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PubMed1998

Interaction of Bovine Myelin Basic Protein with Cholesterol.

The interaction of myelin basic protein with cholesterol and the conformational changes occurring in the protein upon interaction with the lipid were investigated. The myelin basic protein (MBP) plays an important role in stabilizing the multilamellar structure of the myelin membrane. MBP interacts in a specific way with the lipids components of the membrane. The major lipid component is the cholesterol which comprises 40-44 mol% of the lipids. In order to understand the effect of the lipids in the protein conformation we have studied the interaction between MBP and cholesterol. The conformational changes induced in the protein upon interaction with different concentrations of cholesterol were characterized by transmission electron microscopy (TEM) and monolayer studies. Aqueous solution of MBP from bovine brain (obtained by the method of Cheifetz and Moscarello) exhibited a circular dichroism (CD) spectrum characteristic of random coil protein molecules. Upon addition of cholesterol, MBP-cholesterol complexes were observed by TEM. The monolayer compression experiments show plateaus in their surface pressure-area isotherms. The presence of these plateaus has previously been interpreted as alpha-helix conformation. By seeding the MBP onto the aqueous support, we have determined the compression work for the protein on the surface. Experimental areas of the mixtures MBP-cholesterol are smaller than the area calculated by adding the areas of the pure components, indicating that there are attractive forces between both components. The calculated entropy of compression indicates that the highest organization is reached when lipid and protein are almost in the same proportion. Copyright 1998 Academic Press.

Journal Article↗

PubMed1986

Evolutionary divergence in the structure of myelin basic protein: comparison of chondrichthye basic proteins with those from higher vertebrates.

A basic protein has been purified from the CNS myelin of the gummy shark (Mustelus antarticus). Electroblotting was used to examine the capacity of rabbit antisera raised against this electrophoretically pure protein to recognize myelin basic protein from higher vertebrates. The antisera bound to two shark proteins including the original polypeptide antigen and to chicken, bovine, and human myelin basic proteins. Thus, the shark protein appeared to possess antigenic determinants that have been retained through evolutionary divergence of these proteins. Whereas bovine basic protein caused experimental allergic encephalomyelitis in guinea pigs, animals that received injections of the shark protein showed neither clinical nor histological signs of this disease. However, tests for delayed-type hypersensitivity and for Arthus reaction following injection with the shark protein revealed a T-cell-mediated response to this antigen and substantial cross-reactivity with higher vertebrate basic proteins. Analysis of the amino acid composition of the shark protein, and comparison of its tryptic peptide map with that of the bovine protein, revealed substantial changes in the amino acid sequence. Although the shark protein has some antigenic determinants in common with the proteins from higher vertebrates, it appears that much of the structure differs.

Amino Acids↗

PubMed1980

Immunocytochemical method to identify myelin basic protein in oligodendroglia and myelin sheaths of the human nervous system.

To study the distribution of basic protein (BP) and other myelin constituents immunocytochemically in human nervous tissue, we modified the unlabeled antibody enzyme (peroxidase-antiperoxidase) method. The technique is described here. Because the availability of unfixed tissue from human central nervous system is limited, we tested the method on blocks that had been fixed in formalin and embedded in paraffin, fixed and stored in 4% formalin, or frozen at autopsy and stored. We obtained the best results with paraffin blocks. BP antiserum stained oligodendroglia and myelin sheaths in the developing human nervous system. In the adult, myelin sheaths were well stained. Also, abnormalities associated with myelin breakdown could be identified in multiple sclerosis lesions. The results suggest that this method will be useful in studying the cellular distribution of myelin components in human demyelinating diseases.

Adolescent↗

PubMed1997

Role of myelin basic protein and proteolipid protein genes in multiple sclerosis: single strand conformation polymorphism analysis of the human sequences.

