IgG reactivity against citrullinated myelin basic protein in multiple sclerosis.

An increased level of citrullinated myelin basic protein (MBP-C8) has been reported in the brains of multiple sclerosis (MS) patients. However, the involvement of the immune response to post-translational modified MBP in the pathophysiology of MS remains speculative. The aim of this study was to compare the levels of immunoglobulin G antibodies to several MBP epitopes, before and after citrullination, in the cerebrospinal fluid (CSF) and sera of MS patients using enzyme-linked immunosorbent assay (ELISA). We analyzed antibody reactivity against various MBP-peptides in the CSF and sera of 60 MS patients, and 30 patients with other neurological diseases (OND) as controls. The peptides tested were: MBP(75-98) (peptide 1), native (peptide 2) and citrullinated (peptide 3) MBP(108-126) (ARG(122)-->Cit(122)), and native (peptide 4) and citrullinated (peptide 5) MBP(151-170) (ARG(159, 170)-->Cit(159, 170)). All selected peptides could support an immune reactivity in CSF and sera of MS and OND patients. A higher reactivity against peptide 4 was found in the CSF of MS patients compared with OND patients (P<0.0001), but not against citrullinated peptides (peptides 3 and 5). However, we observed that the citrullination state of peptide 2 modified the patterns of immune reactivity more markedly in MS patients (P<0.0001) than in OND patients (P<0.02). Although some MBP epitopes could be a potential target in MS, our data did not demonstrate any difference of antibody response to MBP peptides in their citrullinated forms.     

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

The citrullination and N-terminus acylation of myelin basis protein (MBP) increases the heterogeneity among the MBP isoforms. The present study was undertaken to further characterize the immune response to the citrullinated from (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 acetyl 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.     

A comparison of immunochemical methods for the detection of antibodies to myelin encephalitogenic protein.

The antibody titer to myelin encephalitogenic protein in rabbits and rats has been comparatively studied by double antibody radioimmunoassay, quantitative microcomplement fixation, and radioimmunoelectrophoresis. In general, these immunochemical tests quantitatively paralleled one another but minor differences were sometimes present. The desirability of using a combination of these methods in assesing the antibody response to myelin encephalitogenic protein is emphasized.     

An immunochemical analysis of a myelin basic protein serum factor: cross reactivity with residues 69-71 of the rabbit encephalitogenic sequence 65-74 of myelin basic protein

A partially purified myelin basic protein serum factor (MBP-SF), cross-reactive with residues 65-74 (TTHYGSLPQK) of myelin basic protein, has been employed in an immunochemical study to identify the nature of the cross-reacting determinant more precisely. To probe the structural requirements of this determinant, Scatchard inhibition analyses and competitive peptide inhibition radioimmunoassays were employed with a series of peptide analogs of the 65-74 region and with three different reagent antisera: a rabbit anti-rat myelin antiserum (#My05) and two antisera, one rabbit (#162) and one chicken (#66), raised against synthetic peptide S24 (TTHYGSLPQKG). Scatchard inhibition analyses with MBP-SF revealed specific inhibition of binding of 125I-S24 to #162 and #My05, but not to #66. Further delineation of the structural requirements of the cross-reactive determinant, employing a liquid-phase radioimmunoassay, revealed a unique reactivity pattern for the chicken anti-S24 antiserum which, unlike #162 and #My05, did not cross-react under high-affinity conditions with synthetic peptide S20 (GSLPQK, representing the C-terminal half of S24). This, in concert with the Scatchard data, is suggestive of the presence of a cross-reactive determinant centered around residues 69-71 of MBP.     

Decreased myelin basic protein content of the aged human brain.

Myelin basic protein was extracted from frontal lobe white matter obtained at autopsy from seven individuals ranging in age from 72 to 78 years. Qualitative and quantitative comparisons were made between basic protein from this group and that extracted from another group of individuals ranging in age from 45 to 65 years. The basic protein content of the older brains was considerably lower than that of the younger brains. Qualitative studies that included assay for encephalitogenic activity, disk gel electrophoresis at pH 4.3, and immunoprecipitation did not reveal any difference in the basic protein derived from the two age groups. Possible factors that may help explain the decreased basic protein content of the aged brain are discussed.     

Cation-dependent extraction of basic protein from isolated human myelin

Cation-dependent acid protease activity associated with isolated human myelin was inhibited by pepstatin A, or enzymatic reactions were suppressed altogether by maintaining samples at 0°C rather than 37°C to examine whether or not they are involved in the extraction of myelin basic protein (MBP) by increased ionic strength. These measures largely abolished the degradation of MBP by acid protease activity associated with myelin, whereas the extraction of protein was only slightly diminished. Electrophoresis revealed that soluble protein was exclusively accounted for by undegraded MBP. Acid proteolysis, therefore, appears not to be involved in the cation-mediated removal of MBP from myelin. It is suggested that this mechanism may account for the appearance of undegraded MBP in body fluids, as well as for its pathologically increased degradation once it has become soluble.     

