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PubMed1978

Silica gel radioimmunoassay for myelin basic protein.

1. A radioimmunoassay which relies upon the capacity of silica gel to absorb free human basic protein of myelin (BP) is described. 2. Reagents involved in the assay include unlabelled BP, I-BP, and rabbit anti-BP. The assay is performed in excess normal rabbit serum, and uses 3-4% silica gel for absorption of free I-BP. 3. The assay is simple, reproducible and not particularly time-consuming. It is capable of detecting BP at a level of 1-10 or more nanograms in samples containing varying amounts of serum. The assay is thus highly suitable as a routine test in clinical laboratories interested in detecting BP in cerebrospinal fluid in patients with suspected or proven demyelination.

Gels↗

PubMed1993

Immunological analysis of the amino terminal and the C8 isomer of human myelin basic protein.

The citrullination and N-terminus acylation of myelin basic protein (MBP) increases the heterogeneity among the MBP isoforms. The present study was undertaken to further characterize the immune response to the citrullinated form (C8) of MBP as well as to the variably acylated N-terminus of MBP. Six well-characterized murine monoclonal antibodies (mAbs) to human MBP-C8 or MBP peptides (four mAbs to MBP acetyl 1-9, one mAb to MBP 10-19 and one mAb to MBP 80-89), one murine T cell line (PL11) to human MBP peptide acetyl 1-9 and one Lewis rat T cell line (RT-1) to guinea pig (GP) MBP peptide 68-88 were used to assess reactivity with MBP-C1, MBP-C8, and MBP peptides including a series of MBP peptide 1-21 containing 0, 2, 4, 6, 8 or 10 carbon fatty acids. Enzyme-linked immunosorbent assay (ELISA) results revealed that all of the mAbs reacted with human MBP-C1 and MBP-C8 except anti-MBP 10-19 and anti-MBP-C8. The former reacted only with MBP-C1 and the latter only with MBP-C8. The presence and length of acylation of MBP peptide 1-21 modified reactivity. Three mAbs to MBP acetyl 1-9 reacted only with acetyl 1-21, and one mAb anti-MBP actyl 1-9 reacted with all of MBP 1-21 preparations whether acylated or not. mAb anti-MBP-C8 generally reacted better with acylated MBP 1-21 having longer fatty acids. The PL11 T cell line strongly proliferated to human MBP-C1, MBP-C8 and MBP acetyl 1-9, responded, but less well, to MBP 1-21 with longer fatty acids and failed to respond to nonacylated MBP peptide 1-21. The RT-1 cell line responded strongly to GP MBP peptide 68-88, marginally to MBP-C8 and failed to respond to MBP-C1 or any of the other MBP peptides. Specific immune responses to different MBP charge isomers and different N-terminal acylating groups of MBP may play a role in immune-mediated demyelination.

Acylation↗

PubMed1986

Myelin basic protein ability to organize lipid bilayers: structural transition in bilayers of lysophosphatidylcholine micelles.

Myelin basic protein isolated by a single step with the cationic detergent cethyltrimethylammonium bromide in a lipid-bound form is able to induce structural transition of lysophosphatydilcholine micelles into multi-laminar vesicles. This finding, observed through electron microscopy, is discussed in the light of the assumed ability of the basic protein to organize myelin lipids.

Animals↗

PubMed1986

Gangliosides inhibit phospholipid-sensitive Ca2+-dependent kinase phosphorylation of rat myelin basic proteins.

Gangliosides inhibit the phosphorylation of both small and large rat myelin basic proteins (SMBP, LMBP) by an endogenous phospholipid-sensitive Ca2+-dependent protein kinase (C-kinase). Using a rat brain myelin preparation in an in vitro phosphorylation assay system, we determined the inhibition constants (IC50's) of the gangliosides GM1, GD1a, GD1b, and GT1b to be approximately 160 microM, 65 microM, 65 microM, and 40 microM, respectively. Asialoganglioside GA1, ceramide, and N-acetylneuraminic acid (NANA, sialic acid) failed to produce similar inhibition, suggesting that both the lipid and the sialic acid moieties are necessary, but neither alone is sufficient to produce inhibition. The results indicate that gangliosides may regulate protein kinase C activities in the nervous system.

