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Elliptical erythrocyte membrane skeletons and heat-sensitive spectrin in hereditary elliptocytosis
Abstract
Erythrocyte membranes (ghosts) and membrane skeletons (submembranous reticula of spectrin, actin, and protein 4.1 prepared by extracting ghosts with Triton X-100) from 15 patients with hereditary elliptocytosis (HE) were elliptical, which indicates that the primary defect responsible for the abnormal shape of these cells resides in the skeleton. The protein composition of HE skeletons was normal, but in three kindreds purified spectrin heterodimer from 7/7 HE patients was heat sensitive and denatured at 48.0 +/- 0.1 degrees C instead of 49.0 +/- 0.3 degrees C (P less than 0.0005). Heat sensitivity was detected by precipitation and, in the spectrin from one patient, by changes in circular dichroism. In one other kindred spectrin dimer from 3/3 patients denatured at the normal temperature. In two of the three kindreds with heat-sensitive spectrin, intact erythrocytes exhibited budding and fragmentation at the temperature at which spectrin denatured. In the third kindred spectrin was heat sensitive, but erythrocytes were not. The symptoms in the latter kindred were clinically more severe (hemolytic HE with spherocytosis) than in the other three (mild HE). We conclude that defects in the erythrocyte membrane skeleton may be a common feature of HE. As judged by heat denaturation of erythrocytes and purified spectrin dimer, three phenotypically distinct forms of HE exist, two of which are characterized by defective, heat-sensitive spectrin. It remains to be determined whether the molecular defect in spectrin responsible for heat sensitivity is the primary genetic defect responsible for HE.
... Heated cells were fixed by adding 100 mL of 2% glutaraldehyde in PBS to each tube. The samples were examined for echinocytic and spherocytic cells by phase contrast microscopy and the numbers of normal and abnormal cells counted (19). ...
... Erythrocytes have been shown to have particular sensitivity to temperature (19,31). Although human RBCs maintain their discoid morphology upon heating to 48 C, increasing the temperature to 49-50 C induces rapid shape transition with membrane fragmentation and generation of spherocytes (19). ...
... Erythrocytes have been shown to have particular sensitivity to temperature (19,31). Although human RBCs maintain their discoid morphology upon heating to 48 C, increasing the temperature to 49-50 C induces rapid shape transition with membrane fragmentation and generation of spherocytes (19). Diluted suspensions of RBCs from aIIbI and WT mice were heated to temperatures between 45 and 50 C. ...
Spectrin tetramers of the membranes of enucleated mammalian erythrocytes play a critical role in red blood cell survival in circulation. One of the spectrins, αI, emerged in mammals with enucleated red cells following duplication of the ancestral α-spectrin gene common to all animals. The neofunctionalized αI-spectrin has moderate affinity for βI-spectrin, while αII-spectrin, expressed in non-erythroid cells, retains ancestral characteristics and has a 10-fold higher affinity for βI-spectrin. It has been hypothesized that this adaptation allows for rapid make-and-break of tetramers to accommodate membrane deformation. We have tested this hypothesis by generating mice with high-affinity spectrin tetramers formed by exchanging the site of tetramer formation in αI-spectrin (segments R0 and R1) for that of αII-spectrin. Erythrocytes with αIIβI presented normal hematologic parameters yet showed increased thermostability and their membranes were significantly less deformable: under low shear forces they displayed tumbling behavior, rather than tank-treading. The membrane skeleton is more stable with αIIβI and shows significantly less remodeling under deformation than red cell membranes of wild-type mice. These data demonstrate that spectrin tetramers undergo remodeling in intact erythrocytes and that this is required for the normal deformability of the erythrocyte membrane. We conclude that αI-spectrin represents evolutionary optimization of tetramer formation: neither higher affinity tetramers (as shown here) nor lower affinity (as seen in hemolytic disease), can support the membrane properties required for effective tissue oxygenation in circulation.
... Recent studies indicate that the atypical morphology and predisposition to hemolysis observed in some forms ofhereditary elliptocytosis (HE) may be associated with molecular defects or deficiencies in the RBC membrane skeletal components (6)(7)(8)(9)(10)(11)(12). On a molecular level, several spectrin variants, as well as a deficiency in band 4.1, have been reported to be associated with elliptocytosis (7)(8)(9)(10)(11)(12)(13). Liu et al. have found that the quantity of spectrin heterodimers in 00C extracts is elevated in a subpopulation of HE patients (designated HE type 1 or HE[SpD-SpD]) (8). ...
... The biochemical data presented here are consistent with clinical data that indicate that HE is a heterogenous disease characterized by an autosomal dominant mode of inheritance (7,31). In part, this heterogeneity may result from the fact that several different membrane skeletal defects can give rise to elliptical morphology (7)(8)(9)(10)(11)(12)(13). Thus, it is not surprising that the limited tryptic peptide maps of spectrin produced by some HE patients are indistinguishable from those of normal volunteers, while others are atypical. ...
... In part, this heterogeneity may result from the fact that several different membrane skeletal defects can give rise to elliptical morphology (7)(8)(9)(10)(11)(12)(13). Thus, it is not surprising that the limited tryptic peptide maps of spectrin produced by some HE patients are indistinguishable from those of normal volunteers, while others are atypical. Similarly, some HE spectrins are more sensitive to heat denaturation while others denature normally (13). In these HE individuals, who do not have a spectrin selfassociation defect, the molecular defect may involve another functional domain ofspectrin or a structural or regulatory component of the cytoskeleton other than spectrin. ...
Hereditary elliptocytosis (HE) is a clinically and biochemically heterogenous group of diseases characterized by elliptically shaped erythrocytes and an autosomal dominant mode of inheritance. Whereas the self-association of spectrin heterodimers to tetramers is defective in a subpopulation of HE patients, designated HE[SpD-SpD], it is normal in others. We have examined the peptide pattern produced by limited tryptic digestion of spectrin extracts from patients with HE[SpD-SpD] to determine if the functional defects in spectrin self-association could be correlated with structural changes in the spectrin molecule. Although the peptide pattern produced by limited tryptic digestion of spectrin extracts from those HE patients with normal spectrin self-association was indistinguishable from the pattern from control normal volunteers, digestion of the spectrin extracts from the HE[SpD-SpD] patients showed a reproducible diminution in the 80,000-D domain of the alpha-subunit, which is involved in spectrin dimer self-association. The decrease in the 80,000-D fragment was associated with an increase in a 74,000-D fragment in eight of nine families, or, in one family, with an increase of fragments at 46,000 and 17,000 D. These atypical peptide patterns were similar to those previously reported in two variants of hereditary pyropoikilocytosis (HPP), which also had defective self-association of spectrin. These data indicate that two distinct structural variants of spectrin alpha-subunit are associated with the defective spectrin heterodimer self-association in a subpopulation of HE patients.
... Erythroid precursors in bone marrow of HE patients are round without any morphologic abnormalities which suggests elliptical shape of elliptocytes develop during the normal aging process of red blood cell (RBC) in circulation [3]. The mechanism involves repeated episodes of deformation that occur as RBCs goes through capillary beds; repeated deformation causes permanent damages and changes in the cytoskeleton of RBCs in HE. [4] Normal RBCs recovers their normal biconcave physical shape after they emerge from narrow capillaries. However, RBCs from patient with HE seems to lack normal interactions between cytoskeletal and membrane components that cause the cells gain an elliptocytic shape. ...
... A common feature of HS RBC is loss of membrane surface area and resultant change in cell shape from discocytes to spherocytes [110]. In HE [31,111,112] and hereditary pyropoikilocytosis (HPP), a severe case of HE [113,114], the protein defects occur at membrane cytoskeletal proteins such as a-spectrin, b-spectrin, and protein 4.1, and thus interrupt the selfassociation of spectrin a/b heterodimers, leading to the formation of elliptocytes and poikilocytes. Echinocytes are characterized by small, evenly spaced thorny projections [115,116], also referred as burr cells. ...
We review recent advances in multiscale modeling of the biomechanical characteristics of red blood cells (RBCs) in hematological diseases, and their relevance to the structure and dynamics of defective RBCs. We highlight examples of successful simulations of blood disorders including malaria and other hereditary disorders, such as sickle-cell anemia, spherocytosis and elliptocytosis.
... Zmniejszona oporność osmotyczna występuje u homozygot. W niektórych laboratoriach wykonuje się test wrażliwości termicznej erytrocytów (lub wyizolowanych spektryn) [152]. Prawidłowe krwinki ulegają spontanicznej fragmentacji (wskutek denaturacji spektryn) w temperaturze 49 °C, natomiast w cięższych przypadkach typowej HE erytrocyty rozpadają się już w 44-48 °C [144]. ...
