Journal of Neuroimmunology, 38 (1992) 115-128 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-5728//$05.00

115

JNI 02178

Epitope mapping of polyclonal and monoclonal antibodies against two a-bungarotoxin-binding a subunits from neuronal nicotinic receptors Kathryn E. M c L a n e a, Xiadong W u a, Jon M. Lindstrom b and Bianca M. Conti-Tronconi a a Department of Biochemistry, College of Biological Sciences, University of Minnesota St. Paul, MN, and b Institute of Neurological Sciences, University of Pennsylvania Medical School, Philadelphia, PA, USA

(Received 3 May 1991) (Revised, received and accepted 27 December 1991)

Key words: a-Bungarotoxin-binding protein; Nicotinic acetylcholine receptor; Synthetic peptides; Recombinant fusion protein; Avian nervous system; Epitope mapping

Summary Recently, cDNAs for a subunits of two different neuronal a-bungarotoxin-binding proteins (aBgtBP) were isolated from chick brain, designated aBgtBP a l and t~BgtBP a2. These are now also referred to as subunits or7 and a8, respectively. Expression studies in Xenopus oocytes have indicated that a7 subunits are able to form cation channels that are sensitive to nicotinic ligands, and therefore represent bona fide nicotinic acetylcholine receptor subunits. Polyclonal and monoclonal antibodies (mAbs) have been produced against: (i) affinity-purified chick brain aBgtBP; and (ii) fusion proteins containing the unique cytoplasmic sequences a7(327-412) and a8(293-435). Here, synthetic overlapping peptides corresponding to their deduced amino acid sequences are used to map the epitopes recognized by the different antibodies. The polyclonal response to affinity-purified aBgtBPs and the fusion proteins indicates that sequence segments 290-420 of both subunits contain several major and minor epitopes. mAbs selected for their ability to bind both native and denatured aBgtBPs isolated from chick brain also recognize subunit-specific sequential epitopes within the sequence segment 290-420. The epitopes recognized by the mAbs correspond to the minor epitopes defined using antisera. The mAbs characterized in these studies will provide useful probes for further studies of aBgtBP structure and histological localization.

Introduction The subunits of the nicotinic acetylcholine receptor (AChR) form a superfamily of homoloCorrespondence to: B.M. Conti-Tronconi, Department of Biochemistry, University of Minnesota, 1479 Gortner Ave., St. Paul, MN 55108, USA.

gous proteins that share common structural features (Lindstrom et al., 1987b; Betz, 1990; Stroud et al., 1990). AChRs isolated from the Torpedo electric organ and from vertebrate muscle are composed of four types of subunits in the stoichiometry ot2~'yt~ (Raftery et al., 1980; ContiTronconi et al., 1982). In contrast, molecular genetic approaches have defined several different

116

AChR subunits expressed on neurons (a2, a3, a4, a5, and /32,/33, /34) that can be functionally reconstituted in Xenopus oocytes by coexpression of certain a and /3 subunit combinations (reviewed in Luetje et al., 1990; Deneris et al., 1991). Additionally, two relatively divergent subunits of the neuronal AChR superfamily have been identified in the chick brain, which constitute neuronal a-bungarotoxin-binding proteins (aBgtBPs). These AChR a subunits have been designated a7 and a8 (also referred to as aBgtBP a l and aBgtBP a2, respectively)(Conti-Tronconi et al., 1985; Schoepfer et al., 1990; Couturier et al., 1990). Common to all AChR subunits is a large Nterminal domain (approx. 200 amino acids) that is believed to be largely extracellular, four putative membrane spanning domains (designated M1 to M4) and a large highly variable sequence between M3 and M4 that is believed to be cytoplasmic (Lindstrom et al., 1987b; Stroud et al., 1990). The development of subunit-specific monoclonal antibodies (mAbs) and the precise mapping of their epitopes has provided useful probes to determine the transmembrane distribution of different subunit sequences (LaRochelle, 1985; Lindstrom and Criado, 1985; Criado et al., 1985a, b; Lindstrom, 1987; Lindstrom et al., 1987a, b; Maelicke et al., 1989; Pedersen et al., 1990; Das and Lindstrom, 1991). The most prominent sequential epitopes of the AChR subunits, and those that are recognized by mAbs in both the native and denatured subunits, are located in a large cytoplasmic domain between M3 and M4, whose sequence is virtually unique to each AChR subunit (Ratnam et al., 1986a, b; Das and Lindstrom, 1991). Given the high degrees of heterogeneity and homology between subunits of different neuronal AChR subtypes, it is critical that mAbs used to determine their differential expression be characterized by epitope mapping. In the present study, synthetic overlapping peptides corresponding to the complete a7 subunit and the unique sequences of the cytoplasmic domain of the a8 subunit, a8(290-435), are used to map the epitopes of polyclonal and monoclonal antibodies developed against affinity-purified c~BgtBPs from the chick brain and fusion proteins containing the amino acid sequences a7(327-412) and a8(293-