Animals↗

PubMed1983

Autoimmune encephalomyelitis (EAE) mediated or prevented by T lymphocyte lines directed against diverse antigenic determinants of myelin basic protein. Vaccination is determinant specific.

Lines of T lymphocytes reactive against the basic protein of myelin (BP) were found in previous studies to mediate experimental autoimmune encephalomyelitis (EAE) in rats. Moreover, inoculation of rats with attenuated anti-BP line cells vaccinated them against subsequent attempts to induce active EAE by injection of BP in adjuvant. In the present study, we investigated the effects of T lymphocyte lines reactive to different antigenic determinants on the BP molecule, they are the major encephalitogenic peptide (EP) determinant present on guinea pig BP (G-BP), and minor, non-EP determinants present on bovine BP (B-BP). We found that both lines of T lymphocytes could mediate EAE. Resistance to active EAE acquired by spontaneous recovery from line mediated EAE or by vaccination with attenuated cells, however, was found to be specific for the particular BP determinant. Thus, EAE may be mediated by lines of T lymphocytes reactive to different determinants on the BP molecule, but the resistance to EAE acquired by exposure to line cells is determinant specific. This suggests that acquired resistance to EAE is directed by the receptor specificity of the autoimmune anti-BP T cells.

Animals↗

PubMed1990

Conservation throughout vertebrate evolution of the predicted beta-strands in myelin basic protein.

To identify functionally important parts of the 18.5-kDa myelin basic protein (MBP), the amino acid sequences from 10 species ranging from shark to human were aligned using the SEQHP computer program. The residues that are invariant or very conservatively substituted (Arg/Lys, Ser/Thr, Ile/Leu, Asp/Glu) among all 10 proteins were scored. Of the 72 conserved residues in the 170-residue human protein (42% conserved), 32 are found within the five beta-strands previously predicted (45 residues, 71% conserved), 23 within the small-loops region (42 residues, 55% conserved), but only 17 within the large-loops region (83 residues, 20% conserved). Of the 22 hydrophobic residues within the predicted beta-sheet of human MBP, 20 hydrophobic residues remain in the shark protein, 19 of them in the same positions. In contrast, there are 10 hydrophobic residues elsewhere in the human protein, but only 7 remain in the shark protein and only 1 of them is in the same position. The triprolyl sequence found in all mammalian MBPs and in the chicken MBP is not conserved in the shark protein. The four alternately spliced forms of mouse MBP can be accommodated by the beta-structural model, but not the 17-kDa human MBP, which lacks exon 5. These findings confirm the crucial role of the hydrophobic residues in the predicted beta-sheet for the structure and function of the protein. It seems likely that the conserved portions of the protein make an important contribution to the highly ordered lamellar structure of myelin.

Amino Acid Sequence↗

PubMed1993

Selective expression of foreign genes in glioma cells: use of the mouse myelin basic protein gene promoter to direct toxic gene expression.

We have previously demonstrated that retrovirus-mediated genes were transferred to mouse glioma cells in a meningeal gliomatosis model (Yamada et al.: Japanese Journal of Cancer Research 83:1244-1247, 1992). This retrovirus vector contains the Escherichia coli. beta-galactosidase (beta-gal) gene as a marker for integration of the lacZ gene, which is controlled by the SV40 early promoter. We investigated whether lacZ genes could be specifically controlled in mouse glioma cells by glial-specific promoters, including the 2.5 kb 5' flanking region of the mouse glial fibrillary acidic protein (GFAP) gene, the 1.3 kb 5' flanking region of the myelin basic protein (MBP) gene, and the 1.5 kb 5' flanking region of the myelin proteolipid protein (PLP) gene. Psi-2 packaging cells were transfected with each retrovirus vector (GFAP promoter-, MBP promoter-, and PLP promoter-lacZ) and the infectious virus particles were recovered from the supernatants. Blue staining for beta-gal was detected in various fibroblast, myeloma, and glioma cell lines transduced with the retrovirus BAG vector. On the other hand, blue staining was only detected in glioma cells after transduction with the lacZ gene-bearing retrovirus controlled by glial-specific promoters. The strongest promoter activity was detected after transduction with the retrovirus in which the MBP promoter controlled the lacZ gene. Mouse glioma cells transduced with retrovirus containing the MBP promoter directing the herpes simplex virus type 1 thymidine kinase (HTK) gene were extremely sensitive to ganciclovir, while the parental cells and cells transduced with retrovirus containing the lacZ gene were not sensitive to ganciclovir.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗

PubMed1991

Protein kinase C-dependent activation of a myelin basic protein kinase by gastrin-releasing peptide in Swiss 3T3 fibroblasts.

Addition of gastrin releasing peptide to serum-starved Swiss 3T3 mouse fibroblasts results in a transient appearance of a myelin basic protein-kinase activity in cytosolic extracts. Increased kinase activity is also observed upon stimulation of cells with bradykinin, epidermal growth factor or 4 beta-phorbol dibutyrate. Chromatographic analysis of the cytosolic extracts show that both gastrin-releasing peptide and 4 beta-phorbol dibutyrate induce the appearance of a kinase activity similar to that induced by epidermal growth factor. The response to gastrin-releasing peptide is abolished by down-regulation of protein kinase C and attenuated by acute inhibition of protein kinase C using staurosporine. The effect of epidermal growth factor was also suppressed under these conditions, albeit to a lesser extent. The results indicate (1) that activation of myelin basic protein kinase(s) may be common to different growth factors, and (2) that protein kinase C may participate in this response, at least in the case of gastrin-releasing peptide.

3T3 Cells↗

PubMed1979

Lymphocyte transformation in the presence of myelin basic protein in multiple sclerosis and control subjects.

The transformation of lymphocytes in vitro in the presence of human myelin basic protein has been investigated in normal people, patients with multiple sclerosis (MS) and controls with other neurological diseases. There was little or no response at low concentrations (1--10 microgram/ml) but significant transformation at higher concentrations (100--1000 microgram/ml) in all three groups. There was no significant difference among the groups as a whole, but those MS patients who had had disease for more than 10 years did show greater responses than normal subjects (P less than 0.05). Increased responses could not be correlated with any other aspect of disease activity: in particular they were not increased in patients with acute relapses. The use of autologous serum instead of homologous AB Rhesus positive serum did not significantly alter lymphocyte responsiveness. The absence of any response in the presence of purified calf thymus histone suggests that the response to myelin basic protein indicates a low level of lymphocyte sensitization to this antigen even in normal subjects. The present evidence does not support a primary pathogenetic role for such a reaction in MS. The increased response in patients with a long duration of disease might merely be an effect of white matter damage or might represent an amplification of the normal immune response contributing to myelin breakdown and leading to the emergence of the progressive stage of the disease. The study of lymphocyte responsiveness over a wide range of concentrations of myelin basic protein is considered to resolve some of the controversy surrounding this subject in the literature.

Adolescent↗

PubMed1998

MEBA derepresses the proximal myelin basic protein promoter in oligodendrocytes.

The central nervous system expression of myelin basic protein (MBP) is restricted to oligodendrocytes and is developmentally regulated; these regulatory features are transcriptionally mediated. We have previously shown that the proximal 149 nucleotides of the MBP promoter were both necessary and sufficient to activate the transcription of MBP in cultured oligodendrocytes, but not in other cell types. Sequences within the distal portion of this promoter, which contains a nuclear factor 1 (NF1) binding site, repressed activation of the MBP promoter in Cos-7 cells, but not in oligodendrocytes. We now describe a sequence upstream of and partially overlapping the NF1 site that activates the MBP promoter in oligodendrocytes, but not in Cos-7 cells. A protein complex binds to this site, designated MEBA (myelinating glia-enriched DNA binding activity), and is enriched in nuclear extracts prepared from the brain, oligodendrocytes, and Schwann cells. The amount of MEBA parallels MBP expression and myelinogenesis in the developing brain and parallels new MBP expression as purified oligodendrocytes differentiate. Mutational analyses of binding and function distinguish MEBA, an activator, from NF1, a repressor of MBP transcription, and suggest that MEBA consists of at least two proteins. Because the binding sites of MEBA and NF1 overlap, we suggest that MEBA may either compete with or modify NF1 binding, thereby activating the MBP promoter in oligodendrocytes.

