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Myelin-Associated Oligodendrocytic Basic Protein: Identification of an Encephalitogenic Epitope and Association with Multiple Sclerosis¹

Andreas Holz^2,*, Bibiana Bielekova, Roland Martin and Michael B. A. Oldstone^*

^* Viral-Immunobiology Laboratory, Division of Virology, Department of Neuropharmacology, The Scripps Research Institute, La Jolla, CA 92037; and Neuroimmunology Branch, National Institute of Neurologic Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892

	Abstract

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Myelin-associated oligodendrocytic basic protein (MOBP) is an abundant myelin constituent expressed exclusively by oligodendrocytes, the myelin-forming cells of the CNS. We report that MOBP causes experimental allergic encephalomyelitis and is associated with multiple sclerosis. First, we note that purified recombinant MOBP inoculated into SJL/J mice produces CNS disease. Tests of overlapping peptides spanning the murine MOBP molecule map the encephalitogenic site to amino acids 37–60. MOBP-induced experimental allergic encephalomyelitis shows a severe clinical course and is characterized by a prominent CD4⁺ T lymphocyte infiltration and a lesser presence of CD8⁺ T cells and microglia/macrophages around vessels and in the white matter of the CNS. Second, PBL obtained from patients with relapsing/remitting multiple sclerosis mount a proliferative response to human MOBP, especially at amino acids 21–39. This response equals or exceeds the response to myelin basic protein and an influenza virus hemagglutinin peptide, both serving as internal controls. Thus, a novel myelin Ag, MOBP aa 37–60, plays a role in rodent autoimmune CNS disease, and its human MOBP counterpart is associated with the human demyelinating disease multiple sclerosis.

	Introduction

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T lymphocytes and their products play a pivotal role in the pathogenesis of multiple sclerosis (MS),³ the most common demyelinating disease of humans, and in its laboratory model, experimental allergic encephalomyelitis (EAE) (reviewed in Ref. 1). A distinguishing theme for both MS and EAE is that T cells recognize and respond to specific peptides from myelin presented by selected MHC molecules. However, in MS the environmental event(s) responsible for triggering such T cell responses as well as the specific myelin Ags recognized in vivo by these T cells are largely unknown. Thus, it is important to identify the potential disease-causing Ags (peptides) in myelin and use that data to determine whether such myelin peptides mimic the sequence or confirmations of microbial peptides. Further, different myelin Ags (peptides) may be associated with different subsets of humans having MS (2, 3). Hence, the recognition of all the myelin Ags that cause disease are important and may shed light on strategies to suppress Ag-specific immunopathologic T cell responses (4, 5), give leads for identifying and testing whether a specific infectious agent is involved in the triggering of disease (6, 7, 8, 9), and assist in identifying different subgroups of patients with disease (2). Currently, the major myelin Ags known are myelin basic protein (MBP) (10, 11, 12, 13), proteolipid protein (PLP) (14, 15, 16, 17, 18), and myelin oligodendrocyte glycoprotein (19, 20, 21), although other neural Ags can also cause inflammation of the CNS (22).

To search for other genes specifically expressed by oligodendrocytes that may be involved in demyelinating disease, myelin-associated oligodendrocytic protein (MOBP) was identified from a rat spinal cord library (23, 24). By alternative splicing, a single-copy Mobp gene generated in rodents and humans several different transcripts that encoded MOBP isoforms with identical amino termini but different carboxyl ends. Analysis of the MOBP proteins revealed their structure to be highly basic with isoelectric points larger than 10 and insignificant homologies with other myelin constituents like MBP or PLP. The most abundant MOBP isoform was the 81 aa constituting MOBP81 protein (23, 25). Biologic analysis of MOBP indicated three major differences from MBP and PLP. First, MOBP was located exclusively in oligodendrocytes of the CNS, whereas MBP and PLP appeared both in the CNS and peripheral nervous systems (23, 24, 26). Second, in rodents, transcription of the Mobp gene occurred 2–3 days later than expression of the MBP or PLP genes (23, 27). Third, MOBP was transcriptionally active at the time of myelin compaction (23), and MOBP protein was noted at the major dense line of myelin (24), suggesting a role for this protein in myelin structure. MOBP was estimated as the third most abundant myelin constituent of the CNS (24).

Here we analyzed the role of MOBP in CNS autoimmune disease. We find that this new myelin constituent causes EAE in susceptible mice and map its encephalitogenic peptide sequence. Furthermore, we demonstrate that MOBP is involved in the pathogenesis of MS, and T cells isolated from MS patients show significantly elevated proliferative responses to human MOBP peptides.

	Materials and Methods

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Peptides

Peptide libraries covering the entire murine MOBP81 peptide (25) and human MOBP aa 1–70 (24) were synthesized at Chiron Technologies (San Diego, CA; see Table I and Fig. 3). The encephalitogenic PLP aa 139–151 peptide (VSLGKWLGHPDKF) (28) was used as a positive control for EAE induction. The immunodominant human MBP aa 83–99 peptide (10, 11, 29) and influenza-hemagglutinin (Flu-HA) aa 306–318 peptide (PKYVKQNTLKLAT) (30, 31) served as control Ags for proliferation studies using human T cells.

