Synergy between paclitaxel plus an exogenous methyl donor in the suppression of murine demyelinating diseases.

Progressive demyelination in multiple sclerosis (MS) reflects the negative balance between myelin damage and repair due to physical and molecular barriers, such as astrocytic glial scars, between oligodendrocytes and target neurons. In this paper, we show that combination therapy with paclitaxel (Taxol) plus the universal methyl-donor, vitamin B12CN (B12CN), dramatically limits progressive demyelination, and enhances remyelination in several independent, immune and nonimmune, in vivo and in vitro model systems. Combination therapy significantly reduced clinical signs of EAE in SJL mice, as well as the spontaneously demyelinating ND4 transgenic mouse. Astrocytosis was normalised in parallel to ultrastructural and biochemical evidence of remyelination. The combination therapy suppressed T cell expansion, reduced IFN-gamma, while enhancing IFN-beta and STAT-1 expression, STAT-1 phosphorylation and methylation of STAT-1 and MBP in the brain. Paclitaxel/B12CN has nearly identical effects to the previously described combination of IFN-beta/ B12CN, whose clinical usefulness is transient because of IFN-neutralising antibodies, not observed (or expected) with the present drug combination. This report provides a mechanistic foundation for the development of a new therapeutic strategy in humans with MS.

Multiple sclerosis: an important role for post-translational modifications of myelin basic protein 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.

Primary structure of equine myelin basic protein by mass spectrometry.

Equine myelin basic protein (MBP) has been isolated from spinal cord and shown to consist of a number of components (charge isomers) by alkaline-urea gel electrophoresis. Mass analyses of several of these components showed that each was posttranslationally modified and some have been identified. Component 1, the most cationic charge isomer, was sequenced by a combination of liquid chromatography and mass spectrometry of peptides obtained by proteolytic digestion. At 172 residues it is slightly larger than the bovine (169) and the human (170). A major difference between bovine and equine sequences was the replacement of AQGH (bovine residues 76-79) by SRDG (equine). A number of other replacements involving single amino acids were also found. Methylated arginine (residue 108 equine) was found as both the mono- and the dimethylated derivative and represents the first MS/MS evidence for this modification in any MBP.

Peptidylarginine deiminase: a candidate factor in demyelinating disease.

In earlier studies we demonstrated that an increase in the relative amounts of citrullinated myelin basic protein (MBP) was found in multiple sclerosis (Moscarello et al. 1994). To determine the temporal relationship between the citrullinated MBP and peptidylarginine deiminase (PAD), the enzyme responsible for deiminating arginyl residues in proteins, we studied enzyme activity, enzyme protein, PAD mRNA in a spontaneously demyelinating transgenic mouse model and we correlated the amount of PAD with citrullinated MBP. Both PAD protein as measured in an immunoslot blot method and PAD RNA were elevated. In fractionation studies we showed that the increase in PAD enzyme was due to an increase in the PAD found in membrane fractions and not the soluble PAD (PADII). From our data we concluded that up-regulation of myelin-associated PAD was responsible for the increase in citrullinated MBP in our transgenic mice prior to onset of clinical or pathological signs of demyelination. We postulate that a similar mechanism may be responsible for the increase in citrullinated MBP in multiple sclerosis.

Paclitaxel (Taxol) attenuates clinical disease in a spontaneously demyelinating transgenic mouse and induces remyelination.

Treatment with paclitaxel by four intraperitoneal injections (20 mg/kg) 1 week apart attenuated clinical signs in a spontaneously demyelinating model, if given with onset of clinical signs. If given at 2 months of age (1 month prior to clinical signs), disease was almost completely prevented The astrogliosis, prominent in our model, was reversed by paditaxel as determined by astrocyte counts and quantitation of GFAP. Electron microscopic examination of affected regions at 2.5 months demonstrated that the myelin was generally normal. By 4 months of age, demyelination was common in the superior cerebellar peduncle, maximal at 6 months, but continued to 8 months. In addition to myelin vacuolation and nude axons, the presence of many thin myelin sheaths suggested remyelination or partial demyelination. Although no evidence of oligodendrocyte loss was seen, nuclear changes were observed. To substantiate that remyelination was occurring, we measured MBP (18.5 kDa), MBP-exon II, Golli-MBP, TP8, Golli-MBP-J37, platelet-derived growth factor alpha (PDGFR alpha) and sonic hedgehog (SHH). Of these TP8, PDGFR alpha and SHH were up-regulated in the untreated transgenic. After paditaxel treatment, MBP-Exon II, TP8, PDGFR alpha and SHH were further up-regulated. We concluded that some of the effects of paditaxel were to stimulate proteins involved in early myelinating events possibly via a signal transduction mechanism.

