Monoclonal antibodies to the main immunogenic region of the nicotinic acetylcholine receptor bind to residues 61-76 of the alpha subunit

Article

Reference

Monoclonal antibodies to the main immunogenic region of the nicotinic acetylcholine receptor bind to residues 61-76 of the alpha

subunit

BARKAS, T., et al .

Abstract

Monoclonal antibodies (mAbs) to the main immunogenic region (MIR) bind to fusion proteins containing region 37-200 of the alpha chain of Torpedo, mouse, and chicken nicotinic acetylcholine receptor. In the case of the mouse alpha chain, these mAbs react with sequence 61-216 but not with 74-216. A synthetic peptide M1, containing residues 61-76 of the mouse alpha chain, also binds these anti-MIR mAbs, showing that all or part of their binding site is included in this region. The conformational dependence and epitope specificity of the mAbs are discussed.

BARKAS, T., et al . Monoclonal antibodies to the main immunogenic region of the nicotinic acetylcholine receptor bind to residues 61-76 of the alpha subunit. Journal of Biological Chemistry , 1988, vol. 263, no. 12, p. 5916-5920

PMID : 2451673

Available at:

http://archive-ouverte.unige.ch/unige:85387

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THE JOURNAL OF BIOLOGICAL CHEMISTRY

0 1988 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 263, No. 12, Issue of April 25, pp. 5916-5920, 1988 Printed in U.S.A.

Monoclonal Antibodies to the Main Immunogenic Region

of the Nicotinic Acetylcholine Receptor Bind to Residues 61-76 of the ^a Subunit*

(Received for publication, July 31, 1987)

Thomas Barkas$, Jean-Marc Gabriel, Alex Maurons, Graham J. Hughesll, Beatrice Roths, Christine Alliodg, Socrates J. Tzartosll

,

From the Service of Neurology, Centre Hospitalier Universitaire Vaudois, 1011 Lausanne, Switzerland, the Departments of

§Biochemistry and llMedical Biochemistry, University of Geneva 1205, Geneva, Switzerland, and the IlHellenic Pasteur Institute, Athens 11521, Greece

Monoclonal antibodies (mAbs) to the main immuno- genic region (MIR) bind to fusion proteins containing region 37-200 of the CY chain of Torpedo, mouse, and chicken nicotinic acetylcholine receptor. In the case of the mouse a chain, these mAbs react with sequence 61- 216 but not with 74-216. A synthetic peptide M1, containing residues 61-76 of the mouse a chain, also binds these anti-MIR mAbs, showing that all or part of their binding site is included in this region. The con- formational dependence and epitope specificity of the mAbs are discussed.

In the last 15 years it has been clearly demonstrated that the human neuromuscular disease myasthenia gravis is caused by a humoral immune response to the neuromuscular form of the nAChR’ (reviewed in Refs. 1 and 2). Antibodies to the nAChR, both in myasthenic patients and in animals immu- nized experimentally with nAChR, are heterogeneous. How- ever, the majority of these antibodies are directed against a region on the extracellular part of the a subunit, the MIR (3- 6), and antibodies to this region are implicated in the pathol- ogy of the disease (7). Localization of the MIR is, therefore, an important step toward understanding and treatment of myasthenia.

In a previous study using hybrid proteins containing parts of the a chain of mouse nAChR fused to a fragment of /3- galactosidase (a), we demonstrated that mAbs to the MIR could be divided into two categories. Both bound strongly to constructs containing residues 6-85. Group 1 (e.g. mAbs 6, 198) bound equally well to constructs X4 (residues 6-216) and X8 (residues 37-216), while group 2 (e.g. mAbs 37 and 42), which reacted well with the long construct, showed very little or no reactivity with the shorter protein, suggesting that two

* This work was supported by the Swiss Foundation for Scientific Research (Grants 3.145.0.85, 3.437.0.86 (to T. B.), 3.154.0.85 (to M.

B.), and 3.901.0.85 to Dr. A. Steck), the Sandoz Foundation (to T.

