RESUMO
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.
Assuntos
Processamento Alternativo , Camelídeos Americanos , Forma Celular , Proteínas do Citoesqueleto , Eritrócitos , Animais , Humanos , Actinas/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Eritrócitos/citologia , Eritrócitos/metabolismo , Membranas/metabolismo , Peptídeos/metabolismo , Ligação Proteica , Espectrina/genética , Espectrina/metabolismo , Forma Celular/genéticaRESUMO
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.
Assuntos
Eritroblastos/citologia , Eritroblastos/metabolismo , Eritropoese , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Cães , Humanos , Células K562 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Citoplasmáticos e Nucleares/deficiênciaRESUMO
Protein export from the endoplasmic reticulum (ER) depends on the interaction between a signal motif on the cargo and a cargo recognition site on the coatomer protein complex II. A hydrophobic sequence in the N terminus of the bovine anion exchanger 1 (AE1) anion exchanger facilitated the ER export of human AE1Δ11, an ER-retained AE1 mutant, through interaction with a specific Sec24 isoform. The cell surface expression and N-glycan processing of various substitution mutants or chimeras of human and bovine AE1 proteins and their Δ11 mutants in HEK293 cells were examined. The N-terminal sequence (V/L/F)X(I/L)X(M/L), (26)VSIPM(30) in bovine AE1, which is comparable with ΦXΦXΦ, acted as the ER export signal for AE1 and AE1Δ11 (Φ is a hydrophobic amino acid, and X is any amino acid). The AE1-Ly49E chimeric protein possessing the ΦXΦXΦ motif exhibited effective cell surface expression and N-glycan maturation via the coatomer protein complex II pathway, whereas a chimera lacking this motif was retained in the ER. A synthetic polypeptide containing the N terminus of bovine AE1 bound the Sec23A-Sec24C complex through a selective interaction with Sec24C. Co-transfection of Sec24C-AAA, in which the residues (895)LIL(897) (the binding site for another ER export signal motif IXM on Sec24C and Sec24D) were mutated to (895)AAA(897), specifically increased ER retention of the AE1-Ly49E chimera. These findings demonstrate that the ΦXΦXΦ sequence functions as a novel signal motif for the ER export of cargo proteins through an exclusive interaction with Sec24C.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Sinais Direcionadores de Proteínas , Proteínas de Transporte Vesicular/metabolismo , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Proteína 1 de Troca de Ânion do Eritrócito/genética , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Sítios de Ligação/genética , Ligação Competitiva , Bovinos , Células HEK293 , Humanos , Immunoblotting , Proteínas de Membrana/genética , Microscopia Confocal , Mutação , Peptídeos/metabolismo , Ligação Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMO
The PDZ (PSD-95/Drosophila discs-large protein/zonula occludens protein) domain-containing proteins Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) and NHERF2 interact with the glutamate transporter GLAST. To characterize the roles of these NHERF proteins in the plasma membrane targeting of GLAST, we examined the interaction of green fluorescent protein (EGFP)-tagged GLAST with epitope-tagged NHERF proteins in human embryonic kidney (HEK) 293T cells. Co-expression of either NHERF protein increased the cell surface expression of EGFP-GLAST. Deletion of the C-terminal PDZ domain-binding motif caused an increase in EGFP-GLAST with immature endoglycosidase H-sensitive N-linked oligosaccharides, suggesting impaired exit of EGFP-GLAST from the endoplasmic reticulum (ER). Immunoprecipitation experiments revealed that NHERF1 predominantly bound EGFP-GLAST containing immature N-glycans, whereas NHERF2 co-precipitated EGFP-GLAST with mature N-glycans. Expression of a dominant-negative mutant of the GTPase Sar1 increased the interaction of EGFP-GLAST with NHERF1 in the ER. By contrast, immunofluorescence microscopy showed that NHERF2 co-localized with EGFP-GLAST in ER-Golgi intermediate compartments (ERGICs), at the plasma membrane and in early endosomes, but not in the ER. These results suggest that NHERF1 interacts with GLAST during ER export, while NHERF2 interacts with GLAST in the secretory pathway from the ERGIC to the plasma membrane, thereby modulating the cell surface expression of GLAST.
