Journal of Neuroimmunology, 29 (1990) 81-92

81

Elsevier JNI 00977

Adult thymus expresses an embryonic nicotinic acetylcholine receptor-like protein Scott N e l s o n a n d B i a n c a M. C o n t i - T r o n c o n i Department of Biochemistry, College of Biological Sciences, University of Minnesota, St. Paul, MN 55108, U.S.A.

(Received 17 October 1989) (Revised, received16 March 1990) (Accepted 19 March 1990) Key words: Thymus; Acetylcholinereceptor; Myastheniagravis

Summary The subunit composition of acetylcholine receptor-like protein(s) (AChR-LP) expressed by normal thymus was investigated. In skeletal muscle, the AChR exists in two forms, an embryonic form which contains the y-subunit and an adult form where the "~,-subunit is substituted by a different, homologous subunit called ~. Antibodies against unique sequence segments of the embryonic "/-subunit and of the adult c-subunit of bovine muscle AChR, in addition to antibodies specific for the et-, fl-, and ~-subunits of bovine muscle AChR, were used to probe immunoblots of AChR-LP(s) from bovine thymus. Subunits of approximate M r 41 kDa, 48-54 kDa, 57 kDa and 67-72 kDa were recognized by anti-a, anti-fl, anti-~, and anti-8 antibodies respectively. Anti-E antibodies did not recognize any protein band from bovine thymus. AChR-LP similar or identical to the embryonic muscle AChR is therefore expressed in normal thymus.

Address for correspondence: Bianca M. Conti-Tronconi, Department of Biochemistry, College of Biological Sciences, University of Minnesota, 1479 Gortner Avenue, St. Paul, MN 55108, U.S.A. Abbreviations: MG, myasthenia gravis; AChR, acetylcholine receptor; a-BTX, a-bungarotoxin; T-AChR-LP, thymus acetylcholinereceptor-likeprotein; HPLC, high-pressure hquid chromatography; Tris, tris(hydroxymethyl)aminomethane; EDTA, ethylenediaminetetraacetic acid; EGTA, ethyleneglycol-bis(fl-aminoethylether)-N, N, N ', N '-tetraacetic acid; PMSF, phenylmethylsulfonylfluoride; SDS, sodium dodecyl sulfate; KLH, keyhole limpet hemocyanin; ECDI, 3-ethyl-(3ethyldiamine)-carbodiimide; TBS, 10 mM Tris, pH 7.4, 140 mM NaCI; TBS-T, 10 mM "Iris, pH 7.4, 140 mM NaC1 (TBS) 0.1% Tween-20; BSA, bovine serum albumin; mRNA, messenger ribonuclcic acid; Cys, cysteine; Mr, relative molecular mass; kDa, kilodalton; AChR-LP, acetylchohne receptor-like protein; CNBr, cyanogenbromide; DEAE, diethylaminoethyl; HCI, hydrochloric acid; Torpedo, Torpedo californica; mAbs, monoclonal antibodies.

Introduction In the human disease myasthenia gravis (MG), the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction is the target of an autoimmune response and anti-AChR antibodies are produced (Drachman, 1983; Engel, 1984; Lindstrom, 1985). Anti-AChR antibodies cause accelerated destruction and functional impairment of the AChR, failure of neuromuscular transmission and ultimately paralysis (Engel, 1984; Lindstrom, 1985). Myasthenic symptoms can be induced in a variety of animals by immunization against purified AChR (Patrick and Lindstrom, 1973), or in: jection of anti-AChR antibodies (Toyka et al., 1975; Lennon and Lambert, 1980; Richman et al., 1980; Gomez and Richman, 1983; Tzartos et al., 1987).

0165-5728/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)

82 The origin of the antigenic stimulation which initiates the autoimmune response in MG is unknown. Thymic abnormalities are frequently present in myasthenic patients, suggesting a dysfunction of the thymus (Li et al., 1977; Levine, 1979; Thomas et al., 1982; Engel et al., 1984; Wekerle and Muller-Hermelink, 1986). In most MG cases the thymus is hyperplastic and contains 'germinal centers', which are believed to be morphological markers of active antibody production (Li et al., 1977; Levine, 1979; Thomas et al., 1982; Engel, 1984; Wekerle and Muller-Hermelink, 1986) and which are not present in normal thymuses. A thymoma is present in some patients, sometimes even before myasthenic symptoms appear (Kumura and Van Allen, 1967; Cuenod et al., 1980). The thymus tissue from myasthenic patients contains B-cells which can synthesize anti-AChR antibodies in vitro (Mittag et al., 1974; Scadding et al., 1981; Kamo et al., 1982; Fujii et al., 1984, 1986; Lisak et al., 1986; Heidenreich et al., 1988), and CD4 + cells specific for the AChR can be propagated from myasthenic thymuses (Melms et al., 1988). Because a crucial function of the thymus is induction of tolerance to self constituents (Adkins et al., 1987), the AChR seems to be a unique autoantigen for which an autoimmune response can occur, and possibly be initiated, within the thymus (Wekerle et al., 1981). Immunological crossreactivity between a thymus component and muscle AChR has been shown (Aharonov et al., 1975; Fuchs et al., 1980; Ueno et al., 1980; Schluep et al., 1987; Kirchner et al., 1988). The thymus contains binding sites for a-bungarotoxin (aBTX), which specifically recognizes AChRs from peripheral tissues (Engel et al., 1977; Kao and Drachman, 1977; Kawanami et al., 1987, 1988). In addition, cultured cells from adult human thymus can differentiate into striated muscle fibers which bear AChR (Feltkamp-Vroom, 1966; Van de Velde and Friedman, 1966; Wekerle et al., 1975). We previously demonstrated that the a-BTX binding component from thymus is related both structurally and immunologically to a true AChR (Kawanami et al., 1988), because it contains four peptides with apparent molecular weights of 40 kDa, 51 kDa, 56 kDa and 66 kDa, which cross-

