RESUMEN
Pallid is one of 12 independent murine mutations with a prolonged bleeding time that are models for human platelet storage pool deficiencies in which several intracellular organelles are abnormal. We have mapped the murine gene for protein 4.2 (Epb4.2) to chromosome 2 where it co-localizes with pallid. Southern blot analyses suggest that pallid is a mutation in the Epb4.2 gene. Northern blot analyses demonstrate a smaller than normal Epb4.2 transcript in affected pallid tissues, such as kidney and skin. This is the first gene defect to be associated with a platelet storage pool deficiency, and may allow the identification of a novel structure or biological pathway that influences granulogenesis.
Asunto(s)
Proteínas Sanguíneas/genética , Deficiencia de Almacenamiento del Pool Plaquetario/genética , Animales , Mapeo Cromosómico , Proteínas del Citoesqueleto , ADN/genética , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Proteínas de la Membrana , Ratones , Mutación , Fenotipo , ARN/genéticaRESUMEN
Hereditary spherocytosis (HS) is the most common inherited haemolytic anaemia in Northern Europeans. The primary molecular defects reside in the red blood cell (RBC) membrane, particularly in proteins that link the membrane skeleton to the overlying lipid bilayer and its integral membrane constituents. Ankyrin-1 is the predominant linker molecule. It attaches spectrin, the major skeletal protein, to the cytoplasmic domain of band 3, the RBC anion exchanger. Two-thirds of patients with HS have combined spectrin and ankyrin-1 deficiency; deficiency of band 3 occurs in about 15 to 20% (ref.1). These data suggest that ankyrin-1 or band 3 defects may be common in HS. To test this we screened all 42 coding exons plus the 5' untranslated/promoter region of ankyrin-1 and the 19 coding exons of band 3 in 46 HS families. Twelve ankyrin-1 mutations and five band 3 mutations were identified. Missense mutations and a mutation in the putative ankyrin-1 promoter were common in recessive HS. In contrast, ankyrin-1 and band 3 frameshift and nonsense null mutations prevailed in dominant HS. Increased accumulation of the normal protein product partially compensated for the ankyrin-1 or band 3 defects in some of these null mutations. Our findings indicate that ankyrin-1 mutations are a major cause of dominant and recessive HS (approximately 35 to 65%), that band 3 mutations are less common (approximately 15 to 25%), and that the severity of HS is modified by factors other than the primary gene defect.
Asunto(s)
Ancirinas/genética , Mutación , Esferocitosis Hereditaria/genética , Ancirinas/sangre , Secuencia de Bases , Femenino , Genes Dominantes , Genes Recesivos , Heterocigoto , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Polimorfismo Genético , Polimorfismo Conformacional Retorcido-Simple , Regiones Promotoras Genéticas , Esferocitosis Hereditaria/epidemiología , Esferocitosis Hereditaria/etiologíaRESUMEN
We have recently cloned and characterized ankyrin-3 (also called ankyrin(G)), a new ankyrin that is widely distributed, especially in epithelial tissues, muscle, and neuronal axons (Peters, L.L., K.M. John, F.M. Lu, E.M. Eicher, A. Higgins, M. Yialamas, L.C. Turtzo, A.J. Otsuka, and S.E. Lux. 1995. J. Cell Biol. 130: 313-330). Here we show that in mouse macrophages, ankyrin-3 is expressed exclusively as two small isoforms (120 and 100 kD) that lack the NH2-terminal repeats. Sequence analysis of isolated Ank3 cDNA clones, obtained by reverse transcription and amplification of mouse macrophage RNA (GenBank Nos. U89274 and U89275), reveals spectrin-binding and regulatory domains identical to those in kidney ankyrin-3 (GenBank No. L40631) preceded by a 29-amino acid segment of the membrane ("repeat") domain, beginning near the end of the last repeat. Antibodies specific for the regulatory and spectrin-binding domains of ankyrin-3 localize the protein to the surface of intracellular vesicles throughout the macrophage cytoplasm. It is not found on the plasma membrane. Also, epitope-tagged mouse macrophage ankyrin-3, transiently expressed in COS cells, associates with intracellular, not plasma, membranes. In contrast, ankyrin-1 (erythrocyte ankyrin, ankyrin(R)), which is also expressed in mouse macrophages, is located exclusively on the plasma membrane. The ankyrin-3-positive vesicles appear dark on phase-contrast microscopy. Two observations suggest that they are lysosomes. First, they are a late compartment in the endocytic pathway. They are only accessible to a fluorescent endocytic tracer (FITC-dextran) after a 24-h incubation, at which time all of the FITC-dextran-containing vesicles contain ankyrin-3 and vice versa. Second, the ankyrin-3-positive vesicles contain lysosomal-associated membrane glycoprotein (LAMP-1), a recognized lysosomal marker. This is the first evidence for the association of an ankyrin with lysosomes and is an example of two ankyrins present in the same cell that segregate to different locations.
