RESUMO
Clearance of non-infected red blood cells (nRBCs) is one of the main components of anemia associated with Plasmodium vivax malaria. Recently, we have shown that anemic patients with P. vivax infection had elevated levels of anti-RBCs antibodies, which could enhance in vitro phagocytosis of nRBCs and decrease their deformability. Using immunoproteomics, here we characterized erythrocytic antigens that are differentially recognized by autoantibodies from anemic and non-anemic patients with acute vivax malaria. Protein spots exclusively recognized by anemic P. vivax-infected patients were identified by mass spectrometry revealing band 3 and spectrin as the main targets. To confirm this finding, antibody responses against these specific proteins were assessed by ELISA. In addition, an inverse association between hemoglobin and anti-band 3 or anti-spectrin antibodies levels was found. Anemic patients had higher levels of IgG against both band 3 and spectrin than the non-anemic ones. To determine if these autoantibodies were elicited because of molecular mimicry, we used in silico analysis and identified P. vivax proteins that share homology with human RBC proteins such as spectrin, suggesting that infection drives autoimmune responses. These findings suggest that band 3 and spectrin are potential targets of autoantibodies that may be relevant for P. vivax malaria-associated anemia.
Assuntos
Anemia/complicações , Proteína 1 de Troca de Ânion do Eritrócito/imunologia , Autoanticorpos/imunologia , Eritrócitos/imunologia , Malária Vivax/complicações , Plasmodium vivax/imunologia , Espectrina/imunologia , Adulto , Anemia/imunologia , Humanos , Imunoglobulina G/imunologia , Malária Vivax/imunologiaRESUMO
Adult T-cell leukemia (ATL) is one of the most aggressive hematologic malignancies caused by human T-lymphotropic virus type 1 (HTLV-1) infection. The prognosis of ATL is extremely poor; however, effective strategies for diagnosis and treatment have not been established. To identify novel therapeutic targets and diagnostic markers for ATL, we employed focused proteomic profiling of the CD4(+)CD25(+)CCR4(+) T-cell subpopulation in which HTLV-1-infected cells were enriched. Comprehensive quantification of 14 064 peptides and subsequent 2-step statistical analysis using 29 cases (6 uninfected controls, 5 asymptomatic carriers, 9 HTLV-1-associated myelopathy/tropical spastic paraparesis patients, 9 ATL patients) identified 91 peptide determinants that statistically classified 4 clinical groups with an accuracy rate of 92.2% by cross-validation test. Among the identified 17 classifier proteins, α-II spectrin was drastically accumulated in infected T cells derived from ATL patients, whereas its digestive protease calpain-2 (CAN2) was significantly downregulated. Further cell cycle analysis and cell growth assay revealed that rescue of CAN2 activity by overexpressing constitutively active CAN2 (Δ(19)CAN2) could induce remarkable cell death on ATL cells accompanied by reduction of α-II spectrin. These results support that proteomic profiling of HTLV-1-infected T cells could provide potential diagnostic biomarkers and an attractive resource of therapeutic targets for ATL.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Calpaína/metabolismo , Infecções por HTLV-I , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Leucemia-Linfoma de Células T do Adulto , Adulto , Apoptose/imunologia , Biomarcadores/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/virologia , Calpaína/genética , Calpaína/imunologia , Ciclo Celular/imunologia , Linhagem Celular , Sobrevivência Celular/imunologia , Progressão da Doença , Infecções por HTLV-I/diagnóstico , Infecções por HTLV-I/imunologia , Infecções por HTLV-I/metabolismo , Humanos , Imunofenotipagem , Leucemia-Linfoma de Células T do Adulto/diagnóstico , Leucemia-Linfoma de Células T do Adulto/metabolismo , Leucemia-Linfoma de Células T do Adulto/virologia , Proteômica , RNA Interferente Pequeno/genética , Espectrina/imunologia , Espectrina/metabolismoRESUMO
OBJECTIVES: The Ro ribonucleoprotein particle, targeted in systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS), includes Ro60 (SSA) and La (SSA) autoantigens. Anti-Ro60 occurs in SLE and SS. The importance of α-fodrin and spectrin as well as anti-Ro and anti-fodrin/spectrin antibodies in SS and SLE, led us to hypothesise that rabbit immunisation with Ro60 or 4-hydroxy-2-nonenal-modified Ro60 would induce anti-spectrin. In addition, we hypothesised that antibodies to Ro60 and La will develop in animals immunised with spectrin. METHODS: Two NZW rabbits each were immunised with 4-hydroxy-2-nonenal-modified Ro60 or unmodified Ro60. Methods used included ELISA, including an inside-out RBC membrane ELISA, and Crithidia lucilae assays. RESULTS: Commercial anti-spectrin sera bound significantly to Ro60 (OD 2.6 ± 0.1), Ro60 multiple antigenic peptides (MAPs) (3 out of 21 Ro60 MAPs), La (OD 4.4±0.5), and La fragments as well as to double stranded DNA but not to BSA (OD 0.6±0.1). Anti-spectrin binding to purified spectrin could be inhibited by spectrin (>95%), and Ro60 or La (70%). When the binding of anti-spectrin was tested against a nested set of La fragments we found that a N4 fragment representing the C-terminal 250 aa (aa 159 to 408) bound the strongest (OD=4.12) followed by a N9 fragment (the C-terminal 36aa; aa373 to 408 (OD=1.36). Also, significant anti-spectrin antibody levels were induced by Ro60 and HNE-modified Ro60 immunisation. CONCLUSIONS: We found intermolecular epitope spreading from Ro60/La to spectrin and vice versa, and this may have pathological significance in these animal models of autoimmunity.
