Portal de Periódicos da CAPES

Multiple Sclerosis

2003; Elsevier BV; Volume: 2; Issue: 7 Linguagem: Inglês

10.1074/mcp.m200050-mcp200

1535-9484

Jeongkwon Kim, Fabrizio G. Mastronardi, D. D. Wood, David M. Lubman, Ramin Zand, M.A. Moscarello,

RNA Research and Splicing

Myelin basic protein (MBP) represents a candidate autoantigen in multiple sclerosis (MS). We isolated MBP from normal and MS human white matter and purified six components (charge isomers) to compare the post-translational modifications on each. The sites and extent of methylation, deimination, and phosphorylation were documented for all tryptic peptides by mass spectrometry. We found that mono and dimethylated arginine 107 was increased in MS samples; deimination of arginine occurred at a number of sites and was elevated in MS; phosphorylation was observed in 10 peptides in normal samples but was greatly reduced or absent in most peptides from MS samples. Data obtained with MBP isolated from fresh brain obtained from a spontaneously demyelinating mouse model supported the view that the changes observed in human brain were probably related to pathogenesis of demyelination, i.e. we found decreased phosphorylation and decreased amounts of glycogen synthesis kinase in brain homogenates using specific antibodies. This study represents the first to define post-translational modifications in demyelinating disease and suggest an important role in pathogenesis. Myelin basic protein (MBP) represents a candidate autoantigen in multiple sclerosis (MS). We isolated MBP from normal and MS human white matter and purified six components (charge isomers) to compare the post-translational modifications on each. The sites and extent of methylation, deimination, and phosphorylation were documented for all tryptic peptides by mass spectrometry. We found that mono and dimethylated arginine 107 was increased in MS samples; deimination of arginine occurred at a number of sites and was elevated in MS; phosphorylation was observed in 10 peptides in normal samples but was greatly reduced or absent in most peptides from MS samples. Data obtained with MBP isolated from fresh brain obtained from a spontaneously demyelinating mouse model supported the view that the changes observed in human brain were probably related to pathogenesis of demyelination, i.e. we found decreased phosphorylation and decreased amounts of glycogen synthesis kinase in brain homogenates using specific antibodies. This study represents the first to define post-translational modifications in demyelinating disease and suggest an important role in pathogenesis. Multiple sclerosis (MS) 1The abbreviations used are: MS, multiple sclerosis; MBP, myelin basic protein; GSK, glycogen synthesis kinase; DM20, myelin proteolipid protein isoform; ND4, transgenic mice containing 70 copies of DM20 cDNA; PLP, proteolipid protein. is the most common demyelinating disease of the human central nervous system. It is multifactorial, requiring genetic, environmental (possibly viral), and immunological factors (1Moscarello M.A. Myelin basic protein, the executive molecule of the myelin membrane.in: Devon R.M. Doucette R. Juurlink B.H.J. Nazarali A.J. Schreyer, D.J. Verge V.M.K. Cell Biology and Pathology of Myelin: Evolving Biological Concepts and Therapeutic Approaches. Plenum Press, New York1996: 13-25Google Scholar). Genetic screens of MS populations have failed to uncover a major susceptibility locus (2Chataway J. Feakes R. Coraddu F. Gray J. Deans J. Fraser M. Robertson N. Broadley S. Jones H. Clayton D. Goodfellow P. Sawcer S. Compston A. The genetics of multiple sclerosis: Principles, background and updated results of the United Kingdom systematic genome screen.Brain. 1998; 121: 1869-1887Google Scholar), suggesting that MS is a polygenic disease with each gene of small effect. Myelin basic protein (MBP), a major myelin protein that accounts for 35% of the total myelin protein, is a strong candidate autoantigen (3Steinman L. Multiple sclerosis: A two-stage disease.Nat. Immunol. 