epitope mapping of monoclonal antibodies to torpedo acetylcholine receptor γ subunits, which...

Journal of Neuroimmunology, 36 (1992) 13-27 13 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-5728/92/$05.00

JNI 02101

Epitope mapping of monoclonal antibodies to Torpedo acetylcholine receptor y subunits, which specifically recognize

the e subunit of mammalian muscle acetylcholine receptor

Scott Nelson, G. Diane Shelton *, Sijin Lei, Jon M. Lindstrom ** and Bianca M. Conti-Tronconi

Department of Biochemistry, University of Minnesota, St. Paul, MN 55108, USA, and Receptor Biology Laboratory, The Salk Institute for Biological Sciences, San Diego, CA 92037, USA

(Received 21 May 1991) (Revised, received 7 August 1991)

(Accepted 7 August 1991)

Key words: Muscle acetylcholine receptor; Antibody; Synthetic peptide

Summary

Epitopes for four monoclonal antibodies (mAbs) to the 3' subunit of Torpedo nicotinic acetylcholine receptor (AChR), and one mAb crossreactive with the y and 6 subunits of Torpedo AChR were mapped using overlapping synthetic peptides corresponding to the complete amino acid sequence of Torpedo y subunit. The epitopes for all mAbs were within a 50 residue sequence region, on the cytoplasmic surface of the AChR. Three mAbs crossreacted with mammalian muscle AChRs. Two of them specifically recognized the e subunit of AChRs at adult neuromuscular junction.

Correspondence to: Bianca M. Conti-Tronconi, Department of Biochemistry, University of Minnesota, 1479 Gortner Avenue, St. Paul, MN 55108, USA.

* Current address: Comparative Neuromuscular Laboratory, Off. of Vet. Services, M-104, University of California, San Diego, CA 92093, USA.

** Current address: University of Pennsylvania Medical School, Institute of Neurological Sciences, 215 Medical Education Building, Philadelphia, PA 19104-6074, USA. Abbreviations: HPLC, high pressure liquid chromatography; nm, nanometer; mAb, monoclonal antibody; AChR, acetylcholine

receptor; KLH, keyhole limpet hemocyanine; ECDI, 3-ethyl-(3-ethyldiamine)-carbodimide; Tris, Tris(hydroxymethyl)aminomethane; KP buffer, 10 mM potassium phosphate buffer, pH 7.0; TBS, 10 mM Tris, 140 mM NaCl, pH 7.4; TBS-T, TBS plus 0.1% Tween-20; I25I-Prot A, 125I-radiolabelled Protein A; a-BTX, a-bungarotoxin; 125I-ot-BTX, 12SI-radiolabelled ot-bungarotoxin; SDS, sodium dodecyl sulfate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Mr, relative molecular mobility; SPRIA, solid phase radioimmunoassay; BSA, bovine serum albumin; PBS, 137 mM NaCI, 2.5 mM KCI, 1.4 mM Na2PO 4, 1.5 mM KH2PO4; PBS-T, PBS plus 0.05% Tween-20; Tris/BSA, or low ionic strength buffer, 20 mM Tris, 0.4% BSA; high ionic strength buffer, PBS with 0.4% BSA; buffer 1, 50 mM Tris, 50 mM NaCI, 5 mM EDTA, 1.2 mM EGTA, 20 mM PMSF; cpm, counts per minute; TBS/BSA, TBS plus 0.4% BSA; PBS-Triton, PBS plus 0.1% Triton X-100; EDTA, ethylenediamine-tetraacetic acid; EGTA, ethyleneglycol-bis(fl-aminoethyl ether) N,N,N',N'-tetraacetic acid; PMSF, phenylmethylsulfonyl fluoride; NRS, normal rat serum; MIR, main immunogenic region.

14

The e-specific mAbs were used, in conjunction with mAbs specific for the a and /3 subunits and anti-peptide antisera specific for the •, 3' and 6 subunits, to identify in Western blots the subunit complement of embryonic and adult bovine muscle AChRs.

Introduction

The nicotinic acetylcholine receptor (AChR) of fish electric tissue and vertebrate skeletal muscle is formed by four homologous subunits, in the stoichiometry Cez/3Tt~ or a2/3•~ (review in Lind- strom et al., 1987; Claudio, 1989; Stroud et al., 1990). The AChR structure is highly conserved, and corresponding subunits of AChRs from different animals and tissues are highly homologous (review in Lindstrom et al., 1987; Claudio, 1989; Stroud et al., 1990).

Embryonic (or denervated) muscle and adult (innervated) muscle express different AChRs (review in Schuetze and Role, 1987; Claudio, 1989). Both adult and embryonic AChRs contain a, /3 and 3 subunits (Mishina et al., 1986). In embryonic or denervated muscle AChR, a 3' subunit is also present, which is substituted for, in adult innervated muscle, by a homologous • subunit (Mishina et al., 1986; Witzemann et al., 1987, 1989; Gu and Hall, 1988a, b). Adult and embryonic AChRs differ in their location along the muscle membrane, turnover rate, ion gating func- tion, immunochemical properties and pharmacol- ogy (Hall et al., 1983; Mishina et al., 1986; Schuetze and Role, 1987; Witzemann et al., 1987). Little is known on the structure of embryonic and especially of adult AChR, because of their diffi- cult isolation in sufficient amounts for structural studies, and the lack of probes specific for either form of the muscle AChR proteins, e.g. specific for the embryonic 3' or the adult • subunits.

