&p.1:Abstract Myelin oligodendrocyte glycoprotein (MOG) is a member of the immunoglobulin superfamily ex- pressed exclusively in central nervous system ...
J Mol Med (1997) 75:77–88
© Springer-Verlag 1997
REVIEW
&roles:C.C.A. Bernard · T.G. Johns · A. Slavin · M. Ichikawa C. Ewing · J. Liu · J. Bettadapura
Myelin oligodendrocyte glycoprotein: a novel candidate autoantigen in multiple sclerosis
&misc:Received: 6 June 1996 / Accepted: 20 August 1996
&p.1:Abstract Myelin oligodendrocyte glycoprotein (MOG) is a member of the immunoglobulin superfamily expressed exclusively in central nervous system (CNS) myelin. While the function of MOG is unknown, a number of studies have shown that immune responses to MOG contribute to the autoimmune-mediated demyelination seen in animals immunized with whole CNS tissue. This paper summarizes our recent studies, which unequivocally demonstrate that MOG by itself is able to generate both an encephalitogenic T cell response and an autoantibody response in Lewis rats and in several strains of mice. In Lewis rats the injection of both native MOG and MOG35–55 peptide produces a paralytic relapsing-remitting neurological disease with extensive plaque-like demyelination. The antibody response to MOG35–55 was highly restricted, as no reactivity to either other MOG peptides or myelin proteins could be detected. Fine epitope mapping showed that antibody from serum and cerebrospinal fluid of injected rats reacted strongly to MOG37–46, which is contiguous to the dominant T cell epitope contained within MOG44–55. NOD/Lt and C57BL/6 mice were also susceptible to severe neurological disease following injection with recombinant MOG or MOG35–55 peptide, indicating that this specific CNS autoantigen, or some of its determinants, can induce a pathogenic response across animal species. Severe paralysis and extensive demyelination were seen in both strains, but NOD/Lt mice experienced a chronic relapsing disease whereas C57BL/6 mice had a chronic non-remitting disease. Moreover, transfer of MOG35–55 T cells into naive NOD/Lt mice also produced severe neurological impairment as well as histological lesions. These results emphasize that a synergism between a T cell-response and anti-MOG antibodies may be important for C.C.A. Bernard (✉) · T.G. Johns · A. Slavin · M. Ichikawa C. Ewing · J. Liu · J. Bettadapura Neuroimmunology Laboratory, Faculty of Science and Technology, La Trobe University, Bundoora, Melbourne, Victoria, 3083, Australia Communicated by: H.D. Perez and G. Stock&/fn-block:
the development of severe demyelinating disease. This, together with our demonstration that there is a predominant T cell response to MOG in patients with multiple sclerosis, clearly indicates that MOG is probably an important target autoantigen in this disease. &kwd:Key words Myelin oligodendrocyte glycoprotein · Multiple sclerosis · Experimental autoimmune encephalomyelitis · Demyelination · Autoantigen Abbreviations CFA Complete Freund’s adjuvant · CSF Cerebrospinal fluid · CNS Central nervous system · EAE Experimental autoimmune encephalomyelitis · MAG Myelin associated glycoprotein · MBP Myelin basic protein · MHC Major histocompatibility complex · MOG Myelin oligodendrocyte glycoprotein · rMOG recombinant MOG · MS Multiple sclerosis · PBL Peripheral blood lymphocytes · PLP Proteolipid protein · SI Stimulation index&bdy:
Introduction Multiple sclerosis (MS) is a chronic disease of the central nervous system (CNS), characterized by perivascular inflammation and localized myelin destruction [1]. Despite scientific and clinical research into MS, the target(s) of this inflammatory response have remained elusive. It might be an infectious agent, a component of self or a pathogen that contains a peptide that mimics self. As yet no infectious agent(s) responsible for triggering MS have been identified [2], despite the use of highly sensitive techniques [3, 4]. However, it is plausible that a combination of environmental factors in a susceptible host could lead to the development of an autoreactive phenomenon [5]. The concept that both genes and environment can contribute to the development of MS is best illustrated by molecular mimicry. This idea proposes that the activation of peripheral T cells by viral or bacterial peptides that are homologous to or mimic specific self antigens
