Journal of Neuroscience Research 33: 177-187 (1992)

Myelin/Oligodendrocyte Glycoprotein Is a Unique Member of the Immunoglobulin Superfamily M.V. Gardinier, P. Amiguet, C. Linington, and J.-M. Matthieu Laboratory of Neurochemistry, Department of Pediatrics, CHUV, Lausanne, Switzerland (M.V.G., P.A., J.-M.M.); Laboratory of Neuroimmunology, Max-Planck-Institute for Psychiatry, Planegg-Martinsreid, Germany (C.L.)

Myelin/oligodendrocyte glycoprotein (MOG) is a primary target autoantigen in experimental autoimmune encephalomyelitis, a widely used animal model for autoimmune demyelinating diseases such as multiple sclerosis. We have isolated several rat MOG cDNAs and confirmed their identity by comparision with MOG N-terminal peptide sequence. As expected, MOG mRNA expression is CNS-specific and peaks during active myelination. Our studies show that full length MOG mRNA is -1.6 kb and encodes a signal peptide of 27 amino acids, followed by 218 residues for mature MOG (24,962 MW). A single site for Nglycosylation is found at Asn-31. Rather than the ubiquitous AAUAAA polyadenylation signal, a series of three overlapping, rare poly A signals were identified. The N-terminal half of mature MOG shares 52% identity with bovine butyrophilin, a possible lipid receptor. This same region has 39% identity with chicken B-G antigen, a major histocompatibility complex antigen involved in B cell selection and immune repertoire development. We show that both MOG and butyrophilin, each exhibiting a single Iglike variable region domain, meet criteria for inclusion in the immunoglobulin superfamily. Moreover, MOG appears to represent a unique member of this superfamily in that it possesses two potential transmembrane domains, in contrast to a single membrane-spanning domain or glycophospholipid anchor found in all other members of Ig superfamily members. 0 1992 Wiley-Liss, Inc. Key words: autoimmunity, demyelination, multiple sclerosis, polyadenylation, receptor

may undergo more than a 600-fold increase in plasma membrane (Raine, 1976). This activity can be detected by large increases in gene activity of the major myelin proteins-proteolipid proteins (PLP, DM20), myelin basic proteins (MBP), myelin-associated glycoprotein (MAG), and 2',3'-cyclic nucleotide 3'- phosphodiesterase (CNP). Myelin also contains several quantitatively minor glycoproteins (Poduslo et al., 1976; Zanetta et al., 1977). For the most part, these proteins remain virtually uncharacterized, and their functions are unknown. Undoubtedly, these proteins play significant roles that must be discovered to achieve a real understanding of myelinogenesis. Myelin/oligodendrocyte glycoprotein (MOG) was originally identified and characterized as a minor CNSspecific glycoprotein by the mouse monoclonal antibody, 8- 18C5, directed against rat cerebellar glycoproteins (Linington et al., 1984). Purified MOG migrates primarily as a 26-28 kDa doublet band with a minor band at 53 kDa on SDS-PAGE immunoblots (Matthieu and Amiguet, 1990). Recent evidence has shown that both chemical and enzymatic deglycosylation result in a single 25 kDa peptide, and thus the major 26-28 kDa components reflect differential glycosylation while the minor 54 kDa component is probably an aggregate form (Amiguet et al., 1992). Immunoelectron microscopy localized MOG to oligodendrocyte processes and the external surface of myelin sheaths in adult rat CNS; some MOG expression was also noted at the oligodendrocyte cell body in developing rat CNS (Linington et al., 1988). Further analysis with immunogold electron microscopy, which allows detection of antigens in compact myelin, confirmed MOG's predominantly external localization

INTRODUCTION Formation of the complex multilamellar myelin sheath occurs during early development as a tightly regulated, short, sudden burst of membrane production by oligodendrocytes. During this time, an oligodendrocyte 0 1992 Wiley-Liss, Inc.

Received May 26, 1992; revised June 17, 1992; accepted June 23, 1992. Address reprint requests to Dr. Minnetta V. Gardinier, Department of Pathology, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL.

