A Molecular Model of Myelin Oligodendrocyte Glycoprotein

Abstract: Myelin oligodendrocyte glycoprotein (MOG) is a protein on the surface of myelin sheaths. It is a putative target of the autoimmune attack in the inflammatory and demyelinating CNS disease multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. MOG belongs to the immunoglobulin superfamily (IgSF), and its extracellular N-terminal domain contains many conserved IgSF consensus residues seen in immunoglobulin variable region folds. The aim of the present study was to create a molecular model of the extracellular N-terminal domain of mouse MOG. No crystal structure is yet available of MOG, and thus a molecular model would be useful in providing insight into its structure and binding characteristics. Molecular graphics techniques and molecular dynamics with secondary structure-based restraints were used in the construction and refinement of the MOG model. Regions of high prediction confidence were identified, and possible glycosylation, dimerization, complement binding, and antibody-binding regions in MOG were mapped and analyzed.     

Investigation of Myelin/Oligodendrocyte Glycoprotein Membrane Topology

Abstract: Myelin/oligodendrocyte glycoprotein (MOG) is a CNS-specific integral membrane protein that is an atypical member of the immunoglobulin (Ig) superfamily with two potential transmembrane domains based upon hydropathy analysis. With only one other exception, all Ig family members possess a single or no membrane spanning region. In order to analyze MOG membrane topology, we prepared stably transfected cells that express mouse MOG and used three domain-specific antisera to ascertain the localization of these hydrophilic domains. As expected, MOG's glycosylated N-terminal Ig-like domain was identified as extracellular, because membrane permeabilization was not required for immunoreactivity with the MOG_1–125 antiserum. In contrast, both MOG_154–169 and MOG_198–218 antisera stained cells only upon permeabilization. These data indicate that only MOG's N-terminal hydrophobic domain spans the lipid bilayer, and we propose that MOG's C-terminal hydrophobic domain associates with the cytoplasmic face of the plasma membrane. As for MOG's second hydrophobic domain, it is clear that either orientation (transmembrane versus membrane-associated) would be unique among Ig-like proteins, and the implications of our proposed topology for MOG in oligodendroglial plasma membrane are discussed.     

The Structure and Function of Myelin Oligodendrocyte Glycoprotein

Abstract : Myelin oligodendrocyte glycoprotein (MOG) is a quantitatively minor component of CNS myelin whose function remains relatively unknown. As MOG is an autoantigen capable of producing a demyelinating multiple sclerosis-like disease in mice and rats, much of the research directed toward MOG has been immunological in nature. Although the function of MOG is yet to be elucidated, there is now a relatively large amount of biochemical and molecular data relating to MOG. Here we summarize this information and include our recent findings pertaining to the cloning of the marsupial MOG gene. On the basis of this knowledge we suggest three possible functions for MOG : (a) a cellular adhesive molecule, (b) a regulator of oligodendrocyte microtubule stability, and (c) a mediator of interactions between myelin and the immune system, in particular, the complement cascade. Given that antibodies to MOG and to the myelin-specific glycolipid galactocerebroside (Gal-C) both activate the same signaling pathway leading to MBP degradation, we propose that there is a direct interaction between the membrane-associated regions of MOG and Gal-C. Such an interaction may have important consequences regarding the membrane topology and function of both molecules. Finally, we examine how polymorphisms and/or mutations to the MOG gene could contribute to the pathogenesis of multiple sclerosis.     

The Expression Patterns of Nogo-A,Myelin Associated Glycoprotein and Oligodendrocyte Myelin Glycoprotein in the Retina After Ocular Hypertension

Nogo-A, a major myelin inhibitory protein, inhibits axon growth and synaptic function in the central nervous system. Glaucoma is a progressive neuropathy as a result of retinal ganglion cell (RGC) death. Synaptic degeneration is thought to be an early pathology of neurodegeneration in glaucoma and precedes RGC loss. Here experimental ocular hypertension model was induced in adult rats with laser coagulation of the episcleral and limbal veins. The expression of Nogo-A, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp) in the retina was investigated using immunohistochemistry and Western blotting. We found that Nogo-A was expressed in the RGCs and upregulated after the induction of ocular hypertension. OMgp was only expressed in the inner plexiform layer. There was no MAG expression in the retina. Our data provided, for the first time, the expression patterns of three myelin proteins in the adult retina and suggested an important role of Nogo-A in the RGC death and synaptic degeneration in glaucoma.     

