Journal of Neuroscience Research 28:607413 (1991)

Rapid Communication Myelin/Oligodendrocyte-Specific Protein: A Novel Surface Membrane Protein That Associates With Microtubules C.A. Dyer, W.F. Hickey, and E.E. Geisert, Jr. Department of Neurology, Wayne State University School of Medicine, Detroit (C.A.D.);Department of Pathology, Washington University School of Medicine, S t . Louis (W.F.H.); Department of Cell Biology, Neurobiology Research Center, University of Alabama at Birmingham, Birmingham (E.E.G.) Only a few proteins are known to be exclusively expressed in central nervous system (CNS) myelin. A novel surface membrane protein expressed only in CNS myelin and oligodendrocytes of higher vertebrates has been identified by a monoclonal antibody. This CNS myelin/oligodendrocyte-specific protein, MOSP, has a molecular weight of 48 kDa and a PI of approximately 6.7. In the presence of the monoclonal antibody, MOSP remains on the surface of cultured oligodendrocytes but becomes associated with cytoplasmic microtubules. Our results suggest that MOSP plays an important role in membranekytoskeleton interactions during the formation and maintenance of CNS myelin. MOSP also may play a critical role in the pathogenesis of diseases of CNS myelin. Key words: immunocytochemistry, monoclonal antibody

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INTRODUCTION This paper is the first to describe the novel membrane surface protein found exclusively on oligodendrocytes and myelin in the central nervous sysem (CNS), myelin/oligodendrocyte-specificprotein (MOSP). Myelin, produced by oligodendrocytes in the CNS and Schwann cells in the peripheral nervous system, alters the passive electrical properties of axons, increasing the conduction velocity of action potentials. Although both CNS and PNS myelin share a variety of unique lipids and proteins (Lees and Brostoff, 1984; Lemke, 1988), only a few proteins are known to be selectively expressed in CNS myelin (Lees and Brostoff, 1984; Lemke, 1988; Mikol et al., 1990; Linington et a]., 1984). Defining the differences between CNS and PNS myelin may provide dramatic insights into the process of myelination and the 0 1991 Wiley-Liss, Inc.

pathogenesis of certain idiopathic diseases involving myelin such as multiple sclerosis, certain leukodystrophies, and other myelinopathies. The results presented in this study demonstrate that MOSP has biological properties distinct from other myelin membrane surface components.

MATERIALS AND METHODS Monoclonal Antibody Production The monoclonal IgM antibody CEI was produced from mice immunized with cultured glial membrane proteins and a final boost of rat CNS white matter. Five days following the final boost, the animals were anesthetized with Ketalar (10 mg) and the inguinal lymph nodes were dissected from the mice. Disassociated lymhocytes were prepared and added to an equal number of AG8 myeloma cells; fusion was performed in PEG 1000 (Boehringer Mannheim). CEl was initially detected in an ELISA by using live cultures of mixed glial cells. Tissue Culture Murine oligodendroglial enriched shake-off cultures were grown as described by Dyer and Benjamins ( 1990). Briefly, single-cell suspensions were prepared from 2 day-old Balb/c mouse cerebra. The cells were plated in 75 cm2 flasks and were grown for approximately 8 days in DMEM (Gibco) containing 10% calf serum (Hyclone). Small, dark, process-bearing cells were shaken from the bed layer of predominantly astrocytes and replated on coverslips. The differentiating oli-

Received January 14, 1991: revised February 8, 1991; accepted FebN a y 10, 1991.

Address reprint requests to C.A. Dyer, Department of Neurology. Wayne State University School of Medicine. Detroit, MI 48201.