Asunto(s)
Ancirinas/análisis , Lisosomas/química , Macrófagos/química , Secuencia de Aminoácidos , Animales , Ancirinas/genética , Antígenos CD/análisis , Secuencia de Bases , Células de la Médula Ósea , Células COS , Clonación Molecular , ADN Complementario/genética , Membranas Intracelulares/química , Riñón/química , Proteínas de Membrana de los Lisosomas , Glicoproteínas de Membrana/análisis , Ratones , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos Nucleicos/genética , Análisis de Secuencia de ADN , Espectrina/metabolismo , TransfecciónRESUMEN
Protein 4.1 is a crucial component of the erythrocyte membrane skeleton. Responsible for the amplification of the spectrin-actin interaction, its presence is required for the maintenance of erythrocyte integrity. We have demonstrated a 4.1-like protein in nonerythroid cells. An antibody was raised to erythrocyte protein 4.1 purified by KCl extraction (Tyler, J. M., W. R. Hargreaves, and D. Branton, 1979, Proc. Natl. Acad. Sci. USA, 76:5192-5196), and used to identify a serologically cross-reactive protein in polymorphonuclear leukocytes, platelets, and lymphoid cells. The cross-reactive protein(s) were localized to various regions of the cells by immunofluorescence microscopy. Quantitative adsorption studies indicated that at least 30-60% of the anti-4.1 antibodies reacted with this protein, demonstrating significant homology between the erythroid and nonerythroid species. A homologous peptide doublet was observed on immunopeptide maps, although there was not complete identity between the two proteins. When compared with erythrocyte protein 4.1, the nonerythroid protein(s) displayed a lower molecular weight--68,000 as compared with 78,000-and did not bind spectrin or the nonerythroid actin-binding protein filamin. There was no detectable cross-reactivity between human acumentin or human tropomyosin-binding protein, which are similarly sized actin-associated proteins, and erythrocyte protein 4.1. The possible origin and significance of 4.1-related protein(s) in nonerythroid cells are discussed.
Asunto(s)
Plaquetas/análisis , Proteínas Sanguíneas/aislamiento & purificación , Proteínas del Citoesqueleto , Membrana Eritrocítica/análisis , Proteínas de la Membrana/sangre , Neuropéptidos , Neutrófilos/análisis , Complejo Antígeno-Anticuerpo , Membrana Celular/análisis , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida/métodos , Epítopos/análisis , Humanos , Sueros Inmunes , Proteínas de la Membrana/aislamiento & purificación , Peso MolecularRESUMEN
Mice homozygous for the nb mutation (Chromosome 8) have a severe hemolytic anemia and develop a psychomotor disorder at 6 mo of age. The nb/nb mice are deficient in erythroid ankyrin (Ank-1) but, until the present study, the role of Ank-1 and of Ank-2 (brain ankyrin) in disease genesis was unknown. In normal erythroid tissues, we show that two major transcripts are expressed from Ank-1, and one of these is also present at high levels in the cerebellum. By in situ hybridization and immunocytochemistry, Ank-1 localizes to the cerebellar Purkinje cells and, to a lesser extent, the granule cells. In nb/nb mice, Ank-1 transcripts are markedly reduced in both erythroid and neural tissue, and nb/nb Purkinje cells and granule cells are nearly devoid of Ank-1. The neurological syndrome appears concurrently with a dramatic loss of Purkinje cells. Ank-2 maps to Chromosome 3 and its expression is unaffected by the nb mutation. We conclude that Ank-1 is specifically required for Purkinje cell stability and, in its absence, Purkinje cell loss and neurological symptoms appear.