Demyelinating Antibodies to Myelin Oligodendrocyte Glycoprotein and Galactocerebroside Induce Degradation of Myelin Basic Protein in Isolated Human Myelin

Abstract: Although the specificity of multiple sclerosis (MS) brain immunoglobulins (lgs) remains unknown, the incubation of these lgs with human myelin can lead to myelin basic protein (MBP) degradation mediated by neutral proteases. In this study, we demonstrate that monoclonal antibodies (mAbs) specific to myelin components such as the CNS-specific myelin oligodendrocyte glycoprotein (MOG) and galactocerebroside (GalC) are found to induce a significant loss of MBP mediated by neutral proteases in myelin. By contrast, antibodies to periaxonal and structural components of myelin, such as MBP and myelin-associated glycoprotein, are ineffective in inducing such MBP degradation. Among the 11 different anti-MOG mAbs directed to externally located epitopes of MOG, only two were found to induce a significant degradation of MBP, suggesting that antibody-induced MBP degradation is not only antigen specific but also epitope specific. Based on the inhibition of MBP degradation in the presence of EGTA and the analysis of the degradation products obtained following incubation of myelin with mAbs to GalC and MOG (8-18C5), the neutral protease involved in this antibody-induced degradation of MBP could be calcium-activated neutral protease. Taken together, these results suggest that antibodies to GalC and MOG can play a major role in destabilizing myelin through MBP breakdown mediated by neutral proteases and thus have an important role to play in the pathogenesis of MS.     

不同剂量MOG抗原免疫诱导小鼠自身免疫性脑脊髓炎模型的比较

目的比较不同剂量髓鞘少突胶质细胞糖蛋白(myelin oligodendrocyte glycoprotein,MOG35-55)免疫诱导C57BL/6小鼠实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)的作用。方法将C57BL/6小鼠分为正常组和三组不同剂量MOG35-55诱导的EAE模型组,共4组。模型组分别以每只200、100、50μg的MOG35-55与完全弗氏佐剂(complete Freund s adjuvant,CFA)混合的乳化抗原皮下注射免疫诱导EAE模型,正常组以生理盐水替代。观察不同剂量MOG35-55对C57BL/6小鼠体重、发病率以及神经功能评分等影响,同时取小鼠脑和脊髓,利用光镜和透射电镜观察小鼠病理组织学改变。结果三组不同剂量MOG35-55均能诱导EAE模型,发病率为100%,呈慢性单相病程,病理学观察发现小鼠脑和脊髓有炎性细胞浸润、脱髓鞘及轴突损伤等改变。但小剂量组在体重减轻、临床症状评分及病理学改变等方面均较中、大剂量组明显。结论用MOG35-5550μg剂量免疫诱导的C57BL/6小鼠EAE模型稳定,可在今后的研究中应用。     

Expression, Purification, and Encephalitogenicity of Recombinant Human Myelin Oligodendrocyte Glycoprotein

Abstract: Myelin oligodendrocyte glycoprotein (MOG), a putative autoantigen in multiple sclerosis (MS), is a quantitatively minor component of the CNS. In view of the difficulties associated with the purification of MOG from brain tissues, the extracellular domain of human MOG corresponding to the N-terminal 121 amino acids was expressed in Escherichia coli as a glutathione sulfotransferase fusion protein. The expressed protein was localized to inclusion bodies, and varying the growth parameters resulted in the solubilization of small amounts of GST-MOG that could be affinity purified on glutathione agarose columns. The fusion protein found in the inclusion bodies could be solubilized with urea. The solubilized fusion protein was cleaved with thrombin, and the extracellular domain was purified by CM Sephadex 50 chromatography to homogeneity. Injection of recombinant human MOG into different strains of mice resulted in the induction of an MS-like disease, characterized by severe neurological impairment and extensive CNS demyelinated lesions. Recombinant MOG produced in E. coli should prove to be useful as a highly purified biological reagent for immunological, pathological, functional, and structural studies.     