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godendrocytes were then grown overnight in chemically Liposome ELISA Assay defined medium (CDM) (Bottenstein, 1986). in 9/10 CEl reactivity with mixed ganglioside, ceramide, CDM, 1/10 10% calf serum DMEM for the next 24 hr, sulfatide, galactocerebroside, glucocerebroside, gangliand in 4/5 CDM, 1/5 10% calf serum DMEM for the oside GMI , and asialo GMI was tested in the ELISA remaining days in culture. Cultures were used at about according to Benjamins et al. (1987). 26 days post birth; at this time about 30-50% of the cells are oligodendrocytes and the remainder are predomi- Isolation of MOSP via CE1 Immunoaffinity Column nantly astrocytes with some microglia. Purified CE I (approximately 1 mg) was conjugated Meningeal fibroblast cultures were prepared by us0.5 ml of CNBr-activated Sepharose C L 4 B beads to ing methods similar to those described by McCarthy and according to the protocol supplied by Pharmacia. Oligode Vellis (1980). The cultures were derived from pia dendrocyte-enriched cultures were labeled for 18 hr with dissected from the surface of the developing cerebral 150 pC/ml of (3H)leucine ( 1 11 Ci/mmol) (ICN). A 0.5% cortices of 1-5 day-old rat pups. Triton X100-soluble lysate was prepared and incubated Rat mixed glial cultures were prepared as described with the CEI affinity beads overnight with constant mixby McCarthy and de Vellis (1980). ing at 4°C. Nonadherent proteins were washed from the Neuronal cultures were prepared as described by 7.5. Adherent procolumn with Tris-buffered saline, pH Bartlett and Banker (1984). Timed pregnant SpragueM glycine, pH 2.3, plus 0.2% teins were eluted with 0.2 Dawley rats were deeply anesthetized with sodium penX100; column fractions were immediately neuTriton tobarbital (70 mg/kg) on day 18 of gestation, and the rat tralized with 0.5 M Tris, pH 8.5. The column fractions embryos were removed. The cortices were dissected out; single-cell suspensions were prepared and plated at about containing radioactivity were electrophoresed on a SDS lo4 cells/cm2 as described by Bartlett and Banker (1984). 5-20% polyacrylamide gel (Laemmli, 1970). Gels were Schwann cell cultures were prepared from sciatic sliced and radioactivity was determined. A 48 kDa pronerves from 1-3 day-old Sprague-Dawley rats according tein was isolated by using this method. However, only one out of three CE1 affinity columns effectively isolated to Kreider et al. (1981). MOSP; this may be due to a loss of immunoreactivity of CE 1 after attachment to Sepharose. Immunoprecipitation proved to be the only method which reliably isolated Indirect Immunofluorescent Staining MOSP (see below). Live cultures were immunostained for MOSP by treating them with CEl for 15 rnin and then rhodamineconjugated goat anti-mouse IgM (GAM-TRITC) for 15 min at 37"C, followed by fixation with 4% paraformaldehyde for 10 min. Cells double stained for MOSP and tubulin were first treated while living with CEl for 15 rnin and GAM-TRITC for 15 rnin at 37°C and then fixed with 4% paraformaldehyde for 10 min, and permeabilized with 0.05% saponin. Cultures were then treated with a mouse monoclonal IgG to tubulin followed by GAM-FITC specific for mouse IgG (Organon Teknika). For positive identification of oligodendrocytes and Schwann cells, cultures were double stained for galactocerebroside by addition of rabbit anti-galactocerebroside IgG for 15 rnin followed by fluorescein-conjugated goat antirabbit IgG (GAR-FITC) for 15 min. Schwann cell cultures were stained either within 24 hr of plating or 3 days following plating to examine both galactocerebroside-positive and -negative Schwann cells for expression of MOSP. Immunostaining of tissue sections was performed according to Geisert et al. (1990). Briefly, tissue was fixed in 4% paraformaldehyde, sectioned in a freezing microtome, and immunostained; either fluoresceinconjugated GAM IgG (Boehringer Mannhiem) or GAM IgM (Southern Biotech) was used depending on the class of Drimarv antibody.

Isolation of MOSP via Immunoprecipitation Oligodendroglial-enriched cultures were labeled for 18 hr with 250 pCi/ml ("S)methionine (1,160 Ci/ mmol, New England Nuclear); 0.5% Triton X-100-soluble lysates were prepared and pre-absorbed by incubating with 10 pg of rat IgM monoclonal anti-sulfatide and goat IgG anti-mouse IgM (cross-reactive with rat IgM) bound to protein A-Sepharose CL-4B beads. After constant mixing at 4°C overnight, the beads were sedimented. The lysate was removed and again treated with anti-sulfatide and goat anti-mouse IgM bound to protein A-Sepharose. The beads were removed and the lysate was then treated with goat anti-mouse IgM bound to protein A-Sepharose to ensure that all the anti-sulfatide was complexed and cleared from the lysate. The lysate was then treated with 10 pg CEl and goat anti-mouse IgM bound to protein A-Sepharose overnight as above. The immune complexes bound to the beads were washed 6 times in Tris-buffered saline, pH 7.4, boiled for 3 rnin in Laemmli sample buffer, and electrophoresed on a SDS 5-20% polyacrylamide slab gel. Individual lanes from slab gels were sliced into 1.5 mm pieces which were treated with Protosol (New England Nuclear) overnight, and radioactivity in each slice was determined.