Assuntos
Aldeídos/imunologia , Anticorpos Antinucleares/sangue , Autoantígenos/imunologia , Autoimunidade , Imunização , Ribonucleoproteínas/imunologia , Espectrina/imunologia , Aldeídos/administração & dosagem , Animais , Ligação Competitiva , DNA/metabolismo , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos , Epitopos , Ligação Proteica , Coelhos , Ribonucleoproteínas/administração & dosagem , Espectrina/administração & dosagem , Antígeno SS-BRESUMO
OBJECTIVE: To investigate the full extent of Purkinje cell cytoplasmic autoantibody type 1 autoimmunity (classically associated with paraneoplastic cerebellar degeneration) from clinical, immunohistochemical, and neuropathological perspectives. DESIGN: Case series. SETTING: Mayo Clinics, 3 sites (Minnesota, Arizona, and Florida). PATIENTS: Of 133,138 patients tested over a 21-year period, 83 (0.06%) were identified as seropositive for Purkinje cell cytoplasmic autoantibody type 1 IgG. MAIN OUTCOME MEASURES: The frequency of cerebellar and noncerebellar disorders and the clinical outcomes (neurological and oncological) of the patients. RESULTS: All patients were women. At initial presentation, 64 patients (77%) had a cerebellar disorder, and 19 patients (23%) had an extracerebellar disorder. Over the clinical course, neurological symptoms and signs were multifocal in 50 patients (60%), and they involved the cerebellum (89% of patients), the pyramidal tract (30%), the brainstem (13%), and the spinal anterior horn cells or peripheral nerve (10%; frequently upper limb predominant); 11% of patients did not develop cerebellar ataxia. Serological and neuropathological findings were observed in the cerebellum, the brainstem, the spinal cord, the anterior horn, and the dorsal root ganglion that paralleled the diversity of clinical signs. After a median follow-up of 18 months, 1 or more carcinomas had been detected in 88% of patients: ovarian epithelial cancer (53%), breast cancer (22%), fallopian tubal cancer (11%), primary peritoneal cancer (5%), metastases of unknown primary cancer (4%), and other cancers (4%). Sustained improvement was reported in 15% of patients following oncological or immunological therapies. Voltage-gated calcium channel antibodies coexisted in 23 patients (28%). CONCLUSIONS: Purkinje cell cytoplasmic autoantibody type 1 autoimmunity most commonly affects the cerebellum, but the spectrum of neurological symptoms and presentations is broad. Neurological outcomes are usually poor, even when cancer remission is achieved.
Assuntos
Doenças do Sistema Nervoso Central/patologia , Cerebelo/metabolismo , Fatores de Troca do Nucleotídeo Guanina/imunologia , Imunoglobulina G/metabolismo , Doenças do Sistema Nervoso Periférico/patologia , Disautonomias Primárias/patologia , Espectrina/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Arizona/epidemiologia , Neoplasias da Mama/complicações , Neoplasias da Mama/imunologia , Carcinoma/complicações , Carcinoma/imunologia , Doenças do Sistema Nervoso Central/líquido cefalorraquidiano , Feminino , Florida/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Minnesota/epidemiologia , Exame Neurológico , Doenças do Sistema Nervoso Periférico/líquido cefalorraquidiano , Fosfopiruvato Hidratase/metabolismo , Disautonomias Primárias/líquido cefalorraquidianoRESUMO
PURPOSE: The aim of this study was to characterize the serum antibody reactivities occurring after ocular ischemia reperfusion. The time course of serum antibody responses was examined. METHODS: Wistar rats were exposed to transient ocular ischemia by elevating intraocular pressure to 130 mm Hg for 60 minutes. Axonal damage was evaluated on optic-nerve sections 2 and 4 weeks later. Blood samples collected before and several times after ischemia were used for antibody detection via customized protein microarrays. Different tissue antigens, including heat shock proteins (HSPs) and crystallins, were selected based on previous identification of antibody reactivities in studies on ischemic events or ophthalmic diseases associated with ischemia. Antibody reactivity was compared using multivariate statistical techniques. RESULTS: Significant axonal damage was observed 2 and 4 weeks after ocular ischemia (P < 0.05). Animals showed certain immunoreactivities against antigens even before ischemia, whereas many reactivities increased afterward. Significantly different responses were detected 2, 3, and 4 weeks after ischemia (P < 0.05). Antibody reactivity against actin, glial fibrillary acidic protein, HSP 27, vimentin, or spectrin continually increased. CONCLUSIONS: Ischemia induced by acute intraocular pressure elevation led to complex changes in antibody reactivities in sera of treated animals. Upregulation of serum autoantibodies, especially against heat shock and structural proteins, progressively increased throughout the 4-week follow-up period, whereas others such as ubiquitin decreased. The upregulation of anti-HSP 27 antibodies might be an attempt to protect the tissue from ischemic damage.