2001; 2: 762-764Google Scholar). It is a 170-amino acid protein in the human, containing 19 arginyl and 12 lysyl residues, which accounts for its basic character. It is devoid of cysteinyl residues, with a high proportion of disorder promoting amino acids such as A, R, G, Q, S, P, E, and K. It is a member of an expanding group of proteins that include the amyloid and prion proteins, considered to be intrinsically disordered (4Dunker K.A. Lawson J.A. Brown C.J. Williams R.M. Romero P. Intrinsically disordered protein.J. Mol. Graphics Model. 2001; 19: 26-59Google Scholar), in which the disordered state is the functional state. Because of this, post-translational modifications determine the nature and extent of secondary structure, permitting the protein to adopt multiple conformations for a variety of binding events (5Wright P.E. Dyson H.J. Intrinsically unstructured proteins: Re-assessing the protein structure-function paradigm.J. Mol. Biol. 1999; 293: 321-331Google Scholar). Because interactions between the positive arginyl and lysyl residues and the negatively charged phosphate groups of the membrane phospholipids are essential to the structure of compact myelin, changes in positive charge of MBP would decrease the strength of these interactions (6Cheifetz S. Moscarello M.A. Effect of bovine basic protein charge microheterogeneity on protein-induced aggregation of unilamellar vesicles containing a mixture of acidic and neutral phospholipids.Biochemistry. 1985; 24: 1909-1914Google Scholar). MBP is an unusual protein that has never been crystallized due to the myriad of post-translational modifications it possesses. These include phosphorylation, deamidation, deimination, arginine methylation, and N-terminal acylation. These give rise to a family of microheteromers or "charge isomers," several of which can be resolved by chromatography (7Chou F.C-H. Chou C-H-J Shapira R. Kibler R.F. Basis of microheterogeneity of myelin basic protein.J. Biol. Chem. 1976; 251: 2671-2679Google Scholar). The effects of some of these modifications on protein structure and protein-lipid interactions has been studied in our laboratories. Phosphorylation by protein kinase C in vitro resulted in a large increase in β-structure as determined by circular dichroism. Although several sites were phosphorylated by protein kinase C, phosphorylation at serine 7 was particularly important for the stabilization of the β-structure (8Ramwani J.J. Epand R.M. Moscarello M.A. Secondary structure of charge isomers of myelin basic protein before and after phosphorylation.Biochemistry. 1989; 28: 6538-6543Google Scholar). Strong interaction between arginine 5 and 9 with the phosphate on serine 7 was responsible for the stabilization of structure. Deimination of arginyl residues by the action of peptidylarginine deiminase (EC 7.5-3.15) generates citrulline, an important irreversible modification of MBP. In a series of studies, we demonstrated that 20% of the MBP isolated from normal human adults was citrullinated, compared with 45% in chronic MS and 90% in fulminating MS (9–11). In in vitro studies we showed that citrullinated MBP enhanced T cell responsiveness (12Tranquill L.R. Cao L. Ling N.C. Kalbacher H. Martin R.M. Whitaker J.N. Enhanced T-cell responsiveness to citrulline-containing myelin basic protein in multiple sclerosis patients.Multiple Sclerosis. 2000; 6: 220-225Google Scholar), altered conformation (11Beniac D.R Wood D.D. Palaniyar N. Ottensmeyer F.P. Moscarello M.A. Harauz G. Cryoelectron microscopy of protein-lipid complexes of human myelin basic protein charge isomers differing in degree of citrullination.J. Struct. Biol. 2000; 129: 80-95Google Scholar), and induced vesicle fragmentation (13Boggs J. Rangaraj G. Koshy K.M. Ackerley C. Wood D.D. Moscarello M.A. Highly deiminated isoform of myelin basic protein from multiple sclerosis brain causes fragmentation of acidic lipid vesicles.J. Neurosci. Res. 1999; 57: 529-535Google Scholar). Clearly this modification, which has been studied most extensively, has important implications for myelin membrane stability. The effects of other modifications such as methylation of arginine 107 as the mono-methyl or symmetric dimethyl arginine (14Baldwin G.S. Carnegie E.R. Specific enzymic methylation of an arginine in the experimental allergic encephalomyelitis protein from human myelin.Science. 