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PubMed1985

A serum factor cross-reactive with antibodies to a determinant of rabbit encephalitogenic sequence 65-74 of myelin basic protein.

A serum factor, cross-reactive with antibodies to a defined determinant of myelin basic protein (residues 66-71), has been found in the sera of nine mammalian species where it may function as a specific neuroautotolerogen. In equilibrium competitive inhibition radioimmunoassays the factor appears to be completely competitive with synthetic peptide S24 (TTHYGSLPQKG) at high affinity and is therefore termed MBP-SF-24 (myelin basic protein serum factor of the S24 type). The bulk of the activity can be recovered by ammonium sulfate fractionation at 61.1% saturated ammonium sulfate (SAS), pH 7, (fraction E) after removal by precipitation at pH 7 of the 37.5, 42.6, 47.5, and 51.4% SAS fractions (fractions A-D), including the immunoglobulins, and before removal by precipitation at pH 5 of the albumin fraction (fraction F). The factor, by its retention on XM300 during ultrafiltration of fraction E, can be purified 20-fold from serum proteins without much loss through a combination of SAS fractionation and ultrafiltration. The yield of MBP-SF-S24 in fraction E may range from a low 26 pmol S24 equivalents from 10 ml in sheep serum to a high 1.7 nmoles from 10 ml rat serum. The serum factor is reactive at high affinity with each of two populations of S24-reactive antibodies in one rabbit reagent antiserum and with one of two populations of S24-reactive antibodies in another. It appears to express a determinant involving residues THYGSL (66-71) of myelin basic protein with the same conformation as found in intact S24.

Amino Acid Sequence↗

PubMed1994

Study by infrared spectroscopy of the interaction of bovine myelin basic protein with phosphatidic acid.

The effect of bovine myelin basic protein (MBP) on dimyristoylphosphatidic acid (DMPA) and phosphatidic acid prepared from egg yolk phosphatidylcholine (EPA) has been investigated by transmission and attenuated total reflectance (ATR) Fourier transform infrared spectroscopy. Interaction of MBP with DMPA and EPA dispersions decreases the lipid acyl chain conformational disorder as a consequence of hydrophobic interactions of the protein with the lipids. Since these effects are more important for EPA dispersions than for DMPA, MBP is believed to penetrate more into EPA bilayers. This could be due to the fact that the hydrogen bond network formed by the charged polar headgroups of EPA is weaker than that of DMPA. This is supported by the spectra of the phosphate region showing that the phosphate groups of EPA are less hydrogen bonded than DMPA. In the presence of MBP, the hydrogen bond network is replaced by electrostatic interactions of the protein with the polar headgroups of the lipid. Infrared spectra of the polar headgroup region also show evidence that MBP enhanced the second ionization state of the phosphate group at neutral pH, this effect being more important for EPA than for DMPA bilayers. Also, infrared spectra of the lipid carbonyl stretching region show evidence that MBP limits the accessibility of water molecules to the interfacial part of the lipid bilayer. Finally, ATR measurements on oriented films of lipid/protein complexes indicate that the penetration of the protein into the lipid bilayer is followed by a reorientation of the lipid acyl chains toward the normal to the bilayer in the case of EPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Effects of acyl chain length on the conformation of myelin basic protein bound to lysolipid micelles.

The interactions of myelin basic protein with micelles of lysophosphatidylcholine detergents of different acyl chain lengths were investigated by circular dichroism (CD), small-angle X-ray scattering, Fourier transform infrared spectroscopy (FT-IR), and 1H, 13C and 31P nuclear magnetic resonance spectroscopy (NMR). Circular dichroic, FT-IR, and 1H NMR measurements indicated that the conformational changes induced in the protein molecules by association with micelles depended on the acyl chain length of the detergents. Size is one of the physical properties of micelles which is a function of the length of the acyl chains. The radii of gyration of detergent micelles in complexes with the protein measured by small-angle X-ray scattering indicated that the average size of the micelles was a quadratic function of the acyl chain length. The dependence of the protein conformational changes on micelle size was used to ascertain the order in which different protein segments associate with the detergents. Several procedures were employed to change the fluidity of micelles formed with detergents of given acyl chain lengths. The conformational changes observed on the MBP molecule by varying the micelle properties without changing the length of the chain, suggested that the changes depended on the size and fluidity of the micelles.

