The α-bungarotoxin receptor purified from a human neuroblastoma cell line: Biochemical and immunological characterization

0306-4522/89$3.00+ 0.00 Pergamon Press plc IBRO

NeuroscienceVol. 32, No. 3, pp. 759-161, 1989 Printed in Great Britain

THE a-BUNGAROTOXIN RECEPTOR PURIFIED FROM A HUMAN NEUROBLASTOMA CELL LINE: BIOCHEMICAL AND IMMUNOLOGICAL CHARACTERIZATION C. GOTTI,* A. ESPARIS OGANDO and F. CLEMENTI CNR Center of Cytopharmacology, Department of Medical Pharmacology, University of Milan, via Vanvitelli 32, 201 29 Milan, Italy Abstract-The pharmacological and electrophysiological characteristics of the a-bungarotoxin receptor present on the human neuroblastoma cell line IMR-32 indicate that this receptor is not associated with an acetylcholine-operated ionic channel. In this paper we report its biochemical purification and immunological characterization. This molecule has a standard sedimentation coefficient of 19s and sodium dodecyl-sulphate gel electrophoresis shows that it is made up of three polypeptide chains of molecular weights of 67,000,60,000 and 52,000. Ligand binding to blots of purified receptor revealed that only the polypeptide of molecular weight 52,000 is bound by [1251]a-bungarotoxin. The purified a-bungarotoxin receptor was bound by polyclonal antibodies raised against purified fetal calf, Torpedo and chick optic lobe nicotinic receptors and by the sera of myasthenic patients. Furthermore, despite the fact that a number of different immunological techniques were used, it was impossible to label this cc-bungarotoxin receptor with mAb 35, a monoclonal antibody which binds some neuronal nicotinic receptors. Rabbit antisera against the purified a-bungarotoxin receptor were used to compare this protein with other known nicotinic receptors and, once again, it was demonstrated that there is some immunological cross-reactivity between the a-bungarotoxin receptor present on neuroblastoma cells and Torpedo, fetal calf and chick optic lobe nicotinic receptors. All these immunological data, together with previously published pharmacological and molecular biology data, demonstrate that the a-bungarotoxin receptor present in nerve cells is neither a muscular nor a neuronal nicotinic receptor, although it has similarities with both.

Nicotinic acetylcholine receptors (AChRs) from both skeletal muscle and Torpedo electric organs have been extensively purified and characterized. Much

of the knowledge about these AChRs is due to the presence in nature of a-bungarotoxin (a-BTX), an a-neurotoxin which tightly and specifically binds to them and blocks their function.2*.32 Nicotinic transmission also plays an important role in the functioning of autonomic ganglia and of certain areas of the CNS, although neuronal nicotinic receptors have only recently been characterized.24i”1,42*U This delay was due to the lack of a suitable ligand. In fact, although cl-BTX binds with high affinity to a number of neuronal tissues, in most cases it fails to block their nicotinic receptor function.*4,‘0*22.”Pharmacological evidence and, more recently, several studies using molecular biology techniques and monoclonal antibodies, have revealed that neuronal

*To whom correspondence should be addressed. Abbreviations: AChR, acetylcholine receptor; a-BTX, a-bungarotoxin; BTXR, K-bungarotoxin receptor; BSA, bovine serum albumin; EDTA, ethylendiaminetetraacetic acid; EGTA, ethyleneglycol-bis-(B-aminoethylether)-N,N,N’,N’-tetra-a&tic &id; mAb, monoclonal antibody; PMSF, phenyl-methyl-sulfonyl fluoride; PVP, polyvinyl-pyrrolidone; SDS, sodium dodecyl-sulphate.

AChRs are a large family of molecules with a well defined structure and topographical distribution.1~7J2*‘3*3E~39,45 In this family, at least two groups of molecules can be identified: those that modulate an ionic channel and bind nicotine with high affinity, but do not bind a-BTX; and those which may or may not modulate an ionic channel but which do bind a-BTX, as well as nicotine with low affinity. Autoradiographic studies with (3H]nicotine and [‘251]a-BTX have provided a detailed map of the localization of these two groups of molecules in the mammalian CNS and indicate that these molecules have different discrete distributions.5v27 While the structure, distribution, subtypes and, to a certain extent, also the function of high-affinity nicotine binding AChRs is known, information is still very scanty as far as a -BTX receptors (BTXRs) are concerned. The physiological role of these receptors is unclear and their identification as cholinergic receptors depends exclusively on pharmacological binding experiments. So far, it has been demonstrated that, in neuronal tissues, there are at least two classes of a-BTX binding molecules. One is coupled to a functional acetylcholine-operated ion channel, blocked by the toxin (present in insect,” toad and goldfish nervous systems,” chick optic lobes24 and mammalian cerebellar interneurons’), while the other (present in several mammalian brain areas,24 as well as in rat cervical ganglia,2 chick ciliary ganglia,3 cat carotid 759

