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Results for “Myelin Basic Protein”

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PubMed1985

Low-ultraviolet circular dichroism spectroscopy of oligopeptides 1-95 and 96-168 derived from myelin basic protein of rabbit.

Myelin basic protein (MBP) is a major protein constituent of the myelin sheath of the central nervous system, where it is believed to have functional alpha-helical segments. One element of the function of the protein might be "conformational adaptability" of specific regions of its amino acid sequence, since the purified protein appears to be largely devoid of ordered structure. To pursue this question, low-ultraviolet circular dichroism (CD) spectroscopy was conducted on the sequential thrombic peptides 1-95 and 96-168 of the protein in the presence of 0-92% trifluoroethanol (TFE), a solvent known to promote stable secondary structures in polypeptides. The series of CD spectra of the oligopeptides were subjected to a computerized best-fit analysis of four peptide conformations, the alpha-helix, beta-structure, beta-turn, and nonordered form. Agreement between experimental and best-fit composite spectra was achieved when standard CD curves of peptide conformations were derived from known theoretical spectra and experimental spectra of polypeptides. In dilute buffer alone, oligopeptides 1-95 and 96-168 evidence no alpha-helix but significant beta-structure (18% and 23%, respectively), as well as a predominant, extended nonordered conformation. However, the two parts of the protein differed in conformational adaptability. From 0% to 30% TFE, 96-168 exhibited concomitant transitions to 10% helix and 32% beta-structure from the nonordered form. In contrast, in 10-30% TFE, 1-95 underwent a transition to approximately 21% helix with partial loss of beta-structure as well as nonordered form; higher concentrations of TFE (40-75%) promoted additional transitions to both helix and beta-structure (totaling 33% and 25%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2006

Myelin basic protein functions as a microtubule stabilizing protein in differentiated oligodendrocytes.

Myelin basic protein (MBP) is an oligodendrocyte-specific protein essential for oligodendrocyte morphogenesis at late stages of cell differentiation. There is evidence that the morphogenetic function of MBP is mediated by MBP interaction with the cytoskeleton. Thus, an MBP/cytoplasmic microtubule association has been reported, and MBP has Ca(2+)/calmodulin-regulated microtubule cold-stabilizing activity in vitro. However, the unambiguous demonstration of a microtubule-stabilizing activity for MBP in cells has been difficult because oligodendrocytes contain variants of STOP (stable tubule only polypeptide) proteins, which are responsible for microtubule cold stability in different cell types. Herein, we have used genetic mouse models and RNA interference to assay independently the microtubule cold-stabilizing activities of MBP and of STOP in developing oligodendrocytes. In wild-type oligodendrocytes, microtubules were cold stable throughout maturation, which is consistent with the presence of STOP proteins from early stages of differentiation. In contrast, in oligodendrocytes from STOP-deficient mice, microtubules were cold labile in the absence of MBP expression or when MBP expression was restricted to the cell body and became stable in fully differentiated oligodendrocytes, where MBP is expressed in cell extensions. The suppression of MBP by RNA interference in STOP-deficient oligodendrocytes suppressed microtubule cold stability. Additionally, STOP suppression in oligodendrocytes derived from shiverer mice that lack MBP led to the complete suppression of microtubule cold stability at all stages of cell differentiation. These results demonstrate that both STOP and MBP function as microtubule-stabilizing proteins in differentiating oligodendrocytes and could be important for the morphogenetic function of MBP.

Animals↗

PubMed1999

Myelin basic protein isoforms in myelinating and remyelinating rat brain aggregate cultures.

Recent evidence suggests that myelin basic protein (MBP) exon-2-containing isoforms play a significant role in the onset of myelination because they are more abundant during early development. The pattern of expression of MBP exon-2-containing isoforms was studied in rat brain aggregate cultures during myelination to draw comparisons with the developing brain and at remyelination after demyelinative treatment. The pattern of MBP isoform expression in the aggregate cultures was found to be similar to that of the brain and was recapitulated after demyelination with antimyelin antibodies. Macrophage enrichment, resulting in increased accumulation of total MBP in the cultures, did not alter the isoform distribution. Both control and enriched cultures expressed a 16-kDa protein (26+/-9.8% of total MBP for control samples) that reacted with MBP antisera at the onset of myelination (day in vitro 14) but was barely detectable by day in vitro 21. The expression of this protein, also present in postnatal day 6 rat brain but no longer by day 11, has been predicted by reverse transcription polymerase chain reaction in embryonic mouse brain. The results of the present study reinforce the value of the aggregate culture system as a versatile yet accurate model of myelination and remyelination.

