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1.
J Biol Chem ; 299(2): 102877, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36621628

RESUMEN

The red blood cells (RBCs) of vertebrates have evolved into two basic shapes, with nucleated nonmammalian RBCs having a biconvex ellipsoidal shape and anuclear mammalian RBCs having a biconcave disk shape. In contrast, camelid RBCs are flat ellipsoids with reduced membrane deformability, suggesting altered membrane skeletal organization. However, the mechanisms responsible for their elliptocytic shape and reduced deformability have not been determined. We here showed that in alpaca RBCs, protein 4.1R, a major component of the membrane skeleton, contains an alternatively spliced exon 14-derived cassette (e14) not observed in the highly conserved 80 kDa 4.1R of other highly deformable biconcave mammalian RBCs. The inclusion of this exon, along with the preceding unordered proline- and glutamic acid-rich peptide (PE), results in a larger and unique 90 kDa camelid 4.1R. Human 4.1R containing e14 and PE, but not PE alone, showed markedly increased ability to form a spectrin-actin-4.1R ternary complex in viscosity assays. A similar facilitated ternary complex was formed by human 4.1R possessing a duplication of the spectrin-actin-binding domain, one of the mutations known to cause human hereditary elliptocytosis. The e14- and PE-containing mutant also exhibited an increased binding affinity to ß-spectrin compared with WT 4.1R. Taken together, these findings indicate that 4.1R protein with the e14 cassette results in the formation and maintenance of a hyperstable membrane skeleton, resulting in rigid red ellipsoidal cells in camelid species, and suggest that membrane structure is evolutionarily regulated by alternative splicing of exons in the 4.1R gene.


Asunto(s)
Empalme Alternativo , Camélidos del Nuevo Mundo , Forma de la Célula , Proteínas del Citoesqueleto , Eritrocitos , Animales , Humanos , Actinas/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Eritrocitos/citología , Eritrocitos/metabolismo , Membranas/metabolismo , Péptidos/metabolismo , Unión Proteica , Espectrina/genética , Espectrina/metabolismo , Forma de la Célula/genética
2.
J Cardiothorac Vasc Anesth ; 36(3): 855-861, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-34253445

RESUMEN

OBJECTIVES: Blood-processing techniques and preservation conditions cause storage lesions, possibly leading to adverse outcomes after transfusion. The authors investigated the metabolic changes and deformability of red blood cells (RBCs) during storage and determined the effect of storage lesions on circulating RBCs during cardiac surgery. DESIGN: Prospective study. SETTING: Tertiary care center affiliated with a university hospital. PARTICIPANTS: Adults who underwent elective cardiac surgery requiring cardiopulmonary bypass. INTERVENTIONS: The authors collected aliquots of autologous and irradiated allogeneic RBCs and blood samples from seven patients who received autologous whole blood and nine patients who received irradiated allogeneic RBCs before incision (baseline), at the start and end of cardiopulmonary bypass, and at completion of surgery. MEASUREMENTS AND MAIN RESULTS: The authors analyzed RBC deformability, erythrocyte indices, and density distribution to evaluate blood banking-induced alterations of autologous and allogeneic RBCs and changes in circulating RBCs in recipients, after blood transfusion. Time-dependent biochemical changes and significant decreases in deformability during storage occurred in both groups; however, homologous RBCs had significantly lower deformability than autologous RBCs. Trends in mean corpuscular volume and mean corpuscular hemoglobin concentration differed in both groups. In the homologous transfusion group, during cardiac surgery, RBC deformability, mean corpuscular volume, and mean corpuscular hemoglobin concentration showed significant changes compared with baseline values, and a greater number of denser subpopulations was observed at surgery completion. CONCLUSIONS: Blood-processing techniques contribute to storage lesions, suggesting that transfusion of autologous whole blood, rather than allogeneic RBCs, could maintain the ability of circulating RBCs to deform and lead to potentially better transfusion outcomes.