760

C. GOTTI et al

bodiesI PC12 cells”’ and rat locus coeruleus neurons9) has an unknown physiological role. The exact regional and cellular distribution of these two molecules is not known and attempts to isolate them from the whole brain by means of affinity chromatography with a-BTX column have not produced any convincing evidence as to which of these two molecules has, in fact, been purified. In order to obtain more precise information about these molecules, our group (as well as those of other centers) has looked for cell models to grow in vitro which would express only one type of BTXR. Schoepfer et ~1.~~ and Syapin et ~1.~’ have studied a medulloblastoma cell line which expresses an ionic channel controlled by a-BTX, Kemp has isolated an a-BTX binding protein from PC12 cells2’ while the present authors have concentrated on a human neuroblastoma cell line which expresses the BTXR unrelated to an ionic channel.6,‘7 Neuroblastoma cells have several of the characteristics of neuronal cells and, in addition, they are homogeneous, grow in culture without the addition of any specific growth factor and can be differentiated by pharmacological agents.” We found that one cell line (IMR-32) was particularly suitable for our purpose. In fact, this cell line expresses quite a large number of a-BTX binding sites (26OOjcell) but very few, if any, functional nicotinic AChRs (0-2/ceil), as measured by electrophysiological techniques.” In addition, these AChRs are not blocked by a-BTX. Our previous experiments have indicated that this BTXR has several pharmacological and biochemical characteristics similar to those of BTXRs not associated with ionic channels present in the CNS. We report here the results obtained from the isolation of the BTXR from IMR-32 cells, its biochemical characterization, its subunit composition and the localization of the a-BTX binding subunit. In addition, by using polyclonal antibodies raised against peripheral AChRs, and by using antibodies raised against the BTXR purified from IMR-32 cells, we are able to make an extensive comparison of this protein with other known AChRs. EXPERIMENTAL

PROCEDURES

Methods a-Bungarotoxin purification. Cells (obtained from American Type Culture Collection) were grown at confluency as previously described. ‘,‘s Cells were never kept in continuous culture for more than three months. Cells grown at confluency for 10 days in Petri dishes were detached with buffer A (50mM Tris--HCl pH 7.4, 50mM NaCl, 2mM EDTA, 2 mM EGTA, 2 mM iodoacetamide, 2 mM PMSF and 10 pg/ml of aprotinin) and centrifuged at 10,000g for IOmin. The pellets were washed once again and then homogenized in an excess of buffer A using an Ultra Turrax homogenizer. Pellets collected at 100,OOOg for 1h were homogenized in 20~01 of buffer A plus a mixture of lOpg/ml of each of the following protease inhibitors: leupeptin, bestatin and pepstatin A. Triton X-100 was added at a final concentration of 1% and the mixture was shaken