Susceptibility to multiple sclerosis (MS) is widely held to have a strong genetic component. While the identities of genes conferring susceptibility are currently unknown, possible candidates include those genes coding for proteins which function in central nervous system (CNS) myelin. Two such genes are the human myelin basic protein (MBP) and proteolipid protein (PLP) genes, whose products make up approximately 80% of the total protein in CNS myelin. The association of a variable number tandem repeat (VNTR) 5' to the human MBP gene with MS has been the subject of conflicting reports. Here we test the hypothesis that mutations in the human MBP and PLP genes might be associated with MS by examining the entire expressed sequence of both genes by single strand conformation polymorphism (SSCP) analysis, using a panel of 71 MS patients and 71 controls. We have also re-examined the VNTR region in patients and controls. Three base changes were found in the human PLP gene and nine base changes in the human MBP gene; these were essentially equally distributed between patients and controls. No preferential distribution of various alleles of the VNTR between patients and controls was found. Although intronic and regulatory regions have not been examined, it would appear unlikely that mutations in these genes coding for the two major CNS myelin proteins contribute significantly to genetic susceptibility to MS.

Adult↗

PubMed1989

Phosphorylation of myelin basic protein and peptides by ganglioside-stimulated protein kinase.

Rabbit myelin basic protein (MBP) was phosphorylated by a ganglioside-stimulated protein kinase to a stoichiometry of 1.4 and 2.1 mol phosphate/mol MBP in the presence and absence of GTlb, respectively. Two-dimensional peptide mapping analyses revealed that two of the sites of phosphorylation were distinct from those catalyzed by cAMP-dependent protein kinase or protein kinase C. Phosphorylation of one of these sites by ganglioside-stimulated protein kinase was inhibited by GTlb, suggesting that the inhibitory effect of gangliosides on MBP phosphorylation may be substrate-directed. Although ganglioside-stimulated protein kinase did not phosphorylate MBP at a domain containing residues 82-117, a synthetic peptide Arg-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys corresponding to residues 111-120 was phosphorylated by the kinase in a ganglioside-stimulated manner. These findings suggest that the conformation of MBP may be important in determining its phosphorylatability.

Amino Acid Sequence↗

PubMed1981

Dependence on lipid structure of the coil-to-helix transition of bovine myelin basic protein.

In aqueous solution bovine myelin basic protein has a close-to-random coil structure that is partially transformed to helix on interaction with lipids. Circular dichroism spectra have been used to follow this conformational transition which, with phospholipids, decreases in the order phosphatidylglycerol, phosphatidic acid approximately equal to phospholipids, decreases in the order phosphatidylethanolamine. There appears to be a strong correlation between the extent of alpha-helix formation and the degree of penetration of the hydrophobic region of the bilayer, as assessed by other methods. Cholesterol mixed in bilayers with phosphatidylserine has little effect on the protein secondary structure. Although basic protein binds strongly to cerebroside and to cerebroside sulphate, two of the other major myelin lipids, the intrinsic chirality of these lipids precludes assessment of their effect on the protein conformation. No significant changes in the circular dichroism spectra accompany the protein association with either of the zwitterionic bilayer-forming lipids, phosphatidylethanolamine and phosphatidylcholine. This seems to exclude extensive penetration into bilayers of these lipids and hence to exclude appreciable hydrophobic interactions; on the other hand, it is argued that little evidence exists for ionic attractions to these lipids. The optical activity of peptides derived from the basic protein by cleavage at the 42-43 and 88-89 peptides bonds (with cathepsin D) and at the 115-116 bond (with a skatole derivative) has also been measured in an attempt to locate the helix-forming regions within the primary structure.

Animals↗

PubMed1993

Limited proteolysis of myelin basic protein in a system mimetic of the myelin interlamellar aqueous space.

We have investigated the early steps of myelin basic protein (MBP) degradation in a membrane mimetic system (reverse micelles), resembling the interlamellar aqueous spaces where the protein is located in the myelin sheath. MBP, unfolded in buffer, refolds on incorporation into the micelles, resulting in reduced accessibility to three proteolytic enzymes, trypsin, cathepsin D, and Staphylococcus aureus V8 protease, in comparison with aqueous solution. Eleven cleavage sites seen in buffer are removed from proteolytic attack in micellar solution. These sites delineate a protected protein domain displaying a potential beta-sheet structure capable of interacting with the myelin membrane. An additional site not seen in buffer is attacked in the micelles. Experiments with a structure inducer, 15% 1-propanol in buffer, reveal that the refolding pattern of MBP in reverse micelles is specific to the membrane biomimetic system and is not produced by organic solvent per se. Micellar digestions of MBP generate long peptides, two of which, isolated after tryptic digestion, have been found to be immunodominant in multiple sclerosis patients. The findings suggest the structure induced in MBP by the micelles resembles that leading to production of the self-peptides recognized by T cells during proteolytic breakdown of MBP in autoimmune demyelinating diseases.

