observations indicated that although splenectomy can effectively relieve the symptoms of hereditary spherocytosis, it has little effect on correction of cytoskeletal membrane defects of hereditary spherocytosis. We concluded that atomic force microscopy is a powerful tool to investigate the pathophysiological mechanisms of hereditary spherocytosis and to monitor treatment efficacy in clinical practices. To the best of our knowledge, this is the first report to study hereditary spherocytosis with atomic force microscopy and offers important mechanistic insight into the underlying role of splenectomy.     

Studies on protein kinase activity and the binding of adenosine 3'5-monophosphate by membranes of hereditary spherocytosis erythrocytes.

Evidence has been presented recently of a deficiency of an endogenous membrane-associated protein kinase in erythrocytes of patients with hereditary spherocytosis (HS). We have measured endogenous protein kinase activity in erythrocyte membranes of 4 HS subjects using different membrane isolation and reaction conditions and find that the phosphorylation of the spectrin component (mean ± S.E. 17.1 ± 1.2 pmoles/10 mins per mg protein) is not significantly different to that of 4 normal controls (mean ± S.E. 20.7 ± 1.1 pmoles/10 mins per mg protein). Phosphorylation of exogenous proteins such as casein and protamine is also not deficient in HS erythrocyte membranes. Adenosine 3′5-monophosphate (cyclic AMP) binding to normal and HS erythrocyte membranes was also studied using a Millipore filtration assay. The affinity of cyclic AMP for erythrocyte membranes as determined by Hill plots of binding data from 4 HS subjects (K_D mean ± S.E. = 2.2 ± 0.2 nM) was not significantly different to 4 normal controls (K_D mean ± S.E. = 2.8 ± 0.6 nM). The rate of dissociation of bound cyclic AMP from HS membranes was also similar to control membranes. We thus cannot confirm the prediction by others that an abnormality of cyclic AMP interaction with the erythrocyte membrane underlies HS.     

Saturation transfer EPR studies of membrane alteration in hereditary spherocytosis

Our earlier spin label electron paramagnetic resonance (EPR) studies of hereditary spherocytosis (HS) erythrocyte revealed the existence of structural alteration(s) when the membrane is subjected to heat stress. We have now used saturation transfer EPR to show restricted motion in membrane proteins even without subjecting membrane to stress. The abnormal motional behavior is amplified when membranes are incubated at 47 degrees C and is readily detectable by conventional EPR. Gel electrophoresis and lipid assays show that proteins but not lipids are released upon heating. Thus, the more restricted motions in HS membranes may be due to a different membrane protein organization, ultimately resulting in the abnormal morphology of HS cells.     

Abnormal binding of spectrin to the membrane of erythrocytes in some cases of hereditary spherocytosis

In two cases of hereditary spherocytosis that we have examined, spectrin was bound abnormally tightly to the erythrocyte membrane, and could not be released by low ionic strength dialysis. This type of behaviour occurs in normal red cells only after heating above 50 degrees C. It appears that some cases of spherocytosis may be due to the presence of a protein which is abnormally temperature sensitive.     

Assessment of erythrocyte deformability with the laser-assisted optical rotational cell analyzer (LORCA)

The erythrocyte deformability of 28 patients with anemia was evaluated with the laser-assisted optical rotational cell analyzer (LORCA), an image analyzer that converts into numerical form the degree of refraction of a laser beam induced by red cells subjected to a range of torsional stresses. The patients were 10 thalassemics, including three with intermediate forms (1 HbC/beta degree, 1 homozygote beta for Orkin's haplotype VI, 1 beta degree/beta delta Sicilian type) and seven heteroygotes for beta Th; six with hereditary spherocytosis (including 2 with structural alteration of the spectrin beta chain); three with type II congenital dyserythropoietic anemia (HEMPAS), two hemizygotes and one heterozygote for G-6PD deficiency, and six with severe hypochromic hyposideremic anemia. Red cell deformability was reduced in intermediate thalassemia, hereditary spherocytosis and HEMPAS, normal in heterozygous beta thalassemia and G-6PD deficiency, and increased in hypochromic hyposideremic anemia. These results show that erythrocyte deformability can be impaired by an Hb chain imbalance, membrane and cyto skeleton structure anomalies and changes in the red cell area/volume ratio.     

