Journal of Neuroimmunology, 36 (1992) 13-27 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-5728/92/$05.00
13
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 Lindstrom et al., 1987; Claudio, 1989; Stroud et al., 1990). The A C h R 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 function, immunochemical properties and pharmacology (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 difficult 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. Comparison 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 consistent 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
I
5 i0-
mab #145
5Z
~
0?IOz m~b #154
mab #165 5~ 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 388407, 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-
°1 i1
~J5 o?, 15
~1o
e~ r...) TORPEDO &SUBUNITPEPTIDES
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 346425 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, T8376395. The S peptide sequences recognized by mAb 7 correspond 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, HYLFDTYDPGRRPVQEPEDT; 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, D F C P G V I R RHDGDSAGGPGE; 3,186-205, RHRPAKMLLDEAAPAEEAGH; 8198-213, VNVDPSVPLDSPNRQD; 3,386-405, LVRAALEKLEKGPESGQSPE; e379-398, R H R H G T W T A T L CQNLGAAAP; 8380-397, EKQSERHGLARRLTTARR. 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 (Ultrasphere 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 antipeptide 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 (Nelson 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 assessed by dot blot assay (Nelson and ContiTronconi, 1990). Purified antibodies, at 1-10
TABLE 1 M O N O C L O N A L A N T I B O D I E S U S E D IN THIS S T U D Y mAb
7 132 145 154 162 165 168
Immunogen
TAChR TAChR TACbR TAChR TAChR TAChR TAChR
a
(nat.) (den.) (den.) (den.) (den.) (den.) (den.)
Ig subclass b
ND IgGl IgG2a IgG1 IgM Ig2A IgG1
Subunit specificity c
3', 6 3' y y y y 7
Titer (nM) against A C h R s d Torpedo electric tissue
Bovine muscle Fetal
Adult
18,200 30,000 30,000 13,000 1,800 30,300 28,000
6.22 0 0.6 0 0 1.13 0
3.43 ND 0 170 ND 1.23 750
T A C h R indicates purified Torpedo A C h R , native (nat.) or denatured in SDS (den.). B A C h R indicates purified embryonic bovine AChR. b From Tzartos et al., 1986. c From Tzartos and Lindstrom (1980) for m A b 7, from Tzartos et al. (1986) for the other mAbs. d T h e titers of m A b s 132 and 162 are from Tzartos et al. (1986), the other titers were determined in this study. ND, not determined. a
17 / z g / m l 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 immobilization 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 - B T X (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, 2 5 / z g / m l
Affinity Purified Antibodies to Peptides
'MM
A
-
13
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 Lindstrom et al., 1987; Claudio, 1989; Stroud et al., 1990). The A C h R 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 function, immunochemical properties and pharmacology (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 difficult 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. Comparison 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 consistent 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
I
5 i0-
mab #145
5Z
~
0?IOz m~b #154
mab #165 5~ 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 388407, 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-
°1 i1
~J5 o?, 15
~1o
e~ r...) TORPEDO &SUBUNITPEPTIDES
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 346425 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, T8376395. The S peptide sequences recognized by mAb 7 correspond 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, HYLFDTYDPGRRPVQEPEDT; 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, D F C P G V I R RHDGDSAGGPGE; 3,186-205, RHRPAKMLLDEAAPAEEAGH; 8198-213, VNVDPSVPLDSPNRQD; 3,386-405, LVRAALEKLEKGPESGQSPE; e379-398, R H R H G T W T A T L CQNLGAAAP; 8380-397, EKQSERHGLARRLTTARR. 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 (Ultrasphere 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 antipeptide 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 (Nelson 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 assessed by dot blot assay (Nelson and ContiTronconi, 1990). Purified antibodies, at 1-10
TABLE 1 M O N O C L O N A L A N T I B O D I E S U S E D IN THIS S T U D Y mAb
7 132 145 154 162 165 168
Immunogen
TAChR TAChR TACbR TAChR TAChR TAChR TAChR
a
(nat.) (den.) (den.) (den.) (den.) (den.) (den.)
Ig subclass b
ND IgGl IgG2a IgG1 IgM Ig2A IgG1
Subunit specificity c
3', 6 3' y y y y 7
Titer (nM) against A C h R s d Torpedo electric tissue
Bovine muscle Fetal
Adult
18,200 30,000 30,000 13,000 1,800 30,300 28,000
6.22 0 0.6 0 0 1.13 0
3.43 ND 0 170 ND 1.23 750
T A C h R indicates purified Torpedo A C h R , native (nat.) or denatured in SDS (den.). B A C h R indicates purified embryonic bovine AChR. b From Tzartos et al., 1986. c From Tzartos and Lindstrom (1980) for m A b 7, from Tzartos et al. (1986) for the other mAbs. d T h e titers of m A b s 132 and 162 are from Tzartos et al. (1986), the other titers were determined in this study. ND, not determined. a
17 / z g / m l 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 immobilization 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 - B T X (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, 2 5 / z g / m l
Affinity Purified Antibodies to Peptides
'MM
A
-
