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Myelin basic protein was shown to be a substrate for protein kinase from rabbit muscle. One of the major sites of phosphorylation was the serine residue in the sequence Gly-Arg-Gly-Leu-Ser-Leu. The arginine residue in this sequence is known to be a substrate for a protein methylase.
To examine the basic human brain proteins, we subjected 9 mmol/L urea extracts to non-equilibrium gel electrophoresis. The pattern observed differs distinctly from that with equilibrium gel electrophoresis. With this technique, the myelin proteins (myelin basic protein, proteolipids, and basic Wolfgram proteins) and many other unindentified major basic proteins can be demonstrated. The myelin basic proteins occur as two major polypeptides of different charge and slightly different molecular mass, indicating the action of at least two genes. The proteolipid proteins occur as a long series of charge isomers, suggesting multiple genes or extensive post-transcriptional modification. In one patient with schizophrenia, a charge-change mutation of the larger myelin basic protein (MBL) was observed and is termed "MBL-Duarte."
The nervous system specific proteins glial fibrillary acidic protein (GFAp), S-100 and myelin basic protein (MBP) were determined in 535 human cerebrospinal fluid (CSF) samples. The level of all three proteins was increased in CSF of patients with nonselective destructive central nervous tissue disease such as encephalitis, cerebrovascular disease or tumoural compression. The increases in GFAp were more constant, making it a better marker of CNS pathology. Increases in MBP in CSF of patients with acute demyelinating disease were confirmed. S-100 did not seem to give more information as GFAp. Isolated increases of GFAp could be demonstrated in patients with dementia (Alzheimer type or multi-infarct dementia) or syringomyelia. Since CNS of these patients is very rich in fibrillary astrocytes, containing large amounts of GFAp, it is suggested that GFAp is to be considered as a specific marker of fibrillary gliosis in CSF and can be used as a diagnostic tool in dementia and syringomyelia.
Membrane-bound proteolysis may be implicated in the pathogenesis of demyelinating disorders including multiple sclerosis (MS). We previously found that the extent of myelin basic protein (MBP) degradation by the calcium-activated neutral protease did not differ for isolated human control myelin or MS myelin. Hence we suggested that, if involved in demyelination, the myelin neutral protease must be activated in vivo by an increased availability of free calcium. The postulate was therefore tested that immunoglobulin (Ig) binding to myelin results in activation of the myelin neutral protease, possibly through release of free calcium from calcium-binding sites of myelin. Isolated myelin from the brains of controls and patients with MS were incubated with purified Igs eluted from the brains of patients with MS or controls and degradation of MBP was assessed by quantitative electroimmunoblotting. Such degradation was significantly greater in myelin incubated in the presence of MS Igs than in myelin incubated without added Igs or in the presence of control Igs. Furthermore, the degree of MBP degradation in myelin incubated with control Igs was similar to that observed in myelin incubated without added Igs. Accordingly, it is suggested that Ig in MS brain potentiates myelin breakdown. Moreover activation of membrane-bound proteolysis by Ig binding to myelin appears to represent a hitherto undescribed pathway for demyelination in MS.
The zinc-binding proteins (ZnBPs) in porcine brain were characterized by the radioactive zinc-blot technique. Three ZnBPs of molecular weights about 53 kDa, 42 kDa, and 21 kDa were identified. The 53 kDa and 42 kDa ZnBPs were found in all subcellular fractions while the 21 kDa ZnBP was mainly associated with particulate fractions. This 21 kDa ZnBP was identified by internal protein sequence data as the myelin basic protein. Further characterization of its electrophoretic properties and cyanogen bromide cleavage pattern with the authentic protein confirmed its identity. The zinc binding properties of myelin basic protein are metal specific, concentration dependent and pH dependent. The zinc binding property is conferred by the histidine residues since modification of these residues by diethyl-pyrocarbonate would abolish this activity. Furthermore, zinc ion was found to potentiate myelin basic protein-induced phospholipid vesicle aggregation. It is likely that zinc plays an important role in myelin compaction by interacting with myelin basic protein.
Myelin basic protein (BP) has the capacity to induce experimental allergic encephalomyelitis (EAE) in animals as well as to prevent and suppress EAE. Immunoreactive BP or BP fragments appear in cerebrospinal fluid (CSF) in persons with multiple sclerosis in acute disease periods and in individuals with acute damage to central nervous system myelin. Humoral and cellular immune responses, especially in the CSF, may occur but are of uncertain effect in the etiology and pathogenesis of MS. Attempts to use BP to suppress MS have disclosed no therapeutic benefit.