Asunto(s)
Procedimientos Quirúrgicos Cardíacos , Trasplante de Células Madre Hematopoyéticas , Conservación de la Sangre/efectos adversos , Conservación de la Sangre/métodos , Procedimientos Quirúrgicos Cardíacos/efectos adversos , Puente Cardiopulmonar/efectos adversos , Deformación Eritrocítica , Eritrocitos , Humanos , Estudios Prospectivos
3.
J Biol Chem ; 295(23): 8048-8063, 2020 06 05.
Artículo en Inglés | MEDLINE | ID: mdl-32358067

RESUMEN

TSPO2 (translocator protein 2) is a transmembrane protein specifically expressed in late erythroblasts and has been postulated to mediate intracellular redistribution of cholesterol. We identified TSPO2 as the causative gene for the HK (high-K+) trait with immature red cell phenotypes in dogs and investigated the effects of the TSPO2 defects on erythropoiesis in HK dogs with the TSPO2 mutation and Tspo2 knockout (Tspo2-/-) mouse models. Bone marrow-derived erythroblasts from HK dogs showed increased binucleated and apoptotic cells at various stages of maturation and shed large nuclei with incomplete condensation when cultured in the presence of erythropoietin, indicating impaired maturation and cytokinesis. The canine TSPO2 induces cholesterol accumulation in the endoplasmic reticulum and could thereby regulate cholesterol availability by changing intracellular cholesterol distribution in erythroblasts. Tspo2-/- mice consistently showed impaired cytokinesis with increased binucleated erythroblasts, resulting in compensated anemia, and their red cell membranes had increased Na,K-ATPase, resembling the HK phenotype in dogs. Tspo2-deficient mouse embryonic stem cell-derived erythroid progenitor (MEDEP) cells exhibited similar morphological defects associated with a cell-cycle arrest at the G2/M phase, resulting in decreased cell proliferation and had a depletion in intracellular unesterified and esterified cholesterol. When the terminal maturation was induced, Tspo2-/- MEDEP cells showed delays in hemoglobinization; maturation-associated phenotypic changes in CD44, CD71, and TER119 expression; and cell-cycle progression. Taken together, these findings imply that TSPO2 is essential for coordination of maturation and proliferation of erythroblasts during normal erythropoiesis.


Asunto(s)
Eritroblastos/citología , Eritroblastos/metabolismo , Eritropoyesis , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Proliferación Celular , Células Cultivadas , Perros , Humanos , Células K562 , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Citoplasmáticos y Nucleares/deficiencia
4.
J Cell Mol Med ; 24(23): 13991-14000, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33103382

RESUMEN

Mature human erythrocytes circulate in blood for approximately 120 days, and senescent erythrocytes are removed by splenic macrophages. During this process, the cell membranes of senescent erythrocytes express phosphatidylserine, which is recognized as a signal for phagocytosis by macrophages. However, the mechanisms underlying phosphatidylserine exposure in senescent erythrocytes remain unclear. To clarify these mechanisms, we isolated senescent erythrocytes using density gradient centrifugation and applied fluorescence-labelled lipids to investigate the flippase and scramblase activities. Senescent erythrocytes showed a decrease in flippase activity but not scramblase activity. Intracellular ATP and K+ , the known influential factors on flippase activity, were altered in senescent erythrocytes. Furthermore, quantification by immunoblotting showed that the main flippase molecule in erythrocytes, ATP11C, was partially lost in the senescent cells. Collectively, these results suggest that multiple factors, including altered intracellular substances and reduced ATP11C levels, contribute to decreased flippase activity in senescent erythrocytes in turn to, present phosphatidylserine on their cell membrane. The present study may enable the identification of novel therapeutic approaches for anaemic states, such as those in inflammatory diseases, rheumatoid arthritis, or renal anaemia, resulting from the abnormally shortened lifespan of erythrocytes.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Eritrocitos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Fosfatidilserinas/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfato/metabolismo , Transporte Biológico , Calcio/metabolismo , Micropartículas Derivadas de Células/metabolismo , Senescencia Celular/genética , Activación Enzimática , Membrana Eritrocítica/metabolismo , Humanos , Proteínas de Transporte de Membrana/genética , Potasio/metabolismo
5.
Biochem Biophys Res Commun ; 516(3): 705-712, 2019 08 27.
Artículo en Inglés | MEDLINE | ID: mdl-31253392