Asunto(s)
Proteínas Sanguíneas/deficiencia , Encéfalo/fisiopatología , Cerebelo/patología , Proteínas de la Membrana/deficiencia , Células de Purkinje/patología , Anemia Hemolítica/sangre , Anemia Hemolítica/genética , Anemia Hemolítica/patología , Animales , Ancirinas , Proteínas Sanguíneas/análisis , Proteínas Sanguíneas/genética , Encéfalo/fisiología , Mapeo Cromosómico , Membrana Eritrocítica/fisiología , Ligamiento Genético , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Ratones , Ratones Mutantes , Poli A/análisis , Poli A/genética , ARN/análisis , ARN/genética , ARN Mensajero , Valores de Referencia , Reticulocitos/fisiología , Transcripción GenéticaRESUMEN
We cloned a novel ankyrin, Ank3, from mouse kidney cDNA. The full-length transcript is predicted to encode a 214-kD protein containing an 89 kD, NH2 terminal "repeat" domain; a 65 kD, central "spectrin-binding" domain; and a 56 kD, COOH-terminal "regulatory" domain. The Ank3 gene maps to mouse Chromosome 10, approximately 36 cM from the centromere, a locus distinct from Ank1 and Ank2. Ank3 is the major kidney ankyrin. Multiple transcripts of approximately 7.5, 6.9, 6.3, 5.7, 5.1, and 4.6 kb are highly expressed in kidney where Ank1 and Ank2 mRNAs are barely detectable. The smaller mRNAs (< or = 6.3 kb) lack the entire repeat domain. These transcripts have a unique 5'untranslated region and NH2-terminal sequence and encode a predicted protein of 121 kD. Two small sequences of 21 and 18 amino acids are alternatively spliced at the junction of the repeat and spectrin-binding domains in the larger (> or = 6.9 kb) RNAs. Alternative splicing of a 588 bp sequence (corresponding to a 21.5-kD acidic amino acid sequence) within the regulatory domain also occurs. Ank3 is much more widely expressed than previously described ankyrins. By Northern hybridization or immunocytochemistry, it is present in most epithelial cells, in neuronal axons, in muscle cells, and in megakaryocytes/platelets, macrophages, and the interstitial cells of Leydig (testis). On immunoblots, an antibody raised to a unique regions of the regulatory domain detects multiple Ank3 isoforms in the kidney (215, 200, 170, 120, 105 kD) and in other tissues. The 215/200 kD and 120/105-kD kidney proteins are close to the sizes predicted for the 7.5/6.9- and 6.3/5.7-kb RNAs (with/without the 588-bp acidic insert). Interestingly, it appears that Ank3 exhibits a polarized distribution only in tissues that express the approximately 7.0-kb isoforms, the only isoforms in the kidney that contain the repeat domain. In tissues where smaller transcripts (< or = 6.3 kb) are expressed. Ank3 is diffusely distributed in some or all cells and may be associated with cytoplasmic structures. We conclude that Ank3 is a broadly distributed epithelial ankyrin and is the major ankyrin in the kidney and other tissues, where it plays an important role in the polarized distribution of many integral membrane proteins.
Asunto(s)
Empalme Alternativo , Ancirinas/análisis , Ancirinas/genética , Riñón/química , Secuencia de Aminoácidos , Animales , Ancirinas/química , Ancirinas/aislamiento & purificación , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Cruzamientos Genéticos , ADN Complementario/genética , Epitelio/química , Immunoblotting , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Músculos/química , Neuronas/química , Especificidad de Órganos , ARN Mensajero/análisis , ARN Mensajero/genética , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
The hereditary hemolytic anemias include a heterogeneous class of disorders caused by defects in the proteins that constitute the membrane skeleton of the red blood cell. The combination of classical and molecular genetics together with clinical findings is rapidly improving our understanding of the basis of these defects.