Erythrocyte membrane is a complex, semifluid and dynamic structure composed of lipid components associated with a number of proteins with different functions. Membranopathies - inherited hemolytic anemias caused by the defects in erythrocyte membranes - in terms of genetic and pathophysiology are a very diverse group of hematologic diseases. The most frequently observed are hereditary spherocytosis and hereditary elliptocytosis caused by deficiencies in membrane and cytoskeleton proteins or disturbances in their interactions that lead to a impaired deformability of red blood cells. Less common are the defects caused by increased permeability of erythrocyte membranes to monovalent cations. They are collectively described as hereditary stomatocytosis, their molecular background is still unknown and cause the diagnostic difficulties. Congenital deficiencies of red blood cell blood group antigens, including the Rhnull phenotype, cause mostly mild symptoms of hemolytic anemia and affect a small group of patients. Very rare membranopathies are dyslipidemias - erythrocyte membrane lipid defects caused by metabolic diseases.
... Pyropoikilocytosis is a disorder of erythrocytes characterized by abnormal temperaturesensitive cell fragmentation (15), and the spectrin has been shown to have increased sensitivity to thermal denaturation (16). Some kindreds with elliptocytosis also appear to have spectrin with slightly increased heat sensitivity (17), and spectrin from a similar patient was shown to yield abnormal trypsin digestion fragments (18). Other reports indicate that other forms of elliptocytosis may be due to alterations in spectrin tetramer-dimer equilibrium (19) or reductions in the amount of band 4.1 (20,21). ...
Patients from two families with chronic hemolytic anemia have been studied. The erythrocytes are very fragile and appear microcytic with a great variety of shapes. Clinical evaluation failed to identify traditionally recognized causes of hemolysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed no significant abnormality of the major polypeptide bands. Erythrocytes spectrin-ankyrin and ankyrin-membrane interactions were analyzed with 125I-labeled spectrin, 125I-labeled ankyrin, and inside-out vesicles. Patients' vesicles bound 125I-spectrin normally. Likewise, patients' spectrin and ankyrin competed normally for the binding sites on control membranes. None of the individual components appeared to have abnormal thermal sensitivity. Ankyrin-stripped, inside-out vesicles prepared from the patients bound less 125I-ankyrin than did vesicles prepared from normals (P less than 0.05 for all corresponding points in the high-affinity region). Scatchard analysis showed the most significant abnormality to be a 50% reduction in the high affinity ankyrin binding sites. Similar experiments were performed with blood from patients with spherocytosis and splenectomized controls, but no abnormalities were detected. The water soluble 43,000-dalton fragments of band 3 (the high-affinity ankyrin binding sites) were prepared from one of the patients and competed normally for 125I-ankyrin binding in solution. This suggests that the primary structural defect is a reduction in the number of high affinity membrane binding sites for ankyrin, and is consistent with an abnormal organization of band 3 in the membrane.
Blood formation normally takes part in the bone marrow, where the common pluripotent stem cells give rise to a series of progenitor cells for three main cell lines: red cells, white cells (granulocytes, monocytes, lymphoid cells) and platelets. They mature in the bone marrow and are released into the peripheral blood. The normal blood count measures these cell components. Additionally, a peripheral blood smear is a very important test for corpuscular abnormalities and cell configuration. Thus, in the normal blood test many components can be measured (see Table 27.1).
Blood formation normally takes part in the bone marrow, where the common pluripotent stem cells give rise to a series of progenitor cells for three main cell lines: red cells, white cells (granulocytes, monocytes, lymphoid cells) and platelets. They mature in the bone marrow and are released into the peripheral blood. The normal blood count measures these cell components. Additionally, a peripheral blood smear is a very important test for corpuscular abnormalities and cell configuration. Thus, in the normal blood test many components can be measured (see Table 27.1).
Molecular defects directly affecting a membrane component can modify the shape of the red cell and reduce its survival capacity; the same effect can result from molecular lesions at the level of a cytoplasmic protein or enzyme. In some diseases where the primary defect does not involve a membrane component, a secondary membrane lesion develops, contributing to or causing red cell death. For instance, in some hemoglobinopathies, a less stable anomalous hemoglobin interacts with the membrane and modifies lipid and proteins; in erythroenzymopenias, such as glucose-6-phosphate dehydrogenase (G6PD) deficiency and pyruvate kinase (PK) deficiency, the altered structure of a key enzyme involved in glycolysis or the pentose pathway, impairs the ability of red cells to withstand stress conditions, and there are clues that lead us to suspect a membrane lesion secondary to the enzyme deficiency. In this review, membrane processes found in the three above-mentioned groups of diseases will be described: in particular, we will deal with two hemoglobinopathies, thalassemia and sickle-cell anemia; with the enzymopathies glucose-6-phosphate dehydrogenase and pyruvate kinase deficiency; and with the hereditary membrane skeleton diseases spherocytosis, elliptocytosis, and pyropoikilocytosis. A summary of membrane lesions found in each disease will be presented, more detailed reviews concerning each of these topics being available [see Wagner et al. (1985), Hebbel et al. (1985), and Hebbel (1986) for sickle cell anemia; Rachmilewitz et al. (1985) for thalassemia; Palek and Lux (1983), Becker and Lux (1985), Palek (1985) for hereditary defects of membrane skeleton; Beutler (1983), Valentine et al. (1983) for G6PD and PK deficiencies]. Membrane damage associated with an impaired survival capacity of pathological red cells will be compared with membrane properties of senescent normal red cells (see Bartosz, this volume). The general aim of the review is to analyze which membrane lesions in erythrocyte pathology shorten red cell life span, and to find insights into membrane processes that would be worth investigating in those diseases, where indirect evidence suggests the presence of membrane damage. Structural integrity of the membrane allows the erythrocyte to perform its biological role of oxygen transport from the lungs throughout the body. The ability to deform reversibly during flow is crucial to the red cell for performing its function. This property, known as cellular deformability, is determined by membrane material characteristics, cell surface-area-to-volume ratio, and cytoplasmic viscosity, and is strictly related to the red cell survival capacity (Mohandas et al., 1983). Since structure and function of the membrane are highly involved in determining cell deformability (through the maintenance of ion gradients and water distribution, and through the transport of nutrients and catabolites), the life span of a red cell ultimately depends on the integrity of its membrane.
This chapter discusses the red cell membrane proteins. The human red blood cell (rbc), despite its inability to replace nuclear encoded proteins, is a dynamic entity that must deform multiple times during its 120-day life span and predicted 500,000 circuits of the body. The deformable lattice binds unique cross-linking proteins that interact with proteins embedded in and protruding bidirectionally through the cell membrane. The cytoskeleton is constructed during the differentiation of rbcs from immature precursors. Interactions between the various proteins are well-characterized but the actual mechanics of membrane assembly is not clear because studies have, of necessity, been performed in vitro rather than in vivo. The membrane superstructure maintains the integrity of the rbc since deficiencies or aberrant structure of the cytoskeletal proteins cause hemolytic anemias of varying severity.
Spectrin, the major cytoskeletal protein in erythrocytes, is localized on the inner membrane surface in association with membrane-spanning glycoproteins and with intramembrane particles. The presence of a specific, high-affinity protein binding site for spectrin on the cytoplasmic surface of the membrane has been established by measurement of reassociation of spectrin with spectrin-depleted inside-out vesicles. A 72,000 Mr proteolytic fragment of this attachment protein has been purified, which bound to spectrin in solution and competed for reassociation of spectrin with vesicles. A 215,000 Mr polypeptide has been identified as the precursor of the spectrin-binding fragment. The membrane attachment protein for spectrin was named ankyrin, and has been purified and characterized. Ankyrin has been demonstrated to be tightly associated in detergent extracts of vesicles with band 3, a major membrane-spanning polypeptide, and to bind directly to a proteolytic fragment derived from the cytoplasmic domain of band 3. Ankyrin is thus an example of a protein that directly links a cytoplasmic structural protein to an integral membrane protein. The organization of the erythrocyte membrane has implications for more complex cell types since immunoreactive forms of ankyrin distinct from myosin or filamin have been detected by radioimmunoassay in a variety of cells and tissues. Indirect immunofluorescent staining of cultured cells reveals immunoreactive forms of ankyrin in a cytoplasmic meshwork and in a punctate distribution over nuclei. The staining changes dramatically during mitosis, with concentration of stain at the spindle poles in metaphase and intense staining of the cleavage furrow during cytokinesis.
