Endogenous Basic Protein Phosphatases in the Brain Myelin

Direct treatment of brain myelin with freezing/thawing in 0.2 M 2-mercaptoethanol stimulated the endogenous myelin phosphatase activity manyfold when 32P-labeled phosphorylase a was used as a substrate, a result indicating that an endogenous myelin phosphatase is a latent protein phosphatase. When myelin was treated with Triton X-100, this endogenous latent phosphatase activity was further stimulated 2.5-fold. Diethylaminoethyl-cellulose and Sephadex G-200 chromatography of solubilized myelin revealed a pronounced peak of protein phosphatase activity stimulated by freezing/thawing in 0.2 M 2-mercaptoethanol and with a molecular weight of 350,000, which is characteristic of latent phosphatase 2, as previously reported. Moreover, endogenous phosphorylation of myelin basic protein (MBP) in brain myelin was completely reversed by a homogeneous preparation of exogenous latent phosphatase 2. By contrast, under the same conditions, endogenous phosphorylation of brain myelin was entirely unaffected by ATP X Mg-dependent phosphatase and latent phosphatase 1, although both enzymes are potent MBP phosphatases. Together, these findings clearly indicate that a high-molecular-weight latent phosphatase, termed latent phosphatase 2, is the most predominant phosphatase responsible for dephosphorylation of brain myelin.     

Immunoblot Identification of 13.5 Kilodalton Myelin Basic Protein in Goldfish Brain Myelin

Myelin isolated from goldfish brain shows a multilamellar structure with a major dense line and two intraperiod lines. Sodium dodecyl sulfate gel electrophoresis revealed that the protein profile of goldfish brain myelin is distinctly different from that of rat brain myelin. No protein migrating to the position of proteolipid protein or DM-20 was seen in goldfish myelin. Goldfish acclimated to 5 degrees, 15 degrees, and 30 degrees C showed no qualitative differences in myelin proteins. The 13.5 kD protein in goldfish brain myelin and brain homogenate was intensely immunostained with the antiserum to human basic protein by the immunoblot technique. In contrast, none of the proteins of goldfish myelin were immunostained with antiproteolipid protein serum; however, both proteolipid protein and DM-20 of rat brain myelin were immunostained. The significance of the synthesis of myelin proteins by astrocytes in the goldfish brain is discussed.     

Myelin Basic Protein Is a Zinc-Binding Protein in Brain: Possible Role in Myelin Compaction

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.     

ADP-Ribosylation of Human Myelin Basic Protein

Abstract: When isolated myelin membranes were ADP-ribosylated by [³²P]NAD⁺ either in the absence of toxin (by the membrane ADP-ribosyltransferase) or in the presence of cholera toxin, the same proteins were ADP-ribosylated in both cases and myelin basic protein (MBP) was the major radioactive product. Therefore, cholera toxin was considered a good model for ADP-ribosylation of myelin proteins. Although purified human MBP migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 20 kDa, the microheterogeneity that is masked under these conditions can be clearly demonstrated on alkaline-urea gels at pH 10.6. At this pH, MBP is resolved into several components that differ one from the other by a single charge (charge isomers). These charge isomers can be resolved on CM52 columns at pH 10.6, and several can be ADP-ribosylated. Component 1 (C-1), the most cationic charge isomer, incorporated 1.79 mol of ADP-ribose/mol of protein. C-2 and C-3 (which differ from C-1 by the loss of one and two positive charges, respectively) incorporated slightly less at 1.67 and 1.63 mol of ADP-ribose/mol of protein, respectively, whereas C-8, the least cationic, incorporated less than 0.11 mol/mol of protein. In the presence of neutral hydroxylamine, the ADP-ribosyl bond was shown to have a half-life of about 80 min, suggesting an N-glycosidic linkage between ADP-ribose and an arginyl residue of the protein. As MBP contains several components that are ADP-ribosylated to different specific activities, the use of MBP, ADP-ribosylated in the natural membrane, to identify the sites involved would yield a mixture of peptides difficult to resolve. Therefore, to identify the sites ADP-ribosylated, an endoproteinase Lys-C digest of C-1 ADP-ribosylated by cholera toxin was prepared. Two radioactive peptides were isolated by reversed-phase HPLC. Amino acid and sequence analyses identified the radioactive peptides as residues 5–13 and 54–58 of the human sequence (sp. act., 0.89 and 0.62 nmol of ADP-ribose/nmol of peptide, respectively). The ADP-ribosylated residues were identified as Arg⁹ and Arg⁵⁴ by automated and manual Edman sequencing. Taken together with our previous observation that MBP binds GTP at a single site, these data suggest that MBP functions as part of a signal transduction system in myelin.     