Assuntos
Membrana Celular/metabolismo , Transportador 1 de Aminoácido Excitatório/metabolismo , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Cães , Retículo Endoplasmático/metabolismo , Transportador 1 de Aminoácido Excitatório/genética , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Domínios PDZ/genética , Fosfoproteínas/genética , Trocadores de Sódio-Hidrogênio/genéticaRESUMO
Anion exchanger 1 (AE1) is the most abundant integral membrane protein in red cells and is essential for maintaining red cell mechanical stability. However, the mechanism for the assembly of AE1 into the membrane skeletal network remains unknown. Several mutants of murine AE1 tagged with an N-terminal enhanced green fluorescent protein (EGFP) and/or an extracellular FLAG epitope inserted adjacent to the N-glycosylation site were prepared, and their expression was analyzed in HEK293 or COS-1 cells by immunofluorescence microscopy, biotinylation, and deglycosylation. The EGFP- and FLAG-tagged AE1 mutant, as well as the wild-type AE1, exhibited cell surface expression in transfected cells and showed a rapid internalization that appeared to occur through the early endosome into the Golgi apparatus. Interestingly, the form of the protein with an endoglycosidase H (endo H)-sensitive N-glycan was the major component of EGFP-tagged and wild-type AE1. By contrast, the polypeptide with an endo H-resistant oligosaccharide was the predominant form of FLAG-tagged AE1. These data demonstrate that the processing of N-glycan is not a prerequisite to cell surface expression of AE1 and suggest that the FLAG tag insertion altered the accessibility of the N-glycan to enzymes in the Golgi which facilitate processing of oligosaccharides. Although whether this structural alteration would affect the structural and functional properties of AE1 remains unknown, cell surface expression and endocytic internalization of FLAG-tagged AE1 mutants indicate that these mutants are suitable for studying the mechanisms of the assembly and plasma membrane insertion of AE1.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Epitopos , Regulação da Expressão Gênica/fisiologia , Peptídeos/química , Animais , Proteína 1 de Troca de Ânion do Eritrócito/genética , Membrana Celular/metabolismo , Camundongos , Mutação , OligopeptídeosRESUMO
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.
Assuntos
Aldeídos/metabolismo , Membrana Eritrocítica/metabolismo , Peroxidação de Lipídeos , Espectrina/metabolismo , Aldeídos/farmacologia , Sequência de Aminoácidos/efeitos dos fármacos , Membrana Eritrocítica/efeitos dos fármacos , Humanos , Immunoblotting , Dados de Sequência Molecular , Espectrina/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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.
Assuntos
Cães/genética , Potássio/sangue , Sequência de Aminoácidos , Anemia/sangue , Anemia/veterinária , Animais , Anticorpos Monoclonais , Cátions/sangue , Cães/sangue , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/ultraestrutura , Eritrócitos/metabolismo , Genótipo , Humanos , Proteínas de Membrana/sangue , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Linhagem , Fenótipo , Reticulócitos/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
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.
Assuntos
Receptores da Transferrina/metabolismo , Reticulócitos/fisiologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Cães , Reticulócitos/citologia , Reticulocitose/fisiologia , ATPase Trocadora de Sódio-Potássio/isolamento & purificaçãoRESUMO
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.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/química , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Linhagem Celular , Regulação da Expressão Gênica/fisiologia , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
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.
Assuntos
Proteínas Sanguíneas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas PrPC/metabolismo , Animais , Proteínas Sanguíneas/genética , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Leucemia Eritroblástica Aguda , Camundongos , Chaperonas Moleculares/genética , Proteínas PrPC/genética , Fatores de TempoRESUMO
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.