RESUMEN

Distribution of phosphatidylserine (PS) in the erythrocyte membrane is essential for its activity. Flippase transports phospholipids from the outer to the inner leaflet of the lipid bilayer and maintains asymmetric distribution of phospholipids in the plasma membrane. ATP11C, a flippase, catalyzes PS flipping at the plasma membrane in association with cell cycle control protein 50A (CDC50A). ATP11C T418 N mutation causes 90% decrease in erythrocyte PS-flippase activity. However, the mechanism of the activity reduction remains unknown. To study the endogenous expression of ATP11C in erythrocytes, we produced a monoclonal antibody against human ATP11C. Immunoblotting analyses with this antibody revealed the absence of ATP11C in erythrocyte membranes derived from a patient with the T418 N mutation. Transiently expressed ATP11C wild-type in cultured cells localized in the cell membranes in the presence of CDC50A. Contrastingly, ATP11C T418 N mutants stacked at the endoplasmic reticulum (ER) even in the presence of CDC50A, suggesting improper intracellular trafficking. Expression of the T418 N mutant in cultured cells was lower than that in the wild-type. However, reduced expression of the T418 N mutant was partially restored by treatment with proteasome inhibitors, suggesting ER-associated degradation of the mutant protein. Cells expressing T418 N did not show flippase activity at the plasma membrane. These data show that the loss of PS-flippase activity in erythrocytes carrying ATP11C T418 N mutation is due to impaired enzymatic activity, improper membrane trafficking, and increased proteasome degradation.


Asunto(s)
Adenosina Trifosfatasas/genética , Anemia Hemolítica Congénita/genética , Predisposición Genética a la Enfermedad/genética , Proteínas de Transporte de Membrana/genética , Mutación Missense , Adenosina Trifosfatasas/metabolismo , Anemia Hemolítica Congénita/metabolismo , Animales , Transporte Biológico/genética , Células COS , Chlorocebus aethiops , Retículo Endoplásmico/metabolismo , Membrana Eritrocítica/química , Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Femenino , Células HeLa , Humanos , Immunoblotting , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo
6.
J Cardiothorac Vasc Anesth ; 33(11): 2960-2967, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31072700

RESUMEN

OBJECTIVES: During cardiac surgery, circulating red blood cells (RBCs) are at risk of exposure to environmental factors during extracorporeal circulation and transfusion of stored RBCs. For this study, the authors observed morphological differences, deformability, density distribution, and erythrocyte indices of RBCs during cardiac surgery with cardiopulmonary bypass (CPB). DESIGN: Prospective study. SETTING: Tertiary care center affiliated with a university hospital. PARTICIPANTS: Adults who underwent elective cardiac surgery requiring CPB. INTERVENTIONS: Blood samples were obtained from 13 patients before incision (baseline), at initiation of CPB, after separation from CPB, and at completion of surgery. MEASUREMENTS AND MAIN RESULTS: The morphological index (MI) in RBCs using light microscopy and the maximum deformability index (DImax) using an ektacytometer were evaluated. In addition, the fractionation of RBCs and erythrocyte indices were measured. The MI at initiation of CPB was significantly higher without blood transfusion compared with baseline, although the DImax did not significantly decrease simultaneously. The DImax after separation from CPB and at completion of surgery were significantly lower than that at baseline. This lowered DImax was accompanied by a significantly reduced mean corpuscular volume and elevated mean corpuscular hemoglobin concentration compared with baseline. Dense RBC subpopulations increased after initiating CPB. The MI after separation from CPB and at completion of surgery partially recovered. Administered stored RBCs showed a high MI and the lowest DImax. CONCLUSIONS: Morphological changes at initiation of CPB are considered potentially reversible transformations without loss of the membrane surface area and do not have a significant effect on the DImax. A decrease in deformability likely is due to transfusion of stored RBCs.


