Monoclonal Antibodies FK1 and WF6 Define Two Neighboring

Tm JOURNAL OF BCOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistly and Molecular Biology, Inc

Vol. 269, No. 14, Issue of April 8, pp. 10407-10416,1994 Printed in U S A .

Monoclonal Antibodies FK1 and WF6 Define Two Neighboring Ligand Binding Sites on Torpedo Acetylcholine Receptor a-Polypeptide”

(Received for publication, October 15, 1993, and in revised form, December 17, 1993)

Bernd SchroderS, Sigrid Reinhardt-MaelickeS, Andre SchrattenholzS, Kathryn E. McLane9, Axel Kretschmea Bianca M. Conti-Tronconis, and Alfred MaelickeSII From the $Caboratory of Molecular Neurobiology, Institute of Physiological Chemistry and Pathobiochemistry, Johannes-Gutenberg University Medical School, Duesbergweg 6, 55099 Maim, Germany, the §Department of Biochemistry, College of Biological Sciences, University of Minnesota, St. Paul, Minnesota 55108, and the Ventrale Forschung, Buyer AG, 51375 Leuerkusen, Germany

Previous studies have identified the sequence region flanking the invariant vicinal cysteinyl residues at positions 192 and 193 of the nicotinic acetylcholine receptor a-subunit as containing major elements of the binding site for acetylcholine and its agonists and antagonists, including antibody WF6 (Conti-Tronconi, B. M., Diethelm, B. M., Wu, X, Tang, F., Bertazzon, T., Schriider, B., Reinhardt-Maelicke, A, and Maelicke, A. (1991) Biochemistry 30, 2575-2584). Recently we have shown that the sequence region flanking lysine a125 contains elements of the binding site for physostigmine and related ligands, including antibody FK1 (Schratten- holz, A, Godovac-Zimmerman, J., Schiifer, HA, Albu- querque, E. X, and Maelicke, A. (1993) Eur. J. Biochem. 216, 671-677). Here we report the identification by enzyme-linked immunosorbent assay techniques, employing fragments of the lbrpedo nicotinic acetylcholine receptor a-subunit N-terminal region and a panel of synthetic peptides matching in sequence preselected portions of this subunit, of the sequence regions a118- 145 and al8l-216 as contributing to the FK1 epitope. Of the synthetic peptides employed, a118-137 displayed the highest affinity of FK1 binding. Binding of FK1 and WF6 to single residue-substituted analogs of the sequence al8l-200 indicated that the two antibodies have different attachment point patterns within this sequence region. These results, and those of ligand competition studies, suggest that the binding sites for FK1 and physostigmine, and those of WF6 and acetylcholine, are within the same general region of the receptor’s three- dimensional structure. The sites neighbor each other, with limited overlap in the case of occupation by high molecular weight ligands.

Nicotinic acetylcholine receptors (nAChR)’ of peripheral tis-

(Ma 599/12-3), the Fonds der Chemischen Industrie (to A. M.), Com- * This work was supported by the Deutsche Forschungsgemeinschaft

mission of the European Communities, Human Capital and Mobility Programme Grant ERBCHRXCT 930167 (to A. M.), and United States National Institute on Drug Abuse Program Project Grant 5P01- DA05695 (to B. C.-T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

I( To whom all correspondence should be addressed. Tel.: 49-6131-395-

The abbreviations used are: nAChFt, nicotinic acetylcholine receptor(s); ACh, acetylcholine; Phy, (-)physostigmine (eserine); HPLC, high performance liquid chromatography; TMF, Torpedo membrane fragments; ELISA, enzyme-linked immunosorbent assay; PAGE, polyacryl- amide gel electrophoresis; PBS, phosphate-buffered saline.

911; Fax: 49-6131-393-536.

sues, e.g. skeletal muscle and fish electrocytes, are pseudosym- metric pentameric complexes of homologous polypeptides with the stoichiometry a2/3y8 (Conti-Tronconi et al., 1982). Whereas all five subunits have been shown to participate in the formation of the integral cation channel (Sumikawa et al., 1981; Imoto et al., 1986; Giraudat et al., 1986,1987,1989; Oberthuer et al., 1986; Oberthuer and Hucho, 1988; Revah et al., 1990), the major constituents of the acetylcholine (ACh) binding site are within the a-polypeptide. A large body of studies including covalent affinity labeling (Kao et al., 1984; Pedersen et al., 1986, 1990; Langenbuch-Cachat et al., 1988; Dennis et al., 1986, 1988; Abramson et al., 1989), binding of snake a-neuro- toxins and ACh-competitive antibodies to synthetic peptides (Ralston et al., 1987; Wilson and Lentz, 1988; Conti-Tronconi et al., 1990, 1991; Griesman et al., 1990; McLane et al., 1992) and to a-subunit fragments expressed in Escherichia coli transfor- mants (Gershoni, 1987; Aronheim et al., 1988; Ohana et al., 1990,1991), and site-directed mutagenesis of single amino acid residues (Mishina et al., 1986; Tomaselli et al., 1991) point to a region surrounding and including cysteines 192 and 193 as potential site of ligand interaction. In addition to this major cholinergic subsite, two more sequence regions within the a-subunit were identified by epitope mapping studies as par- ticipating in ligand binding, i.e. a region close to the single N-glycosylation site and a region preceding and partially overlapping with the main immunogenic region (Conti-Tronconi et al., 1990; Wahlsten et al., 1993). Photoaffinity labeling studies (Dennis et al., 1988; Galzi et al., 1990) also suggest a model of the cholinergic binding region composed of at least three distinct and discontinuous segments within the N-terminal sequence region of the a-polypeptide.

