Substrate specificity for myelin basic protein-specific protein methylase I.

The substrate specificity of bovine brain myelin basic protein (MBP)-specific protein methylase I (S-adenosyl-L-methionine:protein-L-arginine N-methyltransferase, EC 2.1.1.23), which methylates arginine residues of protein, has been studied using various MBPs, several synthetic peptides and heterogeneous nuclear ribonucleoprotein complex protein (hnRNP). (1) Among MBPs from different species of brain, the carp MBP was found to be the best substrate for MBP-specific protein methylase I. This high degree of methyl acceptability is most likely due to the fact that carp MBP is not in vivo methylated at the arginine residue (Deibler, G.E. and Martenson, R.E. (1973) J. Biol. Chem. 248, 2387-2391) and that the methylatable amino acid sequence is present in this protein. (2) In order to study the minimum chain length of MBP polypeptide which functions as the methyl acceptor, several synthetic polypeptides whose sequences are identical to the region surrounding the residue 107 of bovine MBP (the in vivo methylation site) were synthesized. It was found that the hexapeptide, Gly-Lys-Gly-Arg-Gly-Leu (corresponding to residues 104-109 of bovine MBP), was the shortest methyl accepting peptide, while the tetrapeptide, Gly-Arg-Gly-Leu (corresponding to residues 106-109) was inactive as a substrate. (3) hnRNP protein is known to contain methylarginine at residue 193 (Williams, K.R., Stone, K.L., LoPresti, M.B., Merrill, B.M. and Plank, S.R. (1985) Proc. Natl. Acad. Sci. USA 82, 5666-5670) which is post-translationally modified. Thus, the RNP protein overproduced in Escherichia coli and therefore did not contain methylarginine was examined for its methyl acceptability. It was found that neither MBP-specific nor histone-specific protein methylase I could methylate this methylarginine-less RNP protein. This suggests a possible existence of a distinct protein methylase I specific for this nuclear protein.     

Expression of a myelin basic protein gene in transgenic shiverer mice: correction of the dysmyelinating phenotype

Mice homozygous for the autosomal recessive mutation shiverer (shi) lack myelin basic protein (MBP) and exhibit a distinct behavioral pattern including tremors (shivering), convulsions, and early death. We have previously demonstrated that shiverer mice have a partial deletion in the gene encoding MBP. We now have introduced the wild-type MBP gene into the germ line of shiverer mice by microinjection into fertilized eggs. Transgenic shiverer mice homozygous for the introduced gene have MBP mRNA and protein levels that are approximately 25% of normal, and produce compacted myelin with major dense lines. Correct temporal and spatial expression of the MBP gene is achieved with a genomic MBP cosmid clone containing 4 kb of 5' flanking sequence and 1 kb of 3' flanking sequence. Moreover, the four different forms of MBP produced by alternative patterns of RNA splicing are present. These homozygous transgenic shiverer mice no longer shiver nor die prematurely.     

Myelin basic protein inhibits histone-specific protein methylase I

Bovine brain myelin basic protein, free of associated proteolytic activity, was found to be a specific inhibitor of histone-specific protein methylase I (S-adenosyl-L-methionine:protein-L-arginine N-methyltransferase, EC 2.1.1.23) purified from bovine brain. 50% of the methyl group incorporation into the histone substrate catalyzed by the methylase I was inhibited by myelin basic protein at a concentration of 0.326 mM. However, neither of the peptide fragments (residues 1-116 and residues 117-170) generated by the chemical cleavage of myelin basic protein at the tryptophan residue retained the inhibitory activity for histone-specific protein methylase I. Proteins such as gamma-globulin, bovine serum albumin, bovine pancreatic ribonuclease and polyarginine did not exhibit significant inhibitory activity toward the enzyme. The Ki value for myelin basic protein was estimated to be 3.42 X 10(-5) M for histone-specific protein methylase I and the nature of the inhibition was uncompetitive toward histone substrate.     