Restricted endogenous proteolysis of myelin basic protein of zinc-treated myelin.

Isolated myelin of bovine spinal cord was saturated with 500 mu ZnCl2 to immobilize myelin basic protein (MBP) and to assess the degradation of endogenous or exogenous MBP by neutral proteinase activity of myelin. While the initial degradation of endogenous MBP was not affected by Zn2+ a single fragment of approximately 17kDa accumulated in zinc-treated myelin as compared to several fragments in the control. In contrast exogenous MBP was fully degraded even by zinc-saturated myelin. In immobilizing MBP zinc appears to limit the access of endogenous proteinases to a terminal portion of the primary structure of myelin-associated MBP.     

An enzyme-linked immunosorbent assay for myelin basic protein-specific protein methylase I.

A sandwich enzyme-linked immunosorbent assay (ELISA) has been developed to determine myelin basic protein (MBP)-specific protein methylase I. Rabbit immunoglobulin anti-bovine MBP-specific protein methylase I, purified by Sepharose-A affinity chromatography, was utilized as the primary antibodies, while the same antibodies which had been conjugated to peroxidase were employed as the indicator antibodies. This assay method was about 280 times more sensitive than the conventional trichloracetic acid (TCA) precipitation method. Employing the ELISA, the level of MBP-specific protein methylase I during mouse brain development was examined; the peak level of the methylase was shown to be at 16th postnatal day, indicating temporal correlation with myelination. Among several species of brains examined, human showed the highest and carp the least amount of MBP-specific protein methylase I; 6.33 micrograms and 0.33 micrograms per mg of brain cytosol protein, respectively. Dysmyelinating jimpy hemizygous mouse brain showed the immunoreactive MBP-specific protein methylase only 60% that of the control at 20 days of age. The high sensitivity of the method together with the fact that MBP-specific protein methylase is present in human cerebrospinal fluid suggests a possible clinical application of this method for evaluating demyelinating disorders.     

Three isoforms of human myelin basic protein: Purification and structure

Myelin basic protein (MBP) occurs in multiple forms. Three of these isoforms from human MBP (HMBP) have been highly purified. HMBP, component 1 (18.5 kDa HMNP-1), was purified by ion-exchange chromatography at pH 10.6 in 2 M urea. During this ion-exchange chromatography, a fraction (Fraction 3), which contained HMBP component 3 (monophosphorylated or deamidated 18.5 kDa) and 17.2 kDa HMBP, was collected and further purified by fast protein liquid chromatography, which separated 17.2 kDa HMBP and HMDP component 3. When the latter was subjected to limited thrombic digestion, all of HMBP component 3 not phosphorylated at theonine 98 was cleaved. This digestion mixture was separated on Sephadex, and yielded pure component 3, monophosphorylated at theonine 98 (HMBP 3pT98), for which phosphate analysis yielded approximately 1 mole P/mole protein, and NMR showed only one phosphorylation site present. Circular dichroism (CD) studies were carried out on dilute solutions of HMBP-1 (18.5 kDa), 17.2 kDa HMBP, and HMBP3pT98 (phosphorylated 18.5 kDa). The CD spectrum of HMBP-1 was similar to that reported for rabbit MBP-1 and bovine MBP-1, but the spectra of 17.2 kDa HMBP and HMBP 3pT98 were distinctly different from HMBP-1. When analyzed by best-fit computations, 17.2 kDa HMBP showed about a 9% increase of ordered structure, and a greater increase, about 12%, was estimated for HMBP3pT98, attributable to β-structure and β turn. © 1995 Wiley-Liss, Inc.     

Epitopes of immunoreactive myelin basic protein in human cerebrospinal fluid

To define in more detail the features of the immunoreactive myelin basic protein (MBP) present in cerebrospinal fluid (CSF) of humans following acute injury to central nervous system myelin, the epitopes of MBP recognized by three different antisera, each capable of detecting immunoreactive MBP in CSF, were examined. All three antisera reacted well with human MBP and human MBP peptide 45-89. Only in radioimmunoassays in which the MBP peptide 45-89 served as the radioligand could clearly elevated values of immunoreactive MBP be measured in CSF specimens from 5 patients with multiple sclerosis during or immediately after an exacerbation. The two antisera that reacted well with MBP peptide 80-89 resulted in higher levels of immunoreactive MBP measured in CSF. An epitope present in human MBP peptide 80-89 but sharing a conformation with both MBP and MBP peptide 45-89 is present in CSF following acute central nervous system myelin damage in multiple sclerosis.     

