The crystal structure of myelin oligodendrocyte glycoprotein, a key autoantigen in multiple sclerosis Craig S. Clements†‡, Hugh H. Reid†‡§, Travis Beddoe†‡, Fleur E. Tynan†, Matthew A. Perugini¶, Terrance G. Johnsʈ, Claude C. A. Bernard§††, and Jamie Rossjohn†,†† †Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3168, Australia; §Neuroimmunology Laboratory, Biochemistry Department, La Trobe University, Bundoora, Victoria 3083, Australia; ¶Department of Biochemistry, Melbourne University, Parkville, Victoria 3010, Australia; and ʈLudwig Institute for Cancer Research, Austin and Repatriation Medical Centre, Level 6, Harold Stokes Building, Studley Road, Heidelberg 3084, Australia Edited by David R. Davies, National Institutes of Health, Bethesda, MD, and approved July 21, 2003 (received for review May 25, 2003) Myelin oligodendrocyte glycoprotein (MOG) is a key CNS-specific tion-dependent (11), whereas antibodies against linear epitopes autoantigen for primary demyelination in multiple sclerosis. Al- of MOG do not induce widespread demyelination (12). though the disease-inducing role of MOG has been established, its MOG is a type I integral membrane protein possessing a single precise function in the CNS remains obscure. To gain new insights extracellular Ig variable domain (Ig-V) (3, 13, 14). The amino into the physiological and immunopathological role of MOG, we acid sequence of MOG is highly conserved among animal species determined the 1.8-Å crystal structure of the MOG extracellular (Ͼ90%), indicative of an important biological function. MOG is domain (MOGED). MOGED adopts a classical Ig (Ig variable domain) specifically expressed in the CNS on the outermost lamellae fold that was observed to form an antiparallel head-to-tail dimer. of the myelin sheath as well as the cell body and processes of A dimeric form of native MOG was observed, and MOGED was also oligodendrocytes (3). The developmentally late expression of shown to dimerize in solution, consistent with the view of MOG MOG correlates with the later stages of myelinogenesis, sug- acting as a homophilic adhesion receptor. The MOG peptide, a gesting that MOG has a role in the completion, compaction, 35-55 ͞ major encephalitogenic determinant recognized by both T cells and and or maintenance of myelin, further suggesting that MOG has demyelinating autoantibodies, is partly occluded within the dimer an adhesive function within the CNS (3). Consistent with MOG’s interface. The structure of this key autoantigen suggests a rela- possible adhesive role in the CNS, a homodimeric form of MOG NEUROSCIENCE tionship between the dimeric form of MOG within the myelin has not only been observed after isolation from the CNS but has sheath and a breakdown of immunological tolerance to MOG that additionally been observed in situ (15). In addition, MOG may is observed in multiple sclerosis. also have an immune-related function because it binds C1q, and thus could be a regulator of the classical complement pathway (16). ultiple sclerosis (MS) is an inflammatory disease of the We have determined the 1.8-Å crystal structure of MOG MCNS characterized by localized myelin destruction and extracellular domain (MOGED) to further our understanding of axonal degeneration (1). An autoimmune reaction against my- the function of this protein and to elucidate its role in the elin antigens of the CNS contributes to the immunopathological pathogenesis of demyelinating diseases of the CNS. mechanisms of this enigmatic disease. This concept is supported by observations in patients with MS such as elevated levels of Methods lymphocytes reactive to myelin antigens, the presence of oligo- Protein Expression and Refolding. MOGED (residues 1–117) of the clonal Ig in the cerebrospinal fluid, and perivascular inflamma- mature mouse protein, containing an amino terminal six- tory cell invasion of the CNS (2). Immune responses to abundant histidine tag, was produced in the Escherichia coli strain M15 proteins of the CNS myelin sheath, proteolipid protein (PLP), (pREP4) by using the pQE9 expression vector (Qiagen, Mel- and myelin basic protein (MBP) are thought to produce some of bourne, Australia). After expression and preparation of a the pathological lesions in MS and its animal model, experimen- cleared lysate per the manufacturer’s instructions, MOGED was tal autoimmune encephalomyelitis (EAE). However, there is purified by using Ni-NTA Superflow (Qiagen) under denaturing another important autoantigen linked to the pathogenesis of conditions (6 M guanidine⅐HCl), per the manufacturer’s instruc- both MS and EAE, the myelin oligodendrocyte glycoprotein tions, on a BioLogic LP Chromatography System (Bio-Rad). The (MOG), which is a minor component of the myelin sheath column was washed to remove nonspecifically bound material, (Ϸ0.05% of total myelin protein) (3). In susceptible animals, and bound protein was refolded