RESUMO
Myelin, the insulating sheath made by extensive plasma membrane wrapping, is dependent on the presence of highly adhesive molecules that keep the two sides of the membrane in tight contact. The Po glycoprotein (Po) is the major component of the peripheral nervous system (PNS) myelin of mammals. The exact role that Po protein has played in the evolution of myelin is still unclear, but several phylogenetic observations suggest that it is a crucial component in the development of myelin as a multi-lamellar membrane structure. Sharks, which appeared in the fossil record about 400 million years ago, are the first fully myelinated organisms. In this study we investigated the expression pattern of shark myelin Po to suggest a way it might have played a role in the evolution of myelin in the central nervous system. We found that sharks have more than two isoforms (32, 28 and 25 kD), and that some of these might not be fully functional because they lack the domains known for Po homophilic adhesion.
Assuntos
Proteína P0 da Mielina/química , Tubarões/metabolismo , Squalus acanthias/metabolismo , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Western Blotting , Sistema Nervoso Central/química , Sistema Nervoso Central/embriologia , Epitopos/imunologia , Evolução Molecular , Glicosilação , Humanos , Dados de Sequência Molecular , Peso Molecular , Proteína P0 da Mielina/análise , Proteína P0 da Mielina/imunologia , Proteína P0 da Mielina/isolamento & purificação , Sistema Nervoso Periférico/química , Sistema Nervoso Periférico/embriologia , Isoformas de Proteínas/análise , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/isolamento & purificação , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Coelhos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Tubarões/embriologia , Tubarões/imunologia , Especificidade da Espécie , Squalus acanthias/imunologia , Vertebrados/metabolismoRESUMO
OBJECTIVE: To purify P0 protein from guinea pig's inner ear by preparative SDS-PAGE and study the possible role it may play in the etiology of autoimmune inner ear disease. METHOD: A mixture of membraneous proteins of inner ear was separated by preparative SDS-PAGE. The corresponding band at 30kd was cut and electrically eluted. The protein collected was identified by analytical SDS-PAGE and Western blot assay. A group of 20 guinea pigs were immunized with P0 protein emulsified in complete Freund's adjuvant, another 10 guinea pigs were immunized with complete Freund 's adjuvant only as control. The guinea pigs' hearing thresholds, serum IgG level and morphological changes in the inner ear were investigated. The distribution of P0 protein in the cochlear was detected by immunohistochemical technique. RESULT: The purity of the protein was demonstrated by a single band at the 30 kD site in SDS-PAGE, which was identified as P0 protein by western blot analysis assay. About 17.5% P0-immunized guinea pigs showed increased hearing thresholds, elevated IgG level (F =6.48, P <0. 01), as well as a decreased number of spiral ganglion cells and inflammatory cell infiltration in the cochlear nerve region. The P0 protein is distributed in the cochlear nerve and spiral ganglion only. CONCLUSION: P0 protein from guinea pig's inner ear can be successfully purified by preparative SDS-PAGE and an animal model of experimental autoimmune inner ear disease induced by P0 protein is successfully established.
Assuntos
Adenoviridae/genética , Cóclea , Genes Reporter , Proteína P0 da Mielina/isolamento & purificação , Animais , Cóclea/metabolismo , Modelos Animais de Doenças , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Técnicas de Transferência de Genes , Genes Homeobox , Vetores Genéticos , CobaiasRESUMO
OBJECTIVE: To purify P0 protein from guinea pig's inner ear by preparative SDS-PAGE and to study the possible role it may play in the etiology of autoimmune inner ear disease. METHOD: A mixture of membraneous proteins of inner ear was separated by preparative SDS-PAGE. The corresponding band at 30,000 was cut and electrically eluted. The protein collected was identified by analytical SDS-PAGE and Western blot assay. A group of 20 guinea pigs were immunized with P0 protein emulsified in complete Freunds adjuvant, another 10 guinea pigs were immunized with complete Freunds adjuvant only as control. The guinea pigs' hearing thresholds, serum IgG level and morphological changes in the inner ear were investigated. The distribution of P0 protein in the cochlear was detected by immunohistochemical technique. RESULT: The purity of the protein was demonstrated by a single band at the 30000 site in SDS-PAGE, which was identified as P0 protein by western blot analysis assay. About 17.5% P0-immunized guinea pigs showed increased hearing thresholds, elevated IgG level (F = 6.48, P<0.01), as well as a decreased number of spiral ganglion cells and inflammatory cell infiltration in the cochlear nerve region. The P0 protein is distributed in the cochlear nerve and spiral ganglion only. CONCLUSION: P0 protein from guinea pigs in ner ear can be successfully purified by preparative SDS-PAGE and an animal model of experimental autoimmune inner ear disease induced by P0 protein was successfully established.
