RESUMO
Animal models are fundamental to advance knowledge of disease pathogenesis and to test/develop new therapeutic strategies. Most of the current knowledge about the pathogenic mechanisms underpinning autoimmune demyelination processes implicating autoantigens has been obtained using the Experimental Autoimmune Neuritis (EAN) animal model. The most widely used EAN model is obtained by active immunization of Lewis rats using a peptide, P0 (180-199), issuing from the major peripheral nervous system myelin protein. But this model mimics only the classical monophasic acute form of demyelinating polyradiculoneuropathy, i.e. Guillain-Barré syndrome (GBS). We developed a new model by immunizing Lewis rats using the same immunodominant neuritogenic peptide P0 (180-199) but this time with its S-palmitoyl derivative, S-palm P0 (180-199). All of the animals immunized with the S-palm P0 (180-199) peptide developed a chronic relapsing-remitting form of the disease corresponding to the electrophysiological criteria of demyelination (slow sensory nerve conduction velocity, prolonged motor nerve latency, partial motor nerve conduction blocks) with axon degeneration. These findings were confirmed by immunohistopathology study and thus, appear to mimic human chronic inflammatory demyelinating polyradiculopathy (CIDP). This new model opens up new avenues of research for testing new anti-inflammatory and neuroprotective therapeutic strategies.
Assuntos
Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/induzido quimicamente , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/tratamento farmacológico , Animais , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/tratamento farmacológico , Doenças Desmielinizantes/fisiopatologia , Modelos Animais de Doenças , Imuno-Histoquímica , Neurônios Motores , Condução Nervosa , Neurite Autoimune Experimental , Ácido Palmítico/química , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/fisiopatologia , Ratos , Ratos Endogâmicos LewRESUMO
Opticospinal demyelinating diseases in humans are mostly characterized by the opticospinal form of multiple sclerosis (MS) and neuromyelitis optica (NMO). Increasing attention has recently focused on astrocyte markers, aquaporin-4 (AQP4) and glial fibrillary acidic protein (GFAP) in these diseases. We induced opticospinal demyelination in Brown Norway rats with soluble recombinant rat myelin oligodendrocyte glycoprotein (1-116) and incomplete Freund's adjuvant. Clinical, MRI, neuropathological and immunological evaluations were performed, with a focus on AQP4 and GFAP. We confirmed the opticospinal phenotype, including extensive myelitis, but also showed the MRI-characterized involvement of the periventricular area. Expression levels of myelin, AQP4 and GFAP showed the early involvement of astrocytes before demyelination in the optic nerve. The overexpression of AQP4 was particularly pronounced in the spinal cord and was concomitant with demyelination and astrocyte apoptosis. The disability scores were correlated with demyelination and inflammation but not with AQP4/GFAP expression. No antibodies against the linear and conformational epitopes of AQP4 were detected. Whereas a NMO-like phenotype was observed in this model, the AQP4/GFAP expression during the disease process was more closely related to opticospinal MS than NMO. However, this model raises the question of a continuum between opticospinal MS and the seronegative NMO subtype.