We measured spectrin "extractability" in erythrocytes which were metabolically depleted by incubation at 37 degrees C in plasma or glucose-free buffers. Membranes were extracted with 1 mM EDTA (pH 8, 40 h, 4 degrees C) and analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This procedure solubilized 85--90% of the spectrin, actin, and residual hemoglobin from ghosts of fresh erythrocytes. In incubated erythrocytes, inextractable spectrin rapidly accumulated when ATP concentrations fell below 0--15% of normal. In severely depleted cells, 60--90% of the total ghost spectrin became inextractable. Inextractability was not abolished by physically disrupting the ghost before extraction, but was reversed when erythrocyte ATP was replenished with adenosine. The accumulation of inextractable spectrin correlated temporally with the increase in apparent membrane deformability and the increases in erythrocyte vicosity, calcium content, sodium gain, and potassium loss characteristic of ATP-depleted erythrocytes. No change in integral membrane protein topography (assessed by the distribution of intramembranous particles and concanavalin A surface-binding sites) was detected in depleted cells. Analogous changes were observed in erythrocytes exposed to extremes of pH and temperature. When the pH in the erythrocyte interior fell below 5.5, a pH where spectrin was aggregated and isoelectrically precipitated, erythrocyte and ghost viscosity increased coincident with a marked decrease in spectrin extractability. Similarly above 49 degrees C, a temperature where spectrin was denatured and precipitated, erythrocyte viscosity rose as inextractable spectrin accumulated. These observations provide direct evidence of a change in the physical state of spectrin associated with a change in erythrocyte shape and deformability. They support the concept that erythrocyte shape and deformability are largely determined by the shape and deformability of the spectrin-actin protein meshwork which laminates the inner membrane surface.
Mammalian erythrocytes that lack cytoplasmic organelles and a nucleus are a useful model for studying the effect of heat on the cell membrane and cytoskeleton. The effect of heat on the membrane bilayer and cytoskeleton of erythrocytes is remarkably similar to that observed in nucleated cells. Some concentrations of D2O and glycerol can effectively protect erythrocytes from heat-induced damage to the membrane and cytoskeleton. These results are similar to observations in nucleated cells. Heating erythrocytes in some concentrations of anisotonic NaCl solutions reduced damage, an observation that does not apply to enhanced killing of nucleated cells. This difference implies that some components of the cytoplasm or nucleus, or both, may contribute to the enhancement of cytotoxicity of nucleated cells when they are heated in the anisotonic NaCl solution. Incremental heating, dividing a heat treatment into two fractions, and preheating of erythrocytes all modify the effect of heat on erythrocytes slightly, but the results suggest little, if any, development of thermotolerance. The response of chicken erythrocytes is similar to that of mammalian erythrocytes, although higher temperatures are required to produce a heat effect in chicken erythrocytes. These observations suggest that the characteristic differences in heat sensitivity in nucleated and enucleated cells involve components other than the cell membrane.
The rules governing the transbilayer reorientation (flip-flop) of long-chain amphiphilic components in biological membranes were further elucidated by studying the flip-flop of palmitoylcarnitine in human erythrocytes. Flip rates were derived from the time-dependent decrease of extractability of palmitoylcarnitine by albumin after primary insertion of trace amounts of the labeled probe into the outer membrane layer. The flip rate (half time 2.6 hr at 37°C in human erythrocytes) is fast enough to be measurable also in membranes exhibiting low flip rates such as that of ox erythrocytes. Flip rate constants for the inward and outward reorientation are similar and the probe equilibrates at a 1∶1 ratio between the two layers.
The flip is a simple, diffusion-like process. It is not inhibited but even enhanced by chemical modification of membrane proteins. It is also enhanced by insertion of channel-forming antibiotics into the membrane and by pre-exposure of the cells to temperatures exceeding 42°C. The extent of this enhancement increases with the duration and the temperature of the pre-exposure. Since spectrin is denatured in this range of temperatures, the finding constitutes a new piece of evidence that the membrane skeleton is involved in the maintenance of bilayer stability and that a decrease of bilayer stability goes along with the formation of local defects acting as flip sites for phospholipids and related compounds.
As a particularity, the flip is enhanced by lowering the pH and exhibits interindividual variability, phenomena not observed for the flip-flop of lysophosphatidylcholine. This suggests that generalizations on the kinetics of nonmediated flip-flop of membrane-intercalated amphiphiles may not be justified.
Two new G6PD variants with severe enzyme deficiency in Switzerland (G6PD Avenches, G6PD I) and in Germany (G6PD Moosburg, G6PD II) are described. One patient had suffered from severe postpartal hyperbilirubinemia, the other one presented with chronic hemolysis and remittent hyperbilirubinemia. Both variants showed diminished electrophoretic mobility, both variants were heat labile. The Michaelis-Menten constants KM for glucose-6-phosphate and for NADP+ were normal. 2-Desoxy-glucose-6-phosphate was utilized by G6PD I in a higher and by G6PD II at a lower rate than by the normal enzyme. Desamino-NADP+ and galactose-6-phosphate were utilized by both variants at a normal rate. The electrophoretic separation of membrane proteins of G6PD II showed both in the presence and in the absence of 6-mercaptoethanol no difference concerning the formation of membrane protein aggregates between patient and normal control.
The lipid bilayer of the adult red cell is supported on its inner surface by a complex arrangement of proteins known as the membrane skeleton. This filamentous network, a major component of which is a multifunctional protein called spectrin, has an essential role in determining the shape, structural integrity, and deformability of the red cell. A significant achievement of modern biochemistry and hematology has been the elucidation of the organization of the components of the membrane skeleton and their relationship to other membrane proteins and lipids. This article reviews current concepts of membrane skeleton structure and function and emphasizes recent advances which have been made in characterizing and classifying molecular defects of the skeleton which manifest clinically with changes in the shape and stability of the red cell. The pathobiology of hereditary skeletal defects associated with hereditary spherocytosis (HS), hereditary elliptocytosis (HE), and hereditary pyropoikilocytosis (HPP) are comprehensively discussed. Secondary defects of the membrane skeleton occurring in glucose-6-phosphate dehydrogenase deficiency and sickle cell anemia are also briefly considered.
The results of hybridization analyses using cDNA probes for mouse and human alpha-spectrin mRNA indicate that a single gene encodes the alpha-subunit of erythrocyte spectrin. Sequencing of the cDNA clones showed that they code for 370 amino acids (aa) covering three repeat domains close to the C terminus of alpha-spectrin. The cloned cDNAs will now permit the isolation of the alpha-spectrin gene and should lead to the characterization of the genetic aspects in human hereditary anemias in which alpha-spectrin has been characterized as the site of the molecular defect.
When the human red cell consumes its ATP, the cell loses its discoid character in favour of a spiculated and eventually a spherical form. This discocyte—echinocyte transformation parallels both degradation of phosphatidylinositol 4,5-bisphosphate and phosphatidic acid but not dephosphorylation of cytoskeletal proteins. Dephosphorylation of both spectrin and band 3 lags behind metabolic crenation.
Exogenous vanadate accelerates both shape changes and lipid dephosphorylation in a parallel manner during metabolic depletion. In contrast to its effect on lipids, vanadate reduces the rate of protein dephosphorylation. These observations strongly support a shape control mechanism in the red cell, based on phosphoinositide metabolism and compatible with a bilayer-couple model.
The clinical features of hereditary elliptocytosis were studied in 25 cases and compared with 20 normal individuals. Based on morphological features, these patients were classified into two groups: those with a rod-shaped type of elliptocytosis (nine cases), and those with a non-rod type (16 cases). Most of the cases with overt haemolysis were detected among cases of the non-rod type. Overt haemolysis typically tended to be accompanied by stomatocytic changes, which appear to be superimposed on elliptic transformation of the red cells. Sodium influx increased in eight of nine HE patients with overt haemolysis, but in none of the HE with rod-shaped red cells. Significant quantitative and qualitative differences in red cell membrane lipids were observed between the two types of HE. No spectrin abnormalities in domain composition, dimer-dimer association, and thermal stability were observed in 11 of the HE patients studied, including four cases with overt haemolysis.