Interaction of myelin basic protein and proteolipid protein

The interaction of myelin basic protein (MBP) and proteolipid protein (PLP) was studied using a microtitre well binding assay and the ligand-blot overlay technique. The binding of iodinated PLP to MBP that was immobilized on microtitre wells was saturable and reversible. Its selectivity was investigated by the ligand-blot overlay technique. Iodinated PLP was found to bind MBP but not any other CNS myelin proteins. This interaction was not dependent on the phosphoryl moiety of MBP. Binding of PLP to histone H4 also occurred, but the amount of PLP bound per unit MBP was greater than for this histone.     

Human myelin basic protein peptide 69-89: immunochemical features and use in immunoassays of cerebrospinal fluid

The characteristics and heterogeneity of the myelin basic protein (MBP)-like material appearing in cerebrospinal fluid (CSF) after acute central nervous system (CNS) myelin injury are unresolved. Antigenic material containing an epitope in the carboxyl-terminal portion of human MBP peptide 45-89 (from the intact molecule of 170 residues) is a prominent species of the MBP-like material present. In an effort to define further the MBP-like material in CSF and to enhance its detection, a modified double-antibody radioimmunoassay has been developed using a radioligand of human MBP synthetic peptide 69-89. This assay is more sensitive with results paralleling those of previously used MBP assays for CNS myelin damage. Results with this assay provide additional confirmation of the presence of an epitope of MBP in the decapeptide of MBP 80-89 but in a conformation simulating that of intact MBP in CSF after CNS myelin injury. Unexpected buffer effects were noted to influence the immunochemical behavior of some of the small peptides of MBP.     

Antibody to peptides of human myelin basic protein in post-rabies vaccine encephalomyelitis sera.

Development of neurologic complications after Semple rabies vaccine is closely linked to development of antibody to myelin basic protein (MBP). The portions of MBP against which the antibodies are directed were analyzed by enzyme immunoassay in sera and cerebrospinal fluid from 27 patients with vaccine complications. Most of the antibody was directed to regions of MBP peptides 45-89 and 90-170. There was no apparent correlation between antibody specificity for MBP peptides 1-44, 45-89 and 90-170 and the type of post-vaccinal neurologic complication. We conclude that the immunoglobulin repertoire in human B lymphocytes for responding to human MBP favors the portion of the MBP molecule containing residues 45-170.     

Antibodies specific for the lipid-bound form of myelin basic protein during experimental autoimmune encephalomyelitis

On the hypothesis that myelin basic protein isolated with surrounding lipids may constitute an autoantigen in demyelinating diseases we studied the antibody response to the lipid-free and lipid-bound form of myelin basic protein during the course of experimental autoimmune encephalomyelitis induced in rats with either form of protein. Immunization with the lipid-bound form of myelin basic protein induced high titres of antibodies directed to the protein, accompanied by no antibodies to cerebroside 30 days after immunization. Antibodies specifically directed to the lipid-bound form of myelin basic protein were revealed after removal of antibodies recognizing the delipidated myelin basic protein. Anti lipid-bound myelin basic protein antibodies could already be detected at day 10 post-immunization, reaching a maximum at day 20 post-immunization. Demonstrations of antibodies entirely specific for the lipid-bound form of myelin basic protein suggests that this molecule may present epitopes not to be found in its already extensively studied primary structure, possibly the result of conformational changes following lipid binding.     

Purification of autoantibodies to myelin basic protein by antigen specific affinity chromatography from cerebrospinal fluid IgG of multiple sclerosis patients. Immunoreactivity studies with human myelin basic protein.

Immunoglobulin G (IgG) was purified by single-step protein A-Sepharose (Pharmacia) affinity chromatography from the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients and controls. Autoantibodies to myelin basic protein (anti-MBP) were isolated from the purified IgG fraction by two-step antigen specific affinity chromatography. Anti-MBP in the context of whole CSF or in purified form reacts equally to MBP prepared from non-MS or MS brain tissue. Kinetic studies of anti-MBP titers demonstrate that when anti-MBP is reacted with increasing amounts of non-MS or MS MBP, the autoantibody is immunoabsorbed by either antigen in vitro. Immunoabsorption of anti-MBP by MBP or its synthetic peptides may also be possible in vivo as a potential therapeutic tool.     

Immunocytochemical method to identify myelin basic protein in oligodendroglia and myelin sheaths of the human nervous system

To study the distribution of basic protein (BP) and other myelin constituents immunocytochemically in human nervous tissue, we modified the unlabeled antibody enzyme (peroxidase-antiperoxidase) method. The technique is described here. Because the availability of unfixed tissue from human central nervous system is limited, we tested the method on blocks that had been fixed in formalin and embedded in paraffin, fixed and stored in 4% formalin, or frozen at autopsy and stored. We obtained the best results with paraffin blocks. BP antiserum stained oligodendroglia and myelin sheaths in the developing human nervous system. In the adult, myelin sheaths were well stained. Also, abnormalities associated with myelin breakdown could be identified in multiple sclerosis lesions. The results suggest that this method will be useful in studying the cellular distribution of myelin components in human demyelinating diseases.