T cells of multiple sclerosis patients target a common environmental peptide that causes encephalitis in mice.

Multiple sclerosis (MS) is a chronic autoimmune disease triggered by unknown environmental factors in genetically susceptible hosts. MS risk was linked to high rates of cow milk protein (CMP) consumption, reminiscent of a similar association in autoimmune diabetes. A recent rodent study showed that immune responses to the CMP, butyrophilin, can lead to encephalitis through antigenic mimicry with myelin oligodendrocyte glycoprotein. In this study, we show abnormal T cell immunity to several other CMPs in MS patients comparable to that in diabetics. Limited epitope mapping with the milk protein BSA identified one specific epitope, BSA(193), which was targeted by most MS but not diabetes patients. BSA(193) was encephalitogenic in SJL/J mice subjected to a standard protocol for the induction of experimental autoimmune encephalitis. These data extend the possible, immunological basis for the association of MS risk, CMP, and CNS autoimmunity. To pinpoint the same peptide, BSA(193), in encephalitis-prone humans and rodents may imply a common endogenous ligand, targeted through antigenic mimicry.

The effects of deimination of myelin basic protein on structures formed by its interaction with phosphoinositide-containing lipid monolayers.

The recombinant 18.5-kDa charge isoform of murine myelin basic protein (rmMBP) is unmodified posttranslationally and was used to study the effects of deimination, i.e., the conversion of arginyl to citrullinyl residues, on the protein's interactions with itself and with lipids. The unmodified species rmMBP-Cit(0) (i.e., containing no citrullinyl residues) interacted with binary monolayers containing acidic (phosphatidylinositol) and nickel-chelating lipids to form paracrystalline arrays with 4.8-nm spacing. A sample of protein was deiminated to an average of 9 citrullinyl residues per molecule of protein, yielding rmMBP-Cit(9). Under both low- and high-salt conditions, this species formed better-ordered domains than rmMBP-Cit(0), viz., planar crystalline assemblies. Thus, deimination of MBP resulted in a significant alteration of its lipid-organizing and self-interaction properties that might be operative in myelin in vivo, especially in progression of the autoimmune disease multiple sclerosis. Comparisons of amino acid sequences indicated significant similarities of MBP with filaggrin, a protein that is deiminated in another autoimmune disease, rheumatoid arthritis, suggesting that comparable epitopes could be targeted in both pathologies. In contrast, binary lipid monolayers consisting of phosphatidylinositol-4-phosphate (or phosphatidylinositol-4,5-bisphosphate) and a nickel-chelating lipid formed helical tubular vesicular structures, which appeared to be induced and/or stabilized by rmMBP, especially in its deiminated form. Sequence comparisons with other actin- and phosphoinositide-binding proteins (vinculin, ActA, MARCKS) suggested that the carboxyl-terminal segment of MBP could form an amphipathic alpha helix and was the phosphoinositide binding site.

Characterization of a recombinant murine 18.5-kDa myelin basic protein.

A recombinant hexahistidine-tagged 18.5-kDa isoform of murine myelin basic protein has been characterized biochemically and immunogenically, by mass spectrometry, by circular dichroism under various conditions (in aqueous solution, with monosialoganglioside G(M1), and in 89% 2-propanol), and by transmission electron microscopy. The preparations of this protein indicated a high degree of purity and homogeneity, with no significant posttranslational modifications. Circular dichroic spectra showed that this preparation had the same degree of secondary structure as the natural bovine 18.5-kDa isoform of myelin basic protein. Incubation of the recombinant protein with lipid monolayers containing a nickel-chelating lipid resulted in the formation of fibrous assemblies that formed paracrystals of spacings 4.8 nm between fibers and 3-4 nm along them.

Cryoelectron microscopy of protein-lipid complexes of human myelin basic protein charge isomers differing in degree of citrullination.