Asunto(s)
Puente Cardiopulmonar/métodos , Deformación Eritrocítica/fisiología , Eritrocitos/patología , Cardiopatías/cirugía , Anciano , Transfusión Sanguínea , Femenino , Cardiopatías/sangre , Humanos , Periodo Intraoperatorio , Masculino , Pronóstico , Estudios Prospectivos
7.
Curr Opin Hematol ; 24(3): 167-172, 2017 May.
Artículo en Inglés | MEDLINE | ID: mdl-28118222

RESUMEN

PURPOSE OF REVIEW: The article summarizes new insights into the molecular mechanisms for the maintenance and regulation of the asymmetric distribution of phospholipids in human erythrocyte membranes. We focus on phosphatidylserine, which is primarily found in the inner leaflet of the membrane lipid bilayer under low Ca conditions (<1 µmol/l) and is exposed to the outer leaflet under elevated Ca concentrations (>1 µmol/l), when cells become senescent. Clarification of the molecular basis of phosphatidylserine flipping and scrambling is important for addressing long-standing questions regarding phosphatidylserine functions. RECENT FINDINGS: ATP11C, a P-IV ATPase, has been identified as a major flippase in analyses of patient erythrocytes with a 90% reduction in flippase activity. Phospholipid scramblase 1 (PLSCR1) has been defined as a Ca-activated scramblase that is completely suppressed by membrane cholesterol under low Ca concentrations. SUMMARY: For survival, phosphatidylserine surface exposure is prevented by cholesterol-mediated suppression of PLSCR1 under low Ca concentrations, irrespective of flipping by ATP11C. In senescent erythrocytes, PLSCR1 is activated by elevated Ca, resulting in phosphatidylserine exposure, allowing macrophage phagocytosis. These recent molecular findings establish the importance of the maintenance and regulation of phosphatidylserine distribution for both the survival and death of human erythrocytes.


Asunto(s)
Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Lípidos de la Membrana/metabolismo , Fosfolípidos/metabolismo , Adenosina Trifosfatasas/metabolismo , Transporte Biológico , Humanos , Proteínas de Transporte de Membrana/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Factores de Tiempo
8.
Biochemistry ; 55(25): 3504-3513, 2016 06 28.
Artículo en Inglés | MEDLINE | ID: mdl-27267274

RESUMEN

An asymmetric distribution of phospholipids in the membrane bilayer is inseparable from physiological functions, including shape preservation and survival of erythrocytes, and by implication other cells. Aminophospholipids, notably phosphatidylserine (PS), are confined to the inner leaflet of the erythrocyte membrane lipid bilayer by the ATP-dependent flippase enzyme, ATP11C, counteracting the activity of an ATP-independent scramblase. Phospholipid scramblase 1 (PLSCR1), a single-transmembrane protein, was previously reported to possess scrambling activity in erythrocytes. However, its function was cast in doubt by the retention of scramblase activity in erythrocytes of knockout mice lacking this protein. We show that in the human erythrocyte PLSCR1 is the predominant scramblase and by reconstitution into liposomes that its activity resides in the transmembrane domain. At or below physiological intracellular calcium concentrations, total suppression of flippase activity nevertheless leaves the membrane asymmetry undisturbed. When liposomes or erythrocytes are depleted of cholesterol (a reversible process in the case of erythrocytes), PS quickly appears at the outer surface, implying that cholesterol acts in the cell as a powerful scramblase inhibitor. Thus, our results bring to light a previously unsuspected function of cholesterol in regulating phospholipid scrambling.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Colesterol/metabolismo , Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Membrana Dobles de Lípidos/metabolismo , Lípidos de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Animales , Transporte Biológico , Calcio/metabolismo , Células Cultivadas , Eritrocitos/citología , Humanos , Ratones , Fosfatidilserinas/metabolismo
9.
Haematologica ; 101(5): 559-65, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-26944472