Intracellular Translocation of Myelin Proteolipid Protein

Brainstem slices prepared from 22-day-old rats were employed to study the intracellular translocation of radioactively labeled myelin proteolipid protein (PLP). Double-isotope and short pulse-chase procedures allowed us to demonstrate the flux of PLP through nine different subcellular membrane fractions that were isolated on the basis of their particle size and buoyant density. Tagged PLP was rapidly depleted from microsomes, showed transient passage through a number of presumably intermediate membranous pools, and accumulated in myelin. On the basis of the kinetics of PLP labeling and isotope ratios, the membranes can be arranged as they participate in the intracellular translocation of PLP and consistently show a pattern indicating possible precursor-product relationships.     

Electron Microscopic Immunocytochemical Localization of Myelin Proteolipid Protein and Myelin Basic Protein to Oligodendrocytes in Rat Brain During Myelination

Electron microscopic immunocytochemical studies were carried out to localize myelin basic protein and myelin proteolipid protein during the active period of myelination in the developing rat brain using antisera to purified rat brain myelin proteolipid protein and large basic protein. The anti-large basic protein serum was shown by the immunoblot technique to cross-react with all five forms of basic protein present in the myelin of 8-day-old rat brain. Basic protein was localized diffusely in oligodendrocytes and their processes at very early stages in myelination. The immunostaining for basic protein was not specifically associated with any subcellular structures or organelles. The ultrastructural localization of basic protein suggests that it may be involved in fusion of the cytoplasmic faces of the oligodendrocyte processes during compaction of myelin. Immunoreactivity in the oligodendrocyte and myelin due to proteolipid protein appeared at a later stage of myelination than did that due to basic protein. Staining for proteolipid protein in the oligodendrocyte was restricted to the membranes of the rough endoplasmic reticulum, the Golgi apparatus, and apparent Golgi vesicles. The early, uncompacted periaxonal wrappings of oligodendrocyte processes were well stained with antiserum to large basic protein whereas staining for proteolipid protein was visible only after the compaction of myelin sheaths had begun. Our evidence indicates that basic protein and proteolipid protein are processed differently by the oligodendrocytes with regard to their subcellular localization and their time of appearance in the developing myelin sheath.     

Metalloendoprotease Cleavage of 18.2- and 14.1-Kilodalton Basic Proteins Dissociating from Rodent Myelin Membranes Generates 10.0- and 5.9-Kilodalton G-Terminal Fragments

Rat and guinea pig myelin membranes were incubated at physiological ionic strength with millimolar concentrations of Ca2+/Mg2+ ions (37 degrees C; pH 7.4). After 1-3 h, electrophoresis of the membranes revealed loss of 50% of 18.2- and 14.1-kilodalton (kDa) forms of myelin basic protein (MBP). Concomitantly, peptides representing 25% of the original membrane-associated MBP were detected in incubation media. Roughly equal amounts of MBP fragments with molecular masses of 10.0 and 8.4 kDa were found in media from guinea pig myelin incubations. Media from rat myelin experiments contained a major 8.4-kDa and minor 10.0- and 5.9-kDa MBP peptides. Kinetic studies implied that proteolysis occurred subsequent to MBP dissociation from the membranes. Immunoblotting studies indicated that both the 18.2- and 14.1-kDa forms of MBP were cleaved near residue 73 to produce a 10.0- and 5.9-kDa C-terminal fragment, respectively. Degradation of MBP in myelin membranes was partially inhibited by only 5-20% using leupeptin (20 microM) but up to 50% by dithiothreitol mM), phenylmethylsulphonyl fluoride (1 mM), and phosphoramidon (50 microM) but up to 50% by dithiothreitol (DDT, 10 mM). Only DDT and 1,10-phenanthroline substantially blocked the formation of the characteristic 10.0-and 5.9-kDa C-terminal fragments. This suggests that MBP, dissociating from myelin membrane preparations, is cleaved near residue 73 by a metalloendoprotease distinct from N-ethylmaleimide/leupeptin-sensitive calpains and phosphoramidon-sensitive endopeptidase 24.11.     