1971; 171: 579-581Google Scholar, 15Brostoff S.W. Eylar E.H. Localization of the methylated arginine in the A-1 protein from myelin.Proc. Nat. Acad. Sci. U. S. A. 1971; 68: 765-769Google Scholar) have not been studied as extensively. Methylation of MBP by vitamin B12 administration reversed the myelinolysis of subacute combined degeneration of the spinal cord, suggesting an important role for methylation in myelination (16Kim S. Lim I.K. Park G.H. Paik W.K. Biological methylation of myelin basic protein: Enzymology and biological significance.Int. J. Biochem. 1997; 29: 743-751Google Scholar). A role for arginine methylation in signaling and transcriptional activation has been postulated (17McBride A.E. Silver P.A. State of the Arg: Protein methylation at arginine comes of age.Cell. 2001; 106: 5-8Google Scholar, 18Mowen K.A. Tang J. Zhu W. Schurter B.T. Shuai K. Herschman H.R. David M. Arginine methylation of STAT 1 modulates IFNα/β induced transcription.Cell. 2001; 104: 731-741Google Scholar, 19Branscombe T.L. Frankel A. Lee J.H. Cook J.R. Yang Z-H. Pestka S. Clarke S. PRMT5 (the Janus Kinase-Binding Protein 1) catalyses the formation symmetric dimethylarginine residues in proteins.J. Biol. Chem. 2001; 276: 32971-32976Google Scholar, 20Pollack B.P. Korlenko S.V. He W. Isotova L.S. Barnoski B.L. Pestka S. The human homologue of the yeast proteins Skb1 and Hsl7p interacts with Jak kinases and contains protein methyltransferase activity.J. Biol. Chem. 1999; 274: 31531-31542Google Scholar). The recent demonstration that the enzyme responsible for methylating arginine, protein methyl transferase-5, acts in concert with Janus kinase (18Mowen K.A. Tang J. Zhu W. Schurter B.T. Shuai K. Herschman H.R. David M. Arginine methylation of STAT 1 modulates IFNα/β induced transcription.Cell. 2001; 104: 731-741Google Scholar, 19Branscombe T.L. Frankel A. Lee J.H. Cook J.R. Yang Z-H. Pestka S. Clarke S. PRMT5 (the Janus Kinase-Binding Protein 1) catalyses the formation symmetric dimethylarginine residues in proteins.J. Biol. Chem. 2001; 276: 32971-32976Google Scholar), which phosphorylates STAT-1 transcription factor in signaling activity of the interferon receptor (18Mowen K.A. Tang J. Zhu W. Schurter B.T. Shuai K. Herschman H.R. David M. Arginine methylation of STAT 1 modulates IFNα/β induced transcription.Cell. 2001; 104: 731-741Google Scholar). Because interferon β is used widely in the treatment of MS, elucidation of the role played by methylation may have therapeutic relevance. Whereas an assessment of the post-translational modifications in the "charge isomers" of MBP from bovine and dogfish have been reported (21Zand R. Li M.X. Jin X. Lubman D. Determination of the sites of postranslational modifications in the charge isomers of bovine myelin basic protein by capillary electrophoresis-mass spectroscopy.Biochemistry. 1998; 37: 2441-2449Google Scholar, 22Zand R. Jin X. Kim J.K. Wall D.B. Gould R. Lubman D.M. Studies of posttranslational modifications in spiny dogfish myelin basic protein.Neurochem. Res. 2001; 26: 539-547Google Scholar), no similar analysis has been carried out for the human MBP "charge isomers." As a first step in defining the roles of these three major modifications, we have isolated several of the MBP charge isomers, protease digested each, resolved the peptides by liquid chromatography, and identified the modification(s) in each by mass spectrometry. We demonstrated that MS MBP was less phosphorylated, more methylated, and more deiminated than MBP from normal tissue. We suggest that these modifications of MBP, which affect charge (phosphorylation, deimination), conformation, and hydrogen bonding (methylation), compromise its ability to form stable myelin multilayers. Because similar changes were found in a spontaneously demyelinating transgenic model, the changes documented are unlikely to represent artifacts of disease or post-mortem autolysis. We suggest that MS may represent a new class of diseases called "post-translational" diseases. All samples were collected from human brain at autopsy. Normal brain was obtained from violent or sudden cardiac death not involving brain injury. The white matter was carefully dissected from the gray matter, frozen in liquid nitrogen, and stored at −70 °C. Only non-involved white matter was used, i.e. plaque tissue was carefully avoided. Ages of normal samples varied from 25 to 75 years, and wherever possible