The basic protein is a major structural protein of the central nervous system myelin in which it constitutes 30% of the total protein.(1)
T lymphocyte cell lines selected with whole MBP typically recognize only 1-3 immunodominant epitopes (Offner et al., 1987, 1988; Trotter et al., 1987: Bourdctte et al., 1988; McCarron and McFarlin, 1988) and these immunodominant MBP epitopes frequently are cncephalitogenic (Vandenbark et al., 1985b, 1989; Hashim et al., 1991). Thus, the identification of immunodominant MBP epitopes is likely to provide an in vitro method of identifying unrecognized enccphalitogenic epitopes of MBP. (2) 12a
Although the cause of multiple sclerosis (MS) is not known, a T cell-mediated autoimmune process has been postulated. Myelin basic protein (MBP) is a potential target antigen because it induces experimental allergic encephalomyelitis (EAE) in susceptible animals. Encephalitogenic epitopes of MBP differ among susceptible strains and correlate with the MHC class II genotype. (3)
Figure1. Structure of Myelin Basic Protein
Figure2. Formula of Myelin Basic Protein (87-99)
1. EYLAR, E. H., in L.Rowland (Editor), Immunologic disorders of the nervous system, Academic Press, New York, 1971, Chapter 5.
2. Jones et al (1992) the synthetic 87-99 peptide of myelin basic protein is encelphalitogenic in Buffalo rats. J.Neuroimmunol. 37 203
3. Fritz, R., and D.E. McFarlin . 1989. Encephalitogenic epitopes 10. Richert, J., E.D. Robinson, G.E . Deibler, R.E. Martenson, of myelin basic protein. Chem. Immunol. 46:101.
A cyclic analogue, [cyclo(87-99)MBP(87)(-)(99)], of the human immunodominant MBP(87)(-)(99) epitope, was designed based on ROESY/NMR distance information and modeling data for linear epitope 87-99, taking into account T-cell (Phe(89), Lys(91), Pro(96)) and HLA (His(88), Phe(90), Ile(93)) contact side-chain information. The cyclic analogue was found to induce experimental allergic encephalomyelitis (EAE), to bind HLA-DR4, and to increase CD4 T-cell line proliferation, like that of the conformationally related linear MBP(87)(-)(99) epitope peptide. The mutant cyclic peptides, the cyclo(91-99)[Ala(96)]MBP(87)(-)(99) and the cyclo(87-99)[Arg(91)Ala(96)]MBP(87)(-)(99), reported previously for suppressing, to a varying degree, autoimmune encephalomyelitis in a rat animal model, were found in this study to possess the following immunomodulatory properties: (i) they suppressed the proliferation of a CD4 T-cell line raised from a multiple sclerosis patient, (ii) they scored the best in vitro TH2/TH1 cytokine ratio in peripheral blood mononuclear cell cultures derived from 13 multiple sclerosis patients, inducing IL-10 selectively, and (iii) they bound to HLA-DR4, first to be reported for cyclic MBP peptides. In addition, cyclic peptides were found to be more stable to lysosomal enzymes and Cathepsin B, D, and H, compared to their linear counterparts. Taken together, these data render cyclic mimics as putative drugs for treating multiple sclerosis and potentially other Th1-mediated autoimmune diseases.
This work reports molecular dynamics studies at the receptor level of the immunodominant myelin basic protein (MBP) epitope 87-99 implicated in multiple sclerosis, and its antagonists altered peptide ligands (APLs), namely [Arg91, Ala96] MBP87-99 and [Ala91,96] MBP87-99. The interaction of each peptide ligand with the receptor human leukocyte antigen HLA-DR2b was studied, starting from X-ray structure with pdb code: 1ymm. This is the first such study of APL-HLA-DR2b complexes, and hence the first attempt to gain a better understanding of the molecular recognition mechanisms that underlie TCR antagonism by these APLs. The amino acids His88 and Phe89 serve as T-cell receptor (TCR) anchors in the formation of the trimolecular complex TCR-peptide-HLA-DR2b, where the TCR binds in a diagonal, off-centered mode to the peptide-HLA complex. The present findings indicate that these two amino acids have a different orientation in the APLs [Arg91, Ala96] MBP87-99 and [Ala91,96] MBP87-99: His88 and Phe89 remain buried in HLA grooves and are not available for interaction with the TCR. We propose that this different topology could provide a possible mechanism of action for TCR antagonism.
Derangement of cellular immunity is central in the pathophysiology of multiple sclerosis (MS) and is often manifested by abnormal cytokine production. We investigated cytokine secretion in peripheral blood mononuclear cells (PBMC) of 18 MS patients and 15 controls and correlated cytokine polarization with the nature of antigenic stimulus. We synthesized two novel citrullinated peptides, linear [Cit(91), Ala(96), Cit(97)]MBP(87-99) and cyclo(87-99)[Cit(91), Ala(96), Cit(97)]MBP(87-99) that resulted from citrullination of 91,97 Arg residues in antagonists, linear [Arg(91), Ala(96)]MBP(87-99) and cyclo(87-99)[Arg(91), Ala(96)]MBP(87-99) peptides. PBMC from MS patients and controls were cultured with citrullinated peptides, and both peptides caused a Th1 polarization in all MS patients studied. In contrast, culture with noncitrullinated MBP peptides resulted in heterogeneous cytokine secretion that differed between individual patients. Thus, citrullination of self-antigens may potentially trigger disease in susceptible individuals. This finding may open new avenues in drug design of new substances that inhibit citrullination and arrest epitope spreading and worsening of MS.
This work proposes a structural motif for the inhibition of experimental autoimmune encephalomyelitis (EAE) by the linear altered peptide ligands (APLs) [Ala91,96] MBP87-99 and [Arg91,Ala96] MBP87-99 of myelin basic protein. Molecular dynamics was applied to reveal distinct populations of EAE antagonist [Ala91,96] MBP87-99 in solution, in agreement with NOE data. The combination of the theoretical and experimental results led to the identification of a putative active conformation. This approach is of value as no crystallographic data is available for the APL-receptor complex. TCR contact residue Phe89 has an altered topology in the putative bioactive conformations of both APLs with respect to the native peptide, as found via crystallography; it is no longer prominent and solvent exposed. It is proposed that the antagonistic activity of the APLs is due to their binding to MHC, preventing the binding of self-myelin epitopes, with the absence of an immunologic response as the loss of some interactions with the TCR hinders activation of T-cells.
A synthetic peptide corresponding to residues 87-99 (S87-99) of myelin basic protein (BP) induced the proliferation of an encephalitogenic, BP-specific T cell line selected in vitro from inbred Buffalo-strain rats (RT1b). Active immunization with guinea pig (GP)-BP or S87-99 in complete Freund's adjuvant (CFA) and intravenous pertussigen induced acute experimental autoimmune encephalomyelitis (EAE) 10-12 days after immunization. Fifty percent of recovered rats developed a single relapse 17-21 days after immunization. T lymphocytes selected in vitro with S87-99 transferred acute, non-relapsing EAE into naive recipients. Histological examination during acute EAE revealed foci of inflammatory cells associated with demyelination in the spinal cords and peripheral nerve roots. Thus, as in several other rodent strains, the 87-99 region of BP is antigenic and encephalitogenic in the inbred Buffalo-strain rat. Additionally, the 87-99 sequence of GP-BP was predicted to be antigenic by two different methods. These results suggest that the 87-99 region of BP, which is highly conserved among mammalian species, may be widely encephalitogenic due to antigen-intrinsic properties.
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