react with antisera raised against Torpedo californica AChR. We called this a-BTX binding component ' t h y m u s AChR-like protein' (TAChR-LP). Two forms of skeletal muscle AChR exist, one expressed in embryonic (or denervated) muscle, the other in adult (innervated) muscle (review in Hall et al., 1983; Schuetze, 1986). These two AChRs differ in one of their constituent subunits. They both contain the a-, /3- and 6-subunits (Mishina et al., 1986). In embryonic AChR, a 7-subunit is also present (Mishina et al., 1986; Gu and Hall, 1988). Upon innervation, synthesis of the y-subunit is turned off and activation of another gene occurs, encoding for a homologous subunit, called ,, which contributes to formation of the adult muscle AChR (Mishina et al., 1986; Gu and Hall, 1988). The genes encoding the ~and y-subunits of bovine muscle AChRs have been cloned and sequenced (Takai et al., 1984; Takai, 1985). Adult and embryonic muscle AChRs differ in their turnover rate, ion gating properties and pharmacology (Ziskind and Dennis, 1978; Weinberg and Hall, 1979; Dwyer et al., 1981; Trautman, 1982; Hall et al., 1983, 1985; Schuetze et al., 1985; Mishina et al., 1986; Scheutze, 1986; Gu and Hall, 1988). An unresolved question is whether the TAChR-LP is most similar to adult or embryonic muscle AChR. The latter is not expressed in adult (innervated) skeletal muscle and, at least in bovine muscle, the corresponding m R N A is already absent at birth (Mishina et al., 1986). The presence within the thymus of a form of AChR normally absent from the other tissues could be related to the break in tolerance which causes MG. In the present study, peptides corresponding to unique sequence segments of bovine y- and ~-subunit were synthesized and used to raise subunitspecific antibodies. These antibodies, and other monoclonal and polyclonal antibodies against the a-, r - and 8-subunits, were used to identify the complement of subunits present in preparations of purified T-AChR-LP from bovine thymus. Subunits immunologically corresponding to the embryonic muscle AChR a-, fl-, y- and ~-subunits were present.

83 Materials and methods

Peptide synthesis and characterization Six peptides, 16-21 residues long, corresponding to sequence segments of the T-, (- and 8-subunits which are highly divergent among these and the other AChR subunits were synthesized by manual parallel synthesis (Houghton, 1985). The peptides corresponded to the following sequence segments; T9-28, 3'186-205, 3,386-405; 8102114, 8198-213, 8382-399; ¢9-28, (186-205, ¢379-398 (Fig. 1). The purity of the peptides was assessed by reverse-phase high-performance liquid chromatography (HPLC) using a C18 column (U1trasphere ODS) and a gradient of acetonitrile in water plus 0.1% trifluoroacetic acid. The composition of all peptides was determined by amino acid analysis using phenylthiocarbamyl derivatives of the amino acids released by acid hydrolysis (Heinrickson and Meredith, 1984). The sequence

¢ y B

KNEELRL~~JTISLKU-TLTHLISLHEKEETLTTSUWIGIDWQDYR RNQEERL~HUSLK -TLINLISLHEREEALTTNUWIEMOWCDVR LNEEERLIRHLFEEKAYHKELRPRRHKESUEISLALILSHLISLKEUEETUTHUWIEQGWTDSB

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6

VEUTYFPFOWQNCSLVFRSQTYNAEEUEFVFAUDDE-GKTISKIDIDTEAY--TENGEWAI~ VSUTFFPFDWQNCSLIFQSQTYSTNEINLQLS-QED-GQTIEWIFIDPEAF--TENGEWAIBI'Jm ISUTYFPFDWOHCSLKFSSLKYTTKEITLSLKQAEEDGRSYPUEWIIIDPEGFTENGEWEIVHRP