Recent electrophysiological and biochemical studies have identified a novel class of nAChR ligands, of which (-)-physostigmine (Phy) and galanthamine are the prototypes (Schrat- tenholz et al., 1993a, 1993b; Pereira et al., 1993a, 1993b; Maelicke et al., 1993; Dunn and Raftery, 1993). Physostigmine activates single channel currents in skeletal muscle cells and reconstituted Torpedo giant vesicles and also in preparations containing mammalian central nervous system nAChR, whose characteristics resemble those evoked by ACh (Shaw et al., 1985; Maelicke et al., 1993; Pereira et al., 1993b). Channel activation by Phy is insensitive to typical cholinergic antagonists, such as a-bungarotoxin and monoclonal antibody WF6 (peripheral nAChR) or methyllicaconitine (central nervous system nAChR), but is antagonized in all of these systems by monoclonal antibody FK1 (Maelicke et al., 1993; Pereira et al., 1993b). Covalent labeling studies employing [benzene ring3H]- (-)-physostigmine as photoactivatable ligand indicated that residue aLys-125 is within, or in close vicinity of, the Phy bind-

10407

10408 FKl Binding to

ing site (Schrattenholz et al., 1993b). The availability of the two monoclonal antibodies WF6 and

FK1, which selectively antagonize channel activation by ACh and Phy (Fels et al., 1986; Pereira et al., 1993a, 1993b), respectively, prompted us to employ them in epitope mapping studies using three different approaches: (i) binding to immobilized nAChR in the presence and absence of the other antibody, which demonstrated limited overlap of the epitopes of FK1 and WF6; (ii) binding to fragments of the nAChR a-subunit (by Western blotting), which showed that the region around aLys- 125 is required for FK1 binding but does not suffice for WF6 binding; and (iii) binding to synthetic peptides matching in their sequences selected portions of the Torpedo nAChR a-subunit primary structure, which identified the regions a118-145 and a181-216 as contributing to the FK1 epitope. Using single residue-substituted synthetic analogs of the peptide a181-200 (Conti-Tronconi et al., 1991), the patterns of attachment points of WF6 and FK1 were found to be different. Taken together, these results indicate that the binding sites for WF6 and FK1, and by inference also those for ACh and Phy, reside in close proximity to each other within the same “receptive region” of the N-terminal extracellular domain of the nAChR a-polypeptide. The sites are distinct and therefore permit ligand-specific recognition and response.

EXPERIMENTAL PROCEDURES Peptide Synthesis and Characterization-Peptides, 19-21 amino ac-

ids long, were synthesized according to Houghten (1985). The purity of the peptides was assessed by reverse phase HPLC using a C18 column (Ultrasphere ODs) and an acetonitrile/water gradient (5-70%) containing 0.1% trifluoroacetic acid. A major peak was consistently present, accounting for 6545% of the total absorbance a t 214 nm. The amino acid composition of all peptides, determined by derivatization of amino acid residues released after acid hydrolysis with phenylisothiocyanate, followed by separation on a reverse phase HPLC column (PIC0.TAG) as described by Heinrickson and Meredith (1984), yielded in each case a good correspondence between experimental and theoretical values. The sequence and purity of peptides corresponding to sequence a181-200 were verified by gas phase sequencing (Applied Biosystems), and only the expected sequences were found. Contaminating sequences, which would be expected from truncated peptides randomly missing amino acids from incomplete coupling, totaled 5-15%, and the contribution of each single sequence was below the level of detectability. Because we have demonstrated previously (Conti-Tronconi et al., 1990, 1991) that this level of purity is fully satisfactory for epitope mapping experiments, the peptides were not purified further. The sequences and codes of the peptides were reported previously (Conti-Tronconi et al., 1991).

Antibodies WF6and FKl-The production and properties of antibody WF6 were described previously (Watters and Maelicke, 1983; Fels et al., 1986; Conti-Tronconi et al., 1991). It was purified from supernatants of mouse hybridoma cells raised in IgG-free medium by chromatography on protein A-Sepharose 4B (Pharmacia LKB Biotechnology Inc.) of the desalted and redissolved pellet. The concentration of WF6 was determined by W absorption at 280 nm, 1 mg/ml = 1.4 A,.

Monoclonal antibody FK1 was raised by Dr. Gregor Fels from our laboratory against membrane preparations from hindleg muscles of newborn rats. Screening of growing hybridomas was performed with purified Torpedo nAChR. The monoclonal antibody used in this study was produced by hybridoma subclone 2E2, it is an IgM ( h e r s h a m isotyping kit). Because the antibody denatures a t low protein content of the medium, all studies reported here were performed with unpurified mouse hybridoma supernatant. Attempts to develop a concentration/ purification procedure maintaining the binding specificity of the antibody were unsuccessful.

Monoclonal antibody PK1 was raised by Dr. Rita Pliimer from our laboratory against the synthetic peptide 01127-132 conjugated to bovine serum albumin by glutaraldehyde (Plumer et al., 1984). The antibody studied here was produced by hybridoma subclone BNIC5, it is an IgM and, similar to FK1, it is very sensitive to low protein content of the medium. PK1 binds to membrane-bound and purified nAChR, inde- pendent of whether these preparations are native or denatured. Bind- ing of PK1 to Torpedo membrane fragments (TMF) is not affected by a-cobratoxin, carbamoylcholine, u-tubocurarine, and decamethonium (Plumer et al., 1984). Because of its lower affinity of binding to Torpedo