Assuntos
Autoanticorpos/sangue , Autoantígenos/imunologia , Proteínas do Olho/imunologia , Proteínas de Choque Térmico HSP27/imunologia , Traumatismo por Reperfusão/imunologia , Doenças Retinianas/imunologia , Animais , Axônios/patologia , Proteína Glial Fibrilar Ácida/imunologia , Imunoglobulina G/sangue , Pressão Intraocular , Masculino , Proteína Básica da Mielina/imunologia , Proteínas da Mielina , Glicoproteína Associada a Mielina/imunologia , Glicoproteína Mielina-Oligodendrócito , Nervo Óptico/patologia , Análise Serial de Proteínas , Ratos , Ratos Wistar , Vasos Retinianos , Espectrina/imunologia , Regulação para CimaRESUMO
Chronic and complex autoimmune diseases, currently treated palliatively with immunosuppressives, require multi-targeted therapy for greater effectiveness. The naturally occurring polyphenol curcumin has emerged as a powerful "nutraceutical" that interacts with multiple targets to regress diseases safely and inexpensively. Up to 8 g/day of curcumin for 18 months was non-toxic to humans. However, curcumin's utility is limited by its aqueous insolubility. We have demonstrated a heat-mediated 12-fold increase in curcumin's aqueous solubility. Here, we show by SDS-PAGE and surface plasmon resonance that heat-solubilized curcumin binds to proteins. Based on this binding we hypothesized that heat-solubilized curcumin or turmeric would prevent autoantibody targeting of cognate autoantigens. Heat-solubilized curcumin/turmeric significantly decreased binding of autoantibodies from Sjögren's syndrome (up to 43/70%, respectively) and systemic lupus erythematosus (up to 52/70%, respectively) patients as well as an animal model of Sjögren's syndrome (up to 50/60%, respectively) to their cognate antigens. However, inhibition was not specific to autoimmunity. Heat-solubilized curcumin/turmeric also inhibited binding of commercial polyclonal anti-spectrin to spectrin (50/56%, respectively). Thus, we suggest that the multifaceted heat-solubilized curcumin can ameliorate autoimmune disorders. In addition, the non-toxic curcumin could serve as a new protein stain in SDS-PAGE even though it is less sensitive than the Coomassie system which involves toxic chemicals.
Assuntos
Reações Antígeno-Anticorpo/efeitos dos fármacos , Doenças Autoimunes/imunologia , Curcumina/química , Curcumina/farmacologia , Temperatura Alta , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Animais , Autoanticorpos/imunologia , Autoanticorpos/metabolismo , Autoantígenos/imunologia , Autoantígenos/metabolismo , Doenças Autoimunes/dietoterapia , Curcuma/química , Curcuma/metabolismo , Curcumina/metabolismo , Suplementos Nutricionais , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Fatores Imunológicos/metabolismo , Indicadores e Reagentes , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Extratos Vegetais/química , Extratos Vegetais/metabolismo , Extratos Vegetais/farmacologia , Síndrome de Sjogren/imunologia , Solubilidade , Espectrina/imunologia , Ressonância de Plasmônio de SuperfícieRESUMO
Serum from a patient with paraneoplastic encephalomyelitis (PEM) and small cell lung cancer (SCLC) showed high titer immunohistochemical staining of the axon initial segment (AIS) on rat and human brain sections. EM studies showed that the antigen was localized in close proximity of the microtubules in the AIS. Double labeling experiments and absence of staining at the nodes of Ranvier excluded the previously identified betaIV spectrin as autoantigen. Screening a rat hippocampal cDNA library resulted in the isolation of ubiquitin-conjugating enzyme E2E1 (UBE2E1). However, blocking and elution experiments excluded UBE2E1 as the AIS autoantigen.