MyelidOligodendrocyte-SpecificProtein

Two-Dimensional Gel Electrophoresis 2-D gel electrophoresis of immunoprecipitates was performed according to O'Farrell (1975). Equilibrium isoelectric focusing was followed by electrophoresis through SDS 5-20% polyacrylamide gels. Radioactivity in the gels was enhanced by fluorography (Bonner and Laskey, 1974) and detected by autoradiography. The pH gradient in the focused gels was determined by slicing duplicate gels into 0.5 cm sections, placing each section into 0.5 ml dH,O, and measuring the pH after 30 min. A pH gradient ranging from 3.2 to 9.3 was reproducibly obtained. The PI of the CEI protein spots detected on the 2-D gel autoradiograph was determined by measuring the distance the proteins migrated through IEF gel and comparing that to the pH gradient obtained from the sliced gels. Lectin Affinity Columns 0.5% Triton X- 100 lysates from ("S)methioninelabeled oligodendrocyte shakeoff cultures were passed through concanavalin A-agarose (Sigma) and wheat germ agglutinin-agarose (Sigma) columns. The columns were washed extensively with 0.2% Triton X-100 Trisbuffered saline (TBS), pH 7.4, until background counts were obtained. Glycoproteins were eluted from the concanavalin A column with 0.2 M D-mannose in 0.2% Triton X-100 TBS and from the wheat germ column with 0.2% N-acetyl D-glucosamine in 0.2% Triton X-100 TBS. The adherent glycoprotein fractions were then immunoprecipitated and analyzed by gel electrophoresis as previously described.

RESULTS AND DISCUSSION MOSP is recognized by the monoclonal IgM antibody, CEI, that was produced from mice immunized with rat CNS white matter. A number of rat tissues were screened by using immunohistochemical methods to determine the distribution of MOSP. In sections of the CNS, only myelin and oligodendrocytes were labeled. No specific staining was observed in sections from any of the other tissues tested, including peripheral nerve, retina, liver, skin, and kidney. The epitope on MOSP recognized by CE1 is present in tissue sections of CNS myelin from chick, mouse, rat, ferret, cat, monkey, and man. Interestingly, no immunoreactivity was observed in sections of frog or goldfish brain. Thus, the CEl epitope appears to be conserved in higher vertebrate species. The distribution of MOSP was further characterized in normal and pathological tissues. Surgically obtained and autopsy human CNS tissue were immunostained with CEI . In sections of normal spinal cord, the distribution of MOSP (Fig. la,b) is observed in brightly

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stained rings, similar to MBP (Fig. Id). In the spinal cords of patients with multiple sclerosis, both the CEI epitope (Fig. lc) and myelin basic protein (Fig. Id) are absent from the sclerotic plaques. This demonstrates that CEl stains normal CNS myelin and that when demyelination occurs, immunoreactivity for MOSP, like MBP, is lost. A variety of culture systems were immunostained to determine the specific cell type(s) expressing MOSP, including enriched mouse oligodendrocytes, mixed rat glial cells, meningeal rat fibroblasts, rat neurons, and Schwann cells. In these cultures, only oligodendrocytes were labeled, with all other cell types negative (not shown). Live oligodendrocytes were briefly treated with CEI ( 5 min at 37"C), followed by fixation and addition of second antibody conjugated to fluorescein; the positive surface staining observed under these conditions indicates that MOSP is constitutively expressed on the surface of oligodendrocytes. In tissue culture. murine oligodendrocytes elaborate extensive membrane sheets that have uniform surface distributions of MOSP (Fig. 2a) and other selected myelin markers (Dyer and Matthieu, unpublished results; Dyer and Benjamins, 1988a,b). These membrane sheets contain an internal network of microtubular veins that surround domains of myelin basic protein (Dyer and Benjamins, 1988b). The normal distribution of surface antigens on cultured oligodendrocytes can be visualized by fixation and then immunocytochemical staining (Dyer and Benjamins, 1988a). When antibodies specific for selected myelin markers bind to live oligodendrocytes, the antibody:antigen complexes reorganize to form characteristic patterns. For example, when antibodies reactive with either galactocerebroside or sulfatide bind to oligodendrocytes, this results in the redistribution of the antibody:glycolipid complexes into surface patches directly over the cytoplasmic domains of myelin basic protein (Dyer and Benjamins, 1988b); see Figure 2b for galactocerebroside patching. Preliminary data show that the binding of a monoclonal antibody to the myelin/oligodendrocyte glycoprotein (MOG) on the surface of cultured murine oligodendrocytes also results in redistribution of the antibody:protein complexes over cytoplasmic domains of myelin basic protein (Dyer and Matthieu, unpublished results). The surface expression of proteolipid protein on cultured oligodendrocytes is diffuse and faint (Dyer and Benjamins, 1988b), unlike MOSP, which is abundantly expressed on the membrane surface. In contrast to the redistribution pattern of the above myelin markers, MOSP redistributes in a lacy surface pattern (Fig. 2c) that directly overlies internal microtubular veins (Fig. 2d). Furthermore, this redistribution pattern is dependent upon the integrity of the microtubular network. Following pretreatment of oligodendrocytes with