Asunto(s)
Anemia Hemolítica/genética , Membrana Eritrocítica/patología , Glicoproteínas de Membrana/genética , Anemia Hemolítica/patología , Animales , Humanos , Modelos GenéticosRESUMEN
Thalassemic red cells show irregular morphology and maldistribution of glycoproteins and sialic acids. These changes are compatible with damage to the red cell membrane skeleton. To test this possibility, we systematically studied the interconnections of skeletal proteins in patients with a form of alpha thalassemia (HbH disease), in patients with beta thalassemia intermedia, and in normal individuals. Alpha- and beta-thalassemic spectrin functions normally in spectrin self-association, binding to normal inside-out vesicles (IOVs), and binding to actin in the presence and absence of normal protein 4.1. Binding of normal spectrin to beta: thalassemic IOVs is normal but alpha-thalassemic IOVs are defective and bind only half the normal amount of spectrin (66 +/- 5 vs. 120 +/- 16 micrograms spectrin dimer/mg IOV protein, respectively). A different defect is detected in beta thalassemia, in which protein 4.1 shows markedly reduced ability (48 +/- 7% of normal) to enhance the binding of normal spectrin to actin and a decreased ability to bind normal spectrin in a binary interaction, compared with normal protein 4.1 (24 +/- 1 and 43 +/- 1 micrograms protein 4.1/mg spectrin, respectively). As no quantitative deficiency of beta-thalassemic protein 4.1 is detected, we assume an acquired lesion is present, which affects about half of the protein 4.1 molecules. These findings indicate that specific, localized, yet different defects exist in the skeletal proteins of alpha- and beta-thalassemic red cells. The different molecular lesions imply that the mechanism of hemolysis and probably the interaction of unpaired globin chains with the membrane differs in the two diseases.
Asunto(s)
Proteínas del Citoesqueleto/análisis , Membrana Eritrocítica/análisis , Neuropéptidos , Talasemia/sangre , Ancirinas , Proteínas Sanguíneas/metabolismo , Electroforesis en Gel de Poliacrilamida , Humanos , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/metabolismo , Espectrina/análisisRESUMEN
A patient with xerocytosis was found to have swimming-induced intravascular hemolysis and shortening of erythrocyte life-span. In a microviscometer, xerocytes were more susceptible than normal erythrocytes to hemolysis by shear stress. Fractionation of normal and abnormal cells on discontinuous Stractan density gradients revealed that increasingly dehydrated cells were increasingly more shear sensitive. This sensitivity was partially corrected by rehydrating xerocytic erythrocytes by means of the cation-ionophore nystatin in a high potassium buffer. Conversely, normal erythrocytes were rendered shear sensitive by dehydrating them with nystatin in a low potassium buffer. This effect of dehydration was entirely reversible if normal cells were dehydrated for less than 4 h but was only partially reversed after more prolonged dehydration. It is likely that dehydration of erythrocytes results in shear sensitivity primarily because of concentration of cell contents and reduced cellular deformability. With prolonged dehydration, secondary membrane changes may potentiate the primary effect. This increased shear sensitivity of dehydrated cells may explain atraumatic exercise-induced hemolysis in xerocytosis as cardiac output is shifted to vessels of exercising muscles with small diameters and high shear rates.