Purification and Partial Structural and Functional Characterization of Mouse Myelin/Oligodendrocyte Glycoprotein

The myelin/oligodendrocyte glycoprotein (MOG) is found exclusively in the CNS, where it is localized on the surface of myelin and oligodendrocyte cytoplasmic membranes. The monoclonal antibody 8-18C5 identifies MOG. Several studies have shown that anti-MOG antibodies can induce demyelination, thus inferring an important role in myelin stability. In this study, we demonstrate that MOG consists of two polypeptides, with molecular masses of 26 and 28 kDa. This doublet becomes a single 25-kDa band after deglycosylation with trifluoromethanesulfonic acid or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, indicating that there are no or few O-linked sugars and that the doublet band represents differential glycosylation. Partial trypsin cleavage, which also gave a doublet band of lower molecular weight, confirmed this idea. MOG was purified by polyacrylamide gel electrophoresis, followed by electroelution. Three N-terminal sequences of eight to 26 amino acids were obtained. By western blot analysis, no binding was found between MOG and cerebellar soluble lectin. MOG does not seem to belong to the signal-transducing GTP-binding proteins. Reduced MOG concentrations were observed in jimpy and quaking dysmyelinating mutant mice, giving further support to its localization in compact myelin of the CNS.     

Ciliary Neurotrophic Factor Role in Myelin Oligodendrocyte Glycoprotein Expression in Cuprizone-Induced Multiple Sclerosis Mice

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system that leads to loss of myelin and oligodendrocytes and damage to axons. Myelin oligodendrocyte glycoprotein (MOG) is a minor component of the myelin sheath, but is an important autoantigen linked to the pathogenesis of MS. Ciliary neurotrophic factor (CNTF) has been shown to enhance the generation, maturation, and survival of oligodendrocytes in culture medium. The aim of this study was to demonstrate the role of CNTF on MOG expression in the cerebral cortex of Cuprizone-induced MS mice. The mice were treated by Cuprizone for five weeks in order to induce MS. The mice were then divided into 3 groups. The first group was injected subcutaneously (SC) by CNTF in the amount of 250 μg/kg BW per day. The second group (SHAM) was injected SC by normal saline and the third group was left without injection as the control group. After four weeks the mice were killed and the cerebral cortex was harvested and the expression of MOG was studied by Western blotting. The data from this study show that the MOG expression was significantly increased in the CNTF-injected group as compared to the other groups. It is concluded that CNTF increases the MOG expression and may be important in the pathophysiology of MS. It is also concluded that CNTF may play a role in the process of remyelination by inducing the MOG expression.     

Demyelination Induced in Aggregating Brain Cell Cultures by a Monoclonal Antibody Against Myelin/Oligodendrocyte Glycoprotein

A monoclonal antibody (8-18C5) directed against myelin/oligodendrocyte glycoprotein (MOG) induced demyelination in aggregating brain cell cultures. With increasing doses of anti-MOG antibody in the presence of complement, myelin basic protein (MBP) concentration decreased in a dose-related manner. A similar, albeit less pronounced, effect was observed on specific activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase. In the absence of complement, anti-MOG antibody did not induce detectable demyelination. In contrast to the effect of anti-MOG antibody and as expected, anti-MBP antibody did not demyelinate aggregating brain cell cultures in the presence of complement. These results provide additional support to the suggestion that MOG, a quantitatively minor myelin component located on the external side of the myelin membrane, is a good target antigen for antibody-induced demyelination. Indeed, they show that a purified anti-MOG antibody directed against a single epitope on the glycoprotein can produce demyelination, not only in vivo as previously shown, but also in cultures. Such an observation has not been made with polyclonal antisera raised against purified myelin proteins like MBP and proteolipid protein, the major protein components of the myelin membrane, or myelin-associated glycoprotein. These observations may have important implications regarding the possible role of anti-MOG antibodies in demyelinating diseases.     

Differences in Brain Morphological Findings between Narcolepsy with and without Cataplexy

Objective

Maps of fractional anisotropy (FA) and apparent diffusion coefficient (ADC) obtained by diffusion tensor imaging (DTI) can detect microscopic axonal changes by estimating the diffusivity of water molecules using magnetic resonance imaging (MRI). We applied an MRI voxel-based statistical approach to FA and ADC maps to evaluate microstructural abnormalities in the brain in narcolepsy and to investigate differences between patients having narcolepsy with and without cataplexy.

Methods

Twelve patients with drug-naive narcolepsy with cataplexy (NA/CA), 12 with drug-naive narcolepsy without cataplexy (NA w/o CA) and 12 age-matched healthy normal controls (NC) were enrolled. FA and ADC maps for these 3 groups were statistically compared by using voxel-based one-way ANOVA. In addition, we investigated the correlation between FA and ADC values and clinical variables in the patient groups.