An abnormal spectrin, in which one subunit is truncated, has been detected in a large German family. The inheritance is autosomal dominant. The affected members of the family suffer in widely varying degree from a microcytic hemolytic anemia. The red cell morphology varies correspondingly from smooth elliptocytes to predominantly poikilocytes. The abnormal spectrin makes up approximately 30% of the total and is almost entirely present as the dimer. The truncated chain is not phosphorylated by the endogenous cAMP-independent kinase, and it has been identified as a chain of beta-type, using monoclonal antibodies. Because a univalent terminal spectrin alpha-chain fragment will bind to normal dimers with an association constant lower by only a factor of two than that for the self-association of the dimers, it would be expected that the mutant dimers (alpha beta') would readily enter into an association with normal (alpha beta) dimers to give alpha 2 beta beta' tetramers (though not with each other). In dilute solution this is indeed observed, and the diminution in tetramer concentration when 30% of normal spectrin is replaced by alpha beta' dimers, amounts to only a small proportion. Moreover, in the membrane skeleton, if there is pairwise apposition of dimer units, only 9% of pairings will be between units that cannot associate. We have shown that the failure of alpha beta' dimers to enter into heterologous associations in situ is not due to the elimination of the ankyrin binding site near the truncated end of the beta-chain: this site is fully functional, as judged by rebinding to spectrin-depleted vesicles. When the spectrin is extracted from the membrane in the cold, the material released initially consists almost entirely of alpha beta' dimers; when the spectrin of normal membranes is partly dissociated to dimers in situ by warming at low ionic strength, extraction in the cold then leads similarly to much more rapid release of the dimer than of the tetramer. The similar rates of liberation of normal and abnormal dimer make it unlikely that the interaction of the latter with the membrane is in any way defective. When mixtures of alpha beta and alpha beta' dimers are bound to spectrin-depleted inside-out membrane vesicles from normal cells and tetramers are allowed to form by equilibration at 30 degrees C, the proportion of the abnormal species appearing in the tetramer is much lower than would be expected on a statistical basis. The relation of the self-association equilibrium on the membrane to that of spectrin in dilute solution is analyzed.
Filipin, a polyene antibiotic, interacts with beta-hydroxy sterols such as cholesterol in most cell membranes, forming bumps and pits that are visible by electron microscopy of freeze-fracture replicas. The markedly reduced perturbability of the red blood cell (RBC) membrane, compared to other cells, has been attributed to the constraining influence of the red cell membrane skeleton, the undercoat composed of spectrin, actin, and protein 4.1. To test the influence of the membrane skeleton on filipin-induced perturbation of the RBC membrane, we studied the interaction of filipin with red cells that were inherently devoid of spectrin and RBC in which spectrin had been crosslinked or denatured. These spectrin-deficient, crosslinked, and denatured cells have a fivefold increase in the number of filipin-induced perturbations as compared to control cells, despite equivalent membrane cholesterol content. These findings confirm that the spectrin-based membrane skeleton strongly influences the organization of the membrane so as to limit perturbation by filipin:cholesterol interaction and that for membranes in which the cholesterol content is known, filipin is a useful probe for testing the avidity of spectrin-based cytoskeletal attachment.
Hereditary elliptocytosis (HE) is, in the heterozygous state, a common mild congenital hemolytic disease. In contrast, homozygous elliptocytosis is a severe transfusion-dependent hemolytic anemia. The major determinant of red cell membrane shape and stability is a two-dimensional proteinaceous meshwork named membrane skeleton. Spectrin, the most important protein of the membrane skeleton, is basically a heterodimer composed of alpha and beta chains. Within the membrane, spectrin dimers self-associate to form tetramers. In type I HE spectrin dimer self-association is defective and an excess of spectrin dimer is present in the patient's red cell membranes. The defective self-association is often correlated with an abnormality of the spectrin alpha chain which is depicted by limited tryptic digest of spectrin. In a family previously studied by us (Dhermy et al., 1984), the search for a spectrin defect in the red cells of the fetus of the pregnant mother was indicated for the following reasons: the diagnosis of heterozygous type I HE with the same spectrin variant had been made in the mother as well as in the father. Moreover, homozygous HE had been recognized in one of the children born two years previously with a persistent and severe transfusion dependent hemolytic anemia. Preliminary studies of normal fetal erythrocytes at twenty weeks gestation have shown that fetal and adult spectrin molecules are identical. The results obtained in the fetus at risk allowed us to diagnose type I HE (though elliptocytes were not present in the blood) for the following reasons: (i) erythrocyte deformability was decreased (ii) spectrin self-association was defective with an excess of dimer species in the membrane (iii) limited tryptic digest of spectrin showed the same abnormal pattern as seen in the heterozygous mother, with a decrease in the 80,000-dalton peptide and a concomitant increase in the 74,000-dalton peptide. The heterozygous state, strongly suspected on the tryptic digest pattern of fetal spectrin, was confirmed when the mother gave birth to a baby who did not have hemolytic anemia during the first 18 months of life.
Human erythrocyte spectrin heated above 49 degrees C could be separated into two fractions by DEAE-Toyopearl column chromatography at room temperature. The first fraction eluting with the salt gradient was predominantly the alpha subunit, indicating a heat-induced dissociation of the spectrin alpha beta dimer into monomers. The second fraction, obtained with 0.5 M NaOH after salt elution, consisted of high-molecular-weight proteins in addition to alpha and beta subunits, which were visualized by gel electrophoresis with sodium dodecyl sulfate. The isolated beta subunit when heated above 48 degrees C could also be separated into two fractions by column chromatography. About 30% of the protein eluted with the salt solution and the rest of the proteins were in the alkali eluate in which high molecular weight protein bands also appeared, indicating a heat-induced aggregation of the beta subunits. Almost all the isolated alpha subunit, however, eluted out with the salt solution, even though the subunit was heated at 52 degrees C. Studies of the binding of subunits to inside-out vesicles indicate that the isolated beta subunit was denatured irreversibly by heating; on the other hand, the alpha subunit kept its binding ability after heating above 50 degrees C. These findings are attributed to the heat-induced dissociation of the spectrin molecules into alpha and beta subunits at 49-50 degrees C, and eventual aggregation of the denatured beta subunits.
A number of abnormalities in cellular physiology have been observed in hereditary spherocytes, including alterations in shape, membrane cation permeability and deformability, intracellular metabolism and tendency for splenic entrapment. Many observations have been observed only in a subset of patients with HS and may studies have not been confirmed. Therefore, it is likely that there is heterogeneity with regard to the specific molecular cause of the disease. The major research problem has been to determine primary molecular defects in HS. Much evidence supports a molecular defect in the erythrocyte membrane skeleton and three abnormalities involving spectrin have been demonstrated to be directly related to HS. First, spectrin deficiency has been shown in autosomal recessive spherocytosis in mouse mutants and partial deficiency observed in all human patients with HS. Second, a specific functional defect in spectrin purified from the red cells of some kindreds with autosomal dominant HS has been identified: lack of binding capacity for protein 4.1. Third, a less well characterized functional abnormality has been described in which spectrin binds more tightly to the erythrocyte membrane. These defects may, by an unidentified mechanism, contribute to the spheroidal shape and haemolytic disease ameliorated by splenectomy.
More definitive studies are necessary in order to determine the origins of HS. Such studies require:
1Use of appropriate controls for splenectomy and young red cell age,
2Tracing a defect through affected family members,
3Verifying that a defect corresponds to the appropriate heredity pattern, for example that a heterozygote for an autosomal dominant defect had 50% abnormal protein,
4Differentiating the effects of splenic or circulatory conditioning from the primary red cell defects,
5Verifying that the defect is present in the intact cell and is not secondary to experimental manipulations,
6Distinguishing an unrelated, linked polymorphism from the primary mutation responsible for the disorder.
Finally, the pathophysiology of the disease will have to be explained on the basis of the primary molecular defect, as well as the mechanism of all secondary physiological changes in the hereditary spherocyte.
We determined whether the membrane defect in hereditary pyropoikilocytosis (HPP) is associated with thermally induced changes in the lipid bilayer, the stability of which was probed by the rate of translocation of phosphatidylcholine (PC) over the two leaflets. [14C]PC was incorporated into the outer leaflet of the lipid bilayer of the intact erythrocytes using a PC-specific phospholipid exchange protein. The transbilayer equilibration of this PC was determined by measuring the time-dependent changes in its accessibility to exogenous phospholipase A2. The rate of transbilayer equilibration of PC was increased in HPP cells at 37 degrees C when compared to normal erythrocytes (rate constants, 0.07 +/- 0.02 and 0.03 +/- 0.01 h-1, respectively). A further dramatic increase in PC transbilayer equilibration was noted in HPP cells incubated at 44 degrees C (rate constant, 0.15 +/- 0.02 h-1). A similar marked acceleration in transbilayer movement of PC was also seen in normal erythrocytes when incubated at 46 degrees C (rate constant, 0.13 +/- 0.03 h-1). Despite the enhanced transbilayer mobility of PC in HPP cells when compared to normal erythrocytes, no major alteration in the asymmetric distribution could be observed when probed with phospholipase A2. Since changes in transbilayer mobility of PC and cell morphology occur in HPP cells at lower temperature than in normal red cells, it may be concluded that the enhanced thermal sensitivity of spectrin is the major factor responsible for these changes. Our results therefore support the view that the structural integrity of the skeletal network is essential for stabilization of the lipid bilayer of the red cell membrane.