normals were age-matched with MS samples. This procedure was routinely done at the Canadian Brain Tissue Bank, from which most of the samples were obtained. A few samples were obtained from the Rocky Mountain Brain Bank in Colorado. Only white matter from autopsies carried out 8–10 h post-mortem was used. In addition to age and sex, clinical histories, including drug therapy and pathological reports, were obtained on all patients. Histopathology was done on each sample at the brain bank to confirm the diagnosis. None of the samples had any evidence of additional neurological disease. Histopathology of the normal samples confirmed the complete absence of disease. Isolation of MBP was carried out essentially as described for bovine MBP (6Cheifetz S. Moscarello M.A. Effect of bovine basic protein charge microheterogeneity on protein-induced aggregation of unilamellar vesicles containing a mixture of acidic and neutral phospholipids.Biochemistry. 1985; 24: 1909-1914Google Scholar). Briefly, the frozen tissue was extracted with chloroform-methanol, followed by acetone, at 4 °C to remove lipid. The solids were immediately extracted with 0.2 N H2SO4 overnight at 4 °C. The acid extracts were combined with an equal volume of absolute ethanol and allowed to precipitate at −20 °C overnight. The precipitated material represents the total MBP fraction, which was subsequently dissolved in 0.1 m glycine buffer, pH 9.6, containing 6 m urea and applied to a CM52 column. The C-8 fraction was collected in the flow-through and further purified on high-pressure liquid chromatography (10Wood D.D. Bilbao J.M. O'Connor P. Moscarello M.A. Acute multiple sclerosis (Marburg type) is associated with developmentally immature myelin basic protein.Ann. Neurol. 1996; 40: 18-24Google Scholar). The charge isomers C-5 → C-1 were eluted from the column with a sodium chloride gradient 0 → 0.2 M in the glycine-urea buffer, pH 10.6. The least cationic C-5 eluted first, followed by C-5 → C-1 in that order. The most cationic, least modified charge isomer was C-1. Protein concentrations were determined by amino acid analyses after hydrolysis in 5.7 N HCl. All "charge isomers" were derived from the 18.5-kDa isoform. Therefore "charge isomer" is used in the biological sense and not the mass spectrometric sense, because they vary in mass depending on the nature of the post-translational modification. "Charge isomers" from normal human brain are abbreviated as C-1N, C-2N, C-3N, etc. The corresponding "charge isomers" from MS tissue are represented as C-1MS, C-2MS, C-3MS, etc. Western blots were carried out by the method of Towbin et al. (23Towbin H. Staehelin T. Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc. Nat. Acad. Sci. U. S. A. 1979; 76: 4350-4354Google Scholar). The antibodies used in this study included a polyclonal anti-MBP IgG, a monoclonal antibody P12 reactive with phosphorylated threonine 98 (24Yon M. White P. Groome N. Preparation of a novel monoclonal antibody specific for myelin basic protein phosphorylated on Thr 98.J. Neuroimmunol. 1995; 58: 121-129Google Scholar), a monoclonal antibody that recognizes phosphoserine, and a monoclonal that recognizes all subunits (α, β, γ) of glycogen synthesis kinase (GSK). The transgenic mice (ND4) used in these studies were CD-1 containing 70 copies of the cDNA for DM20, a myelin proteolipid. They are normal at birth, but at 3 months of age they demyelinate spontaneously, which becomes progressively worse from 3 to 10 months, after which the animals are moribund (25Mastronardi F.G. Ackerley C A. Arsenault L. Roots B.I. Moscarello M.A. Demyelination in a transgenic mouse: A model for multiple sclerosis.J. Neurosci. Res. 1993; 36: 315-324Google Scholar). Each charge isomer (1 mg) was digested with trypsin (Promega, Madison, WI) at a ratio of 1:50 trypsin:protein overnight at 37 °C in 100 mm ammonium bicarbonate buffer, pH 8.0. The digests were vacuum dried and reconstituted in deionized water. Separation of the tryptic peptides was performed on an in-house constructed polybrene capillary electrophoresis apparatus with a 50-cm long capillary (26Jin X. Kim J. Parus S. Lubman D.M. Zand R. On-line capillary electrophoresis/microelectrospray ionization-tandem mass spectrometry using an ion trap