Results

Compared to the NC group, the NA/CA group showed higher ADC values in the left inferior frontal gyrus and left amygdala, and a lower ADC value in the left postcentral gyrus. The ADC value in the right inferior frontal gyrus and FA value in the right precuneus were higher for NA/CA group than for the NA w/o CA group. However, no significant differences were observed in FA and ADC values between the NA w/o CA and NC groups in any of the areas investigated. In addition, no correlation was found between the clinical variables and ADC and FA values of any brain areas in these patient groups.

Conclusions

Several microstructural changes were noted in the inferior frontal gyrus and amygdala in the NA/CA but not in the NA w/o CA group. These findings suggest that these 2 narcolepsy conditions have different pathological mechanisms: narcolepsy without cataplexy form appears to be a potentially broader condition without any significant brain imaging differences from normal controls.     

Characterization and Expression of the cDNA Coding for the Human Myelin/Oligodendrocyte Glycoprotein

Abstract: We report here the characterization of a full-length cDNA encoding the human myelin/oligodendrocyte glycoprotein (MOG). The sequence of the coding region of the human MOG cDNA is highly homologous to that of other previously cloned mouse, rat, and bovine MOG cDNAs, but the 3' untranslated region differs by an insertion of an Alu sequence between nucleotides 1,590 and 1,924. Accordingly, northern blot analyzes with cDNA probes corresponding to the coding region or the 3' untranslated Alu-containing sequence revealed a single band of 2 kb, rather than the 1.6 kb of bovine, rat, or mouse MOG cDNA(s). Immunocytochemical analysis of HeLa cells transfected with human MOG cDNA, which was performed using a specific antibody raised against whole MOG, clearly indicated that MOG is expressed at the cell surface as an intrinsic protein. These data are in accordance with the predicted amino acid sequence, which contains a signal peptide and two putative transmembrane domains. The knowledge of the human MOG sequence should facilitate further investigations on its potential as a target antigen in autoimmune demyelinating diseases like multiple sclerosis.     

Human Myelin/Oligodendrocyte Glycoprotein: A New Member of the L2/HNK-1 Family

Abstract— Myelin/oligodendrocyte glycoprotein (MOG) is a quantitatively minor component of CMS myelin. In this study, human MOG was found to express the L2/HNK-1 epitope on N-linked oligosaccharide structures. This carbohydrate epitope has been found previously in three other characterized human myelin glycoproteins: the my-elin-associated glycoprotein, P₀, and the oligodendrocyte-myelin glycoprotein. It seems, therefore, that the L2/HNK-1 epitope is expressed frequently in human myelin glycoproteins. Serial lectin affinity chromatography of ¹⁴C-glycopeptides indicated that MOG N -oligosaccharide structures are mainly of the complex type, accounting for 77.8% of total radioactivity. In contrast with myelin-asso-ciated glycoprotein and P₀, which express the L2/HNK-1 epitope on fucosylated structures, in MOG the epitope was detected on all glycopeptide fractions obtained by serial lectin affinity chromatography, although a preferential expression of the L2/HNK-1 epitope was observed on fucosylated structures. Finally, the data indicated that, as for other human myelin glycoproteins, only a subpopulation of MOG molecules expresses the L2/HNK-1 epitope.     

Antibodies to Myelin/Oligodendrocyte-Specific Protein and Myelin/Oligodendrocyte Glycoprotein Signal Distinct Changes in the Organization of Cultured Oligodendroglial Membrane Sheets

Abstract: Cultured murine oligodendrocytes elaborate extensive membrane sheets that, unlike multilamellar myelin in vivo, allow the study of interactions between myelin proteins and cytoskeletal elements. This article describes the events that occur due to the interaction of specific antibodies with their respective antigens, myelin/oligodendrocyte-specific protein (MOSP) and myelin/oligodendrocyte glycoprotein (MOG), which are expressed uniquely by oligodendrocytes. After antibody binding, surface anti-MOSP:MOSP complexes redistribute over those cytoplasmic microtubular veins that have 2',3'-cyclic nucleotide 3'-phosphohydrolase colocalized along them. In contrast, surface anti-MOG-MOG complexes redistribute over internal myelin basic protein domains. Long-term anti-MOSP IgM exposure results in an apparent increase in number as well as thickness of microtubular structures in oligodendrocyte membrane sheets, whereas long-term anti-MOG exposure causes depolymerization of microtubular veins in membrane sheets. These data suggest that antibody binding to these two surface proteins elicits signals that have opposite effects on the cytoskeleton in oligodendroglial membrane sheets. Thus, it is possible that signals transduced via antibody binding may contribute to the pathogenesis of diseases affecting CNS myelin.     