6 C y 6

IIRBKPLFYUINIIUPCULISGLVLLAYFLPAQAGGQKCTU LIQRKPLFYUIHIIRPCULISSUAILIYFLPAKAGGQKCTV IIRRKPLFYUINILUPCULISFMINLUFYLPADCGE-KTSM SINVLLRQTVFLFLIAQKTPETSLSVPLLGRYLIFUMVUATLIVMNCUIVLNUSLR~PTTHAMS AINVLLAQTVFLFLVAKKUPETSQAUPLISKYLTFLLUUTILIUVNAVVULNVSLRSPHTHSMA AISULLRQSUFLLLISKRLPATSMAIPLIGKFLLFGMULUTMUUUICUIVLHIHFRTPSTHULS

PRLRYULLELLPQLLGSG .... RPPEIPRAASPPRRRSSLGLLLBA-EELILKKPRSELVFEQQ RGURKUFLRLLPQLLRMHURPLAPUAUQORHPRLQHGSSSGWPITAGEEURLCLPRSELLFBOR

6

EPUKKLFLETLPEILHMS-RPAEDGPSPGTLIRR---SSSLGYISKREEYFSLKSRSDLMF~mll

6

~ I B C C U D A U H F U A S S T R O Q E A T G E E ORH CC-$-LKOAA-PAIQACUEACNLIARARHQQTHFOSG IMmmlRBII[~PAGSEQAQQELFS ELKPRUDGAHFIUNHMKDQNNYHEEKDCWNRUA

¢

USDWURMGKALDS ICFWAALULFL UG SSL IFLGAYFNRUPQLPYPPCM

"J 5

NKEWFLUGRULDRUCFLRMLSLFU CGTAGI FLI1RHYHRUPRLPFPGOPRSYLPSSD RTUDRLCLFUUTPLMUUGTRWI FL DQGRYNQPPPQPFPGDPFSVLEKDKRFI

Fig. 1. Sequence alignment of bovine AChR "t-, 8-, and (-subm~Jts (from Takai et a]., 1984, 1985; Kubo et aL, 1985).

The sequence segments corresponding to the peptides synthetized and used in this study are indicated by shaded segments. Theycorrespond to the sequencesegments 9-28, 188207, 379-398 of the (-subunit, 9-28, 186-205, 386-405 of the 3,-subunit and 102-114, 198-213, 382-399 of the 8-subunit.

and the purity of randomly selected peptides from each synthesis was further verified by gas-phase sequencing (Herrick et al., 1981).

Preparation of affinity resin Purified a-BTX was conjugated to CNBractivated Sepharose 2-B following the procedure for 'moderate activation' as described by Porath et al. (1973). The ability of the a-BTX/Sepharose resin to bind AChR was assessed as described by Gotti et al. (1982) using Torpedo AChR solubilized with Triton X-100. Different batches of aBTX/Sepharose bound 7-9 nmol of A ChR/ m l packed resin (expressed as a-BTX binding sites, as measured by DEAE assay (Schmidt and Raftery, 1973)).

Preparation of thymus A ChR-LP Thymus tissue was obtained from a local slaughterhouse from calves approximately 6 months of age. The tissue was obtained within minutes after death, packed on ice and transported to the laboratory. All the following procedures were performed at 4 ° C unless otherwise specified. The thymuses were dissected clean of all adventitial tissue and homogenized with an equal volume of a buffer containing 50 mM Tris (tris(hydroxymethyl)aminomethane), 50 mM NaC1, 5 mM ethylenediarnine tetraacetic acid (EDTA), 1.2 mM ethyleneglycol-bis(fl-aminoethylether)-N, N, N ' , N'-tetraacetic acid (EGTA) (buffer 1) plus 20 mM phenylmethylsulfonyl fluoride (PMSF) and 10 mM iodoacetamide (IAA) in a Waring blender at full speed in a 1-gallon container until a smooth slurry was obtained. The homogenate was centrifuged in a Sorvall GS3 rotor at 11,000 × g for 2 h. The pellets were resuspended in two-thirds of their volume of buffer 1 plus 20 m M PMSF and 10 mM IAA, and Triton X-100 was added to a final concentration of 1%. The mixture was extracted with vigorous agitation for 1-3 h. The Triton X-100 extract was centrifuged for 1 h at 11,0(30 x g in a Sorvall GS-3 rotor. The supernatant was recovered and further clarified by ultracentrifugation at 150,000 × g in a Beckman 35 rotor for 90 min. The lipid-rich layer on the surface was removed and the clarified supernatant was gently retrieved and passed through glass wool to remove additional fines and

84

lipids. The supernatant was then either incubated batchwise with 2 - 4 mls of a-BTX Sepharose for approximately 48 h or passed through an a-BTX Sepharose affinity column (2-4 ml bed volume) for 1-3 days (flow rate - 0.5 ml/min). The resin was recovered by filtration on a sintered glass funnel and washed with 100 ml each of the following buffers (all ice cold): 10 mM Tris, 1 m M E D T A pH 7.4, 0.1% Triton X-100; buffer 1 plus 1% Triton X-100; buffer 1 plus 0.5 M NaC1 and 0.1% Triton X-100. The resin was further washed with a small volume of distilled water and stripped for 1 h at room temperature with 3 - 4 m l / g resin of 1% sodium dodecyl sulfate (SDS) in water. The SDS present in the eluate was eliminated by cryoprecipitation on ice for 1 - 2 h, followed by centrifugation for 3-5 min in an International Clinical Centrifuge at approximately one-half of full speed, to remove precipitated SDS. The resultant supernatant was lyophilized.