View larger version (26K): [in this window] [in a new window]   FIGURE 3. T lymphocytes from several MS patients show a vigorous and specific proliferative response to MOBP. MOBP-, MBP-, and Flu-HA-peptide-specific T cell proliferation was analyzed in MS patients (n = 8) and healthy donors (n = 7) using IL-7-modified primary proliferation. This assay expands Ag-specific memory and in vivo-activated T cells (34 ). Briefly, a total of 60 x 105 PBMC from each individual was tested per Ag (1 µM). PBMC were stimulated for 7 days in the presence of peptide and IL-7 (10 ng/ml). After this period, cells were pulsed with [3H]thymidine and cpm were measured by scintillation counting. A, Raw data for a representative MS patient. The lines for the cut-off and the background are indicated. The cut-off line represents two stringent criteria: first, a stimulation index larger than 2 and, second, a cpm (cpm) larger than background level plus 3 SD. Note that the cut-off for Ag-specific proliferation was high above background values. Between zero and five positive wells were detected for each Ag, which is consistent with precursor frequency estimates for these autoreactive cells in the range of one to five per million. These data are characteristic of those observed with the other six positively responding MS patients studied. B, Proliferation data for lymphocytes obtained from MS patients and healthy donors and normalized for proliferation to Flu-HA (internal control). The differences of MOBP-specific proliferation between MS patients and healthy donors were statistically significant (2 calculated based on the number of positive wells per each Ag, p = 0.008). Furthermore, although the proliferative response to MOBP peptides was comparable to or higher than those to Flu-HA in the MS patients, the responses to Flu-HA were greater in the control population (p < 0.01; Mann-Whitney rank sum test).

EAE induction was performed according to established protocols (18, 32). Sixty micrograms MOBP fusion protein, 100 µg MOBP peptides, or 150 µg PLP aa 139–151 peptide were used as inducing agents. These Ags were emulsified in CFA containing 5 mg/ml Mycobacterium tuberculosis H37RA (Difco Laboratories, Detroit, MI) and injected s.c. at the base of the tail of 6- to 8-wk-old female SJL/J mice (The Jackson Laboratory, Bar Harbor, ME). Simultaneously, 150 ng pertussis toxin (List Biological Laboratories, Campbell, CA) was given i.v. and 48 h later. Animals were scored and weighed daily. Clinical scoring was according to the following scale: 0, normal animal, no clinical signs of EAE; 1, completely limp tail; 2, impaired righting reflex with completely limp tail; 3, partial hind limb paralysis; 4, complete hind limb paralysis; 5, hind and fore limb paralysis, moribund.

Generation of MOBP-GST fusion protein

The open reading frame encoding rat MOBP81 protein (23) was PCR amplified using the primers 5'-CGGAATTCTGATGAGTCAAAAAGTGGCC and 5'-CATCAGCAGGTGTCCAC. The PCR product was EcoRI/XhoI digested and ligated unidirectionally into the EcoRI/XhoI sides of plasmid pGEX-KG (33) (kindly provided by Dr. John Patterson, The Scripps Research Institute, La Jolla, CA). DNA sequencing confirmed the integrity of the resulting GST-MOBP plasmid expressing rat MOBP aa 1–81 in frame with the GST protein. Recombinant fusion protein was made in Escherichia coli strain BL21 after induction with 0.5 mM isopropyl -D-thiogalactoside (IPTG) and affinity-purified with glutathione beads as described (33). In brief, exponentially growing bacterial cultures were IPTG induced, and after a 2-h incubation period cells were collected by centrifugation. Bacteria were lysed with lysozyme (10 µg/ml) in 25 mM sodium-HEPES buffer, pH 7.5, containing 0.1% Nonidet P-40, 1 mM ETDA, and a mixture of various protease inhibitor (Boehringer Mannheim, Mannheim, Germany). After sonication and centrifugation at 10,000 x g, soluble proteins of the supernatant were incubated with glutathione beads (Pharmacia, Piscataway, NJ) for 30 min. Beads were washed several times with phosphate buffer. GST-MOBP was released from the beads by incubation with 10 mM reduced glutathione (Sigma, St. Louis, MO) in 50 mM Tris, pH 8.0. GST protein that was used as a control protein for animal inoculations was prepared following the same procedure.

Protein fractions were analyzed by 16% PAGE and Western blotting as described (23). For GST-MOBP cleavage, fusion protein was incubated with 1 U thrombin protease (Pharmacia) for 15 min at room temperature.

Histology and immunohistochemistry

Tissues used for histologic analysis were fixed in 10% Bouin’s solution and embedded in paraffin. Tissue sections were stained with either hematoxylin-eosin or Luxol fast blue before mounting and photography.