Animals↗

PubMed1999

Highly deiminated isoform of myelin basic protein from multiple sclerosis brain causes fragmentation of lipid vesicles.

Myelin basic protein (MBP) occurs as a number of charge isomers due to phosphorylation, deamidation, and deimination of arginine to citrulline. All of these modifications decrease the net positive charge of the protein and its ability to cause aggregation of negatively charged lipid vesicles. This is used as a model system for the ability of MBP to cause adhesion of the cytosolic surfaces of myelin. Therefore, the effect of two deiminated forms of MBP on lipid vesicles was compared with that of the unmodified, most positively charged isomer, C1, to determine how loss of positively charged arginines would affect the function of MBP. The deiminated forms were the isomer isolated from normal human brains, in which only 6 Arg are deiminated to citrulline (MBP-Cit(6)), and an isomer isolated from the brain of a patient who died with acute, fulminating multiple sclerosis (Marburg type), in which 18 of the 19 Arg were deiminated (MBP-Cit(18)). Whereas C1 caused aggregation of lipid vesicles, resulting in an increase in absorbance due to light scattering, MBP-Cit(18) caused a decrease in absorbance of the lipid vesicles. Size exclusion chromatography and negative staining electron microscopy showed that this was due to fragmentation of the large multilayered vesicles into much smaller vesicles. MBP-Cit(6) caused less aggregation of lipid vesicles than did C1. However, no fragmentation of the vesicles into smaller ones in the presence of C1 and MBP-Cit(6) was detected by size exclusion chromatography or electron microscopy. The membrane fragmentation caused by MBP-Cit(18) is dramatically different from the effects of other forms of MBP from normal brain and may indicate a pathogenic effect of this charge isomer, which may have contributed to the severity of the Marburg type of multiple sclerosis. Alternatively, the deimination may have been a secondary effect resulting from the disease process. Regardless of the role of MBP-Cit(18) in multiple sclerosis, the effect of this modification indicates that, when most of the arginines of MBP are modified to an uncharged amino acid, the protein acquires properties similar to an apolipoprotein; thus, it may take up an amphipathic structure when bound to lipid. A partly amphipathic character may also be related to the role of MBP-Cit(6) in normal immature myelin, where it is the predominant charge isomer.

Animals↗

PubMed1984

Myelin basic protein immunoreactivity in serum of neurosurgical patients.

Patients admitted to the neurosurgical wards for the management of nervous system tumours, subarachnoid and intracerebral haemorrhage, head injury, spinal and peripheral nerve lesions, and other miscellaneous neurosurgical conditions, were studied by assay of serum immunoreactivity for myelin basic protein. Of 171 patients, 70% proved to have elevated myelin basic protein activity. In cerebral cases the extent of brain damage assessed by clinical methods appeared to correlate with the appearance of elevated serum myelin basic protein. In spinal and peripheral nerve cases no similar elevation of myelin basic protein was observed.

Adolescent↗

PubMed2002

Study of expression of myelin basic proteins (MBPs) in developing rat brain using a novel antibody reacting with four major isoforms of MBP.