A large library of mAbs, specific for each of the Torpedo AChR subunits, has been developed in one of our laboratories (review in Lindstrom, 1986). MAbs against the a, /3 and fi subunits have been characterized and used for structural studies of AChRs from piscine electric tissue and vertebrate muscle. Although several mAbs versus the Torpedo y subunit are included in this library, their epitopes and binding characteristics have not been investigated yet. The necessity for probes

specific for mammalian muscle 3' and • subunits makes such investigations worth doing, because, in spite of its nomenclature, the Torpedo 3' subunit, so called because of its apparent molecular weight in SDS gel electrophoresis (larger than the /3 subunit and smaller than the ~ subunit), is part of a mature, junctional AChR, and should be the equivalent of the adult muscle • subunit. Com- parison of the subunit sequences does not clarify this dilemma, because the Torpedo 3, subunit has an almost equal degree of sequence identity with both mammalian muscle • and 3' subunits (Takai et al., 1984, 1985).

In the present paper we have mapped the segments of the Torpedo 7 sequence recognized by five mAbs specific for the Torpedo 3' subunit. All mAbs recognized epitopes within a 50 residue sequence on the cytoplasmic surface of the AChR. Three mAbs crossreacted with muscle AChR from different mammalian species. Two of them uniquely recognized the • subunit, both in its native and in its denatured form, and could be used to demonstrate the presence of • subunit in the AChRs at the neuromuscular junction of adult bovine, canine, and human muscle. This is consis- tent with the idea that Torpedo AChR 3' subunit corresponds to adult vertebrate muscle AChR • subunit. The mAbs to •, 3' and 6 subunits, in conjunction with other mAbs specific for the a and /3 subunits, and sequence-specific polyclonal sera specific for the mammalian 7, •, or 6 subunit, directly demonstrated the presence of the expected subunit complement in purified AChRs from fetal and adult bovine muscle.

Materials and methods

Peptide synthesis and characterization Peptides 14-20 residues long and overlapping

each other by 4-6 residues, corresponding to the entire sequence of the Torpedo 3' subunit (Noda et al., 1983), and to the sequence region 346-425

15

of Torpedo S subunits (Noda et al., 1983) were synthesized (Houghton, 1985). The peptides corresponded to the sequence segments of Torpedo 3, and S subunits indicated along the abscissae of Fig. 1 and Fig. 2 (inset). For mapping of mAb epitopes by solid phase radioimmunoassay (see below), we also used existing panels of similar overlapping synthetic peptides corresponding to the complete sequences of the 3, and 8 subunits of human muscle AChR. The human synthetic peptide panels have been previously described (Protti et al., 1991a, b), and they closely corresponded to the homologous sequence segments of the Torpedo 3, and 8 subunit (for the human 3, peptide sequences, see also the abscissa of Fig. 2). Nine peptides, 16-21 residues long, corresponding to sequence segments of the bovine 3,,

lO mab #7

5

i0- - mab #145

5Z

0?-

~ IOz m~b #154

mab #165

5 ~

I

07 . . . . . 3-_ . .

10- mab#168

5

PEPTIDE Fig. 1. Direct binding of mAbs to synthetic peptides corresponding to the entire Torpedo 3" subunit, measured by SPRIA. The sequences of the peptides are indicated along the abscissa. Each mAb recognized only one peptide, mAb 7 recognized peptide 3,373-392, mAb 145 recognized peptide 388- 407, and mAbs 154, 165 and 168 all recognized peptide 3,358-377. All peptides recognized were within the sequence

segment 3'358-407. See text for experimental details.

20-

o?, 15

e~ r...)

°1 i1 ~J5

~1o

TORPEDO &SUBUNIT PEPTIDES

5

H U M A N 8 - S U B U N I T PEPTIDES

Fig. 2. Direct binding of mAb 7 to synthetic peptides corresponding to the entire human 8 subunit, and, in the inset, to synthetic peptides corresponding to the sequence region 346- 425 of the Torpedo S subunit, measured by SPRIA. The sequences of the peptides are indicated along the abscissae. MAb recognized only one human peptide, HS373-392, and the largely overlapping homologous Torpedo peptide, T8376- 395. The S peptide sequences recognized by mAb 7 corre- spond well to the homologous region of the Torpedo 3" subunit which is recognized by this mAb (see Fig. 1). See text for

experimental details.

E, and S subunits (Takai et al., 1984, 1985; Kubo et al., 1985), which are highly divergent among these and the other AChR subunits, were also synthesized. The latter peptides corresponded to the following sequence segments: e9-28, H Y L F D T Y D P G R R P V Q E P E D T ; 3,9-28, G D L M Q G Y N P H L R P A E H D S D V ; 8102-114, QISYSCNVLIYPS; e186-205, DFCPGVIR- RHDGDSAGGPGE; 3,186-205, RHRPAKM- LLDEAAPAEEAGH; 8198-213, VNVDPSV- PLDSPNRQD; 3,386-405, LVRAALEKLEKG- PESGQSPE; e379-398, R H R H G T W T A T L - CQNLGAAAP; 8380-397, EKQSERHGLAR- RLTTARR. The peptides are indicated by a code which includes the letter T, B or H for Torpedo, bovine and human AChRs, the symbol 3, or e or S for the corresponding subunits, and two numbers, which indicate the position, on the AChR subunit sequence, of the amino terminal and carboxyl terminal residue of the peptide. Reverse phase high performance liquid chromatography (HPLC) analysis, using a C~8 column (Ultra- sphere ODS, Beckman Instruments, Fullerton, CA, USA) and a gradient of acetonitrile in water plus 0.1% trifluoroacetic acid, consistently yielded

16

a major peak, accounting for 65-85% of the total optical density (214 nm) for all peptides. This approach underestimates the purity of the peptides, because low molecular weight contaminants absorb at the wavelength used. The purity and sequence of randomly selected peptides for each synthesis were verified by gas-phase amino-terminal sequencing (Applied BioSystems, Foster City, CA, USA). Consistently, only the expected sequence was found, as expected from peptide preparations containing contaminating truncated homologous peptides, where one or more residues are randomly missing along the sequence because of incomplete coupling, each present at a level below detectability (< 2-3%). Amino acid composition analysis (Heinrickson and Meredith, 1984) yielded a satisfactory correspondence between experimental and expected values for all peptides.