Clonal diversity of myelin basic protein-specific T lymphocytes

A panel of myelin basic protein (MBP)-specific, class II major histocompatibility complex (A^s)-restricted T-cell clones were established from SJL/J mice. Three clonotypes, based on their responses to guinea pig MBP and its peptide fragments, were observed. Clonotype I cells, represented by clones HS.6, HS.D2, HS.8, HS.E10, and HS.C1, were reactive to the encephalitogenic C-terminal fragment of MBP, amino acid residues 89–169. Clonotype II, represented by clone HS.E3, was reactive to fragments containing residues 43–88, and clones HS.D12 and HS.C7, representing clonotype III cells, responded to the whole molecule only. Three clones from clonotype I were capable of transferring both clinical and histological signs of experimental allergic encephalomyelitis (EAE) into naive mice. Southern blot analysis of T-cell receptor -chain genes using J 1- and J 2-specific probes showed that the rearrangement pattern was unique in each of the clones. These results suggest that the development of EAE may represent an autoaggressive polyclonal T-cell response.     

Epigenetic factors up-regulate expression of myelin proteins in the dysmyelinating jimpy mutant mouse

Proteolipid protein (PLP) is a major structural component of central nervous system (CNS) myelin. Evidence exists that PLP or the related splice variant DM-20 protein may also play a role in early development of oligodendrocytes (OLs), the cells that form CNS myelin. There are several naturally occurring mutations of the PLP gene that have been used to study the roles of PLP both in myelination and in OL differentiation. The PLP mutation in the jimpy (jp) mouse has been extensively characterized. These mutants produce no detectable PLP and exhibit an almost total lack of CNS myelin. Additionally, most OLs in affected animals die prematurely, before producing myelin sheaths. We have studied cultures of jp CNS in order to understand whether OL survival and myelin formation require production of normal PLP. When grown in primary cultures, jp OLs mimic the relatively undifferentiated phenotype of jp OLs in vivo. They produce little myelin basic protein (MBP), never immunostain for PLP, and rarely elaborate myelin-like membranes. We report here that jp OLs grown in medium conditioned by normal astrocytes synthesize MBP and incorporate it into membrane expansions. Some jp OLs grown in this way stain with PLP antibodies, including an antibody to a peptide sequence specific for the mutant jp PLP. This study shows that: (1) an absence of PLP does not necessarily lead to dysmyelination or OL death; (2) OLs are capable of translating at least a portion of the predicted jp PLP; (3) the abnormal PLP made in the cultured jp cells is not toxic to OLs. These results also highlight the importance of environmental factors in controlling OL phenotype. © 1996 John Wiley & Sons, Inc.     

Studies of posttranslational modifications in spiny dogfish myelin basic protein

The objective of this investigation was to determine whether nonmammalian myelin basic protein contained charge isomers resulting from extensive posttranslational modifications as seen in mammalian MBP. Four charge isomer components from dogfish MBP have been isolated. These forms arise by phosphorylation and deamidation modifications. Components C1, C2 and C3 have been characterized. We are currently characterizing component C8. Dogfish MBP is less cationic than mammalian MBP and has about 50% lower mobility on a basic pH gel electrophoresis relative to human and to bovine MBP. The mammalian component C1, which is unmodified, is modified in the dogfish by phosphorylation. The reduced electrophoretic mobility is largely attributable to the charge reduction resulting from phosphorylation in serine 72, 83, and 120 or 121 in C1, and C3. In component C2, two or three phosphate groups were distributed among residues 134, 138 and 139. It was found that dogfish amino acid residue 30 was a lysine residue and not a glutamate residue as reported in the literature.     

A myelin basic protein-specific T lymphocyte line that mediates experimental autoimmune encephalomyelitis

A T lymphocyte line, BP-1, expressing the T helper phenotype was selected from Lewis rats immunized with guinea pig myelin basic protein (GP-BP) in complete Freund's adjuvant (CFA). The BP-1 line responded specifically to GP-BP but not to PPD after the first round of selection, and responded to rat but not human or bovine BP. When injected i.p. into histocompatible Lewis or F1 (Lewis X P2) recipients, the BP-1 line induced both clinical signs of experimental autoimmune encephalomyelitis (EAE) and delayed type hypersensitivity (DTH) reactions in ears challenged intradermally with GP-BP but not PPD. The severity of clinical signs and the degree of ear swelling were dependent on the dose of BP-1 cells injected. Both activities were detectable with as few as 0.1 X 10(6) BP-1 line cells and required prior activation of the line cells with GP-BP presented by accessory cells. Lewis rats that had recovered from EAE induced by injection of GP-BP in CFA were more susceptible than naive rats to BP-1 line-mediated disease, requiring as few as 0.03 X 10(6) line cells. Clinical EAE and DTH could be serially transferred into F1 (Lewis X P2) recipients with BP-1 cells and back to nonirradiated Lewis parents with activated splenocytes, suggesting that BP-1 cells persist in recipient rats. These results demonstrate the potent biologic activities of an autoreactive BP-specific T lymphocyte line. This line possesses properties similar to BP lines described previously as well as to culture-conditioned splenic T effector cells; thus, the data presented here bridge the gap between these two approaches for studying T effector lymphocyte functions.     