Five patients with hereditary elliptocytosis (HE) from two unrelated black families were studied. The patients had prominent elliptocytosis and a decreased erythrocyte resistance to heat treatment. In one infant blood smears showed elliptocytosis and poikilocytosis; his erythrocytes fagmented at a lower temperature than those of his mother and sister, both having typical mild HE. Defective dimer-dimer association was present in all patients. Limited tryptic digestion of spectrin and subsequent analysis by one- and two-dimensional electrophoresis revealed a similar and reproducible decrease in the 80,000-dalton peptide (alpha I domain) and the concomitant appearance of a 46,000-dalton peptide. All the patients had the polymorphism of the spectrin alpha II domain commonly observed in black populations. In addition, modifications relative to the alpha III domain were detected; similar variants were found in one black control subject out of 136 and are likely related to a genetic polymorphism of the alpha III domain. No differences were observed between the peptide patterns in the infant with poikilocytosis and those of his HE sister and mother.
The relationship between membrane structural properties and functions has been generally inferred from observed thermotropic phenomena. By the use of 16-dinyloxyl stearic acid spin probe we investigated the red blood cell membrane components involved in three characteristic thermotropic structural transitions occurring at 8, 20, and 40 degrees C. The transition at 8 degrees C is removed by chymotrypsin treatment at the cytoplasmic membrane layer. The 20 degrees C phase transition is unmodified after chymotrypsin treatment and occurs at 15 degrees C after complete proteolysis of intramembrane chymotrypsin-insensitive peptides. Liposomes from the total lipid extract of RBC show only one thermotropic transition at 15 degrees C. The 40 degrees C phase transition is absent in vesicles free of skeletal proteins, in vesicles obtained after RBC storage, and in low-ionic-strength resealed ghosts. Transitions at 8 degrees C and 40 degrees C appear to be due to the interactions of cytoplasmic exposed proteins with membrane, whereas the 20 degrees C transition is intrinsic to the lipid component.
The interaction of spectrin with spectrin-depleted inside-out membrane vesicles was studied in a kindred with an atypical variant of hereditary elliptocytosis inherited in a recessive manner. The probands are characterized by prominent elliptocytosis, decreased erythrocyte thermal stability, an altered limited tryptic peptide pattern of spectrin digested at low ionic strength, and defective spectrin dimer-dimer association. The parents are normal. The spectrin/band 3 ratio determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of isolated membranes of the probands was decreased to approximately 70% of control values, and total erythrocyte spectrin content in one proband was also decreased on SDS-PAGE. When a monospecific antispectrin antibody was used, a faintly labeled fragment of molecular weight approximately 28,000 was detected on immunoblots of whole cell lysates of one proband and a control, but could not account for the decreased erythrocyte spectrin content of the proband on SDS-PAGE. Binding and competitive inhibition studies revealed an alteration in the spectrin-ankyrin interaction due to an abnormality of spectrin in the probands. No defect was found in the mother; the father's spectrin showed decreased binding affinity, although it was not so severe as in the probands. Separation of bound and unbound spectrin dimers from one proband and subsequent conversion to tetramers showed that the self-association defect was detectable only on the bound subpopulation of her spectrin. These findings demonstrate a hitherto undescribed functional abnormality of spectrin in this kindred which could result in decreased stability of the membrane skeleton and contribute to the elliptocytic shape of these erythrocytes.
The membrane proteins of normal and hereditary spherocytosis have been labelled with a maleimide-analog nitroxide spin label and studied by electron paramagnetic resonance techniques. The spectral amplitude ratios from weakly and strongly immobilized labels differed slightly at 20° and 40°. Increasing the temperature to 47° and incubating for long time periods markedly accentuated the difference. It is suggested that the apparent differences in heat sensitivity between normal and hereditary spherocytosis erythrocyte membrane proteins reflect a latent structural alteration(s) of hereditary spherocytosis erythrocyte membrane proteins. Such structural alterations may result in altered functional behavior when the membrane is subjected to stress.
According to recent works, hereditary elliptocytosis (HE) appears to be related in some instances, to a defective self-association of spectrin (type I HE). We report a new case of type I HE observed in a white patient. Study of limited tryptic digestion of a spectrin dimer showed modification of a peptide involved in the dimer self-association process.
The interaction of erythrocyte ghosts and vesicles with chromatographed hemoglobin (Hb) A and Hb S was studied under various conditions. Although no binding of either Hb A or Hb S to inside-out vesicles was detected, under conditions of physiological ionic strength and pH, several properties of white membrane ghosts were effected by the presence of Hb. Addition of Hb A and Hb S (2 g/dl) to membrane ghosts in 6 mM MgATP, 150 mM NaCl, 10 mM Tris-HCl buffer, pH 7.4, was found to effect the echinocyte-discocyte transition, the extent of endocytosis, the volume, and the sealing of ghosts. Our observations suggest that the structure of membrane ghosts is influenced by cytosol proteins and that the environment of the red cell membrane plays an important role in the definition and the control of the membrane structure and function.
Patients with abetalipoproteinemia have an inborn absence of the major apoprotein of low density plasma lipoproteins, an abnormal serum and red cell lipid profile, and spiny erythrocytes, called acanthocytes. We now show that these deformed cells are reversibly converted to a normal shape, that of a biconcave disk, by incubation with 3 to 10 X 10(-5) M chlorpromazine. We suppose that chlorpromazine acts by expanding the cytoplasmic leaflet of the bilayer, thus promoting inward curvature. Ghosts isolated from the acanthocytes are themselves spiny but are also converted to normal, concave disks by chlorpromazine or merely by a brief incubation at 37 degrees C in low ionic strength buffer. We attribute the latter to a redistribution of lipids between the two leaflets of the membrane bilayer. Similar observations were made with red cells and ghosts from a patient with benign echinocytosis. These observations suggest that the morphological abnormality in acanthocytes and echinocytes can be ascribed to the same mechanism as crenation in vitro; that is, a bilayer couple effect in which an excess of surface area in the outer leaflet over the inner leaflet of the membrane bilayer drives outward curvature. It is striking that cells which were chronically abnormal in shape in vivo contain the information to become biconcave disks immediately upon simple chemical treatment in vitro.
During its 120 day life span, the red cell meets the extraordinary demands of deformability and structural integrity necessary for it to squeeze through small capillaries, negotiate the beating heart, and survive the suffocating environment of the spleen. Increased knowledge of the parts of the red cell membrane that control these functions may allow investigators to classify inherited disorders of the red cell membrane by their molecular defects. In addition, many acquired red cell problems may have a disrupted red cell architecture as the final pathway for the abnormal morphology or shortened red cell life span. This review presents briefly, current knowledge of red cell membrane structure and function and discusses the pathophysiology, diagnosis, and treatment of three red cell membrane disorders: hereditary spherocytosis, hereditary elliptocytosis, and hereditary pyropoikilocytosis.
A 10-year-old black girl with hereditary elliptocytosis had a transient increase in hemolysis and unusual red cell morphologic changes during an episode of acute hepatitis. Changes in membrane lipids of these elliptocytes with defective membrane skeletal protein and abnormal distribution of cholesterol may be responsible for this phenomenon.
We present our experience with two patients, the daughters of related but normal parents, who had nearly fatal hemolytic anemia requiring early splenectomy. Both had striking clinical improvement, but spherocytosis persisted. Red-cell-membrane ghosts were at least 50 per cent deficient in spectrin when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with band 1 reduced more than band 2. The spectrin deficiency was also detected when whole-red-cell lysates were analyzed by radioimmunoassay. Great care was taken to avoid membrane proteolysis, and there was no evidence of spectrin fragmentation. No defect in membrane binding of spectrin or in the membrane-binding sites (ankyrin) was identified. These patients had a severe form of spherocytosis that was probably due to an inadequate amount of spectrin, and the manner of inheritance was probably mendelian recessive.
The proportion of spectrin tetramers and dimers in 4 degrees C low ionic strength extracts of red cell membranes of 9 subjects with 4 different variants of hereditary elliptocytosis (HE) and 2 subjects with hereditary spherocytosis (HS) was determined by nondenaturing gel electrophoresis. Such extracts reflect the native oligomeric state of spectrin in the red cell membrane. In two hemolytic HE variants (an unclassified adult with increased thermal sensitivity of red cells and an infant also showing increased thermal sensitivity of red cells), the proportion of dimers was increased, whereas the remaining subjects had values within the control range. Conversion of spectrin tetramers to dimers under isotonic conditions at 37 degrees C, or spectrin dimers to tetramers at 30 degrees C, resulted in a high proportion of dimers in the above two HE variants, as well as in a third variant with probable mild HE and sporadic hemolysis. The mother of the infant with elliptocytosis and increased thermal sensitivity of red cells, although hematologically normal, had an increased proportion of dimers in 4 degrees C low ionic strength extracts of her red cell membranes. These findings reflect an underlying primary or secondary abnormality of spectrin in these subjects that affects the association state of spectrin in the red cell membrane. Their exact relationship to the pathogenesis of the elliptical shape of the red cell, or to the presence of hemolysis, is at present unclear.