at 4°C for 2 h. The supematant (100,OOOg for 1h) was shaken at 4°C for 5 h with 3 ml Sepharosea-BTX (concentration of coupled toxin 1mg/ml of gel). The beads were rapidly washed in a funnel with 125 vol of buffer A containing 0.1% Triton X-100. then with 250 vol of the latter medium containing 1 M’ NaCl and finally with 125 vol of buffer A plus 0.1% Triton X-100. The beads were then poured into a column and incubated with 1.5 vol of 1 M carbamylcholine chloride in buffer A plus 0.1% Triton X100 with rotation for 12 h at 4°C. After the carbamylcholine eluate was recovered the column was washed with 20 ml of bidistillated water and then 1.5 vol of 1% sodium dodecylsulphate (SDS) was added and the slurry was left for 2 h at room temperature and then recovered. The carbamvlcholine eluate, called BTXRI, was then dialysed three times against 3 I of 10 mM Tris-HCl DH 7.4. 0.5 mM EDTA. 0.5 mM EGTA, 1 mM PMSF plus 0.1%’ Triton X-100, for at least 8 h each time, while the SDS eluate, called BTXRII, was immediately lyophilized. BTXRI was labeled with 125I using a modification of the chloramine T procedure adapted for small amounts of protein as previously described.” The purification procedure described above gave consistent and reproducible results for at least 10 purifications. In order to detect possible contaminating polypeptides from true BTXR subunits the following experiment was done twice. The supematant, obtained after extraction of the membrane pellet with 1% Triton X-100 and centrifugation at 100,000g for 1h, was divided into three portions. Cold a-BTX to a final concentration of 5 x 1O-5 M was added to the first portion; nicotine was added at a final concentration of 0.5 x IO-’ M to the second portion while no drug was added to the third. The three supernatants were preincubated for 2 h at 4°C and then I ml of Sepharose a-BTX was added to each of them and they were shaken for 5 h at 4°C. The samples were then washed and processed as described above. PuriJcation of Torpedo, fetal calf‘ and chick optic lobe acetylcholine receptors. Torpedo AChR was routinely purified from IOOg of frozen Torpedo electric organs by affinity chromatography on cobra toxin-Sepharoseaccordima to Gotti et al..” with some minor modifications. The specific activity of the purified receptor was between 4300 and 6500pmol of a-BTX binding sites per mg of protein. Fetal calf AChR was routinely purified from 5OOg of frozen fetal calf muscles according to Gotti et al.‘S The specific activity of the purified receptor was between 4000 and 5OOOpmol a-BTX binding sites per mg of protein. Chick optic lobe AChR was routinely purified from 36 g of frozen optic lobe of newborn chicken by affinity chromatography on Sepharose-cl-BTX according to the method described by Conti-Tronconi et al.’ with some minor modifications. The specific activity of this last purified receptor ranged from 3500 to 4800pmol G(-BTX binding sites per mg of protein. cc-Bungarotoxin binding assay. [‘2SI]a-BTX binding to membrane, as well as to solubilized and purified receptors, was determined by means of the DE81 DEAE disk assay as previously described,‘5 using a saturating concentration of 1’2511a-BTX(20 nM) and 12 h incubation at 4°C. Nonspekicz binding was determined in parallel using 5 PM cold a-BTX. At the end of the incubation every set of filters was washed four times for 5 min each, using cold wash buffer. Sucrose gradient centrifugation. Aliquots of detergent extracts from IMR-32 cells and Torpedo membrane or “‘I-labeled purified BTXR were layered onto a 12-ml linear gradient from 5 to 20% sucrose in buffer A plus 0. I % Triton X-100. The gradients were centrifuged for 12 h at 36,000rpm in a Beckmann SW 40.1 rotor. Fractions of 0.3 ml were collected from the top and, in the case of the samples obtained from detergent extracts, analysed for [‘221]a-BTX binding using the method described above.

a-Bungarotoxin

while for the fractions obtained from i251-labeled BTXR, radioactivity was directly determined by y counting. Protein determination. The protein concentration of the samples was determined by using either the BCA protein assay reagent from Pierce as in the method of Lowry et ~i.~ Bovine serum albumin (BSA) was used as standard for both methods. Electrophoresis. SDS-polyacrylamide gel electrophoresis was performed according to Laemmliz3 Gels were either stained with Coomassie Brilliant Blue or silver stained for proteins according to Sammons et ai? Polyacrylamide gels of radioiodinated proteins were autoradiographed for 4-24 h at -70°C using preflashed Kodak X-Omat AR film and intensifying screen. Autoradiograms were standardized for molecular weight by using Bio-Rad low molecular weight standards resolved on the same gel and stained with Coomassie Blue. ~lectropho~tic transfer of proteins to nitr~llulose paper and subsequent probing with antibodies were carried out as described elsewhere.“’ Isoelectric focusing. Isoelectric focusing of ‘251-labeled BTXR was performed as described previously” with the following changes: focusing was done for 5 h at 400 V and the gel slices were counted in a Beckman y counter. [‘251]a-Bungarotoxin binding to electroblotted a-b~garotoxin receptor. Lanes of electroblotted

purified

purified BTXR were incubated with 200 nM [‘2SI]a-BTX for 12 h at room temperature in incubation buffer (10 mM Tris-HC! pH 7.4, 0.9% NaCl, 15% BSA, 0.2% PVP and 0.2% ficoll). Non-specific binding was measured in parallel using 20 p M cold a-BTX. After the incubation, lanes were quickly washed with 10 mM Tris-HC! pH 7.4 plus 0.9% NaCl and exposed for autoradiography. Production of antisera against Torpedo, fetal co& and chick optic lobe acetylcholine receptors. Three rabbits were immunized three times at two-week intervals with intact AChR purified from Torpedo electric organs. For every immunization 100 pg of Torpedo AChR was emulsified in 500~1 of complete Freund’s adjuvant and given by intradermal injection. Fifteen days after the third injection the rabbits were bled and the sera tested for anti-Torpedo AChR titer. A similar procedure was followed for the preparation of antisera against both purified fetal calf AChR and chick optic lobe AChR. The only differences were that only two rabbits were immunized for each receptor and every rabbit received 70 pg of either fetal calf AChR or chick ootic lobe AChR for ev&y immunization. The titers of the podled sera, tested in an immunop~pitation assay as described below, were as follows: 700 nM for anti-Torpedo AChR antisera, 400nM for anti-fetal calf AChR antisera and 350nM for anti-chick optic lobe AChR antisera. Production of anfisera against a-bungurotoxin

761

receptor from human neuroblastoma

receptor.