Myelin basic proteins (MBPs) from 6-day-old, 10-day-old, 20-day-old and adult normal mouse brain were compared with those from 20-day-old jimpy (dysmyelinating mutant) mouse brain to determine the effect of reduced levels of proteolipid protein (PLP) on MBPs. Alkaline-urea-gel electrophoresis showed that 6-day-old and 10-day-old normal and jimpy MBPs lacked charge microheterogeneity, since C8 (the least cationic of the components; not be confused with complement component C8) was the only charge isomer present. In contrast, MBPs from 20-day-old and adult normal mouse brain displayed extensive charge microheterogeneity, having at least eight components. A 32 kDa MBP was the major isoform observed on immunoblots of acid-soluble protein from 6-day-old and 10-day-old normal and 20-day-old jimpy mouse brain. There were eight bands present in 20-day-old and adult normal mouse brain. Purified human MBP charge heteromers C1, C2, C3 and C4 reacted strongly with rat 14 kDa MBP antiserum, whereas the reaction with human C8 was weak. This suggested that MBPs from early-myelinating and jimpy mice did not react to MBP antisera because C8 was the major charge isomer in these animals. Purification of MBPs from normal and jimpy brain by alkaline-gel electrophoresis showed that both normal and jimpy MBPs have size heterogeneity when subjected to SDS/PAGE. However, the size isoforms in normal mouse brain (32, 21, 18.5, 17 and 14 kDa) differed from those in jimpy brain (32, 21, 20, 17, 15 and 14 kDa) in both size and relative amounts. Amino acid analyses of MBPs from jimpy brain showed an increase in glutamic acid, alanine and ornithine, and a decrease in histidine, arginine and proline. The changes in glutamic acid, ornithine and arginine are characteristic of the differences observed in human C8 when compared with C1.
Two guinea pig myelin basic protein preparations which differed markedly in their contents of high pH electrophoretic or chromatographic forms were studied in an attempt to elucidate the causes of their microheterogeneity. Both total preparations and components isolated therefrom were examined for their amino acid compositions, NH2-terminal and COOH-terminal residues, total phosphorus contents, amd contents of phosphamino acids. The results showed that the five components differed sequentially by a single charge and that the microgeterogeneity arose as a result of secondary modifications of a single secies (Component 1) Of basic protein. Two modifications were demonstrated; viz. phosphorylation of serine and threonine and loss of COOH-terminal arginine. These two modifications were insufficient to account completely for the observed microheterogeneity; an additional cause, deamidation, was postulated. From the relationship between the number of components present in the total basic protein, the phosphorus and phosphoamino acid contents of the components, and the changes in relative electrophoretic mobility of the components which accompanied their phosphorylation and dephosphorylation we conclude that in the native basic protein no more than two sites in any polypeptide chain are phosphorylated.
The myelin basic protein gene produces two families of proteins, the golli proteins and the 'classic' myelin basic proteins from three transcription start sites (tsp). The golli proteins are expressed from the first tsp, and little is known about genetic elements that control its activity. We have examined elements that may regulate the expression of the golli products produced from this promoter in neural cell lines with constructs containing upstream portions of the first tsp by transient transfection assays. Three putative regulatory elements were identified, among them a 345bp novel silencer region, termed the golli silencer region (GSR), which was characterized in detail. This silencer was responsible for a significant (approx. 60%) inhibition of luciferase expression in PC12 cells. It was orientation-dependent and a double dose of this GSR completely abolished expression of the luciferase reporter activity. Transfections with deleted constructs identified three critical sites that bind at least two repressor proteins. We postulate that the silencer activity is the result of synergistic interactions between these repressor proteins and might involve the formation of a high-ordered protein-DNA structure.