Animals↗

PubMed2003

A combinatorial network of evolutionarily conserved myelin basic protein regulatory sequences confers distinct glial-specific phenotypes.

Myelin basic protein (MBP) is required for normal myelin compaction and is implicated in both experimental and human demyelinating diseases. In this study, as an initial step in defining the regulatory network controlling MBP transcription, we located and characterized the function of evolutionarily conserved regulatory sequences. Long-range human-mouse sequence comparison revealed over 1 kb of conserved noncoding MBP 5' flanking sequence distributed into four widely spaced modules ranging from 0.1 to 0.4 kb. We demonstrate first that a controlled strategy of transgenesis provides an effective means to assign and compare qualitative and quantitative in vivo regulatory programs. Using this strategy, single-copy reporter constructs, designed to evaluate the regulatory significance of modular and intermodular sequences, were introduced by homologous recombination into the mouse hprt (hypoxanthine-guanine phosphoribosyltransferase) locus. The proximal modules M1 and M2 confer comparatively low-level oligodendrocyte expression primarily limited to early postnatal development, whereas the upstream M3 confers high-level oligodendrocyte expression extending throughout maturity. Furthermore, constructs devoid of M3 fail to target expression to newly myelinating oligodendrocytes in the mature CNS. Mutation of putative Nkx6.2/Gtx sites within M3, although not eliminating oligodendrocyte targeting, significantly decreases transgene expression levels. High-level and continuous expression is conferred to myelinating or remyelinating Schwann cells by M4. In addition, when isolated from surrounding MBP sequences, M3 confers transient expression to Schwann cells elaborating myelin. These observations define the in vivo regulatory roles played by conserved noncoding MBP sequences and lead to a combinatorial model in which different regulatory modules are engaged during primary myelination, myelin maintenance, and remyelination.

Animals↗

PubMed1996

Immunoglobulins reactive with myelin basic protein promote CNS remyelination.

We tested the hypothesis that immunoglobulins directed against a CNS autoantigen, myelin basic protein, may promote remyelination in the course of a chronic, immune-mediated demyelinating disease. SJL/J mice infected chronically with Daniel's strain of Theiler's virus served as an experimental model of MS. The spinal cords of these mice exhibit extensive primary demyelination and inflammation with minimal spontaneous remyelination. Treatment with whole antiserum or affinity-purified mouse immunoglobulins directed against rat or rabbit myelin basic protein increased new myelin synthesis as measured by quantitative morphometry. Electron microscopy revealed numerous oligodendrocytes in remyelinated CNS lesions and a relative lack of inflammatory cells. Viral antigen persisted in the spinal cord despite enhanced CNS-type remyelination. These findings indicate that immunoglobulins reactive with myelin autoantigens have the potential to promote myelin repair.

Animals↗

PubMed1989

The thermodynamically stable state of myelin basic protein in aqueous solution is a flexible coil.

Conformational studies of myelin basic protein (MBP) in solution generally have used protein purified in organic solvents and acid. The use of such conditions raises the possibility that the secondary structure reported for the basic protein represents a denatured state. Therefore we have purified this protein by using a procedure that avoids denaturants. Bovine myelin was extracted with 0.2 M-CaCl2 and the protein was purified from the supernatant by chromatography on Sephadex G-75. The conformation of the basic protein was characterized by using c.d. and 1H-n.m.r. spectroscopy. In solution, it appeared to be predominantly randomly coiled, with only small segments of persistent structure. However, in the presence of myristoyl lysophosphatidylcholine the secondary structure of MBP became more ordered, and sedimentation-velocity experiments showed that MBP aggregated. Comparison of our results with published data indicates that Ca2+-extracted basic protein behaves similarly to the protein purified by traditional methods with respect to its ordered conformation in solution in the absence and in the presence of lipid and with respect to its self-association. Thus its thermodynamically stable structure in aqueous solution appears to be a highly flexible coil.