Torpedo nAChR

1 2 3 4 5 6 i

6 - 0 - 67 kD Y - - n - -- -52kD

.“ FIG. 1. Location of the binding sites for antibodies F K l and

WF6 on the nAChR from T. murmorata. Shown is an immunoblot of TMF and of two fragments of I: californica nAChR a-subunit (al-246 and al-approximately 170), incubated with antibodies FK1 and WF6. The second antibody was a horseradish peroxidase-labeled anti-mouse Ig; antibody binding was visualized by enhanced chemiluminescence (see “Experimental Procedures” for details). Lune 1, Coomassie Blue- stained SDS-PAGE of the TMF preparation; lane 2, binding of WF6 to TMF; lane 3, binding of WF6 to fusion proteins; lane 4, binding of FKl to TMF; lane 5, binding of FKl to fusion proteins; lane 6, Coomassie Blue-stained SDS-PAGE of purified E. coli extract containing the two fusion proteins. From these data, antibody WF6 selectively binds to the a-polypeptide and only the longer one of the two fusion proteins, FK1 binds to the a-polypeptide and to both fusion proteins, suggesting that the main prototope of WF6 resides downstream from that of FK1, within the sequence region a170-246.

nAChR, as compared with FK1, PK1 was not studied in further detail. In the present study it is applied only in competition ELISAs aimed at testing for overlapping antigenic sites.

nAChR-rich Membrane Fragments-TMF were prepared from l’br- ped0 marmorata electric tissue according to Duguid and Raftery (1980), with the modifications described by Reinhardt et al. (1984). The nAChFt concentration of the membrane suspension was approximately 10 PM in terms of a-bungarotoxin binding sites, at a protein concentration of 3 mg/ml .

Fragments of Torpedo a-Polypeptide N-terminal Sequence-A cDNA encoding amino acids 1-246 of nAChR a-polypeptide from Torpedo californica was inserted by Dr. A. Kretschmer of Bayer AG Leverkusen into pMAL vector (Guan et al., 1987) downstream from the malE gene which encodes maltose-binding protein. From DNA sequencing of the construct, the inserted nAChR sequence was identical to that published by Noda et al. (1982), with the C-terminal sequence of the fusion protein expected to be Lys-Met-Thre-Leu-Ser, terminated by TGA TAA in the expression construct. The fusion protein was expressed in E. coli and was purified by affinity chromatography (Kellerman and Ferenci, 1982) following the protocol of the kit supplier (New England Biolabs). SDS- PAGE yielded two major protein bands (67 and 52 kDa), in addition to small amounts of various E. coli proteins that bind to the amylose column. The two major protein bands were isolated by preparative SDS-PAGE (Schrattenholz et al., 1993b) and were proteolytically cleaved by human serum protease factor Xa. Protein sequencing by Edman degradation of the affinity-purified nAChR fragments showed that their primary structures began with the sequence Ile-Ser-Glu-Phe (from the EcoRI cloning site), followed by the N-terminal sequence of Torpedo a-polypeptide (Noda et al., 1982). The proteins therefore con- stitute fragments of the N-terminal extracellular region of T californica nAChR a-subunit, with the longer one containing the first 246 amino acid residues and the shorter one containing approximately 60-70 fewer residues.

Zmmunoblottiw-TMF (50 pl) were solubilized in 2% SDS, 2% 2-mercaptoethanol, 10% glycerol, 62.5 m Tris-HCI, pH 6.8, and were submitted to SDS-PAGE using discontinuous slab gels according to Laemmli (1970). One slot each of protein standards and TMF was stained with Coomassie Blue, and the remaining slots were blotted onto Trans-Blot nitrocellulose membranes (Bio-Rad) using a Pegasus semi- dry blot apparatus (Phase). Again, one slot each of protein standards and membrane fragments was stained for comparison. The unreacted sites of the other slots were blocked by a 15-min incubation at room

FK1 Binding to Torpedo d C h R 10409

100

80

- E , P

3 0 n

20

0

- 20 [ m m phyostigmine ~

* 100 [mMl acetylcholine

without ligand

- 50 [mM] physostigmine

1 2 3 4 S 6 7 8 9 10 11

reciprocal antibody dilution[-log 2 )

. ~

- 20 [ m m phyostigmine

+ 1 [mm acetylcholine

-X- without ligaod ~. . ~~ ~J

I 2 3 4 5 6 7 8 9 10 11

reciprocal antibody dilution [-log 21

FIG. 2. Ligand competition ELISA with TMF and antibodies F K 1 and WF6. The competition between antibodies FK1 and WF6, respectively, and low molecular weight ligands (ACh, Phy) for binding to immobilized TMF was measured by ELISA under standardized conditions. For

m~ Phy; 0, 100 m~ ACh. Panel B, competition ELISA with WF6: x, no ligand; A, 20 m~ Phy; m, 100 m~ Phy; 0,l m~ ACh. With the reservation experimental details, see “Experimental Procedures.” Panel A, competition ELISA with FK1: x, no ligand; A, 20 m~ Phy; + , 50 m~ Phy; m, 100

that this is a very insensitive assay, there is little, if any, competition of ACh with FK1, and of Phy with WF6, for binding to membrane-bound Zbrpedo nAChR, suggesting that the ACh and FK1 binding sites and the Phy and WF6 binding sites do not overlap.

temperature with defatted milk powder (3% w/v) in 150 m~ NaCl, 10 m~ Tris-HC1, pH 7.4 (TBS), followed by bovine serum albumin (3% w/v), in TBS. The blot was then washed twice with TBS, followed by incubation for 1 h at room temperature with WF6 (5 pg/ml) and FK1 (hybridoma supernatant, diluted 10-fold), respectively, in 150 m~ NaCl, 0.5 or 3% Tween 20 (the higher concentration only in the case of FKl), 10 m~ Tris-HC1, pH 7.4 (TBST), and washed (three times for 10 min each with TBST). After incubation for 1 h at room temperature with the second antibody (horse radish peroxidase-linked rabbit anti-mouse Ig, diluted 1:200 in 1% w/v bovine serum albumin in TBST) and three washes (as above), the enhanced chemiluminescence detection kit (ECL, Amer- sham Corp.) was applied according to the procedure given by the supplier.