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with minor labeling in compact myelin (Brunner et al., 1989). The developmental pattern of MOG expression parallels that of other myelin proteins, but lagging about 2 days behind MBP expression (Matthieu and Amiguet, 1990). A similar observation was made using cultured neonatal rat optic nerve oligodendrocytes (Scolding et al., 1989). This late appearance of MOG relative to other myelin proteins suggests that MOG may function in maintenance and completion of the myelin sheath. MOG is the major target autoantigen for demyelinating autoantibodies in experimental allergic encephalomyelitis (EAE), which is an autoimmune mediated inflammatory demyelinating disease of the CNS. EAE has been used extensively as an animal model for multiple sclerosis (MS) and is induced in susceptible species following immunization with CNS tissue (or purified myelin) and Freund’s complete adjuvant. Using the guinea pig EAE model, Lebar and coworkers deduced the presence of an immunodominant myelin protein autoantigen, M,, that was responsible for the induction of a potent demyelinating antibody response (Lebar et al., 1976, 1979). Subsequently, M, was identified as MOG (Lebar et al., 1986). MOG localization on the outer surface of the myelin sheath provides an ideal target antigen for attack during autoimmune demyelination. In vitro studies have also demonstrated that treatment of aggregating brain cell cultures with anti-MOG antiserum in the presence of complement results in significant demyelination (Kerlero de Rosbo et al., 1990). Its importance in the immunopathogenesis of demyelination was demonstrated by correlation of MOG-specific autoantibody titer with in vivo demyelinating activity of sera obtained from guinea pigs with chronic relapsing EAE (Linington and Lassmann, 1987). In addition, intravenous injection of MOG-specific monoclonal antibodies massively augmented CNSspecific demyelination in animals with acute EAE induced by either active immunization with MBP or passive transfer of MBP-specific T cells (Schluesener et al., 1987; Lassmann et al., 1988; Linington et al., 1988; Piddlesden et al., 1991). Moreover, repeated cotransfer of MBP-specific T cells and anti-MOG antibodies induced the formation of persistently demyelinated lesions similar to those seen in MS (Linington et al., submitted). Recently, MOG was also implicated as a target autoantigen in the pathogenesis of MS (Xiao et al., 1991). In order to extend our understanding of MOG’s function and role as an autoantigen in the pathogenesis of demyelinating diseases, we have isolated several rat MOG cDNA clones. The identity of these clones was confirmed by comparison of the deduced amino acid sequence in rat with mouse MOG N-terminal peptide sequence (Amiguet et al., 1992). CNS-specific MOG mRNA is approximately 1.6 kb, and its pattern of de-

velopmental expression coincides with that seen for other myelin protein mRNAs, peaking between postnatal days 15 and 25 in rodents. MOG mRNA does not contain the ubiquitous AAUAAA polyadenylation signal, but rather a series of overlapping, rare variants of this signal. Finally, we show that MOG shares significant partial homologies with bovine butyrophilin, a lactating mammary gland-specific glycoprotein involved in lipid interactions, and chicken B-G antigen, a major histocompatibility complex (MHC) antigen. This same region of homology fits the criteria for an immunoglobulin-like domain, and thus MOG joins the immunoglobulin superfamily. Surprisingly, MOG appears to be a unique member of this superfamily in that MOG contains two putative transmembrane domains in contrast to the single transmembrane domain (or glycophospholipid anchor) exhibited by other superfamily members.

MATERIALS AND METHODS Library Screening A rat oligodendrocyte-enriched library was the kind gift of Dr. Wendy Macklin (University of California, Los Angeles); mRNA from cultured oligodendrocytes (McCarthy and de Vellis, 1980) was used for synthesis of this library. A hgtll cDNA library was screened (=8 X lo5 recombinants) for MOG fusion protein expression with a mixture of MOG mAbs (8-l8C5, Y 1, Y 10; 1:1,000 dilution each), followed by incubation with alkaline phosphatase-conjugated goat anti-mouse IgG (1 5,000 dilution) per manufacturer’s instructions (ProtoBlot immunoscreening system, Promega). Normal goat serum (20%) was used as the blocking agent. Three clones were obtained following three to four rounds of plaque purification. Inserts were excised by EcoRI digestion and subcloned into pBSII(KS-). Additional MOG cDNA clones were obtained by screening another 8 x lo5 clones from the oligodendrocyte-enriched library in AZAPII using ”P-labeled purified insert from pMOG3. Transfers and hybridizations were performed with standard techniques (Sambrook et al., 1989). Hybridization solution contained 6 X SSC/5 X Denhardt’s/O.S% SDS/O.l mg per ml salmon sperm DNA, and hybridizations were performed at 55°C with ~ 0 . 5X lo6 c p d m l 32P-pMOG3 insert. Filters were washed in 2 x SSC/O.l% SDS, 1 x SSC/O.l% SDS, and 0.5 X SSCIO. 1% SDS (30 min each) at 50°C. Kodak XAR-5 films were exposed at -80°C for 1-3 days with DuPont Cronex intensifying screens. Twenty-nine MOG cDNA clones were isolated following three to four rounds of plaque purification. Rescue of pBSII(SK-) plasmids containing MOG inserts was performed by in vivo excision protocol (Stratagene).