The abnormal phosphorylation of spectrin in human hereditary spherocytosis

The phosphorylation of the proteins of the erythrocyte membrane of patients suffering from hereditary spherocytosis is investigated in intact erythrocytes by their incubation in the presence of radioactive inorganic phosphate. Examination of the phosphorylated components by high-resolution two-dimensional gel electrophoresis reveals only one defect in the pathological membranes, a depressed phosphorylation of the smaller polypeptide of spectrin; band 2. The phosphorylation of band 2 is measured with reference to the phosphorylation of syndein ($\text{2.1 + 2.2 + 2.3}$). In patients showing overt clinical symptoms and for whom splenectomy is advocated the phosphorylation of band 2 is depressed by approx. 70%. After splenectomy the phosphorylation of membrane proteins is restored to normal levels.     

Capillary gel electrophoresis: separation of major erythrocyte membrane proteins

A new separation method of human erythrocyte membrane proteins by sodium dodecyl sulfate capillary gel electrophoresis (SDS–CGE) is described. In this method, a replaceable gel matrix was used. Seven major erythrocyte membrane proteins, α-and β-spectrin, ankyrin 2.1, band 3 (anion-exchanger), 4.1a and b, and 4.2 (pallidin), were separated and identified by SDS–CGE method. High reproducible migration times of these proteins (inter-assay coefficients of variation less than 2%), as well as quantification (inter-assay coefficients of variation less than 11%) were obtained. This new SDS–CGE method may provide important diagnostic evidence for hereditary spherocytosis. It can be a powerful diagnostic tool in place of SDS polyacrylamide gel electrophoresis for erythrocyte membrane protein analysis.     

Increased methyl esterification of altered aspartyl residues in erythrocyte membrane proteins in response to oxidative stress.

Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT; EC 2. 1.1.77) catalyses the methyl esterification of the free alpha-carboxyl group of abnormal L-isoaspartyl residues, which occur spontaneously in protein and peptide substrates as a consequence of molecular ageing. The biological function of this transmethylation reaction is related to the repair or degradation of age-damaged proteins. Methyl ester formation in erythrocyte membrane proteins has also been used as a marker reaction to tag these abnormal residues and to monitor their increase associated with erythrocyte ageing diseases, such as hereditary spherocytosis, or cell stress (thermal or osmotic) conditions. The study shows that levels of L-isoaspartyl residues rise in membrane proteins of human erythrocytes exposed to oxidative stress, induced by t-butyl hydroperoxide or H2O2. The increase in malondialdehyde content confirmed that the cell membrane is a primary target of oxidative alterations. A parallel rise in the methaemoglobin content indicates that proteins are heavily affected by the molecular alterations induced by oxidative treatments in erythrocytes. Antioxidants largely prevented the increase in membrane protein methylation, underscoring the specificity of the effect. Conversely, we found that PCMT activity, consistent with its repair function, remained remarkably stable under oxidative conditions, while damaged membrane protein substrates increased significantly. The latter include ankyrin, band 4.1 and 4.2, and the integral membrane protein band 3 (the anion exchanger). The main target was found to be particularly protein 4.1, a crucial element in the maintenance of membrane-cytoskeleton network stability. We conclude that the increased formation/exposure of L-isoaspartyl residues is one of the major structural alterations occurring in erythrocyte membrane proteins as a result of an oxidative stress event. In the light of these and previous findings, the occurrence of isoaspartyl sites in membrane proteins as a key event in erythrocyte spleen conditioning and hemocatheresis is proposed.     

Biochemical studies on abnormal erythrocyte membranes protein abnormality of erythrocyte membrane in biliary obstruction

Biochemical studies on erythrocyte membranes from eleven obstructive jaundice patients (due to various disorders) have been undertaken. By scanning electron microscopic observation these erythrocytes were spur and target in appearance. The lipid composition showed a marked increase in both cholesterol and phosphatidylcholine. In addition to these changes, it was unexpectedly demonstrated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate that a specific membrane protein component 4.2 was reduced or absent in all cases tested. This membrane protein abnormality was identical with that of hereditary spherocytosis erythrocyte membranes. It is of particular interest to note that after surgical relief of biliary obstruction in a typical case of common duct cholelithiasis, the disc electrophoretic pattern of erythrocyte membranes became normal and both lipid composition and red cell morphology returned to normal.     