Asunto(s)
Anemia Hemolítica/sangre , Eritrocitos Anormales/fisiología , Esfuerzo Físico , Natación , Adulto , Desecación , Envejecimiento Eritrocítico , Hemólisis , Humanos , Masculino , Reología , Estrés Mecánico , Equilibrio HidroelectrolíticoRESUMEN
Hereditary spherocytosis (HS) is an inherited disorder of erythrocyte shape associated with spectrin deficiency and hemolytic anemia. In a subset of patients with the autosomal dominant form of HS, spectrin displays a reduced capacity to bind protein 4.1 and, therefore, actin; both functions that are critical to the membrane skeleton. A specific structural defect has not been identified in the spectrin from these patients. Chymotryptic digestion of the isolated spectrin chains shows impaired cleavage of the distal peptide of the beta subunit, the beta IV domain. In previous work, we have shown that mild oxidation markedly diminishes the binding capacity of normal spectrin for protein 4.1. Here we observe that chemical reduction of freshly isolated, untreated HS spectrin dramatically improves its function. Thus, a primary structural defect in the beta subunit of spectrin in this subtype of HS may lead to oxidant sensitivity, and secondarily, to a functional defect in the binding of spectrin to protein 4.1 and actin.
Asunto(s)
Proteínas Sanguíneas/metabolismo , Proteínas del Citoesqueleto , Proteínas de la Membrana , Neuropéptidos , Espectrina/fisiología , Esferocitosis Hereditaria/fisiopatología , Disulfuros , Humanos , Sustancias Macromoleculares , Peso Molecular , Mutación , Oxidación-Reducción , Mapeo Peptídico , Unión Proteica , Conformación Proteica , Espectrina/genética , Esferocitosis Hereditaria/patología , Relación Estructura-ActividadRESUMEN
The mechanism by which sickle cells and xerocytic red cells become depleted of cations in vivo has not been identified previously. Both types of cells exhibit elevated permeabilities to sodium and potassium, in the case of sickle cells, when deoxygenated. The ouabain-insensitive fluxes of sodium and potassium were equivalent, however, in both cell types under these conditions. When incubated 18 hours in vitro, sickle cells lost cations but only when deoxygenated. This cation depletion was blocked by ouabain, removal of external potassium, or pretreatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonate, which blocks the increase in cation permeability induced by deoxygenation. The loss of cation exhibited by oxygenated xerocytes similarly incubated was also blocked by ouabain. These data support the hypothesis that the elevated "passive" cation fluxes of xerocytes and deoxygenated sickle cells are not directly responsible for cation depletion of these cells; rather, these pathologic leaks interact with the sodium pump to produce a net loss of cellular cation.
Asunto(s)
Anemia Hemolítica Congénita/sangre , Anemia de Células Falciformes/sangre , Eritrocitos/metabolismo , Canales Iónicos/fisiología , Potasio/metabolismo , Sodio/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/análogos & derivados , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Transporte Biológico , Calcio/fisiología , Deshidratación/sangre , Hemoglobinas/análisis , Humanos , Ouabaína/farmacologíaRESUMEN
Although functional abnormalities of the sickle erythrocyte membrane skeleton have been described, there is little quantitative data on the function of the proteins that compose the skeleton. We have examined the association of spectrin, the major skeletal protein, with ankyrin, its high-affinity membrane binding site, and found sickle erythrocytes to have markedly reduced binding. Binding is assayed by incubation of purified 125I-spectrin with spectrin-depleted inside-out vesicles (IOVs) and measurement of the label bound to IOVs. Sickle IOVs bind approximately 50% less ankyrin than do controls IOVs (P less than 0.001). Control experiments show that this reduced binding is not a function of faulty composition or orientation of sickle IOVs, or of reticulocytosis per se. Our least symptomatic patient has the highest binding capacity, suggesting that this abnormality may be related to clinical severity. This trend is supported by experiments showing that asymptomatic subjects with sickle trait, sickle cell anemia and high fetal hemoglobin, and sickle beta +-thalassemia have normal binding, whereas a symptomatic patient with sickle beta zero-thalassemia has abnormal binding. In contrast to what we see with ankyrin in situ on the IOV, when isolated and studied in solution, sickle ankyrin binds normally to spectrin. This discrepancy may be related to preferential purification of the normal ankyrin species or to an abnormal topography of the membrane near the spectrin attachment site. We hypothesize that sickle hemoglobin or perhaps the metabolic consequences of sickling damage the protein skeleton. This damage may alter the surface of the erythrocyte and result in abnormal cell-cell interactions which may be related to clinical severity.