For immunohistochemistry, brains and spinal cords of SJL/J mice were rapidly dissected and fresh frozen in OCT Tissue Tek compound (Fisher Scientific, Pittsburgh, PA). Next, 8-µm thin cryomicrotome sections were collected on SuperFrost/Plus slides (Fisher Scientific, Pittsburgh, PA), postfixed in ice-cold 95% ethanol and air-dried. Slides were blocked with 2% FBS/phosphate buffer for 1 h and with avidin/biotin solutions for 10 min each (Vector Laboratories, Burlingame, CA). Tissue sections were incubated for 1 h at room temperature with primary Abs directed against several cell-surface markers (rat anti-mouse CD4⁺, rat anti-mouse CD8⁺, rat anti-mouse B220 (all obtained from PharMingen, San Diego, CA, and used at 1:200 dilutions), and rat anti-mouse F4/80 (1:100 diluted; Serotec, Oxford, U.K.)). After washes with phosphate buffer, slides were covered for 30 min with FBS/phosphate buffer solution containing 1:200 diluted biotinylated anti-rat secondary Ab (Vector Laboratories). Tissue sections were washed again and incubated with a streptavidine-HRP complex for 30 min (Vector Laboratories). After PBS washes, slides were stained for 5 min with diaminobenzidine (Zymed, South San Francisco, CA), counterstained with hematoxylin (Sigma), and embedded in AquaMount before photography. Control experiments omitting the primary Ab produced no staining, indicating the absence of background.

T cell proliferation studies

PBMC were obtained from MS patients and healthy donors. All patients had relapsing/remitting MS and did not receive immunosuppressive or immunomodulatory therapy within 3 mo prior testing. Between groups were no significant age or MHC class II differences.

The T lymphocyte proliferation assay was performed as described (34). A total of 6 x 10⁶ PBMC were tested for each Ag in 96-well microtiter plates. PBMC from each individual were plated in 60 wells each containing 1 x 10⁵ PBMC. PBMC were stimulated with 1 µM of each peptide in IL-7-enriched (10 ng/ml) T cell medium. On day 7, cells were pulsed with [³H]thymidine for 6 h, and incorporated radioactivity was determined by scintillation counting. Sixty negative control wells (seeded without Ag) were included for each patient. An unrelated Ag, Flu-HA aa 306–318, which induces T cell recall responses in almost all individuals (30, 31), was included as a positive control and served as an internal standard. Wells were considered positive when both of the following criteria were reached: 1) the stimulation index is >2 (cpm greater than twice the average cpm of all negative control wells); and 2) the cpm is greater than the average cpm of negative control wells + 3 SD. The combination of these criteria have been reported to be optimal to ensure a high degree of specificity while maintaining the sensitivity of the assay (34).

To compare proliferative responses of PBMC from MS patients directly with those from controls, stimulation indexes of each Ag were calculated individually and normalized against the proliferative response of the Flu-HA peptide.

	Results

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Recombinant rodent MOBP81 protein is encephalitogenic in SJL/J mice

To characterize MOBP functionally, we engineered a cDNA expressing a fusion protein of the major rodent MOBP (MOBP81) isoform and the GST protein. The fusion gene was expressed in E. coli, and the soluble product was purified by affinity chromatography (Fig. 1). Subsequently, several MOBP-specific bands were observed by using anti-MOBP Ab (Fig. 1A). This result likely indicates an altered migration behavior of the highly basic fusion protein or its partial degradation. However, a unique protease cleavage site within the fusion protein (33) allowed the enzymatic cleavage and removal of MOBP from GST after incubation with thrombin protease (Fig. 1B); the resulting recombinant 12-kDa MOBP was consistent with the full-length MOBP molecule (23), and no MOBP-specific degradation products were detected.

View larger version (68K): [in this window] [in a new window]   FIGURE 1. Recombinant GST-MOBP induces lymphocytic infiltration in the CNS of SJL/J mice. A, Bacteria containing a GST-MOBP-expressing plasmid were induced with IPTG, and recombinant fusion protein was affinity purified using glutathione beads (33 ). During the purification process, fractions were monitored by PAGE and Western blotting using an anti-MOBP peptide Ab (23 ). Lane 1, No induction. Lane 2, Induction with 0.5 mM IPTG. Lane 3, Total soluble proteins of the induced fraction. Lane 4, Affinity-purified GST-MOBP. B, GST-MOBP fusion protein incubated with or without 1 U of thrombin protease. A 12-kDa protein corresponding to full-length MOBP was detectable by Western blotting (23 ). C, Photomicrograph of CNS tissue taken from an SJL/J mouse 14 days after inoculation with 60 µg GST-MOBP emulsified in CFA. A monoclonal anti-murine CD4+ Ab shows perivascular accumulation of CD4+ T lymphocytes in the white matter region of the cerebellum (counterstained with hematoxylin). Similar results were obtained in all five mice inoculated with purified MOBP.

MOBP peptides spanning aa 37–60 induce severe clinical EAE in SJL/J mice

We next sought the peptide sequence(s) of the MOBP responsible for the encephalitogenic activity. Overlapping peptides, each containing 15 aa, were synthesized that covered the entire rodent MOBP molecule (25). These peptides were combined into six separate pools (Table I); individual pools were emulsified in CFA and the separate emulsions injected into SJL/J mice (seven to eight mice per group; two separate experiments). Of these six pools, only pool 4, consisting of MOBP aa 37–60, induced acute EAE (Table I). The pool 4 inoculum produced characteristic clinical signs (Table I) of weight loss, limp tail, paresis, ataxia, and paraplegia. The mean time of disease onset was 10 days after the initial inoculation, and the mean maximum severity of disease was 3.5 on a scale of 5 (Table I). The incidence rate of EAE induced by the MOBP peptides was 75% (six of eight mice).