Animals↗

PubMed2002

Effects of the osmolyte trimethylamine-N-oxide on conformation, self-association, and two-dimensional crystallization of myelin basic protein.

The osmolyte trimethylamine-N-oxide (TMAO) is a naturally in vivo occurring "chemical chaperone" that has been shown to stabilise the folding of numerous proteins. Myelin basic protein (MBP) is a molecule that has not yet been suitably crystallized either in three dimensions for X-ray crystallography or in two dimensions for electron crystallography. Here, we describe lipid monolayer crystallization experiments of two species of recombinant murine MBP in the presence of TMAO. One protein was unmodified, whereas the other contained six Arg/Lys-->Gln substitutions to mimic the effects of deimination (i.e., the enzymatic modification of Arg to citrulline), which reduces the net positive charge. Planar arrays of both proteins were formed on binary lipid monolayers containing a nickel-chelating lipid and a phosphoinositide. In the presence of TMAO, the diffraction spots of these arrays became sharper and more distinct than in its absence, indicating some improvement of crystallinity. The osmolyte also induced the formation of epitaxial growth of protein arrays, especially with the mutant protein. However, none of these assemblies was sufficiently ordered to extract high-resolution structural information. Circular dichroic spectroscopy showed that MBP gained no increase in ordered secondary structure in the presence of TMAO in bulk solution, whereas it did in the presence of lipids. Dynamic light-scattering experiments confirmed that the MBP preparations were monomodal under the optimal crystallization conditions determined by electron microscopy trials. The salt and osmolyte concentrations used were shown to result in a largely unassociated population of MBP. The amino acid composition of MBP overwhelmingly favours a disordered state, and a neural-network-based scheme predicted large segments that would be unlikely to adopt a regular conformation. Thus, this protein has an inherently disordered nature, which mitigates strongly against its crystallization for high-resolution structure determination.

Amino Acid Sequence↗

PubMed1984

The effect of age on the microheterogeneous pattern of human myelin basic protein.

Dry weight, total basic protein and myelin basic protein (MBP) in human corpus callosum tissue were found to be significantly reduced with age, confirming other reports. The heterogeneous patterns of the MBP samples were investigated and could be classified into three groups: a normal one with peak 1 larger than peak 3; an old-age pattern with peak 1 smaller than peak 3; and a pathological pattern with peak 1 greatly reduced. Because peak 1 is the only one which is significantly reduced with age, it is suggested that future investigations could be simplified. The physiological and pathophysiological role of MBP heterogeneity is discussed.

Adult↗

PubMed2000

Deimination of myelin basic protein. 1. Effect of deimination of arginyl residues of myelin basic protein on its structure and susceptibility to digestion by cathepsin D.

The effect of deimination of arginyl residues in bovine myelin basic protein (MBP) on its susceptibility to digestion by cathepsin D has been studied. Using bovine component 1 (C-1) of MBP, the most unmodified of the components with all 18 arginyl residues intact, we have generated a number of citrullinated forms by treatment of the protein with purified peptidylarginine deiminase (PAD) in vitro. We obtained species containing 0-9.9 mol of citrulline/mol of MBP. These various species were digested with cathepsin D, a metalloproteinase which cleaves proteins at Phe-Phe linkages. The rate of digestion compared to component 1 was only slightly affected when 2.7 or 3.8 mol of citrulline/mol of MBP was present. With 7.0 mol of citrulline/mol of MBP, a large increase in the rate of digestion occurred. No further increase was observed with 9.9 mol of citrulline/mol of MBP. The immunodominant peptide 43-88 (bovine sequence) was released slowly when 2.7 and 3.8 mol of citrulline/mol of MBP was present, but it was released rapidly when 7.0 mol of citrulline/mol of MBP was present. The dramatic change in digestion with 7.0 mol of citrulline/mol of MBP or more could be explained by a change in three-dimensional structure. Molecular dynamics simulation showed that increasing the number of citrullinyl residues above 7 mol/mol of MBP generated a more open structure, consistent with experimental observations in the literature. We conclude that PAD, which deiminates arginyl residues in proteins, decreases both the charge and compact structure of MBP. This structural change allows better access of the Phe-Phe linkages to cathepsin D. This scheme represents an effective way of generating the immunodominant peptide which sensitizes T-cells for the autoimmune response in demyelinating disease.

Animals↗

PubMed1996

Modifications of myelin basic protein in DM20 transgenic mice are similar to those in myelin basic protein from multiple sclerosis.