Assuntos
Autoanticorpos/imunologia , Axônios/imunologia , Neoplasias Pulmonares/imunologia , Síndromes Paraneoplásicas do Sistema Nervoso/imunologia , Carcinoma de Pequenas Células do Pulmão/imunologia , Idoso , Animais , Encéfalo/imunologia , Humanos , Neoplasias Pulmonares/complicações , Masculino , Proteínas do Tecido Nervoso/imunologia , Síndromes Paraneoplásicas do Sistema Nervoso/complicações , Ratos , Carcinoma de Pequenas Células do Pulmão/complicações , Espectrina/imunologia , Enzimas de Conjugação de Ubiquitina/imunologiaRESUMO
Autoantibodies specific for alpha-fodrin fragments are found in the tissues of persons afflicted with Sjögren's syndrome (SS). However, the mechanism for alpha-fodrin degradation remains elusive. The following experiments utilized Par C5 cells to examine the role of P2X7 receptor (P2X7R) in apoptosis, particularly in the cleavage and release of alpha-fodrin, an apparent SS autoantigen. Five mM ATP stimulation induced apoptotic cell death with a sustained Ca2+ influx, which was mimicked in HEK cells transfected with P2X7R. ATP also induced cleavage of alpha-fodrin mediated by caspase-3 and calpain, releasing alpha-fodrin fragments through membrane blebs. However, both apoptotic cell death and alpha-fodrin cleavage were inhibited in the presence of 300 microM oxidized-ATP (ox-ATP), an irreversible blocker of P2X7R, or in Ca(2+)-free solution. We concluded that P2X7R plays an important role in apoptosis and alpha-fodrin degradation in salivary epithelial cells, providing an important clue elucidating the presence of alpha-fodrin fragments in SS tissues.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Receptores Purinérgicos P2/metabolismo , Espectrina/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Apoptose/fisiologia , Autoantígenos/metabolismo , Cálcio/metabolismo , Cálcio/farmacologia , Calpaína/farmacologia , Proteínas de Transporte/antagonistas & inibidores , Caspase 3/farmacologia , Morte Celular/fisiologia , Linhagem Celular , Extensões da Superfície Celular/metabolismo , Extensões da Superfície Celular/ultraestrutura , Células Epiteliais/metabolismo , Humanos , Proteínas de Membrana/imunologia , Proteínas dos Microfilamentos/antagonistas & inibidores , Neuropeptídeos/antagonistas & inibidores , Neuropeptídeos/metabolismo , Glândula Parótida/metabolismo , Glândula Parótida/patologia , Antagonistas do Receptor Purinérgico P2 , Ratos , Receptores Purinérgicos P2X4 , Receptores Purinérgicos P2X7 , Transdução de Sinais/efeitos dos fármacos , Síndrome de Sjogren/imunologia , Síndrome de Sjogren/metabolismo , Espectrina/imunologiaRESUMO
Spectrin was first described in erythrocytes where it forms a filamentous network in the cytoplasmic face of the plasma membrane and participates in the membrane's structural integrity in addition to controlling the lateral mobility of integral membrane proteins. In fungi, spectrin-like proteins have been described in the plasma membrane, concentrated mainly in the region of maximum apical expansion. This localization led to the idea of a spectrin based membrane skeleton in fungi participating in mechanical integrity of the plasma membrane, generating and maintaining cell polarity. The occurrence of spectrin-like proteins in filamentous fungi, yeasts and Oomycetes, however, is questionable since the presence of such proteins has only been demonstrated with immunochemical methods using antibodies whose specificity is unclear. There is no evidence of a gene coding for the high molecular weight alphabeta-spectrin in the genome of these organisms. Mass spectrometric analysis of the anti alphabeta-spectrin immunoreacting peptides from Neurospora crassa and Phytophthora infestans identified them as elongation factor 2 (NCU07700.4) and Hsp70 (PITG_13237.1), respectively. An attempt was made to correlate the reactivity of anti-spectrin antibody to a common feature of these three proteins i.e., spectrin, elongation factor 2 and heat shock protein 70, in that they all have a hydrophobic region implicated in chaperon activity.