MOG Is a Unique Ig Superfamily Member

Sequence Analysis Nucleotide sequence was obtained by standard dideoxy nucleotide sequencing reactions (Sanger et al., 1980) with 35S-dATP and Sequenase 2.0 per rnanufacturer's instructions (Stratagene). Sequencing reactions were performed using double-stranded plasmid DNA from cesium chloride minipreparations (Saunders and Burke, 1990). Standard 8 M urea denaturing polyacrylamide gels (6%) were used for separation of sequencing reaction products. Autoradiographic exposures of fixed and dried gels were carried out for 1-7 days at room temperature with Kodak XAR film. Sequencing films were read with a CBS Scientific gel reader, and the sequence was assembled using IBI Pustell sequence analysis package. T3 and T7 oligonucleotide primers (Promega) were used to obtain 5' and 3' terminal sequence for 17 MOG clones. Overlapping sequences were identified, and a full length strand of MOG cDNA was determined. An apparent full length cDNA clone, pMOG24, was used to ascertain the sequence of the opposite strand. This sequence was obtained using five MOG-specific oligonucleotides (plus and minus designations indicate strand synthesis during sequencing reactions):

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lo6 c p d m l 32P-labeled pMOG3 insert. Unhybridized probe was removed following three washes (15-20 min each, 50°C) in 2 x SSC/O.l% SDS, 1 X SSC/O.l% SDS, and 0.5 X SSC/O.l% SDS. Kodak XAR-5 films were exposed at -80°C for 1-13 days with DuPont Cronex intensifying screens.

Primer Extension Analyses Primer extension assays (Sambrook et al., 1989) were performed to map the 5' end of MOG mRNA. An oligonucleotide was designed for hybridization to MOG mRNA approximately 100 nucleotides from the putative 5' end: oMOG12

~

(nt 96-1 15)

5'-CAGAGAAAGGCTCCACACAC-3'

This probe (=lo pmol) was radiolabeled at the 5' end with 32P-ATP (50 pCi) and T4 polynucleotide kinase ( 5 U) (Sambrook et al., 1989). Following inactivation of the kinase, -20% of the labeling reaction was coprecipitated in ethanol with 20 p g total cellular RNA. The primer/RNA mixture was resuspended in 20 p1 2.5 X MMLV reverse transcriptase buffer ( 5 X stock = 250 mM Tris, pH 8.3/375 mM KC1/15 mM MgCl,), denatured for 10 min at 70"C, and hybridized for 2 hr at 55°C. Subsequently, the extension reaction was diluted (50 p1 final volume) to obtain 1 X MMLV buffer in the presoMOG5 - (nt 305-324) 5'-CGGTACAGATGAACCACTCT-3' oMOG6 - (nt 624-643) 5'-ACGGAGTTTTCCTCTCAGTC-3' ence of 10 mM DTT, 0.5 mM dNTPs, and 200 U MMLV reverse transcriptase (Bethesda Research LabooMOG7 + (nt 799-8 18) 5'-TCTTGAAGAGCTAAGAAACC-3' oMOG8 - (nt 913-932) 5'-GTTTCTGTAGATGTAGATTG-3' ratories). Following incubation for 2 hr at 37°C and one phenokhloroform extraction, nucleic acids were precipoMOG9 + (nt 1,105-1,124) 5'-GATTCTGCCAACCAGGACAC-3' itated overnight (-20°C). Extension products were sepoMOG13 + (nt 1,395-1,414) 5'-TGAGAAGCAAGGGTCGAGAA-3' arated in 8 M urea 6% polyacrylarnide sequencing gels. Film exposure was overnight as described above. SeNorthern Blot Studies quencing reactions of known plasmid DNAs were used Total cellular RNA was isolated as described pre- as size markers. viously (Gonda et al., 1982; Gardinier and Macklin, 1988). Briefly, single whole brain homogenates were digested with Proteinase K in SDS (37"C, 1-2 hr), fol- RESULTS lowed by phenoUchloroform extraction and ethanol pre- Clone Isolation and Sequence Analysis cipitation. DNA was removed by digestion with RNase A rat oligodendrocyte-enriched cDNA hgt 1 1 lifree-DNase (37"C, 2 X 15 min), followed by phenol/ brary was screened for MOG expression with a mixture chloroform extraction. RNA was precipitated and stored of three mAbs (8-18C5, Y I and Y lo), and three clones in ethanol at -20°C. (pMOG3, pMOG4, pMOG5) were identified and puriMOG mRNA was studied using standard denatur- fied. The inserts (each -1 kb) were removed and subing agarose gels (Davis et al., 1986) and Northern blot cloned into the EcoRI site of pBluescript plasmid for analysis (Thomas, 1980). Total cellular RNA (20 pg) subsequent studies. The library was rescreened with rawas separated in 1 % denaturing agarose gels. RNA was diolabeled insert from pMOG3, and 29 additional clones transfered to Zetaprobe (BioRad) nylon membranes in 10 were isolated. Sequence analysis of the inserts at 5' and x SSC and irreversibly fixed by laying membrane on 3' termini allowed us to determine that the clones confilter saturated with 50 mM NaOH for 5 min. Filters were sisted of multiple isolates for five groups of overlapping neutralized by rinsing three times in 2 X SSC. Hybrid- clones (Fig. 1). Comparisons between deduced amino acid seization solution contained 6 X SSC/5 X Denhardt'd 0.5% SDSO.1 mg per ml salmon sperm DNA. Hybrid- quence from rat MOG cDNAs and previously deterizations were performed overnight at 55°C with 1-2 X mined mouse MOG N-terminal peptide sequence con-