Eryptosis in hereditary spherocytosis and thalassemia: role of glycoconjugates

The present work is aimed to study the mechanism of faster erythrocyte clearance in hereditary spherocytosis (HS), a heterogeneous disorders characterized by alterations in the proteins of the red cell membrane skeleton along with different kinds of thalassemia. The maximum exposure of phosphatidylserine (PS) is found in HS compared to those in both α- and β-thalassemia. Interestingly, in HS more PS exposed cells were found in younger erythrocytes compared to normal and the thalassemics where aged cells showed higher loss of PS asymmetry. Loss of sialic acid and GlcNAc bearing glycoconjugates, presumably the glycophorins, was also found upon aging. The loss of PS asymmetry together with the cell surface glycoproteins mediated by membrane vesiculation, seemed to play key role in early clearance of erythrocytes from circulation following a mechanism similar to HbEβ-thalassemia.     

Membrane proteins in hereditary spherocytosis, elliptocytosis and stomatocytosis

Cell membrane proteins of patients with hereditary spherocytosis, elliptocytosis and stomatocytosis were analyzed by SDS acrylamide gel electrophoresis. From 7 patients with hereditary spherocytosis only one can be supposed to have a reduction of a band with a molecular weight of about 80,000 daltons. 3 members of a family with hereditary elliptocytosis and 1 patient with hereditary stomatocytosis did not differ from the donors. The question of the nature of membrane defect in haemolytic anaemias could not be answered by the results obtained.     

Erythrocyte membrane deformability and stability: two distinct membrane properties that are independently regulated by skeletal protein associations

Skeletal proteins play an important role in determining erythrocyte membrane biophysical properties. To study whether membrane deformability and stability are regulated by the same or different skeletal protein interactions, we measured these two properties, by means of ektacytometry, in biochemically perturbed normal membranes and in membranes from individuals with known erythrocyte abnormalities. Treatment with 2,3-diphosphoglycerate resulted in membranes with decreased deformability and decreased stability, whereas treatment with diamide produced decreased deformability but increased stability. N-ethylmaleimide induced time-dependent changes in membrane stability. Over the first minute, the stability increased; but with continued incubation, the membranes became less stable than control. Meanwhile, the deformability of these membranes decreased with no time dependence. Biophysical measurements were also carried out on pathologic erythrocytes. Membranes from an individual with hereditary spherocytosis and a defined abnormality in spectrin-protein 4.1 association showed decreased stability but normal deformability. In a family with hereditary elliptocytosis and an abnormality in spectrin self-association, the membranes had decreased deformability and stability. Finally, membranes from several individuals with Malaysian ovalocytosis had decreased deformability but increased stability. Our data from both pathologic membranes and biochemically perturbed membranes show that deformability and stability change with no fixed relationship to one another. These findings imply that different skeletal protein interactions regulate membrane deformability and stability. In light of these data, we propose a model of the role of skeletal protein interactions in deformability and stability.     

Relative deficiency of Ca2 plus-dependent adenosine triphosphatase activity of red cell membranes in hereditary spherocytosis

The Ca²⁺-dependent adenosine triphosphatase activity associated with the plasma membrane of normal human erythrocytes is similar to that of erythrocytes from patients with hereditary spherocytosis. When spherocytic ghosts are compared to age-matched controls, however, they show a significantly decreased Ca²⁺-dependent adenosine triphosphatase activity. The role of the relative deficiency of Ca²⁺-dependent adenosine triphosphatase in spherocytic ghosts is discussed in the light of the effects of intracellular [Ca²⁺] on the deformability and the rigidity of the cell membrane. This enzyme may be involved in the molecular mechanism of hereditary spherocytosis.     