High resolution 13C and 1H NMR spectra of myelin basic protein over a range of pH and concentrations indicate that intramolecular folding of the polypeptide chain occurs in aqueous solution in the region of residues 85 to 116. At pH 4 in D2O solution, the 13C resonances due to nonprotonated carbons of phenylalanine and tryptophan are broadened and chemically shifted compared to the same resonances when the protein is dissolved in 6M guanidinium hydrochloride. These residues occur in the region of the polypeptide chain in which the intramolecular folding may occur. As the pH is raised and the positive charge on the protein reduced from 28 to 18, intermolecular aggregation occurs, which appears to involve these same folded regions. Data on T1 (longitudinal relaxation times) of protons indicate also that amino-acid sidechains vary considerably in their motional freedoms. The concentration dependence of the proton NMR spectra provides further information on association of protein monomers. The region of the protein involved in folding, polymerization and substrate specificities is conservative in various species and we can surmise that it may have a specialized role in protein-lipid interactions in the myelin membrane. We suggest that the protein forms dimers across the cytoplasmic apposition during the formation of myelin. Estimates of the repulsive energies of interaction between approaching membranes suggest that some special mechanism of this kind is required to overcome the repulsive forces due to breakdown of water structure and electrostatic interaction.
Chemically and immunologically, myelin basic protein (MBP) is very similar with the basic protein extracted from animal and human tumors. The results of this study demonstrated that splenocytes from C57BL/6 mice bearing B16 melanoma cells are sensitized to MBP, suggesting that this protein may share common antigenic determinants with antigens from B16 melanoma cells. The RNA preparations isolated from lymphoid tissues of normal or immunized guinea pigs with bovine MBP are referred to as N-RNA or MBP-RNA, respectively. It was also found that MBP-RNA is active in transferring MBP reactivity to normal splenocytes whereas N-RNA had no effect. To investigate whether this transfer to MBP immunoreactivity could result in a protective immunity, C57BL/6 mice transplanted with B16 melanoma received normal splenocytes treated with N-RNA or MBP-RNA. Two weeks after injection of B16-F10 cells, the mice were sacrificed and the tumor of each animal was removed and weighed. A significant inhibition of B16 melanoma growth was only achieved in C57BL/6 mice treated by splenocytes incubated with MBP-RNA which acts as an anti-tumor RNA. In this context, MBP could be considered as a tumor antigen.
Recent reports that myelin basic protein (MBP) can be ADP-ribosylated and contains specific sites that bind GTP and GM1 ganglioside, have suggested an analogy to the properties of cholera toxin. Comparisons of pairs of sequences between these two proteins yielded two regions of homology between MBP and the cholera toxin B (chol B) subunit, and one region of homology with the cholera toxin A (chol A) subunit. The matching sites within chol B consisted of a 17 amino acid residue sequence (residues 30-46 in chol B and residues 102-118 in human-MBP, hMBP, p less than 0.0007) and an 11 residue span (residues 31-41 in chol B and sequence 29-39 in hMBP, p less than 0.0004). The homologous site within chol A corresponded to an 11 residue span (residues 130-140 in chol A and 67-77 in hMBP sequence, p less than 0.00007). Since portions of the cholera toxin sequence are virtually identical to sections of the sequence in E. coli toxin, the homology is also valid for the same sequences in this toxin. The highly antigenic behavior of MBP that is related to the induction of experimental allergic encephalomyelitis may be paralleled by comparable neural pathology from the homologous regions of cholera toxin.
Myelin basic protein (MBP) is a commonly used substrate for in vitro determination of numerous protein kinase activities. Herein we describe a rapid method for isolating relatively large amounts of MBP from bovine brain with a purity greater than that currently available from commercial sources. Lipids were first extracted from the CNS tissue by homogenization in sec-butanol. Washes under neutral and mildly basic conditions were employed to remove neutral and acidic proteins from the defatted residue. MBP was subsequently extracted under acidic conditions and further purified by chromatography on CM Sephadex C-25. Potential contaminating enzyme activities were destroyed by heart treatment. This method typically yields a recovery of 1.0-1.5 mg MBP per gram of starting material with a purity of greater than 95%. The MBP prepared in this manner was suitable for determination of kinase activities by both solution and the "in gel" kinase assay systems.
The mouse myelin basic protein promoter was transcribed in brain nuclear extracts. The distal promoter region from -253 to -54 directed preferential transcription in brain extracts, whereas the same region repressed transcription activity in liver extracts. Stimulation of transcription was observed when the distal region was located only in a native orientation. The proximal region downstream from -53 alone still directed preferential transcription. It is suggested that cooperative function by the two promoter regions may be required for higher specificity.