Asunto(s)
Anemia de Células Falciformes/patología , Membrana Eritrocítica/ultraestructura , Proteínas de la Membrana , Espectrina/metabolismo , Ancirinas , Membrana Eritrocítica/análisis , Hemoglobinas/análisis , Humanos , Canales Iónicos/metabolismo , Membrana Dobles de Lípidos , Proteínas de la Membrana/metabolismo , Unión Proteica , Reticulocitos/fisiologíaRESUMEN
During liquid preservation under blood bank conditions, red cell membranes inexorably undergo damage that decreases erythrocyte survival after transfusion. Accordingly, we have surveyed membrane skeletal protein interactions during storage. We uncovered a decrease in the in vitro formation of spectrin-actin complex in the absence (50%) or presence (60%) of protein 4.1. Actual formation of the spectrin-actin-protein 4.1 complex fell in a linear fashion during the storage period. This fall in spectrin-actin interaction tightly correlated with the decline in total red cell phospholipid (R = 0.9932) measured simultaneously. This decrement of spectrin-actin association could be restored to greater than 70% of normal values by preincubation of stored spectrin with 50 mM dithiothreitol. This storage injury to spectrin-actin interaction might weaken the membrane skeleton and lead to decreased red cell survival. In vitro reversibility of the damage by reducing agents suggests a possible new direction for prolonging the shelf life of stored blood.
Asunto(s)
Actinas/metabolismo , Conservación de la Sangre , Proteínas Sanguíneas/metabolismo , Proteínas del Citoesqueleto , Membrana Eritrocítica/análisis , Neuropéptidos , Espectrina/metabolismo , Adenosina Trifosfato/análisis , Bancos de Sangre , Electroforesis en Gel de Poliacrilamida , Envejecimiento Eritrocítico , Humanos , Proteínas de la Membrana/análisis , Oxidación-Reducción , Fosfolípidos/análisisRESUMEN
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.
Asunto(s)
Adenosina Trifosfato/farmacología , Eritrocitos/efectos de los fármacos , Proteínas de la Membrana/aislamiento & purificación , Espectrina/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Membrana Eritrocítica/anatomía & histología , Membrana Eritrocítica/efectos de los fármacos , Membrana Eritrocítica/metabolismo , Eritrocitos/anatomía & histología , Eritrocitos/metabolismo , Hemólisis , Humanos , Concentración de Iones de Hidrógeno , Microscopía Electrónica , Desnaturalización Proteica , Espectrina/metabolismo , Temperatura , ViscosidadRESUMEN
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.
Asunto(s)
Actinas , Anemia de Células Falciformes/sangre , Eritrocitos Anormales , Anemia de Células Falciformes/etiología , Eritrocitos Anormales/análisis , PolietilenglicolesRESUMEN
High density lipoproteins (d 1.063-1.210 g/ml) were isolated from the plasma of normal individuals (HDL) and seven homozygous patients with Tangier disease (HDLt). In Tangier patients, the concentration of protein in the high density region (HDLt) was only 0.5-4.5% of normal. Immunochemical studies, including mixing experiments conducted in vivo and in vitro, indicated that HDLt was different from HDL. HDLt was the only high density lipoprotein detectable in the plasma of Tangier homozygotes. In heterozygotes both HDL and HDLt were present. HDLt was not detected in the plasma of over 300 normal persons and 10 patients with secondary high density lipoprotein deficiency and appeared to be a unique marker for Tangier disease.ApoHDL contained two major apoproteins designated apoLp-Gln-I and apoLp-Gln-II; together they comprised 85-90% of the total protein content. Both of the major HDL apoproteins were present in apoHDLt; but apoLp-Gln-I was disproportionately decreased with respect to apoLp-Gln-II, the ratio of their concentrations being 1: 12 in apoHDLt as compared with 3: 1 in apoHDL. Several minor apoprotein components which together comprise 5-15% of apoHDL were present in approximately normal proportions in apoHDLt. In the HDL of Tangier patients it was estimated that, compared with normal individuals, the concentration of apoLp-Gln-I was decreased about 600-fold and the concentration of apoLp-Gln-II about 17-fold. The decrease in these apoproteins was not due to preferential segregation with the lipoprotein fractions of d < 1.063 g/ml or with the plasma proteins of d > 1.21 g/ml. Tangier apoLp-Gln-I and apoLp-Gln-II appeared to be immunochemically identical with their normal counterparts, and no differences between the two sets of apoproteins were detected on polyacrylamide gel electrophoresis at pH 9.4 or 2.9. These results are most compatible with the hypothesis that the hereditary defect in Tangier disease is a mutation in an allele-regulating synthesis of apoLp-Gln-I.