RESUMEN

Phosphatidylserine is localized exclusively to the inner leaflet of the membrane lipid bilayer of most cells, including erythrocytes. This asymmetric distribution is critical for the survival of erythrocytes in circulation since externalized phosphatidylserine is a phagocytic signal for splenic macrophages. Flippases are P-IV ATPase family proteins that actively transport phosphatidylserine from the outer to inner leaflet. It has not yet been determined which of the 14 members of this family of proteins is the flippase in human erythrocytes. Herein, we report that ATP11C encodes a major flippase in human erythrocytes, and a genetic mutation identified in a male patient caused congenital hemolytic anemia inherited as an X-linked recessive trait. Phosphatidylserine internalization in erythrocytes with the mutant ATP11C was decreased 10-fold compared to that of the control, functionally establishing that ATP11C is a major flippase in human erythrocytes. Contrary to our expectations phosphatidylserine was retained in the inner leaflet of the majority of mature erythrocytes from both controls and the patient, suggesting that phosphatidylserine cannot be externalized as long as scramblase is inactive. Phosphatidylserine-exposing cells were found only in the densest senescent cells (0.1% of total) in which scramblase was activated by increased Ca(2+) concentration: the percentage of these phosphatidylserine-exposing cells was increased in the patient's senescent cells accounting for his mild anemia. Furthermore, the finding of similar extents of phosphatidylserine exposure by exogenous Ca(2+)-activated scrambling in both control erythrocytes and the patient's erythrocytes implies that suppressed scramblase activity rather than flippase activity contributes to the maintenance of phosphatidylserine in the inner leaflet of human erythrocytes.


Asunto(s)
Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Anemia Hemolítica Congénita/genética , Anemia Hemolítica Congénita/metabolismo , Eritrocitos/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Adenosina Trifosfatasas/deficiencia , Adolescente , Anemia Hemolítica Congénita/diagnóstico , Transporte Biológico Activo , Biomarcadores , Calcio/metabolismo , Índices de Eritrocitos , Eritrocitos/efectos de los fármacos , Humanos , Masculino , Proteínas de Transporte de Membrana/deficiencia , Mutación , Fosfatidilserinas/metabolismo , Fosfatidilserinas/farmacología
10.
Biochemistry ; 52(34): 5760-9, 2013 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-23889086

RESUMEN

Oxidative damage and clustering of band 3 in the membrane have been implicated in the removal of senescent human erythrocytes from the circulation at the end of their 120 day life span. However, the biochemical and mechanistic events leading to band 3 cluster formation have yet to be fully defined. Here we show that while neither membrane peroxidation nor methemoglobin (MetHb) formation on their own can induce band 3 clustering in the human erythrocytes, they can do so when acting in combination. We further show that binding of MetHb to the cytoplasmic domain of band 3 in peroxidized, but not in untreated, erythrocyte membranes induces cluster formation. Age-fractionated populations of erythrocytes from normal human blood, obtained by a density gradient procedure, have allowed us to examine a subpopulation, highly enriched in senescent cells. We have found that band 3 clustering is a feature of only this small fraction, amounting to ∼0.1% of total circulating erythrocytes. These senescent cells are characterized by an increased proportion of MetHb as a result of reduced nicotinamide adenine dinucleotide-dependent reductase activity and accumulated oxidative membrane damage. These findings have allowed us to establish that the combined effects of membrane peroxidation and MetHb formation are necessary for band 3 clustering, and this is a very late event in erythrocyte life. A plausible mechanism for the combined effects of membrane peroxidation and MetHb is proposed, involving high-affinity cooperative binding of MetHb to the cytoplasmic domain of oxidized band 3, probably because of its carbonylation, rather than other forms of oxidative damage. This modification leads to dissociation of ankyrin from band 3, allowing the tetrameric MetHb to cross-link the resulting freely diffusible band 3 dimers, with formation of clusters.


Asunto(s)
Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Envejecimiento Eritrocítico/fisiología , Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Metahemoglobina/biosíntesis , Proteína 1 de Intercambio de Anión de Eritrocito/inmunología , Humanos , Cinética , Peroxidación de Lípido , Metahemoglobina/metabolismo , Oxidación-Reducción , Estrés Oxidativo , Multimerización de Proteína
12.
Cancer Gene Ther ; 30(7): 973-984, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-36932197