435). The results of these studies indicate that certain regions of the N-terminal extracellular segment of the a7 subunit contain sequential epitopes. In addition, the cytoplasmic segments a7(327-412) and a8(293-435) are shown to contain several sequences that are highly immunogenic, as well as other less prominent epitopes. It is these less prominent epitopes that are recognized by the mAbs characterized in the present study, which were selected for their ability to bind both native and denatured aBgtBPs isolated from chick brain.

Materials and methods

Peptide synthesis and characterization Peptides, 19-21 amino acids long, were synthesized by manual parallel synthesis (Houghten, 1985). The purity of the peptides was assessed by reverse-phase HPLC (high pressure liquid chromatography) using a C18 column (Ultrasphere ODS) and an acetonitrile/water gradient (570%) containing 0.1% trifluoroacetic acid. A major peak was consistently present, which accounted for 65-85% of the total absorbance at 214 nm. This analysis may lead to misleadingly low estimates of the peptide purity because contamination from low molecular mass reagents used during cleavage and extraction of the peptide also absorb at this wavelength. The amino acid composition of all peptides, determined by derivatization of amino acid residues released by acid hydrolysis with phenylisothiocyanate, followed by separation on a reverse-phase HPLC column (PICO.TAG) as described by Heinrickson and Meredith (1984), yielded a satisfactory correspondence between experimental and theoretical values. The sequence and purity of peptides corresponding to sequences 181-200 of different a subunits, the a7 and a8 subunits, and other randomly selected peptides, were verified by gasphase sequencing (Applied Biosystems), which indicated that contamination by truncated peptides was less than 5-15%. The sequence and codes of the peptides corresponding to the a7 and o~8 subunits are reported in Figs. 1 and 2. Overlapping peptides were also synthesized, cor-

117 responding to the region between amino acid residues 290-420 of the mouse muscle a l subunit (Boulter et al., 1985), the rat neuronal a3 subunit (Boulter et al., 1986), and the rat neuronal a4 subunit (Goldman et al., 1987), as follows: the sequence segments a1(277-296), a1(280-297), a1(293-308), a1(304-322), a1(304-323), a1(320337), a1(322-341), a1(339-347), te1(331-350), a1(343-356), a1(343-362), a1(352-368), a1(364380), a1(373-392), a1(376-393), a1(387-406), a1(389-408), and a1(403-421) of the mouse a l subunit; a3(287-306), a3(302-321), a3(319-338), a3(334-353), a3(349-368), a3(364-383), a3(379-398), a3(394-414), and a3(409-429) of the rat a3 subunit; and a4(338-357), a4(391410), a4(411-430), a4(451-470), a4(471-490), and a4(491-510) of the rat a4 subunit.

Affinity-purified aBgtBPs and fusion proteins protCh34-2 and protCh31-5 Chick brain aBgtBPs were isolated by affinity chromatography using a-cobratoxin as affinity ligand (Whiting and Lindstrom, 1986, 1987). In order to generate subunit-specific imunogens, fusion proteins were constructed corresponding to the sequence segments a7(327-412) and a8(293435), designated protCh34-2 and protCh31-5, respectively (Schoepfer et al., 1990). Subcloning of cDNA fragments encoding the amino acid sequences a7(327-412), protCh34-2, and a8(293435), protCh31-5, and the construction of bacterial expression plasmids has been previously described in detail (Schoepfer et al., 1990). protCh31-5 was purified from inclusion bodies in 8 M urea, whereas protCh43-2 was purified by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis.