Two preparations of myelin basic protein (MBP) were derived from an acid excretion of chloroform-methanol defatted bovine spinal cord. The first was purified by ion-exchange chromatography using guanidine-HCl; the second, by high performance liquid chromatography (HPLC) using a triethylamine eluant. Both methods of preparation yield MBP which is identical on acid-urea polyacrylamide gel electrophoresis and which has identical encephalitogenic potency. Because of the greater time-efficiency of the HPLC system with no deleterious side effects due to buffer contamination, this latter method can be recommended for MBP purification.
Rabbit myelin basic protein (BP) contains several Arg-X bonds with differing susceptibilities to thrombic cleavage as measured by the yields of the various cleavage products obtained under three different conditions. Under conditions where the thrombin-to-substrate ratio was very low (1 NIH unit/mg BP), the concentration of substrate was relatively low (4 mg BP/ml), and the incubation time was short (2 h), the rabbit BP was cleaved essentially completely and specifically at a single site, the Arg(95)-Thr(96) bond. The BPs of other species (beef, pig, guinea pig, rat) were similarly cleaved, no doubt because all have the same amino acid sequence in this region of the protein. Under conditions in which the enzyme-to-substrate ratio and the substrate concentration were higher (2 NIH units/mg BP, 8 mg BP/ml) and the incubation time was long (24 h), additional, partial cleavages occurred, principally at the Arg(43)-Phe(44) and Arg(128)-Ala(129) bonds, but with some cleavage at the Arg(31)-His(32) and Arg(63)-Thr(64) bonds as well. Under conditions in which all three variables were elevated (5 NIH units/mg peptide, 20 mg peptide/ml, 24 h), more extensive cleavage occurred at the above sites. In peptide (96-168), which we examined in detail, nearly complete cleavage of the Arg(128)-Ala(129) bond occurred, with partial cleavage at the unmethylated Arg(105)-Gly(106), Arg(111)-Phe(112), Arg(150)-Leu(151), and Arg(160)-Ser(161) bonds. The susceptibilities to cleavage of the Arg-X bonds in the BP can be explained with varying degrees of success in terms of the known specificity of thrombin. Cleavage of two of the bonds, Arg(128)-Ala(129) and Arg(160)-Ser(161), suggests the occurrence of a chain reversal or beta-turn in the sequence preceding the scissile bonds. Most cleavages of the BP with thrombin do not occur in the more hydrophobic regions; in particular, the hydrophobic region in the center of the molecule that includes the Phe-Phe(87-88) sequence is left intact.
In this study myelin basic protein (MBP) was tested for its effect on chemotaxis of human peripheral blood leukocytes (PBL) and neutrophils from multiple sclerosis (MS) patients. MBP appeared to inhibit specifically the formyl-methionyl-leucyl-phenylalanine (FMLP)-induced chemotaxis of both the total leukocyte population and neutrophils. In comparison, no inhibition of chemotaxis was observed in healthy donors and patients with other neurological diseases. From MS patients we collected neutrophil supernatants obtained by incubation of the cells in a serum-free medium at 37 degrees C for 60 min and evaluated their effect on chemotaxis of neutrophils from healthy donors. Chemotaxis of healthy donor neutrophils was inhibited specifically in the presence of MBP after treatment with these supernatants, presumably relating to the presence of immune complexes on the surface of neutrophils from MS patients. Those complexes can be eluted into the incubation medium and coat healthy donor neutrophils, thus arming them specifically.
The interaction between myelin basic protein (MBP) and G-actin was studied under nonpolymerizing conditions, i.e.,2mM HEPES, pH 7.5, 0.1 mM CaCl2 and 0.2 mM ATP. Fluorescence studies using pyrenyl-actin and the measurements of ATP hydrolysis rate show that MBP induces changes in the structure of the actin monomer similar to those occurring during polymerization by salt. Electron microscope observations of the MBP-G-actin complex reveal the presence of filamentous structures which appear as separate filaments or as bundles of filaments in lateral association. These filaments are polar as visualized by attachment of heavy meromyosin. The biochemical data together with electron microscope observations suggest that the binding of MBP to G-actin under non-polymerizing conditions induces an interaction between actin monomers leading to the formation of filamentous structures which may be similar to F-actin filaments. The effects of MBP on G-actin can be reversed by calmodulin in the presence of Ca2+.