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ing frame encodes a mature MOG peptide of 218 amino acids with a calculated MW of 24,962. This figure is in , excellent accordance with the estimated MW of deglyp MOG 7 I cosylated MOG at 25,000. Asn-31 was identified as the only potential attachment site for N-linked carbohyp MOG 14 1 drates. Finally, two hydrophobic regions (Ile- 122 to Gln150; Val-I75 to Asn-200) indicate the presence of two p MOG 19 I possible transmembrane domains (Fig. 4). A canonical p MOG 24 1 AAUAAA polyadenylation signal was not found, and the poly A tail was limited to a short stretch of 13 A residues. At 16-31 nucleotides above the short poly A 800 1200 1600 NT 0 400 stretch, an AU-rich area was found to contain four over0 noncodlngieglOnS sequence (inoiys~s lapping hexanucleotide sequences that have been previsignal peptide Coding reglo" ously identified as alternative, less efficient polyadenylation signals (AAUUAA, AAUAAU, and AAUACA) Fig. 1 . A schematic diagram of rat MOG cDNA clones and (Wickens, 1990). This poly A motif was found in all rat sequencing strategy. Open box, noncoding region; hatched cDNA clones containing this region; preliminary data box, putative signal peptide; closed box, coding region for shows that this same sequence is also conserved in mature MOG. Arrows indicate direction and length of se- mouse MOG mRNA (data not shown). quence analysis. Sequencing oligonucleotides are shown as A database search revealed that MOG cDNA has small boxes at ends of arrows. NT, nucleotide. not been identified previously. A FASTA comparison (Pearson and Lipman, 1988) showed striking partial sequence homologies with bovine butyrophilin and chicken firmed the identity of our rat MOG clones (Fig. 2). B-G antigens (Fig. 5). Butyrophilin, a 56 kDa membrane Mouse peptide sequence had yielded 18 unambiguous glycoprotein, is expressed specifically in lactating mamassignments, and the rat deduced sequence matched at 16 mary gland (Mather et al., 1980). The first 1 15 amino positions. Furthermore, probable amino acid assign- acids of butyrophilin (Jack and Mather, 1990) and MOG ments were confirmed in four out of six ambiguous po- exhibit 52% identity. In addition, this same region is sitions (Arg-13, Glu-19, Glu-21, and Arg-25). Only four -39% identical to chicken B-G antigens (Miller et al., clear differences were found; however, preliminary 1991), which are a very polymorphic group of MHC mouse cDNA sequence indicates that three of these dif- antigens. A stretch of 22 amino acids from Glu-64 to ferences are attributable to errors in the mouse peptide Ile-85 shows very strong sequence similarity between sequence analysis (data not shown). A species difference MOG and B-G antigens with 55% identity and 41% condoes occur at residue 10 (Tyr, mouse; His, rat). In con- servative substitutions. We noted that Cys-24 and Cys-98 are spaced apclusion, the identity of our rat MOG cDNA clones was confirmed by comparison with N-terminal mouse MOG propriately for variable (V) region domains (65-75 amino acid sequence, and ambiguities within the peptide amino acids) of the immunoglobulin superfamily (Fig. 6). Closer inspection and comparison with other memsequence were eliminated. Sequence analysis of 5' and 3' termini from all bers of this subfamily revealed that MOG also contained MOG clones (note arrows in Fig. 1) revealed regions of several conserved amino acids within its potential Ig-like identical overlapping sequences and allowed us to as- domain. A PROFILE analysis (Gribskov et al., 1987, semble a first strand MOG cDNA sequence (Fig. 3 ) . 1990) was performed to confirm the significance of the This sequence was 1.6 kb in length and corresponded alignment between MOG and other members of the Ig in size to pMOG24. Five oligonucleotides were designed superfamily . Significant alignments are identified as from this sequence information and were used to obtain those sequences with z (SD units) > 6. Both butyrothe opposite strand of sequence from pMOG24 only. All philin, not identified previously as an Ig superfamily sequence obtained from pMOG24 was identical to se- member, and MOG yield highly significant z values of quence from the other partial MOG clones. 7.85 and 10.80, respectively. Thus MOG and butyroFollowing a 5' noncoding region of 88 nucleotides, philin clearly qualify as members of the variable region an open reading frame of 735 nucleotides was identified. subfamily of the Ig superfamily. Based on the N-terminal peptide sequence and the deduced amino acid sequence from the open reading frame, MOG mRNA Expression Northern blot analysis of total cellular RNA from we predict a signal peptide of 27 amino acids, encoded by nucleotides 89-169. The remainder of the open read- various mouse and rat tissues identified a single mRNA 1