Two rabbits were subdermally immunized three times at two- to three-weekly intervals with 20-4Opg of affinity purified BTXR obtained bv the elution of the a-BTX receptor from the affinity cohunn with ~rbamylcholine. The BTXR was emulsified in 400 ~1 of Freund’s complete adjuvant. After three injections, sera were collected and tested for titer against the immunogen. Every month a booster of the receptor in incomplete Freund adjuvant was given to these rabbits. Since a-BTX dissociates quickly from the BTXR it was impossible to determine the titer of the sera against the immuno~n by using [‘25i]a-B~-Ia~I~ receptor. Consequently we used the chloramine T method” to label the receptor and used this receptor in the immunoprecipitation assay. In this case the titer is expressed as cpm of i2’I-labeled BTXR precipitated per JI! of serum. The two rabbits had similar titers with 3000 and 4000 cpm precipitated per pl of serum, while the preimmune serum of both rabbits gave a precipitation of 300 cpm/p 1of serum. Probing of pur$ed a-bungarotoxin receptor with PO&chmal antibodies. Lanes of electroblotted BTXR were

probed with anti-l”orpedo, fetal calf and chick optic lobe AChR polyclonal antisera and myasthenic sera. Lanes were incubated overnight at 4°C with polyclonal antisera at dilution of 1: 100 and then washed. The antibodies bound were revealed by the addition of [i2SI]Prot. A. Lanes were then washed and exposed for autoradiography. Immunolocalization. IMR-32 cells, grown on gelatincoated coverslips, were fixed in 1% paraformaldehyde for 20min at room temperature and were then washed three times with Dulbecco’s phosphate buffered saline plus 0. I % BSA and 0.1 M glycine (pH 8) to quench residual aldehyde activity. The cells were incubated for 2 h at room temperature with mAb 35 (monoclonal antibody 35) (diluted 1: 300) and further washed three times. The cells were then incubated with a biotinylated anti-rat secondary antibody for 30 min (dilution I : 100) washed and incubated with avidin-Texas Red (diluted I : 100)for 15 min. After three further washes, the cells were mounted on a drop of Moviol and observed in a Zeiss Axiophot microscope. equipped for epiguorescence. Immunoprecipifation assay. Immunoprecipitation assays were performed as previously described.16 Aliquots of 1% Triton extracts of Torpedo, fetal calf or chick optic lobe membranes. labeled with lu511a-BTX or 1251-labeledourified BTXR, were incubated overnight with varying amounts of the serum to be tested. These samples were diluted with normal rabbit serum to a final volume of 10 pl in order to reach the same immunoglobulin concentration in all samples. Goat anti-rabbit IgG were added and, after 2 h, at room temperature, the samples were centrifuged for 5 min in a microcentrifuge (10,OOOg). The pellets were washed twice with phosphate buffered saline and then counted in a y counter. The titer of the sera obtained by using [1251]aBTX-labeled receptor is expressed as nmol of [‘251]a-BTX precipitated/liter of serum. Materials

Minimum essential medium, FCS and antibiotics were obtained from Flow Lab., U.K.; plastic Petri dishes were from Corning, France; lyophilized Bungarus multicinctus venom, anti protease inhibitors and car~mylcholine was obtained from Sigma, U.S.A.; Sepharose ConA and activated Sepharose 4BCL were from Pharmacia, Sweden; Na lzsI and [rZ51]Prot.A were obtained from Amersham. U.K.: and reagents for gel electrophoresis were obtained from Bio-Rad Laboratories, U.S.A. All other chemicals were of the highest purity commercially available. RESULTS

a-Bungarotoxin

pur$cation

For the purification experiments of the BTXR we selected a subclone of IMR32 cell line which expresses a high number (0.2-0.4pmol) of a-BTX binding sites/mg of protein. A summary of the yield of the BTXR at the major steps of purification, in a representative experiment, is given in Table 1.

Table 1. Representative purification of a-bungarotoxin receptor from IMR-32 cells

Cell homogenate Membrane fraction Detergent extract of membrane fraction Carbamylcholine eluate from a-BTX column

Protein (n-in) .-.

Receptor (nmolt

Specific activitv

520 336

160 140

0.3 0.41

80

130

1.62

0.015

28

_

:--

*

1870

Yield 100

20

762

C.