Polyclonal and monoclonal antibodies The immunization schedules, preparation of hybridomas and screening of mAbs were described in detail in Schoepfer et al. (1990). EAMG 150 and EAMG 151 are polyclonal antisera raised in female Lewis rats against native and denatured affinity-purified chick brain aBgtBPs, and mAb 305 was produced from the same immunization as EAMG 150. mAb 306 and mAb 307 were obtained from a female Balb/c mouse immunized with chick and rat brain affinity-purified

aBgtBPs. EAMG 157 is a polyclonal antiserum from a female Lewis rat immunized with the a8 fusion protein, and mAbs 308-312 were produced from hybridomas resulting from that immunization. EAMG 163 is the polyclonal antiserum from a female Lewis rat immunized with the a7 fusion, and mAbs 318-320 were derived from that immunization. The titers of polyclonal antisera and mAbs were determined by immunoprecipitation of aBgtBPs in chick brain extracts labelled with [125I]aBgt (Schoepfer et al., 1990).

Enzyme-linked immunosorbent assay (ELISA) Peptide solutions (50 /zl of 100 /.Lg/ml in 10 mM potassium phosphate buffer, pH 7.4 (KP)) were added to each well of a 96-well polystyrene plate (Nunc, Immunolon) and incubated for 1 h at 37°C. The plates were washed three times with KP containing 100 mM NaC1 and 0.05% Tween (PBS/Tween), and blocked with 2% bovine serum albumin (BSA) in KP (1 h at 37°C). The plates were incubated overnight at 4°C with 0.4-8 nM antibodies in 1% BSA in KP. After three washes with PBS/Tween the plates were incubated with peroxidase-conjugated goat-anti-rat IgG or goatanti-mouse IgG (BioRad) diluted 1:1000 in 1% BSA in KP for 30 min at room temperature. After two washes with PBS/Tween, 100 /xl substrate was added (0.15 M 1,2-phenylenediamine, 0.05% H202, 0.1 M sodium citrate, pH 5.0). After 10 min the reaction was stopped by addition of 25 /xl of 2.5 M H2SO4, and the absorbance was read at 490 nm in an automated microplate reader (Model EL312, Biotek).

Solid-phase radioimmunoassay (SPRIA ) SPRIA were performed essentially as described for the ELISA, with the following modifications. The second antibody step was eliminated for mouse mAbs, and for rat antibodies was substituted with rabbit-anti-rat IgG (absorbed on human IgG, Sigma). Following incubation with the second antibody, the plates were washed three times with PBS/Tween. [ 125I]Protein A was added to each well (105 cpm in 75/~1 1% BSA in PBS) for 30 rain at room temperature and the plates were washed three times with PBS/Tween, followed by addition of 200 /xl of 2% SDS. The solution was removed and counted in a y counter.

118

Inhibition of immunoprecipitation of [~25I]aBgtlabelled native protein by synthetic peptides mAbs were preincubated with peptides (at 1 mg/ml or 0.2 mg/ml) for 2 h at room temperature using 0.4 nM mAb to the a7 subunit and 0.15 nM mAb to the a8 subunit in 100 mM NaCI, 10 mM NAN3, 10 mM Na phosphate, pH 7.5. A 2% Triton X-100 extract of embryonic day 18 chicken brain in the same buffer and containing 2 nM in aBgt-binding sites (approx. 2 nM in sites on binding proteins containing the a7 subunit and approx. 0.6 nM on proteins containing the a8 subunit) was preincubated with [xzsI]aBgt (80 nM) for 2 h at 4°C. Then 50/~1 of mAb plus or minus peptides were mixed in triplicate with 50 /xl of [125I]aBgt-labelled brain extract and 4 ~1 of normal rat serum (except for the mouse mAbs 306 and 307, which used carrier serum). After overnight at 4°C, 100/~1 of a dilution of goat-anti-rat IgG was added for 2 h at 4°C (except for the mouse mAbs 306 and 307, which used 15 ~1 of fixed Staphlococcal Protein A (Pansorbin, Bio Rad)). After adding 1 ml of 0.5% Triton X-100 buffer, the samples were centrifuged for 2 min in a microcentrifuge. The supernate was aspirated, and 1 ml of 0.5% Triton X-100 buffer was added. Centrifugation and washing were repeated again

before [125I]aBgt in the pellet was determined by y-counting.