Effects of centrifugation on transmembrane water loss from normal and pathologic erythrocytes

Plasma 125I-albumin was used as a marker of extracellular dilution in order to study the effect of high-speed centrifugation on transmembrane water distribution in several types of human red cells, including normal (AA), hemoglobin variants (beta A, AS, SC, beta S, and SS), and those from patients with hereditary spherocytosis. SS and AA erythrocytes were also examined for changes in intracellular hemoglobin concentration of three different density fractions and with increasing duration of spin. The minimum force and duration of centrifugation required to impair water permeability were found to vary with the red cell type, the anticoagulant used (heparin or EDTA), the initial hematocrit of the sample centrifuged, as well as among the individual erythrocyte fractions within the same sample. When subjecting pathologic erythrocytes to high-speed centrifugation, the 125I-albumin dilution technique can be used to determine whether the centrifugation procedure has led to an artifactual red cell water loss and to correct for this when it does occur. An abnormal membrane susceptibility to mechanical stress was demonstrated in erythrocytes from patients with hereditary spherocytosis and several hemoglobinopathies.     

Polymorphism of the erythrocyte band 3 gene (EPB3) in ethnic groups of Costa Rica

Band 3 (AE1) is one of the most abundant proteins in the membrane of the human erythrocyte. This protein works as an anionic CI - and HCO3- exchanger and it also functions as an anchor for several proteins of the erythrocyte's cytoesqueleton. Several mutations have been described and many polymorphic variants have been associated to hereditary spherocytosis. The identification of a genetic marker at position 5' of the AEl gene could be associated to several molecular defects of the erythrocyte. This genetic marker is a restriction fragment length polymorphism (RFLP) produced by restriction enzime Pst I. For this polymorphism a total of 216 individuals belonging to seven different populations were analyzed: one from the Central Valley, two African descendants (Lim6n and Guanacaste) and four Amerindians (Bribri, Cabecar, Maleku and Guaymi). The most frequent allele in the Amerindian population was no 1. No significant differences were found with respect to the Hardy-Weinberg equilibrium in six of the populations, although the Guaymi group does present significative differences     

The effect of cryogenic storage on human erythrocyte membrane proteins as determined by polyacrylamide-gel electrophoresis

A study was conducted to determine the effects of freezing on the major membrane proteins of isolated human erythrocyte membranes. Membranes in low or normal ionic strength medium were frozen at slow or fast freezing rates. The membrane protein composition and elution of proteins from the membranes were studied utilizing polyacrylamide-gel electrophoresis in a sodium dodecyl sulfate or an acetic acid-urea-phenol solvent system. Neither a change in the composition of the membrane proteins nor any elution of membrane protein during freezing and thawing was observed. The data indicate that any human erythrocyte membrane damage during freezing and thawing was not related to a change in major membrane protein composition. Human red cell membranes were stable at ?80 or ?196 °C in the absence of a cryoprotective agent.     

A spin-label study of membrane proteins and internal microviscosity of erythrocytes in hereditary spherocytosis

Electron spin resonance (ESR) spectra of erythrocyte membranes of patients with hereditary spherocytosis (HS) and of healthy controls labeled with a maleimide spin label did not differ significantly both before and after prolonged incubation at 37 degrees C. It suggests that the different behavior of spin-labeled HS erythrocyte membranes upon incubation at a higher temperature reported previously is due indeed to structural abnormalities of HS red cell membranes and not to alterations in their proteolytic activity. Measurements of the rotational correlation time of Tempamine spin probe demonstrated a significant elevation of internal microviscosity of erythrocytes in HS, more pronounced in non-splenectomized patients.     