Animals↗

PubMed2001

"Classic" myelin basic proteins are expressed in lymphoid tissue macrophages.

"Classic" myelin basic proteins (MBPs) are demonstrated in lymph nodes of SJL mice by western blot and RT-PCR. Interestingly, expression of these "classic" MBPs was increased during the late relapsing phase of adoptive experimental autoimmune encephalomyelitis (EAE). When splenocytes from SJL mice were separated into macrophage versus B lymphocyte-enriched populations, intact MBP isoforms were demonstrated in the macrophage-enriched population while undetectable in the B lymphocyte-enriched population. RT-PCR demonstrated "classic" MBP transcripts in splenic macrophages, as well as in a macrophage cell line (RAW). The expression of "classic" MBPs in lymphoid tissue macrophages raises the possibility that MBP-specific T cells may be exposed to autoantigen outside the central nervous system (CNS).

Animals↗

PubMed1990

Localization of an encephalitogenic epitope for the SJL mouse in the N-terminal region of myelin basic protein.

T cells from SJL mice reactive with myelin basic protein peptide 1-38 have been reported to be encephalitogenic when adoptively transferred into naive syngeneic recipients. To determine whether the encephalitogenic epitope recognized by peptide 1-38-specific SJL T cells was different from those recognized by H-2u-restricted MBP peptide 1-38-specific T cells, peptide 1-38-specific SJL T cell lines were developed following immunization with guinea pig MBP peptide 1-38. Following a period of in vitro selection in the presence of peptide 1-38 and syngeneic antigen-presenting cells, one of two T cell lines transferred severe clinical disease adoptively. The second line was not encephalitogenic. When the fine specificity for antigen of the two T cell lines was determined by the use of overlapping synthetic peptides, the encephalitogenic epitope recognized by the encephalitogenic line was localized to residues 17-27. This epitope is clearly distinct from that recognized by H-2u mice. The non-encephalitogenic line was found to react only with peptide 1-38, and did not react with mouse MBP.

Animals↗

PubMed1990

Identification of MARPP (14-20), morphine- and cyclic AMP-regulated phosphoproteins of 14-20 kDa, as myelin basic proteins: evidence for their acute and chronic regulation by morphine in rat brain.

MARPP(14-20), morphine- and cyclic AMP-regulated phosphoproteins of 14, 17-18, and 20 kDa, were identified originally by one-dimensional electrophoresis as a group of proteins whose state of phosphorylation was decreased by acute morphine and increased by chronic morphine in the rat locus coeruleus. We now show that MARPP(14-20) represent myelin basic proteins based on biochemical and immunochemical criteria. First, MARPP(14-20) were found to have isoelectric points of about 11 based on their migration on non-equilibrium pH gradient electrophoresis. Second, MARPP(14-20) were greatly enriched in myelin fractions of brain, and were not detectable, or present at very low levels, in other subcellular fractions of brain. Third, analysis of phosphorylated MARPP(14-20) by one- and two-dimensional gel electrophoresis demonstrated precise comigration with immunolabeled myelin basic proteins. In contrast, MARPP(14-20) were distinguished from histones, another group of low molecular weight, highly basic phosphoproteins, in these subcellular fractionation and immunochemical studies. Finally, we confirmed using two-dimensional electrophoresis, that changes observed previously by one-dimensional electrophoresis in the phosphorylation of MARPP(14-20) in response to acute and chronic morphine, and to acute forskolin, occur in myelin basic proteins. It was also found that changes in the state of phosphorylation of myelin basic proteins in response to chronic morphine occur without a change in the total amount of the proteins as determined by immunoblot analysis. The results demonstrate that the phosphorylation of myelin basic proteins is regulated by morphine in the nervous system, and raise the possibility that regulation of these proteins contributes to mechanisms underlying some of the acute and chronic actions of opiates in brain.