Characterization of cloned cDNA representing rat myelin basic protein: absence of expression in brain of shiverer mutant mice

A cDNA library was constructed from mRNA isolated from the brains of 18-day-old rats, the age at which myelin biosynthesis is maximal. A synthetic DNA probe synthesized based on reverse translation of the amino acid sequence of rat myelin basic protein (MBP) was used to select two cDNA clones encoding MBP. A 1.5 kb Eco RI fragment from one clone was completely sequenced. When translated, a portion of this sequence was identical at 126 of 127 positions with the reported amino acid sequence for small MBP from the rat. Brains from mice of the homozygous shiverer genotype contained neatly reduced amounts of MBP mRNA relative to wild type. A deletion of MBP sequences in the genome of shiverer mice was also demonstrated. cDNAs for MBP will allow molecular investigation of the role this gene plays in both dysmyelinating and demyelinating diseases, as well as questions of MBP biosynthesis.     

Phosphorylation on basic amino acids in myelin basic protein.

Isolated rat brain myelin when incubated with γ³²P labelled ATP yields proteins bearing acid labile, base stable phosphoryl groups. Phosphorylated myelin basic protein can be isolated and degraded with trypsin and pronase to yield principally phosphoarginine and phosphohistidine. Only a very small amount of phosphorerine survives the base treatment used in the isolation procedure.     

D-aspartic acid in purified myelin and myelin basic protein

The presence of the biologically uncommon D-isomer of aspartic acid in the white matter of human brains has been reported previously from this laboratory (1). We now report that the level of D-aspartate in human brains is higher in purified myelin than in white matter and is even higher in the myelin basic protein fraction. There also appears to be a difference in the level of D-aspartate found in human brain as compared to bovine brain, possibly a species or age-related difference.     

Detection of various forms of brain myelin basic protein in vertebrates by electroimmunoblotting

An electroimmunoblot technique was used to detect various forms of myelin basic protein (MBP) in brain homogenates of 14 vertebrate species. Three antibodies were used to probe the immunoblots: a monoclonal anti-human MPB reacting with an antigenic determinant located at amino acid residues 131 to 136; a polyclonal anti-human MBP and a polyclonal anti-chicken MBP. Because no processing of the tissue is required prior to electrophoresis, in vitro artifacts are minimized. The 18.5 K form of MBP was present in all species except the shark. A 21.5 K MBP was observed in ovine, bovine, pig, rabbit, mouse, rat, monkey, but not in human, guinea pig, shark, toad and marsupial brains. A variant with a molecular weight between 17 K and 18 K was found in mouse, rat, bovine, human, monkey, pig, and chicken brains, and was the sole component in the shark brain. Marsupial brains had five or six forms of MBP between 14.5 K and 18.5 K.Special Issue dedicated to Dr. Elizabeth Roboz-Einstein.This research was supported by grants from the National Multiple Sclerosis Societies of Australia and U.S.A., National Health and Medical Research Council and by the Jack Brockhoff Foundation of Australia. D.S.L. is a scholar of the Leukemia Society of America.     

Myelin deficient mice: expression of myelin basic protein and generation of mice with varying levels of myelin

Mice homozygous for the mutation myelin deficient (mld), an allele of shiverer, exhibit decreased CNS myelination, tremors, and convulsions of progressively increasing severity leading to an early death. In this report we demonstrate in mld mice that the gene encoding myelin basic protein (MBP) is expressed at decreased levels and on an abnormal temporal schedule relative to the wild-type gene. Southern blot analyses, field-inversion gel electrophoresis studies, and analyses of mld MBP cosmid clones indicate that there are multiple linked copies of the MBP gene in mld mice. We have introduced an MBP transgene into mld mice and found that myelination increases and tremors and convulsions decrease. Mld and shiverer mice with zero, one, or two copies of the MBP transgene express distinct levels of MBP mRNA and myelin. The availability of a range of mice expressing graded levels of myelin should facilitate quantitative analysis of the roles of MBP in the myelination process and of myelin in nerve function.     