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1 GGCTTCTTGG GGTAAGGGAC ATGCAGCCAG AGGGCCTTAG CTTGGCCTGA TCCTGCTGTC AGGGACCCTG TCCCCAGTAA CCATAAAG 89 ATG GCC GGT GTG TGG AGC CTT TCT CTG CCC AGC TGC CTC CTG TCC CTG CTC CTC CTC CTC CAG TTG TCA CGC

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233 CTG CCG TGC CGT ATA TCT CCT GGG AAG AAT GCC ACG GGC ATG GAG GTG GGG TGG TAC CGT TCT CCC 'lTT TCA 2 2 Leu Pro C y s Arg Ile ser Pro Gly L y s a A l a Thr Gly Met Glu Val Gly Trp Tyr Arg ser Pro Phe ser 305 AGA GTG GTT CAT CTG TAC CGA AAT GGC AAG GAC CAA GAC GCA GAG CAA GCG CCT GAA TAC CGG GGA CGC ACA 46 Arg Val Val H i s Leu Tyr Arg Asn Gly Lys Asp Gln Asp Ala Glu Gln Ala Pro Glu Tyr Arg Gly Arg Thr 377 GAG CTT CPG AAA GAG TCT ATC GGC GAG GGA AAG GTT GCC CTC AGG ATC CAG AAC GTG AGG TTC TCG GAT GAA 70 Glu Leu Leu Lys Glu Ser Ile Gly Glu Gly Lys Val Ala Leu Arg Ile Gln Asn Val Arg Phe Ser Asp Glu 449 GGA GGC TAC ACA TGC TTC TTC AGA GAC CAC TCC TAC CAA GAA GAA GCC GCC GTG GAG Tl!G AAA GTA GAA GAT 94 Gly Gly Tyr Thr Cys Phe Phe Arg Asp H i s Ser Tyr Gln Glu Glu Ala Ala Val Glu Leu Lye Val GlU Asp 521 CCC TTC TAC TGG ATC AAC CCT GGC GTG CTG GCT CTC ATT GCC CTT GTG CCT ATG CTG CTC CTG CAG GTC TCT 118 Pro Phe Tyr Trp Jle Asn Pro GIv Val Leu Ala Leu Ile Ala Leu Val Pro Met Leu Leu Leu Gln Val Ser

593 GTA GGC CTT GTA TTC CTC TTC CTG CAG CAC AGA CTG AGA GGA AAA CTC CGT GCA GAA GTC GAG AAT CTC CAT 142 Val Glv Leu Val Phe Leu Phe Leu Gln H i s Arg Leu Arg Gly Lys Leu Arg Ala Glu Val Glu Asn Leu H i s 665 CGG ACT TTT GAT CCT CAC TTC CTG AGA GTG CCC TGC TGG AAG ATA ACA CTG TTT GTT ATT GTC CCT GTT CTT 166 Arg Thr Phe Asp Pro H i s Phe Leu Arg Val Pro Cvs TrD Lvs IIe Thr Leu Phe Val Ile Val Pro Val Leq

737 GGA CCC CTG GTT GCT TTG ATC ATC TGC TAC AAC TGG CTG CAC CGA AGA CTG GCA GGA CAG TTT CTT GAA GAG 190 G1v Pro Leu Val Ala Leu Ile 11e Cvs Tvr Asn Trp Leu H i s Arg Arg Leu Ala Gly Gln Phe Leu G1U Glu 809 CTA AGA AAC CCC TIT TGAGTGATGG TGCGTCTTCC TGGAGCGAAG GAAAGCCTGG CAGGTCCPTG GGGATGTCTC TCACXGCCT 214 Leu Arg Asn Pro Phe End 894 CTCTGTTATG GACACATTCC AATCTACATC TACAGAAACA CTCGGGATCC TGAGTAGAAC TTGATPTTCC TCCCACAACT GTCTTTCTTT 984 CCTCAGCTCC TTCCTCTAAA TCI'TTTTTGA CTCCTGTCTT AGTCTGCCTC TAGAAAACAC CCCATGACTA AGGCCAGGGA GTCTATCAGG 1074 AGACACCTAT GTACCAGAGT AATACAGGAG TGATTCTGCC AACCAGGACA CCCAGCGCTT CAACATTACG ATCTACAGAA CATCAGCTCT 1164 GCAAATGGTG GATATTTCTT CCAGAGCCAC AGTCTTGACT GGGAAGAATA TCAGTGGGGA GAGTAAGGGG GTGAGGGACA GAAGAACCCA 1254 CAACATGAGG GAAAGCTAAA TGAGCCAGGG TATGAATATG GGAGCCTGAA CCAAAACCTG GAGATTAAAA AAAAAAGTCA AAAGAATACT 1344 GACTGGATCA GAGCAACACC ATCTTGCTTT GGCAGCCTGG TGGGAGGACC TTGAGAAGCA AGGGTCGAGA ACTGGGTCTG TTCTCTCATT 1434 TTTCTCCTAT CTTATTTGAG TACCCACTGT TTGGTTTTTT TGTTTTTTTT TTTTTCTTCT T C G m T T T C AACAATTACT ACTTGGTGGT 1524 GTATCAGACA CAAACAAGCT AGGTGCTAAT TAATAATAAT ACAAGTTTCT TTGTGCACAA AAAAAAAAAA A 4**44t* .E ..D ...