Assuntos
Esclerose Múltipla/fisiopatologia , Neuromielite Óptica/patologia , Nervo Óptico/patologia , Medula Espinal/patologia , Animais , Aquaporina 4/metabolismo , Modelos Animais de Doenças , Encefalite/patologia , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/complicações , Ensaio de Imunoadsorção Enzimática , Feminino , Regulação da Expressão Gênica/fisiologia , Proteína Glial Fibrilar Ácida/metabolismo , Imageamento por Ressonância Magnética , Esclerose Múltipla/patologia , Proteína Básica da Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito/metabolismo , Glicoproteína Mielina-Oligodendrócito/toxicidade , Neuromielite Óptica/induzido quimicamente , Neuromielite Óptica/metabolismo , Nervo Óptico/metabolismo , Fragmentos de Peptídeos/toxicidade , Ratos , Medula Espinal/metabolismo , Estatísticas não Paramétricas , Fatores de TempoRESUMO
To study the effects of a point mutation found in Pelizaeus-Merzbacher disease (PMD) on the physicochemical and structural properties of the extracellular loop 4 of the myelin proteolipid protein (PLP), we synthesized the peptide PLP(181-230)Pro215 and one mutant PLP(181-230)Ser215 with regioselective formation of the two disulphide bridges Cys200-Cys219 and Cys183-Cys227. As conventional amino acid building blocks failed to give crude peptides of good quality we had to optimize the synthesis by introducing pseudoproline dipeptide building blocks during the peptide elongation. In peptide Pro215 the first bridge Cys200-Cys219 was obtained after air oxidation, but in peptide Ser215 because of aggregation, dimethyl sulfoxide (DMSO) oxidation had to be used. The second bridge Cys183-Cys227 was obtained by iodine oxidation of both Cys (acetamidomethyl, Acm)-protected peptides. The secondary structures of the parent and mutant loops were analysed by circular dichroism (CD) in the presence of trifluoroethanol (TFE) and sodium dodecyl sulphate (SDS) as a membrane mimetic. Analysis of the spectra showed that the content of alpha-helix and beta-sheet varied differently for both peptides in TFE and SDS solutions, demonstrating the sensitivity of their conformation to the environment and the differences in their secondary structure. The ability of both peptides to insert into the SDS micelles was assayed by intrinsic tryptophan fluorescence.
Assuntos
Proteína Proteolipídica de Mielina/síntese química , Doença de Pelizaeus-Merzbacher/genética , Mutação Puntual , Sequência de Aminoácidos , Substituição de Aminoácidos , Dicroísmo Circular , Dimetil Sulfóxido , Humanos , Iodo/metabolismo , Micelas , Dados de Sequência Molecular , Proteína Proteolipídica de Mielina/química , Oxirredução , Fragmentos de Peptídeos , Peptídeos/síntese química , Peptídeos/química , Prolina/química , Estrutura Secundária de Proteína , Serina/química , Dodecilsulfato de Sódio/química , Trifluoretanol/químicaRESUMO
Our interest is focused on the induction of allergic contact dermatitis (ACD) by the strong skin sensitizer, methyl octanesulfonate, which is a potent methyl transfer agent, especially to histidine and methionine residues. We are particularly interested to study the effect of methylation on the presentation and recognition of the ovalbumin (OVA) T-cell epitope, OVA323-339, by the T-cell receptor (TCR). Here we report the synthesis of the modified monomer N-alpha-Fmoc-N-tau-methyl-L-histidine and its incorporation by solid phase synthesis into the three possible methylated analogues of OVA323-339, that were needed as references for the subsequent studies. Native OVA was haptenized by methyl octanesulfonate. Using classical protein chemistry techniques (trypsin digestion, gel permeation, HPLC, MS and Edman sequencing) we were able to show that OVA323-339 was selectively methylated at His331. Circular dichroism (CD) studies showed that the methylation has no influence on the secondary structure of the peptide.