Animals↗

PubMed1986

Multiple epitopes in a dodecapeptide of myelin basic protein determined by monoclonal antibodies.

Three custom synthesized myelin basic protein (MBP) peptides, bovine peptide 79-88, human peptide 80-89, and human peptide 82-91, were used to produce four murine monoclonal antibodies (MAb) that were selected on the basis of reaction in a solid phase radioimmunoassay (SRIA) with human MBP. The MAb were compared with respect to antigen specificity against intact MBP and 10 overlapping MBP peptides. One MAb recognized an epitope near the amino-terminus of bovine MBP peptide 79-88. A second MAb was directed towards an epitope that is more reactive in human MBP peptide 45-89 than in intact MBP, but is not recognized in any of the small MBP peptides examined. The third MAb detected an epitope near the middle of human MBP peptide 80-89, whereas the fourth MAb reacted with the carboxyl-terminal portion of human MBP peptide 82-91. Epitopes recognized in SRIA were sometimes not detected by the same MAb in a fluid phase double antibody radioimmunoassay. These results demonstrate the multiplicity of potential epitopes in a dodecapeptide of MBP and do not support the concept of a single, dominant epitope in the region of MBP peptide 80-89.

Amino Acid Sequence↗

PubMed1988

A new epitope on human myelin basic protein arising from cleavage by a metalloendoprotease associated with brain myelin membranes.

Human brain myelin membranes were incubated to allow activity of an associated metalloendoprotease which cleaves myelin basic protein (MBP). A 10.3 kDa C-terminal fragment of MBP, peptide C, isolated from the incubation medium had a blocked N-terminal. After treatment with pyroglutamyl aminopeptidase, N-terminal sequencing indicated that Gln74 of MBP formed the N-terminal residue of peptide C. A rabbit antiserum was raised to a synthetic peptide containing the sequence Pyroglu-Lys-Ser-His-Gly-Arg, corresponding to the first six residues of peptide C. By immunoblotting this serum reacted with peptide C but not with intact MBP. The data indicate that cleavage of MBP by a myelin-associated protease engenders a new epitope.

Amino Acid Sequence↗

PubMed1995

In vivo clonal expression of T lymphocytes specific for an immunodominant N-terminal myelin basic protein epitope in healthy individuals.

The role of myelin basic protein (MBP) T cell recognition in the induction of experimental allergic encephalomyelitis (EAE) has been well established in mice and rats. A remarkable restriction has been observed in T cell receptor (TCR) genes utilized by encephalitogenic T cell lines (TCLs) specific for immunodominant epitopes in these species. Pathological similarities between this animal model and multiple sclerosis (MS) has led to consider MBP as a major candidate autoantigen in this human disorder. Unlike in inbred strains of animals, the T cell response to MBP in humans is quite heterogenous with regard to fine epitope specificity. The existence of V alpha and/or V beta restriction in MBP-specific T cells, from MS patients and healthy controls, is still a matter of debate. In this study we generated 77 MBP-specific TCLs from nine healthy donors and showed that peptide 7-27 is one of the most frequently recognized epitopes. 37% of all epitope-specific TCLs recognized this peptide and p7-27 specific TCLs were generated from seven out of the nine subjects studied. A high level of in vivo clonal expansion was observed in p7-27-specific TCLs in several subjects, which however is not specific of this epitope since this phenomenon was also observed in p85-104- and 149-162-specific TCLs.

Adult↗

PubMed2000

A polymorphism in the repetitive (TGGA)n sequence 5' to the human myelin basic protein gene in Italian multiple sclerosis patients.