Assuntos
Doenças Autoimunes/imunologia , Modelos Animais de Doenças , Doenças do Labirinto/imunologia , Proteína P0 da Mielina/imunologia , Animais , Cobaias , Proteína P0 da Mielina/isolamento & purificaçãoRESUMO
Pituitary gland is a well-known endocrine tissue. The hypothalamo-neurohypophysial system, containing arginine vasopressin and oxytocin, shows a reversible morphological reorganization of both neurons and glial cells during chronic physiological stimulations. Since many signal transducing and cell adhesion molecules (CAMs) are recovered in membrane microdomain (MD) fractions, MDs are considered as signaling platforms of cells. In order to know the molecular background for these endocrine systems, we characterized MD-components derived from rat pituitary and found specific enrichment of several proteins in the fraction. One of them was identified as myelin protein zero (P0) with mass analysis and this result was further confirmed by a result that a specific antibody to this protein reacted to the authentic P0 protein in the myelin fraction of rat sciatic nerve. P0 is one of type-I transmembrane CAMs and a major structural component of mammalian peripheral nerve myelin. In mammals, expression of P0 has been considered to be restricted to peripheral nervous system. This result however indicates that P0 expresses more widely and its enrichment in the MD-fraction from rat pituitary suggests the participation in cell-cell communications.
Assuntos
Microdomínios da Membrana/metabolismo , Proteína P0 da Mielina/biossíntese , Hipófise/metabolismo , Transdução de Sinais/fisiologia , Animais , Anticorpos Monoclonais/química , Arginina Vasopressina/metabolismo , Imuno-Histoquímica , Microdomínios da Membrana/química , Microdomínios da Membrana/imunologia , Proteína P0 da Mielina/química , Proteína P0 da Mielina/imunologia , Proteína P0 da Mielina/isolamento & purificação , Ocitocina/metabolismo , Hipófise/química , Hipófise/citologia , Hipófise/imunologia , Ratos , Ratos Wistar , Nervo Isquiático/química , Nervo Isquiático/citologia , Nervo Isquiático/imunologia , Nervo Isquiático/metabolismoRESUMO
The glycoprotein P0, the major structural protein of the peripheral nerve myelin, plays a critical role in holding myelin lamellae together via interaction of both extracellular and cytoplasmic domains. Mutations in the human P0 gene give rise to severe and progressive forms of dominantly inherited peripheral neuropathies like CMT1B. Here we report on the characterization of a bovine P0-derived protein of nearly 26 kD that corresponds to the P0 protein truncated in its cytoplasmic domain. Matrix assisted laser desorption ionization (MALDI)-time-of-flight/time-of-flight (TOF/TOF) mass spectrometry (MS) analysis on its tryptic digest has provided a peptide mapping, the main difference of which from the normal P0 analog was represented by the absence of the cluster of peaks at m/z 1513.7501, 1530.7701, and 1546.7651. The latter corresponds to the P0 fragment QTPVLYAMLDHSR and to its pyroglutamic and methionine-oxidized derivatives. The species at 1530.7701 covering the sequence 186-198 of P0 is not an artifact and might have a functional role in the myelin architecture.