Assuntos
Alcanossulfonatos/química , Alérgenos/química , Epitopos de Linfócito T/química , Haptenos/química , Histidina/análogos & derivados , Histidina/química , Ovalbumina/química , Alcanossulfonatos/imunologia , Alérgenos/imunologia , Animais , Galinhas , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Haptenos/imunologia , Histidina/síntese química , Irritantes/química , Irritantes/imunologia , Metilação , Ovalbumina/imunologia , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
Proteolipid protein (PLP) is the most abundant protein of CNS myelin, and is posttranslationally acylated by covalent attachment of long chain fatty acids to cysteine residues via a thioester linkage. Two of the acylation sites are within epitopes of PLP that are encephalitogenic in SJL/J mice (PLP(104-117) and PLP(139-151)) and against which increased immune responses have been detected in some multiple sclerosis patients. It is known that attachment of certain types of lipid side chains to peptides can result in their enhanced immunogenicity. The aim of this study was to determine whether thioacylated PLP peptides, as occur in the native protein, are more immunogenic than their nonacylated counterparts, and whether thioacylation influences the development of autoreactivity and experimental autoimmune encephalomyelitis. The results show that in comparison with nonacylated peptides, thioacylated PLP lipopeptides can induce greater T cell and Ab responses to both the acylated and nonacylated peptides. They also enhanced the development and chronicity of experimental autoimmune encephalomyelitis. Synthetic peptides in which the fatty acid was attached via an amide linkage at the N terminus were not encephalitogenic, and they induced greater proportions of CD8+ cells in initial in vitro stimulation. Therefore, the lability and the site of the linkage between the peptide and fatty acid may be important for induction of encephalitogenic CD4+ T cells. These results suggest that immune responses induced by endogenous thioacylated lipopeptides may contribute to the immunopathogenesis of chronic experimental demyelinating diseases and multiple sclerosis.
Assuntos
Adjuvantes Imunológicos/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Epitopos Imunodominantes/imunologia , Epitopos Imunodominantes/metabolismo , Proteína Proteolipídica de Mielina/imunologia , Proteína Proteolipídica de Mielina/metabolismo , Ácidos Palmíticos/metabolismo , Acilação , Adjuvantes Imunológicos/administração & dosagem , Sequência de Aminoácidos , Animais , Autoanticorpos/biossíntese , Linhagem Celular , Células Cultivadas , Encefalomielite Autoimune Experimental/etiologia , Feminino , Epitopos Imunodominantes/administração & dosagem , Imunofenotipagem , Injeções Subcutâneas , Lipoproteínas/administração & dosagem , Lipoproteínas/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Proteína Proteolipídica de Mielina/administração & dosagem , Ácidos Palmíticos/imunologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/imunologiaRESUMO
The unique role of interleukin (IL)-5 in eosinophil production, activation, and localization makes this cytokine a prime target for therapeutic intervention in diseases characterized by a selective blood and tissue eosinophilia. In an attempt to block the effects of IL-5 on eosinophils, a strategy was developed to suppress the expression of the IL-5 receptor alpha chain (IL-5Ralpha) by antisense oligonucleotides (ASOs). IL-5Ralpha ASOs were identified which selectively and specifically suppress the expression of messenger RNA and proteins of both the membrane and the soluble form of the receptor in constitutively IL-5R-expressing murine BCL-1 cells in vitro. Moreover, these IL-5Ralpha-specific ASOs were able to selectively inhibit the IL-5-induced eosinopoesis from murine fetal liver and bone marrow cells in vitro, suggesting that these molecules may affect the development of IL-5-mediated eosinophilia in vivo. Indeed, intravenous administration of IL-5Ralpha-specific ASOs not only suppressed the bone-marrow and blood eosinophilia in mice after short-term treatment with recombinant murine IL-5 but also inhibited the development of blood and tissue eosinophilia in a ragweed-induced allergic peritonitis model. Thus, blocking the expression of IL-5Ralpha on eosinophil using ASOs may have therapeutic benefits in eosinophilic diseases such as asthma.