Asunto(s)
Errores Innatos del Metabolismo Lipídico/sangre , Lipoproteínas/sangre , Alelos , Aminoácidos/análisis , Reacciones Antígeno-Anticuerpo , Apoproteínas/aislamiento & purificación , Centrifugación por Gradiente de Densidad , Cromatografía de Afinidad , Cromatografía DEAE-Celulosa , Cromatografía en Gel , Carbohidratos de la Dieta , Electroforesis , Heterocigoto , Homocigoto , Humanos , Hiperlipidemias/sangre , Inmunodifusión , Inmunoelectroforesis , Lipoproteínas HDL/análisis , Lipoproteínas HDL/sangre , Lipoproteínas HDL/aislamiento & purificación , Hepatopatías/sangre , MutaciónRESUMEN
We analyzed the DNA sequence of the cDNA encoding the NH2 terminal region of beta spectrin from members of a kindred with autosomal dominant hereditary spherocytosis associated with defective protein 4.1 binding. We found a point mutation at codon 202 within the 272 amino acid NH2-terminal region of beta spectrin. TGG was changed to CGG, resulting in the replacement of tryptophan by arginine. The base change eliminates a normally occurring PvuII restriction site and creates a new MspI site. This finding enabled rapid detection or exclusion of the mutation at the DNA level among the family members, including one member for whom this analysis was performed prenatally. The mutation was found only in the affected family members and occurred as a de novo mutation in the proband. It has not been found in 20 other kindreds. The recombinant peptide derived from the normal cDNA retains the capacity to sediment with protein 4.1 and F-actin. The mutant peptide spontaneously degrades. This variant represents both the first point mutation and the first beta spectrin mutation demonstrated in autosomal dominant hereditary spherocytosis. Furthermore, the mutation is located within a conserved sequence among spectrinlike proteins and may define an amino acid critical for protein 4.1 binding activity.
Asunto(s)
Proteínas del Citoesqueleto , Variación Genética , Proteínas de la Membrana/metabolismo , Neuropéptidos , Mutación Puntual , Espectrina/genética , Esferocitosis Hereditaria/genética , Adulto , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Niño , ADN/sangre , Drosophila/genética , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Linaje , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Longitud del Fragmento de Restricción , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Espectrina/metabolismo , Esferocitosis Hereditaria/sangreRESUMEN
The precise pathogenic mechanism of platelet destruction in immune thrombocytopenias is not known, although many investigators have found that platelet-associated IgG is increased in these diseases. We report here the differentiation between specific binding of anti-platelet antibody, associated with platelet destruction, and the ubiquitous presence of nonspecific, platelet-associated IgG. Using an electrophoretic separation and antibody overlay technique, we have identified a specific membrane protein that bears target platelet antigens in immune thrombocytopenias. When posttransfusion purpura serum was studied, antibody binding to the PlA1 antigen on glycoprotein IIIa was readily distinguished from the nonspecific binding of immunoglobulin to a protein of 200,000 mol wt. After reduction of disulfide bonds, the PlA1 antigenicity was not observed, and IgG bound nonspecifically to a protein band with an apparent molecular weight of 45,000. We have also identified anti-platelet antibodies in patients with idiopathic thrombocytopenic purpura and determined their antigenic specificity. Antibodies which bind to a 100,000-mol wt protein were found in nine of thirteen patients with chronic disease. The antigens in three of these cases were studied in detail by using both reduced and nonreduced control and Glanzmann's thrombasthenic platelets. Target antigens were localized to glycoprotein IIIa, but are different from PlA1. The immune thrombocytopenic purpura antigenic system is clearly distinguished from nonspecific platelet-associated IgG. Sera from eight children with acute idiopathic thrombocytopenic purpura were also studied. In all cases, the nonspecific IgG binding to the 200,000-mol wt protein was observed. However, we were unable to demonstrate antibody binding to glycoprotein IIIa, which suggested that the acute childhood form of this disease may have a different pathogenic mechanism than that of the autoimmune chronic cases.