Assuntos
Proteínas de Algas/análise , Proteínas Fúngicas/análise , Neurospora crassa/química , Phytophthora/química , Espectrina/análise , Proteínas de Algas/metabolismo , Animais , Especificidade de Anticorpos , Extratos Celulares/análise , Galinhas , Reações Cruzadas , Técnica Indireta de Fluorescência para Anticorpo , Proteínas Fúngicas/imunologia , Proteínas Fúngicas/metabolismo , Soros Imunes/análise , Soros Imunes/imunologia , Neurospora crassa/metabolismo , Fator 2 de Elongação de Peptídeos/análise , Fator 2 de Elongação de Peptídeos/genética , Fator 2 de Elongação de Peptídeos/metabolismo , Phytophthora/metabolismo , Coelhos , Sensibilidade e Especificidade , Espectrina/imunologia , Espectrina/metabolismoRESUMO
BACKGROUND: Hypergammaglobulinemia and polyclonal B-cell activation commonly occur in Plasmodium sp. infections. Some of the antibodies produced recognize self-components and are correlated with disease severity in P. falciparum malaria. However, it is not known whether some self-reactive antibodies produced during P. falciparum infection contribute to the events leading to cerebral malaria (CM). We show here a correlation between self-antibody responses to a human brain protein and high levels of circulating TNF alpha (TNFalpha), with the manifestation of CM in Gabonese children. METHODOLOGY: To study the role of self-reactive antibodies associated to the development of P. falciparum cerebral malaria, we used a combination of quantitative immunoblotting and multivariate analysis to analyse correlation between the reactivity of circulating IgG with a human brain protein extract and TNFalpha concentrations in cohorts of uninfected controls (UI) and P. falciparum-infected Gabonese children developing uncomplicated malaria (UM), severe non-cerebral malaria (SNCM), or CM. RESULTS/CONCLUSION: The repertoire of brain antigens recognized by plasma IgGs was more diverse in infected than in UI individuals. Anti-brain reactivity was significantly higher in the CM group than in the UM and SNCM groups. IgG self-reactivity to brain antigens was also correlated with plasma IgG levels and age. We found that 90% of CM patients displayed reactivity to a high-molecular mass band containing the spectrin non-erythroid alpha chain. Reactivity with this band was correlated with high TNFalpha concentrations in CM patients. These results strongly suggest that an antibody response to brain antigens induced by P. falciparum infection may be associated with pathogenic mechanisms in patients developing CM.
Assuntos
Malária Cerebral/imunologia , Malária Falciparum/imunologia , Espectrina/imunologia , Criança , Estudos de Coortes , Gabão , Humanos , Imunoglobulina G/imunologia , Espectrometria de Massas , Análise Multivariada , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Spectrin tetramers are cytoskeletal proteins required in the formation of complex animal tissues. Mammalian alphaII- and betaII-spectrin subunits form dimers that associate head to head with high affinity to form tetramers, but it is not known if this interaction is regulated. We show here that the short C-terminal splice variant of betaII-spectrin (betaIISigma2) is a substrate for phosphorylation. In vitro, protein kinase CK2 phosphorylates Ser-2110 and Thr-2159; protein kinase A phosphorylates Thr-2159. Antiphospho-Thr-2159 peptide antibody detected phosphorylated betaIISigma2 in Cos-1 cells. Immunoreactivity was increased in Cos-1 cells by treatment with forskolin, indicating that phosphorylation is promoted by elevated cAMP. The effect of forskolin was counteracted by the cAMP-dependent kinase inhibitor, H89. In vitro, protein kinase A phosphorylation of an active fragment of betaIISigma2 greatly reduced its interaction with alphaII-spectrin at the tetramerization site. Mutation of Thr-2159 to alanine eliminated inhibition by phosphorylation. Among the processes that require spectrin in mammals is the formation of neurites (incipient nerve axons). We tested the relationship of spectrin phosphorylation to neuritogenesis by transfecting the neuronal cell line, PC12, with enhanced green fluorescent protein-coupled fragments of betaIISigma2-spectrin predicted to act as inhibitors of spectrin tetramer formation. Both wild-type and T2159E mutant fragments allowed normal neuritogenesis in PC12 cells in response to nerve growth factor. The mutant T2159A inhibited neuritogenesis. Because the T2159A mutant represents a high affinity inhibitor of tetramer formation, we conclude that tetramers are requisite for neuritogenesis. Furthermore, because both the T2159E mutant and the wild-type allow neuritogenesis, we conclude that the short C-terminal betaII-spectrin is phosphorylated during this process.
Assuntos
Neuritos/metabolismo , Neurônios/metabolismo , Espectrina/química , Espectrina/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/farmacologia , Células COS , Caseína Quinase II/metabolismo , Chlorocebus aethiops , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dimerização , Humanos , Dados de Sequência Molecular , Neurônios/ultraestrutura , Células PC12 , Fosforilação , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrina/genética , Espectrina/imunologia , Treonina/metabolismo , TransfecçãoRESUMO
The cortical cytoskeleton of the mammalian red cell, composed of spectrin, actin, protein 4.1, adducin, and protein 4.9, is generally regarded as a homogeneous structure that maintains the integrity of the membrane and the lateral disposition of integral membrane proteins. The major component of this structure is a hetero-oligomer of alphaI and betaISigma1 spectrin. In other tissues, most notably muscle and brain, a transcript of the betaI spectrin gene is generated by alternative exon utilization, yielding a protein that has the COOH-terminal 19 residues of betaISigma1 spectrin replaced by 210 novel residues to generate betaISigma2 spectrin. This new transcript contains a pleckstrin homology (PH) domain and may even exist under some conditions in a homopolymeric form. Using antibodies specific for the COOH-terminal domains of either betaISigma1 or betaISigma2 spectrin, we find that contrary to previous understandings, mature human erythrocytes contain a subpopulation of spectrin that is immunoreactive with antibodies to the betaISigma2 isoform, and that this spectrin is distributed into distinct plasma membrane patches. These results suggest that the native mammalian erythrocyte membrane skeleton, rather than being homogeneous, contains discrete submicron-scale microdomains that differ in both their composition and dispersion across the cell surface. The precise nature and role of these putative microdomains is under active investigation.