Assuntos
Eosinofilia/prevenção & controle , Eosinófilos/metabolismo , Interleucina-5/farmacologia , Oligonucleotídeos Antissenso/farmacologia , Receptores de Interleucina/antagonistas & inibidores , Animais , Northern Blotting , Western Blotting , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Primers do DNA/química , Eosinofilia/metabolismo , Feminino , Humanos , Técnicas In Vitro , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Peritonite/genética , Peritonite/patologia , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Receptores de Interleucina/metabolismo , Receptores de Interleucina-5 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transfecção , Células Tumorais CultivadasRESUMO
The tyrosine kinase Syk has been proposed to play a critical role in the antiapoptotic effect of interleukin (IL)-5 in human eosinophils. However, little is known about the involvement of Syk in other IL-5-mediated activation events. To further address these questions, the role of Syk in IL-5-induced eosinophil differentiation, activation, and survival was analyzed using cells obtained from Syk-deficient mice. We could demonstrate that Syk-deficient fetal liver cells differentiate into mature eosinophils in response to IL-5 at the same rate as wild-type fetal liver cells and generate the same total number of eosinophils. Moreover, no difference in IL-5-induced survival of mature eosinophils between Syk(-/-) and wild-type eosinophils could be demonstrated, suggesting that the antiapoptotic effect of IL-5 does not require Syk despite the activation of this tyrosine kinase upon IL-5 receptor ligation. In contrast, eosinophils derived from Syk-deficient but not wild-type mice were incapable of generating reactive oxygen intermediates in response to Fcgamma receptor (FcgammaR) engagement. Taken together, these data clearly demonstrate no critical role for Syk in IL-5-mediated eosinophil differentiation or survival but underline the importance of this tyrosine kinase in activation events induced by FcgammaR stimulation.
Assuntos
Diferenciação Celular , Sobrevivência Celular , Precursores Enzimáticos/deficiência , Eosinófilos/enzimologia , Interleucina-5/farmacologia , Proteínas Tirosina Quinases/deficiência , Receptores Fc/fisiologia , Animais , Apoptose , Ativação Enzimática , Precursores Enzimáticos/fisiologia , Eosinófilos/citologia , Peptídeos e Proteínas de Sinalização Intracelular , Fígado/citologia , Fígado/embriologia , Fígado/enzimologia , Camundongos , Proteínas Tirosina Quinases/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória , Quinase SykRESUMO
In order to test the effect of thiopalmitoylation on the encephalitogenic properties of two proteolipid protein (PLP) T-cell epitopes, we have studied the on-resin S-palmitoylation of peptides, synthesized using the Fmoc/tBu strategy. The use of two Cys protecting groups was investigated: the tert-butylsulfenyl (StBu) and the methoxytrityl (Mmt). Our studies show that the ease of deprotection of the thiol protected with StBu was sequence dependent. The deprotection of Cys(StBu) was difficult in the case of the two peptides PLP(104-117) and PLP(139-151). Neither of the two Cys(StBu) (Cys108 and Cys140, respectively) could be deprotected with tributylphosphine. Beta-mercaptoethanol was only efficient for the deprotection of Cys(StBu)140 at 85 degrees C and at 135 degrees C for Cys108. The two palmitoylated peptides could be obtained in good yield starting from Cys protected with Mmt. Our conclusion is that the Mmt group is the more versatile protecting group of the thiol for use in the on-resin synthesis of thiopalmitoylated peptides.
Assuntos
Epitopos de Linfócito T/química , Proteína Proteolipídica de Mielina/química , Peptídeos/química , Peptídeos/síntese química , Acilação , Humanos , Ácidos Palmíticos , Resinas Vegetais/química , Compostos de SulfidrilaRESUMO
The low levels of complement receptor 1 (CR1) on erythrocytes in autoimmune diseases and AIDS may be due to accelerated loss in the circulation, or to a diminished expression of CR1 on the red cell lineage. Therefore, we analyzed the expression of CR1 on reticulocytes (R) vs erythrocytes (E). Healthy subjects had a significant higher CR1 number per cell on R (919 +/- 99 CR1/cell) than on E (279 +/- 30 CR1/cell, n = 23), which corresponded to a 3. 5- +/- 1.3-fold loss of CR1. This intravascular loss was confirmed by FACS analysis, which showed that all R expressed CR1, whereas a large fraction of E was negative. The systemic lupus erythematosus (SLE), HIV-infected, and cold hemolytic Ab disease (CHAD) patients had a CR1 number on R identical to the healthy subjects, contrasting with a lower CR1 on their E. The data indicated a significantly higher loss of CR1 in the three diseases, i.e., 7.0- +/- 3.8-, 6.1- +/- 2.9-, and 9.6- +/- 5.6-fold, respectively. The intravascular loss was best exemplified in a patient with factor I deficiency whose CR1 dropped from 520 CR1/R to 28 CR1/E, i.e., 18.6-fold loss. In one SLE patient and in the factor I-deficient patient, the FACS data were consistent with a loss of CR1 already on some R. In conclusion, CR1 is lost progressively from normal E during in vivo aging so that old E are almost devoid of CR1. The low CR1 of RBC in autoimmune diseases and HIV-infection is due to a loss occurring in the circulation by an active process that remains to be defined.