Enzyme-linked Zmmunosorbent Assays-ELISAs were performed as described previously (Conti-Tronconi et al., 1991) except that the coating and reaction volumes and the incubation times were changed to those described below.

Antcgeen Inhibition ELZSA-In this assay the competition between immobilized TMF and dissolved synthetic peptide for binding to the antibody under study is tested under standardized conditions (Catty and Raykundalia, 1989). Briefly, first the binding capacity of the wells for TMF was determined using ELISA and WF6 as primary antibody. Once the optimal TMF coating concentration was known, we determined the respective dilutions of WF6 and FK1 at which, under stan-

dardized conditions of incubation, the ELISA produced an extinction reading of 1.0 A4w. Using the TMF coating and antibody concentrations determined in this way, the optimal concentration ranges for competition studies were obtained by competition ELISA for each individual synthetic peptide.

Antigen inhibition ELISAs were performed in flexible microtiter plates (Falcon). 30 pl of coating solution (TMF, diluted 1:1,000 in 0.1 M NaHCO,, pH 9.0) was added to each well, and coating was achieved by overnight incubation at 37 “C in a water-saturated atmosphere. In par- allel, the reaction mixtures consisting of serial dilutions of a synthetic peptide and a constant concentration of antibody were prepared and incubated under the same conditions. From the preceding standardiz- ing experiments, WF6 was applied as 1,600-fold diluted stock solution (600 ng of IgG/ml), and FK1 as 128-fold diluted hybridoma supernatant (80-240 ng of IgM/ml). In control incubations, the synthetic peptide solution was replaced by buffer (0% inhibition), or the antibody solution was replaced by a bovine serum albumin solution of identical protein concentration (100% inhibition). The following day, the coating solution was removed from the wells, 180 pl of 1% gelatin in phosphate-buffered saline (PBS) was added to each well (to mask remaining coating sites in the wells) and, after a 1-h incubation at 37 “C, the wells were washed three times with 200 pl of PBS each. Next, 30 pl of peptide-antibody reaction mixture was added to each well, according to an exact time protocol, and after an incubation of 1 h at 37 “C, the reaction solution

FK1 Binding to Torpedo nAChR 10411

i 10

0 00 0.05 0 10 0.15 0 20 0.21 concentrat la" o f peptlde [uM]

concentrat ion of peptide [uM]

0

100 /- B o

0

concentratlon of peptlde [uM]

0 0 25 50 75 100 125 150

concentratlon of peptide [uM]

100- C

0 - Y x

0 1 2 3 4 5 6 7 concentratlon o f peptide [uM]

100 ,9 D

0

i I concentration o f peptide [uM]

i 5 0

FIG. 3. Inhibition binding isotherms of the interaction of antibodies Fgl and ~6 with their mdor prototopes on nAChR from

by standardized ELISA (for details see "Experimental Procedures"). Inhibition binding data were fitted by rectangular hyperbolas employing the Ibrpedo. The competition between varying concentrations of dissolved synthetic peptide and immobilized TMF for binding of antibody was studied

PC program GraphPAD InPlot tm. Two ranges of the binding curves are shown, with the insets representing enlargements of the initial parts. Binding of antibody FK1 to peptides (~118-137 (panel A) and a184-203 (panel E ) is shown. Ki(u118-137) = (1.68 + 0.26).104 M; Ki(n184-203) =

M; Ki(a18P2O3) = (3.89 + 0.60).104 M. (5.08 + 1.49).10-' M. Binding of antibody WF6 to peptides (~118-137 (panel C ) and a184-203 (panel D ) is shown. K,(a118-137) = (4.66 + 1.47)*10"

nAChR a-subunit. In contrast to WF6, however, FK1 also recognized a fusion protein approximately 70 residues shorter (Fig. 1). These data suggest that important constituents of the epitope of FK1 reside significantly closer to the N terminus than the major prototope of WF6, which is known to flank the vicinal cysteines a192, a193 (Conti-Tronconi et al., 1990). Since the two fusion proteins contain rather large fragments of the nAChR a-subunit, they are better models of the receptor's three-dimensional structure than the much shorter synthetic peptides employed in epitope mapping (see below). Their selec- tivity in regard to the binding of FK1 and WF6 therefore supports the notion that the epitopes of the two antibodies are distinct and locally separated from each other.

Competition of FKl and WF6 with Acetylcholine and Physo- stigmine for nAChR Binding-Previous studies have indicated that FK1 blocks the binding of Phy, but not of ACh, to Torpedo nAChR, suggesting that these two channel-activating ligands may bind to separate sites at the receptor (Maelicke et al., 1993; Pereira et al., 1993b; Schrattenholz et al., 1993a, 1993b). The present results of ligand competition ELISAs further support this conclusion. As shown in Fig. 2, coincubation with 100 lll~ ACh did not reduce the binding of FK1 to immobilized TMF under the experimental conditions applied, whereas 50 mM Phy reduced FK1 binding by 50% or more. In contrast, binding of WF6 to immobilized TMF was unaffected by coincubation with 100 l ~ l ~ Phy and was strongly inhibited by 1 l ~ l ~ ACh (Fig. 2). The immunoassays clearly demonstrate preferential inhibition of FK1 binding by Phy and of WF6 binding by ACh, consistent with separate locations of the epitopes for FK1 and WF6, as was suggested by Western blots with fusion proteins (Fig. 1).