Amino Acid Sequence↗

PubMed1987

Cyclic AMP decreases the phosphorylation state of myelin basic proteins in rat brain cell cultures.

Previous work has suggested that myelin basic proteins are phosphorylated prior to their appearance in the myelin sheath (Ulmer, J. B. and Braun, P. E. (1984) Dev. Neurosci. 6, 345-355). In order to corroborate this finding we have examined the phosphorylation of myelin basic proteins in rat brain cell cultures containing 14-17% oligodendrocytes. Incorporation of 32P into the 14-, 17-, 18.5-, and 21.5-kDa myelin basic proteins was observed in cells incubated with 32P at 7, 14, and 21 days in culture. Myelin basic proteins in 14-day cells incorporated 32P linearly until at least 120 min after the addition of isotope. The apparent half-life of myelin basic protein phosphate groups was determined to be approximately 80 min in pulse-chase experiments. However, this value may be an overestimation due to the presence of significant levels of acid-soluble radioactivity in the cells throughout the chase period. The presence of dibutyryl cAMP or 8-bromo-cAMP in the incubation medium substantially inhibited the incorporation of 32P into the myelin basic proteins at all time points studied. The presence of dibutyryl cAMP in the chase medium in pulse-chase experiments resulted in an increase in the turnover rate of [32P] phosphate in the myelin basic proteins. These results indicate that cAMP decreases the phosphorylation state of myelin basic proteins in oligodendrocytes by inhibiting the phosphorylation and/or stimulating the dephosphorylation of myelin basic proteins.

Animals↗

PubMed1986

A rapid method for purification of myelin basic protein.

A rapid procedure for purification of myelin basic protein has been developed. White matter is delipidated with 2-butanol, and the residue is extracted at pH 7.5 and 8.5. Myelin basic protein is solubilized by extraction in acetate buffer, pH 4.5. The entire procedure requires less than 4 h, and gives homogeneous protein essentially free of protease activity. This procedure can be scaled down to process milligram amounts of white matter; thus it can be very useful for purification of myelin basic protein from very limited amounts of human white matter obtained during surgery.

Acetates↗

PubMed1995

Basic fibroblast growth factor down-regulates myelin basic protein gene expression and alters myelin compaction of mature oligodendrocytes in vitro.

The effects of basic fibroblast growth factor (bFGF) on myelin basic protein (MBP) gene expression and myelin-like membrane formation were investigated in oligodendrocyte cultures containing mainly mature oligodendrocytes expressing MBP. These cultures were obtained by selective detachment of the cells of the oligodendrocyte lineage from 40-day-old mixed cultures derived from newborn rat brain. They were further purified by a 3-day pretreatment with cytosine arabinoside (ARA-C) in order to kill cycling cells. After withdrawal of ARA-C, daily treatment of the cells with bFGF for 3 days induced a drastic decrease in MBP mRNA level compared to control cultures treated only with ARA-C. Moreover, the percentage of oligodendrocytes labelled with anti-MBP antibodies decreased by 50%, as well as that of oligodendrocytes expressing myelin oligodendrocyte glycoprotein (MOG), whereas proteolipid protein (PLP) immunolabelled cells were less affected. At the ultrastructural level, myelin-like membranes were still abundant in the ARA-C- and bFGF-treated cultures, but they were conspicuously uncompacted compared to cultures only pretreated with ARA-C. These results bring the first evidence that bFGF is able to down-regulate myelin protein gene expression in mature oligodendrocytes and to alter myelin structure. They imply that if bFGF is secreted after a demyelinating lesion of the central nervous system (CNS), this plasticity of mature oligodendrocytes will allow final remyelination of axons to complete only after this factor has returned to low levels.