Prediction of antigenic determinants A prediction of the antigenic determinants of the complete a7 subunit was made for comparison with those sites determined experimentally using the P C / G E N E program ANTIGEN, which is based on the method of Hopp and Woods (1981). This predictive model uses the hydrophilicity as an index of antigenicity and calculates the average hydrophilicity of sequence segments of six amino acids.

Results

Rationale We have previously demonstrated using subunit-specific antibodies that the a7 subunit is contained in > 90% of the aBgt-binding complexes in chick brain extracts, whereas the a8 subunit is contained in < 15% of these sites (Schoepfer et al., 1990). The a7 and a8 subunits are relatively homologous (approx. 62% identity), with the most divergent sequences contained in a large putative cytoplasmic domain between

TABLE 1 MONOCLONAL AND POLYCLONAL ANTIBODIES USED IN THIS STUDY Name

Type a

Immunogen

Species

Isotype b

EAMG 150 EAMG 151 mAb 305 mAb 306 mAb 307 EAMG 157 mAb 308 mAb 309 mAb 310 mAb 311 mAb 312 EAMG 163 mAb 318 mAb 319 mAb 320

pAb pAb mAb mAb mAb pAb mAb mAb mAb mAb mAb pAb mAb mAb mAb

Purified aBgtBPs Purified otBgtBPs Purified aBgtBPs Purified aBgtBPs Purified aBgtBPs protCh31-5 a8(293-435) protCh31-5 a8(293-435) protCh31-5 a8(293-435) protCh31-5 a8(293-435) protCh31-5 a8(293-435) protCh31-5 a8(293-435) protCh34-2 a7(327-412) protCh34-2 a7(327-412) protCh34-2 a7(327-412) protCh34-2 a7(327-412)

Rat Rat Rat Mouse Mouse Rat Rat Rat Rat Rat Rat Rat Rat Rat Rat

ND ND IgG IgG IgG ND IgG IgG IgG IgG IgG ND IgG IgG IgG

pAb, polyclonal antibodies; mAb, monoclonal antibody. b ND, not determined. a

2C 1 1 2B

2B 2A

119

residues 329-414, which is virtually unique to each a subunit subtype of the AChR supergene family (Schoepfer et al., 1990; Boulter et al., 1990). In the present study, synthetic peptides corresponding to the complete a7 subunit and the non-homologous cytoplasmic domain of the a8 subunit are used to: (i) identify the continuous, or sequential epitopes of the a7 and a8 subunits recognized in the polyclonal antibody response to affinity-purified aBgtBPs and recombinant fusion proteins, and (ii) define the epitope specificities of mAbs raised against these different aBgtBP preparations. The methods used in the construction of the fusion proteins, the immunization protocols and the properties of the antibody preparations used in the present studies have been described previously (Schoepfer et al., 1990). A summary of the antibodies used in the present paper is given in Table 1. Synthetic peptides used to map the epitopes are given in Figs. 1 and 2. Recognition of peptides by different antibodies was determined using solid-phase radioimmuno assays (SPRIA), and enzyme-linked immunosorbent assays (ELISA) (Conti-Tronconi et al., 1990, 1991), as described in Materials and Methods. Both types of assays were performed (3-4 times) for each antibody preparation, and the overall results obtained using both detection systems were virtually identical. For some antibodies of low titer, however, the ELISA method yielded better signal/noise ratio. Results using both types of assay detection systems are reported here, and the results are directly comparable.

Epitope mapping of the antibodies against affinitypurified otBgtBPs Antibodies directed towards affinity-purified native and denatured chick brain aBgtBPs were tested using overlapping peptides corresponding to the complete a7 subunit and the putative cytoplasmic region of the a8 subunit (depicted in Figs. 1 and 2). Polyclonal antisera from two different rats, designated EAMG 150 and EAMG 151, yielded similar recognition patterns using both ELISA and SPRIA assays. A typical experiment (n = 4) as shown in Fig. 3B indicated that continuous epitopes were contained within several sequence regions of the a7 subunit - a7(1-

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20), a7(61-90), a7(106-155), a7(185-219), a7(245-264), and a7(290-420). The most prominent epitopes were contained within the N-terminal putative extracellular segment a7(136-155) and the sequence region within a7(305-400), which forms a cytoplasmic domain in the Torpedo electroplax and vertebrate muscle nAChRs (Ratnam et al., 1986a, b), and that is highly