Brain nucleic acids and protein in various neurological mutant mice

A study was made to compare alterations in the cerebral contents of nucleic acids and protein of several mouse strains affected by different neurological mutations: jimpy, msd, quaking, reeler, weaver, and dwarf. In normal and affected jimpy and msd mice the brain components analyzed were very similar. On the other hand, the cerebral hemispheres of quaking mice showed significant decreases in total RNA and DNA, when compared with those of normal littermates. In the affected reeler and weaver mice, total protein, RNA, and DNA in the cerebellum differed markedly from controls. Protein decreased slightly, whereas nucleic acids showed no significant variation in the cerebral hemispheres of the same mutants. The cerebella and cerebral hemispheres of affected dwarf mice had wet weights and total protein contents that were about 20% lower than those of their controls; DNA did not vary significantly in the various brain regions analyzed. The decrease of DNA we report in reeler and weaver mutant cerebellum in toto quantifies the lack of cell number, in contrast to histological studies which give only semiquantitative information.     

Cross-reactivity of myelin basic protein-specific T cells with multiple microbial peptides: experimental autoimmune encephalomyelitis induction in TCR transgenic mice.

Activation of autoreactive T cells is a crucial event in the pathogenesis of autoimmune diseases. Cross-reactivity between microbial and self Ags (molecular mimicry) is one hypothesis that could explain the activation of autoreactive T cells. We have systematically examined this hypothesis in experimental autoimmune encephalomyelitis using mice bearing exclusively myelin basic protein (MBP)-specific T cells (designated T+ alpha-). A peptide substitution analysis was performed in which each residue of the MBPAc1-11 peptide was exchanged by all 20 naturally occurring amino acids. This allowed the definition of the motif (supertope) that is recognized by the MBPAc1-11-specific T cells. The supertope was used to screen protein databases (SwissProt and TREMBL). By the search, 832 peptides of microbial origin were identified and synthesized. Of these, 61 peptides induced proliferation of the MBPAc1-11-specific transgenic T cells in vitro. Thus, the definition of a supertope by global amino acid substitution can identify multiple microbial mimic peptides that activate an encephalitogenic TCR. Peptides with only two native MBP-residues were sufficient to activate MBPAc1-11-specific T cells in vitro, and experimental autoimmune encephalomyelitis could be induced by immunizing mice with a mimic peptide with only four native MBP residues.     

Cell-mediated immunoreactivity of tumor-bearing mice with myelin basic protein and related peptides

Spleen cells (SC) from tumor-bearing mice and mice immunized with porcine myelin basic protein (MBP) reacted in vitro in E-rosette augmentation assays with MBP and certain of its constituent peptides. Peptides 1-115, 43-169, 64-83, 113-121, and 153-161 reacted significantly with both types of SC, while peptide 1-19 reacted only with SC from MBP-immunized mice. The phenomenon of specific inhibition of peptide reactivity by a moderate excess of a related protein was used to identify peptides as accessible epitopes of that protein. Peptide 113-121 was specifically inhibited by excess MBP when reacted with both types of SC, whereas peptide 64-83 was inhibited by excess MBP only when reacted with SC from MBP-immunized mice. These reactions suggest that the immunizing antigen in tumor-bearing mice is related to MBP but differs in epitopes associated with peptides 1-19 and 64-83.     

Lipid and basic protein interaction in myelin

1. Purified myelin labelled with [(3)H]myo-inositol or [1-(14)C]acetate was incubated with trypsin or acetylated trypsin at 37 degrees C, pH8.0 for 30min. 2. After incubation and centrifugation analysis of the myelin pellet showed marked digestion of basic protein on polyacrylamide-gel electrophoresis. Proteolipid and Wolfgram proteins remained unchanged. 3. A loss of 15% of total protein and loss of all classes of lipids was also found. Most significant lipid losses were phosphoinositides, phosphatidylserine and sulphatide. 4. A low-density material containing more phospholipid than cholesterol and galactolipid was isolated from the supernatant obtained after centrifugation of trypsin-treated myelin. 5. Interaction of sulphatide and myelin basic protein was shown to take place in a biphasic system. Basic protein does not form any complex either with cerebroside or cholesterol in the same solvent system. 6. The release of acidic lipids from myelin suggests that they may be linked to basic protein by ionic forces and the neutral lipids may be by lipid-lipid interactions. 7. The relevance of these studies as a model of brain degeneration is discussed.