GOTTI f?t d.

A fraction ranging between 60 and 80% of the BTXR contained in the Triton extract was absorbed to the affinity column and about 15-20% of the absorbed BTXR was specifically eluted by carbamylcholine. After elution the proteins still absorbed to the column were recovered by incubation with 1% SDS. Different preparations of the BTXR eluted with carbamylcholine, extensively dialysed to remove the cholinergic ligand, had a specific activity ranging between 1.5 and 2.3 nmol [‘251]cc-BTXbinding sites/mg of protein, indicating an overall purification of 7500-11,500 from the membranes. This purified BTXR is a glycoprotein which binds ConA, and when run on a 5-20% sucrose gradient, has a sedimentation coefficient of 10s. Isoelectric focusing of the purified ‘251-1abeledBTXR gave a single peak with a PI of 5.3. Subunit composition

SDS gel electrophoresis of the BTXR obtained either from the carbamylcholine eluate (BTXRI) or from the SDS eluate of the column (BTXRII), showed a very similar peptide pattern. The two BTXRs were analysed on SDS gel electrophoresis either by means of autoradiography of the radiolabeled receptors or by means of silver or Coomassie Blue staining. Different preparations consistently contained three major components, whose molecular weights were 67,000 + 1000, 60,000 + 2000 and 52,000 + 1000 (Fig. 1). Sometimes a band of molecular weight 48,000 was present and migrated in a diffuse faint band. For this reason we cannot definitively conclude whether the 48,000 mol. wt band is a true BTXR subunit or a degradation product of other peptides.

.

67 60 52

ABCDEF Fig. I Analysis of BTXR purified from IMR-32 cells by means of SDS gel electrophoresis carried out in 9% acrylamide. (A) Coomassie Blue staining of standard proteins. (B) Coomassie Blue staining of 20 fig of purified Torpedo AChR. (C) Coomassie Blue staining of 6ng of purified BTXR eluted from the affinity column with carbamylchohne (BTXRI). (D) Coomassie Blue staining of 12 pg of purified BTXR eluted from the affinity column with 1% SDS (BTXRII). (E) Coomassie Blue staining of affinity column eluate of a Triton extract preincubated with saturating concentration of a-BTX. (F) Autoradiogram of ‘251-labeled purified BTXR.

Faint bands of lower and higher molecular weight were sometimes present, possibly because of partial degradation or cross-linking of BTXR subunits: in fact the number of bands increased with the aging of the preparation or the length of the purification process. In order to investigate whether the polypeptides that we purified were true BTXR subunits or contaminating polypeptides, we divided the Triton X-100 extract of the membrane pellet into three equal portions: one was incubated with saturating concentration of cold a-BTX, the second was incubated with an excess of nicotine, while no drug was added to the third portion. After a preincubation of 2 h these portions were incubated with identical volumes of a-BTXSepharose, washed and eluted; they were then analysed by SDS gel electrophoresis and by binding of [‘251]a-BTX. The SDS gel electrophoresis, stained either with the silver staining or the Coomassie Blue technique, showed that the sample obtained from the extract to which no drug was added contained all three peptides of molecular weights 67,000, 60,000 and 52,000, while the sample obtained from the extract that was preincubated with saturating concentration of a-BTX did not contain any detectable amounts of these peptides (see Fig. 1E). In the sample preincubated with nicotine the three peptides of molecular weights 67,000, 60,000 and 52,000 were detectable but the amount present in the gel was approximately half of that detected in the drug-free sample. We also did binding experiments on the sample purified under the three different conditions by means of [‘251]a-BTX with similar results. No binding was detectable in the sample preincubated with an excess of cold cr-BTX, while the amount of the binding present in the sample preincubated with nicotine was approximately 4&50% of the binding present in the untreated sample. Localization of [‘251]~-bungarotoxin binding to the isolated receptor subunit

In order to determine which subunit(s) of the purified receptor bind [‘z51]u-BTX both purified BTXRs (BTXRI and BTXRII) and purified Torpedo AChR were run on SDS gel electrophoresis under reducing conditions and then electroblotted onto with membrane and incubated nitrocellulose [“51]~-BTX. The results of the binding of [i2’I]a-BTX to purified BTXRI, BTXRII and to purified Torpedo AChR are shown in Fig. 2. In those experiments where the Coomassie Blue staining of the purified only three major peptides of BTXRs showed molecular weights 67,000, 60,000 and 52,000, only the peptide of molecular weight 52,000 was labeled by [i251]a-BTX. In the same experiment the Torpedo AChR was labeled only on the 42,000 mol. wt (c() subunit. In those BTXR preparations where, in addition to the three peptides of molecular weights 67,000, 60,000 and 52,000 the peptide of molecular

a-Bungarotoxin

A

B

receptor from human neuroblastoma

C

Fig. 2. Localization of [‘251]a-BTX binding to Electra blots of Torpedo AChR (lane A), BTXRI (lane B) and BTXRII (lane C). Receptors were separated in 8% acrylamide and then blotted onto a nitrocdllulose membrane incubated with 200 nM [‘251]a-BTX. After washing, the nitrocellulose membrane was autoradiographed for 24 h.