Transgenic mice containing different numbers of transgenes (2-70) of the myelin proteolipid protein DM20 were phenotypically normal up to 3 mo of age, after which the mice containing 70 copies of the transgene spontaneously demyelinated and died at 10-12 mo. Since we demonstrated that demyelination in multiple sclerosis involved specific chemical changes in myelin basic protein (MBP), we investigated the MBP in our transgenic line for similar changes. Both the total amount of MBP in brain and the MBP mRNA levels were unaffected at the different ages. All the isoforms (14-21 kD) of MBP were present, but the microheterogeneity (a posttranslational event) was changed resulting in a higher proportion of the less cationic components reminiscent of the changes in MBP found in multiple sclerosis. An increased amount of the citrullinated form of MBP was found by Western blot analysis. Immunogold labeling of cryosections of brain revealed a greater density of particles with the anticitrulline antibody at 10 mo and that the levels of peptidylarginine deiminase (which deiminates protein-bound arginine to citrulline) were increased. This stable transgenic line represents a useful animal model for the human disease multiple sclerosis.

Animals↗

PubMed1994

Limited postnatal ethanol exposure permanently alters the expression of mRNAS encoding myelin basic protein and myelin-associated glycoprotein in cerebellum.

Experiments were designed to test the hypothesis that ethanol exposure during development can selectively affect the expression of specific isoforms of myelin protein gene expression in the rat cerebellum. We focused on myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) gene expression. Both of these genes are alternatively spliced to yield 4 (MBP) or 2 (MAG) mRNA isoforms. Prenatal ethanol exposure, delivered to the dams in a liquid diet, did not significantly alter the expression of MBP or MAG gene expression in the cerebellums of 15-day-old pups, as measured by quantitative in situ hybridization using specific oligodeoxynucleotide probes. In contrast, postnatal ethanol exposure delivered directly to the pups over a 6-day period by gastrostomy tube (PN days 4-10) reduced the expression of specific MBP and MAG isoforms in the cerebellum of animals in adulthood. These data demonstrate that ethanol exposure, especially during the period of rapid myelination, has selective effects on mRNA isoforms encoding specific MBPs and MAG.

Animals↗

PubMed1997

Nuclear transport of myelin basic protein.

The multiple myelin basic protein (MBP) isoforms expressed by myelinating cells are now known to have different expression patterns. The relative abundance of the isoforms containing exon II is greater early in myelinogenesis, whereas in compact myelin the isoforms lacking this exon are more abundant. Further, the individual MBPs exhibit different intracellular localizations, indicating that the isoforms may not be functionally equivalent in myelinating cells. The major MBPs (14 kD and 18.5 kD) have strong affinity for membranes, while on the other hand, the less abundant isoforms (17 kD and 21.5 kD) localize to the nucleus of young oligodendrocytes, suggesting a regulatory role in the myelination program. The same intracellular distribution patterns have been observed when the MBPs are expressed in Hela cells and in shiverer oligodendrocytes. Thus, the intracellular fate of these proteins seems to be generally directed through alternative expression of exon II. Furthermore, the extent of MBPexII entry into the nucleus was found to be directly related to the growth state of host cells. In this paper, we demonstrate that nuclear proteins constitutively expressed by Hela cells also exhibit an apparently growth-related nucleo-cytoplasmic distribution revealing that MBPexII exhibits the same behavior as bona fide nuclear proteins. Also, to further characterize MBP nuclear transport, we explored various parameters of the translocation of MBP into the nucleus using an in vitro system. This experimental paradigm permits the uncoupling of synthesis and translocation events; thus, the transport of MBP into cell nuclei can be studied as a function of time. We also evaluated how changes in temperature as well as energy depletion affect the in vitro nuclear transport of MBP.

Biological Transport↗

PubMed1977

Conformational changes induced by ionic strength and pH in two bovine myelin basic proteins.

The structures of two biologically different myelin proteins, A1 from the central nervous system and P2 from the peripheral nervous system, were investigated. Both proteins were isolated from nerve tissues. Conformational changes in the homogeneous proteins were examined in aqueous solutions by means of circular dichroism measurements. The secondary structures of both proteins proved to be very stable between pH 2.5 and pH 11.7. Unlike the P2 protein, the A1 protein is stable up to pH 13 without detectable conformational changes. The stereochemistry of the polypeptide chains of both proteins is markedly different in the presence of urea. While the value of theta222 for the A1 protein changes linearly with increasing urea concentration, a sigmoidal curve was obtained for the P2 protein. The observed differences in the dichroic properties of the basic myelin proteins A1 and P2 indicate the possibility of further structure - function correlations.