Assuntos
Membrana Eritrocítica/química , Glicoforinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células da Medula Óssea/química , Bovinos , Clonagem Molecular , DNA Complementar/genética , Cães , Eletroforese em Gel de Poliacrilamida/veterinária , Feminino , Glicoforinas/química , Glicoforinas/imunologia , Humanos , Immunoblotting/veterinária , Dados de Sequência Molecular , Técnicas de Amplificação de Ácido Nucleico/veterinária , Isoformas de Proteínas , Alinhamento de Sequência/veterinária , Especificidade da EspécieRESUMO
BACKGROUND: Interleukin-13 receptor alpha 2 (IL13Rα2) is considered a prognostic marker for gliomas according to the World Health Organization (WHO) grade. However, the expression levels of the marker vary from case to case, even within the same grade. We investigated whether IL13Rα2 could serve as a predictor of poorer prognosis in gliomas. METHODS: mRNA expression of IL13Rα2 was measured using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) in 52 formalin-fixed paraffin-embedded glioma samples (4 pilocytic astrocytomas, 9 diffuse astrocytomas, 12 anaplastic astrocytomas, and 27 glioblastomas, grade IV). The expression levels were compared with regard to WHO grade, MIB-1 index, patient's age, and overall survival. RESULTS: Real time qRT-PCR showed that IL13Rα2 is expressed in a subset of cases with a progressive increase from low- to high-grade astrocytomas (HGAs). The expression had a significant positive correlation with the MIB-1 index and advanced patient age at diagnosis. The overall survival (OS) of patients who had HGAs with higher levels of IL13Rα2 expression was significantly lower than the OS of those with HGAs with lower levels of IL13Rα2. In the 39 HGA cases studied, the median survival benefit in the lower expression group was 167.4 months. The median OS (mOS) in HGA group with lower IL13Rα2 expression was 186.4 months, while the mOS in the group with higher IL13Rα2 expression was 18.6 months (P=0.033). The hazard ratio, adjusted by fitting the Cox proportional hazard models for the mOS in the HGAs with higher IL13Rα2 levels and the HGGs with lower IL13Rα2 levels, was 5.97 (95% CI: 1.76 to 20.32). CONCLUSIONS: The results showed that IL13Rα2 may be used as a marker of poorer prognosis in HGAs, even among tumors of the same grade.
Assuntos
Astrocitoma/metabolismo , Neoplasias Encefálicas/metabolismo , Subunidade alfa2 de Receptor de Interleucina-13/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Astrocitoma/mortalidade , Astrocitoma/patologia , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Prognóstico , Taxa de Sobrevida , Adulto JovemRESUMO
An R664X nonsense mutant AE1 is responsible for dominant hereditary spherocytosis in cattle and is degraded by the proteasomal endoplasmic reticulum-associated degradation. The present study demonstrated that R664X AE1 translated in vitro had the trypsin-sensitve site identical to that of the wild-type AE1. The P661S/R664X mutant containing a possible N-glycosylation site at Asn660 showed an increase in size by 3 kDa both in the cell-free translation system and in transfected HEK293 cells. Moreover, steady state levels of R664X and P661S/R664X in HEK293 cells were markedly increased in the presence of a proteasome inhibitior. These findings indicate that the truncated C-terminal region of R664X AE1 has lumenal localization in the endoplasmic reticulum and is not accessible to proteasomal machineries in the cytosol.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Doenças dos Bovinos/metabolismo , Retículo Endoplasmático/metabolismo , Esferocitose Hereditária/veterinária , Animais , Proteína 1 de Troca de Ânion do Eritrócito/genética , Bovinos , Doenças dos Bovinos/genética , Doenças dos Bovinos/patologia , Mutação Puntual , Esferocitose Hereditária/genética , Esferocitose Hereditária/metabolismo , Esferocitose Hereditária/patologiaRESUMO
The role of macrophages in the erythrocyte membrane oxidative damage and the pathogenesis of anemia in Babesia gibsoni-infected dogs with low parasitemia were investigated. Macrophages derived from peripheral blood monocytes (PBM) from B. gibsoni-infected dogs produced significantly higher chemiluminescent responses, indicating the release of reactive oxygen intermediates, than those from non-infected dogs when the cells were subjected to non-specific stimulation with phorbol 12-myristate 13-acetate (PMA) and opsonized zymosan (OZ), or infected dog erythrocyte membranes opsonized with infected dog serum. These results indicate that PBM of B. gibsoni-infected dogs with low parasitemia were highly activated compared to those of non-infected dogs. Furthermore, the membrane lipid peroxidation of normal dog erythrocytes incubated with PBM from B. gibsoni-infected dogs was significantly higher (p<0.05) than that of erythrocytes incubated with PBM from non-infected dogs when the PBM were stimulated with the opsonized membranes. These results suggest that the oxidative damage of erythrocytes observed in B. gibsoni-infected dogs with low parasitemia might be induced, in part, by reactive oxygen species released from the activated PBM. On the other hand, the present study also showed a significant increase (p<0.001) of IgG-bound erythrocytes in B. gibsoni-infected dogs compared with such erythrocytes in non-infected dogs. The increase of IgG-bound erythrocytes in infected dogs might reflect the increase of erythrocytes with oxidative damage induced by the infection with B. gibsoni. The results of the present study suggest that the increase of IgG-bound erythrocytes in the circulation of infected dogs induce a high degree of erythrocyte loss via immunological phagocytosis by activated macrophages, resulting in severe anemia in spite of low parasitemia.