An electrophoretically fast-moving variant of the spectrin beta-chain was discovered in the erythrocyte membranes of a woman and her father who both exhibited elliptocytosis and mild hemolytic anemia. This abnormal beta'-subunit (Mr = 214,000) co-existed with a decreased normal beta-chain and represented about half of the total beta-chains in the membrane. In contrast to the spectrin beta-chain, the beta'-chain was phosphorylated neither in the membrane by endogenous protein kinases nor in solution by pure membrane casein kinase whether or not the spectrin was dephosphorylated by erythrocyte cytosolic spectrin phosphatase. The presence of the beta'-chain was associated with a defective self-association of spectrin dimer to form tetramer as manifested by: (a) an excess of spectrin dimer in the 4 degrees C spectrin crude extract, (b) a defective self-association of the spectrin dimer in the 37 degrees C crude spectrin extracts. Gel electrophoretic analysis of the tetramer and dimer species isolated from the proband's 4 degrees C extract showed that the tetramer contained trace amounts of the beta'-chain, whereas in contrast, a large proportion of beta'-chain was present in the dimer. These results demonstrated the responsibility of the beta'-chain for the defective reassociation of spectrin dimer into tetramer. The study of this abnormal spectrin confirms the participation of spectrin beta-chain in dimer-dimer association and strongly suggests that the phosphorylation sites of the normal beta-chain are located at the end of the molecule involved in the dimer-dimer interactions.
To test the capability of the atomic force microscope for distinguishing membrane proteins with/without cytoskeletal associations, we studied the pull-out mechanics of lipid tethers from the red blood cell (RBC). When wheat germ agglutinin, a glycophorin A (GLA) specific lectin, was used to pull out tethers from RBC, characteristic force curves for tether elongation having a long plateau force were observed but without force peaks which are usually attributed to the forced unbinding of membrane components from the cytoskeleton. The result was in agreement with the reports that GLA is substantially free of cytoskeletal interactions. On the contrary, when the Band 3 specific lectin, concanavalin A, was used, the force peaks were indeed observed together with a plateau supporting its reported cytoskeletal association. Based on these observations, we postulate that the state of cytoskeletal association of particular membrane proteins can be identified from the force profiles of their pull-out mechanics.
Alow-salt extract prepared from human erythrocyte membranes forms a solid gel when purified rabbit muscle G- or F-actin is added to it to give a concentration of- 1 mg/ml . This extract contains spectrin, actin, band4.1, band4.9, hemoglobin, and several minor components . Pellets obtained by centrifugation of the gelled material at 43,000 g for 10 min contain spectrin, actin, band 4.1, and band 4.9 . Although extracts that are diluted severalfold do not gel when actin is added to them, the viscosity of the mixtures increases dramatically over that of G-actin alone, extract alone, or F-actin alone at equivalent concentrations . Heat-denatured extract is completely inactive . Under conditions of physiological ionic strength
Treatment of isolated human erythrocyte membranes with Triton X-100 at ionic strength ⋍0.04 preferentially released all the glycerolipid and glycoprotein species. At low ionic strength, certain nonglycosylated polypeptides were also selectively solubilized. The liberated polypeptides were free of lipids, but some behaved as if associated into specific oligomeric complexes. Each detergent-insoluble ghost residue appeared by electron microscopy to be a filamentous reticulum with adherent lipoid sheets and vesicles. The residues contained most of the membrane sphingolipids and the nonglycosylated proteins. The polypeptide elution profile obtained with nonionic detergents is therefore nearly reciprocal to that previously seen with a variety of agents which perturb proteins. These data afford further evidence that the externally-oriented glycoproteins penetrate the membrane core where they are anchored hydrophobically, whereas the nonglycosylated polypeptides are, in general, bound by polar associations at the inner membrane surface. The filamentous meshwork of inner surface polypeptides may constitute a discrete, self-associated continuum which provides rather than derives structural support from the membrance.
Membrane protein phosphorylation was measured in intact human erythrocytes with an assay which simultaneously monitored 32PO4 incorporation into ATP and membrane protein substrates. The pattern of phosphorylation in intact cells differed from that seen with isolated ghosts and likely reflected contributions of cytoplasmic protein kinases and phosphatase(s) as well as membrane protein kinases. Band 2 of spectrin, band 3, and a band in the 4.5 region were labeled. Dibutyryl cAMP (1 mM) did not alter intact cell phosphorylation. The data indicate that spectrin contains approximately 1 to 2 mol of exchangeable phosphorus/mol of spectrin, depending on the fraction of intracellular ATP available for isotopic exchange. The degree and pattern of phosphorylation were normal in intact circulating erythrocytes from nine patients with hereditary spherocytosis (HS) and in the splenic cordal erythrocytes of one HS patient. The membrane protein phosphorylation pattern of isolated erythrocyte ghosts varied, depending on the assay conditions. In the Greenquist and Shohet assay (Greenquist, A. C., and Shohet, S. B. 1974) phosphorylation of HS ghosts was normal after a 5-min incubation and was variably abnormal at 60 min. In the Avruch and Fairbanks assays (Avruch, J., and Fairbanks, G. 1974 and Fairbanks, G., and Avruch, J. 1974), phosphorylation of HS ghosts was normal under all incubation conditions. The authors conclude the intact erythrocyte assay may be the optimal screening test for detecting pathologically significant dysfunctions of membrane protein phosphorylation and that studies in erythrocyte ghosts may lead to erroneous conclusions about the status of membrane protein phosphorylation in the intact erythrocyte. This problem is illustrated by hereditary spherocytosis where phosphorylation of intact erythrocytes, presumably the physiologically relevant situation, is normal.
Two-dimensional tryptic and chymotryptic analyses of all the major bands in a sodium dodecyl sulfate/polyacrylamide gel of the human erythrocyte membrane show that each band has a characteristic map. However, band 2.1 (nomenclature of T. L. Steck) and several polypeptides below this band exhibit similar tryptic and chymotryptic peptide maps and thus appear to be a family of closely related proteins or degradation products. Furthermore, they all contain a subset of peptides that are accounted for by the peptides from two known spectrin-binding fragments. We show that both fragments derive from 2.1-related proteins and conclude that band 2.1 and its related proteins, which we name "syndeins", bind spectrin and connect it to the erythrocyte membrane.
One-dimensional and two-dimensional peptide-mapping techniques are used to identify the protein which gives rise to the 72,000 dalton alpha-chymotryptic fragment previously shown to be the membrane attachment site for spectrin. Peptide maps of the 72,000 dalton fragment are very different from maps of Bands 1, 2, 2.9, 3, 3.1, 4.1, and 4.2 and very similar to maps of the apparently closely homologous polypeptides, Bands 2.1, 2.2, 2.3, and 2.6. Limited proteolysis of erythrocyte membranes is shown to generate Band 3', another polypeptide which has been associated with spectrin-binding activity. Peptide maps of Band 3' are very similar to maps of Band 2.1, suggesting that Band 3' is also a proteolytic fragment of Band 2.1. It is concluded that Band 2.1 and possibly some or all of the other, related polypeptides which electrophorese in the 2 region is (are) the spectrin-binding protein(s) of the human erythrocyte.
The polypeptides of the human erythrocyte membrane were analyzed by polyacrylamide gel electrophoresis in 1% sodium dodecyl sulfate. Six major bands (I-VI) together make up over two-thirds of the protein staining profile. Component III (mol wt 89,000) predominates in the ghost membrane; it constitutes 30% of the protein and numbers over 106 chains/ghost. Components I and II form a slow-moving doublet (approximate mol wt 250,000) containing 25% of the protein. The molar amounts of I + II, IV (mol wt 77,500), V (mol wt 41,300), and VI (mol wt 36,200) are similar, falling in the range 3.4-4.6 × 105 chains/ghost. Four bands were recognized in gels stained by the periodic acid-Schiff procedure. A broad Schiff-positive zone just behind the tracking dye corresponds to membrane lipids. Three bands of lower mobility are sialoglycoproteins. The most prominent of these has an apparent molecular weight of 83,500 and contains at least 57% of the sialic acid of ghosts. The Schiff-positive bands were not colored by protein stains. Sialidase treatment of ghosts selectively increased the mobilities of the sialoglycoproteins without affecting the protein-staining profile. Attempts to produce subunits from the large polypeptides by treatment with various denaturing agents were unsuccessful. Normally, no polypeptides of size less than 15,000 were seen in ghost electrophorograms. However, heating ghosts with low levels of sodium dodecyl sulfate and high levels of salt produced diffuse bands of low average molecular weight. This highly variable effect is attributed to degradation by proteinases. Components I, II, and V were solubilized by incubating ghosts at low ionic strength. Component VI was released by washing with buffered saline at concentrations above 0.1 M. Both elution procedures were rapid (15 min), complete, and selective; they were also conservative in that new bands were not created and the electrophorograms of released and retained material were complementary. The eluted material contained negligible sialic acid and no Schiff-positive lipids. Two classes of membrane protein were distinguished by their response to the elution procedures. Components I, II, V, and VI compose one class. They make up 30-35 % of the protein and are tenuously related to the membrane, possibly by predominantly ionic bonds. The second class, which includes components III, IV, and the sialoglycoproteins, together with various minor components, constitutes 65-70% of the protein. These polypeptides are tightly bound; their properties may reflect participation in hydrophobic protein-protein and protein-lipid interactions.