*******

Fig. 3. Rat MOG cDNA sequence. The full-length cDNA sequence of pMOG24 is given. A putative signal peptide sequence is underlined. Two potential transmembrane domains are double underlined. A probable N-glycosylation site (Asn-

31) is boxed. Four overlapping, alternative polyadenylation signals are indicated: AATTAA (+), AATAAT (m), and AATACA (*).

species of -1.6 kb (Fig. 7A). MOG mRNA expression was restricted to CNS tissue (brain); it was not detectable in other tissues, including PNS sciatic nerve mRNA.

MOG mRNA was first detected at day 10 (Fig. 7B). Peak expression occurred between 15 and 25 days of age, coincident with the period of active myelination in

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Fig. 5. Rat MOG, bovine butyrophilin, and chicken B-G antigen amino acid homologies. Identical amino acids are denoted by vertical lines. Positive amino acid relationships are denoted by a colon; zero-value relationships, by a period; and negative relationships, by no marking. These relationships are defined by the PAM250 (percent accepted mutation) scoring matrix used in standard sequence alignment programs. Positive relationships indicate a higher probability that the residues are evolutionarily related, whereas negative relationships indicate a higher probability for chance mutation.

mice and rats. Also MOG mRNA concentrations decreased significantly by 40 days of age, yet a substantial signal remained stable through day 80. These results are consistent with the expression patterns for mRNAs of the other major myelin proteins.

Primer Extension Analysis Primer extension studies were undertaken to map the 5‘ end of MOG mRNA and confirm that pMOG24 represents a full length cDNA clone (Fig. 8). A 20-mer oligonucleotide was designed to hybridize to MOG mRNA =I00 nucleotides from the putative 5‘ terminus (nt 96-1 15). Hybridizations were performed with 5’

end-labeled primer and rat total cellular RNA from whole brain, brain stem, spinal cord, and sciatic nerve. Primer extension products were observed in each lane of CNS tissue (whole brain, brain stem, and spinal cord) RNA. An intense doublet band of 119 and 120 nucleotides was noted with a minor band at 116 nucleotides. These results indicate that pMOG24 may lack only 1-5 nucleotides at the 5’ end. This minimal difference may also be due to nucleotide composition differences between MOG mRNA and the sequencing markers used. No extension products were seen using PNS sciatic nerve RNA as a negative control.

DISCUSSION The isolation of MOG cDNAs has allowed us to determine an amino acid sequence for MOG and has provided us with powerful tools to begin a more in-depth characterization of this myelin membrane glycoprotein, which is the primary target autoantigen for demyelinating antibodies in EAE. As expected, we found that MOG mRNA expression is CNS-specific and is maximal during peak myelination from 15-25 days of age in rats and mice. Only a single 1.6 kb MOG mRNA is seen, and its expression remains significant throughout development, albeit reduced at later ages, presumably for maintenance of the myelin sheath. Sequence analysis of pMOG24 reveals an open reading frame that encodes a putative signal peptide of 27 amino acids followed by mature MOG peptide of 2 1 8 amino acids (MW,,,, = 24,962). Deglycosylated MOG has a rather basic calculated PI of 8.87. Our deduced amino acid sequence for rat MOG agreed well with Nterminal peptide sequence of mouse MOG. In agreement with our glycosylation studies indicating N-linked carbohydrates (Amiguet et al., 1992), a single potential Nglycosylation site (Asn-Xxx-Ser/Thr) at Asn-3 1 was identified. Finally, possible transmembrane domains were identified for two extremely hydrophobic stretches of 29 (Ile-122 to Gln-150) and 26 amino acids (Val-I75 to Asn-200). The lack of a canonical AAUAAA polyadenylation signal in MOG cDNAs was surprising. Normal polyadenylation results from an internal cleavage of unprocessed mRNA transcripts, followed by addition of a poly A tail to this new 3’ end that is approximately 10-30 nucleotides downstream from a polyadenylation signal (Proudfoot and Brownlee, 1976; Nevins, 1983). MOG mRNA contains an AU-rich area with a series of overlapping, alternative polyadenylation signalsAAUCAA, AAUAAIJ (twice), and AAUACA. Each of these variant signals occurs with a frequency of 6 are significant. Stippled areas denote identical residues shared by at least four of nine V-set representatives.