B.), and the Muscular Dystrophy Society of America (to S. J. 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.

2 To whom correspondence should be addressed Laboratoire de Neurobiologie, BH19/208, Centre Hospitalier Universitaire Vaudois, Rue de Bugnon, 1011, Lausanne, Switzerland.

The abbreviations used are: nAChR, nicotinic acetylcholine receptor; MIR, main immunogenic region; mAb, monoclonal anti- body; KSCN, potassium thiocyanate; ELISA, enzyme-linked immu- nosorbent assay; HPLC, high pressure liquid chromatography; a- BTX, a-bungarotoxin.

spatially distinct epitopes might be recognized by these two groups. Using similar constructs from Torpedo californica (mAb 6 was raised against Torpedo nAChR; mAbs 37 and 42 are anti-Electrophorus nAChR and react better with Torpedo nAChR than with mammalian nAChRs (3, 4)), it became clear that group 2 also binds, albeit weakly, to residues 37- 200. This has led us to re-evaluate the binding of these anti- MIR antibodies and to define precisely the epitopes recog- nized. By the use of fusion proteins derived from the a subunits of nAChRs from Torpedo, mouse, and chick, and by the use of synthetic peptides, we can now show that part or all of the binding site(s) for both groups of antibodies resides in residues 61-76 of the a chain.

MATERIALS AND METHODS

Anti-AChR mAbs-mAbs were derived from rats immunized with intact nAChR from different species (3-6). Hybridoma supernatants were used in all tests without prior concentration.

Construction of Expression Plasmids-Expression clones contain- ing mouse a chain-derived fragments 6-216 (X4) and 37-216 (X8) have been previously described (8).

Clones B31N-55 and B31N-59 were derived from mouse expression clone p a x 4 in the following way: p a x 4 was linearized by EcoRI digestion and treated with exonuclease Ba131. The “nibbled” DNA ends were repaired with Klenow polymerase and ligated to dodecamer EcoRI linkers. The shortened mouse-derived fragments were freed by digestion with EcoRI and HindIII and recloned in pUC8 cut with EcoRI and HindIII. The transformed colonies were screened for a- BTX binding by our solid-phase colony assay (S), and several posi- tives (i.e. clones deleted by 3n nucleotides) were characterized by Maxam-Gilbert sequencing (9). Two of them, B31N-55 and B31N- 59, were determined to encode, respectively, positions 61-216 and 74- 216 of the mouse AChR a subunit sequence.

Clone pCaX2 was constructed by subcloning a PstI-Hind11 frag- ment encoding residues 35-216 of chicken a subunit cDNA clone pa114L7’ into pUC8 cut with PstI and HindIII (the latter site having been filled by Klenow polymerase). Maxam-Gilbert sequencing con- firmed the correct frame relationship of the construct.

The following expression clones were derived from Torpedo AChR a subunit cDNA clone aT3 (a gift from Dr. J. Boulter, see Ref. 10 for restriction map); pTaXl is a subclone of the MaeI-EcoRV fragment encoding residues -2 to 200 (position 1 corresponding to the NHz- terminal serine in the mature a subunit); pTaXlQ includes the fl fragment (11) cloned at the BamHI site in the pUC8 vector, down- stream from the insertion, resulting in a shorter COOH-terminal extension of just 6 residues (see Ref. 8 for similar constructs); pTaX2 was constructed by inserting a PvuII-EcoRV fragment of clone aT3 encoding residues 37-200 of the Torpedo a subunit sequence at the Hind11 site in pUC8; pTaX2Q was derived by insertional mutagenesis (the Q fragment bounded by filled EcoRI sites was inserted in the filled HindIII site of pTaX2), resulting in a 5-residue COOH-terminal extension.