Specific associations of spectrin with Bands 2.1 and 4.1 have been examined by measuring the binding of purified 125I-Band 2.1 and 125I-Band 4.1 to [32P]spectrin in solution. Binding of Bands 2.1 and 4.1 to spectrin was measured as 125I radioactivity precipitated by an anti-spectrin. Staphylococcus aureus complex. The association between spectrin and Band 2.1 is characterized by relatively high affinity (Kd congruent to 10(-7) M at pH 7.6) and saturation of available binding sites at a molar ratio of 1:1 (Band 2.1/spectrin heterodimer). Band 4.1 binding to spectrin is characterized by a similar affinity (Kd congruent to 10(-7) M at pH 7.6) with saturation of available sites occurring at a stoichiometric ration of 2:1 (Band 4.1/spectrin heterodimer). Scatchard plots of Band 4.1 binding to spectrin are curvilinear and consistent with a positively cooperative interation. Bands 2.1 and 4.1 bind to different sites on the spectrin molecule: unlabeled Band 4.1 does not competitively displace 125 I-Band 2.1 from spectrin in solution, and low angle rotary-shadowed platinum-carbon replicas of these polypeptides reveal two discrete binding sites.
A complex of spectrin and actin, isolated from sheep erythrocyte ghosts, accelerates the polymerization of actin in buffer containing 0.3 mM MgCl 2. The rate of actin polymerization is similarly increased by sonicated F-actin nuclei. At steady state, the critical concentration of actin is lower when polymerization occurs in the presence of spectrin/actin complex than in its absence. Polymerization of actin in the presence of spectrin/actin complex is inhibited by substoichiometric concentrations of cytochalasin D, which is thought to block the net polymerizing ends of growing actin filaments, in the same way that cytochalasin D inhibits actin polymerization in the absence of complex. However, actin filaments formed in the presence of complex are more stable to cytochalasin D added after polymerization has occurred than are filaments formed in the absence of complex. We conclude from these data that spectrin/actin complex accelerates actin polymerization by simple nucleation and, therefore, that the complex consists of short actin oligomers cross-linked by spectrin tetramers which stabilize the net depolymerizing ends of the actin oligomers. The isolated spectrin/actin complex is a fragment of the erythrocyte cytoskeleton. From our proposal for the structure of the complex and estimates of others for the relative amounts of actin, spectrin, and spectrin/actin complex-related high affinity cytochalasin binding sites in human erythrocyte ghosts, we suggest that the cytoskeletal network contains short oligomers of actin consisting on average of about ten subunits each, with one of every two actin subunits cross-linked by a spectrin tetramer to a subunit of another actin oligomer.
The phosphorylation of spectrin polypeptide 2 is thought to be involved in the metabolically dependent regulation of red cell shape and deformability. Spectrin phosphorylation is not affected by cAMP. The reaction in isolated membranes resembles the cAMP-independent, salt-stimulated phosphorylation of an exogenous substrate, casein, by enzyme(s) present both in isolated membranes and cytoplasmic extracts. Spectrin kinase is selectively eluted from membranes by 0.5 M NaCl and co-fractionates with eluted casein kinase. Phosphorylation of band 3 in the membrane is inhibited by salt, but the band 3 kinase is otherwise indistinguishable operationally from spectrin kinase. The membrane-bound casein (spectrin) kinase is not eluted efficiently with spectrin at low ionic strength; about 80% of the activity is apparently bound at sites (perhaps on or near band 3) other than spectrin. Partitioning of casein kinase between cytoplasm and membrane is metabolically dependent; the proportion of casein kinase on the membrane can range from 25% to 75%, but for fresh cells is normally about 40%. Dephosphorylation of phosphorylated spectrin has not been studied intensively. Slow release of 32Pi from [32P] spectrin on the membrane can be demonstrated, but phosphatase activity measured against solubilized [32P] spectrin is concentrated in the cytoplasm. The crude cytoplasmic phosphospectrin phosphatase is inhibited by various anions – notably, ATP and 2,3-DPG at physiological concentrations. Regulation of spectrin phosphorylation in intact cells has not been studied. We speculate that spectrin phosphorylation state may be regulated (1) by metabolic intermediates and other internal chemical signals that modulate kinase and phosphatase activities per se or determine their intracellular localization and (2) by membrane deformation that alters enzyme–spectrin interaction locally. Progress in the isolation and characterization of spectrin kinase and phosphospectrin phosphatase should lead to the resolution of major questions raised by previous work: the relationships between membrane-bound and cytoplasmic forms of the enzymes, the nature of their physical interactions with the membrane, and the regulation of their activities in defined cell-free systems.
The organization of erythrocyte membrane lipids and proteins has been studied following the release of cytoplasmic components with the non-ionic detergent Triton X-100. After detergent extraction, a detergent-resistant complex called the erythrocyte cytoskeleton is separated from detergent, solubilized lipid and protein by sucrose buoyant density sedimentation. In cytoskeletons prepared under isotonic conditions all of the major erythrocyte membrane proteins are retained except for the integral protein, glycophorin, which is quantitatively solubilized and another integral glycoprotein, band 3, which is only 60% removed. When cytoskeletons are prepared in hypertonic KCl solutions, band 3 is fully solubilized along with bands 2.1 and 4.2 and several minor components. The resulting cytoskeletons have the same morphology as those prepared in isotonic buffer but they are composed of only three major peripheral proteins, spectrin, actin and band 4.1. We have designated this peripheral protein complex the ‘shell’ of the erythrocyte membrane, and have shown that the attachment of band 3 to the shell satisfies the criteria for a specific interaction. Although Triton did affect erythrocyte shape, cytoskeleton lipid content and the activity of membrane proteases, there was no indication that Triton altered the attachment of band 3 to the shell. We suggest that band 3 attaches to the shell as part of a ternary complex of bands 2.1, 3 and 4.2.
At least two kinds of enzymes are active in the proteolytic self-digestion of erythrocyte membranes. The specific activities of these enzymes do not decrease with repeated washings of purified stroma. The effects of a variety of inhibitors on the membrane preparation's capacity to digest 125-I-labelled casein, covalently linked to latex beads, have been examined. Pepstatin-inhibitable enzyme, active at low pH, digests the membrane extensively to small polypeptide fragments. Spectrin, located at the internal part of the membrane, is readily degraded. Diisopropylfluorophosphate-inhibitable enzyme, active at pH 8-9, has only limited digestive capacity. Some of the membrane components, such as the small molecular weight glycoproteins, are resistant to digestion. The restricted capacity of digestion is due to the membrane molecular arrangement; increased disaggregation removes the restriction and increases the activity. Spectrin is not digested unless the membrane topography is disrupted by NP-40 neutral detergent. These observations suggest that the enzymes active at basic pH are located external to the cell. Intact cells do possess a limited capacity to degrade 125-I-labelled casein when their surfaces are brought into contact with substrate-coated beads.
In this paper we have assumed that recombination is a random process. However, if recombination in the human female involved a process similar to copy choice, then El1 could easily lie between Rh and αFUC. If subsequent investigators are able to show that El1 does not lie distal to both Rh and αFUC then family MRC 1817 provides the first human example of triple recombination between markers on a single chromosome arm. Moreover this would have occurred in less than the distal half of 1p which would suggest that the human genome is capable of rather more recombination during female meiosis than some cytogenic observations imply. A family which segregates simultaneously for PGD, elliptocytosis, Rh, αFUC and PGM1 contains a recombinant suggesting that the loci lie in this order.
Erythrocytes from neonates with elliptocytosis were studied for their pattern of heat-induced fragmentation. Membrane alterations began at 44 degrees C. There was a gradual progression in shape changes as the temperature was increased to 47 degrees C, at which point frank fragmentation occurred. Normal erythrocytes show no morphologic changes until the critical temperature of fragmentation, 49 degrees C is reached. Heat studies were repeated a few months later, at a time when the patients' erythrocyte morphology had become typical of elliptocytosis. Morphologic changes occurred abruptly at 48 degrees C with complete fragmentation. Increased thermal sensitivity of the red cell membrane has previously been demonstrated for pyropoikilocytes, and these studies suggest that some cases of elliptocytosis may be mild expressions of a similar membrane defect.