Histologic and immunohistochemical evaluation of MOBP aa 37–60 induced EAE

Analysis of brains and spinal cords revealed widespread infiltration of mononuclear cells throughout the CNS (Fig. 2), but no infiltration in the peripheral nervous system or other peripheral tissues. In the CNS, mononuclear cells were noted in perivenular, periventricular, and subpial areas (Fig. 2, A and B), as well as in the CNS parenchyma, primarily in white matter. CNS inflammation was observed in all animals inoculated with the peptide pool spanning MOBP aa 37–60. The amount of infiltration observed in animals correlated with the severity of clinical disease. A myelin-specific stain (Luxol fast blue) revealed moderate myelin breakdown (data not shown). The degree of demyelination in MOBP-inoculated animals was similar to that observed in mice in which EAE was induced with PLP aa 139–151.

View larger version (146K): [in this window] [in a new window]   FIGURE 2. Amino acids 37–60 is an encephalitogenic site on the murine MOBP. Photomicrographs of spinal cords taken from a SJL/J mouse inoculated with a pool of peptides containing MOBP aa 37–60. Hematoxylin-eosin staining documents infiltration of mononuclear cells near the ventricle (A) and around a blood vessel near the surface of the spinal cord (B). C–E, Immunohistochemical staining on adjacent sections of the white matter of the spinal cord counterstained with hematoxylin; C shows the presence of CD4+ T lymphocytes, D shows F4/80-positive macrophages/microglia, and E shows CD8+ T lymphocytes. F, Perivascular infiltration of CD4+ T cells in the white matter of the brain stem parenchyma is shown. Similar infiltration was noted in spinal cord, cerebellum, and cortex. In contrast, there was no infiltration in peripheral tissues. Corresponding results were seen in mice in two separate experiments. G, The lack of CD4+ T cells (or any other mononuclear cells; not shown) infiltrating into the CNS of animals inoculated with MOBP pool 3 (MOBP aa 25–48; histologic section obtained from the spinal cord; study of sections from brain stem, cerebellum, and cortex also failed to reveal any CD4+ T cell infiltration) is demonstrated. A similar absence of T cell infiltration was noted in SJL/J mice inoculated with peptides from other regions of MOBP comprising aa 1–24 (pool 1), 13–36 (pool 2), 49–72 (pool 5), and 61–81 (pool 6; see Table I).

Four overlapping peptides of the encephalitogenic site were individually administered to SJL/J mice (five mice per group). All four peptides initiated infiltration of mononuclear cells, primarily CD4⁺, in the CNS. Although peptides aa 37–51 and aa 40–54 caused more CNS infiltration of CD4⁺ T lymphocytes than MOBP peptides aa 43–57 and aa 46–60, none of the mice inoculated developed clinical EAE.

T cells from MS patients respond to human MOBP peptides by specific proliferation

Lastly, we turned our attention to whether MOBP is a potential target Ag for lymphocytes from MS patients. Human MOBP (24) is 97% homologous with the common amino-terminal part of murine MOBP. Sequence analysis identified the presence of putative peptide-binding motifs for the MS-associated HLA-DR alleles DRB5*0101 (DR2a), DRB1*0401, and also a partial motif for DRB1*1501 (also referred to as DR2b) in human MOBP aa 21–39 (data not shown) that mapped near the murine MOBP encephalitogenic region. This observation of a shared encephalitogenic region in SJL/J mice and DR2-positive MS patients had also been observed for MBP aa 83–99 (1, 10, 35). To test the immunogenicity of human MOBP, a set of six overlapping peptides spanning human MOBP aa 1–70 was made and used to assay the proliferative response of lymphocytes obtained from eight MS patients suffering from active r/r disease and from seven healthy donors (Fig. 3). Except for one, each patient responded strongly to at least one of the MOBP peptides (Fig. 3A), and the overall response was comparable to that of a standard recall Ag, Flu-HA aa 306–318 (30, 31) (Fig. 3). Equal numbers of the healthy donors and patients with MS responded by specific proliferation to both MBP aa 83–99 and Flu-HA aa 306–318. The proliferative response against MBP in the healthy control group was expected and agrees with previous findings (36, 37, 38). Accordingly, we detected proliferation against MOBP peptides also in the control group. To directly compare the MOBP-specific proliferation between MS patients and controls, we normalized the response to each peptide to that of Flu-HA aa 306–318 (Fig. 3B). The difference in MOBP-specific proliferation between MS patients and healthy donors was significant (p = 0.008; Fig. 3B), particularly the stimulation by MOBP aa 21–39, MOBP aa 31–49, and MOBP aa 1–19. Responses were 15-, 12- and 11-fold higher in the patients than in controls, respectively (Fig. 3B).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The main findings of this report are, first, the mapping of a MOBP peptide sequence encephalitogenic in SJL/J mice, and, second, the association of human MOBP with MS. The link of MOBP with CNS autoimmune disease by these two lines of evidence might lead to further the understanding of the multifactorial disease MS, the cause of which is unknown.