Preparation of bovine muscle A ChR Bovine muscle AChR was prepared from frozen bovine fetuses as described by Gotti et al. (1982). The AChR adsorbed onto the washed affinity resin was stripped with SDS, cryoprecipitated, and lyophilized. It has been previously demonstrated (Gotti et al., 1982) that, following extensive washes of the affinity resin, the SDS-gel pattern of SDSstripped AChR is very similar or identical to the pattern of the AChR competitively eluted by carbachol, which typically has a specific activity of 5.9 nmol a-BTX binding sites/mg protein and is therefore at least 74% pure. Early bovine fetuses, 12 inches long or less, were used to purify denervated AChR. A near-term bovine fetus ( - 5 5 lb) was used to isolate AChR from innervated muscle.

Anti-peptide antibody production Peptides were coupled to keyhole limpet hemocyanin (KLH) using 3-ethyl-(3-ethyldiamine)-carbodiimide (ECDI). 5 mg of each of the peptides corresponding to sequence segments of one subunit were dissolved in a minimal volume of water and mixed with K L H solution (625/~1; 40 m g / m l in water). ECDI was added as a freshly prepared 2 M solution in water, to a final concentration of 0.1 M. The p H was adjusted to 3-5 with dilute HC1

and the reaction allowed to proceed overnight at room temperature. The K L H / p e p t i d e conjugates were stored frozen. For animal immunization, the concentration of the K L H / p e p t i d e conjugate was adjusted to 1 m g / m l with water. Equal volumes of K L H / p e p t i d e conjugate and incomplete Freund's adjuvant were emulsified and used to immunize rabbits for anti-y and anti-c antisera production and rats for anti-8 antiserum production. The rabbits were injected at several sites along the back with a total of 1 mg of p e p t i d e / K L H mixture, the rats were injected with 0.5 mg. The injections were repeated at 2-week intervals until a satisfactory antipeptide antibody titer was obtained (see below), then repeated every 1-2 months to maintain a stable high titer. Blood was obtained by ear vein bleeds from the rabbits and from tail vein bleeds from anesthetized rats and allowed to clot overnight to obtain sera. The monoclonal antibodies (mAbs) used in this study were the kind gift of Dr. Jon Lindstrom (Salk Institute). They were raised using SDS-denatured Torpedo AChR as immunogen. Two of them (mAbs Nos. 152 and 155) recognized Torpedo a-subunit. The other two (mAbs Nos. 118 and 124) recognized Torpedo fl-subunit. They all crossreact with fetal bovine A C h R (Tzartos et al., 1986).

Affinity purification of antibodies Antibodies specific for individual peptides were purified from the antisera by affinity chromatography. The peptides were coupled to AH- or CHSepharose (Sigma), through carboxyl or amino groups respectively, using ECDI as a crosslinking reagent, following the manufacturer's instructions. 10 mg of p e p t i d e / m l of packed resin were used. Anti-y and anti-c rabbit antisera (10 ml) were passed through an affinity column (1.9 cm length x 0.8 cm diameter, 0.5 ml bed volume) at a flow rate of approximately 6 m l / h . The resin was washed with 10 m M Tris, 140 mM NaC1 p H 7.4 (TBS) until the absorbance at 280 nM was less than 0.01. The colunm was eluted with 1 ml aliquots of 0.1 M glycine, p H 2.5. The eluted aliquots were immediately neutralized with 60 #1 of 1 M Tris base. Anti-8 rat serum was treated similarly. Because rat immunoglobulins were found to bind non-specifically to the column to a much larger extent than rabbit antibodies, the column

85 was washed with several volumes of 10 mM Tris, 1 M NaC1 p H 7.4 to reduce non-specific binding. After neutralization, the optical density at 280 nm of the eluted fractions was measured. The antibody concentration was estimated using a value OD280,0.1~ = 1.4.