Monoclonal antibodies The production and characteristics of the mAbs

used have been described previously (Tzartos and Lindstrom, 1980; Tzartos et al., 1986). Most mAbs were raised using SDS-denatured Torpedo AChR as immunogen. Two mAbs (152 and 155) recognized Torpedo a subunit, two others (mAbs 118 and 124) the Torpedo [3 subunit, and crossreacted with fetal bovine AChR (Tzartos et al., 1986).

The other seven mAbs (Table 1) reacted with the Torpedo y or bovine 3' subunit. One of the anti-y mAbs (mAb 7) was raised against the Torpedo 6 subunit, and crossreacted with both Torpedo 3' and /~ subunits (Tzartos and Lindstrom, 1980).

Production of and affinity purification of anti- peptide polyclonal antibodies

Polyclonal antibodies against unique sequence segments of the e, 3' and 3 subunits of bovine muscle AChRs were obtained by immunization with the peptides described above, coupled to keyhole limpet hemocyanin (KLH, Sigma, St. Louis, MO, USA) as described previously (Nel- son and Conti-Tronconi, 1990). Anti-y and e antisera were raised in rabbits, anti-~ antisera in rats. Peptides y386-405, E186-205, e379-398 and 3382-399 were the best immunogens.

Anti-peptide antibodies were purified by affinity chromatography, using peptides coupled to AH- or CH-Sepharose (Sigma, 10 mg peptide/ml packed resin) as described previously (Nelson and Conti-Tronconi, 1990). Antibody concentration was estimated assuming OD280.0.1% = 1.4. 2-3 mg of antibody were obtained from 10 ml of anti-y and anti-e rabbit serum, 0.2-0.3 mg from 4 ml of anti-~ rat serum. Antibody specificity was as- sessed by dot blot assay (Nelson and Conti- Tronconi, 1990). Purified antibodies, at 1-10

T A B L E 1

M O N O C L O N A L ANTIB ODIE S US E D IN THIS S T UDY

mAb Immunogen a Ig Subunit subclass b specificity c

Titer (nM) against A C h R s d

Torpedo Bovine muscle electric Fetal Adult tissue

7 T A C h R (nat.) ND 3', 6 18,200 6.22 3.43 132 T A C h R (den.) IgGl 3' 30,000 0 ND 145 T A C b R (den.) IgG2a y 30,000 0.6 0 154 T A C h R (den.) IgG1 y 13,000 0 170 162 T A C h R (den.) IgM y 1,800 0 ND 165 T A C h R (den.) Ig2A y 30,300 1.13 1.23 168 T A C h R (den.) IgG1 7 28,000 0 750

a T A C h R indicates purified Torpedo AChR, native (nat.) or denatured in SDS (den.). BAChR indicates purified embryonic bovine AChR.

b From Tzartos et al., 1986. c From Tzartos and Lindstrom (1980) for mAb 7, from Tzartos et al. (1986) for the other mAbs. d The titers of mAbs 132 and 162 are from Tzartos et al. (1986), the other titers were determined in this study. ND, not determined.

/ zg /ml dilutions, recognized only the relevant peptide (Fig. 3). Antibody anti-8382-399 only weakly - - although specifically - - recognized this peptide. Because the antibodies were purified using the relevant peptide as affinity ligand, they all must be able to bind to the peptide. Weak recognition in dot blot assay of peptide 8382-399 must be due to poor ability of this peptide to bind nitrocellulose, or to its immobi- lization in a conformation unsuitable for antibody binding.

Iodination and calibration of lesI-a-BTX a-Bungarotoxin (a-BTX) was purified from

Bungarus multicinctus (Biotoxins, St. Cloud, FL, USA), as described by Clark et al. (1972), and radiolabelled with 1251 as described by Lindstrom et al. (1981). The specific activity of radiolabelled a-BTX (125I-a-BTX), determined as described by Blanchard et al. (1979), was 200,000-250,000 cpm/pmol .

Mapping of mAb epitopes on the Torpedo y subunit sequence by SPRIA

The assay was carried out at room temperature. Peptide solutions (300/zl aliquots, 25 / zg /ml

Synthetic Peptides

Affinity Purified Antibodies to Peptides

A 'MM -<cg

"~ 37g-3g8* 37g-3g8 188-207

g-28 y 38B-4os

y g-28 8 380-3g7 8 Ig8-213

Fig. 3. Dot-blot assay of three anti-e, one anti-y, and one anti-6 anti-peptide polyclonal antibodies, affinity purified using the peptides indicated at the top of the figure, tested for their reactivity to the peptides indicated on the right of the figure. Peptide e379-398" contained an additional tyrosine

residue at the carboxyl terminus.