The knowledge of association of MOBP (or any other autoantigen) with CNS autoimmune disease may result in uncovering MS triggering factors/agents because self-Ags may act as molecular mimics for a specific and critical pathogen involved in disease initiation (7). Studies of monozygotic twins, of case clusters, and of MHC types document that individuals who develop MS must have an appropriate immunogenetic background influenced by several MHC genes likely involved in presenting a limited number of "self" myelin Ags (39, 40, 41, 42, 43, 44). However, the discordance among monozygotic twins (39, 45), the epidemiologic studies of susceptibility or resistance to MS in individuals migrating to areas with high or low incidence of disease before their 15th year of age (46), the association of MS with viral infections (47, 48), as well as outbreaks of MS in the Faroe Islands (49) indicate that environmental factors, presumably infections, are also important for disease pathogenesis. Hence, it is important to know about all molecules involved in MS. Our data links MOBP with MS. Therefore, it will be of interest to identify sequences from microbial agents that have linear or conformational fits (6, 7, 8, 9) with human MOBP and testing their abilities to elicit cross-reactive immune responses.

The disease patterns of MS are variable, and the pathology of multiple sclerosis is highly diverse (50). Hence, several immunopathogenetic mechanisms may contribute to disease onset and its progression (2). The fact that autoreactivity against MBP, PLP, myelin oligodendrocyte glycoprotein, and, as demonstrated here, MOBP, is observed during the course of MS indicates by itself the complexity of immunopathological mechanisms involved in CNS autoimmune disease. To fully understand them is ultimately required for development of effective treatments. Therefore, it will be of great interest to evaluate and compare the immune responses to MOBP and other MS-associated autoantigens in the heterogeneous disease phenotypes in both the animal model and MS (2, 22, 50) and the time when T lymphocytes recognize these molecules during the course of MS. Current studies evaluate such responses prospectively in MS patients.

We report that EAE can be induced with recombinant rodent MOBP81 protein and, by systematically assaying overlapping peptides spanning the entire murine MOBP81 protein, we mapped a MOBP encephalitogenic epitope to aa 37–60. The clinical, histological, and immunohistochemical evaluation of MOBP-induced EAE revealed no significant differences when compared with a PLP encephalitogenic peptide: clinical signs were acute, and disease included severe hind leg paralysis, CD4⁺ T lymphocytes and macrophages dominated the mononuclear cell infiltrate, and a focal myelin breakdown was observed. Recently, it was shown that another isoform of rat MOBP is able to cause clinical and histological signs of EAE in SJL/J mice (51). Rat MOBP170 (e.g., rOPRP1; Ref. 24) was expressed and purified in a baculovirus expression system, and, similar to our findings (Table I and Fig. 2), EAE was observed when recombinant MOBP170 was administered to SJL/J mice (51). We used recombinant rat MOBP81 protein for EAE induction in SJL/J mice (Fig. 1). Because the rodent MOBP81 and MOBP170 protein isoforms are identical in their first amino-terminal 69 aa (23, 24, 25), it can be concluded that at least one encephalitogenic domain must be contained in MOBP aa 1–69. In agreement with this consideration, we find that an encephalitogenic domain is located in the MOBP peptide sequence aa 37–60 (Table I and Fig. 2). In our and in Maata et al. (51) reports, recombinant rat protein was used for EAE induction in mouse. Rat and mouse MOBP81 are highly conserved and show only a single conserved amino acid change at position 5 (Val Met) (25). This amino acid substitution is located outside the identified encephalitogenic site in SJL/J mice (MOBP aa 37–60). Accordingly, the synthesized mouse peptides at positions aa 37–60 and the bacterially expressed recombinant rat protein both caused EAE in SJL/J mice.

MS is an autoimmune disease affecting the CNS. Therefore, potential autoantigens associated with MS must reside within brain and spinal cord. It is important to note that MOBP is specifically expressed by oligodendrocytes (23). Expression of MOBP outside the CNS has not been observed (23, 24). In contrast, the MS-associated autoantigens MBP and PLP both are expressed by oligodendrocytes, but also in the peripheral nervous system (52, 53, 54) as well as by cells in the thymus (55). In agreement with the CNS-specific expression of MOBP, we do not detect mononuclear cell infiltration in peripheral organs or the peripheral nervous system after inoculation of SJL/J mice with the encephalitogenic MOBP peptide pool. The strict target-organ-specific expression of MOBP makes this protein an attractive autoantigen for MS, in particular after the demonstration of an association of MOBP with CNS autoimmune disease.

	Acknowledgments

 
The technical help of Bryan Coon and the assistance by Dr. Mathias Kalbus in identifying the DR-associated MOBP peptide motifs is acknowledged. This is publication number 12064-NP from the Division of Virology, Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California.

	Footnotes

 
¹ This work was supported in part by U.S. Public Health Service Grants NS38719 and AI09484 and the J. D. and Iva Leiper Trust. A.H. is funded by fellowships of the Swiss National Foundation and the National Multiple Sclerosis Society.