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Fig. 1. Immunofluorescent staining of human CNS tissue from normal spinal cord (a,b) and from an active multiple sclerosis plaque in the spinal cord (c,d). a: CEl staining of transversely cut myelin tubes in the periphery of the spinal cord. x 125. b: CEI staining of the intraparenchymal portion

of a spinal nerve root with tangentially cut axons. c: Loss of

colchicine, a drug which depolymerizes microtubules, the redistribution of MOSP to produce the lacy surface pattern does not occur; the membrane sheets remain solidly stained for MOSP (Fig. 2e,f). These observations suggest that MOSP is capable of forming stable transmembrane associations with the cytoskeletal network of oligodendrocytes and that this association is dependent upon the presence of polymerized microtubules. To define the antigen recognized by C E l , the reactivity of the antibody with myelin lipids and proteins was examined. A variety of glycolipids, including the two highly antigenic myelin-enriched glycolipids galactocerebroside and sulfatide (Sommer and Schachner, 1981; Ranscht et al., 1982; Neike et al., 1988; Benjamins et al., 1987), were screened in a liposome ELISA assay. No CEl immunoreactivity was observed with any of the lipids tested. Western blotting with CEl is problematic. While a number of blots have been negative, a faint band in the vicinity of the 43 kDa molecular weight marker has been observed on some occasions. Blotting

with monoclonals may be difficult for a number of reasons including l) the loss of epitopes after denaturation or 2) the binding of reactive epitopes to the nitrocellulose, thus not exposed for antibody binding. Therefore, immunoaffinity column chromatography (see Materials and Methods) and immunoprecipitation with CE 1 were performed to isolate MOSP. Immunoprecipitations were performed on Triton X- 100-soluble lysates of cultured oligodendrocytes radiolabeled with (3sS)methionine. The lysate was first treated with anti-sulfatide IgM to remove labeled proteins that nonspecifically bind to IgM molecules and then was immunoprecipitated with CEl . This procedure resulted in the isolation of a major peak of 48 kDa (Fig. 3a). When a second sample, immunoprecipitated in an identical manner, was examined by 2-D gel electrophoresis, a pair of labeled proteins with isoelectric points of approximately 6.7 were observed at 48 kDa (Fig. 3b.c). Despite the extensive preclearing of the lysates prior to precipitation with CEl (see Materials and Methods), proteins which nonspecifically adhere to IgM

MOSP staining in a multiple sclerosis plaque (right side of photograph). d: Loss of MBP staining on a multiple sclerosis lesion (lower left portion of photograph) is similar to that observed with CEI in c. x 250.