Myelin basic proteins (MBPs) are the major protein components of myelin. MBP isoforms are known to have different expression patterns. In order to distinguish the different expression patterns on myelination, we have developed a novel antibody reacting with the four major isoforms of MBPs with molecular masses of 21.5 kDa, 18.5 kDa, 17.0 kDa, and 14.0 kDa. These MBPs were initially separated by acid urea gel and sodium dodecyl sulfate polyacrylamide gel electrophoreses and detected with the luminol reaction. Then the antibody developed was used to determine the relative amounts of MBP isoforms. The MBPs of oligodendrocytes were detected by the enhanced luminol reaction using Renaissance (Dupont NEN, Boston, MA). From the immunological aspect, the MBP monoclonal antibody (Sires et al. [1981] Science 214:87-89) was revealed to recognize MBPs with molecular masses of 21.5 kDa and 18.5 kDa. Furthermore, we found that Ile-166 in the rat 18.5-kDa MBP isomers was replaced by methionine. The 14.0-kDa and 18.5-kDa isoforms of MBP are the most abundant MBP species and comprise more than 70% of the total MBPs in 3.5-and 24-month-old rats. MBPs are expressed during development and the compositions of MBPs in mature (3.5 months old) and aged (24 months old) rats were almost the same. The expression of the 14.0-kDa and 18.5-kDa MBPs occurred earlier in the cerebellum and the spinal cord than in the cerebrum by approximately 1 week. MBPs are also expressed upon oligodendrocyte maturation by interacting with astrocytes. The above results suggest that the regulation of MBP isoforms during development and oligodendrocyte differentiation may indicate the point of occurrence of both the unique patterns of isoform expression and the shift in intracellular localization of MBPs with the maturation of oligodendrocytes.

Amino Acid Sequence↗

PubMed1986

Evidence for specific polypeptide chain folding in myelin basic protein from reactions between fragments of the protein and monoclonal antibodies.

The specificities of two monoclonal IgM antibodies (18.25 and 21.14.2) evoked in mice with guinea pig myelin basic protein were examined and interpreted in terms of a specific folding of the protein's polypeptide chain. Studies with guinea pig and rabbit myelin basic protein fragments showed that a region encompassing the central Phe-Phe (87-88) sequence is obligatory, but not sufficient, for reactivity with antibody 18.25. Appreciable reactivity was observed for rabbit peptides 22-95 and 45-151, and lower, but significant, reactivity was shown by peptide 32-95. Only very weak reactivity was seen with peptide 44-95. No reactivity was observed with peptide 1-95 after its lysine residues were acetylated, acetamidinated, or guanidinated. These results have been interpreted in terms of a polypeptide chain folding that creates an epitope within sequence Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val (84-92). The specific conformation of this epitope, which includes probably the Lys-89 and possibly the Asn-90 and Val-92 side chains, could be formed by the association of sequence 84-92 with either sequence Ile-Leu-Asp-Ser-Ile-Gly-Arg-Phe-Phe (37-45) or with sequence Val-Leu-Ser-Arg-Phe (108-112) to form beta-sheet structures essentially identical with those that appear to be present in the intact BP [Martenson R.E.J. Neurochem. 46, 1612-1622 (1986)]. The second monoclonal antibody, no. 21.14.2, reacts only with guinea pig myelin basic protein and fragments containing the species-restricted sequence Arg-Ala-Asp-Tyr-Lys-Ser-Lys (129-135).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1983

Highly sensitive-beta-D-galactosidase-linked immunosorbent assay for anti-myelin basic protein antibody.

A highly sensitive enzyme-linked immunosorbent assay (ELISA) procedure has been devised for the determination of anti-myelin basic protein antibodies. First, rabbit anti-myelin basic protein (MBP) sera to be assayed were incubated with MBP immobilized on small pieces of silicone rubber. Next, anti-MBP bound specifically to the solid phase was allowed to react either with Fab' of guinea pig anti-rabbit IgG conjugated with beta-D-galactosidase from Escherichia coli or with protein A conjugated with beta-D-galactosidase. The amount of fixed anti-MBP antibodies was assayed by fluorometric determination of the enzyme activity using 4-methylumbelliferyl-beta-D-galactoside as a substrate. The sensitivity of the present assay was at least an order of magnitude higher than the hitherto available radioimmunoassay and ELISA.

Animals↗

PubMed1989

The duplicated myelin basic protein gene in mld mutant mice does not impair transcription.

Myelin basic protein (MBP) gene organization and expression were analyzed in wild type and myelin deficient (mld) mutant mice. Southern analysis demonstrated MBP gene duplication in mld mice. In addition, we present evidence that one MBP gene in mld mice is normal for at least 14 kilobases (kb) upstream from exon I, whereas the second gene is normal for at least 3.5 kb but not more than 7 kb upstream from exon I. Run-on experiments showed that the rate of MBP gene transcription in mld mice is similar to that seen in normal mice. Detailed analysis of the transcriptional activity of various regions of the gene led us to conclude that all portions of the MBP gene are transcribed in mld mice. Consequently, we propose that the low levels of MBP mRNA observed in these mice (2-5% of the wild-type level) are not due to deficient transcriptional activity.