48,000 was also present, the last peptide showed, a very faint [‘251]cc-BTXbinding, indicating that this band is probably a degradation product of the peptide of molecular weight 52,000. When the binding was performed in the presence of an excess of cold a-BTX a labeled band was never observed.

weight

Immunological receptor

characterization

of a-bungarotoxin

Analysis with polyclonal antibodies raised against acetylcholine receptors purified from dzyerent tissues.

In an attempt to identify subunits of the BTXR which are homologous to other known AChRs, Western blots of purified BTXR were probed with polyclonal antisera raised in rabbits against purified Torpedo, fetal calf and chick optic lobe AChRs and human sera from myasthenic patients. Western blots of affinity purified BTXR were incubated with the antisera and revealed by [‘251]Prot A. We found that all three antisera (Fig. 3B-D) were able to recognize the peptides of molecular weights 60,000 and 52,000 while myasthenic serum (Fig. 3F) recognized only the peptide of molecular weight 60,000. In the same experiment normal rabbit sera and normal human sera were not able to recognize any BTXR peptides (Fig. 3A and E). To test whether there were immunological similarities between BTXR and neuronal AChR, we did some experiments with mAb 35, an mAb raised against the AChR purified from Eiectrophorus E. which recognizes, in chick and rat brains,24,41 a neuronal nicotinic AChR that does not bind a-BTX. We used this mAb in an immunoprecipitation assay using ‘251-labeled purified BTXR, in an immunolabeling experiment using blots of purified BTXR and in an immunolocalization study in order to label the

ABCDEF Fig. 3. Western blots of purified BTXR probed with different polyclonal antisera. Purified BTXRs were run on SDS polyacrylamide gel electrophoresis, 8% acrylamide, transferred onto nitrocellulose membrane and then probed with antisera diluted 1: 100. Antisera were incubated overnight at 4”C, then washed and bound antibodies were revealed by [‘251]Prot.A. The following antisera were tested: (A) normal rabbit; (B) rabbit anti-Torpedo; (C) rabbit anti-fetal calf; (D) rabbit anti-chick optic lobe; (E) normal human; (F) myasthenic patient. present on the surface of our IMR-32 cells. We never found any immunoprecipitation of BTXR by means of mAb 35, nor did we find any binding of mAb 35 to electroblotted BTXR (perhaps due to the fact that mAb 35 mainly recognizes native receptors), nor did immunocytochemistry experiments reveal any binding to native cells. BTXR

Analysis with anti-a-bungarotoxin receptor antibodies. Two rabbits were immunized with the affinity

purified BTXR. The rabbit antisera were tested in an immunoprecipitation assay using ‘251-labe1ed BTXR as antigen and then pooled. The antiserum specifically immunoprecipitated the [‘251]BTXR and, on Western blots of purified BTXR, recognized the peptides of molecular weights 60,000 and 52,000 (see Fig. 4A). In order to see whether BTXR shares any antigenic determinants with other known AChRs, the antiserum was tested in: (a) an immunoprecipitation assay using [‘251]a-BTX-labeled Torpedo, fetal calf or chick optic lobe AChRs as antigen; and (b) a study of the binding of this antiserum to blots of purified Torpedo, fetal calf or chick optic lobe AChRs. This antiserum was able to specifically immunoprecipitate [‘251]a-BTX-labeled Torpedo, fetal calf and chick optic lobe AChRs as is shown in Fig. 5. In the same experiments, pre-immune serum was unable to show any immunoprecipitation. The titers of the antiserum against these three antigens were very similar, being 28 nM against fetal calf AChR, 48 nM against Torpedo AChR and 45 nM against chick optic lobe AChR. These titers were low but reproducible.

764

C. GOTTI PI ul

BTXR purified from IMR-32 cells has some epitopes in common with the peripheral AChR, despite the phylogenetic distance between them.

60 52

DISCUSSION

This is the first report dealing with the purification and biochemical characterization of an cr-BTX receptor taken from a human nerve cell. We used neuroblastoma cells because these cells have many of the properties of neurons and, in addition, they are a homogeneous cell population which expresses only one type of a-BTX binding site. They thus provide a convenient and clean model for the purification and characterization of this molecule.