M. Ballivet, unpublished data.

5916

Main Immunogenic Region of Acetylcholine Receptor 5917

Isolation of Fusion Proteins, Electrophoresis, and Immurwbbtting- This was performed as previously described (8) with the following modifications. Centrifugation of bacterial pellets was at 14,000 X g for 15 min. Certain fusion proteins insoluble in 8 M urea were dissolved directly in sodium dodecyl sulfate gel sample buffer after extraction of the bacterial pellets with Triton X-100 and KSCN.

ELISA Tests-The proteins used in these tests were nAChR pu- rified from the electric organ of Torpedo marmorata (12) and fusion proteins X4 and X8 (8). Plates were coated overnight a t 4 "C with protein (2.5 pg/ml) or, for peptides, with poly-DL-alanyl-poly-L-lysine (20 pg/ml) in 0.1 M sodium bicarbonate, 0.02% sodium azide. Volumes throughout the tests were 100 pl. For peptide testing, the PO~Y-DL- alanyl-poly-L-lysine-coated plates were washed 3 times with phos- phate-buffered saline (0.15 M NaCl, 10 mM potassium phosphate buffer, pH 7.1), and then a freshly prepared mixture of peptide (1 pg/

ml) and glutaraldehyde (0.125%) in phosphate-buffered saline was added and incubated for 1 h a t 22 "C. All plates were washed 3 times prior to use with phosphate-buffered saline containing 0.05% Tween 20 (buffer A), and all further incubations were a t 22 'C. Dilutions of hybridoma supernatants in buffer A were added and incubated for 2 h. The plates were washed 3 times with buffer A, and then bound mAbs were detected using horseradish peroxidase coupled to rabbit anti-rat IgG (H

+

Peptide Isolation and Characterization-Peptides PI, P3, and P9 (residues 151-169, 425-437, and 378-391, respectively, of the a sub- unit of Torpedo nAChR) were synthesized, purified, and characterized as described (13-15).

Peptide M1 (residues 61-76 of the a subunit of mouse nAChR) was synthesized by Dr. J-P. Corradin (Institute of Biochemistry, Lausanne) using the solid-phase method and isolated by gel filtration (Sephadex G-25 in 10% acetic acid) and ion-exchange chromatogra- phy (DEAE-Sephadex A-25 in 50 mM ammonium acetate, pH 5.4).

Trace impurities were removed by reverse-phase HPLC (gradient of 0-50% acetonitrile in 20 mM sodium phosphate, 10 mM sodium perchlorate, pH 7.0). A second system of reverse-phase HPLC (gra- dient of 0-60% acetonitrile in 0.1% trifluoroacetic acid) revealed a single peak. The amino acid analysis, performed by precolumn deri- vatization with 4-dimethylaminoazobenzene-4'-sulfonyl chloride (16) was as expected for the pure peptide: Asp 5.2 (5), Gly 2.10 (2), Pro 0.94 ( l ) , Val 2.00 (2), Leu 1.02 ( l ) , Lys 1.90 (2), Tyr 2.10 (2), Trp not determined (1). NH2-terminal sequence analysis (17) gave the unique sequence Val-Asp-Tyr-Asn-Leu-Lys-Trp-Asn-Pro-Asp-Asp-Tyr- Gly-Gly-Val-Lys.

RESULTS

Binding on Immunoblots to Short Constructs from Three Species

Both groups of anti-MIR mAbs were tested for binding to similar short constructs from the a subunit of Torpedo, mouse, and chick nAChRs. Equivalent amounts of fusion proteins containing residues 37-200 of Torpedo, 37-216 of mouse, and 35-216 of chicken nAChRs were tested by im- munoblotting. As expected, mAbs from group 1 (6, 198) re- acted strongly with all three constructs with mAb 6 (anti- Torpedo nAChR) showing a preferential reactivity with Tor- pedo and chick proteins. However, in contrast with the pre- vious results using only mouse-derived sequences (8), binding of mAbs from group 2 (37, 42, anti-Electrophorus nAChR) could also be detected, especially using the Torpedo construct (Fig. 1). a-BTX bound better to chick and Torpedo constructs.