Sphingomyelin phase transition in the sheep erythrocyte membrane

The dependence of membrane dynamics on the mole ratio of lecithin to sphingomyelin (L/S) was examined by the fluorescence depolarization of the fluidity probe DPH in membranes isolated from sheep and human erythrocytes. In these membranes L/S is the main variable of lipid composition (0.02 and 1.7, respectively). The sheep erythrocyte membrane, which is rich in sphingomyelin, displays a higher lipid microviscosity than the human erythrocyte membrane in addition to a broad gel/liquid-crystal phase transition in the range of 26–35°C. Single-walled lipid vesicles of high sphingomyelin content, when studied by the same technique, exhibited dynamic characteristics similar to those found in the sheep erythrocyte membrane. Both the apparent microviscosity and the transition temperature decreased with increasing the L/S. Membrane proteins of human and sheep erythrocytes were fluorescently labeled with the sulfhydryl reagent N-dansylaziridine and the emission spectrum was recorded as a function of temperature. In the human erythrocyte membranes a gradual increase in the ratio of emission maxima at 520 and 490 nm was observed between 6 and 40°C. At this temperature range the ratio of the above emission maxima in sheep erythrocyte membranes displayed a break between 20 and 28°C, which partially overlapped the phase transition observed for the lipid core. The effect of the lipid phase transition on membrane proteins for the lipid core. The effect of the lipid phase transition on membrane proteins was further assessed by comparing the activity of the membrane bound phospholipase A2 in the intact and detergent-solubilized sheep erythrocyte membranes. Below 31°C the lipids suppress the enzyme activity by about 90%, whereas above this temperature this suppression is progressively abolished.     

Protein-dependent lipid lateral phase separation as a mechanism of human erythrocyte ghost resealing

The hypothesis of a correlation between a 10°–20°C lipid phase transition and the resealing process of human erythrocyte membrane has been investigated. The conditions required to reseal human erythrocyte ghosts have been studied by measuring the amount of fluorescein-labeled dextran (FD) that is trapped into the membrane. Temperature per se was sufficient to induce membrane resealing: (1) at 5 mM sodium phosphate, pH 7.8 (5P8), resealing began at 12°C; (2) at salt concentrations above 8 mM sodium phosphate, it occurred at lower temperature; and (3) in isotonic saline was detected just above 5°C. The removal of peripheral membrane proteins from unsealed membranes by chymotrypsin at 0°C in 5P8 was followed by membrane resealing. This seems to imply that the presence of proteins is necessary to maintain the membrane unsealed. Protein-induced lateral phase separation of lipids may be a reasonable mechanism for the observed phenomena. In fact, the permeability of phosphatidylserine-phosphatidylcholine mixed liposomes to FD is modified by lipid lateral phase separation induced by pH or poly-L-lysine. Electron spin resonance studies of membrane fluidity by a spin labeled stearic acid showed a fluidity break around 11°C, which may be due to a gel–liquid phase transition. Fluidity changes are abolished by chymotrypsin treatment. It is suggested that a lateral phase separation is responsible for the permeability of open ghosts to FD. Accordingly, disruption of phase separation apparently produces membrane reconstitution. In this respect peripheral proteins and particularly the spectrin-actin network, may play a major role in membrane resealing.     

Erythrocyte membrane defects in hemolytic anemias found through derivative thermal analysis of electric impedance

Hereditary hemolytic anemias originate mainly from defects in hemoglobin and plasma membrane proteins. Here, we propose a new method, thermal analysis of impedance, sensitive to membrane defects. It detects three processes in erythrocyte membrane; fall in membrane capacity at 49.5 °C and activation of passive PO₄²⁺ permeability at 37 °C and inorganic ions at 61.5 °C. The denaturation of spectrin is involved in the first process whilst the anion channel is involved in latter processes. Using this method three persons with xerocytosis were found whereby the fall in membrane capacity and spherization of erythrocytes were both postponed (53 °C) compared to control (49.5 °C). In contrast to control cells, strong activation of passive permeability for Cl⁻ at 37 °C and sucrose at 61 °C were detected that were both eliminated by pre-inhibition of the anion channel with 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid (DIDS). In addition, erythrocytes from 15 patients with various forms of anemia were studied in intact state and after refreshment. The results were compared with the data of clinical laboratory and osmotic fragility test. The final conclusion is that this method detects membrane defects with altered spectrin and anion channel syndrome (hereditary xerocytosis, spherocytosis, poikilocytosis and pyropoikilocytosis, elliptocytosis and stomatocytosis) and, after refreshment, helps differentiate them from the anemia with hemoglobinopathy.