ABCDEFG Fig. 4. Western blots of different purified receptors probed with anti-BTXR antiserum or pre-immune serum. Purified receptors were run on SDS-polyacrylamide gel electrophoresis 8% acrylamide, transferred onto nitrocellulose membrane and then probed with the pre-immune serum (lanes C, E, G) or the anti-BTXR antiserum (lanes A, B, D, F) diluted I : 100. Antisera were incubated for 3 h at room temperature, then washed and bound antibodies were revealed by [‘251]Prot. A. (A) BTXR purified from IMR32 cells (7 pg); (B, C) AChR purified from Torpedo electric organs (IOpg); (D, E) AChR purified from fetal calf muscles (IO pg); (F. G) AChR purified from chick optic lobes (10 pg).

The antiserum, probed on Western blots of the purified Torpedo, fetal calf and chick optic lobe AChRs, was able to bind the subunits of molecular weights 65,000 (6) and 60,000 (y) of Torpedo AChR (Fig. 4B), the subunit of molecular weight 52,000 (/?) of fetal calf AChR (Fig. 4D) and the subunits of molecular weights 52,000 and 58,000 plus a higher molecular weight component of the chick optic lobe AChR (Fig. 4F). These last experiments, together with the results of the immunoprecipitation studies, suggest that the

‘m X ‘h Z b VI ol

100

5

E

0

2

L

6

8

10

pl Of serum

Fig. 5. lmmunoprecipitation curves of [‘251]~-BTX labeled Torpedo (A), chick optic lobe (B) and fetal calf(C) AChRs precipitated by different amounts of anti-BTXR antiserum produced in rabbits, Pre-immune serum was also tested with the same receptors (curves D, E. F).

a-Bungarotoxin receptor structure

Based on the results of the SDS gel electrophoresis of both carbamylcholine and SDS eluants and on control experiments, we identified three polypeptide constituents of the BTXR present on neuroblastoma cells. The subunit patterns of the BTXRs eluted with carbamylcholine or SDS were essentially identical, thus confirming that, after extensive washes of the affinity column, BTXR is the only protein which remains absorbed. From the staining intensities of the bands on Coomassie Blue gels, it seems that the bands of molecular weight 60,000 or 52,000 are more prevalent that the band of molecular weight 67,000. However, it is difficult to obtain the stechiometry of the subunits from the polypeptide pattern of the SDS gel. While the subunit of molecular weight 67,000 seems to be present in smaller quantities in the purified receptor, it is certainly a subunit of the BTXR. This is demonstrated by the fact that this subunit, as well as those of molecular weights 52,000 and 60,000, is absent from the affinity purified sample obtained from the Triton extract preincubated with a saturating amount of a-BTX. The reasons why this subunit is not recognized by the antiserum raised in rabbits against the purified BTXR are not known but a possible explanation is that this subunit, which also seemed to be present in smaller quantities in pure BTXR, is also less immunogenic than the other two subunits of molecular weights 52,000 and 60,000. The specific activity of our purified carbamylcholine eluted BTXR is about 20-30% of the theoretical value calculated by assuming the same cl-BTX binding sites/molecular weight ratio for this BTXR as for the Torpedo AChR. This lower value could be due to many factors: a possible incomplete dialysis of the massive amount of carbamylcholine used for the elution of the BTXR from the column; the presence of a high concentration of iodoacetamide, which at 2 mM has been shown to partially inactivate mammalian AChR;” an incorrect estimate of the cr-BTX binding to the purified receptor (in fact, our previous studies have clearly shown that the dissociation of