directly on the column by using immunization with native or recombinant MOG, or MOG- linear gradients of denaturing versus nondenaturing solutions. derived peptides, or passive transfer of MOG-specific T cells and After elution with 300 mM imidazole in sodium phosphate͞Tris ͞ autoantibodies against MOG elicits a severe neurological disease (pH 8.0), MOGED was dialyzed against 50 mM NaCl 5 mM Tris ͞ that mimics many of the clinical, pathological, and immunolog- (pH 8.0) and concentrated to 1.3 mg ml. ical features of MS (2, 4, 5). A number of encephalitogenic Crystallization. epitopes of MOG (MOG1-22, MOG35-55, and MOG92-106) have Crystallization trials were conducted by using the hanging-drop vapor diffusion technique at room temperature. been identified (4, 6, 7). MOG35-55, in particular, is a potent encephalitogen that, in contrast to MBP and PLP, elicits striking inflammatory and demyelinating immune responses in the CNS This paper was submitted directly (Track II) to the PNAS office. not only in various species but also across different strains of Abbreviations: DTSSP, 3,3Ј-dithiobis(sulfosuccinimidylpropionate); EAE, experimental au- mice (5, 6, 8). toimmune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; MOGED, MOG The ensuing MS-like disease is characterized by relapses and extracellular domain; MS, multiple sclerosis; P0, myelin protein zero. remissions, extensive neural inflammation and demyelination, Data deposition: The atomic coordinates and structure factors have been deposited in the axonal injury, and the production of antibodies to MOG (5, Protein Data Bank, www.rcsb.org (PDB ID code 1PY9). 7–10). Moreover, antibodies to MOG are directly associated with ‡C.S.C., H.H.R., and T.B. contributed equally to this work. myelin damage in the marmoset model of EAE as well as in the ††To whom correspondence may be addressed. E-mail: [email protected]. CNS of patients with MS (10). The epitopes recognized by mAb edu.au or [email protected]. 8-18C5 and other demyelinating mAbs to MOG are conforma- © 2003 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.1833158100 PNAS ͉ September 16, 2003 ͉ vol. 100 ͉ no. 19 ͉ 11059–11064 Downloaded by guest on September 25, 2021 Table 1. Data collection and refinement statistics spersed with refinement within CNS (21). When a sufficient core of the model was built, ARP/WARP was reused and proceeded to Data collection successfully build and refine the model. A final round of Temperature, K 100 refinement was undertaken in CNS. Space group I4 1 The final model, comprising residues 2–117, 215 water mol- Cell dimensions, Å (a, b, c) 65.7, 65.7, 67.6 ecules, and two sulfate ions, has an R of 20.1% and an R Resolution, Å 1.8 factor free of 23.7% (Table 1). One residue, His-103, although in the Total no. of observations 33,022 disallowed area of the Ramachandran plot, fits the electron No. of unique observations 13,229 density very well. Multiplicity 2.5 Data completeness, % 99.0 (100.0) Western Blot Analysis. Myelin was prepared from the brains of Data Ͼ2 ␴ ,% 79.6 I adult wild-type and MOG-deficient mice (unpublished data) and I͞␴ 6.9 (1.8) I human brain by using sucrose gradient centrifugation. Native R ,* % 6.5 (24.6) merge MOG was purified from human brain by affinity chromatogra- Refinement phy with the MOG-specific monoclonal antibody 8-18C5 (22). Nonhydrogen atoms Myelin proteins (2.5 ␮g per lane) and purified native MOG were Protein 934 resolved on 12% SDS͞PAGE gels. MOG was detected by Water 215 Western blot analysis as described (15), using 8-18C5 and Sulfate ions 2 enhanced chemiluminescence detection per the manufacturer’s Resolution, Å 50–1.8 † instructions (ECL, Amersham Biosciences). Rfactor, % 20.1 R ,† % 23.7 free Cross-Linking. Protein for cross-linking was buffer-exchanged into rms deviations from ideality PBS (50 mM phosphate͞150 mM NaCl, pH 7.2) by running the Bond lengths, Å 0.005 protein on a Superdex 75 10͞30 column (Amersham Bio- Bond angles, ° 1.35 sciences). Increasing amounts of MOG were incubated with Dihedrals, ° 26.3 ED 0.1 mM DTSSP [3,3Ј-dithiobis(sulfosuccinimidylpropionate); Impropers, ° 0.7 Pierce] for 30 min at room temperature. The reaction was Ramachandran plot quenched with 1 M Tris, and the whole reaction was analyzed by Most favored and allowed region, % 99 SDS͞PAGE. B factors, Å2 Average main chain 20.3 Blue Native PAGE. Blue native PAGE (31) was used to resolve Average side chain 24.1 MOG . Briefly, MOG was resuspended in Coomassie blue Average water molecule 40.3 ED ED G250 (Serva) loading buffer and resolved on 4–20% native rms deviation in bonded B factors 1.8 PAGE gels (Bio-Rad) at 15 mA for2hat4°C. The gels were then The values in parentheses are for the highest-resolution bin (approximate stained in Coomassie blue R250. Chymotrypsinogen A (Ϸ26 interval 0.1 Å). kDa) and ribonuclease A (Ϸ14 kDa) were used as molecular ϭ͚͉ Ϫ ͗ ͉͚͘͞ *Rmerge Ihkl Ihkl Ihkl.