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may inadvertently produce an autoimmune response [6, 7]. Thus, molecular mimicry depends on the pathogens an individual is exposed to (the environmental factor) and how this particular individual processes and presents the resulting peptides to T cells (the genetic factor). Renewal interest in the relevance of molecular mimicry to MS has been stimulated by a recent report by Wucherpfenning and Strominger [6]. This study demonstrated that structurally related peptides from various pathogens including influenza, herpes virus, Epstein-Barr virus, papilloma virus and Pseudomonas are able to stimulate MBP-specific T cell clones established from MS patients. Importantly, the peptides chosen did not necessarily have strict sequence homology to the 94–101 myelin basic protein (MBP) peptide used to generate the T cell clones but rather contained conserved amino acids at several key positions, know to be important in both major histocompatibility complex (MHC) molecule and T cell receptor interactions [6]. The hypothesis that MS is an immunopathological disease mediated by an immune reaction against CNS antigens, in particular to the quantitatively major myelin proteins, MBP and proteolipid protein (PLP) derives largely from a morphological resemblance with the animal model, experimental autoimmune encephalomyelitis (EAE) [8]. Although the importance of cellular immunity to these specific myelin antigens in the pathogenesis of EAE is uncontested, there is a lack of consistent evidence for sensitization to these autoantigens in MS. Nevertheless, it is known that active sites of demyelination contain perivascular cells consisting predominantly of macrophages, B cells, antigen-presenting cells expressing MHC class II antigens, and CD4+-activated T lymphocytes secreting various cytokines [9]. Consistent with the demonstration that B cells are present in MS brain lesions is the finding that intrathecally produced immunoglobulins are a biochemical marker of the disease [10, 11]. Indeed, approximately 90% of patients with MS have oligoclonal IgG in their cerebrospinal fluid (CSF), with some of these having reactivity to myelin antigens [12–14]. The accumulation of activated T cells in MS lesions, the periplaque area and in surrounding normal-appearing white matter [9, 15] seemingly points to the importance of cell-mediated immune reactions in the pathogenesis of the disease. Although T cell responses to MBP and PLP are likely to be important in the course of MS, it is now recognized that autoimmune sensitization to quantitatively minor myelin autoantigens may play an equivalent or more important role in the disease [16–18]. Since 1976, Lebar and his colleagues [16, 19, 20] have argued that the demyelinating effect observed in animals injected with whole CNS homogenate is mediated by antibodies to a myelin component, termed M2, rather than to myelin proteins such as MBP and PLP. On the basis of immunological reactivity, tissue and cellular localization and molecular weight, M2 has now been identified as myelin oligodendrocyte glycoprotein (MOG) [17, 20–22]. The observations of Lebar et al. [16, 19, 20], together with studies of the demyelinating effects of
anti-MOG antibodies in vitro [23] and in vivo [24–26] suggested to us that MOG could be one of the CNS antigens responsible for the initiation of primary autoimmune-mediated demyelination. MOG is an attractive primary target antigen for autoimmune attack in MS since it is, to the best of our knowledge, the first myelin antigen which meets all known pathogenic requirements for eliciting demyelination of the CNS. Firstly, MOG is directly accessible to an humoral immune response because of its external location on oligodendrocyte surfaces at the outermost lamellae of myelin sheaths [22]. Secondly, it appears to be a specific antigen of CNS myelin as MOG has not been detected at the protein or mRNA level in peripheral nerve or other tissues analysed [21, 27]. Thirdly, purified anti-MOG antibody can cause demyelination when introduced into the CNS, either directly or indirectly [24–26, 28]. Animals with acute EAE induced by injection of MBP (or transfer of MBP-specific T cells) show demyelination in the CNS after anti-MOG antibody is given intravenously because the antibody is able to enter the CNS through the breached blood-brain barrier [24–26]. Intrathecal injection of anti-MOG antibody into normal rats also produces CNS demyelination [28]. Demyelination by anti-MOG antibody has also been demonstrated in vitro using CNS aggregating cell cultures [23]. Fourthly, demyelinating anti-MOG antibodies are present in sera from animals with chronic relapsing EAE provoked by whole CNS tissue homogenate, but antibodies to the more abundant myelin antigens (MBP and PLP) are absent [16, 19, 20]. Finally, in chronic relapsing EAE the serum demyelinating activity, assayed in vivo by intrathecal injection into normal rats, appears to be proportional to anti-MOG antibody titre [29]. The aforementioned studies also suggest that, although MOG is present in low amounts in myelin (only 0.05% of the total myelin proteins) [30, 31], it must have a high immunogenic potential since immunization of rats and guinea pigs with whole CNS tissue homogenate generates detectable levels of anti-MOG antibodies [16, 28]. The mechanisms by which such antibodies produce demyelination are varied but include the activation of myelin proteases leading to MBP degradation [32], the activation of complement [33] and the enhanced phagocytosis of myelin by macrophages [33]. This paper reviews the literature with regard to the immune responses elicited by MOG in both MS and EAE and summarizes the extensive work carried out in our laboratory.