Human myelin basic protein (hMBP) gene is one of the candidate genes in the complex mosaic of multiple sclerosis (MS) susceptibility. In this study we verified the distribution of the polymorphism of the region 5' flanking the first exon of the hMBP gene, in 97 relapsing remitting, 74 primary progressive Italian MS patients, and in 236 healthy controls, using polymerase chain reaction (PCR) and gel electrophoresis analysis in this region from 1116 - 1540 nt. Three different band patterns were observed: one homozygote with a 354 bp long fragment, one homozygote with 424 bp long fragment and one heterozygote with both bands present. The short fragment was statistically more frequent in RRMS patients than in HC (P<0.05). The long fragment was more present in HC. Similarly the short homozygous pattern (354 bp/354 bp) was significantly higher in the RRMS patients versus the healthy controls (P<0.01). The sequence analysis of the hMBP alleles showed that while the long fragments matched the prototype sequence completely, all the short fragments showed a deletion of 70 bp from nt 1177 to nt 1247, which explains the short 354 bp allele detected by PCR. Moreover two single mismatches in positions 1386 (T-->C) and 1431 (G-->A), were present only in the short hMBP fragment.

Alleles↗

PubMed1987

Cerebrospinal myelin basic protein in multiple sclerosis. Identification of two groups of patients with acute exacerbation.

The myelin basic protein concentration in the cerebrospinal fluid (CSF) of 125 patients with multiple sclerosis was measured using a radioimmunoassay technique with a detection level of 200 pg/mL and was correlated with the clinical course of the disease. Myelin basic protein was detected in the CSF of some patients with an active progressive form of the disease and in the CSF obtained during exacerbations with the presence of signs or symptoms not previously experienced by the patient (26 of 29 cases were positive during the period of maximal symptoms). Myelin basic protein was not detected in any patient with an inactive or slowly progressive form of the disease, nor in any patient during exacerbations with only recurrence of old signs or symptoms. These results are consistent with the hypothesis that the two clinical forms of exacerbation defined above may be associated respectively with the absence or presence of an acute demyelination.

Humans↗

PubMed1991

Molecular basis of thyroid hormone regulation of myelin basic protein gene expression in rodent brain.

Regulation of myelin basic protein (MBP) gene expression by thyroid hormone has been investigated in rodent brain. Quantitation of the 4 major alternatively spliced transcripts by RNase protection assay showed that the individual mRNAs, corresponding to MBP isoforms 21.5, 18.5, 17, and 14 kDa, were decreased from 2- to 17-fold at all ages studied (4-60 days) in hypothyroid animals when compared to euthyroid, but the timing of onset of expression was not altered. MBP mRNA was also reduced in young adult rats thyroidectomized at the age of 5-6 weeks and was restored to normal by thyroxine administration. Nuclear run-off assays showed that the rate of MBP gene transcription is dependent on thyroid state. Co-transfection of MBP (-256/+1)-chloramphenicol acetyltransferase chimeric gene with a plasmid expressing thyroid hormone receptor alpha, and in the presence of 3,5,3'-triiodothyronine, into NIH3T3 or NG108-15, increased chloramphenicol acetyltransferase expression 4-fold. Using a footprinting technique and Spodoptera frugiperda 9 (Sf9) nuclear extract infected with baculovirus expressing TR alpha, we have identified a single DNA-binding site (-186/-163) for the receptor. A part of this region contains the AGGACA sequence found in thyroid hormone-responsive elements of other 3,5,3'-triiodothyronine-regulated genes. Our finding of a specific hormone-receptor interaction with the MBP promoter region is the first direct demonstration of a thyroid hormone-responsive element in a brain-specific gene.

Animals↗

PubMed1993

Epitope specificity and V gene expression of cerebrospinal fluid T cells specific for intact versus cryptic epitopes of myelin basic protein.