Assuntos
Anemia/parasitologia , Babesia/crescimento & desenvolvimento , Babesiose/veterinária , Doenças do Cão/parasitologia , Membrana Eritrocítica/metabolismo , Macrófagos/imunologia , Anemia/imunologia , Anemia/metabolismo , Anemia/veterinária , Animais , Babesia/imunologia , Babesiose/imunologia , Babesiose/metabolismo , Babesiose/parasitologia , Doenças do Cão/imunologia , Doenças do Cão/metabolismo , Cães , Membrana Eritrocítica/imunologia , Membrana Eritrocítica/patologia , Citometria de Fluxo/veterinária , Imunoglobulina G/biossíntese , Imunoglobulina G/metabolismo , Peroxidação de Lipídeos , Peróxidos Lipídicos/biossíntese , Medições Luminescentes , Macrófagos/metabolismo , Estresse Oxidativo , Parasitemia/sangue , Parasitemia/imunologia , Parasitemia/metabolismo , Parasitemia/veterinária , Espécies Reativas de Oxigênio/metabolismoRESUMO
To examine substrate specificity and susceptibility to lead, erythrocyte 5'-nucleotidase was measured in dogs, cats, cattle and humans, and its relationship to the reticulocyte count in these species was determined. The reticulocyte count in dogs was similar to that in humans, but the count in cats was higher than that in humans. Reticulocytes were not observed in cattle. The activities of canine erythrocyte 5'-nucleotidase measured using cytidine and uridine 5'-monophosphates, which are preferentially catalyzed by one of the human pyrimidine 5'-nucleotidase isozymes (P5N-I), were similar to those of the human enzyme. The canine enzyme preferentially catalyzed thymidine 3'-monophosphate, which is catalyzed only by human P5N-II, more strongly than the human enzyme. This suggests that canine erythrocytes have two isozymes similar to human P5N-I and P5N-II, and a higher P5N-II-like activity than human erythrocytes. Feline erythrocytes had the highest level of P5N-I-like activity among the species examined, and the bovine enzymic activities including those of P5N-I and II were the lowest among these species. According to these observations, the reticulocyte count was approximately proportional to the P5N-I-like activity in these species. Therefore, the P5N-I-like activity may be involved in the morphological maturation of mammalian erythrocytes. The canine and feline erythrocytes had markedly high activity and preferentially catalyzed purine 5'-monophosphate suggesting the presence of a purine-specific 5'-nucleotidase as in human erythrocytes. In addition, the canine and feline P5N-I-like activity showed less susceptibility to lead than the human P5N-I. This may be a reason why there are few case reports of lead-induced anemia in dogs and cats.
Assuntos
5'-Nucleotidase/metabolismo , Eritrócitos/enzimologia , Reticulócitos/citologia , Animais , Gatos , Bovinos , Células Cultivadas , Cães , Eritrócitos/efeitos dos fármacos , Humanos , Isoenzimas/metabolismo , Chumbo/farmacologia , Contagem de Reticulócitos , Especificidade da Espécie , Especificidade por SubstratoRESUMO
To explore the roles of the conserved YXXΦ-type motif in the erythroid-specific N-terminal stretch of anion exchanger 1 (AE1), cell surface expression and internalization of various mutants derived from murine erythroid AE1 tagged with an N-terminal enhanced green fluorescent protein and an extracellular FLAG (EGFP-mAE1Flag) were explored in K562 and HEK293 cells. EGFP-mAE1Flag showed rapid internalization, in association with the internalizations of transferrin and the endogenous AE1 chaperone-like protein glycophorin A in K562 cells. Disruption of the conserved Y72VEL sequence markedly reduced the internalization and increased the relative abundance of cell-surface AE1, whereas substitution of the N-terminal region from bovine AE1 that lacks the relevant motif for the corresponding region had less of an effect on internalization. Deletion or substitution mutations of the Y7EDQL sequence in the bovine N-terminal stretch resulted in the decreased internalization of the AE1 proteins. Cell surface biotinylation and deglycosylation studies showed that approximately 30% of the cell-surface EGFP-mAE1Flag and several other mutants was sorted to the plasma membrane without N-glycan maturation in the Golgi apparatus. These findings indicate that the conserved YXXΦ sequence or a noncanonical YXXXΦ sequence in the N-terminal region facilitates the endocytic recycling of erythroid AE1 through a clathrin-mediated pathway.