The formation of a high-molecular weight complex between spectrin and F-actin depends on the presence of a third cytoskeletal constituent, protein 4.1. Electron microscopy shows that in this ternary complex the actin filaments are linked by bridges, which have the appearance of spectrin. The spectrin must be in the tetrameric state for such bridges to form: the dimer is evidently univalent, for it binds but forms no cross-links. G-actin also fails to form extended complexes. It is inferred that in the native cytoskeleton the spectrin is tetrameric and associated with 4.1 and probably oligomers of actin.
Molecules of human erythrocyte spectrin have been examined by electron microscopy after low-angle shadowing. Spectrin heterodimers and tetramers were first purified and characterized by polyacrylamide gel electrophoresis and analytical ultracentrifugation under conditions which minimize proteolysis and aggregation. The heterodimers and tetramere were separated for low-angle shadowing by gel filtration in ammonium acetate buffer at physiological ionic strength, in which they showed sedimentation coefficients of 8.9 S and 12.5 S, respectively, similar to those values reported for heterodimers and tetramers in non-volatile buffers. The ammonium acetate buffer promoted the dissociation of spectrin tetramers into heterodimers under conditions in which tetramers in NaCl or KCl buffers are stable. When visualized by low-angle unidirectional and rotary shadowing, spectrin heterodimers appeared as long flexible molecules with a mean shadowed length of 97 nm. Each heterodimer, composed of the two polypeptide chains, band 1 (240,000 Mr) and band 2 (220,000 Mr), often appeared as two separate strands which lay partially separated from one another or coiled round each other in a loose double helix. The association between these polypeptides appears to be weak, except at both ends of the molecule where there are sites of strong binding. Tetramers are formed by the end-to-end association of two spectrin heterodimer molecules without measurable overlap, and have a mean shadowed length of 194 nm. This association to form tetramers probably involves head-to-head binding of the heterodimers, since the higher oligomers to be expected from a head-to-tail binding mode are not observed. The molecular shape of spectrin is quite distinct from that of myosin, to which it has often been likened.
In contrast to the disease in humans, hereditary spherocytosis in the common house mouse produces an extreme spherocytosis. The cells show a broad distribution in size ranging from microcytic to macrocytic. Of particular interest is the finding of a substantial reduction in the major membrane polypeptide called spectrin, supporting a critical role for this protein in the control of erythrocyte shape and membrane stability.
After treatment of intact human erythrocytes with SH-oxidizing agents (e.g. tetrathionate and diamide) phospholipase A2 cleaves approx. 30% of the phosphatidylserine and 50% of the phosphatidylethanolamine without causing hemolysis (Haest, C.W.M. and Deuticke, B (1976) Biochim. Biophys. Acta 436, 353--365). These phospholipids are scarcely hydrolysed in fresh erythrocytes and are assumed to be located in the inner lipid layer of the membrane (Verkleij, A.J., Zwaal, R.F.A., Roelofsen, B., Comfurius, P., Kastelijn, D. and van Deenen, L.L.M. (1973) Biochim. Biophys Acta 323, 178--193). The enhancement of the phospholipid cleavage is now shown to be accompanied by a 50% decrease of the membrane SH-groups and a cross-linking of spectrin, located at the inner surface of the membrane, to oligomers of less than 10(6) dalton. Blocking approx. 10% of the membrane SH groups with N-ethylmaleimide suppresses both the polymerization of spectrin and the enhancement of the phospholipid cleavage. N-Ethylmaleimide, under these conditions, reacts with three SH groups per molecule of spectrin, 0.7 SH groups per major intrinsic 100 000 dalton protein (band 3) and 1.1 SH groups per molecule of an extrinsic protein of 72 000 daltons (band 4.2). Blocking studies with iodoacetamide demonstrate that the SH groups of the 100 000-dalton protein are not involved in the effects of the SH-oxidizing agents. It is suggested that a release of constraints imposed by spectrin enables phosphatidylserine and phosphatidylethanolamine to move from the inner to the outer lipid layer of the erythrocyte membrane and that spectrin, in the native erythrocyte, stabilizes the orientation of these phospholipids to the inner surface of the membrane.
Depending on conditions of extraction from the membrane, spectrin may be recovered either as a dimer or a tetramer. It is demonstrated that these species are linked by a simple equilibrium, and can be readily interconverted. At low temperature, however, either species is kinetically trapped, and no interconversion occurs over periods of up to several days. In the range 25-40 °C, equilibrium is achieved on a time scale of hours to minutes. First-order and second-order rate constants and thermodynamic parameters for the equilibrium have been determined. The large activation energy indicates that conformational effects are rate-limiting. In the absence of other proteins, no associated states higher than the tetramer are formed. Neither phosphorylation of the spectrin with endogenous kinase, nor dephosphorylation with phosphatase have any effect on the dimer-tetramer equilibrium, neither do they promote formation of higher associated states. It is therefore improbable that phosphorylation-dependent shape changes in the erythrocyte are related to the association of spectrin per se, and are likely instead to be the direct result of a spectrin-actin interaction. The dimer-tetramer equilibrium is strongly affected by ionic strength, and at low salt concentrations, such as those used to extract spectrin from membranes, the dimer becomes strongly favoured. Thus the recovery of tetramer by extraction at low temperature implies that this is the basic unit present in the membrane. Calcium and magnesium ions cause further association to higher oligomers, though only at high concentrations (in the millimolar range) of the cation.
Spectrin molecules are distributed uniformly throughout the submembranous regions of intact human erythrocytes. Spectrin does not appear to extend into the red blood cell cytoplasm to any significant extent. Thus, it does not form a recognizable internal scaffolding nor does it seem to connect distant segments of the cell membrane. Spectrin retains its submembranous location in the spiny processes of echinocytes produced by ATP depletion. Thus, these processes do not seem to form by a simple extrusion mechanism powered by contraction of the spectrin network. Spectrin seems to be important for the stability of the lipid bilayer of the red cell membrane, and it probably also plays a role in regulating red cell shape. How it performs either function is still unknown.
Irreversibly sickled cells (ISC's) are circulating erythrocytes in patients with sickle cell disease that retain a sickled shape even when oxygenated. Evidence points to a membrane defect that prevents the return of these cells to the normal biconcave shape. The erythrocyte membrane protein spectrin is believed to help control erythrocyte shape and deformability. Recent studies suggest that normally spectrin and an erythrocyte actin form a self-supporting, fibrillar, lattice-like network on the cytoplasmic membrane surface. When normal erythrocyte ghosts are extracted with Triton X-100 all the integral membrane proteins and most of the membrane lipids are removed, leaving a ghost-shaped residue composed principally of spectrin and actin. We concentrated ISC's from patients with sickle cell anemia and compared the morphology and protein composition of ghosts and Triton-extracted ghost residues prepared from these ISC's with similar preparations of reversibly sickable cells and normal cells. (a) Many ISC's formed ISC-shaped ghosts. (b) All ISC-shaped ghosts formed ISC-shaped Triton residues. (c) Spectrin, erythrocyte actin (Band 5), an unidentified Band 3 component, and Band 4.1 were the major protein components of the Triton residues. All membrane-associated sickle hemoglobin was removed by the Triton treatment. (d) No ISC-shaped ghosts or ISC-shaped Triton residues were formed when deoxygenated, sickled RSC's were lysed or Triton-extracted. ISC-shaped ghosts and Triton residues were never formed from normal cells. These observations suggest that a defect of the "spectrin-actin lattice" may be the primary abnormality of the ISC membrane. Since ISC's are rigid cells, the data support the postulate that spectrin is a major determinant of membrane deformability. Finally, they provide direct evidence that spectrin is important in determining erythrocyte shape.
Microspherocytes, measuring 2-3 mum in diameter, and cells with blunted projections or triangular in shape characterized the erythrocoyte morphology in three children with congenital haemolytic anaemia. Since the erythrocyte morphology resembled that associated with thermal injury, heat-induced changes in erythrocyte morphology and membrane composition were studied. Erythrocytes developed filaments and spheroid bodies which fragmented, resulting in microspherocyte transformation. Normal cells required exposure to 49 degrees C, whereas the patients' cells fragmented at 45 degrees C. Fragmentation was also observed during incubation of patients' cells at 37 degrees C for 17h. The heat-induced transformation of the patients' cells was associated with an increase in the membrane cholesterol:phospholipid and cholesterol:protein ratios. The phospholipid:protein ratio was unchanged. This suggests that fragmentation produces a selective loss of membrane components. Splenectomy ameliorated the haemolytic process. We propose that the patients' red-cell morphology is the result of in vivo fragmentation, and that the spleen is the major site of microspherocyte and poikilocyte destruction.
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