Myelin basic protein (MBP) is considered to be essential for the maintenance of stability of the myelin sheath. Reduction in cationicity of MBP, especially due to conversion of positively charged arginine residues to uncharged citrulline (Cit), has been found to be associated with multiple sclerosis (MS). Here, the interactions of an anionic phosphatidylserine/monosialoganglioside-G(M1) (4:1, w:w) lipid monolayer with 18.5-kDa MBP preparations from age-matched adult humans without MS (no Cit residues), with chronic MS (6 Cit), and with acute Marburg-type MS (18 Cit) were studied by transmission and ultralow dose scanning transmission electron microscopy under cryogenic conditions. Immunogold labeling and single particle electron crystallography were used to define the nature of the complexes visualized. These electron microscopical analyses showed that the three different MBP charge isomers all formed uniformly sized and regularly shaped protein-lipid complexes with G(M1), probably as hexamers, but exhibited differential association with and organization of the lipid. The least cationic Marburg MBP-Cit(18) formed the most open protein-lipid complex. The data show a disturbance in lipid-MBP interactions at the ultrastructural level that is related to degree of citrullination, and which may be involved in myelin degeneration in multiple sclerosis.

Highly deiminated isoform of myelin basic protein from multiple sclerosis brain causes fragmentation of lipid vesicles.

Myelin basic protein (MBP) occurs as a number of charge isomers due to phosphorylation, deamidation, and deimination of arginine to citrulline. All of these modifications decrease the net positive charge of the protein and its ability to cause aggregation of negatively charged lipid vesicles. This is used as a model system for the ability of MBP to cause adhesion of the cytosolic surfaces of myelin. Therefore, the effect of two deiminated forms of MBP on lipid vesicles was compared with that of the unmodified, most positively charged isomer, C1, to determine how loss of positively charged arginines would affect the function of MBP. The deiminated forms were the isomer isolated from normal human brains, in which only 6 Arg are deiminated to citrulline (MBP-Cit(6)), and an isomer isolated from the brain of a patient who died with acute, fulminating multiple sclerosis (Marburg type), in which 18 of the 19 Arg were deiminated (MBP-Cit(18)). Whereas C1 caused aggregation of lipid vesicles, resulting in an increase in absorbance due to light scattering, MBP-Cit(18) caused a decrease in absorbance of the lipid vesicles. Size exclusion chromatography and negative staining electron microscopy showed that this was due to fragmentation of the large multilayered vesicles into much smaller vesicles. MBP-Cit(6) caused less aggregation of lipid vesicles than did C1. However, no fragmentation of the vesicles into smaller ones in the presence of C1 and MBP-Cit(6) was detected by size exclusion chromatography or electron microscopy. The membrane fragmentation caused by MBP-Cit(18) is dramatically different from the effects of other forms of MBP from normal brain and may indicate a pathogenic effect of this charge isomer, which may have contributed to the severity of the Marburg type of multiple sclerosis. Alternatively, the deimination may have been a secondary effect resulting from the disease process. Regardless of the role of MBP-Cit(18) in multiple sclerosis, the effect of this modification indicates that, when most of the arginines of MBP are modified to an uncharged amino acid, the protein acquires properties similar to an apolipoprotein; thus, it may take up an amphipathic structure when bound to lipid. A partly amphipathic character may also be related to the role of MBP-Cit(6) in normal immature myelin, where it is the predominant charge isomer.

Marburg's variant of multiple sclerosis correlates with a less compact structure of myelin basic protein.

Multiple sclerosis (MS) is an autoimmune disease in which the myelin sheath of the central nervous system is degraded, and the 18.5 kDa isoform of myelin basic protein (MBP) is reduced in cationicity. In a unique case of acute, fulminating MS (Marburg's variant), MBP is considerably less cationic than MBP from both normal, and chronic MS-afflicted individuals. This electron microscopical study has identified that, in vitro, the less cationic Marburg MBP isomer forms a more extended protein-lipid complex than MBP from healthy or chronic MS-afflicted individuals. This correlation implies that chemical modifications to MBP in vivo contribute directly to the structural instability of myelin, and subsequent autoantigenic presentation of this protein, observed in vivo in MS.