Comparative Binding to Long and Short Constructs As shown by immunoblotting (Fig. 2) and as previously reported (8), group 2 mAbs 37 and 42 bound much more strongly to the longer mouse construct X4 (6-216) than to the shorter construct X8 (37-216) when tested a t equivalent amounts of transferred protein, while group 1 mAbs 6 and

_ _ _ - _ _ _ - ~

118 42 37 6 198 ot BTX

FIG. 1. Immunoblots of short fusion proteins. Equivalent amounts of fusion proteins Tax2 (left), X8 (center) and CaX2 (right) were electrophoresed and transferred by diffusion as described (8).

Replicates were probed with control mAb 118 (anti+ chain of Tor- pedo nAChR), anti-MIR mAbs 42, 37, 6, and 198, and a-BTX. The molecular masses of the fusion proteins are approximately 30 kDa;

CaX2 migrates with an apparent mass of 20 kDa.

~~~

118 42 37 6 198 a BTX

FIG. 2. Immunoblots of long and short constructs. Equivalent amounts of fusion proteins TaXlR, TaXZR, X4, X8, and CaX2 (left to right) were electrophoresed and transferred by diffusion (8). Rep- licates were probed as in Fig. 1. The masses of the fusion proteins range from 20 to 30 kDa; CaX2 migrates with an apparent mass of 20 kDa.

4 - 1

3 - 1

)-I

0 -

'-1

I

Dilution 01 mAb Log 10

FIG. 3. ELISA tests using X4 and X8. Binding of control mAb 118 (0) and anti-MIR mAbs 6 (O), 37 (A), 42 (A), and 198 (0) to X4 (left) and X8 (right) was tested by ELISA as described under "Ma- terials and Methods." Results shown are the mean and standard error or three separate determinations.

198 reacted a t least as well with the short construct as with the longer protein. Similar, but more quantitative, results were obtained by ELISA tests (Fig. 3).

On immunoblots of Torpedo constructs, group 2 mAbs 37 and 42 again reacted better with the longer protein (-1 to 200) than the shorter (37-200), whereas the converse was true both for mAb 6 and a-BTX. mAb 198 reacted equally well with both constructs.

Identical results were obtained using two different prepa- rations of each protein. The presence or absence of COOH- terminal P-galactosidase-derived amino acids did not influ- ence the result.

5918 Main Immunogenic Region of Acetylcholine Receptor

Localization of the Binding Site of Both Groups of mAbs Using Fusion Proteins-Blots of fusion proteins containing residues 61-216 (construct B31N-55) and 74-216 (construct B31N-59) of the mouse a chain were tested for binding of mAbs. Group 1 mAbs 6 and 198 reacted strongly with 61-216 but showed no binding to 74-216 (Fig. 4). Group 2 mAbs 37 and 42 showed very weak binding to 61-216 and no binding to 74-216 (data not shown). The positive control, a-BTX, bound to both constructs (Fig. 4). These results suggested that the nonoverlapping region, residues 61-73, contains all or most of the binding site(s) for group 1 mAbs and possibly also for group 2 mAbs.

Using Synthetic Peptide-As shown in Fig. 5, both groups of mAbs, tested a t similar concentrations, bound well in ELISA tests to the synthetic peptide M1 (residues 61-76 of the mouse a chain). Five of five control mAbs (111, 118, 141, 168, and 172), specific for other chains of the Torpedo nAChR (5), failed to react with this peptide (data shown for mAb 118, Fig. 5). Neither the anti-MIR nor the control mAbs bound to plates coated with control peptides P1, P9, and P3 (residues 151-169, 378-391, and 425-437, respectively, of the a chain of Torpedo nAChR).

The titers of the different mAbs used for fusion protein and peptide antigens are summarized in Fig. 6. It should be noted that these titers are arbitrarily defined in nonequivalent con- ditions for the different antigens, so only the pattern of reactivity of the mAbs should be compared and not the titer itself.

DISCUSSION