² Address correspondence and reprint requests to Dr. Andreas Holz, Division of Virology, Department of Neuropharmacology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037. E-mail address:

³ Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental allergic encephalomyelitis; MOBP, myelin-associated oligodendrocytic basic protein; PLP, proteolipid protein; MBP, myelin basic protein; Flu-HA, influenza-hemagglutinin; IPTG, isopropyl -D-thiogalactoside.

Received for publication July 27, 1999. Accepted for publication November 1, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Martin, R., H. F. McFarland, D. E. McFarlin. 1992. Immunological aspects of demyelinating diseases. Annu. Rev. Immunol. 10:153.[Medline]
2. Lassmann, H.. 1998. Neuropathology in multiple sclerosis: new concepts. Multiple Sclerosis 4:93.[Abstract/Free Full Text]
3. Noseworthy, J. H.. 1999. Progress in determining the causes and treatment of multiple sclerosis. Nature 399:A40.[Medline]
4. Oldstone, M. B. A., M. von Herrath, H. Lewicki, D. Hudrisier, J. L. Whitton, J. E. Gairin. 1999. Use of a high-affinity peptide that aborts MHC-restricted cytotoxic T lymphocyte activity against multiple viruses in vitro and virus-induced immunopathologic disease in vivo. Virology 256:246.[Medline]
5. Ruiz, P. J., H. Garren, D. L. Hirschberg, A. M. Langer-Gould, M. Levite, M. V. Karpuj, S. Southwood, A. Sette, P. Conlon, L. Steinman. 1999. Microbial epitopes act as altered peptide ligands to prevent experimental autoimmune encephalomyelitis. J. Exp. Med. 189:1275.[Abstract/Free Full Text]
6. Oldstone, M. B.. 1987. Molecular mimicry and autoimmune disease. Cell 50:819.[Medline]
7. Oldstone, M. B. A.. 1998. Molecular mimicry and immune mediated diseases. FASEB J. 12:1255.[Abstract/Free Full Text]
8. Brusic, V., G. Rudy, L. C. Harrison. 1997. Molecular mimicry—from hypothesis towards evidence. Immunol. Today 18:95.[Medline]
9. Wucherpfennig, K. W., J. L. Strominger. 1995. Molecular mimicry in T cell-mediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein. Cell 80:695.[Medline]
10. Ota, K., M. Matsui, E. L. Milford, G. A. Mackin, H. L. Weiner, D. A. Hafler. 1990. T-cell recognition of an immunodominant myelin basic protein epitope in multiple sclerosis. Nature 346:183.[Medline]
11. Martin, R., M. D. Howell, D. Jaraquemada, M. Flerlage, J. Richert, S. Brostoff, E. O. Long, D. E. McFarlin, H. F. McFarland. 1991. A myelin basic protein peptide is recognized by cytotoxic T cells in the context of four HLA-DR types associated with multiple sclerosis. J. Exp. Med. 173:19.[Abstract/Free Full Text]
12. Zamvil, S. S., P. Nelson, J. Trotter, D. Mitchell, R. Knobler, R. Fritz, L. Steinman. 1985. T-cell clones specific for myelin basic protein induce chronic relapsing paralysis and demyelination. Nature 317:355.[Medline]
13. Mokhtarian, F., D. E. McFarlin, C. S. Raine. 1984. Adoptive transfer of myelin basic protein-sensitized T cells produces chronic relapsing demyelinating disease in mice. Nature 309:356.[Medline]
14. Sun, J. B., T. Olsson, W. Z. Wang, B. G. Xiao, V. Kostulas, S. Fredrikson, H. P. Ekre, H. Link. 1991. Autoreactive T and B cells responding to myelin proteolipid protein in multiple sclerosis and controls. Eur. J. Immunol. 21:1461.[Medline]
15. Trotter, J. L., W. F. Hickey, R. C. van der Veen, L. Sulze. 1991. Peripheral blood mononuclear cells from multiple sclerosis patients recognize myelin proteolipid protein and selected peptides. J. Neuroimmunol. 33:55.[Medline]
16. Pelfrey, C. M., J. L. Trotter, L. R. Tranquill, H. F. McFarland. 1993. Identification of a novel T cell epitope of human proteolipid protein (residues 40–60) recognized by proliferative and cytolytic CD4⁺ T cells from multiple sclerosis patients. J. Neuroimmunol. 46:33.[Medline]
17. Markovic-Plese, S., H. Fukaura, J. Zhang, A. al-Sabbagh, S. Southwood, A. Sette, V. K. Kuchroo, D. A. Hafler. 1995. T cell recognition of immunodominant and cryptic proteolipid protein epitopes in humans. J. Immunol. 155:982.[Abstract]
18. Tuohy, V. K., Z. J. Lu, R. A. Sobel, R. A. Laursen, M. B. Lees. 1988. A synthetic peptide from myelin proteolipid protein induces experimental allergic encephalomyelitis. J. Immunol. 141:1126.[Abstract]