Human proteolipid protein (PLP) mediates winding and adhesion of phospholipid membranes but prevents their fusion.

Proteolipid protein (PLP or lipophilin) is a highly conserved, strongly hydrophobic, integral membrane protein, and is the major protein component of central nervous system myelin. Although PLP has been implicated in many functions, its in vivo role is still uncertain. Here, we report the investigation of PLP's putative adhesive function using purified PLP and reconstituted phospholipid vesicles made of either 100% phosphatidylcholine (PC), or a mixture of 92% PC and 8% phosphatidylserine (PS), by weight. PLP-induced changes in the phospholipid bilayer surfaces were directly examined by transmission electron microscopy. We found that upon the introduction of PLP, larger lipid vesicles became smaller and unilamellar. At the PLP:lipid molar ratio of 1:20, vesicle membranes rolled onto themselves forming 'croissant'-like structures that subsequently adhered to each other. The phenomena of PLP-induced bilayer rolling and adhesion were dependent on the concentration of PLP and the period of incubation, but were independent of the presence of calcium and types of phospholipids (PC or PC:PS). Furthermore, the presence of PLP in the lipid bilayers prevented the fusion of membranes. These findings show that PLP can induce membrane 'winding' while preventing the fusion of adjacent lipid bilayers. Hence, our data provide direct evidence for PLP's suspected function of membrane adhesion, and also suggest that PLP could potentially play a role in the formation of the myelin sheath.

Minimizing fatigue during repetitive jobs: optimal work-rest schedules.

Twenty women were asked to generate forces using a dynamometer that were consistent with one of three different work-rest schedules (a low-, medium-, and high-force schedule). Each work-rest schedule consisted of 6 identical blocks of 10 work-rest cycles. Each of the 10 work-rest cycles lasted 1 min. The first work-rest cycle in each block consisted of a 6-s maximal voluntary contraction and a 54-s rest. The remaining 9 work-rest cycles in each block consisted of a submaximal contraction and a rest period. The desired force of the submaximal contraction, the length of this contraction, and the duration of the rest period remained constant within schedules but varied across schedules. The amount of physiological work was kept constant among schedules. The fatigue that developed in the medium-force schedule was significantly lower than that developed in either the low- or high-force schedule. A model was developed that predicted the amount of fatiguable strength at the beginning and end of each contraction of a work-rest cycle. When fit to the results from the experiment, the model explained 94% of the variance. The model can be used to predict the work-rest schedule that minimizes fatigue in a given repetitive job, thereby potentially increasing productivity and reducing the incidence of cumulative trauma disorders.

Acute multiple sclerosis (Marburg type) is associated with developmentally immature myelin basic protein.

We have studied a case of acute, fulminating multiple sclerosis (MS) (Marburg type) at the pathological and biochemical levels. Postmortem examination of the brain revealed extensive areas of gross rarefaction in the hemispheric white matter. Histologically, well-demarcated areas of demyelination with a large influx of macrophages and a subtle perivascular infiltration of lymphocytes were seen with relative preservation of the axis cylinders. Myelin basic protein (MBP) was isolated and purified [correction of purifed] from noninvolved white matter. It was slightly larger in molecular weight than MBP from normal brain or from chronic MS brain. The increase in mass was accounted for, in part, by the deimination of 18 of 19 arginyl residues to citrulline, making the patient's MBP much less cationic than MBP from normal white matter. When expressed as the ratio of least cationic form of MBP to the most cationic (C-8/C-1), the normal ratio was 0.82, chronic MS 2.5, and the patient in this study 6.7. Because the ratio of 6.7 was similar to 7.5 found for a 15-month-old infant, MBP was considered to be of the immature form. The data are consistent with a genetic factor influencing the charge microheterogeneity of MBP. The resulting less cationic MBP cannot carry out its normal function of compacting multilayers.

Localization and partial characterization of a 60 kDa citrulline-containing transport form of myelin basic protein from MO3-13 cells and human white matter.