storage/time-of-flight mass spectrometer with SWIFT technology.Anal. Chem. 1999; 74: 3591-3597Google Scholar). The capillary electrophoresis buffer solution consisted of 100 mm formic acid that was adjusted to pH 2.7 with ammonia. The tryptic peptides were loaded into the capillary using an electrokinetic injection method that applied -2.5 kV for 10 s. A voltage of 12 kV was applied across the capillary to perform the separation. The separated peptides were fed directly into the mass spectrometer as described previously. An ion trap storage/reflectron time-of-flight mass spectrometer was used to detect the separated peptides (27Michael S.M. Chien B.M. Lubman D.M. Detection of electorspray-ionization using a quadrupole ion-trap storage reflectron time-of-flight mass-spectrometer.Anal. Chem. 1993; 65: 2614-2620Google Scholar). A three-dimensional contour map, which displays individual mass spectra along with elution profiles, was used to pinpoint and integrate specific peaks (28Li M.X. Wu J.T. Parus S. Lubman D.M. Development of a three-dimensional topographic map display for capillary electrophoresis mass spectrometry with an ion trap/reflectron time-of-flight mass spectrometer detector: Applications to tryptic digests of isoforms of myelin basic protein.J. Am. Soc. Mass Spectrom. 1998; 9: 701-709Google Scholar). Initial assignments of each peptide peak were performed using the FindMod tool (28Li M.X. Wu J.T. Parus S. Lubman D.M. Development of a three-dimensional topographic map display for capillary electrophoresis mass spectrometry with an ion trap/reflectron time-of-flight mass spectrometer detector: Applications to tryptic digests of isoforms of myelin basic protein.J. Am. Soc. Mass Spectrom. 1998; 9: 701-709Google Scholar), which is available at www.expasy.ch/tools/findmod/. The experimental [M+H]+ values were compared with the theoretical average peptide masses with ±150 ppm mass tolerance. To confirm the assignments, tandem mass spectrometry fragmentation of each peak was performed as described in previous work (27Michael S.M. Chien B.M. Lubman D.M. Detection of electorspray-ionization using a quadrupole ion-trap storage reflectron time-of-flight mass-spectrometer.Anal. Chem. 1993; 65: 2614-2620Google Scholar). In addition, in those instances in which the post-translational modifications resulted in a decrease or increase of 1 or 2 atomic mass units, the assignment was confirmed by the mobility of the ion undergoing electrophoresis. Details of all the mass spectral methods can be found in the various publications indicated above. The proportions of the various charge isomers (Table I) are expressed as the amount of each (μg) divided by the column charge (OD280 units). The amounts of C-2, C-3, C-4, and C-5 between normal and MS material were not significantly different. C-1 from MS was decreased compared with normal, whereas C-8 from MS was increased. This was consistent with our previous observations (10Wood D.D. Bilbao J.M. O'Connor P. Moscarello M.A. Acute multiple sclerosis (Marburg type) is associated with developmentally immature myelin basic protein.Ann. Neurol. 1996; 40: 18-24Google Scholar).Table IRelative proportions of components isolated by cation exchange chromatographyMBPCharge Isomer μg/OD280nC1C2C3C4C-5C8Normal60.22 ± 0.010.12 ± 0.010.18 ± 0.0150.11 ± 0.020.11 ± 0.0170.22 ± 0.05MS80.15 ± 0.0350.11 ± 0.0050.15 ± 0.020.10 ± 0.020.12 ± 0.0150.35 ± 0.03 Open table in a new tab Only arginine 107 occurs as the unmethylated, monomethylated, and symmetrically dimethylated forms. The relative abundance (intensity of methylated peptide/intensity of non-methylated peptide) was obtained for each species. The intensities from dimethylated forms were approximately twice those of monomethylated peptides. The number of samples reflects the number of different brains used. Each number in Table II is the mean of all the brains. Some variation was observed from brain to brain and was due to the differences in duration and severity of disease of the MS patients, which varied from 4 years (severe) to 30 years (benign).Table IIMono- and dimethylation of arginine 107 in MBP charge isomers from normal and MS brains (relative intensities)ComponentMonomethylation (GR*G SLSR)Dimethylation (GR*G SLSR)Number of BrainsC-1 (N)1.61.83C-1 (MS)1.11.12C-2 (N)1.312.43C-2 (MS)0.670.972C-3 (N)0.981.683C-3 (MS)0.691.282C-4 (N)1.232.4416C-4 (MS)2.474.207C-5 (N)0.852.148C-5 (MS)2.494.296C-8 (N)1.141.93C-8 (MS)1.261.942 Open table in a new tab The integrated total ion counts of monomethylated, dimethylated, and unmethylated peptides have been calculated to compare the relative intensities of methylated peptides. The integration was performed on the three-dimensional contour map (28Li M.X. Wu J.T. Parus S. Lubman D.M. Development of a three-dimensional topographic map display for capillary electrophoresis mass spectrometry with an ion trap/reflectron time-of-flight mass spectrometer detector: Applications to tryptic digests of isoforms of myelin basic protein.J. Am. Soc. Mass Spectrom. 1998; 9: 701-709Google Scholar). GLSLSR (residues 108–113) was used for the unmodified standard, while GRGLSLSR (residues 106–113) with arginine 107 methylated was used for the methylated standards. Peptides containing methylated arginine are not cleaved by trypsin (14Baldwin G.S. Carnegie E.R. Specific enzymic methylation of an arginine in the experimental allergic encephalomyelitis protein from human myelin.Science. 1971; 171: 579-581Google Scholar), probably due to steric hindrance by the bulky methyl group. In the case of C-1, C-2, and C-3, the intensities of the mass spectral peaks were low, so these numbers must be interpreted with caution. In C-4 and C-5 of the MS samples, the intensities of the methylated peaks were 1–3 times higher than the corresponding normal charge isomers, and more samples were available, giving considerable strength to these numbers. In both C-4 and C-5 of the MS samples, the ratios (methylated/unmethylated) were 2–3 times greater than the corresponding ratios from normal C-4 and C-5, suggesting that a greater portion of the MBP from MS samples was methylated. We have recently completed a study of the activity of the enzyme protein methyl transferase 5, which methylates MBP symmetrically. This could only be done in the spontaneously demyelinating transgenic mouse model. In a developmental study from 0.5 to 6 months of age, a 3-fold increase in methylation of MBP was observed before signs of disease, suggesting that this post-translational modification was affected early and may have a role in pathogenesis (development) of disease (data not shown). When all phosphorylated peptides derived from normal and MS samples are considered, phosphorylation appears in the normal samples much more frequently than in the MS samples. We recognize that the signals for the phosphorylated peptides were low and even lower for diphosphorylated peptides. However, the signals were sufficiently clear so the distinction was unambiguous. The comparisons of all peptides are collected in Table III. It is clear that normal samples have higher intensities than MS samples, and in several peptides only the normal samples were phosphorylated, e.g. RPSQR (5–9), HGSK (10–13), RGSGK (54–58), TAHYGSLPQK (66–75), NIVTPR (92–97), and FSWGAEGQR (114–122).Table IIIPhosphorylation sites on MBP charge isomers from normal and MS white matter (relative intensities)ComponentPhosphorylation Sites5RPS*QR910HGS*K1314YLAT*AS*T*MDHAR2555GS*GK5866TAHYGS*LPQK7592NIVT*PR9798T*PPPSQGK105114FS*WGAEGQR122143GVDAQGT*LS*K152163S*GS*PMAR169C-1 (N)0 (3Steinman L. Multiple sclerosis: A two-stage disease.Nat. Immunol. 2001; 2: 762-764Google Scholar)0 (3Steinman L. Multiple sclerosis: A two-stage disease.Nat. Immunol. 2001; 2: 762-764Google Scholar)0.52 (3Steinman L. Multiple sclerosis: A two-stage disease.Nat. Immunol. 2001; 2: 762-764Google Scholar)†0.11 (1Moscarello M.A. Myelin basic protein, the executive molecule of the myelin membrane.in: Devon R.M. Doucette R. Juurlink B.H.J. Nazarali A.J. Schreyer, D.J. Verge V.M.K. Cell Biology and Pathology of Myelin: Evolving Biological Concepts and Therapeutic Approaches. Plenum Press, New York1996: 13-25Google Scholar)0 (2Chataway J. Feakes R. Coraddu F. Gray J. Deans J. Fraser M. Robertson N. Broadley S. Jones H. Clayton D. Goodfellow P. Sawcer S. Compston A. The genetics of multiple sclerosis: Principles, background and updated results of the United Kingdom systematic genome screen.Brain. 