Assuntos
Infecções por HIV/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Receptores de Complemento 3b/biossíntese , Reticulócitos/metabolismo , Aglutininas/sangue , Envelhecimento/sangue , Envelhecimento/imunologia , Anemia Hemolítica Autoimune/sangue , Anemia Hemolítica Autoimune/imunologia , Crioglobulinas , Ensaio de Imunoadsorção Enzimática , Eritrócitos/metabolismo , Infecções por HIV/sangue , Humanos , Immunoblotting , Lúpus Eritematoso Sistêmico/sangue , Receptores de Complemento 3b/sangue , Receptores de Complemento 3b/fisiologia , Reticulócitos/imunologia , Reticulócitos/fisiologiaRESUMO
An in vivo magnetic resonance (MR) imaging study was performed on experimental allergic encephalomyelitis (EAE) induced in Lewis rats through proteolipid protein (PLP). PLP was solubilized in water or in an aqueous solution of 1% 10-tridecyl ether (TDE), a non-ionic detergent used in membrane protein research. All 16 rats immunized with 500 microg of TDE-solubilized PLP developed clinical signs and MR abnormalities fully comparable to those observed in MBP-induced EAE. Total paraplegia was observed in 12.5% of rats, mild or moderate paraparesis in 68.8% of rats and tail paralysis in the remaining 18.7% of rats. Whereas only 37.5% of the eight rats immunized with 500 microg of water-solubilized PLP developed minor clinical signs (tail weakness or paralysis). Our observations confirm that the difficulties encountered when trying to induce EAE by means of PLP arise from the highly hydrophobic nature of this protein. Accordingly, if a reproducible model is to be developed, it seems more judicious to use non-ionic detergents in both the extraction and solubilization phases of PLP preparation, this would allow maximal solubilization of the protein while avoiding aggregates, which may otherwise form during either of the PLP preparation.
Assuntos
Apoproteínas/imunologia , Encefalomielite Autoimune Experimental/diagnóstico , Encefalomielite Autoimune Experimental/imunologia , Imageamento por Ressonância Magnética , Proteína Proteolipídica de Mielina/imunologia , Animais , Apoproteínas/efeitos dos fármacos , Encéfalo/patologia , Bovinos , Encefalomielite Autoimune Experimental/patologia , Éteres/farmacologia , Feminino , Imunização , Proteína Proteolipídica de Mielina/efeitos dos fármacos , Ratos , Ratos Endogâmicos Lew , Solubilidade , Medula Espinal/patologia , ÁguaRESUMO
The monoclonal antibody M6-7, which recognizes both native and denatured immunopurified M6a antigen, was used in the present immunocytochemical study to localize its corresponding antigen in young rat brain. Strong labelling was observed in the cerebellar molecular layer, which corresponds to heavily stained axon terminals originating from granule cells. The immunodeposit, as observed by electron microscopy, is present only on the cytoplasmic side of the presynaptic membrane and on the membrane of synaptic vesicles. In contrast, the Purkinje cells and their processes are unstained. Stained synapses are also found, although less frequently, in several other cerebral areas. The pattern of staining at these synapses is similar to that observed in the cerebellar molecular layer. It is hypothesized, on the basis of its restricted distribution in certain neuronal endings and its high homology with myelin proteolipids, that the M6a antigen revealed by the M6-7 antibody is probably involved in a specific biological function in these structures.