The Extracellular Domain of Myelin Oligodendrocyte Glycoprotein Elicits Atypical Experimental Autoimmune Encephalomyelitis in Rat and Macaque Species

Atypical models of experimental autoimmune encephalomyelitis (EAE) are advantageous in that the heterogeneity of clinical signs appears more reflective of those in multiple sclerosis (MS). Conversely, models of classical EAE feature stereotypic progression of an ascending flaccid paralysis that is not a characteristic of MS. The study of atypical EAE however has been limited due to the relative lack of suitable models that feature reliable disease incidence and severity, excepting mice deficient in gamma-interferon signaling pathways. In this study, atypical EAE was induced in Lewis rats, and a related approach was effective for induction of an unusual neurologic syndrome in a cynomolgus macaque. Lewis rats were immunized with the rat immunoglobulin variable (IgV)-related extracellular domain of myelin oligodendrocyte glycoprotein (IgV-MOG) in complete Freund’s adjuvant (CFA) followed by one or more injections of rat IgV-MOG in incomplete Freund’s adjuvant (IFA). The resulting disease was marked by torticollis, unilateral rigid paralysis, forelimb weakness, and high titers of anti-MOG antibody against conformational epitopes of MOG, as well as other signs of atypical EAE. A similar strategy elicited a distinct atypical form of EAE in a cynomolgus macaque. By day 36 in the monkey, titers of IgG against conformational epitopes of extracellular MOG were evident, and on day 201, the macaque had an abrupt onset of an unusual form of EAE that included a pronounced arousal-dependent, transient myotonia. The disease persisted for 6–7 weeks and was marked by a gradual, consistent improvement and an eventual full recovery without recurrence. These data indicate that one or more boosters of IgV-MOG in IFA represent a key variable for induction of atypical or unusual forms of EAE in rat and Macaca species. These studies also reveal a close correlation between humoral immunity against conformational epitopes of MOG, extended confluent demyelinating plaques in spinal cord and brainstem, and atypical disease induction.     

Hoxd1 is Expressed by Oligodendroglial Cells and Binds to a Region of the Human Myelin Oligodendrocyte Glycoprotein Promoter in vitro

(1) Little information exists on the role of clustered Hox genes in oligodendrocyte (OG) development. This study examines the expression profile of Hoxd1 and identifies a potential downstream target in the OG lineage. (2) Immunocytochemical analysis of primary mixed glial cultures demonstrated Hoxd1 was expressed throughout OG development. (3) A human myelin protein gene, myelin oligodendrocyte glycoprotein (MOG), was identified as a putative downstream target of Hoxd1 through Genbank searches utilizing the Hoxd1 homeodomain consensus binding sequence. (4) The dissociation coefficient constant (K _D) and dissociation rate constant (k _d) of the Hoxd1–MOG complex, determined using electrophoretic mobility shift assays (EMSAs), were estimated to be 1.9 × 10⁻⁷ M and 1.3 × 10⁻³ s⁻¹, respectively. The DNA–Hoxd1 homeodomain complex had a half-life (t _1/2) of 15 min. (5) Mutational analysis of Hoxd1–MOG complexes revealed the binding affinity of M1 (with mutation from ⁻¹⁰⁵⁴5′-TAAT-3′⁻¹⁰⁵¹ to TACT within the consensus binding site) and M2 (with mutation from ^-10545′-TAATTG-3′^-1049 to TAATCC within the consensus binding site) probes to the MOG promoter was severely affected. Thus the TAATTG core of the binding sequence appears important for Hoxd1 specificity. (6) Analysis of the involvement of TAAT sites adjacent to the consensus sequence in Hoxd1 binding showed the binding affinity of the M3 probe was affected, but not as severely as the M1 and M2 probes. These in vitro results suggest the TTTAATTGTA sequence is involved in Hoxd1 binding to the MOG promoter but neighboring TAAT sites may also be needed. Thus, MOG may be a target of Hoxd1.