a-Bungarotoxin receptor from human neuroblastoma

765

authors. In fact, the a-BTX binding protein purified from rat brain by Lindstrom’s group” is made up of four peptides and is not immunoprecipitated by antibodies raised against muscle AChR,43 while BTXR from IMR-32 cells is bound by different polyclonal antibodies raised against Torpedo, fetal calf and chick optic lobe AChRs. However, our immunological data are in agreement with the results published by Wonnacott et af.46 and Mills and Wonnacott29 who found that the a-BTX binding protein partially purified from rat brain is immunoprecipitated by antisera raised against muscle AChR. Wonnacott et aZ.&also found an immunoprecipitation of those BTXRs purified from rat brain by an antiserum raised in rabbit against Torpedo AChR. Our findings are more similar to that obtained by Kemp et al.*’ for an a-BTX binding protein purified from rat brain. He found that this receptor is made of three subunits of molecular weights 55,000, 53,000 and 49,000 and that the Neuroblastoma cc-bungarotoxin receptor and muscle subunit of molecular weight 55,000 is labeled by the acetylcholine receptor affinity ligand [3H]MBTA. Finally, Conti-Tronconi et al.’ have purified The BTXR we purified has several properties in an a-BTXR from both the chick optic lobe and common with AChRs from electric organs,32 mammalian skeletal muscles” and chick optic lobes.’ Like from the chick brain; these receptors seem to be very similar, both being composed of three major the BTXR, all these three receptors are integral peptides of molecular weights 48,000, 57,000 and glycoproteins negatively charged at physiological pH 67,000 and two minor polypeptides of molecular and have similar sedimentation coefficients. In addiweights 62,000 and 72,000. Like our BTXR, which tion, all these receptors have nicotinic pharmacology is recognized by antisera raised against Torpedo, and have similar sedimentation coefficients. In addition, all these receptors have nicotinic pharmacology fetal calf and chick optic lobe AChRs, the BTXR electrophoresis. The purified BTXR also shares some purified from chick optic lobe could also be speciantigenic determinants with these AChRs, as was fically immunoprecipitated by an mAb raised against demonstrated by: (a) the binding of antisera raised in muscle AChR. In a previous paper Norman et aL3’ found that rabbits against Torpedo, fetal calf and chick optic among 10 rabbit sera obtained from rabbits immulobe AChRs to purified BTXR; and (b) an antiserum nized with pure AChR purified from cat denervated raised the purified BTXR is able to specifically muscles or Torpedo electric organs, only one raised immunoprecipitate a small, but still significant, portion of the [‘2SI]a-BTX-labeled Torpedo, fetal calf against cat denervated AChR gave appreciable titers against the chick optic lobe pure receptor. and chick optic lobe AChRs and the same antiserum All these studies, in addition to the results is able to bind the same receptors in Western blot presented in this paper, would seem to suggest that experiments. However, there are clear differences between the neuronal BTXR is made of different subunits and neuroblastoma BTXR and AChR of the muscle type. that from an immunological point of view it has low The pharmacology is nicotinic6 but the affinity for but consistent antigenic homology with muscular cholinergic agonists and antagonists is similar to that AChRs. of the CNS BTXR and unlike that of muscle AChR. This molecule is also different from the nicotinic The subunit composition is different both in terms AChRs purified from chick4’ and raP brains deof the number of peptides and in terms of their scribed by several authors. In fact: (a) our BTXR molecular weight. In addition this molecule is not binds neuronal bungarotoxin, a toxin known to associated with an ionic channel.” Furthermore, the specifically label the nerve nicotinic AChR,24 but not IMR-32 cell line does not have mRNAs which hy- to a specific site (unpublished observations); and (b) bridize with probes obtained from the sequence of the it is not recognized by mAb 35 which recognizes other muscular al subunit.” Our BTXR is also different neuronal AChRs. from AChR of the muscle type isolated from a In conclusion, the receptor that we have described human medulloblastoma by Lindstrom’s group.25*36 is not a muscle or neuronal AChR, although it has some similarities with these molecules. We think that Neuroblastoma a-bungarotoxin receptor and brain it may be similar to the BTXR present in the CNS, acetylcholine receptor a receptor which is not related to an ionic channel The BTXR we purified has similarities and differ- and whose function has yet to be discovered. We ences with the BTXRs purified from brain by several hope that our cell line, IMR-32, could be a relevant n-BTX from its receptor is very fast, being reduced to 50% in 30 mitt at room temperature,6 and in our binding assay, we use 20-min cold buffer washes during which some dissociation of the [‘251]a-BTXreceptor complex can occur); an a-BTX binding site/ protein ratio different from that of Torpedo AChR, for which there is as yet no experimental proof; and the presence in the purified receptors of possible contaminating polypeptides. This receptor when run in sucrose density gradient, either in a Triton X-100 extract or in a pure form labeled by lz51 sediments as a single peak with a sedimentation coefficient of approximately 10s. This value is slightly larger than that found for the monomeric form of Torpedo AChR or for muscular AChR but it is in agreement with the larger molecular sizes that have been reported for other neuronal BTXRs’*” or neuronal AChRs4’l”

166

C. GOTTlet

model for finally approaching tion of this molecule.

the study of the func-

Acknowlednements-This work has been sunnorted in Dart by a CNRgrant within the special project “Neurobiology”.

al.

The authors are indebted to all of the members of the Clementi laboratory for their many constructive suggestions, to Mrs Ida Ruffoni for her aid with the manuscript, and P. Tinelli and F. Crippa for their technical assistance. A.E.O. is the recipent of a fellowship from Xunta de Galicia, Spain.

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