Assuntos
Proteína P0 da Mielina/química , Bainha de Mielina/química , Proteômica , Sequência de Aminoácidos , Animais , Bovinos , Eletroforese , Hidrólise , Dados de Sequência Molecular , Proteína P0 da Mielina/isolamento & purificação , Conformação Proteica , Nervo Isquiático/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/químicaRESUMO
Protein zero (P0) glycoprotein is the major integral membrane protein of the peripheral nervous system myelin in higher vertebrates. Previous findings indicate the formation of tetrameric assemblies from studies on isolated P0. To determine whether in intact myelin the P0 exists as oligomers, we isolated myelin from sciatic nerve of Xenopus laevis and analyzed it using sodium dodecyl sulfate and urea gel electrophoresis. P0 oligomerization was confirmed using Western blotting, which showed monomeric P0 at approximately 30 kDa and oligomeric P0 at approximately 60 kDa and approximately 120 kDa. A variety of denaturing conditions failed to convert any appreciable amount of oligomer to monomer. Instead, the addition of these denaturants further increased the amount of dimer and tetramer while decreasing the amount of monomer. Native gels showed dimeric P0 without the appearance of monomer or tetramer, suggesting that dimeric P0, the most prominent form of the protein, is the most stable and likely occurs in the native myelin membrane array.
Assuntos
Proteína P0 da Mielina/análise , Proteína P0 da Mielina/química , Nervo Isquiático/química , Animais , Western Blotting , Dimerização , Eletroforese em Gel de Poliacrilamida , Feminino , Concentração de Íons de Hidrogênio , Proteínas de Membrana/análise , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Proteína P0 da Mielina/isolamento & purificação , Desnaturação Proteica , Temperatura , Xenopus laevisRESUMO
Myelin protein zero (P0), an adhesion protein of the immunoglobulin superfamily, is the major protein of peripheral nervous system myelin in higher vertebrates. Protein zero is required for the formation and maintenance of myelin structure in the internode, likely through homophilic interactions at both the extracellular and the intracellular domains. Mutations and deletions in the P0 gene correlate with hereditary peripheral neuropathies of varying severity. Comparisons between the human and rat isoforms, whose three-dimensional structure has been determined by X-ray crystallography, suggest that these disease-associated genetic alterations lead to structural changes in the protein that alter P0-P0 interactions and hence affect myelin functionality. Knowing the crystal structures of native and altered human P0 isoforms could help to elucidate the structural changes in myelin membrane packing that underlie the altered functionality. Alterations of P0 extracellular domain (P0-ED) are of additional interest as previous X-ray diffraction studies on myelin membrane packing suggest that P0-ED molecules can assume distinct adhesive arrangements. Here, we describe an improved method to express and purify human P0-ED (hP0-ED) suitable for crystallographic analysis. A fusion protein consisting of maltose binding protein fused to hP0-ED was secreted to the periplasm of Escherichia coli to allow an appropriate folding pathway. The fusion protein was extracted via osmotic shock and purified by affinity chromatography. Factor Xa was used to cleave the fusion protein, and a combination of affinity and ion-exchange chromatography was used to further purify hP0-ED. We document several significant improvements to previous protocols, including bacterial growth to approximately 15 OD using orbital shakers and the use of diafiltration, which result in yields of approximately 150 mg highly pure protein per liter of medium.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Escherichia coli , Proteínas de Transporte de Monossacarídeos , Proteína P0 da Mielina/química , Proteína P0 da Mielina/isolamento & purificação , Sequência de Aminoácidos , Soluções Tampão , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Fator Xa/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Proteínas Ligantes de Maltose , Proteína P0 da Mielina/genética , Proteína P0 da Mielina/metabolismo , Pressão Osmótica , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismoRESUMO
P0 is an abundant myelin glycoprotein of peripheral nerves of vertebrates. Various point mutations of this protein are responsible for hereditary neuropathies. In this paper we described purification of P0 glycoprotein using SDS and a metal chelate affinity chromatography. Purified myelin fraction from bovine spinal roots in 0.5% SDS, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.4 is filtered and applied directly to the Cu2+-immobilized affinity chromatography column, equilibrated with the same buffer. After eluting a void volume (or pass through) fraction, P0 protein was eluted by the same buffer but without salt. To remove contamination from the eluent, further purification is continued on a Concanavalin-A coupled agarose column. We purify within two days, 30 mg of P0 protein of apparent molecular weight 27 kDa. The method can be used to purify recombinant or mutated P0 protein found in severe pathologies.