have shown a ten-fold to 50-fold reduction in polyadenylation efficiency (Wickens, 1990). Previous studies of globin mRNAs have shown that shortened poly A tails affect mRNA stability. Stability of globin mRNAs with poly A tracts of A,, was reduced by at least tenfold and near that of deadenylated mRNA, as compared to globin mRNAS with A,,-,,, (Nude1 et al., 1976). All MOG clones that primed from the true poly A tail had a maximum tail length of A15. All other known myelin-specific mRNAs possess the ubiquitous AAUAAA polyadenylation signal. Thus MOG mRNA may exhibit a significantly shorter half life than mRNAs for PLP/DM20 protein, MBP, CNP, and MAG. A highly significant partial sequence homology was found between rat MOG and bovine butyrophilin. Butyrophilin is an acidic membrane glycoprotein expressed specifically in lactating mammary gland (Mather et al., 1980; Franke et al., 1981). This hydrophobic protein comprises >40% of membrane proteins in a structure known as milk fat globule membrane (MFGM). Milk fat droplets are encapsulated in MFGM and “bud” from apical plasma membrane of mammary epithelial cells. Franke and coworkers suggest that butyrophilin

may serve as a membrane-associated receptor for cytoplasmic lipid droplets. Butyrophilin (Jack and Mather, 1990) and MOG cDNAs exhibit a striking 60% identity from MOG nt 73-521, beginning just 16 nucleotides before the Met initiation site. Their respective N-terminal 115 amino acids are 52% identical, and moreover the remaining nonidentical sequence shows 62% conservative amino acid substitutions. Within this region, the probable Nglycosylation site at Asn-31 in MOG is not conserved in butyrophilin. Such a strong homology between bovine and rat sequences suggests that MOG and butyrophilin may share exons from the same gene. Alternatively, a common ancestral gene may have undergone a relatively recent duplication event, giving rise to separate MOG and butyrophilin genes. Nonetheless, this striking partial homology with butyrophilin suggests that MOG may play a role in lipid interactions of the myelin sheath. In addition, this same region shares nearly 39% identity with chicken B-G antigens. These antigens comprise the third class of the tripartite B blood group system (B complex; B-L, B-F, and B-G) forming the chicken MHC-in contrast to the bipartiie mammalian MHC

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BHLiLuSK T

1.6

1234

Sn

-

G T A C G

120116’

kb

A.

1 2 3 4 5 6 7 8

1.6 t kb B. Fig. 7. Northern blot analysis of MOG mRNA expression. A: Tissue specificity was studied using total cellular RNA (20 p-g) from various tissues: brain (B), heart (H), liver (Li), lung (Lu), spleen (S), kidney (K), thymus (T), and sciatic nerve (Sn). B: Developmental MOG mRNA expression in brain was studied at postnatal days 5 , 10, 15, 20, 25, 40, 60, and 80 (lanes 1-8, respectively). A, B: Except sciatic nerve (rat), all other tissues were from mouse. Kilobase, kb. Exposure time: 13 days. class I and class I1 molecules (Kaufman et al., 1991). Although B-G antigen function is undefined, these molecules are involved in immune repertoire development and B cell selection (Salomonsen et al., 1991a,b). B-G molecules are unglycosylated dimeric cell surface proteins with a high degree of polymorphism. Miller and coworkers (1991) recently isolated five B-G cDNAs and demonstrated that they contained N-terminal variable region Ig-like domains and that the polymorphisms arose from heterogeneity of C-termini. Although mammalian homologs of B-G antigens remain unidentified, ‘‘natural

Fig. 8. Mapping 5’ end of MOG mRNA by primer extension analysis. An end-labeled MOG-specific oligonucleotide was mixed with 20 p.g total cellular RNA from spinal cord, sciatic nerve, brain stem, or whole brain (lanes 1-4, respectively). Three bands (120, 1 19, and 116 nucleotides) were observed as extension products in CNS tissue only. Sizes were determined from sequencing reactions (G,T,A,C) of a known plasmid DNA shown on the right. Exposure time: 1 day.