The localization of myelin basic proteins (MBPs) in an immortalized human-human hybrid cell line (MO3-13) formed by fusion of rhabdomyosarcoma TE671-TG6 with primary human oligodendrocytes, cultured from surgical specimens, demonstrated an intracellular localization in vesicles and vacuoles with an intricate internal membranous network and to the external surface of the cell by immunogold electron microscopy. The availability of antibodies to one of the components of MBP, i.e., the citrulline containing component ("C-8"), permitted us to localize this component of MBP to intracellular vacuoles and also on the external surface of the MO3-13 cells. Since the apposition of the external surfaces of the oligodendrocyte is responsible for the intraperiod line of the myelin sheath, localization of C-8 to the external surface of non-permeabilized cells by immunogold scanning electron microscopy is consistent with our observations that C-8 is localized to the intraperiod line of myelin (McLaurin et al.: J Neurosci Res 35:618-628, 1993). Western blots of isolated MBP from MO3-13 cells, probed with an antibody reactive with residues 130-137 of MBP, recognized a protein in the 60 kDa range. No immunoreactivity was found in the 18.5 kDa range. This 60 kDa protein also reacted with a monoclonal antibody raised with residues 70-84 of MBP, 2 different polyclonals raised with whole bovine MBP, an antibody to human MBP raised in monkeys, and the anti-citrulline antibody. These data strongly suggested that the 60 kDa protein contained MBP sequences within its primary structure. A similar protein has been isolated from human myelin-containing fractions but not from compact myelin demonstrating that the 60 kDa protein from MO3-13 cells was not an artefact related to fusion. Sequence determination of peptides obtained from enzymic and chemical cleavages revealed that the 60 kDa protein contained MBP sequences and peptides with 55-60% homology with dynamin, a protein involved in intracellular transport. These data suggest that the externalization of MBP in this cell involves transport by fusion of MBP with another protein. By sequestering MBP in a larger protein, the possibility of inducing autoimmune disease by MBP released, due to cell death, is minimized.

Participation of acetylpseudouridine in the synthesis of a peptide bond in vitro.

Uracil, uridine, and pseudouridine were acetylated by refluxing in acetic anhydride, and the products of acetylation were incubated with a synthetic peptide (1-21) that corresponds to the N-terminal 21 amino acid residues of human myelin basic protein. Peptide bond formation, at the N alpha terminus in peptide 1-21, was obtained with acetyluracil and acetylpseudouridine, but not with acetyluridine. Transfer of an acetyl group from acetyluracil and acetylpseudouridine depended on acetylation in the N-heterocycle. X-ray crystallographic analysis definitively established N-1 as the site of acetylation in acetyluracil. Mass spectrometry of the acetylation products showed that one acetyl group was transferred to peptide 1-21, in water, by either acetyluracil or acetylpseudouridine at pH approximately 6. Release of the acetyl group by acylaminopeptidase regenerated peptide 1-21 (mass spectrometry) and automated sequencing (for five cycles) of the regenerated (deacetylated) peptide demonstrated that the N terminus was intact. The findings are discussed in the context of a possible role for pseudouridine in ribosome-catalyzed peptidyltransfer, with particular reference being made to similarities between the possible mechanism of acyl transfer by acetyluracil/pseudouridine and the mechanism of carboxyl transfer by carboxylbiotin in acetyl CoA carboxylase. The possibility that idiosyncratic appearance of a wide range of acyl substituents in myelin basic protein could be related to a peculiar involvement of ribosomal pseudouridine is mentioned.

Myelin in multiple sclerosis is developmentally immature.

The etiology of multiple sclerosis (MS) is considered to involve genetic, environmental, infective, and immunological factors which affect the integrity of a normally assembled myelin sheath, either directly or indirectly resulting in demyelination. In a correlative study involving protein chemical, mass spectrometric, and electron microscopic techniques we have determined that myelin obtained from victims of MS is arrested at the level of the first growth spurt (within the first 6 yr of life) and is therefore developmentally immature. The data supporting this conclusion include (a) the pattern of microheterogeneity of myelin basic protein (MBP); (b) the NH2-terminal acylation of the least cationic component of MBP ("C-8"); (c) the phase transition temperature (Tc) of myelin isolated from victims of MS correlated with the increased proportion of the least cationic component of MBP; and (d) immunogold electron microscopy using an antibody specific for "C-8" showed that the distribution of gold particles in a 2-yr-old infant was similar to the distribution found in a victim of MS. We postulate that this developmentally immature myelin is more susceptible to degradation by one or a combination of factors mentioned above, providing the initial antigenic material to the immune system.