Dot-blots The presence of antipeptide antibodies was assessed by a dot-blot assay using peptides spotted onto nitrocellulose at a concentration of 400 g g / m l . Aliquots (0.5 gl) of solutions of the different peptides (400 g g / m l in 10 mM potassium phosphate, pH 7.0) were spotted onto nitrocellulose strips and allowed to dry. Before use, the strips were wetted with 10 mM Tris, pH 7.4, containing 140 m M NaC1 (TBS) and 0.1% Tween20 (TBS-T). Antibodies were then added at an appropriate dilution in TBS-T in a total volume of 1 ml and incubated for 2 h at room temperature with continuous agitation. The incubation fluid was removed and the strips were washed twice for 5-10 min each time in 2-3 ml of TBS-T. When rabbit antibodies were used, Protein A (1 × 10 6 cpm) in TBS-T containing 1 m g / m l BSA was added and incubated for 1-2 h and washed 3 times as above in TBS-T. When rat antibodies were used, affinity-purified rabbit anti-rat IgG antibody was added at a 1 : 1000 dilution in TBSTween (1 ml/strip), incubated for 2 h, washed as above, and processed with Protein A as for the rabbit antibodies. The dried strips were mounted on cardboard and exposed for 12-24 h at - 70 ° C using a Quanta III intensifying screen and Kodak RP film.

Western blots SDS-eluted, lyophilized preparations of muscle AChR or T-AChR-LP were resuspended in 1 ml of glass distilled water and the protein assayed as described by Lowry et al. (1951) using BSA as a standard. The sample was mixed with Laemmli's sample buffer (Laemmli, 1970) and loaded on 8.75% polyacrylamide gels using a 4% stacking gel (Laemmli, 1970) to give 15 #g of protein (for T-AChR-LP) or 10-20 pmol binding sites (for muscle A C h R ) / 3 mm strip of gel. After electrophoresis, the protein bands present in the gel were transferred onto 0.2 # m or 0.45 g m nitrocellulose at 20-25 V (2.6-3.3 V / c m ) overnight as described

by Towbin et al. (1979). Following protein transfer, the nitrocellulose was stained with 0.2% Acid Red 150 (Ponceau SS) in 0.3% trichloroacetic acid for 30-60 s and destained in glass distilled water until the bands became apparent. The strip containing the molecular weight markers was cut away, fixed in 25% isopropyl alcohol, 10% acetic acid and air dried. The remaining nitrocellulose sheet was cut into 3 mm strips and blocked for 30-60 min in TBS-T containing 1 m g / m l BSA (2-3 ml/strip) in Accutran disposable trays (Schleicher & Schuell). Antibodies were then added at appropriate dilutions in TBS-T in a total volume of 700 gl and incubated for 2 h at room temperature with continuous agitation. The strips were washed twice for 5-10 rain in 2-3 ml of TBS-T. When rat antibodies were used, affinity purified rabbit anti-rat IgG antibody (Sigma) was added at a 1:1000 dilution in TBS-Tween (1 ml/strip), incubated for 2 h and washed as above. Both rat and rabbit primary antibodies were then incubated with Protein A (1 × 10 6 cpm) in 1 m g / m l B S A / T B S - T for 1-2 h and washed 3 times in TBS-T as described above. The strips were mounted on cardboard and exposed for 2 - 4 days at - 7 0 ° using a Quanta III intensifying screen and Kodak RP film. Results

Peptide purity The purity of the synthetic peptides, as determined by reverse-phase HPLC, was between 65% and 85%. By this approach peptide purity was underestimated due to the presence of low molecular weight contaminants which absorb at the wavelength used (214 nm). When the actual purity and sequence of some randomly selected peptides was verified by gas-phase amino-terminal sequencing, the expected sequence was consistently found and only 10-15% or less contaminating sequences were present, which were shorter homologous peptides where one or more residues were randomly missing along the sequence because of incomplete coupling.

Production of antipeptide antisera Each animal was injected with a mixture of the peptides corresponding to a particular subunit,

86

conjugated to KLH. Not all of the three peptides used for each subunit induced a good antibody response. Peptides V386-405, d88-207, ~379-398 and 8382-399 were the best immunogens. Antibodies against these peptides were affinity purified and used for the Western blot experiments with T-AChR-LP and bovine muscle AChR, as it is described below.

Affinity purification of anti-peptide antibodies Peptide-Sepharose affinity columns were used as described in the Materials and Methods section. Typical yields from anti-v and anti-c rabbit antisera were 2-3 mg of purified anti-peptide antibody/10 ml serum. Typical yields from 4 ml of anti-8 rat sera were 0.2-0.3 mg of purified antipeptide antibody. The antibody specificity was verified by dot-blot. All the affinity-purified antibodies showed a consistent, specific reaction with the relevant peptide at dilutions up to 1-10/xg/ml. For most peptides, the reaction of the peptide with the corresponding anti-peptide antibodies was strong (Fig. 2). One exception was peptide 8382399, which, although consistently and specifically recognized by the corresponding affinity-purified antibody, gave a very weak signal (Fig. 2). Because the antibodies used for the dot-blot experiments were affinity purified using the relevant peptide as the affinity ligand, they all must be able to bind to the peptide attached to the Sepharose matrix. Therefore the weak signal obtained in dot-blots with peptide 8382-399 must be due either to poor ability of this peptide to stick to the nitrocellulose support, or to its immobilization in a conformation non-agreable with antibody binding. Fig. 2 shows the results of a typical experiment where the peptide specificity of affinity purified anti-v, anti-c and anti-8 antibodies was tested.