Animals↗

PubMed1984

Comparison of metastable phase behavior of the complexes of dipalmitoyl phosphatidylglycerol with Mg+2 and myelin basic protein.

Both divalent cations and myelin basic protein (BP) induce metastable phase behavior in phosphatidylglycerol (PG). The stable phase of the divalent cation--PG complex is dehydrated cylinders or sheets of lipid in which adjacent bilayers are probably bridged by the divalent cation. The structure of the stable phase of the BP-PG complex is unknown. To help understand the metastable phase behavior in the BP-PG complex, it was compared with that of the MG+2-PG complex using fatty-acid spin labels and differential scanning calorimetry. The stable state of the BP-PG complex melts at a similar temperature and with a similar enthalpy as the Na+-salt form of the lipid, while the stable state of the Mg+2-PG complex melts 35 degrees higher than and with an enthalpy 63% greater than the Na+-salt form. BP causes pronounced motional restriction of a fatty acid spin labeled near the terminal methyl group in the metastable and stable states of its complex with PG. Computer resolution of spectra of fatty-acid spin labels in the Mg+2-PG complex revealed that the spectra characteristic of the stable state were exchange broadened, showing that the probes are not soluble in the stable dehydrated state of this complex. The much different behavior of the BP-PG complex suggests that its stable phase is not dehydrated, that the probes are not displaced from the complex, and that their behavior reflects the ability of hydrophobic residues of basic protein to penetrate into the bilayer while its basic residues bind electrostatically to the lipid polar head groups.

Calorimetry, Differential Scanning↗

PubMed1990

Identification by mass spectrometry of threonine 97 in bovine myelin basic protein as a specific phosphorylation site for mitogen-activated protein kinase.

Bovine myelin basic protein (MBP) was found to be an excellent in vitro substrate (apparent Km = 50 microM) for MAP (mitogen-activated protein) kinase and can be used in lieu of microtubule-associated protein 2 for purification and functional studies of the enzyme. MBP phosphotransferase activity co-purified with MAP kinase during sequential DE52, phenyl-Superose, and gel filtration chromatography, and kinase activities for the two substrates were co-regulated by mitogen stimulation. MAP kinase phosphorylated MBP exclusively on threonine, and only one major phosphopeptide was generated by digestion with trypsin or endoproteinase Lys-C. Using mass spectrometry, we determined that the phosphorylation site is threonine 97, present in the conserved triproline loop of MBP, with (partial) sequence -Thr-Pro-Arg-Thr97-Pro-Pro-Pro-. Thr97 is a known in vivo phosphorylation site in MBP although enzymes capable of phosphorylating this site have not been identified previously. MAP kinase phosphorylated peptide 88-109 from rabbit MBP and a synthetic peptide 91-109 from human MBP but did not phosphorylate either the histone H1 peptide, utilized by p34cdc2, or the peptide substrate for the recently described proline-directed kinase. Thus, the sequence surrounding threonine 97 in bovine MBP may contain essential features of a recognition sequence for MAP kinase.

Amino Acid Sequence↗

PubMed1986

Region-specific immunoassays for human myelin basic protein.

Three monoclonal antibodies reactive with human myelin basic protein have been used to develop immunoradiometric assays for this protein. Clone 1, a mouse IgG2a, is reactive with an epitope in the region 129-138. Clone 2, a mouse IgG1, is reactive with the region 119-131. Clone 12, a rat IgG, is reactive with the region 86-96. Competition experiments show that the reactions of Clone 1 and Clone 2 are mutually exclusive, probably because of steric effects. In contrast, when either Clone 1 or Clone 2 react they cause minimal interference with the subsequent binding of Clone 12. Less than 1 ng/ml of myelin basic protein can be detected in each of the two immunoradiometric assays developed. Clone 12 can also be used on its own in a competitive immunoassay to detect around 2 ng/ml. Using an extraction technique before the assay, serum or plasma can also be investigated. Assays for defined regions of myelin basic protein should prove valuable in identifying the products of myelin catabolism in patients with demyelinating disease.

Amino Acid Sequence↗

PubMed1992

Cell and tissue-specific expression of a heterologous gene under control of the myelin basic protein gene promoter in transgenic mice.