17

in 10 mM potassium phosphate, pH 7.4 (KP buffer)) were added in triplicate to 96-well plates (Immuno Plate Maxisorp, Nunc, Karnstrug, Den- mark), incubated 1 h (or overnight at 4°C), washed 3 times with KP buffer, 'blocked' with 300 p.1/well of 3% bovine serum albumin (BSA) in PBS with 0.05% Tween-20 (PBS-T) for 30 min, washed once with PBS-T and once with 20 mM Tris-HC1 buffer, pH 7.6. The mAb, diluted 1 : 1000 in 20 mM Tris with 0.4% BSA (Tris /BSA, low ionic strength buffer) or in PBS with 0.4% BSA (high ionic strength buffer), was added (300 ~ l /wel l ) and incubated for 2.5 h. The plates were washed 3 times with PBS-T, incubated for 45 min with affinity purified rabbit anti-rat IgG ( ~ 2.3 mg/ml , Sigma, St. Louis, MO, USA, 300/z l /wel l of a 1:1000 dilution in Tr is /BSA), washed 3 times with PBS-T, incubated for 30 min with lzsI-Protein A in T r i s /BS A (100,000 cpm in 300 /.d/well), and washed 3 times with PBS-T. Bound radioactivity was removed by 2% SDS and counted (Gamma 5500, Beckman Instruments, Fullerton, CA, USA). To test the effect of ionic strength on antibody binding to peptides, mAbs were diluted (1:2000) in T r i s /BS A containing up to 2 M NaCl and incubated in the wells for 0-25 min to measure the rate of association of the complex, or 2.5 h for equilibrium binding.

Competition between peptide and non-denatured Torpedo AChR for mAb binding

The assay was carried out at room temperature. Peptide solution (25 ~1, 0.25 m g /m l in KP buffer) plus 25 /zl of a 5 nM solution of the relevant mAb in TBS containing 0.4% BSA (TBS/BSA) were incubated for 1 h. Aliquots (25 p.1) of 5 nM Torpedo AChR (measured as I25I-a- BTX binding sites by the disk assay of Schmidt and Raftery (1973)), pre-labelled with 7.5 nM I25I-a-BTX, and 80 /z l /ml of normal rat serum (NRS) in TBS with 1% Triton X-100 were added and incubated for 1 h. Goat anti-rat IgG anti- serum (50 #1 of a dilution in PBS sufficient to precipitate 2 ~1 of NRS) was added and incubated for 2 h. 1 ml of PBS containing 0.1% Triton X-100 (PBS-Triton) was added and the precipitated AChR/125I-a-BTX complexes were pel- leted at 10,000 rpm for 5 rain in a Sorvall rotor SH-MT (DuPont de Nemours and Co., Wilming-

18

ton, DE, USA), washed with 1 ml of PBS-Triton and counted. In positive controls (100% precipitation), precipitation of 125I-a-BTX/Torpedo AChR complexes occurred in the absence of peptide. In blank samples the mAb was omitted. Significance of specific inhibition was evaluated by a two-tailed Student t-test.

Immunoprecipitation assay of mAb titers versus embryonic and adult mammalian muscle AChR

Bovine adult AChR was from innervated cheek and facial muscles of adult cattle, or from skeletal muscles of a near-term calf fetus (8-9 months' gestation (Arthur, 1975)), embryonic AChR was from muscle of bovine fetuses at 3-4 months' gestation. All procedures, unless noted, were carried out at 4 ° C. Muscle was homogenized in 1-1.5 volumes (w/v) of 50 mM Tris, 50 mM NaC1, 5 mM EDTA, 1.2 mM EGTA, pH 7.4, 20 mM PMSF (buffer 1), and centrifuged for 60 min at 37,000 xg . The pellets were resuspended in buffer 1 containing 10 mM iodoacetamide and 1.5% Triton X-100, extracted for 2 h, and centrifuged for 60 min at 130,000 x g for embryonic muscle, at 37,000 × g for adult muscle. The AChR in the supernatants, measured as 125I-a-BTX binding sites (Gotti et al., 1982), was labelled with a25I-a-BTX. Adult (~ 0.5 pmol) or embryonic (1 pmol) AChR plus 4/zl of NRS (embryonic muscle) or 2.5 /xl of NRS (adult muscle) were incubated overnight (embryonic muscle) or for 8 h (adult muscle) with increasing concentrations of test or control mAb. AChR/mAb complexes were precipitated by an excess of anti-rat IgG anti- serum, washed twice with PBS containing 0.1% Triton X-100 (embryonic muscle) or 30 mM sodium phosphate buffer, 0.2% Triton X-100, pH 7.6 (adult muscle) and counted (Beckman 5500, Beckman Instruments, Fullerton, CA, USA).

Canine muscle was obtained from animals killed for other purposes by a protocol approved by the Animal Subjects Committee, University of California, San Diego. AChR was extracted from the muscle membranes of near-term canine fetuses and adult intercostal muscle in 1% Triton X-100 (Lindstrom et al., 1981). Rat muscle AChR was from denervated tissue, extracted in 1% Tri- ton X-100 (Lindstrom et al., 1981). Human AChR

was from TE671 cells, and from muscle tissue of amputated limbs. The former was extracted in 1% Thesit with 0.05% SDS (Luther et al., 1989), the latter in 1% Triton X-100 (Lindstrom et al., 1981). Aliquots (250 ~1) of 125I-a-BTX-labelled canine or human muscle extract (0.1-0.3 nM AChR plus 20 nM a-BTX) or TE671 extract (100 /xl, 8 nM AChR plus 20 nM a-BTX) were incubated overnight with 5 /,d of test and control mAbs. Complexes were precipitated with an excess of goat anti-rat IgG for 1 h, then the samples were diluted with 1 ml of 0.5% Triton X-100 buffer prior to centrifugation. The pellets were washed twice with 1 ml of 0.5% Triton X-100 buffer prior to counting for precipitated radioactivity.