Assuntos
Encéfalo/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Anticorpos Monoclonais , Encéfalo/ultraestrutura , Imuno-Histoquímica , Microscopia Imunoeletrônica , RatosRESUMO
Myelin proteolipid protein (PLP) contains 2 immunodominant encephalitogenic epitopes in SJL mice, namely PLP residues 139-151 and 178-191. DM20, a minor isoform of PLP, lacks residues 116-150 and consequently contains only the single major encephalitogenic epitope 178-191. However, it has been found previously that bovine DM20 is not encephalitogenic in SJL mice. Since residue 188 within peptide 178-191 is phenylalanine (F) in murine DM20 and alanine (A) in bovine DM20, we tested the effect of this difference on the immune responses and induction of EAE. SJL mice were immunized with either highly purified murine or bovine DM20. Residues 178-191 were found to be immunodominant for each, but only murine and not bovine DM20 was encephalitogenic. A synthetic peptide corresponding to the murine 178-191 sequence (F188) was also encephalitogenic, whereas the peptide corresponding to the bovine sequence (A188) was not. Both F188 and A188 bind with high affinity to I-As and both are recognized by the SJL T cell repertoire. A188-specific T cell lines reacted to both A188 and F188, but F188-specific T cell lines were not stimulated by A188. F188-specific T cell lines produced mRNA for the Th1 cytokines IL2 and IFN gamma and, in passive transfer experiments, were encephalitogenic upon stimulation with F188, but not A188. In contrast, A188-specific T cell lines produced mRNA for IL4, IL5 and Il10, in addition to IL2 and IFN gamma, and were not encephalitogenic after stimulation with either F188 or A188. Cotransfer of A188-specific T cell lines with F188-specific T cell lines resulted in protection from EAE. Thus, A188 induces a functionally different phenotype of T cells from that induced by F188. Taken together these data suggest that the failure of bovine DM20 to induce EAE may be attributable to induction of protective rather than pathogenic T cell by the immunodominant epitope.
Assuntos
Epitopos/química , Epitopos/imunologia , Proteína Proteolipídica de Mielina/química , Proteína Proteolipídica de Mielina/imunologia , Sequência de Aminoácidos , Animais , Bovinos , Encefalomielite Autoimune Experimental/imunologia , Feminino , Antígenos de Histocompatibilidade Classe II/imunologia , Imunização , Imunização Passiva , Camundongos , Camundongos Endogâmicos C3H , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Relação Estrutura-Atividade , Linfócitos T/imunologiaRESUMO
Lewis rat polyclonal antibodies raised against pure PLP and DM-20 were characterized by indirect and competitive ELISA assays. Anti-PLP antibodies showed a good cross-reactivity with DM-20 and vice versa, suggesting that the chief B-cell epitopes are the same on both proteolipids. Three antigenic determinants, PLP 57-70, PLP 200-218 and the carboxyl-terminal sequence PLP 262-276, were identified for both proteolipids. Peptide PLP 57-70 represents a new B-cell epitope. The major hydrophilic loop (PLP 116-150), deleted in DM-20, did not show any antigenic activity. The study of polyclonal immune response to PLP and DM-20 will be helpful in resolving their potential role in the development and course of experimental allergic encephalomyelitis (EAE) and multiple sclerosis (MS).