Assuntos
Citoesqueleto/ultraestrutura , Membrana Eritrocítica/ultraestrutura , Espectrina/metabolismo , Anticorpos Monoclonais , Eritrócitos/química , Eritrócitos/ultraestrutura , Humanos , Microdomínios da Membrana , Microscopia de Fluorescência , Isoformas de Proteínas , Espectrina/análise , Espectrina/imunologiaRESUMO
Spectrin is the major component of the erythrocyte membrane skeleton and exists as a 526 kDa alphabeta heterodimer. The 246 kDa beta-chain of human spectrin is phosphorylated near the C-terminus, but the exact phosphorylation sites are unknown and the role of this phosphorylation is not fully characterized. In this study, we produced a monoclonal antibody, Sp316, capable of recognizing the C-terminal region of beta-spectrin regardless of its phosphorylation state and used it to purify the phosphorylated region after 2-nitro-5-thiocyanobenzoic acid cleavage of spectrin. Two-dimensional gels, mass spectrometry, and reversed-phase high-performance liquid chromatography were used to characterize these phosphorylation states. Only about 1.5% of spectrin isolated from fresh blood is unphosphorylated, about 9% has more than four phosphates per molecule, and the majority of the protein has one to four phosphates per molecule. A total of six phosphorylation sites were identified by tandem mass spectrometry. Quantitative analysis of the phosphorylation states by reversed-phase high-performance liquid chromatography revealed that phosphorylation of beta-spectrin occurs in a sequential manner where each specific site is completely phosphorylated before the next site is modified. The first phosphorylation event occurs on Ser-2114, followed by Ser-2125, Ser-2123, Ser-2128, Ser-2117, and Thr-2110. The identification of the specific phosphorylated beta-spectrin residues and the ordered sequence of phosphorylation events in vivo should provide an invaluable basis for further studies of the role of these posttranslational modifications in spectrin function in situ.
Assuntos
Membrana Eritrocítica/metabolismo , Espectrina/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel Bidimensional , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Hidrólise , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Mapeamento de Peptídeos , Fosforilação , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Espectrina/imunologia , Espectrina/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Tiocianatos/metabolismo , Tripsina/metabolismoRESUMO
We report on the in vivo uptake of antibodies into plant protoplasts. When protoplasts of sunflower, Arabidopsis or tobacco were incubated in vivo with an antibody, this antibody was detected by immunofluorescence in the cytoplasm and/or the nucleus, depending on the location of the target protein. Furthermore, when protoplasts were cultured in the presence of antibodies, specific effects were observed. Incubation with antibodies raised against p34cdc2 led to a strong inhibition of the division rate, and a decrease in the average DNA content of protoplasts. With antibodies against HaWLIM1, a LIM domain protein of the CRP type, a negative effect on actin organisation was observed. We conclude that antibodies can penetrate plant protoplasts in vivo, and thus may be used as powerful tools for the study of protein function.