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Fig. 2 . Association of surface MOSP with cytoplasmic microtubules following antibody binding. a: Representative oligodendrocyte fixed prior to staining demonstrates that MOSP is uniformly distributed on the surface of membrane sheets under these conditions. b: Treatment of live oligodendrocytes with anti-galactocerebroside IgG for 15 min followed by GARFITC for 15 min at 37°C results in redistribution of the antib0dy:galactocerebroside complexes into surface patches. c: Following treatment of live cultures with CEI for 15 min and then GAM-TRITC for 15 min at 37"C, MOSP redistributes in a lacy network on the membrane surface. d: Same cell in c

double stained for tubulin. Cells were fixed with parformaldehyde, permeabilized with saponin, and treated with mouse anti-tubulin IgG and then GAM-FITC. Note the similar staining patterns of MOSP and microtbules. e: Cultures were exposed to 10 p.g/ml of colchicine for 5 hr and then stained live for MOSP as above. MOSP does not redistribute, but remains evenly distributed on the membrane surface. f: Same cell in e following fixation, permeabilization, and tubulin staining shows that following colchicine treatment, the majority of microtubules are depolymerized within membrane sheets. X 280.

and/or goat anti-mouse IgM and/or Sepharose were still precipitated by CEl second antib0dy:Sepharose. The same pattern of nonspecific proteins appears in the CEl and control 2-D gels except for the 48 kDa proteins in the CEI gel. These two radiolabeled proteins probably represent a single protein, since they migrate as a doublet in 2-D gels and share the CEl epitope. The slight difference in isoelectric points suggests that MOSP may be posttranslationally modified. To determine if MOSP contains carbohydrate side chains containing mannose or sialic acid, the radiolabeled lysates were passed through two lectin affinity columns. MOSP was not retained on either column (not shown), indicating that carbohydrates con-

taining high mannose (concanavalin A) or polysialic acid (wheat germ agglutinin) are not covalently attached to MOSP. Taken together, the available information indicate that MOSP is a 48 kDa protein with a PI of about 6.7. MOSP has physical and biological properties that are distinct from MOG and proteoloipid protein, the only other known integral membrane proteins described to date that are specific to CNS myelin (Lees and Brostoff, 1984; Mikol et al., 1990; Linington et al., 1984; Lebar et al., 1986; Brunner et al., 1989; Nave and Milner, 1989). The molecular weight of MOSP (48 kDa) is considerably higher than MOG (26 and 28 kDa) or proteolipid protein

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Fig. 3. Isolation of MOSP. a: CEI immunoprecipitated a significant 48 kDa protein peak (MOSP) which was not immunoprecipitated by anti-sulfatide (control). b,c: Many proteins were precipitated nonspecifically by anti-sulfatide (b): however, a doublet of 48 kDa and a PI of about 6.7 (arrow) were

specifically precipitated by CEl (c). (Some proteins easily enter IEF gels, while others do not; MOSP may have difficulty entering IEF gels; thus the likely reason why less MOSP is observed in the 2-D gel than the I-D gel.)

(27 kDa) (Lees and Brostoff, 1984; Nave and Milner, 1989; Matthieu and Amiguet, 1990). Proteolipid protein is also present in the cytoplasm of Schwann cells but is not inserted into the plasma membrane ( h c k e t t et a]. , 1987; Griffiths et al., 1989; Baron et al., 1989). Or pathogenic In vivO, the physiologic ment of MOSP remains undefined. It is possible that the effects caused by binding Of the monoclonal antibody CE1 to MoSP On oligodendrocytes mimic the effects elicited by interaction of MOSP with an in vivo endogenous ligand present on other cell types or in myelin itself. The association of MOSP with cytoplasmic microtubules of cultured oligodendrocytes after antibody binding suggests an important role for MOSP in membranekytoskeletal interactions during assembly and maintenance of the multiple myelin sheaths elaborated by individual oligodendrocytes. Abnormalities in the metabolism of MOSP may be central to the pathogenesis of specific myelinopathies or leukodystrophies. Moreover, since MOSP is expressed on the extracellular surface of cultured oligodendrocytes, and presumably myelin, it is possible that MOSP serves as an antigenic target in certain idiopathic diseases that selectively affect CNS myelin.

ACKNOWLEDGMENTS We thank Dr. Joyce Benjamins for helpful discussion and review of this manuscript. we are grateful to ~~~~~l~ Bealmear and Dr. Robert Lisak for providing us with Schwann cell cultures. We thank Ms. Kainette Jones for her secretarial expertise. Anti-sulfatide (A007) and anti-tubulinwere generous gifts from Dr. Kari StefanSSOn and Dr. Lester Binder, respectively. This work was supported by National Institutes of Health grants NS-13143, NS-27321, and NS-236,3 and by National Multiple Sclerosis Society grant ppo172.

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