Competition between FKl and WF6 for Antigenic Sites-"0 obtain a rough estimate of the topography of the epitopes for FK1 and WF6 on Torpedo nAChR, we tested whether binding of

FK1 to the nAChR is affected by WF6, or vice versa. Antibody PK1 was included in our study because it inhibits binding of Phy to Zbrpedo nAChR in a fashion similar to FK1 (Okonjo et al., 1991) but does not compete with acetylcholine, tubocurarine, or a-bungarotoxin for receptor binding (Pliimer et al., 1984). PK1 was raised against the synthetic peptide Zbrpedo a127-132, conjugated to bovine serum albumin (Plumer et al., 1984). As shown in Table I, the average addition index for the pair FKl/PKl was 30.4%, consistent with binding to identical or strongly overlapping antigenic sites. In contrast, the addition index of 73.2% for the pair PKl/WF6 suggests nonoverlap- ping epitopes. For the pair F K m 6 an addition index of 52% was found, consistent with limited overlap of their epitopes. These data suggest that all three antibodies bind to distinct locations within the same general area of the extracellular region of the nAChR a-subunit.

Identification of Sequence Segments Recognized by FKl and WF6-Using a panel of synthetic peptides as representative structural elements for the Torpedo nAChR a-polypeptide, we have previously identified sequence regions containing subsites for the binding of WF6 (Conti-Tronconi et al., 1990; Maelicke et al., 1991; McLane et al., 1992). As the most reliable of the assays employed, antibody and synthetic peptide were prein- cubated, and the mixture was applied to immobilized TMF, thereby measuring the inhibition by peptide of antibody binding to nAChFt (Conti-Tronconi et al., 1991). Under these conditions, the interaction of antibody and peptide occurs in solution, excluding artifacts caused by immobilization of a reaction part- ner. In the present study, this assay was improved further by determining the actual concentration of synthetic peptide in solution, thereby accounting for their different solubilities. Furthermore, we studied in each case the concentration depen- dence of the inhibition, which allowed us to determine apparent

10412 FK1 Binding to Torpedo nAChR TASLE I1

Apparent inhibition constants K, of synthetic peptides interfering with the binding of antibodies FKl and WF6 to TMF The competition between immobilized TMF and dissolved synthetic peptide for binding of the respective antibody was measured under

standardized conditions (for details see “Experimental Procedures”). Apparent inhibition constants K, were obtained from least squares fits of the inhibition binding curves for each synthetic peptide. Four sets of experiments were performed, yielding essentially the same results. The assay is limited to the determination of K, values below 50 p ~ , the latter being indistinguishable from background readings. Relative affinities were calculated based on the synthetic peptide with the lowest K, value in FK1 and WF6 binding, respectively.

Peptide Relative affinity FK1

Relative affinity WF6

a1-20

a3047 a15-33

a4340 a55-74 a6340 a(67-76)2 a75-94 a91-110

a118-137 a106-122

a126-145 a134-153

a150-169 a165-184 a168-185 a181-200 a184-203 a197-216 a230-249 a261-280 01276-295 01291-308 a306322 a313-331

01149-169

a318-336 ~~332-350 a346-364 a360-378 a374-394 a376393 a390-409 a397416 a40M23 a420-437

P.W >50 >50

2 2

>50 1 5 1 4 4 0.016 0.1 5

>50 17 10 2 0.5 0.051 0.05 0.5 9 5 3 5

>50 24 12 11

>50 >50 >50

35 30 18 7

P M

>50 >50 >50 >50 >50 >50 >50 >50 >50 >50

>50 >50 >50 >50 >50 >50

0.466

0.3 0.039

>50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50

<0.002 <0.002 0.008 0.008

<0.002 0.016 0.0032 0.016 0.004 0.004 1 0.16 0.0032

<0.002 0.0009 0.0016 0.008 0.032 0.3137 0.32 0.032 0.0018 0.0032 0.0053 0.0032

0.0007 0.0013 0.0015

<0.002

<0.002 <0.002 <0.002

0.0005 0.0005 0.0009 0.0023

<0.002 <0.002 <0.002 >0.002 <0.002 <0.002 <0.002 <0.002 <0.002 <0.002

<0.002 <0.002 <0.002 <0.002 <0.002 <0.002

0.130 1

<0.002 <0.002 <0.002 <0.002 <0.002 <0.002 <0.002 <0.002 <0.002 <0.002 <0.002 <0.002 <0.002 <0.002 <0.002 <0.002 <0.002

0.0837

K, values for each peptide (see “Experimental Procedures” for details).

The K, values were obtained by quantitative analysis of binding isotherms, as representatively shown for four peptides in Fig. 3. Four series of experiments were performed, with essentially the same results. The K, values of one set of experiments are listed in Table 11, and their reciprocal values are graphi- cally displayed in Fig. 4. In agreement with our previous find- ings (Conti-Tronconi et al., 1990), the sequence regions a184- 203 and all8-137 (ordered according to apparent binding affinities) contained subsites for WF6 binding. Antibody FK1 recognized the sequence regions all8-145 (two peptides) and (31184-216 (two peptides), which overlap with those for WF6 binding. As is evident from Fig. 4, the dominating antigenic subsite of FK1 is different from that of WF6 and is located closer to the N terminus than the latter. From the results with synthetic peptides, antibodies FK1 and WF’6 have overlapping prototopes, suggesting that their distinct inhibitory properties in regard to ACh and Phy may be caused by different attachment point patterns within these sequence regions.