A New Form of Myelin Basic Protein Found in Human Brain

Human myelin basic protein was subjected to ion-exchange chromatography at high pH to separate the differently charged components. Polyacrylamide gel electrophoretic patterns of the fractions showed that the less basic fractions 3, 4, and 5 contained significant amounts of a protein somewhat smaller than the more common 18.5-kDa form. Fraction 3 consisted of approximately equal amounts of this smaller polypeptide and component 3, the 18.5-kDa form found in other mammalian myelin basic protein preparations. The two proteins in fraction 3 were separated by fast protein liquid chromatography. Both have blocked N termini and identical C termini (-Met-Ala-Arg-Arg). When the tryptic digests of the two proteins were fractionated by HPLC, the elution profiles were similar, except that four peaks found in the chromatogram of the larger protein were missing from the chromatogram of the smaller one. In addition, an extra peak was found in the elution pattern of the latter chromatogram. Amino acid analysis of the individual tryptic peptides indicated that the smaller protein lacked residues 106-116 (-Gly-Arg-Gly-Leu-Ser-Leu-Ser-Arg-Phe-Ser-Trp-). The deleted portion corresponds exactly to the amino acid sequence encoded by exon 5 of the mouse basic protein gene. This new form of myelin basic protein has a molecular weight of 17,200, calculated from its amino acid composition.     

Presence of Proteolipid Protein in Coelacanth Brain Myelin Demonstrates Tetrapod Affinities and Questions a Chondrichthyan Association

The protein and glycoprotein compositions of CNS myelin from the living coelacanth (Latimeria chalumnae) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An unglycosylated component of 25 kilodaltons showed substantially stronger immunoblot reactivity with antibodies against mammalian proteolipid protein (PLP) than lungfish glycosylated PLP. DM-20 (intermediate protein) was not detectable in either fish. The presence of unglycosylated PLP in CNS myelin of the actinistian coelacanth contradicts an association with cartilaginous fishes but supports tetrapod affinities closer than those of lungfish.     

人脑髓鞘碱性蛋白的分离纯化和鉴定

 本文介绍了从人脑中分离纯化髓鞘碱性蛋白的方法,人脑组织匀浆经甲醇—氯仿脱脂、酸提取、硫酸铵沉淀和羧甲基纤维素柱层析,得到了纯化的髓鞘碱性蛋白。该蛋白在SDS聚丙烯酰胺凝胶电泳中为单一带,分子量为21kD。在聚焦电泳中测得其等电点在pH10以上,氨基酸组成分析结果也与文献值接近。这为进一步研究人脑髓鞘碱性蛋白的抗原性创造了条件。     

Acylation of Rat Brain Myelin Proteolipid Protein with Different Fatty Acids

The acylation of rat brain proteolipid protein (PLP) with tritiated palmitic, oleic, and myristic acids was studied in vivo and in vitro and compared with the acylation of lipids. Twenty-four hours after intracranial injection of [3H]myristic acid, only 16% of the PLP-bound label appeared as myristic acid, with 66% as palmitic, 9% as stearic, and 6% as oleic acid, whereas greater than 63% of the label in total or myelin phospholipid was in the form of myristic acid. In contrast, after labelling with [3H]palmitic or oleic acids, 75% and 86%, respectively, of the radioactivity in PLP remained in the original form. When brain tissue slices were incubated for short periods of time, the incorporation of palmitic and oleic acids into PLP exceeded that of myristic acid by a factor of 8. In both systems and with all precursors studied, the label associated with PLP was shown to be in ester linkage. The results suggest a preferential acylation of PLP with palmitic and oleic acids as compared with myristic acid. This is consistent with the fatty acid composition of the isolated PLP.     

A Rapid Method for Purification of Myelin Basic Protein

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

An Antibody Specific for Component 8 of Myelin Basic Protein from Normal Brain Reacts Strongly with Component 8 from Multiple Sclerosis Brain