Assuntos
Anticorpos/metabolismo , Calreticulina/imunologia , Plantas/imunologia , Protoplastos/imunologia , Espectrina/imunologia , Animais , Anticorpos/farmacologia , Arabidopsis/imunologia , Transporte Biológico , Proteína Quinase CDC2/imunologia , Linhagem Celular , Células Cultivadas , Galinhas , Helianthus/fisiologia , Humanos , Protoplastos/efeitos dos fármacos , Nicotiana/imunologia , Tubulina (Proteína)/imunologiaRESUMO
Spectrin tetramers form by the interaction of two alpha-beta dimers through two helices close to the C-terminus of a beta subunit and a single helix at the N-terminus of an alpha subunit. Early work on spectrin from solid tissues (typified by alphaII and betaII polypeptides) indicated that it forms a more stable tetramer than erythroid spectrin (alphaI-betaI). In the present study, we have probed the molecular basis of this phenomenon. We have quantified the interactions of N-terminal regions of two human alpha polypeptides (alphaI and alphaII) with the C-terminal regions of three beta isoforms (betaISigma1, betaIISigma1 and betaIISigma2). alphaII binds either betaII form with a much higher affinity than alphaI binds betaISigma1 ( K (d) values of 5-9 nM and 840 nM respectively at 25 degrees C). betaIISigma1 and betaIISigma2 are splice variants with different C-terminal extensions outside the tetramerization site: these extensions affect the rate rather than the affinity of alpha subunit interaction. alphaII spectrin interacts with each beta subunit with higher affinity than alphaI, and the betaII polypeptides have higher affinities for both alpha chains than betaISigma1. The first full repeat of the alpha subunit has a major role in determining affinity. Enthalpy changes in the alphaII-betaIISigma2 interaction are large, but the entropy change is comparatively small. The interaction is substantially reduced, but not eliminated, by concentrated salt solutions. The high affinity and slow overall kinetics of association and dissociation of alphaII-betaII spectrin may suit it well to a role in strengthening cell junctions and providing stable anchor points for transmembrane proteins at points specified by cell-adhesion molecules.
Assuntos
Espectrina/metabolismo , Sítios de Ligação , Humanos , Cinética , Substâncias Macromoleculares , Concentração Osmolar , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrina/química , Espectrina/imunologia , Ressonância de Plasmônio de Superfície , Temperatura , TermodinâmicaRESUMO
Spectrin, a component of the membrane skeleton in erythrocytes and other animal cells, has also been identified in plant and fungal cells. However, its postulated role, i.e., the maintenance of shape and elasticity of the plasma membrane, is probably not exerted in walled cells. To study spectrin in these cells, we chose yeasts because of a high morphological variability of their life cycle. The localization of spectrin in the cells and protoplasts of Saccharomyces cerevisiae and Schizosaccharomyces japonicus var. versatilis was detected by immunoblotting, indirect immunofluorescence, and immunogold electron microscopy techniques with the use of anti-chicken and anti-human erythrocyte spectrin antibodies. A protein band of 220-240 kDa and some bands of lower relative mass were detected in cell and protoplast extracts of both yeast strains. Spectrin-like proteins were revealed by fluorescence microscopy at cell surfaces and in vacuolar membranes. Immunogold-labelling showed spectrin-like proteins in the plasma membrane, endoplasmic reticulum, vacuoles, nuclei, vesicles, mitochondria, and cell walls. The topology of spectrin was not affected by actin depolymerization with Latrunculin B nor was it changed in either act1-1 or cdc42 mutants, under restrictive conditions. Under osmotic stress, both spectrin and actin were delocalized and appeared in the form of large clusters in the cytoplasm. It is concluded that a protein cross-reacting with spectrin antibodies is present in fission and budding yeasts. Generally, it is located in the proximity of the plasma membrane and other intracellular membranes, probably as a part of the membrane skeleton. No evidence of its relationship to either actin or growth zones of the cell can be provided.
Assuntos
Proteínas Fúngicas/análise , Saccharomyces cerevisiae/química , Schizosaccharomyces/química , Espectrina/análise , Actinas/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Extratos Celulares/imunologia , Meios de Cultura , Imunofluorescência , Proteínas Fúngicas/imunologia , Humanos , Immunoblotting/métodos , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/ultraestrutura , Mutação , Pressão Osmótica , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/classificação , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Espectrina/imunologia , Tiazóis/farmacologia , TiazolidinasRESUMO
Autoimmune haemolytic anaemia (AIHA) can be induced in mice by repeated injections with rat red blood cells (RBC). Here we describe the identification of rat and murine RBC antigens recognized by T-cells from mice with this disease. Splenic T-cells from mice with AIHA proliferated in response to multiple murine RBC membrane components, each of which is recognized by rat RBC induced autoantibodies. Thus, there were responses to murine autoantigen fractions that correspond in apparent molecular mass with the anion channel Band 3, with spectrin from the membrane skeleton and with the high and low molecular mass glycophorins, and the equivalent fractions from rat RBC also stimulated proliferation by T-cells. It was confirmed that purified Band 3 from murine and rat RBC also elicited responses. In contrast with the results in AIHA, T-cells from healthy control mice failed to respond to the antigens from either species, with the exception of proliferation induced by murine spectrin in one experiment and weak responses elicited by rat Band 3. It is suggested that T-cells activated by multiple cross-reactions between rat and murine RBC proteins, and by epitope spreading, are necessary to drive autoantibody production in this model of AIHA.