FKl and WF6 Interact with Different Amino Acid Residues within the Same Sequence Region-To test the above hypoth- esis, we employed a set of homologous synthetic peptides (Conti-Tronconi et al., 1991), differing from a181-200 by the exchange of single amino acid residues along the sequence, to measure the influence of single residues in antibody binding. As we did before for the identification of peptides interacting

with the antibodies, the peptide competition assay and the same procedure of data analysis were used. Four series of experiments were performed, with the results of one of them presented in Table I11 and in Fig. 5. As may be deduced from these data, essential residues for the interaction of WF6 with this sequence region were Trp-187, Thr-191, Pro-194, Asp-195, and Tyr-198; less important residues were Tyr-190 and Cys- 193, in good agreement with our previous studies (Conti-Tron- coni et al., 1991). In contrast, the interaction of FK1 with this sequence region was dominated by residues Tyr-181, Trp-184, Trp-187, Cys-192, Asp-195, and Asp-200. The other residues indicated to be involved in FK1 binding (His-186, Val-188, Tyr- 189, Tyr-190, Thr-191, and Cys-193) did not show up reproduc- ibly in all four series of experiments, suggesting that they may represent, at least in part, nonspecific binding of FK1 which is an IgM. Of the essential residues identified in this study, only two are recognized by both FK1 and WF6.

Summarizing these data, binding of antibodies FKl and WF6 to the sequence region (~181-200 appears to involve 5-7 residues that are discontinuously distributed. Because FK1, but not WF6, bound to peptide a197-216 (Fig. 41, this antibody probably interacts with additional residues beyond sequence position 200. Although the assay used here is largely qualita- tive, it clearly supports a model of distinct attachment point patterns within the same prototope. The selective pharmaco- logical properties of FK1 and WF6 therefore are probably due to (i) their different preference in binding to the identified

FKl Binding to Torpedo nAChR 10413 U

synthetic peptide

FIG. 4. Semiquantitative ELISAof the binding of antibodies FK1 and WF6 to synthetic peptides matching in sequence preselected portions of the !7brpe& nAChR a-subunit. ELISAs (triplicates) were performed under standardized conditions (see “Experimental Procedures” for details) with varying dilutions of synthetic peptides, titration curves were constructed, and the corresponding K, values were determined (Table 11). The figure depicts reciprocal K, values. Because antibody FK1 is an IgM, the level of nonspecific binding is higher than in the case of antibody WF6, which is an IgG. We therefore consider only K, values for FKl below 1 as evidence for specific competition. Peptides indicated to contain elements of the antibody binding sites; for FK1: a118-137, a126-145, a181-200, a184-203, and a197-216; for WF6: a118-137, a181-200, 0184-203. Binding to peptide a230-249 is a common experimental artifact (Conti-Tronconi et al . , 1990).

TABLE I11 Apparent inhibition constants K, for single residue substituted analogs of a181-200 interfering

with the binding of antibodies FKl and WF6 to TMF Semiquantitative ELISA of the binding of antibodies FK1 and WF6 to the panel of analogs of a181-200. ELISAS (triplicates) were performed for

several different concentrations of antibody, and titration curves were constructed. K, values were obtained from least squares fits of the respective competition binding curves. The assay is limited to the determination of K, values below 100 w. Relative affinities are expressed in regard to the parent peptide a181-200.

Peptide K, (FK1) K, (W6) Relative affinity Relative affinity FK1 W 6

P flM

Unmodified 3 0.2 1 1 Tyr-181 Arg- 182 0.1 0.2 30 1 Gly-183 1 0.2 3 1 Trp-184 2 100 0.4 50.01 0.5 LYS-185 0.1 0.2 30 1

2 100 0.4 50.01 0.5

His-186 13 0.8 0.23 0.25 75 2 100 0.04

Val-188 6 50.01

Tyr-189 25 0.6 0.5 0.3 0.5

9 0.12

4 0.4

0.3 15

0.05 2 100 0.2 50.01

50.01 0.5 0.05

ASP-195 2 100 2 100 50.01 50.01 50.01

0.5 0.5

50.01 0.5

2 100 0.6 50.01 0.3

Trp-187

Tyr-190 Thr-191 cys-192 2100 0.4 cys-193 8 4 0.375

0.5 2 100 6

0.6 0.4 5 0.6 0.4 5 0.8 >50 0.5 0.4 6

Pro-194

Thr-196 Pro-197 Tyr-198 3.75 Leu-199 ASP-200

prototopes, and (ii) different attachment point patterns within 1990). Different amino acid residues within these subsites are these prototopes. involved in the binding interaction with different ligands, re-

sulting in ligand-specific recognition (Conti-Tronconi et al., DISCUSSION 1991; McLane et al., 1992). The two antibodies WF6 and FK1

We have shown previously that the cholinergic binding re- are interesting examples of ligand-specific recognition, as they gion on the a-subunit of Torpedo nAChR is not a single con- have been reported to function as selective antagonists, the tinuous sequence region but rather is formed by several dis- former blocking the binding and response to ACh (Fels et al., continuous sequence segments folded together in the three- 1986), the latter to Phy (Pereira et al., 1993a). Elucidation of dimensional structure of the receptor (Conti-Tronconi et al., the location and structure of the epitopes for WF6 and FK1 may

10414 FKl Binding to Torpedo nAChR

FIG. 5. Semiquantitative ELISA of the binding of antibodies F K l and WF6 to single residue-substituted analogs of synthetic peptide al8l- 200. ELISAs (triplicates) were performed under standardized conditions (see “Ex- perimental Procedures” for details) with varying dilutions of single residue-substituted analogs of synthetic peptide a181- 200 (Conti-”ronconi et al., 1991), titration curves were constructed, and the corresponding K, values determined by least squares fitting of the binding isotherms (Table 111). Four sets of experiments with essentially the same results were performed. From these data, FK1 and WF6 bind with distinct attachment point patterns to the a181-200 peptide, the only common attachment points being Trp-187 and Asp-195.0, FK1; m, WF6.