Biochemical analysis of MOG While the work of Lebar et al. [16, 19, 20] had identified M2 as a novel CNS protein and showed its importance as an immune target in chronic relapsing EAE, it was Linington et al. [21] who first defined this protein by the 818C5 mouse monoclonal antibody. Immunocytochemical studies using this antibody demonstrated that MOG is specific to CNS myelin and is expressed preferentially at the extracellular surface of both oligodendrocytes and
79 Table 1 Biochemical properties of MOG&/tbl.c:& * CNS specific * Located mostly in outermost lamellae of myelin sheath and on the surface of oligodendrocytes * Surface marker of oligodendrocyte maturation * Expression parallels myelination * Quantitatively minor component of myelin (0.05%) * Glycoprotein; MW: 26/28 kD and 53/55 kD on SDS-PAGE * pI=8.87 (non-glycosylated) * Asn31-site for N-glycosylation * Thr167-possible phosphorylation site * Highly conserved between species * Maps to the MHC region in mouse, rat and human &/tbl.:
the myelin sheath [22, 34]. MOG shows a caudo-rostral gradient of expression throughout development, similar to that of other myelin proteins including MBP and PLP, although its appearance is delayed by some 1–2 days [34, 35]. The known biochemical properties of MOG are described in Table 1. The literature contains various reports for the molecular weight of MOG. It was originally claimed that rat MOG is a 51-kDa protein that degraded to 20- to 26-kDa products [21]. Later reports, however, described murine MOG as a 26/28-kDa doublet resulting from the differential glycosylation of a 25-kDa protein core that dimerizes to a protein of 54 kDa [31]. We showed that human MOG consists of two major bands of molecular weight 28 and 58 kDa with a minor band at 35 kDa [30]. The Nterminal sequence of the two major species is identical [30], further supporting the notion that the upper band is a dimer of MOG. A similar result to that of human MOG was obtained by Birling et al. [36] using purified bovine MOG. Preliminary experiments performed in our laboratory with recombinant MOG (rMOG) protein expressed in baculovirus-infected insect cells suggest that the upper band seen on SDS-PAGE is indeed a MOG dimer. The cloning of cDNA for MOG from mouse, rat, marsupial cat, bovine and human [27, 37–39] revealed that MOG is 218 amino acids in length, is a member of the immunoglobulin superfamily and is highly homologous (>90%) between species. MOG is a somewhat unusual member of this family in that it contains two putative transmembrane regions, suggesting its C-terminal tail is also extracellular [27]. As indicated by biochemical studies, the MOG protein contains an N-glycosylation site found at the Asn at amino acid 31. There is also a putative phosphorylation site at the intracellular Thr, found at the position 167, but this has yet to be confirmed. Initial mRNA studies of MOG detected only a single mRNA transcript [27]. Recently, more sensitive methods have detected several mRNA transcripts resulting from a single MOG gene [37, 40]. These mRNA splice variants would result in either truncated or C-terminal modified MOG species, but their expression as protein isoforms has not yet been reported.