antibodies” against chicken B-G antigens have been identified in rodents and humans (Longenecker and Mossman, 1980). Does this partial homology between MOG and chicken B-G antigens relate in some fashion to MOG’s involvement with autoimmune demyelination? Closer inspection of the amino acid sequence revealed another significant finding related to MOG’s origin. The spacing between Cys-24 and Cys-98 of MOG fits a primary requirement for V-set subfamily members of the Ig superfamily (Williams and Barclay, 1988). The N-terminal side of the second Cys has a consensus sequence of AspXxx-GlyiAla-Xxx-Tyr; in addition, an Arg/Lys residue lies =20 amino acids further upstream. These elements are entirely conserved among subfamily members, and Arg-68 and Asp-92 of MOG match this motif. In addition, Leu is often found two residues before the first Cys residue; Leu-22 follows this pattern. Trp-39 matches another highly conserved alignment 1 116 residues downstream from the first Cys. Bovine butyrophilin was not identified as a member of the Ig superfamily, yet many of these same elements are conserved between butyrophilin, MOG, and V-set subfamily members. A PROFILE analysis of MOG and butyrophilin against Ig V region superfamily members yielded statistically significant alignments. MOG and butyrophilin were 10.80 and 7.85 SD units ( z value) above the mean, respectively; both scores are highly significant. In a serious departure from characteristics of the immunoglobulin superfamily-MOG has two potential

MOG Is a Unique Ig Superfamily Member

MOG, tentative model

Fig. 9. Tentative model for MOG topology. A single variable region Ig-like domain is located on the extracellular surface of the lipid bilayer. The disulfide cysteine linkage is between residues 24 and 98. Two possible transmembrane domains are found between residues 122-150 and 175-200. The C-terminal domain is at the extracellular surface.

transmembrane domains (see Fig. 4). All other members of this superfamily (both V- and C-set subfamilies) possess either a single transmembrane domain or membrane linkage via a glycophospholipid anchor. Hydropathic profiles (Kyte and Doolittle, 1982), using a standard window of nine amino acids (Fig. 4), clearly predict a second possible membrane spanning region from residues 175-200. In addition, both Chou-Fasman (Chou and Fasman, 1978) and Robson-Garnier (Garnier et al., 1978) secondary structure analyses predict extended beta sheets in both putative transmembrane domains. An extended p strand would require only nine residues to span the lipid bilayer (Luthy and Eisenberg, 1991). Vectorial labeling and in situ proteolysis studies will yield experimental proof concerning these possible transmembrane regions. Preliminary evidence from the demyelinating activities of various MOG mAbs suggests that the Nterminal portion containing a potential glycosylation site is on the extracellular face of the membrane (unpublished data). At present, polyclonal antisera against synthetic peptide fragments are in production. Immunohistochemical analyses with these antisera will provide information concerning MOG insertion in the plasma membrane. The simplest topological model of MOG exhibits an extracellular Ig-like domain with both transmembrane domains passing through the plasma membrane, leaving both N- and C-termini on the extracellular surface also (Fig. 9). Thus only a short stretch of 24 amino acids would be localized cytoplasmically . The concentration

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of basic residues on the C-terminal side of the first transmembrane domain also suggests a cytoplasmic localization for residues 151-174 (Hartmann et al., 1989). Also a potential phosphorylation site is found in this region at Thr- 167. Alternatively, the second hydrophobic domain, containing three proline residues, may form a bend, thus entering and exiting the plasma membrane intracellularly. This second model suggests placement of the Cterminus on the intracellular side of the bilayer. Although precedent is lacking, we also offer the possibility that the second transmembrane domain may associate with the tightly apposed adjacent plasma membrane, acting to further anchor MOG into the myelin sheath. Given the exceptional nature of these compacted lipid bilayers in myelin, this possibility should not be overlooked. Finally, MOG may form a dimeric structure. Early studies identified MOG as a 50-54kDa glycoprotein, and we have found that 26-28 kDa MOG readily aggregates to a 54 kDa form. Some Ig superfamily members exist as dimers, including B-G antigens, which MOG shares significant partial homology. Future studies of MOG expression and its cellular localization will address these topological questions. The sequence analysis of MOG cDNAs has provided us with the primary structure of this myelin membrane glycoprotein. Comparisons against other known sequences have presented clues toward possible functions for MOG. The strong identity with butyrophilin suggests its involvement with lipids. Also, this same homology with chicken B-G MHC antigens may relate to MOG involvement in autoimmune demyelination. MOG clearly contains a single Ig-like domain, indicative of a receptor function. We are pursuing the possibility that MOG plays a recognition role in lipid interactions in myelin. Its late expression and localization on the outer myelin surface may serve as a signal to arrest further myelination. Studies with overlapping synthetic MOG peptides will allow us to dissect out potential immunodominant epitopes that may be involved in autoimmune mediated demyelination.

ACKNOWLEDGMENTS The authors thank Ms. Sylvie Cherbuin for her excellent technical assistance. Dr. Wendy B. Macklin is gratefully acknowledged for providing the oligodendrocyte cDNA library and numerous helpful discussions. We appreciate the help of Dr. Roland Luthy with the PROFILE analysis. Y 1 and Y 10 mAbs were kindly provided by Dr. Sara Piddlesden. This research was supported by the Swiss National Fund (grant 31-28544.90), the Swiss Multiple Sclerosis Society, and the European Charcot Foundation.

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