Assuntos
Encéfalo/metabolismo , Epitopos/análise , Proteína Proteolipídica de Mielina , Proteínas do Tecido Nervoso , Proteolipídeos/imunologia , Sequência de Aminoácidos , Animais , Bovinos , Mapeamento de Epitopos , Soros Imunes/análise , Imunoensaio , Técnicas In Vitro , Dados de Sequência Molecular , Proteolipídeos/química , Ratos , Ratos Endogâmicos LewRESUMO
The Ser(P)-containing peptide corresponding to phospholamban 11-19, Ac-Ala-Ile-Arg-Arg-Ala-Ser(P)-Thr-Ile-Glu-NH2, was prepared by the use of Boc-Ser(PO3Ph2)-OH in Boc/solid-phase peptide synthesis followed by HF cleavage of the peptide from the polystyrene resin and subsequent platinum-mediated hydrogenolytic cleavage of the phenyl phosphate groups. A study of the HF deprotection step showed that extensive dephosphorylation of the Ser(PO3Ph2)-residue occurred using three commonly used HF conditions and gave rise to large quantities of the Ser-containing peptide. The subsequent study of model peptide systems under standard HF conditions established firstly that the extent of dephosphorylation was dependent on the HF-contact time, and secondly that the Ser(PO3Ph2) residue underwent dephosphorylation at a slightly higher rate than the Thr(PO3Ph2) residue.
Assuntos
Ésteres do Ácido Fórmico/química , Peptídeos/síntese química , Fosfosserina/química , Fosfotreonina/química , Sequência de Aminoácidos , Dados de Sequência Molecular , FosforilaçãoRESUMO
The interaction of antipeptide antibodies with the corresponding peptide and the cognate protein has been compared using a novel biosensor technology (BIAcore, Pharmacia). The peptide corresponds to residues 110-135 of the coat protein of tobacco mosaic virus, known to encompass an alpha-helical region reactive with antiprotein antibodies. A panel of 33 monoclonal antibodies raised against the peptide was studied and the epitope recognized by these antibodies was determined by the pepscan method. Further discrimination between the antibodies was performed by measurements of association and dissociation kinetic constants. Several antibodies showed an heterogeneous binding profile when reacting with the 25 residue long peptide but not with a shorter 10 residue peptide suggesting that they recognized different conformational states in the longer peptide. Equilibrium affinity constants were calculated for five of the antibodies and were found to be 10-50 times higher for the peptide than for the protein, the difference being caused mainly by a lower association rate constant.
Assuntos
Anticorpos Monoclonais/imunologia , Técnicas Biossensoriais , Peptídeos/imunologia , Proteínas Virais/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Afinidade de Anticorpos , Diversidade de Anticorpos , Sítios de Ligação de Anticorpos , Epitopos/química , Cinética , Camundongos , Camundongos Endogâmicos BALB C/imunologia , Dados de Sequência Molecular , Conformação Proteica , Vírus do Mosaico do Tabaco/imunologiaRESUMO
Phospholamban (PLB), an integral membrane protein of cardiac sarcoplasmic reticulum (SR), is described as the regulator of the Ca(2+)-ATPase pump, via its phosphorylation-dephosphorylation of Ser-16. Recently it has been shown that a direct interaction between the N-terminal hydrophilic domain of PLB and Ca(2+)-ATPase may be one of the mechanisms of regulation. In order to show that this interaction could be modulated by a phosphorylation-induced conformational change in PLB, we ran CD studies on the synthetic peptide PLB(2-33) in its phosphorylated and non-phosphorylated forms, at various pHs, concentrations and in the absence or presence of trifluoroethanol. The results show a clear difference in structure of the phosphorylated and non-phosphorylated peptide.
Assuntos
Adenosina Trifosfatases/química , Proteínas de Ligação ao Cálcio/química , Sequência de Aminoácidos , Dicroísmo Circular , Citoplasma/química , Dados de Sequência Molecular , Fosforilação , Conformação Proteica , Serina/metabolismoRESUMO
The C-peptide of Locusta insulin-related peptide, which is a 50 residue peptide originally isolated from the corpora cardiaca of the insect Locusta migratoria and to which we refer as 5-kDa peptide, has been synthesised chemically by the solid-phase method, using a BOC strategy. Since this peptide contains in its sequence a potential monobasic cleavage site, we also synthesised its 1-38 residue-related fragment, named 4-kDa peptide, although we have no hints of its natural occurrence in the corpora cardiaca. Electrophysiological studies have shown that both the 5-kDa and 4-kDa peptides depolarise the membrane and increase the membrane conductance of neurones freshly isolated from the thoracic ganglia of Locusta. Under voltage-clamp conditions, the current underlying these effects was inwardly directed and could be resolved into 2 components. One component, I(5-kDa)1, activated at potentials more hyperpolarised than -50 mV, peaked at about -75 mV and was blocked by the potassium channel blockers cesium and rubidium. The second component, I(5-kDa)2 was activated at potentials more depolarised than -50 mV, increased with depolarisation and was not blocked by cesium and rubidium. The effects of the 5-kDa and 4-kDa peptides on the membrane potential and membrane conductance of Locusta neurones suggest that these peptides may have a physiological role in the central nervous system of insects.