100

20

U

substituted amino acid

Species Tvoe .. 118 119 120 121 122 123 I 2 !4 125 126 127 128 129 130 131 132 133 I34 135 136 137 138 139 140 141 142 143 1 4 4 145

Amino acid sequence in the regiona&l18-145

e r T y r C y s G l u Ile Ile ValThrHisPheRopheAspGlnGlnAsnCysThrMetLys er Tyr Cys Glu Ile ne Val Thr His Phe Ro We Asp Glu Gln Asn Cys Ser Met Lys - - -. _. ” _“ - -. . -. .̂ . - ” ...-

Torpedo Human , Bovine a1 ~ r p ~ h r RO RO la 11e q~ ~ ’ys cilu ne ne v a ~ ~ n r HIS m e m me ~ s p l r ~ u clm ~611 LY ber Met ~ y s Mouse al Trp Thr Pro Ro Ala ne Tyr Cys Glu Ile ne Val Thr His Phe Ro We Asp Glu Gln Asn Cys Ser Met Lys Chick al Trp Thr Ro Ro Ala ne Tyr Cys Glu Ile ne Val Thr Tyr Phe Pro Phe Asp Gln Gln Asn Cys Ser Met Lys Frog al Trp Thr Pro Ro Ala ne Tyr Cys GIU Ile ne Val Thr Tyr Phe Pro We Asp Gln Gln Asn Cys Ser Met Lys

Rat a2 TrpValProProAlaUeTyr y s S e r S e r C y s S e r U e A s p V a l ~ P h e P h a P r o W e ~ G l n G l n A s n C y s L y s . ~ L g ; s Rat a3 Trp Ile RoRoAla ne P h e L y s S e r SerCysLys Ile AspValThrTyrPheProWeAspTyrGlnAsnCysThiMetLys Rat a4 TrpThrProRoAlaI leTyr iLysSsrSerCysSer ]le AspValThrPhepheProPheAspGlnGlnAsnCysThrMstLys Rat a5 Trp Thr Gln Ro Ala Asn Tyr bys Ser Ser Cys Ile Ile Asp Val Thr Phe Phe Pro We Asp Leu Gln Asn Cys Ser Met Lys Chick a4 Trp Val Pro Ro Ala Ile Tyr 7 iLys Ser Ser Cys Ser Ile Asp Val Thr Phe Phe Pro Phe Asp Gln Gln Asn Cys Lys Met Lys

A

PhY

Species Type Amino acid sequ- in the reeion sU81-216 181 182 183 184 185 186 187 188 189 190 191 192 193 1’ 7 208 209 210 211 212 213 214 215 216

Torpedo Human Bovine

Rat a2 Ala Thr Gly Thr Tyr Asn Ser Lys Lys Tyr Asp Cys Ala Glu - Ile Tyr Pro Asp Val Thr Tyr Tyr Pha Val Ile Arg Arg Leu Pro Lea Phe Tyr Thr I k Rat a3 Ala Pro Gly Tyr Leu His Glu Ile Leu Tyr Asn Cy8 Cys Glu Glu - Ile Tyr Gln Asp Ile Thr Tyr Ser Leu Tyr Ile Arg Arg Leu Pro Leu Phe Tyr Thr Ile Rat a4 Ala Val Gly Thr Tyr Asn Thr Arg Lys Tyr Glu Cys Ala Glu - Ile Tyr Pro Asp ne Thr Tyr Ala Phe-Ile Ile Arg Arg Leu Pro Leu Phe Tur Thr Ile Rat a5 Ala Met Gly Ser Lys Gly Asn Arg Thr Asp Ser Cys rp T p - Pro Tyr Ile Thr Tyr Ser Phe Val Ile Lys Arg Leu Pro Leu Phe Tyr Thr Leu Phe Leu Chick a4 Ala Val Gly Asn Tyr Asn Ser Lys Lys Tyr Glu d -9 hr Glu - Ile Tyr Pro Asp ne Thr Tyr Ser Phe ne Ile Arg Arg Leu Pro Leu Phe Tyr Thr Ile

r- A A

Ach FIG. 6. Comparison of amino acid sequences of different nAChR a-subunits in the regions al18-145 and al81-216. The sequences are

aligned with respect to the Z californica a-subunit sequence. Homologous amino acid residues are indicated by a dark background. The positions of amino acid residues photoafiinity-labeled by Phy (Schrattenholz et al., 1993b) and by bromoacetylcholine (Kao et al., 1984) are indicated by arrows. The level of homology between different nAChR a-subunits is higher in the region a118-145 than in the region 01181-216 and also extends to neuronal subunits.

help to understand the mechanisms of ligand recognition and be distributed over a wider sequence range, even though they channel gating of the nAChR. partially overlap with those of WF6 (Fig. 4). The sequence

Like WF6, antibody FK1 binds to the innervated surface of regions identified here as contributing to the epitopes of FK1 Torpedo electrocytes (not shown) and specifically recognizes the and WF6 may be involved in the formation of a specific struc- nAChR a-polypeptide in immunoblots (Fig. 1). The FK1 epitope tural motif of the nAChR, e.g. the lining of a binding gorge is located within the N-terminal extracellular region of the (Sussman et al., 1991) or two adjacent loop structures. a-subunit (Fig. 1) and consists of at least two discontinuous Several of our results support the notion of distinct locations sequence segments, a118-145 (two overlapping peptides) and of the epitopes of FK1 and WF6. Thus, the Western blots with a181-216 (two overlapping peptides) (Fig. 4). In noteworthy two fusion proteins containing fragments of different length of contrast to the antigenic sites for WF6, those for FK1 appear to the N-terminal sequence of Torpedo a-subunit (Fig. 1) indicated

FKl Binding to

that the FK1 epitope is located upstream from that of WF6. Antigenic site competition studies suggested that FK1 and WF6 bind to antigenic sites that overlap only slightly (Table I). The competition binding studies with low molecular weight ligand (Phy, ACh) suggest that only the sites for ACh and WF6 and those for Phy and FK1 overlap, but not vice versa. In contrast, the epitope mapping studies with synthetic peptides suggest that both antibodies bind to largely the same sequence regions and with similar affinities.