Recent evidence supports the possible involvement of myelin basic protein (BP) as one of the target autoantigens in multiple sclerosis (MS), including elevated frequencies of MS blood and cerebrospinal fluid (CSF) T cells, and the presence in MS plaque tissue of V beta gene sequences and CDR3 motifs characteristic of BP-reactive T cells. Because of its proximity to the target organ, the CSF has long been thought to harbor T cells involved in the pathogenic process. In order to evaluate their frequency and response characteristics, BP-reactive T cells were isolated by limiting dilution from the CSF of patients with MS and other neurological diseases (OND) for quantitation and determination of epitope specificity and V alpha and V beta gene expression. In addition to isolates responsive to intact BP epitopes that were present at a significantly higher frequency in MS versus OND CSF, we here describe a second clonotype responsive to 'cryptic' BP epitopes that is present at approximately equal frequencies in MS and OND patients. In spite of their difference in recognition of intact versus 'cryptic' BP determinants, both clonotypes predominantly recognized epitopes in the N terminal half of human BP, using a similar V gene repertoire that included biased use of V alpha 2 and to a lesser degree V beta 7 and V beta 18. These V gene biases were not related to the epitope specificity of the T cells, indicating that V gene selection is not epitope-driven. These data suggest that there is differential recognition of intact versus 'cryptic' BP determinants in MS versus OND patients that may be related to the processing and presentation of BP to the immune system.

Adult↗

PubMed1994

Protein kinase FA/glycogen synthase kinase-3 predominantly phosphorylates the in vivo site Thr97-Pro in brain myelin basic protein: evidence for Thr-Pro and Ser-Arg-X-X-Ser as consensus sequence motifs.

In a previous study, protein kinase FA/glycogen synthase kinase-3 (FA/GSK-3) was identified as a myelin basic protein (MBP) kinase associated with intact brain myelin. In this report, the phosphorylation sites of MBP by kinase FA/GSK-3 were further determined by two-dimensional electrophoresis/TLC, phosphoamino acid analysis, tryptic peptide mapping, Edman degradation, and direct sequencing. Kinase FA/GSK-3 phosphorylates MBP on both threonine and serine residues. Three tryptic phosphopeptide peaks were resolved by C18 reverse-phase HPLC. Sequential manual Edman degradation together with direct sequence analysis revealed that T(p)PPPSQGK is the phosphorylation site sequence for the first major phosphopeptide peak. When mapping with the bovine brain MBP sequence, we finally demonstrate Thr97-Pro, one of the in vivo phosphorylation sites in MBP, as the major site phosphorylated by kinase FA/GSK-3, implicating a physiologically relevant role of FA/GSK-3 in the regulation of brain myelin function. By using the same approach, we also identified NIVT94(p)PR as the phosphorylation site sequence in the second major tryptic phosphopeptide derived from [32P]MBP phosphorylated by kinase FA/GSK-3, further indicating that kinase FA/GSK-3 represents a Thr-Pro motif-directed MBP kinase involved in the phosphorylation of brain myelin.

Amino Acid Sequence↗

PubMed1989

Resolution and solution behavior of crosslinked myelin basic protein.

The use of chemical crosslinking methodologies for the study of the solution structure and folding of the myelin basic protein required the development of a specific protocol for separating the various reaction products. Myelin basic protein treated with the crosslinking reagent dithiobis(succinimidylpropionate) was subjected to analysis by urea-SDS polyacrylamide gel electrophoresis. This permitted the identification of dimer and higher oligomeric crosslinked products. The dissociating conditions of this method precluded the dimerization of the basic protein observed in systems with SDS and without urea. Similar samples analyzed by gel filtration-fast protein liquid chromatography exhibited a complex elution pattern in contrast to the protein not reacted with the crosslinker. The electrophoretic analysis of the different eluted fractions revealed that at least three monomeric forms of modified myelin basic protein had been separated by gel filtration.

Animals↗