Myelin basic protein (MBP) is the second most abundant protein in CNS myelin. We have used transgenic mice to investigate the ability of the 5' flanking sequence of the mouse MBP gene to regulate the cell-type-specific- and temporal expression of a heterologous gene under its control. Transgenic mice were produced with a construct containing the bacterial chloramphenicol acetyltransferase (CAT) gene down-stream of the MBP 5' flanking sequence and CAT expression was monitored both enzymatically and histochemically. The results indicate that 1323 bp of 5' flanking sequence is sufficient to direct CAT expression specifically to the tissue and cell-type, in which MBP is normally synthesized. Additionally, this length of sequence also retains the ability to temporally regulate CAT levels in a manner analogous to endogenous MBP levels.

Animals↗

PubMed2005

Myelin basic protein accumulation is impaired in a model of protein deficiency during development.

During the development of the central nervous system (CNS) there is a great possibility of permanent effects in consequence of environmental disturbances. Nutritional deficiency is one of the factors that impair the normal CNS formation. In general, the protein deficiency evokes, beyond the damages in the maturation of nervous system, several consequences in body growth, biochemical maturation, motor function and the major cognitive functions. These effects were observed in undernourished children all over the world. Even in a restricted period, the malnutrition status may evoke permanent impairments in feeding behavior and in metabolism. Rats submitted to malnutrition during development, showed a marked decrease in the number of myelinated fibers. This condition may reflect a failure in the beginning of the wrapping of axons by oligodendroglial processes and/or a delay in the myelin synthesis. Myelin basic protein (MBP) is an intracellular oligodendrocyte protein that is directly related to the formation of the myelin sheath. In this study we verified the temporal pattern of MBP expression, by immunohistochemical and immunoblotting analyses, in a model of protein malnutrition induced during the first half of the lactation period. We showed that MBP expression was impaired in our malnutrition model and that some of the effects were maintained in adulthood, with possible consequences in the maturation of myelin sheath.

Aging↗

PubMed1989

Lewis rat lymphoid dendritic cells can efficiently present homologous myelin basic protein to encephalitogenic lymphocytes.

Lymphocytes isolated from Lewis rats immunised with protein antigen in adjuvant were stimulated to proliferate in vitro by splenic dendritic cells (DC) which had been pulsed with purified homologous myelin basic protein (MBP). By contrast, in parallel experiments, lymphocytes did not respond to ovalbumin unless the protein was first processed by macrophages by a chloroquine-sensitive mechanism. DC, pulsed with rat MBP at concentrations as low as 6 micrograms/ml, activated lymphocytes for transfer of severe experimental autoimmune encephalomyelitis (EAE). MBP dissociating from myelin membranes in physiological medium, as well as MBP purified from highly acidic extracts of myelin, was effective for pulsing DC; preincubating myelin with macrophages led to a reduction rather than an enhancement in the severity of the EAE transferred. It is concluded that macrophage-mediated antigen processing is not required for immunogenic presentation of the determinants of MBP which cause EAE in Lewis rats. Furthermore, MBP-pulsed DC may prove useful in experiments requiring activation of encephalitogenic T cells.

Animals↗

PubMed1985

Conformation of two antigenic regions in myelin basic protein.

Four different regions of myelin basic protein from various species have been reported to be the antigenic sites (epitopes) for seven monoclonal antibodies evoked in rats or mice by guinea pig or monkey basic protein. The structures of the epitopes located in the amino-terminal region and in the eight-residue sequence including S-133, were examined by proton n. m. r at 400 MHz in aqueous solutions of peptides obtained by enzymatic cleavage of the rabbit protein. The data suggest conformational similarities between the two regions.

Amino Acid Sequence↗

PubMed1989

Myelin basic protein transcriptional activity in myelin-deficient mutant mice.

Myelin-deficient (mld) mice present two tandem myelin basic protein genes, with the upstream gene containing an inversion (Popko et al., Neuron 1: 221, 1988). Detailed analysis of the transcriptional activity of various regions of the gene showed that all portions of the MBP gene are transcribed in mld mice. Consequently, we propose that the low levels of MPB mRNA observed in mld mice are due to post-transcriptional events.

Animals↗