Identification of the subunit complement of purified T-A ChR-LP The protein components present in T-AChRLP, purified by a-BTX-affinity chromatography as described in the Materials and Methods section, were investigated by SDS-gel electrophoresis. A complex pattern of silver-stained bands was consistently present, as expected from the relatively low degree of purification achieved by the procedure used here. A large amount of a con-

t'q I

t,O

¢q t'q , ~ t 2 ~ I

1'4 ~

~,O

i1'9

I

-~ ~

I

t~

I~ 3 7 9 - 3 9 8 * 379-398 E 188-207 9- 28 386-405 '~ 1 8 6 - 2 0 5 9- 28 ¢~ 3 8 2 - 3 9 9 ¢~ 1 9 8 - 2 1 3 ¢~ 1 0 2 - 1 1 4 Fig. 2. Dot-blot assay of affinity-purified antibodies directed against three different c-subunit peptides (sequence segments c8-28, d88-207, and c379-398), one "/-subunit peptide (sequence segment c386-405) and one 8-subunit peptide (sequence segment 8382-399). The antibodies were purified by affinity chromatography using the corresponding peptide conjugated to Sepharose (see the Materials and Methods section for details), and they were tested using both the corresponding peptide and with other synthetic peptides used in this study. One of the c-subunit peptides (c379-398) was synthesized either with an extra tyrosine residue at the carboxyl end (indicated by an asterisk) or without any extra residue.

taminant with apparent M r 40,000 (presumably actin, see ref. Kawanami et al., 1988) co-purified with T-AChR-LP. It had been previously determined (Kawanami et al., 1988) that this contaminant can be removed by alkaline extraction, and that in the absence of alkaline treatment the T-AChR-LP was about 4% pure (Kawanami et al., 1988). Because a high degree of purification was not necessary for this study, where identification of the T-AChR-LP subunits was achieved by antibody binding rather than by protein gel patterns, the alkaline extraction step, which is labor intensive and reduces the total yield of T-AChR-LP, was omitted. In spite of the complex protein band pattern of T-AChR-LP, several bands could be consistently identified in the molecular mass (Mr) range of 36-66 kDa, which other studies have indicated as the range of M r for purified TAChR-LP subunits (Kawanami et al., 1988).

87 In Western blots of T - A C h R - L P , the anti-a m A b s Nos. 152 and 155 recognized a b a n d of approximately 41 k D a (Fig. 3A, lanes 1 and 2). The two anti-/3 m A b s (Nos. 118 and 124) recognized two bands of approximately 48 k D a and 53 k D a (Fig. 3A, lanes 3 and 4). The affinity-purified anti-7387-406 antibodies consistently recognized a sharp b a n d of 57.5 k D a (Fig. 3A, lane 5 and Fig. 3B, lane 1). Affinity-purified anti-~ 188-207 antibodies did not recognize any b a n d detectably (Fig. 3A, lane 6). Affinity-purified anti-8380-398 antibodies recognized a double b a n d whose two components had Mr approximately 67 k D a and 72 k D a respectively (Fig. 3B, lane 2). Table 1 reports the results obtained for four different preparations. Fig. 3 shows the results of typical experiments.

A

B

2o5lJ

11697-

I

66-

66-

45-

45 36-

I ~

~

I

2

29-

1 2

34

567

3

Fig. 3. Western blots of T-AChR-LP probed with different subunit-specific monoclonal and polyclonal antibodies (see text for details). (A) Lanes 1 and 2: anti-a-subunit mAbs Nos. 155 and 152. Lanes 3 and 4: anti-O-subunit mAbs Nos. 124 and 118. Lane 5:anti-7386-405 antibody. Lane 6: anti-d88207 antibody. Lane 7: negative control (without primary antibody). (B) Lane 1:anti--/386-405 antibody. Lane 2: anti8382-399 antibody. Lane 3: negative control (without primary antibody). The molecular weight markers used were: carbonic anhydrase (29 kDa), egg albumin (45 kDa), bovine serum albumin (66 kDa), phosphorylase b (97.4 kDa), fl-galactosidase (116 kDa), and myosin (205 kDa).