Myelin basic protein (MBP) consists of several components or charge isomers (C-1 through C-8) generated by one or a combination of posttranslational modifications. One of these, C-8, has been shown to contain citrulline (Cit) at defined sites formed by deimination of six arginyl residues. This unusual modification has allowed us to raise antibodies specific for this charge isomer only. To do this, a synthetic peptide, Gly-Cit-Cit-Cit-Cit, was coupled to keyhole limpet hemocyanin and injected into rabbits. The antibodies so generated reacted only with C-8 and not with any of the other charge isomers. A second antibody fraction was raised against the synthetic peptide ACitHGFLPCitHR naturally occurring between residues 24 and 33 of C-8 (all other charge isomers contain R instead of Cit at positions 25 and 31). These antibodies preferred C-8 but reacted with the other charge isomers, to the extent of approximately 25-30% of the reactivity shown with C-8. In studies with C-8 from multiple sclerosis (MS) MBP, much greater reactivity was obtained with these antibodies when compared with their reactivity with C-8 from normal MBP. Because the total number of Cit residues in C-8 from MS and normal MBP is the same, the difference in reactivity may be related to structural factors. The antibodies raised with the tetra-Cit peptide were reacted with three pairs of synthetic peptides: 24ARHGFLPRHR33 and ACitHGFLPCitHR; 120GQRPGFGYGGRAS132 and GQCitPGFGYGGCitAS; and 157GGRDSRSGSPMARR170 and GGCitDSRSGSPMACitR. They reacted only with the Cit-containing peptides in the order 157-170 greater than 120-130 greater than 24-33.(ABSTRACT TRUNCATED AT 250 WORDS)     

In Vivo Methylation of an Arginine in Chicken Myelin Basic Protein

Abstract: The amino acid sequence around the sole methylarginine residue in chicken myelin basic protein was determined and was found to be similar to that previously reported for mammalian myelin basic protein. The ratio N ^G, N '^G-dimethylarginine: N ^G-monomethylarginine:arginine was approximately 1.3:0.9:1.0. No N ^G, N ^G-dimethylarginine was detected in the protein. The in vivo incorporation of methyl groups from [methyl-³H]methionine into methylarginines in myelin was found to occur readily in 2-day-old chickens. Radioactively labelled N ^G, N '^G-dimeth-ylarginine and N ^G-monomethylarginine in myelin were derived solely from myelin basic protein. Radioactivity was also incorporated into N ^G, N ^G-dimeth-ylarginine, although this was not derived from myelin basic protein. As N ^G-monomethylarginine was easily separated from the dimethylarginines, and as it was derived from myelin basic protein, it may be a good marker for myelin basic protein turnover in vivo. A time course study of the incorporation showed that radioactivity was incorporated into N ^G-monomethylarginine up to 6 h after injection, and decayed slowly, with an apparent half-life of about 40 days.     

Preparation and Properties of an Immunosorbent Column Specific for the Myelin Basic Protein

Abstract: An immunosorbent column specific for the myelin basic protein (BP) was prepared by coupling purified anti-BP antibodies to cyanogen bromide (BrCN)-activated Sepharose 4B. The BP-immunosorbent column bound BP between pH 4.5 and pH 6.8. In its working range the column bound approximately 400-475 μg of BP at pH 6.8 and 250 μg at pH 4.5 with recoveries of 72-77%. The BP-immunosorbent column could effectively separate BP from simple mixtures of BP and proteins of similar size and charge and from acid extracts of bovine brain. The results indicate that the BP-immunosorbent column can be used to isolate BP from a mixture of proteins and may be adapted for use in the small-scale purification of the myelin basic proteins involving a minimum number of steps.     

Rearrangement and Reactivation of the Myelin Basic Protein Locus in Myelin-Deficient (mld) Mouse Brain

Abstract: Myelin-deficient ( mld ) is a complex mutation affecting the myelin basic protein (MBP) locus of the mouse. It consists of duplication and partial inversion of the MBP gene and results in a dysfunctional MBP locus. The mutant phenotype is reversed, both in vivo and in vitro, in ∼5% of mld oligodendrocytes. One possible mechanism for the somatic reversion is recombination between homologous sequences of the duplicated gene copies to reconstitute a functional MBP locus. There are several possible recombination events that could reconstitute a functional MBP locus by DNA rearrangement. Two of these would result in reinversion and circularization of specific MBP gene sequences, respectively. In this work polymerase chain reaction analysis was used to detect both reinverted and circularized MBP gene sequences in mld mouse tissues, indicating that DNA rearrangement at the MBP locus does occur. Analysis of individually harvested cells showed that in revertant MBP-positive mld oligodendrocytes DNA rearrangement at the MBP locus was correlated with reactivation of the MBP gene. Fluctuation analysis showed that reactivation of the MBP locus is a stochastic event occurring with a frequency of ∼1.4 × 10⁻⁶ per cell per cell cycle during oligodendrocyte development. The frequency of rearrangement and reactivation of the MBP locus was comparable in double mutant ( mld/mld , scid/scid ) and single mutant ( mld/mld , + ^scid /+ ^scid ) mice, indicating that the scid factor is not required for MBP gene reactivation in mld . The significance of DNA rearrangement in mammalian development is discussed.     