Immunological analysis of the amino terminal and the C8 isomer of human myelin basic protein.

The citrullination and N-terminus acylation of myelin basic protein (MBP) increases the heterogeneity among the MBP isoforms. The present study was undertaken to further characterize the immune response to the citrullinated form (C8) of MBP as well as to the variably acylated N-terminus of MBP. Six well-characterized murine monoclonal antibodies (mAbs) to human MBP-C8 or MBP peptides (four mAbs to MBP acetyl 1-9, one mAb to MBP 10-19 and one mAb to MBP 80-89), one murine T cell line (PL11) to human MBP peptide acetyl 1-9 and one Lewis rat T cell line (RT-1) to guinea pig (GP) MBP peptide 68-88 were used to assess reactivity with MBP-C1, MBP-C8, and MBP peptides including a series of MBP peptide 1-21 containing 0, 2, 4, 6, 8 or 10 carbon fatty acids. Enzyme-linked immunosorbent assay (ELISA) results revealed that all of the mAbs reacted with human MBP-C1 and MBP-C8 except anti-MBP 10-19 and anti-MBP-C8. The former reacted only with MBP-C1 and the latter only with MBP-C8. The presence and length of acylation of MBP peptide 1-21 modified reactivity. Three mAbs to MBP acetyl 1-9 reacted only with acetyl 1-21, and one mAb anti-MBP actyl 1-9 reacted with all of MBP 1-21 preparations whether acylated or not. mAb anti-MBP-C8 generally reacted better with acylated MBP 1-21 having longer fatty acids. The PL11 T cell line strongly proliferated to human MBP-C1, MBP-C8 and MBP acetyl 1-9, responded, but less well, to MBP 1-21 with longer fatty acids and failed to respond to nonacylated MBP peptide 1-21. The RT-1 cell line responded strongly to GP MBP peptide 68-88, marginally to MBP-C8 and failed to respond to MBP-C1 or any of the other MBP peptides. Specific immune responses to different MBP charge isomers and different N-terminal acylating groups of MBP may play a role in immune-mediated demyelination.

The N terminus of human myelin basic protein consists of C2, C4, C6, and C8 alkyl carboxylic acids.

Peptide 1-21, generated by cyanogen bromide cleavage of each of two highly purified components of human myelin basic protein, components 1 and 8, gave a series of peaks in the fast atom bombardment mass spectra with m/z 2299, 2327, 2355, 2383, and 2411, indicating additions of 42, 70, 98, 126, and 154 atomic mass units respectively with m/z 2327 and 2355 as the dominant species. The pentafluorobenzyl esters prepared from an acid hydrolysate analyzed by negative ion chemical ionization gas chromatography mass spectrometry confirmed that C6, C8, and C10 fatty acids were present. These data demonstrated (i) that the N terminus of a myelin basic protein is not simply acetylated but contains C2, C4, C6, C8, and C10 fatty acids with C4 and C6 as the dominant species, (ii) the two components studied (C-1 and C-8) showed different relative amounts of C2 and C8 in particular, and (iii) human myelin basic protein is the first protein to be reported with a complex N terminus consisting of several alkyl carboxylic acid species.

An immunochemical comparison of human myelin basic protein and its modified, citrullinated form, C8.

An immunochemical analysis was conducted to compare the C1 isomer of human myelin basic protein (MBP) with the newly described and less cationic, citrullinated isomer of MBP referred to as C8. Ten polyclonal antisera directed at multiple epitopes or restricted regions of MBP were used in radioimmunoassays to examine MBP-C1 and MBP-C8. Antisera reactive with MBP peptide 1-14 clearly distinguished MBP-C1 from MBP-C8. Antisera to human MBP peptides 10-19 and 90-170, but not to MBP peptide 69-89, showed modest differences between MBP-C1 and MBP-C8. The MBP-C8s from multiple sclerosis (MS) and non-MS brain reacted essentially the same. With murine monoclonal antibodies and enzyme-linked immunosorbent assay (ELISA), differences between MBP-C8 and other isomers were shown for anti-MBP 10-19 but not for anti-MBP 1-9 or anti-MBP 80-89. These findings imply differences in sequence or conformation in the structure of MBP-C7 compared to MBP-C1, most notably near the amino terminus.