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Fig. 2. Synthetic peptides used to m a p epitopes contained within sequence segments of the a 7 and a 8 subunits of a large putative cytoplasmic domain. The d e d u c e d amino acid sequences of a 7 and a 8 subunits between residues 293-435 (Schoepfer et al., 1990) and synthetic peptides used in this study corresponding to sequence segments of this large putative cytoplasmic domain are indicated. Regions of homology are indicated by shading. The peptides were synthesized and characterized as described in Materials and Methods. Fusion proteins were produced corresponding to the a7(327-412) and a8(293-435) as described in Schoepfer et al. (1990).

divergent in sequence between the corresponding subunits of different species and nAChRs of various subtypes (Luther et al., 1989; Schoepfer et at., 1990). For sake of comparison, the regions of antigenicity based on the the method of Hopp and Woods is included in Fig. 3A. The analysis is predicted based on the hydrophilicity profile at intervals of six amino acid residues. It is apparent from comparison of the real and theoretical epitope sequences that hydrophilicity is not a good predictive index for the pattern of epitopes in this sequence. Peptides corresponding to sequence segments of the a8 subunit between amino acid residues 293-435 (shown in Fig. 2) were also tested for recognition by polyclonal rat antisera, EAMG 150 and EAMG 151. Peptides a8(293312), a8(308-327), and a8(323-342), which share amino acid sequence homology with the a7 subunit, were found to react with EAMG 150 and EAMG 151 (data not shown). The mouse mAbs 306 and 307, raised against affinity-purified aBgtBPs from chick and rat

brains, were previously shown to bind native chick aBgtBPs containing the o~7 subunit, but did not react detectably with the low amounts of protein available on Western blots of purified aBgtBP (Schoepfer et al., 1990). However, both mAbs could be shown to recognize sequential epitopes of the a7 subunit in the sensitive solid phase assays used in the present study which permitted reaction with synthetic peptides at relatively high concentrations. As shown in Fig. 3C, both mAbs were found to recognize similar or identical epitopes within the sequence a7(380-400). Peptides a8(368-387) and ol8(383-401), corresponding to the same region of the a8 subunit, were not recognized by mAbs 306 and 307, indicating that their inability to cross-react with the a7 subunit is due to divergence between the a7 and a8 subunits in this sequence (See Fig. 2). mAb 305 was derived from the rat immunized with affinity-purified aBgtBPs, which produced the antiserum EAMG 150 used in Fig. 3B. mAb 305 did not recognize aBgtBPs on Western blots and also did not recognize peptides corresponding to either the a7 or a8 subunits. Previous immunoprecipitation studies indicated that mAb 305 is directed against a minor aBgtBP subtype containing a8 (Schoepfer et al., 1990). The failure to detect binding of mAb 305 to synthetic peptides corresponding to a8 suggests that its epitope is strictly conformation-dependent, either because it involves several non-sequential a8 sequences or because it involves sequences from both the a8 and another subunit. However, because the complete sequence of a8 was not tested, it is possible that mAb 305, like mAb 306 or mAb 307, did not bind to the low concentrations of denatured aBgtBP present on Western blots but would have bound detectably to an appropriate a8 sequence if it had been presented at high concentration in a solid phase assay.

Epitope mapping of antibodies against a7 and a8 fusion proteins In order to generate subunit-specific antibodies for the a7 and a8 subunits, fusion proteins were constructed corresponding to a7(327-412) and o~8(293-435), designated protCh34-2 and protCh31-6, respectively (Schoepfer et al., 1990). Comparison of the polyclonal response of rats

121

immunized with either the a7 or a8 fusion proteins also provided an opportunity to compare the antigenicity of the cytoplasmic sequences of the a7 and a8 subunits directly. Because the a8 subunit represents < 15% of the total aBgtBPs of the chick brain, epitopes identified by antisera directed against affinity-purified aBgtBPs from chick brain (as reported above) may underestimate the antigenicity of unique a8 sequences as a result of the low levels contained in the immunogen. The fusion protein sequences and the synthetic peptides used to map the epitopes within this region are indicated in Fig. 2. The epitopes recognized by rat polyclonal antiserum, EAMG 157, raised against protCh31-5 containing a8(293-435) are shown in Fig. 4. Five of the overlapping peptides corresponding to the

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