Assuntos
Cromatografia de Afinidade/métodos , Proteína P0 da Mielina/isolamento & purificação , Animais , Carboidratos/química , Bovinos , Quelantes/química , Cobre/química , Detergentes/química , Eletroforese em Gel de Poliacrilamida , Histidina/química , Lipídeos/química , Peso Molecular , Proteína P0 da Mielina/química , Desnaturação Proteica , Dodecilsulfato de Sódio/químicaRESUMO
Mutations found in the two major glycosylated transmembrane proteins of the PNS myelin, the peripheral myelin protein zero (P0) and peripheral myelin protein 22 (PMP22), have been independently associated with the most common hereditary demyelinating peripheral neuropathies. Genotype-phenotype correlations in humans and transgenic animals have provided functional evidence that P0 and PMP22 are involved in formation and maintenance of compact myelin. Here, we demonstrate for the first time that P0 and PMP22 proteins form complexes in the myelin membrane, as shown by coimmunoprecipitation experiments, and that glycosylation is not involved in mediating these interactions. Complex formation was also detected when the two proteins were coexpressed in heterologous cells. In transfected cells, P0 and PMP22 are recruited and colocalize at the apposed plasma membranes of expressors as shown by confocal microscopy. These findings provide a new basis for a better understanding of myelin assembly and of the pathomechanisms involved in demyelinating peripheral neuropathies. Furthermore, these results propose a possible explanation why alterations in either of these molecules are sufficient to destabilize the myelin structure and cause a similar disease phenotype.
Assuntos
Proteína P0 da Mielina/metabolismo , Proteínas da Mielina/metabolismo , Bainha de Mielina/química , Nervo Isquiático/química , Animais , Sequência de Bases , Western Blotting , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Glicosilação , Células HeLa , Humanos , Dados de Sequência Molecular , Proteína P0 da Mielina/genética , Proteína P0 da Mielina/isolamento & purificação , Proteínas da Mielina/genética , Proteínas da Mielina/isolamento & purificação , Ligação Proteica , Ratos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
The feasibility of targeting liposomes reconstituted with P0 protein (P0-liposomes) to melanoma cells with high level of intercellular adhesion molecule-1 (ICAM-1) expression was investigated. P0 protein, an immunoglobulin superfamily (IgSF) cell adhesion molecule from peripheral nerve myelin, was used as a model targeting ligand. Liposome uptake by the cells was quantitated using radioactive lipids. The presence of intact P0 protein in the liposome bilayer increased the extent of interaction of liposomes with human M21 (7.80 fold) and A-375 (4.62 fold) melanoma cells compared to control liposomes of same lipid composition but no P0 protein, whereas with MeM 50-10 melanoma cells no significant increase was found (1.70 fold). The extent of binding of P0-liposomes to the melanoma cells correlated with the level of ICAM-1 expression on cells (r2 = 0.9996). M21 and A-375 cells express ICAM-1 (the percentage of stained cells, PSC, was 95% and 85%, respectively), whereas, MeM 50-10 cells do not. P0 protein also increased the interaction of liposomes with P0 protein expressing CHO-X2 cells (4.36 fold, as a positive control) compared to control liposomes. The indirect flow cytometry experiments using biotinylated P0 protein showed that P0 protein itself in solution also binds to M21 cells but does not bind to MeM 50-10 cells. Preincubation of M21 cells with anti-ICAM-1 monoclonal antibody decreased the binding of biotinylated P0 protein to the M21 cells by 35%. P0 protein or its binding domain(s) that mediate the targeting of liposomes through adhesive interactions may be useful for the development of novel types of drug delivery systems. This approach may have relevance in the treatment of metastatic cancers, inflammation and viral diseases, where cell adhesion proteins are over expressed.