At first view, the results from Western blotting (Fig. l), antigenic site competition studies (Table I), and ligand competition (Fig. 2) may appear to be at odds with those from epitope mapping studies (Table I1 and Fig. 4). The former suggest little, if any, overlap of the FK1 and WF6 epitopes, the latter suggest a significant overlap of antibody binding sites. The results are reconciled, however, when the affinities of binding are consid- ered. The studies with native antigen, whole a-subunits, and large fragments (ligand binding, antigenic site competition, Western blots) certainly discriminate better the differences in binding affinities of FK1 and WF6 to their prototopes. In contrast, the epitope mapping studies with synthetic peptides stress the similarities in location of the two subsites. It therefore was crucial to test whether and to what extend the attachment point patterns of the two antibodies overlap.

Using single residue-substituted synthetic analogs of the sequence a181-200, we have collected information in regard to the attachment point patterns of FK1 and WF6 in one of the identified sequence regions. The data summarized in Fig. 5 and Table I11 indicate a limited overlap of the attachment points within this subsite. We do not know though whether all of the attachment points identified in this assay are also used by the native receptor (see below), e.g. the 2 residues suggested to participate in the binding of both antibodies, Trp-187 and Asp- 195, and/or to what extend the patterns are sensitive to con- formational adjustments. Although limited in its quantitative aspects, the competition ELISA with single residue-substituted peptides provides additional evidence in favor of distinct binding sites, with individual attachment point patterns, within a common receptive region.

Like any approach that uses a sequence region excised from the structural context of the cognate native protein, the use of synthetic peptides has important caveats. Among these (i) isolated fragments of a larger binding domain may not be able to bind the ligand in the same way as the native protein because they may contain too few attachment points, or they may fold into aberrant conformations; (ii) synthetic peptides may miss important structural features (disulfide bonds, noncovalent in- teractions with neighboring domains, post-translational modifications). Such caveats apply also to the sequence regions identified as antigenic sites in this study. In native Torpedo nAChR, the region a118-145 contains a disulfide bridge between cysteines 128 and 142 and the only N-glycosylation site. The region a181-216 contains a disulfide bridge between the vicinal cysteines 192 and 193. Although we are aware of these limita- tions of synthetic peptides as structural models, we also note that studies of the kind reported here have demonstrated reliable predictive value in the sequence identification of, among others, the main immunogenic region (Lindstrom et al., 1988) and the a-bungarotoxin binding site (Neumann et al., 1986; Ralston et al., 1987; Wilson and Lentz, 1988; Conti-Tronconi et al., 1990) of the nAChR.

A significant difference between FK1 and WF6 is that FK1 cross-reacts with nAChR from many if not all species, whereas WF6 has only limited cross-reactivity. These properties can be traced down to the primary structures of the dominating subsites for the two antibodies. WF6 probably is capable of cross- reacting with muscle al-subunits of many species by virtue of

Torpedo nAChR 10415

conservation of several critical amino acid residues within the sequence segment 190-198, whereas these residues are not conserved in neuronal a-subunits (McLane et al., 1992). In contrast, the sequence region a118-145 displays the highest level of structural conservation outside the putative transmem- brane domains of a-subunits, with more than 60% identity and an additional 20% of conservative substitutions (Fig. 6; Maelicke, 1992; Pereira et al., 1993b). This strong homology, which applies to all nAChR a-subunits sequenced so far, probably accounts for the unusually high level of cross-reactivity observed for antibody FK1.

The sequence region a128-142 has been suggested previously as being involved in ligand binding (Noda et al., 1982; Smart et al., 19841, although direct evidence for ACh binding so far is lacking. The present study supports the notion that the two regions of the N-terminal extracellular domain of nAChR a-polypeptide stabilized by disulfide bridges (a118-145 and a181-215) participate in the binding of channel-activating ligands and of their antagonists. It is suggestive in this regard that the region a120-145 is slightly hydrophobic and may form a P-pleated sheet domain (Stroud et al., 19901, whereas the region a180-215 is hydrophilic (Noda et al., 1982). These general properties may already have some selective value as to the ligand specificity of each subsite.

The significance of the present study is not confined to the two antibodies investigated and their competitive ligands ACh and Phy. A structural arrangement providing for the recognition of different classes of ligands within a relatively large receptive region may be a general principle in neuroreceptor- ligand interaction. Other examples are the modulating action of glycine on N-methybaspartate type glutamate receptors (Barnard and Henley, 1990) and of benzodiazepines and /3-car- bolines on y-aminobutyric acid receptors (Burt and Kamatchi, 1992).

cal assistance by Vesna Pondeljak, Claudia Schwitter, Helga Taschner Acknowledgments-We acknowledge gratefully the excellent techni-

(Maim), and Xiadong W u (St. Paul).

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Documents

Monoclonal Antibodies FK1 and WF6 Define Two Neighboring