1998; 121: 1869-1887Google Scholar)0.042 (1Moscarello M.A. Myelin basic protein, the executive molecule of the myelin membrane.in: Devon R.M. Doucette R. Juurlink B.H.J. Nazarali A.J. Schreyer, D.J. Verge V.M.K. Cell Biology and Pathology of Myelin: Evolving Biological Concepts and Therapeutic Approaches. Plenum Press, New York1996: 13-25Google Scholar)0 (2Chataway J. Feakes R. Coraddu F. Gray J. Deans J. Fraser M. Robertson N. Broadley S. Jones H. Clayton D. Goodfellow P. Sawcer S. Compston A. The genetics of multiple sclerosis: Principles, background and updated results of the United Kingdom systematic genome screen.Brain. 1998; 121: 1869-1887Google Scholar)0 (2Chataway J. Feakes R. Coraddu F. Gray J. Deans J. Fraser M. Robertson N. Broadley S. Jones H. Clayton D. Goodfellow P. Sawcer S. Compston A. The genetics of multiple sclerosis: Principles, background and updated results of the United Kingdom systematic genome screen.Brain. 1998; 121: 1869-1887Google Scholar)0.037 (1Moscarello M.A. Myelin basic protein, the executive molecule of the myelin membrane.in: Devon R.M. Doucette R. Juurlink B.H.J. Nazarali A.J. Schreyer, D.J. Verge V.M.K. Cell Biology and Pathology of Myelin: Evolving Biological Concepts and Therapeutic Approaches. Plenum Press, New York1996: 13-25Google Scholar)C-1 (MS)0 (3Steinman L. Multiple sclerosis: A two-stage disease.Nat. Immunol. 2001; 2: 762-764Google Scholar)0 (3Steinman L. Multiple sclerosis: A two-stage disease.Nat. Immunol. 2001; 2: 762-764Google Scholar)0.18 (1Moscarello M.A. Myelin basic protein, the executive molecule of the myelin membrane.in: Devon R.M. Doucette R. Juurlink B.H.J. Nazarali A.J. Schreyer, D.J. Verge V.M.K. Cell Biology and Pathology of Myelin: Evolving Biological Concepts and Therapeutic Approaches. Plenum Press, New York1996: 13-25Google Scholar)0 (3Steinman L. Multiple sclerosis: A two-stage disease.Nat. Immunol. 2001; 2: 762-764Google Scholar)0 (2Chataway J. Feakes R. Coraddu F. Gray J. Deans J. Fraser M. Robertson N. Broadley S. Jones H. Clayton D. Goodfellow P. Sawcer S. Compston A. The genetics of multiple sclerosis: Principles, background and updated results of the United Kingdom systematic genome screen.Brain. 1998; 121: 1869-1887Google Scholar)0 (1Moscarello M.A. Myelin basic protein, the executive molecule of the myelin membrane.in: Devon R.M. Doucette R. Juurlink B.H.J. Nazarali A.J. Schreyer, D.J. Verge V.M.K. Cell Biology and Pathology of Myelin: Evolving Biological Concepts and Therapeutic Approaches. Plenum Press, New York1996: 13-25Google Scholar)0 (2Chataway J. Feakes R. Coraddu F. Gray J. Deans J. Fraser M. Robertson N. Broadley S. Jones H. Clayton D. Goodfellow P. Sawcer S. Compston A. The genetics of multiple sclerosis: Principles, background and updated results of the United Kingdom systematic genome screen.Brain. 1998; 121: 1869-1887Google Scholar)0 (2Chataway J. Feakes R. Coraddu F. Gray J. Deans J. Fraser M. Robertson N. Broadley S. Jones H. Clayton D. Goodfellow P. Sawcer S. Compston A. The genetics of multiple sclerosis: Principles, background and updated results of the United Kingdom systematic genome screen.Brain. 1998; 121: 1869-1887Google Scholar)0 (1Moscarello M.A. Myelin basic protein, the executive molecule of the myelin membrane.in: Devon R.M. Doucette R. Juurlink B.H.J. Nazarali A.J. Schreyer, D.J. Verge V.M.K. Cell Biology and Pathology of Myelin: Evolving Biological Concepts and Therapeutic Approaches. Plenum Press, New York1996: 13-25Google Scholar)C-2 (N)0 (2Chataway J. Feakes R. Coraddu F. Gray J. Deans J. Fraser M. Robertson N. Broadley S. Jones H. Clayton D. Goodfellow P. Sawcer S. Compston A. The genetics of multiple sclerosis: Principles, background and updated results of the United Kingdom systematic genome screen.Brain. 1998; 121: 1869-1887Google Scholar)0 (3Steinman L. Multiple sclerosis: A two-stage disease.Nat. Immunol. 2001; 2: 762-764Google Scholar)0.69 (2Chataway J. Feakes R. Coraddu F. Gray J. Deans J. Fraser M. Robertson N. Broadley S. Jones H. Clayton D. Goodfellow P. Sawcer S. Compston A. The genetics of multiple sclerosis: Principles, background and updated results of the United Kingdom systematic genome screen.Brain. 1998; 121: 1869-1887Google Scholar)0.091 (1Moscarello M.A. Myelin basic protein, the executive molecule of the myelin membrane.in: Devon R.M.