RESUMEN

The tumor-elicited inflammation is closely related to tumor microenvironment during tumor progression. S100A8, an endogenous ligand of Toll-like receptor 4 (TLR4), is known as a key molecule in the tumor microenvironment and premetastatic niche formation. We firstly generated a novel multivalent S100A8 competitive inhibitory peptide (divalent peptide3A5) against TLR4/MD-2, using the alanine scanning. Divalent peptide3A5 suppressed S100A8-mediated interleukin-8 and vascular endothelial growth factor production in human colorectal tumor SW480 cells. Using SW480-transplanted xenograft models, divalent peptide3A5 suppressed tumor progression in a dose-dependent manner. We demonstrated that combination therapy with divalent peptide3A5 and bevacizumab synergistically suppressed tumor growth in SW480 xenograft models. Using syngeneic mouse models, we found that divalent peptide3A5 improved the efficacy of anti-programmed death (PD)1 antibody, and lung metastasis. In addition, by using multivalent peptide library screening based on peptide3A5, we then isolated two more candidates; divalent ILVIK, and tetravalent ILVIK. Of note, multivalent ILVIK, but not monovalent ILVIK showed competitive inhibitory activity against TLR4/MD-2 complex, and anti-tumoral activity in SW480 xenograft models. As most tumor cells including SW480 cells also express TLR4, S100A8 inhibitory peptides would target both the tumor microenvironment and tumor cells. Thus, multivalent S100A8 inhibitory peptides would provide new pharmaceutical options for aggressive cancers.


Asunto(s)
Calgranulina B , Receptor Toll-Like 4 , Animales , Ratones , Humanos , Calgranulina B/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Calgranulina A/metabolismo , Péptidos/farmacología , Péptidos/metabolismo
13.
Elife ; 112022 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-36511780

RESUMEN

Collapsin response mediator proteins (CRMPs) are key for brain development and function. Here, we link CRMP1 to a neurodevelopmental disorder. We report heterozygous de novo variants in the CRMP1 gene in three unrelated individuals with muscular hypotonia, intellectual disability, and/or autism spectrum disorder. Based on in silico analysis these variants are predicted to affect the CRMP1 structure. We further analyzed the effect of the variants on the protein structure/levels and cellular processes. We showed that the human CRMP1 variants impact the oligomerization of CRMP1 proteins. Moreover, overexpression of the CRMP1 variants affect neurite outgrowth of murine cortical neurons. While altered CRMP1 levels have been reported in psychiatric diseases, genetic variants in CRMP1 gene have never been linked to human disease. We report for the first-time variants in the CRMP1 gene and emphasize its key role in brain development and function by linking directly to a human neurodevelopmental disease.


Asunto(s)
Trastorno del Espectro Autista , Discapacidad Intelectual , Trastornos del Neurodesarrollo , Animales , Humanos , Ratones , Trastorno del Espectro Autista/genética , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/metabolismo , Proyección Neuronal , Neuronas/metabolismo , Hipotonía Muscular/genética
14.
Biochem Biophys Res Commun ; 391(3): 1543-7, 2010 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-20036642

RESUMEN

Spectrin strengthens the red cell membrane through its direct association with membrane lipids and through protein-protein interactions. Spectrin loss reduces the membrane stability and results in various types of hereditary spherocytosis. However, less is known about acquired spectrin damage. Here, we showed that alpha- and beta-spectrin in human red cells are the primary targets of the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) by immunoblotting and mass spectrometry analyses. The level of HNE adducts in spectrin (particularly alpha-spectrin) and several other membrane proteins was increased following the HNE treatment of red cell membrane ghosts prepared in the absence of MgATP. In contrast, ghost preparation in the presence of MgATP reduced HNE adduct formation, with preferential beta-spectrin modification and increased cross-linking of the HNE-modified spectrins. Exposure of intact red cells to HNE resulted in selective HNE-spectrin adduct formation with a similar preponderance of HNE-beta-spectrin modifications. These findings indicate that HNE adduction occurs preferentially in spectrin at the interface between the skeletal proteins and lipid bilayer in red cells and suggest that HNE-spectrin adduct aggregation results in the extrusion of damaged spectrin and membrane lipids under physiological and disease conditions.