Animals↗

PubMed1985

Interaction of myelin basic protein and polylysine with synthetic species of cerebroside sulfate.

The effect of myelin basic protein on the myelin lipid cerebroside sulfate was studied by differential scanning calorimetry and use of the fatty acid spin label, 16-S-SL, in order to determine (i) the effect of basic protein on the metastable phase behavior experienced by this lipid, and (ii) to determine if basic protein perturbs the lipid packing as it does with some acidic phospholipids. The effects of basic protein on the thermodynamic parameters of the lipid phase transition were compared with those of polylysine which has an ordering effect on acidic phospholipids as a result of its electrostatic interactions with the lipid head groups. Different synthetic species of cerebroside sulfate of varying fatty acid chain length and with and without a hydroxy fatty acid were used. The non-hydroxy fatty acid forms of cerebroside sulfate undergo a transition from a metastable to a more ordered stable state while the hydroxy fatty acid forms remain in the metastable state at the cation concentration used in this study (0.01 M Na+ or K+). The non-hydroxy fatty acid forms were still able to go into a stable state in the presence of both basic protein and polylysine. At low concentrations, basic protein increased the rate of the transition to the stable state, while polylysine decreased it for the longest chain length form studied. However, at high concentrations, basic protein probably prevented formation of the stable state. The hydroxy fatty acid forms did not go into the stable state in the presence of basic protein and polylysine. It is argued that the increased rate of formation of the stable state in the presence of basic protein and decreased rate in the presence of polylysine are consistent with interdigitation of the lipid acyl chains in the stable state. Basic protein also had a small perturbing effect on the lipid. It decreased the total enthalpy of the lipid phase transition. When added to the non-hydroxy fatty acid forms it increased the temperature of the liquid crystalline to metastable phase transition and decreased the temperature of the stable to liquid crystalline phase transition. It significantly decreased the transition temperature of the hydroxy fatty acid forms but only a portion of the lipid was affected. In contrast, polylysine increased the transition temperature of the metastable and stable states of all forms of cerebroside sulfate but had a greater effect on the non-hydroxy fatty acids forms than on the hydroxy fatty acid forms.(ABSTRACT TRUNCATED AT 400 WORDS)

Calorimetry, Differential Scanning↗

PubMed1990

Myelin basic protein: interaction with calmodulin and gangliosides.

The structural characteristics of myelin basic protein (MBP) involved in protein-protein and protein-lipid interactions were investigated. Rabbit MBP could bind calmodulin (CaM) in the presence of Ca2+ to form a complex that remained undissociated in 8 M urea. However, no tight complex formation was observed when the divalent cation was absent. These results suggest that MBP may contain a hydrophobic domain similar to those in the other well-characterized CaM-binding proteins. The stoichiometry of calmodulin binding to MBP was approximately 1:1. Prior limited proteolysis of MBP with trypsin abolished the formation of the MBP-CaM complex, indicating that the entire MBP polypeptide may be involved in the recognition of the hydrophobic clefts in CaM. MBP also formed tight complexes with gangliosides, but the presence of Ca2+ was not required. Binding of gangliosides to MBP-CaM complex released CaM from the complex. The ganglioside-binding sites in MBP were determined after trisecting the protein at two glutamic acid residues with Staphylococcus aureus V8 protease. Subsequent binding studies revealed that a 9.5-kDa polypeptide, which may correspond to the NH2-terminal domain (residues 1-83) of MBP, had higher affinity for the binding of lucifer yellow CH-labeled GM1 than did the other two polypeptides, of apparent molecular mass (Mr) 5,500 and 4,500, respectively. Among the various proteins in purified guinea pig brain myelin, synaptosomes, and synaptosomal membranes, MBP was found to have the highest affinity in binding lucifer yellow CH-GM1.

Animals↗