Double-immunolabelling of adult canine and human muscle with fluorescein and rhodamine

Frozen sections (6 ~M) of normal adult canine or human intercostal muscle were air dried then fixed in acetone for 10 min. The sections were blocked for 15 min with 5% goat serum and 2% BSA in PBS containing 0.1% thimerosal, fol- lowed by incubation for 45 min with mAb 210 (0.66 p~M against Torpedo AChR) or mAb 145 (0.3 /zM against Torpedo AChR, negative control), mAb 168 (0.4 tzM against Torpedo AChR), or mAb 7 (0.27 tzM against Torpedo AChR). The sections were rinsed in PBS for 30 min on ice with gentle shaking, incubated for 45 min with a 1/200 dilution of fluorescein-conjugated a-BTX and a 1/100 dilution of rhodamine-conjugated goat anti-rat IgG (Kirkegaard and Perry Labora- tories, Gaithersburg, MD, USA), rinsed for 30 min in PBS, and mounted.

Purification of bovine muscle AChR Bovine muscle AChR was purified from frozen

bovine fetuses by affinity chromatography, using Naja siamensis a-neurotoxin-Sepharose, as described by Einarson et al. (1982) and Gotti et al. (1982). Adsorbed AChR was stripped with SDS, cryoprecipitated, and lyophilized. Embryonic AChR was purified from early bovine fetuses (< 12 inches), innervated AChR from innervated muscle of a near-term bovine fetus.

Western blots Purified, lyophilized AChR from embryonic or

adult bovine muscle was diluted in 1 ml of dis- tilled water and the protein assayed (Lowry et al., 1951). The samples were electrophoresed (1.25- 2.5 /~g/3 mm gel) on a 8.75% polyacrylamide slab gel with 4% stacking gel (Laemmli, 1970), and Western blots were carried out as described previously (Nelson and Conti-Tronconi, 1990), using subunit-specific antibodies. Briefly, the electrophoresed protein bands were transferred onto 0.45 ~m nitrocellulose sheet (Towbin et al., 1979). The nitrocellulose sheet was cut into 3 mm strips, blocked with 1 mg/ml BSA in TBS-T and incubated for 2 h at room temperature with the test antibody, diluted as needed in TBS-T and washed twice in T~3S-T. For rat mAbs, which bind Pro- tein A poorly, the strips were further incubated with rabbit anti-IgG for 2 h with antibody (Sigma), diluted 1:1000 in TBS-T, then washed as above. Radioiodinated Protein A (1 × 106 cpm in 1 ml of BSA/TBS-T) was added, and incubated for 1-2 h at room temperature. After 3 washes in TBS-T the strips were dried and autoradiographed for 2-4 days at - 70 ° using a Quanta III intensifying screen and Kodak RP film.

Results

Mapping of epitopes for mAbs against Torpedo 3" subunit

Identification of the sequence segments of Torpedo 3" subunit recognized by seven anti- Torpedo 3" mAbs listed in Table 1 was attempted by SPRIA, using 33 synthetic peptides corresponding to the complete Torpedo 3" subunit sequence. Five mAbs (7, 145, 154, 165 and 168) specifically recognized a peptide sequence (Fig. 1). All mAbs recognized epitopes within the sequence segment T3"358-407. MAbs 154, 165, and 168 recognized peptide Ty358-377, mAb 145 recognized peptide T3"388-407. MAb 7 recognized peptide T3'373-392.

Because mAb 7 crossreacts with To,pedo 3" and 6 subunits, we tested its ability to recognize the sequence region of the Torpedo 8 subunit homologous to T3'373-392. Results of a typical

19

SPRIA assay using overlapping synthetic peptides screening the sequence region T8346-425 are reported in the inset of Fig. 2. MAb 7 clearly and consistently bound to the synthetic peptide T8376-395, which largely overlaps the sequence region homologous to T3"373-392 (T8376-395, KSRSELMFEKQQSERHGLVPR; T3'373-392, ELMFEEQKDRHGLKRVNKMT; please notice that, because of the longer sequence of the 8 subunit, with insertion of extra residues, homologous residues of the 3' and ~ subunit have different numbers). MAbs 154 and 168 were also tested for binding to the Torpedo ~ peptides, and they did not recognize any synthetic T8 sequence (data not shown).

Three mAbs (mAbs 7, 154 and 168) crossreacted with mammalian muscle AChRs, and two of them (mAbs 154 and 168) specifically recognized the bovine e subunit (see below), mAbs 154 and 168 crossreacted with adult muscle AChRs from different mammalian species (see below), and it is reasonable to conclude that they in all cases recognized the e subunit. To verify this conclusion, in the absence of synthetic sequences of the mammalian E subunit, we investigated whether these mAbs recognized, in SPRIA experiments, any synthetic sequence of the 3' and 8 subunits of human muscle AChR. Also mAb 7 was tested with the human 3" and 8 peptides. No human peptide was recognized by mAbs 154 and 168, thus excluding the possibility that their ability to recognize adult muscle AChRs, including human muscle AChR, is due to crossreactivity with the 8 subunit, and indirectly confirming their specifity for the adult ~ subunit. MAb 7 did not recognize any H3' peptide, but it consistently, strongly recognized the synthetic sequence H~373-392 (Fig. 2) (KSRSDLMFEKQSERHG- LARR).

The specificity of the SPRIA mapping on the Torpedo 2/ subunit was verified by testing the ability of the T3" peptide recognized by the different rlaAbs to compete with native AChR for mAb binding. Binding to native Torpedo AChR of all mAbs was strongly inhibited by preincubation of the mAb with 125 /zg/ml of the peptide recognized in the SPRIA assay, prior to addition of native AChR. In different experiments the following extents of inhibition were obtained for

20

Adult Canine Muscle Double Labeled Sections

Rhodamine Goat Anti-Rat IgG

site: £358-377 probe: mAb168

Fluorescein o~Bgt

site: ACh site probe: o~Bgt

~: + 'y 373-392 mAb7 o~Bgt

~66-76(MIR) mAb210 o~Bgt

Fig. 4. Sections of adult canine intercostal muscle were double-labelled with fluorescein-conjugated a -BTX for localization of the A C h R binding site and with rhodamine-conjugated goat anti-rat IgG for localization of mAb binding, mAb 145 was used as a negative control and showed no labelling with rhodamine conjugate (results not shown). Localization of the MIR was demonstra ted

by binding of mAb 210.

these mAbs: mAb 7:95 + 4.5% (n = 5); mAb 145: 95 + 6.2% (n = 4); mAb 154:90.8 + 12.2% (n = 6); mAb 165:90.25 + 10.6% (n = 6); mAb 168: 98.4 + 2.1% (n = 4).