19. Sun, J., H. Link, T. Olsson, B. G. Xiao, G. Andersson, H. P. Ekre, C. Linington, P. Diener. 1991. T and B cell responses to myelin-oligodendrocyte glycoprotein in multiple sclerosis. J. Immunol. 146:1490.[Abstract]
20. Kerlero de Rosbo, N., R. Milo, M. B. Lees, D. Burger, C. C. Bernard, A. Ben-Nun. 1993. Reactivity to myelin antigens in multiple sclerosis. Peripheral blood lymphocytes respond predominantly to myelin oligodendrocyte glycoprotein. J. Clin. Invest. 92:2602.
21. Amor, S., N. Groome, C. Linington, M. M. Morris, K. Dornmair, M. V. Gardinier, J. M. Matthieu, D. Baker. 1994. Identification of epitopes of myelin oligodendrocyte glycoprotein for the induction of experimental allergic encephalomyelitis in SJL and Biozzi AB/H mice. J. Immunol. 153:4349.[Abstract]
22. Berger, T., S. Weerth, K. Kojima, C. Linington, H. Wekerle, H. Lassmann. 1997. Experimental autoimmune encephalomyelitis: the antigen specificity of T lymphocytes determines the topography of lesions in the central and peripheral nervous system. Lab. Invest. 76:355.[Medline]
23. Holz, A., N. Schaeren-Wiemers, C. Schaefer, U. Pott, R. J. Colello, M. E. Schwab. 1996. Molecular and developmental characterization of novel cDNAs of the myelin-assosiated/oligodendrocytic basic protein. J. Neurosci. 16:467.[Abstract/Free Full Text]
24. Yamamoto, Y., R. Mizuno, T. Nishimura, Y. Ogawa, H. Yoshikawa, H. Fujimura, E. Adachi, T. Kishimoto, T. Yanagihara, S. Sakoda. 1994. Cloning and expression of myelin-associated oligodendrocytic basic protein. A novel basic protein constituting the central nervous system myelin. J. Biol. Chem. 269:31725.[Abstract/Free Full Text]
25. Holz, A., M. Frank, N. G. Copeland, D. J. Gilbert, N. A. Jenkins, M. E. Schwab. 1997. Chromosomal localization of the myelin-associated oligodendrocytic basic protein and expression in the genetically linked neurological mouse mutants ducky and tippy. J. Neurochem. 69:1801.[Medline]
26. Hudson, L. D.. 1990. Molecular biology of myelin proteins in the central and peripheral nervous systems. Semin. Neurosci. 2:483.
27. Holz, A., M. E. Schwab. 1997. Developmental expression of the myelin gene MOBP in the rat nervous system. J. Neurocytol. 26:467.[Medline]
28. Tuohy, V. K., Z. Lu, R. A. Sobel, R. A. Laursen, M. B. Lees. 1989. Identification of an encephalitogenic determinant of myelin proteolipid protein for SJL mice. J. Immunol. 142:1523.[Abstract]
29. Smith, K. J., J. Pyrdol, L. Gauthier, D. C. Wiley, K. W. Wucherpfennig. 1998. Crystal structure of HLA-DR2 (DRA*0101, DRB1*1501) complexed with a peptide from human myelin basic protein. J. Exp. Med. 188:1511.[Abstract/Free Full Text]
30. Gelder, C. M., J. R. Lamb, B. A. Askonas. 1996. Human CD4⁺ T-cell recognition of influenza A virus hemagglutinin after subunit vaccination. J. Virol. 70:4787.[Abstract]
31. Gelder, C., M. Davenport, M. Barnardo, T. Bourne, J. Lamb, B. Askonas, A. Hill, K. Welsh. 1998. Six unrelated HLA-DR-matched adults recognize identical CD4⁺ T cell epitopes from influenza A haemagglutinin that are not simply peptides with high HLA-DR binding affinities. Int. Immunol. 10:211.[Abstract/Free Full Text]
32. Zamvil, S. S., D. J. Mitchell, A. C. Moore, K. Kitamura, L. Steinman, J. B. Rothbard. 1986. T-cell epitope of the autoantigen myelin basic protein that induces encephalomyelitis. Nature 324:258.[Medline]
33. Guan, K. L., J. E. Dixon. 1991. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal. Biochem. 192:262.[Medline]
34. Bielekova, B., P. A. Muraro, L. Golestaneh, H. McFarland, and R. Martin. 1999. Preferential expansion of autoreactive T-lymphocytes from the memory T-cell pool by IL-7. J. Neuroimmunol. In press.
35. Sakai, K., S. S. Zamvil, D. J. Mitchell, M. Lim, J. B. Rothbard, L. Steinman. 1988. Characterization of a major encephalitogenic T cell epitope in SJL/J mice with synthetic oligopeptides of myelin basic protein. J. Neuroimmunol. 19:21.[Medline]
36. Burns, J., A. Rosenzweig, B. Zweiman, R. P. Lisak. 1983. Isolation of myelin basic protein-reactive T-cell lines from normal human blood. Cell. Immunol. 81:435.[Medline]
37. Pette, M., K. Fujita, B. Kitze, J. N. Whitaker, E. Albert, L. Kappos, H. Wekerle. 1990. Myelin basic protein-specific T lymphocyte lines from MS patients and healthy individuals. Neurology 40:1770.[Abstract/Free Full Text]