TABLE 1 RESULTS FOR FOUR DIFFERENT PREPARATIONS Antibody

Subunit specificity

Bands (kDa)

152 155 118

et a /3

124

/3

Anti-7186-205 Anti-d88-207 Anti-8382-399

Bovine "t Bovine c Bovine 8

41 +1.35(n=3) 41.5 + 1.4 (n = 3) 47.8+2.46 (n = 4) 52.8+2.97 (n = 3) 48.5 + 3.81 (n = 4) 53.9+4.43 (n = 3) 57.5 5:1.1 (n = 4) No bands 67 (n = 1) 72 (n = 1)

Identification of subunits present in embryonic and innervated A ChR The m A b s used in this study had been raised using the Torpedo A C h R and they recognized epitopes on Torpedo A C h R ct-subunit ( m A b s Nos. 152 and 155) and fl-subunit ( m A b s Nos. 118 and 124). Because they crossreact with bovine A C h R (Tzartos et al., 1986), it is reasonable to assume that they recognize the ct and /3 bovine subunits. To further verify this assumption, and further verify the subunit specificity of the anti-peptide antisera used in this study, we used all the m o n o clonal and polyclonal antibodies used for the TA C h R - L P studies to p r o b e Western blots of A C h R s prepared from muscle tissue of early bovine fetuses ( - 1 2 inches long) and from a near-term fetal calf. We expected the latter preparation to contain adult-type A C h R , and therefore the c-subunit (Takai et al., 1985; Mishina et al., 1986), because at this stage of development the muscles are innervated and m R N A for the embryonic ~,-subunit is absent at birth in bovine fetuses (Mishina et al., 1986). O n the other hand, the early fetuses used in this study (12 inches long or less) do not yet express c-subunit or do so to a very small extent (Mishina et al., 1986). In both embryonic and innervated muscle, the anti-a m A b s No. 152 and 155 recognized a single b a n d of apparent M r 45 k D a (Fig. 4A, lanes 2 and 3 and Fig. 4B, lanes 1 and 2), the anti-/3-subunit m A b s Nos. 118 and 124 recognized a single b a n d of apparent M r 48 k D a (Fig. 4A, lanes 4 and 5 and Fig. 4B, lanes 3 and 4) and the affinity-purified anti-8380-398 antibody recognized a sharp b a n d

88

A

B

97-

97-

66-

66-

45-

19

45-

29-

29-

1 2 3 4 5 6 7

1 234

5

67

Fig. 4. Western blot of bovine muscle AChRs. (A) Fetal muscle AChR. Lane 1: negative control (without primary antibody). Lanes 2 and 3: anti-a-subunit mAbs Nos. 155 and 152. Lanes 4 and 5: anti-/3-subunit mAbs Nos. 124 and 118. Lane 6:anti-7386-405 antibody. Lane 7: anti-6382-399. (B) Western blot of innervated muscle AChR. Lanes 1 and 2: anti-a-subunit mAbs Nos. 155 and 152, Lanes 3 and 4: anti-/3subunit mAbs Nos. 124 and 118. Lane 5: anti-d88-207. Lane 6:anti-;c386-405 antibody. Lane 7: negative control (without primary antibody). The molecular weight markers used were carbonic anhydrase (29 kDa), egg albumin (45 kDa), bovine serum albumin (66 kDa), phosphorylase b (97,4 kDa), /3galactosidase (116 kDa), and myosin (205 kDa).

of apparent M r 65 kDa (Fig. 4A, lane 7). In innervated AChR preparations the affinity-purified anti-d88-207 peptide antibodies recognized primarily a 50 kDa band (Fig. 4B, lane 5) while the affinity-purified anti-y386-405 peptide antibodies did not recognize any protein band. In Western blots done with similar amounts of AChR from early bovine fetuses, the anti-y386-405 peptide antibodies consistently recognized a single band of apparent M r 50 kDa (Fig. 4A, lane 6). The affinity-purified anti-d88-207 antibodies occasionally recognized a faint band of Mr 50 kDa in AChR from embryonic muscle, but the signal was much less than that obtained with anti-y386405 antibodies.

Discussion In the present study we demonstrate that mammalian thymus expresses small amounts of an a-BTX binding protein containing peptide compo-

nents immunologically related or identical to all the subunits forming the AChR from mammalian embryonic muscle. The T-AChR-LP contains subunits which react with monoclonal antibodies specific for the a- and /~-subunits, and subunits which react with polyclonal antibodies against sequence segments unique to the 8-subunit and the embryonic 7-subunit. N o component reacted with polyclonal antibodies specific for sequence segments unique to the innervated ~-subunit. Our demonstration that an embryonic AChRLP is expressed in the thymus agrees with reports that myoid epithelial cells in the thymus express a membrane protein(s) which can bind a-BTX (Engel et al., 1977; Kao and Drachman, 1977; Wekerle, 1981). These cells also express membrane structures - - presumably the same protein recognized by a-BTX - - which are recognized by mAbs specific for extrajunctional (i.e. embryonic) muscle AChR, and by mAbs specific for determinants common to both forms of the AChR (Schluep et al., 1987; Kirctmer et al., 1988). The mAbs used by Schluep et al. (1987) recognized a set of epitopes corresponding to those recognized by most anti-AChR autoantibodies in myasthenic patients' sera (Vincent et al., 1986), and this further supports the T-AChR-LP as a good candidate for the original autoimmunogen in MG. Expression in the thymus of a protein similar to embryonic AChR, which is normally absent from the adult muscle, may well explain the appearance of an autoimmune response against this molecule, with later involvement of the muscle AChR, because of its structural similarities with T-AChRLP. An indirect proof that an embryonic type of AChR is involved in initiating the anti-AChR response in M G is the frequent presence of antiembryonic A C h R antibodies in these patients (Weinberg et al., 1979). In a study which used junctional (i.e. adult) and extrajunctional (i.e. embryonic) rat AChR, sera from myasthenic patients recognized determinants either unique to the embryonic form, or common to both forms of the AChR, but never determinants unique to junctional AChR (Weinberg et al., 1979). Indeed, a case of M G has been described where the antiAChR antibodies recognized only the embryonic form of rat AChR (Schuetze et al., 1985). Because embryonic AChR is normally absent from adult