Assuntos
Peptídeo C/fisiologia , Gafanhotos/química , Potenciais da Membrana/efeitos dos fármacos , Neuropeptídeos/fisiologia , Animais , Peptídeo C/química , Peso Molecular , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neuropeptídeos/química , Tórax/inervaçãoRESUMO
A number of continuous epitopes of tobacco mosaic virus protein (TMVP) have been defined by the pepscan technique using polyclonal and monoclonal antibodies to TMVP as well as antisera raised against synthetic peptides. In general, the location of continuous epitopes agreed with the results of earlier studies with peptides synthesized by classical methods although there were some notable exceptions. Results obtained with the different types of antibodies used in this study indicated that a homology of three residues was sufficient to give rise to antigenic cross-reactions. In the case of antibodies raised against a peptide conjugated to ovalbumin, some unexpected cross-reactivities could be explained by assuming that antibodies to the carrier molecule recognized homologous tripeptide sequences in TMVP and ovalbumin.
Assuntos
Capsídeo/imunologia , Reações Cruzadas , Vírus do Mosaico/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais , Capsídeo/genética , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Epitopos/imunologia , Técnicas In Vitro , Dados de Sequência Molecular , Vírus do Mosaico/genética , Ovalbumina/genética , Ovalbumina/imunologia , Homologia de Sequência do Ácido NucleicoRESUMO
We have isolated the smallest bc1 complex subunit from an acidic chloroform/methanol extract of bovine cardiac muscle. The identification of the polypeptide was made possible by classical Edman degradation and amino acid analysis. The measurement of its exact molecular weight by fast atom bombardment mass spectrometry (m/z 6519.8), the characterization of a tryptophan cleavage peptide and pepsic peptides by mass measurements and by mass-analysed ion kinetic energy spectrum analysis allow rectification of the amino acid sequence of the smallest bc1 complex subunit. We found a serine residue instead of Gln 22 and tryptophan residues in place of Ser 34 and Ser 38.
Assuntos
Complexo III da Cadeia de Transporte de Elétrons/química , Miocárdio/química , Sequência de Aminoácidos , Animais , Bovinos , Técnicas In Vitro , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Análise Espectral/métodosRESUMO
In a developmental study, we have shown that DM-20 is present before proteolipid protein (PLP) in the fetal bovine cerebral hemispheres. When the white matter appears (27-30 weeks of gestation), the amount of DM-20 drastically increases. DM-20 remains the major proteolipid until birth. PLP is detected only 2-4 weeks after the appearance of white matter, that is, more than 4 weeks after the appearance of DM-20. The early appearance of DM-20 at the beginning of myelination raises the question of its particular function. In the adult bovine cerebral hemispheres, PLP is the major proteolipid but DM-20 remains quantitatively important because the PLP/DM-20 ratio ranges from 1.5 to 1.7. In the same developmental study we have, in the fetal cerebral hemispheres, isolated and characterized a novel proteolipid (apparent Mr 20,000), which appears even before DM-20 and is not detected in the adult brain. It is structurally related to PLP and DM-20 because the first 31 N-terminal amino acid residues are the same. However, in immunoblot, it did not react either with the antitridecapeptide 117-129 antiserum of PLP or with the anti-C-terminal hexapeptide antiserum of PLP.