Animals↗

PubMed1988

Hydrolysis of myelin basic protein in human myelin by terminal complement complexes.

The participation of terminal complement complexes (TCC) in demyelination has been shown in rodent cerebellar cultures. Since TCC modulates activities of various membrane-associated enzymes and increases the level of cellular Ca2+ we investigated whether TCC could activate Ca2+-dependent neutral proteases in myelin that would lead to hydrolysis of myelin basic protein (BP). Addition of antibody and C7-deficient serum plus C7 to sealed myelin vesicles of two to six bilayers caused significant BP hydrolysis compared to the hydrolysis caused by antibody and C7-deficient serum. Significant hydrolysis occurred at the stage of C5b6,7 assembly, which increased in magnitude at the C5b6-8 stage. C5b6-9 formation did not enhance the effect of C5b6-8. BP hydrolysis by C5b6,7 did not require Ca2+ whereas the effect of C5b6-8/C5b6-9 was, in part, Ca2+-dependent. We postulated that TCC formation in myelin membranes causes activation of myelin-associated neutral proteases with subsequent hydrolysis of BP as a consequence of complement peptide insertion and channel formation. Such processes may alter the structure of myelin and augment the action of other inflammatory cells and their products in demyelinating diseases that could ultimately lead to the loss of myelin.

Complement Membrane Attack Complex↗

PubMed1988

Identification as synapsin of a synaptosomal protein immunoreacting with anti-myelin basic protein antiserum.

Rat brain proteins able to react with anti-myelin basic protein antiserum, raised under conditions to induce experimental allergic encephalomyelitis in rabbits, were examined by immunoblot methods after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Apart from the four forms of myelin basic protein present in rat brain, the antiserum detected other proteins of higher molecular weight. Subcellular fractionation shows that these high-molecular-weight proteins are relatively concentrated in a synaptosome-enriched fraction compared to a myelin fraction. A major protein fraction immunorelated to myelin basic protein migrated in the gels as a doublet with apparent molecular weights of approximately 80K and 86K; these proteins were tentatively identified as synapsin Ia and Ib. A purified synapsin preparation analyzed by immunoblot after two-dimensional gel electrophoresis also reacted with anti-myelin basic protein antisera. When the serum was purified by affinity chromatography on a myelin basic protein-conjugated Sepharose column the nonadsorbed material lost this activity whereas the eluted antibodies reacted with myelin basic protein and synapsin. In addition, sequence amino acid comparison of decapeptides showed some homology between these two proteins. A possible implication of immunological agents against myelin basic protein cross-reacting with extra-myelin proteins in the process of experimental allergic encephalomyelitis is considered.

Amino Acid Sequence↗

PubMed1998

MyEF-3, a developmentally controlled brain-derived nuclear protein which specifically interacts with myelin basic protein proximal regulatory sequences.

Myelin basic protein (MBP) gene contains the upstream regulatory sequence that confers cell type- and stage-specific transcription to MBP expression in oligodendrocytes during brain development. The MB1 regulatory motif located between -14 to -50 with respect to transcription start site binds to a brain derived nuclear protein and plays an important role in transcriptional activation of the MBP promoter in transfection assay. Here, we report the isolation of a recombinant cDNA clone, termed myelin expression factor-3, (MyEF-3) from a mouse brain expression library that encodes a novel protein which interacts with the MBP MB1 domain. Computer assisted evaluations of the MyEF-3 sequence revealed several interesting features including four sites for phosphorylation by casein kinase II, a transmembrane domain at the N-terminus, a nuclear localization signal and a Zinc finger domain at the carboxyl terminal. Results from Western and band shift assays indicate that MyEF-3 binds efficiently to double-stranded MB1 as well as the single-stranded non-coding strand of MB1. The use of short DNA fragments encompassing the nucleotide base substitutions across the MB1 domain in competition band shift assay revealed that the ten nucleotide sequence, 5'-GCCTGTCTTT-3' is important for binding of MyEF-3 to DNA. Results from Northern blot studies demonstrate that expression of MyEF-3 is restricted to brain and developmentally regulated during brain maturation. The biological importance of MyEF-3 in the cell type- and stage-specific expression of MBP during brain development is discussed.

Amino Acid Sequence↗

PubMed1986

An immunochemical analysis of a myelin basic protein serum factor: cross reactivity with residues 69-71 of the rabbit encephalitogenic sequence 65-74 of myelin basic protein.