Increasing ion concentrations (0-0.5 M NaCI, for some mAb 0-1.2 M NaCI) had no effect on the total binding of mAbs 7, 154, 65, and 168 to the relevant T y peptides, or on the rate of binding to the Ty peptide (tested for mAbs 154, 65 and 168 using 0-0.525 M NaCI).

Anti-Torpedo mAbs crossreact with mammalian muscle AChRs; mAb 154 and mAb 168 specifically recognize adult muscle AChR

The relative abilities of anti-Torpedo y mAbs to precipitate native AChR solubilized from bovine or human muscle, and their preference for fetal or adult AChR are summarized in Table 1. A similar pattern of reaction but at greatly re- duced titers ( < 2nM) was detected with AChRs from muscle of rats, dogs, and humans.

Immunochemical localization of adult muscle AChR

Despite the similarly low degree of crossreac- tion detected by immune precipitation with AChRs from both dogs and humans, both mAb 7 and mAb 168 were able to specifically label AChRs at both neuromuscular junctions in adult canine and human muscle. Indirect immunofluo- rescence labelling was used to localize bound mAbs. The positions of neuromuscular junctions were identified by the use of fluorescent a-BTX. As a positive control we used mAb 210 (specific for the main immunogenic region (MIR) on the a subunit) which binds within the sequence segment a68 -76 (Tzartos et al., 1988; Bellone et al., 1989; Das and Lindstrom, 1989; Saedi et al., 1990). As a negative control we used mAb 145 (anti-Torpedo y) which does not crossreact with mammalian muscle AChR. In both human and canine muscle mAb 210 clearly identified the same synapses localized by ce-BTX, while mAb 145 did not bind at all. MAbs 168 and 7 strongly and consistently recognized the AChR present at the neuromuscular junctions of adult canine and

21

Embryonic Muscle M r ( k O )

- I 1 6

-97.4

- 6 6

- 4 5

- 2 9

! z LL,. TTTTT oOo o o I . ~ C ' q T - - . . . . . .

- EE E , , el el e:~ c:~. ~ m m m

. . . . . . . . . . ; ~ ¢¢~ OO r -

- - ~ c - c - t~ t~ t~

Innervated Muscle

Mz( D) -116

-97.4

- 6 6

- 4 5

- 2 9

Fig. 5. Western blots of embryonic and innervated bovine muscle AChRs, probed with the mAbs and anti-peptide antibodies as indicated. In the negative control (lane 'Control') the first antibody had been omitted, mAbs 154 and 168 are the anti-Torpedo mAbs which we demonstrated to crossreact specifically with the E subunit from mammalian muscle of different species. Polyclonal anti-peptide antibodies to ,/386- 405, ~380-398, and e186-205 were raised in rabbits or rats by immunization with KLH-conjugated synthetic peptides and purified by affinity chromatography using the corresponding

peptides. See text for experimental details.

22

human muscle. Fig. 4 reports typical results obtained with sections of adult canine muscle.

Identification of subunits present in embryonic and innervated bovine muscle AChR

The immunolocalization a n d / o r immunoprecipitation results reported above strongly indicate that mAbs 154 and 168 are specific probes of the adult muscle • subunit. We verified this assump- tion by directly studying the binding of these mAbs to mammalian muscle • subunits in West- ern blots of AChRs purified from embryonic or innervated bovine muscle. Identification of the a subunit was obtained by the use of mAbs 152 and 155 and /3 subunits were identified using mAbs 118 and 124. These mAbs, although raised with Torpedo AChR, crossreact with bovine AChR (Tzartos et al., 1986). Identification of the 3' and 6 subunits, and verification of the • subunit, were done using the subunit-specific anti-peptide antibodies described above. MAb 7, in spite of its ability to recognize the synthetic sequence segment of the human 8 subunit and the homologous synthetic sequences of Torpedo 3" and S subunits, did not detectably recognize any denatured bovine AChR subunits in Western blots. Both mAbs 154 and 168, and the anti-e peptide antibodies, detected a protein having the M r expected for the • subunit only in AChR from innervated muscle AChR, while the 3' subunit was detected by the anti-3, peptide antibody only in blots of embryonic AChR (Fig. 5). Similar a , /3 and S subunits were detected in both early and near-term fetal muscle (Fig. 5). In blots of innervated muscle AChR the anti-6 serum antibodies consistently, but very weakly, recognized a band of apparent M r ~ 61 kDa, barely discernible above the high background present for this antibody (data not shown). Anti-/3 and anti-e antibodies occasionally recognized minor bands of low apparent M r (Fig. 5). They are probably degrada- tion products of the mature subunits, because of their lower Mr, sporadic presence, and lower amounts than the main protein band consistently recognized by these probes. The apparent M r of the immunologically identified constituent sub, units of bovine AChRs were as follows: for innervated AChR: a: 41 _+ 3.9 kDa (n = 10);/3:52 _+ 6.8 kDa (n = 10); e: 57.5 _+ 8.1 kDa (n = 9); and S:

61 + 3.6 kDa (n = 3). The M r for subunits of embryonic AChR were: a: 43.5 + 0.2 kDa (n = 4), /3:52.9 + 0.3 kDa (n = 4), 3': 55 kDa (n -- 1), and S: 65 kDa (n = 1).