Multiple Sclerosis Brain Immunoglobulins Stimulate Myelin Basic Protein Degradation in Human Myelin: A New Cause of Demyelination

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.     

Myelin Basic Protein and Myelin Basic Protein Peptides Induce the Proliferation of Schwann Cells Via Ganglioside GM1 and the FGF Receptor

Myelin basic protein (MBP) and two peptides derived from MBP (MBP_1–44 and MBP_152–167) stimulated Schwann cell (SC) proliferation in a cAMP-mediated process. The two mitogenic regions of MBP did not compete with one another for binding to SC suggesting a distinctive SC receptor for each mitogenic peptide. Neutralizing antibodies to the fibroblast growth factor receptor blocked the mitogenic effect of the myelin-related SC mitogen found in the supernatant of myelin-fed macrophages. The binding of ¹²⁵I-MBP to Schwann cells was specifically inhibited by basic fibroblast growth factor (bFGF) and conversely the binding of ¹²⁵I-bFGF was competitively inhibited by MBP. These data suggested that the mitogenic effect of one MBP peptide was mediated by a bFGF receptor. The binding of MBP to ganglioside GM1 and the ability of MBP peptides containing homology to the B subunit of cholera toxin (which binds ganglioside GM1) to compete for the binding of a mitogenic peptide (MBP_1–44) to SC, identified ganglioside GM1 as a second SC receptor. Based on these results, we conclude that MBP_1–44 and MBP_152–167 associate with ganglioside GM1 and the bFGF receptor respectively to stimulate SC mitosis.     

Sites in Myelin Basic Protein that React with Monoclonal Antibodies

The epitopes (antigenic sites) for seven monoclonal antibodies (MAbs) evoked in rats or mice by guinea pig or monkey myelin basic protein (BP) have been located in four different sequences of the BPs extracted from various species. Six of the MAbs were evoked by guinea pig BP. (1) One epitope, possibly a pair, is included within residues 1-14 of all BPs tested and reacts with two rat IgG MAbs. (2) A definite pair of overlapping epitopes includes the central Phe91-Phe92 sequence. One epitope is contained entirely within sequence 90-99 and reacts with a rat IgG MAb. The substitution of Ser in chicken BP for Thr97 destroys this epitope. The other epitope appears to include residues on the amino side of Phe44 and even of His32 and suggests some tertiary structure in BP. This epitope reacts with a mouse IgM MAb that does not recognize the chicken substitution. (3) The third epitope lies within residues 114-121, specifically including Trp118, and reacts with a rat IgG MAb. A cross-reacting epitope probably includes residues 44-45 in certain species (guinea pig and bovine but not rabbit). (4) Another pair of epitopes is located within residues 131-140 but is severely species-restricted. This region in guinea pig BP evoked a species-specific mouse IgM MAb. The same region in monkey BP evoked the seventh MAb, a mouse IgG, which reacts with human, chimpanzee, monkey, bovine, and rat-18.5 kDa BPs and to a lesser extent rabbit BP but not with guinea pig, pig, or chicken BPs. Some tertiary structure in guinea pig BP is also suggested by the reactivities with the IgM MAb. All of the MAbs react with myelin in histologic preparations, but the optimum method of preparation of the tissue varies with each.     

Identification of the New Isoforms of Mouse Myelin Basic Protein: The Existence of Exon 5a

Myelin basic protein (MBP) is a major constituent in the myelin of the CNS. In mice, five forms of MBPs (14 kDa, two types of 17 kDa, 18.5 kDa, and 21.5 kDa) encoded by separate mRNAs have been identified based on cDNA cloning studies. These mRNAs are considered to be produced by alternative splicing from a single gene composed of seven exons. Here we report the existence of two novel MBP mRNAs encoding 19.7-kDa and 21-kDa MBPs identified by cDNA cloning using the polymerase chain reaction. Both of these MBPs contain a sequence of a previously unidentified exon of 66 nucleotides, which was mapped to be just 5' of exon 5 in the MBP gene. MBP mRNAs containing this novel exon (exon 5a) belong to a minor population in the whole brain and PNS and are somewhat enriched in the spinal cord. Exon 5a encodes a very hydrophobic segment rich in valine residues, which presumably forms a beta-pleated sheet.