Asunto(s)
Aldehídos/metabolismo , Membrana Eritrocítica/metabolismo , Peroxidación de Lípido , Espectrina/metabolismo , Aldehídos/farmacología , Secuencia de Aminoácidos/efectos de los fármacos , Membrana Eritrocítica/efectos de los fármacos , Humanos , Immunoblotting , Datos de Secuencia Molecular , Espectrina/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
15.
J Vet Med Sci ; 72(7): 893-901, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-20215716

RESUMEN

Dogs can be divided into two genetic groups (a minor HK phenotype and a major LK phenotype) based on erythrocyte monovalent cation concentrations, which are controlled by the putative hk and lk allelic genes. HK dogs retain Na,K-ATPase in their erythrocytes due to the high activity of the enzyme in their precursor cells, whereas total loss of reticulocyte Na,K-ATPase occurs in LK dogs. Here, we report that the levels of the lipid raft-associated membrane protein stomatin decrease in parallel with those of Na,K-ATPase during reticulocyte maturation due to its extrusion in exosomes. The stomatin content of HK reticulocytes is higher than that of LK reticulocytes, and remains in the erythrocytes at levels compatible with that in human erythrocytes. However, it is almost absent from LK erythrocytes with the lk/lk genotype; similar to the deficiency seen in human red cells with overhydrated stomatocytosis. LK erythrocytes from hk/lk genotype dogs show reduced, but not negligible, levels of stomatin. These results indicate that the erythrocyte stomatin level is a suitable genotypic marker for the HK/LK red cell phenotype, and suggests a functional association between stomatin and Na,K-ATPase. The absence of morphological abnormalities in the erythrocytes of stomatin-deficient LK dogs also confirms that stomatin deficiency and stomatocytic shape change are independent from each other.


Asunto(s)
Perros/genética , Potasio/sangre , Secuencia de Aminoácidos , Anemia/sangre , Anemia/veterinaria , Animales , Anticuerpos Monoclonales , Cationes/sangre , Perros/sangre , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/ultraestructura , Eritrocitos/metabolismo , Genotipo , Humanos , Proteínas de la Membrana/sangre , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Linaje , Fenotipo , Reticulocitos/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , ATPasa Intercambiadora de Sodio-Potasio/metabolismo
16.
Jpn J Vet Res ; 58(1): 17-27, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20645582

RESUMEN

The present study characterizes canine reticulocyte exosomes. Exosomes are small membrane vesicles involved in membrane remodeling that are released from reticulocytes during the final maturation step of red blood cells. The vesicles collected from reticulocyte culture supernatants by differential centrifugation contained major exosomal proteins including heat shock protein cognate 70 (Hsc70) and transferrin receptors (TfR), consistent with the definition of the exosome. In addition, the Na,K-ATPase alpha-subunit and stomatin, a lipid raft-associated protein, were extruded by the exosome pathway, possibly leading to the absence of these proteins in erythrocytes, while the major protein constituents of erythrocyte membranes, spectrin and band 3 were retained in reticulocytes and not expelled into exosomes. The Na,K-ATPase alpha-subunit, as well as TfR and about half of the stomatin contained in exosomes, was obtained in a detergent-soluble fraction that was distinct from the lipid raft microdomain. Moreover, Na,K-ATPase and a portion of stomatin were distributed differently to Hsc70, TfR, stomatin, and ganglioside GM1 in vesicles separated with sucrose density gradient centrifugation. These results demonstrate that a heterogeneous group of exosomes participates in the loss of Na,K-ATPase and membrane remodeling during reticulocyte maturation in dogs.


Asunto(s)
Receptores de Transferrina/metabolismo , Reticulocitos/fisiología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Perros , Reticulocitos/citología , Reticulocitosis/fisiología , ATPasa Intercambiadora de Sodio-Potasio/aislamiento & purificación
17.
Jpn J Vet Res ; 57(3): 135-46, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-20025122