Possible functions of MOG Comparison of MOG with sequence databases revealed extensive homology between the immunoglobulin domain of MOG with the immunoglobulin domains of bovine butyrophilin and the chicken B-G antigen [27]. This homology, however, failed to identify a function for MOG as it appears to have arisen from the duplication of a common ancestral exon encoding the immunoglobulin domain and does not extend to the functionally important transmembrane and cytoplasmic regions [38]. On the other hand, the extracellular location of MOG [22, 27], its identification as a member of the immunoglobulin superfamily [27] and the presence of a L2/HNK-1 carbohydrate epitope [41] all suggest that MOG functions as an adhesion molecule or cellular receptor. Furthermore, as MOG is expressed late in development compared to other myelin proteins [34], it is generally considered that it has a role in the completion and/or compaction of the myelin sheath. In this context, the L2/HNK-1 epitope found on MOG has also been identified on two other myelin-specific glycoproteins, myelin-associated glycoprotein and Po, both of which are known to have adhesive properties [41]. As CNS myelin sheaths appear to be in contact with each other, as opposed to PNS myelin sheaths where MOG is absent, it is attractive to hypothesize that MOG may play a role in the adhesion between neighbouring myelinated fibres, functioning as the “glue” in the maintenance of axon bundles. At this time, however, there is no direct in vitro or in vivo evidence supporting this view and a potential ligand for MOG has not been identified. Dyer and Matthieu [42] are the only investigators to have reported experimental evidence suggesting a possible function of MOG. In this study they showed that the 8-18C5 anti-MOG antibody may mimic a putative ligand for MOG, as its addition to oligodendrocytes in culture causes depolymerization of microtubules. As MBP regulates the stability of microtubules in oligodendrocytes [43], it was suggested that MBP has a role in the depolymerization process. Our work showing that the 8-18C5 antibody stimulates the breakdown of MBP in isolated myelin [32] establishes a possible mechanism for this observation. Since ongoing microtubule turnover in oligodendrocytes is important for the myelination process, one of the functions of MOG may be to regulate microtubule stability, particularly as levels of MBP increase during myelination. Finally, the mapping of the MOG gene within the MHC [38], its homology to the B-G antigens of the chicken MHC [27] and its ability to elicit strong immune responses [29] have led some to speculate that MOG has an immune function within the CNS [44]. However, there are no direct data supporting this hypothesis although it remains an interesting possibility, since such a function may be important in the pathogenesis of MS. Preliminary data from our laboratory indicates that both native and recombinant human MOG are able to bind Clq. If this can be confirmed, then MOG may be the molecule that gives CNS myelin its intrinsic ability to
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activate the complement cascade [45], a mechanism that may contribute to the demyelination process. Thus, MOG may not only be a putative autoantigen in MS but may have a direct influence on the immune response.
T cell responses to MOG in MS The realization that autoimmune recognition of quantitatively minor CNS components in MS (either myelin or non-myelin specific) could also have a prevalent role in disease initiation or progression, prompted us to investigate the relevance of MOG as a novel autoantigen in MS. For this, we examined the reactivity of peripheral blood lymphocytes (PBLs) of MS patients and control individuals to MOG and concomitantly compared such reactivity to other putative myelin antigens including MBP, PLP and myelin-associated glycoprotein (MAG). As a prerequisite for these studies we developed a procedure which allowed us to purify human MOG to homogeneity in sufficient quantities to perform these investigations [30]. As described in detail elsewhere [46], the greatest incidence of proliferative responses by MS PBLs was to MOG, with 12 out of the 24 (50%) patients tested reacting to this antigen. Of these, 8 reacted to MOG exclusively. Only 5 and 2 MS patients responded to MBP and PLP, respectively, and none were significantly positive for MAG. The only individual of the control group (n=16) who reacted positively to MOG had a stimulation index (SI) of 2.3. Interestingly, the PBLs from this control individual also reacted to MBP (SI=2.3). Statistical analysis of the association between MS and responsiveness to each of the specific myelin antigens tested revealed that relationship between MS and responsiveness to MOG was the only one which was statistically significant (P