38. Muraro, P. A., M. Vergelli, M. Kalbus, D. E. Banks, J. W. Nagle, L. R. Tranquill, G. T. Nepom, W. E. Biddison, H. F. McFarland, R. Martin. 1997. Immunodominance of a low-affinity major histocompatibility complex-binding myelin basic protein epitope (residues 111–129) in HLA-DR4 (B1*0401) subjects is associated with a restricted T cell receptor repertoire. J. Clin. Invest. 100:339.[Medline]
39. Ebers, G. C., D. E. Bulman, A. D. Sadovnick, D. W. Paty, S. Warren, W. Hader, T. J. Murray, T. P. Seland, P. Duquette, T. Grey. 1986. A population-based study of multiple sclerosis in twins. N. Engl. J. Med. 315:1638.[Abstract]
40. Wucherpfennig, K. W., H. L. Weiner, D. A. Hafler. 1993. The cellular and molecular pathology of multiple sclerosis. ed. The Molecular Pathology of Autoimmune Diseases 575. Harwood Academic Publishers, Chur, Switzerland.
41. Haines, J. L., M. Ter-Minassian, A. Bazyk, J. F. Gusella, D. J. Kim, H. Terwedow, M. A. Pericak-Vance, J. B. Rimmler, C. S. Haynes, A. D. Roses, et al 1996. A complete genomic screen for multiple sclerosis underscores a role for the major histocompatability complex. The Multiple Sclerosis Genetics Group. Nat. Genet. 13:469.[Medline]
42. Sawcer, S., H. B. Jones, R. Feakes, J. Gray, N. Smaldon, J. Chataway, N. Robertson, D. Clayton, P. N. Goodfellow, A. Compston. 1996. A genome screen in multiple sclerosis reveals susceptibility loci on chromosome 6p21 and 17q22. Nat. Genet. 13:464.[Medline]
43. Vogt, A. B., H. Kropshofer, H. Kalbacher, M. Kalbus, H. G. Rammensee, J. E. Coligan, R. Martin. 1994. Ligand motifs of HLA-DRB5*0101 and DRB1*1501 molecules delineated from self-peptides. J. Immunol. 153:1665.[Abstract]
44. Wucherpfennig, K. W., A. Sette, S. Southwood, C. Oseroff, M. Matsui, J. L. Strominger, D. A. Hafler. 1994. Structural requirements for binding of an immunodominant myelin basic protein peptide to DR2 isotypes and for its recognition by human T cell clones. J. Exp. Med. 179:279.[Abstract/Free Full Text]
45. Utz, U., W. E. Biddison, H. F. McFarland, D. E. McFarlin, M. Flerlage, R. Martin. 1993. Skewed T-cell receptor repertoire in genetically identical twins correlates with multiple sclerosis. Nature 364:243.[Medline]
46. Dean, G., J. F. Kurtzke. 1971. On the risk of multiple sclerosis according to age at immigration to South Africa. Br. Med. J. 3:725.
47. Kurtzke, J. F.. 1993. Epidemiologic evidence for multiple sclerosis as an infection. Clin. Microbiol. Rev. 6:382.[Abstract/Free Full Text]
48. Sibley, W. A., C. R. Bamford, K. Clark. 1985. Clinical viral infections and multiple sclerosis. Lancet 1:1313.[Medline]
49. Kurtzke, J. F., K. Hyllested. 1979. Multiple sclerosis in the Faroe Islands. I. Clinical and epidemiological features. Ann. Neurol. 5:6.[Medline]
50. Lucchinetti, C. F., W. Bruck, M. Rodriguez, H. Lassmann. 1996. Distinct patterns of multiple sclerosis pathology indicates heterogeneity on pathogenesis. Brain Pathol. 6:259.[Medline]
51. Maatta, J. A., M. S. Kaldman, S. Sakoda, A. A. Salmi, A. E. Hinkkanen. 1998. Encephalitogenicity of myelin-associated oligodendrocytic basic protein and 2',3'-cyclic nucleotide 3'-phosphodiesterase for BALB/c and SJL mice. Immunology 95:383.[Medline]
52. Roach, A., K. Boylan, S. Horvath, S. B. Prusiner, L. E. Hood. 1983. Characterization of cloned cDNA representing rat myelin basic protein: absence of expression in brain of shiverer mutant mice. Cell 34:799.[Medline]
53. Kamholz, J., M. Sessa, S. Scherer, H. Vogelbacker, K. Mokuno, P. Baron, L. Wrabetz, M. Shy, D. Pleasure. 1992. Structure and expression of proteolipid protein in the peripheral nervous system. J. Neurosci. Res. 31:231.[Medline]
54. Garbern, J. Y., F. Cambi, X. M. Tang, A. A. Sima, J. M. Vallat, E. P. Bosch, R. Lewis, M. Shy, J. Sohi, G. Kraft, et al 1997. Proteolipid protein is necessary in peripheral as well as central myelin. Neuron 19:205.[Medline]
55. Pribyl, T. M., C. Campagnoni, K. Kampf, V. W. Handley, A. T. Campagnoni. 1996. The major myelin protein genes are expressed in the human thymus. J. Neurosci. Res. 45:812.[Medline]

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