Assuntos
Anemia Hemolítica Autoimune/imunologia , Linfócitos T/imunologia , Anemia Hemolítica Autoimune/etiologia , Animais , Proteína 1 de Troca de Ânion do Eritrócito/imunologia , Proteína 1 de Troca de Ânion do Eritrócito/isolamento & purificação , Autoantígenos/isolamento & purificação , Reações Cruzadas , Eritrócitos/imunologia , Glicoforinas/imunologia , Glicoforinas/isolamento & purificação , Técnicas In Vitro , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos CBA , Ratos , Ratos Wistar , Especificidade da Espécie , Espectrina/imunologia , Espectrina/isolamento & purificaçãoRESUMO
Calpain activity and expression at the protein level were examined in inflammatory cells, activated microglia, and astrocytes prior to or at onset of symptomatic experimental allergic encephalomyelitis (EAE), an animal model for the human demyelinating disease multiple sclerosis (MS). EAE was induced in Lewis rats by injection of guinea pig spinal cord homogenate and myelin basic protein (MBP) emulsified with Complete Freund's Adjuvant (CFA). Calpain translational expression, determined by Western blot and immunocytochemistry, was correlated with calpain activity, infiltration of inflammatory cells, and myelin loss at 2-11 days following challenge with antigen. Controls (CFA only) did not show any changes over time in these parameters and very few changes (CD11+ microglia/mononuclear phagocytes) were seen in either group from days 2 to 8 post-induction. In contrast, from days 9 to 11, the animals that developed the disease (at least grade 1) demonstrated extensive cellular infiltration (CD4+, CD25+, and CD11+ as well as increased calpain expression (content) and activity. This study demonstrates that cell infiltration and increased calpain activity do not begin in the CNS until the onset of clinical signs.
Assuntos
Antígenos CD , Antígenos de Neoplasias , Antígenos de Superfície , Proteínas Aviárias , Proteínas Sanguíneas , Calpaína/metabolismo , Sistema Nervoso Central/imunologia , Quimiotaxia de Leucócito/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Neuroglia/metabolismo , Fagócitos/metabolismo , Linfócitos T/metabolismo , Animais , Basigina , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Calpaína/imunologia , Sistema Nervoso Central/patologia , Sistema Nervoso Central/fisiopatologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/fisiopatologia , Imunofluorescência , Adjuvante de Freund/farmacologia , Masculino , Glicoproteínas de Membrana/metabolismo , Proteína Básica da Mielina/imunologia , Proteína Básica da Mielina/metabolismo , Proteínas de Neurofilamentos/imunologia , Proteínas de Neurofilamentos/metabolismo , Neuroglia/imunologia , Fagócitos/imunologia , Ratos , Ratos Endogâmicos Lew , Receptores de Interleucina-2/imunologia , Espectrina/imunologia , Espectrina/metabolismo , Linfócitos T/imunologia , Regulação para Cima/imunologiaRESUMO
alpha-, beta- Spectrin is ubiquitously distributed in neuronal compartments, i.e. at the plasma membrane of neuronal cell bodies, axonal and dendritic processes, pre- and postsynaptic nerve terminals and around synaptic vesicles, "Brain Res. Bull. 50 (1999) 345". Ultrastructural analyses in the rat retina using the monoclonal antibody AA6, "Hear. Res. 43 (1990) 199", against non-erythroid alpha-spectrin, alphaSPII (spectrin nomenclature according to "Blood 81 (1993) 3173"), revealed that the antibody intensively labeled the cytoplasmic face of the plasma membrane in virtually all neuronal processes. However, no significant immunolabel was observed at the presynaptic plasma membrane, around synaptic vesicles, at presynaptic densities and synaptic ribbons. Therefore, synaptic non-erythroid alpha-spectrin differs immunologically from extrasynaptic non-erythroid alpha-spectrin. This heterogeneity might contribute to the generation of distinct retinal microdomains.
Assuntos
Retina/química , Espectrina/análise , Sinapses/química , Animais , Anticorpos Monoclonais , Membrana Celular/química , Microscopia Eletrônica , Neurópilo/química , Neurópilo/ultraestrutura , Ratos , Retina/ultraestrutura , Espectrina/imunologia , Sinapses/ultraestruturaRESUMO
We report the purification of a presynaptic "particle web" consisting of approximately 50 nm pyramidally shaped particles interconnected by approximately 100 nm spaced fibrils. This is the "presynaptic grid" described in early EM studies. It is completely soluble above pH 8, but reconstitutes after dialysis against pH 6. Interestingly, reconstituted particles orient and bind PSDs asymmetrically. Mass spectrometry of purified web components reveals major proteins involved in the exocytosis of synaptic vesicles and in membrane retrieval. Our data support the idea that the CNS synaptic junction is organized by transmembrane adhesion molecules interlinked in the synaptic cleft, connected via their intracytoplasmic domains to the presynaptic web on one side and to the postsynaptic density on the other. The CNS synaptic junction may therefore be conceptualized as a complicated macromolecular scaffold that isostatically bridges two closely aligned plasma membranes.