RESUMEN

While the C-terminal cytoplasmic tail of anion exchanger 1 (AE1, band 3) has been reported to possess important physiological roles, including one for proper membrane trafficking, its precise characteristics remain unclear. To clarify the overall structural consequences of the conserved sequence EL(K/Q)(L/C)LD(A/G)DD, containing the core binding sequence LDADD for carbonic anhydrase II, in the C-terminal region, we analyzed the membrane expression and turnover of bovine AE1 with a series of truncation and substitution mutations in HEK293 cells. Immunofluorescence microscopy and cell-surface biotinylation demonstrated that truncation mutants missing 18 C-terminal residues targeted the plasma membrane, but the one lacking the conserved region, by truncation of 28 amino acid residues, was retained inside the cells. Substitutions of Ala for Glu901, Leu902, Leu905, and Asp906 in the sequence E901L(K/Q)(L/C)LDADD909 of bovine AE1 or those in the corresponding murine sequence also caused intracellular retention, though these mutants had half-lives comparable to that for wild-type AE1. These data demonstrate that the conserved amino acid residues Glu1, Leu2, Leu5, and Asp6 in the EL(K/Q)(L/C)LD(A/G)DD region have essential structural consequences in stable expression of AE1 at the plasma membrane regardless of the ability in binding to carbonic anhydrase II of this region.


Asunto(s)
Proteína 1 de Intercambio de Anión de Eritrocito/química , Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Línea Celular , Regulación de la Expresión Génica/fisiología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína
19.
Jpn J Vet Res ; 56(2): 75-84, 2008 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-18828445

RESUMEN

Alpha-Hemoglobin stabilizing protein (AHSP) functions as the erythroid-specific molecular chaperon for alpha-globin. AHSP gene expression has been reported to be downregulated in hematopoietic tissues of animals suffering from prion diseases though the mechanism remains to be clarified. Herein, we demonstrate that MELhipod8 cells, a subclone of murine erythroleukemia (MEL) cells, have prion protein (PrPc) on the cell surface and have highly inducible expression of the AHSP and alpha- and beta-globin genes, resembling the expression pattern of the PrP and AHSP genes in bipotential erythroid- and megakaryocyte-lineage cells followed by erythroid differentiation in normal erythropoiesis. Moreover, MELhipod8 cells exhibit greater effective erythroid differentiation with a population of hemoglobinized normoblast-like cells than that observed for the parental MEL cells. These findings suggest that MELhipod8 cells could provide a mechanism for downregulation of the AHSP gene in prion diseases.


Asunto(s)
Proteínas Sanguíneas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas PrPC/metabolismo , Animales , Proteínas Sanguíneas/genética , Línea Celular Tumoral , Regulación de la Expresión Génica , Leucemia Eritroblástica Aguda , Ratones , Chaperonas Moleculares/genética , Proteínas PrPC/genética , Factores de Tiempo
20.
Jpn J Vet Res ; 55(4): 103-14, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18380152

RESUMEN

Glycophorins are the major sialoglycoproteins in red blood cell membranes, possessing various physiological and pathological roles. We examined membrane glycoproteins in canine red cells and cloned cDNAs for two major glycophorins, glycophorins A (GPA) and C (GPC) from bone marrow cells. Periodic acid-Schiff staining and immunoblotting analyses showed that canine red cell membranes contained several glycoproteins immunoreactive to an anti-bovine GPC antibody, whereas the most abundant sialoglycoproteins, the candidates for GPA, did not react with an anti-human GPA antibody. The amino acid sequences of the extracellular domains of GPA and GPC had no significant homology to those from other mammalian species, including humans, and had O-linked and/or N-linked glycosylation sites. On the other hand, the C-terminal cytoplasmic domain and/or the transmembrane helices of GPA and GPC were conserved among species, indicating some functional significance of those regions in red cell membranes that include dimerization of GPA in the membrane-spanning region, and association of GPC with membrane skeletal proteins through binding with protein 4.1 and p55 in the cytoplasmic domain. These findings provide insights for clinical studies to evaluate the involvement of GPA and GPC in the pathogenesis of red cell diseases.


Asunto(s)
Membrana Eritrocítica/química , Glicoforinas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células de la Médula Ósea/química , Bovinos , Clonación Molecular , ADN Complementario/genética , Perros , Electroforesis en Gel de Poliacrilamida/veterinaria , Femenino , Glicoforinas/química , Glicoforinas/inmunología , Humanos , Immunoblotting/veterinaria , Datos de Secuencia Molecular , Técnicas de Amplificación de Ácido Nucleico/veterinaria , Isoformas de Proteínas , Alineación de Secuencia/veterinaria , Especificidad de la Especie
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