A partially purified myelin basic protein serum factor (MBP-SF), cross-reactive with residues 65-74 (TTHYGSLPQK) of myelin basic protein, has been employed in an immunochemical study to identify the nature of the cross-reacting determinant more precisely. To probe the structural requirements of this determinant, Scatchard inhibition analyses and competitive peptide inhibition radioimmunoassays were employed with a series of peptide analogs of the 65-74 region and with three different reagent antisera: a rabbit anti-rat myelin antiserum (#My05) and two antisera, one rabbit (#162) and one chicken (#66), raised against synthetic peptide S24 (TTHYGSLPQKG). Scatchard inhibition analyses with MBP-SF revealed specific inhibition of binding of 125I-S24 to #162 and #My05, but not to #66. Further delineation of the structural requirements of the cross-reactive determinant, employing a liquid-phase radioimmunoassay, revealed a unique reactivity pattern for the chicken anti-S24 antiserum which, unlike #162 and #My05, did not cross-react under high-affinity conditions with synthetic peptide S20 (GSLPQK, representing the C-terminal half of S24). This, in concert with the Scatchard data, is suggestive of the presence of a cross-reactive determinant centered around residues 69-71 of MBP.

Animals↗

PubMed1993

The distribution of myelin basic protein mRNAs within myelinating oligodendrocytes.

The nervous system contains oligodendrocytes with processes that are greatly extended in space. It is now clear that there are numerous complex, poorly understood mechanisms by which polypeptides are synthesized and delivered to their sites of function in these cells. One mechanism is by the active positioning of mRNAs encoding certain proteins to restricted intracellular subdomains. Perhaps the best studied example of this in the vertebrate CNS is the translocation of myelin basic protein mRNAs to the forming myelin sheath, where the newly synthesized polypeptides, which avidly associate with membranes, can be directly incorporated into the myelin membrane. Evidence for this conclusion is presented here in the context of related work on the general phenomenon of mRNA translocation that is under analysis in other systems.

Animals↗

PubMed1976

Lipid-protein interactions with native and modified myelin basic protein.

The basic protein of central nervous system myelin has been shown to form complexes with acidic lipids in vitro. We measured the interaction of myelin basic protein with several charged and neutral lipids in a biphasic chloroform/methanol/water system and investigated the effect of decreasing the electrical charge of the basic amino groups of the myelin basic protein by acetylation. The modified myelin basic protein, which has an average of eight acetyl residues incorporated, was characterised by gel electrophoresis and circular dichroism. Complexes formed between the acetylated myelin basic protein and acidic lipids exhibited a reduction in the amount of lipids bound, a value that could be correlated with the number of modified amino groups. The significance of these experiments with reference to protein-lipid interaction in the myelin membrane is discussed.

Animals↗

PubMed1993

Genetic susceptibility in familial multiple sclerosis not linked to the myelin basic protein gene.

The myelin basic protein (MBP) gene is a candidate locus for disease susceptibility in familial multiple sclerosis. Amplification of a polymorphic tetranucleotide repeat region immediately 5' to MBP exon 1 demonstrated the presence of eight different alleles among members of 14 multiplex multiple sclerosis families (36 affected individuals). Linkage analysis was performed with autosomal dominant and autosomal recessive models, normal individuals with abnormal magnetic resonance scans being scored as either unknown or affected. Cumulative LOD scores were negative for both models of inheritance. The results do not demonstrate linkage between the MBP gene region and multiple sclerosis.

Alleles↗

PubMed2003

Multiple sclerosis: an important role for post-translational modifications of myelin basic protein in pathogenesis.

Myelin basic protein (MBP) represents a candidate autoantigen in multiple sclerosis (MS). We isolated MBP from normal and MS human white matter and purified six components (charge isomers) to compare the post-translational modifications on each. The sites and extent of methylation, deimination, and phosphorylation were documented for all tryptic peptides by mass spectrometry. We found that mono and dimethylated arginine 107 was increased in MS samples; deimination of arginine occurred at a number of sites and was elevated in MS; phosphorylation was observed in 10 peptides in normal samples but was greatly reduced or absent in most peptides from MS samples. Data obtained with MBP isolated from fresh brain obtained from a spontaneously demyelinating mouse model supported the view that the changes observed in human brain were probably related to pathogenesis of demyelination, i.e. we found decreased phosphorylation and decreased amounts of glycogen synthesis kinase in brain homogenates using specific antibodies. This study represents the first to define post-translational modifications in demyelinating disease and suggest an important role in pathogenesis.

Animals↗