Discussion

In this study we have identified two mAbs, raised against the 7 subunit of Torpedo AChR, as specific probes for the homologous subunit of the AChR from innervated vertebrate muscle, and we have mapped their epitopes on the Tor- pedo y subunit sequence. In addition, we have mapped epitopes of two mAbs to the 3' subunit which are species-specific for Torpedo, and for one mAb which reacts with both 3' and 6 subunits of Torpedo, and which Crossreacts with AChR from both embryonic and adult mammalian muscle, probably because of its ability to recognize the homologous epitope on the mammalian 8 subunit. The anti-• mAbs characterized here recognized adult AChR at the neuromuscular junctions in innervated muscle of mammals, and bound the muscle • subunit in Western blots of purified bovine AChRs, while they did not recognize in Western blots the bovine 3' subunit, nor, in SPRIA assays, any synthetic sequence region of the human 3' subunit. The anti-• mAbs, and other monoclonal and polyclonal antibodies specific for the a, /3, 3' and S subunits of mammalian muscle AChR, were used to define the subunit complement and subunit molecular weights of AChRs from embryonic and innervated bovine muscle. Because of their crossreactivity with adult AChRs from mammalian species, despite their limited crossreacting titers, the •- specific mAbs may be useful tools for selectively labelling adult mammalian muscle AChRs.

The epitopes for the five mAbs which were mapped (Figs. 1, 2 and 6) are clustered within the sequence T3'358-407, which is part of the sequence segment between the hydrophobic segments M3 and M4 (reviewed in Lindstrom et al., 1987; Claudio, 1989; Stroud et al., 1990). Homol- ogous sequences on the a , /3 and 6 subunits were previously found to contain epitopes for mAbs (Ratnam et al., 1986a, b; Das and Lindstrom, 1991). In the a, /3 and 6 subunits, much or all of

the sequence between the hydrophobic segments M3 and M4 is known to be on the cytoplasmic surface (Ratnam et al., 1986a, b). Four of the five mAbs to the Torpedo 3 ̀ subunit studied here (mAbs 7, 154, 165, and 168), which all bind both native and denatured AChR, were previously found to bind to the cytoplasmic surface of AChR in the frog neuromuscular junction (Sargent et al., 1984). Thus, as in the case of the homologous a, /3 and ~ subunits, the sequence 7358-407 is exposed on the cytoplasmic surface, in a conformation which must be similar in both native and denatured AChR.

Identification of the sequences containing the epitopes for these five mAbs was confirmed by the competit ion experiments. The excellent inhibition of mAb binding to native Torpedo AChR by the relevant peptides is probably because four of these mAbs were raised using denatured AChR, which may have favored production of antibodies against linear determinants. Resis- tance of the mAb binding to the relevant peptides to increased salt concentrations also sug-

23

gests that the mAbs recognize linear segments of the 3 ̀ subunit sequence, and their complete con- tact with the flexible synthetic peptide, to the exclusion of any water molecule, makes their binding impervious to destabilization by increased osmolarity. Because these mAbs effi- ciently recognize native AChRs, several of the residues in this sequence segment (the ones form- ing the epitopes for the mAbs) must be linearly arranged on the AChR surface. The abundance of charged and hydrophilic residues in this sequence region (Fig. 6) supports its involvement in formation of the AChR surface.

The epitopes for mAbs 154, 165 and 158 are all within residues 7358-377. This region of Tor- pedo 3' subunit is much more similar to the homologous region of mammalian muscle e subunit than to the embryonic 3' subunit (Fig. 6). Nine- teen residues are identical or conservatively sub- sti.tuted in Torpedo 3, and bovine E subunits, and this explains why mAbs 154 and 168, which crossreact with mammalian AChR, specifically recognize the e subunit. The third mAb did not bind to

T¥ 358-377 T¥ 373-392 T¥ 388-407 i , i

Torpedo F I.I.Mr~,~mm:GmI~--~VNKMTSD i DIST~DLYI~L Bovine c ~i.',i~"~"n"~-R~aBrw~I~rLcoJ~-~AAPEI~CCVI~

I 360 370 380 390 400

Proposed epitope - - - T - - - I m.Ab 165 I mAb 7 I

m.Ab 154 m.Ab 145 m.Ab 168

Torpedo y T o r p e d o 5 Human y Human 6 Bovine e Bovine Bovine 6

Proposed epitope

T,/373-392

I m.Ab 7

Fig. 6. Alignment of the sequence segment of the Torpedo AChR y subunit containing the epitopes for the mAbs characterized and used in this study, with the homologous segments of the y and E subunits from bovine muscle AChR (top panel), and of the Torpedo y and 6 subunits; the y and ~ subunits of human muscle AChR, and the y, ~ and ~ subunits of bovine muscle AChR

(bottom panel). The proposed location of the epitopes recognized by the mAbs is indicated below the sequences.

24

mammalian AChR. Because of their specific crossreactivity with adult mammalian AChRs, their epitope(s) should contain residues ILKK, and possibly some more residues on either side of ILKK. The epitope(s) should not extend farther than residue 372, because peptide 3'373-392 was not recognized, nor did it inhibit mAb binding to AChR. Furthermore, the sequence 371-375 is identical in Torpedo 3" and 8 subunits, while these mAbs are specific for 3'. mAb 165, which does not recognize mammalian AChR, should map within the amino terminal part of 3'358-377, which is quite divergent between Torpedo and mammalian subunits.