89 muscle, the paradox exists that myasthenic patients are making autoantibodies against a non-existing antigen. This dilemma is obviously solved by the presence of embryonic AChR in the thymus, i.e. in an organ whose involvement in MG pathogenesis is most probable, although still obscure in its nature. The results of this study also directly demonstrate the presence of c-subunit in AChR from innervated muscle (Fig. 4, lane 5) and of y-subunit in preparations of embryonic muscle (Fig. 4, lane 6). This confirms previous reports where this conclusion had been reached by studying the pattern of appearance of the two different m R N A species (Mishina et al., 1986), the functional properties of AChR expressed in oocytes upon injection of different combinations of RNA for the AChR subunits (Mishina et al., 1986), and the ability of anti-c and anti-~, antisera to recognize and precipitate AChRs from innervated and denervated muscle (Gu and Hall, 1988). Occasionally, in our preparations of embryonic AChR some c-subunit was present, but in amounts much smaller than the 3,-subunit. This finding can be well explained by the beginning of the innervation at this stage of fetal development (Arthur, 1975; Mishina et al., 1986). As expected, Western blots of bovine muscle AChR also demonstrated the presence of a-, fl-, and 8-subunits (Fig. 4). Sometimes the antibodies recognized doublets of bands. In the case of the anti-fl mAbs used, whose epitope within the fl-subunit sequence is not known, this could be due to either crossreaction between homologous subunits or to partial degradation of one subunit during the purification procedure, with appearance of lower M r components. The latter possibility is supported by the fact that frequently a given antibody recognized one band only in one preparation, two bands in another. In addition the anti-8382-399 antiserum, raised against a sequence segment unique to the 6-subunit, and which is unlikely to crossreact with other subunits, also recognized a double band, and faint bands at low M r could be frequently detected when antibodies recognized only one band. AChR subunits are - - with the possible exception of the a-subunit - - very susceptible to proteolysis (Lindstrom et al., 1980; Conti-Tronconi et al., 1982). It is, therefore, not surprising that

some AChR degradation may occur during the lengthy procedures necessary to purify AChR from thymus and muscle. AChR-like structures able to bind a-BTX and different antibodies against the a-subunit of muscle AChR have been demonstrated on the myoid cells of thymuses from both MG patients and normal subjects (Kirchner et al., 1988). These cells also possess immunohistochemical and ultrastructural features of striated muscle cells (Schluep et al., 1987). Indeed, it has been suggested for a long time (Van de Velde and Friedman, 1966; Kao and Drachman, 1977; Wekerle and Katelsen, 1977) that these cells, rather than skeletal muscle, are the initial target of the autoimmune attack in MG. Also the epithelial cells of normal and MG thymuses have binding sites for a-BTX and for some anti-a-subunit antibodies (Engel et al., 1977; Kirchner et al., 1988). If the T-AChR-LP is the primary sensitizing autoantigen, it needs to be presented to the specific anti-AChR T helper cells in association with class II molecules of the major histocompatibility complex. Because myoid cells do not express class II molecules, they are unlikely candidates as the original autosensitizing element. On the other hand, numerous other cells in the thymus can express class II molecules, and these could process and present AChR epitopes released upon damage of T-AChR-LP bearing myoid cells. In this respect, it is of interest that AChR-positive/class II-positive cells have been consistently found in thymuses from MG patients, and they were not myoid cells (Schluep et al., 1987). These cells could be antigen-presenting cells, in the process of active A C h R presentation. The presence in the thymus of a protein with strong structural and antigenic similarities with muscle embryonic AChR supports the notion of a primary anti-AChR sensitization within the thymus as one of the first steps in the pathogenesis of MG.

Acknowledgments We are grateful to Dr. Jon Lindstrom for his generous gift of the monoclonal antibodies used in this study. We thank Ms. Bonnie Allen for skillful

90 p r o c e s s i n g o f t h e text. S u p p o r t e d b y t h e U . S . NINCDS Grant NS 29919 (to B.M.C.-T.) and the U.S. N S F g r a n t B N S - 8 6 0 7 2 9 8 ( t o B . M . C . - T . ) .

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