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Plaquetas/inmunología , Glicoproteínas/inmunología , Proteínas de la Membrana/inmunología , Trombocitopenia/inmunología , Adolescente , Adulto , Especificidad de Anticuerpos , Antígenos de Superficie/inmunología , Autoanticuerpos/inmunología , Niño , Preescolar , Femenino , Humanos , Masculino , Glicoproteínas de Membrana PlaquetariaRESUMEN
Protein 4.2 is a major component of the red blood cell (RBC) membrane skeleton. We used targeted mutagenesis in embryonic stem (ES) cells to elucidate protein 4.2 functions in vivo. Protein 4. 2-null (4.2(-/-)) mice have mild hereditary spherocytosis (HS). Scanning electron microscopy and ektacytometry confirm loss of membrane surface in 4.2(-/-) RBCs. The membrane skeleton architecture is intact, and the spectrin and ankyrin content of 4. 2(-/-) RBCs are normal. Band 3 and band 3-mediated anion transport are decreased. Protein 4.2(-/-) RBCs show altered cation content (increased K+/decreased Na+)resulting in dehydration. The passive Na+ permeability and the activities of the Na-K-2Cl and K-Cl cotransporters, the Na/H exchanger, and the Gardos channel in 4. 2(-/-) RBCs are significantly increased. Protein 4.2(-/-) RBCs demonstrate an abnormal regulation of cation transport by cell volume. Cell shrinkage induces a greater activation of Na/H exchange and Na-K-2Cl cotransport in 4.2(-/-) RBCs compared with controls. The increased passive Na+ permeability of 4.2(-/-) RBCs is also dependent on cell shrinkage. We conclude that protein 4.2 is important in the maintenance of normal surface area in RBCs and for normal RBC cation transport.
Asunto(s)
Proteínas Sanguíneas/fisiología , Eritrocitos/metabolismo , Esferocitosis Hereditaria/metabolismo , Animales , Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Proteínas Sanguíneas/genética , Cationes , Permeabilidad de la Membrana Celular , Proteínas del Citoesqueleto , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/ultraestructura , Eritrocitos/ultraestructura , Marcación de Gen , Transporte Iónico , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Potasio/metabolismo , Sodio/metabolismo , Espectrina/metabolismo , Esferocitosis Hereditaria/sangre , Esferocitosis Hereditaria/etiología , Esferocitosis Hereditaria/genéticaRESUMEN
We studied a French kindred with typical hereditary spherocytosis (HS). Studies of erythrocytes and erythrocyte membranes from HS individuals revealed abnormal erythrocyte membrane mechanical stability as well as 15-20% deficiency of band 3, the anion transporter. Anion transport studies of red cells from two affected individuals revealed decreased sulfate flux. Nucleotide sequence of cDNA encoding the distal third of the cytoplasmic domain and the entire transmembrane domain of band 3 obtained by RT-PCR of reticulocyte RNA of an affected family member was normal. Sequence analysis of genomic DNA from an HS individual identified a nonsense mutation of the band 3 gene, Q330X, near the end of the band 3 cytoplasmic domain. This mutation was present in genomic DNA of all HS family members and absent in DNA of unaffected family members. Using an RT-PCR-based assay, a marked quantitative decrease in accumulation of the mutant band 3 RNA was detected. Thus the codon 330 nonsense mutation is responsible for the decreased accumulation of mutant band 3 RNA and the deficiency of band 3 protein in this kindred. These results have important implications for the role of band 3 defects in the membrane pathobiology of HS as well as for the techniques used in detection of HS mutations.