The epitope for mAb 7, which crossreacts with both Torpedo 8 and 3' subunits, and with both embryonic and adult mammalian AChRs, and which recognizes the homologous synthetic sequences from Torpedo 3" and S subunits, and from the human 8 subunit, but not from the human 3' subunit, should include the amino terminal region of peptide T3'373-392 (residues E / D , L, M, E, X, Q), which is conserved in all subunits and in the Torpedo 3" subunit, while it contains gaps and non-conservative substitutions in the mammalian 3" and • subunits (Fig. 6). The lack of reactivity of mAb 7 with the overlapping synthetic sequence T3'358-377 may be due to improper folding of this peptide. The amino terminal part of T3'373-392 contains a second clus- ter of residues conserved between the 3' and S subunits ( D / E , R, H, G, L). The epitope for mAb 7 could be constructed by these two discon- tinuous segments. The location of the epitope for mAb 145 within the sequence segment 3,388-407 explains its lack of reactivity with mammalian AChRs, as this region is highly divergent in Tor- pedo and mammalian AChRs.

The specificity of mAbs 154 and 168 for the adult E subunit, both in its native form and after full denaturation, and their broad crossreactivity with the e subunit from different vertebrate species, is proven by: (i) their ability to uniquely recognize the denatured E subunit, and not the 3' subunit, in Western blots of embryonic and adult bovine AChRs (Fig. 5); (ii) their specificity for the adult form of bovine and canine AChR after solubilization in non-denaturing detergents, while they do not recognize at all AChRs from bovine,

canine or rat denerva ted / fe ta l muscle (Table 1 and Results section); (iii) their inability to recognize any synthethic peptide sequence of the human muscle AChR y and 6 subunits; and (iv) their labelling of adult AChRs in their native, membrane-bound state, at the neuromuscular junctions of adult muscle from humans and ca- nines (Fig. 4 and Result section). In a previous study these mAbs were shown to specifically label frog neuromuscular junction (Sargent et al., 1984). They may therefore be useful probes for the study of the adult form of muscle AChR from many vertebrate species.

The finding that antibodies against Torpedo 3' subunit preferentially recognize the muscle • subunit, raises an interesting problem of nomenclature. The subunits of Torpedo AChR were ini- tially named a , /3 , y, and S in order of increasing molecular weight (review in Lindstrom et ai., 1987; Claudio, 1989; Stroud et al., 1990). When corresponding homologous subunits were found and sequenced in embryonic mammalian muscle AChR - - the more easily and intensely studied because of its relative abundance - - the same nomenclature was used (Einarson et al., 1982; Conti-Tronconi et al., 1982; Noda et al., 1983; Tanabe et al., 1984; Takai et al., 1984; Kubo et al., 1985). Subsequently, the E subunit was found and sequenced (Takai et al., 1985), and evidence was obtained that it substitutes for the embryonic 3' subunit to form mature, adult junctional AChR (Mishina et al., 1986; Witzemann et al., 1987, 1989; Gu and Hall, 1988a, b). The Torpedo 7 subunit, which contributes to the mature, adult AChR at the electroplax synapses, is, in fact, functionally an E subunit, and this accounts for the shared epitopes between this subunit and the AChRs at the vertebrate neuromuscular junction. In support of this possibility, Torpedo 3" subunit has a phosphorylation site at position Ser353 (Yee and Huganir, 1987), which is conserved in the E subunit, and absent in the mammalian muscle y subunits (review in Steinbach and Zempel, 1987), and it has been reported that antibodies specific for the region of Torpedo y subunit which contain this phosphorylation site (residues 346-359) reacted strongly with adult Torpedo 3" subunit, and minimally or not at all with embryonic Tor- pedo 3" subunit (Souroujon et al., 1990).

Purified bovine muscle AChRs, probed with the different subunit-specific antibodies, contained a, /3 and S subunits of the expected molecular weights in both its embryonic and adult forms, while a 3' subunit was present only in embryonic AChR, and an E subunit was present only in adult AChR (Fig. 5). The a and B subunits of embryonic AChR had slightly higher apparent M r than the corresponding adult subunits. Although this small difference may well be due to the vagaries of M r determination by SDS- PAGE analysis, it raises the possibility that the mRNAs or the precursors of the a and S subunits may be processed differently in adult and in embryonic tissues, resulting in final products of slightly different molecular mass. Different a subunits of human and frog muscle AChRs (Bee- son et al., 1990; Hartman and Claudio, 1990), /3 subunits of mouse muscle AChR (Goldman and Tamai, 1989), and several a subunits of neuronal AChRs (review in McLane et al., 1990) have been described.

The mAbs characterized in the present study, specific for the e subunits of adult muscle AChR, and still broadly reactive with junctional AChRs from many vertebrate species, may be useful probes to study junctional AChRs, the mecha- nisms of assembly of their subunits, and their localization within neuromuscular junctions.

Acknowledgements

We wish to thank Dr. Andrew Engel for the generous hospitality in his laboratory and his precious advice for the experiments on the AChR immunolocalization, and Ms. Bonnie Allen for expert and patient word processing.

This study was supported by the NSF grants BNS-8607289 (to B.M.C.-T.) and BNS-8819911 (to J.M.L.), the NIDA project grant 1 P01- DA05695 (to B.M.C.-T.), the NIH grant NSl1323 (to J.M.L.), Research Grants from the Muscular Dystrophy Association of America (to B.M.C.-T. and J.M.L.), from the California Chapter of the Myasthenia Gravis Foundation (to J.M.L.), and from the Council for Tobacco Research and

Council for J.M.L.).

25

Smokeless Tobacco Research (to

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epitope mapping of monoclonal antibodies to torpedo acetylcholine receptor γ subunits, which...

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