Previously, we had demonstrated that anti-MIR antibodies bound to residues 6-85 of the a chain of mouse nAChR and that they could be subdivided into two groups based on their relative reactivity with fusion proteins containing long (resi- dues 6-216) and short (residues 37-216) sequences of the a subunit of the mouse nAChR (8). Based on our present results using fusion proteins derived from the a subunit of three different species and a synthetic peptide from the mouse sequence, we can demonstrate that all or part of the binding

mAb

198 BTX

FIG. 4. Immunoblots of mouse fusion proteins B31N-55 and B31N-59. Similar amounts of fusion proteins B31N-55 and B31N- 59 (residues 61-216 and 74-216 of the mouse CY chain) were electro- phoresed and blotted as in Ref. 8 using anti-MIR mAb 198 or a-BTX.

””-

1.5 2 2.5 3 3.5 4 45 5 5.5

Dilution of mAb Log 10

FIG. 5. ELISA tests using Torpedo nAChR or peptide M1.

ELISA tests using plates coated with either whole Torpedo nAChR (dotted lines) or peptide M1 (residues 61-76 of the mouse a chain, solid line) were performed using control mAb 118 (O), anti-MIR mAbs 6 (O), 37 (A), 42 (A), and 198 (0) as described under “Materials and Methods.” Results shown are the mean and standard error of three separate assays.

site(s) for both groups of mAbs is carried on residues 61-76 of the mouse a chain. This peptide (which is carried on a short exon coding for residues 59-94 in the human (18) and chick genomes (19)) is identical in mouse, human, and calf nAChRs (18,20); the mammalian sequence shows 63 and 88%

identity of residues with the corresponding Torpedo and chick peptides (see Table I). Within the peptide, the NH2- terminal region shows most variation across species. It would seem probable that anti-MIR antibodies such as mAb 198 which cross-react strongly across species would be directed against the COOH-terminal region, whereas those such as mAbs 6, 37, and 42, which react preferentially with Torpedo constructs, would be directed against the NH,-terminal part.

Binding of anti-MIR mAbs to a synthetic peptide correspond- ing to residues 63-80 of the human a chain has also been o b s e r ~ e d . ~

Although all the anti-MIR mAbs tested clearly bind to the same peptide sequence, it is obvious that they do not show identical specificities, neither for equivalent fusion proteins derived from different species (Figs. 1 and 2) nor for different constructs within a single species (Figs. 3 and 6). Using fusion proteins containing sequences of mouse and Torpedo origin,

M. Ballivet, unpublished results.

S. J. Tzartos and B. Conti-Tronconi, unpublished results.

Main Immunogenic Region of Acetylcholine Receptor 5919

5

4

3

8

e

4 n

E h F

5 2

1

0

A B C D

FIG. 6. Titers of mAbs for Torpedo nAChR, X4, XS, and peptide M1. The results shown in Figs. 3 and 5 were used to calculate titers of hybridoma supernatants for the different antigens: whole Torpedo nAChR ( A ) , X 4 ( B ) , X 8 (C), and peptide M 1 (D). Titers were arbitrarily defined as the dilution of mAb giving an optical density at 490 nm of 1.0 under the conditions defined under “Mate- rials and Methods.” Supernatants used were anti-Torpedo p chain

), anti MIR mAbs 6 (O), 37 (Ed), 42 (W), and 198

(a).

Results shown are the mean and standard error of three separate determinations.

TABLE I

Sequences of a chain residues 61-76 of nAChRs from different species Underlined residues differ from the mammalian sequence. Data from Refs. 10, 18, and 20 and M. Ballivet, unpublished results.

Mouse VDYNLKWNPDDYGGVK

Human VDYNLKWNPDDYGGVK

Calf VDYNLKWNPDDYGGVK

Chick - TDZNLKWNPDDYGGVK

Torpedo - IDSLEWNP@YGGLK

three patterns of reactivity of anti-MIR.mAbs can be distin- guished (Fig. 2). mAb 198 (anti-human nAChR) reacts equally well with fusion proteins from either species while mAbs 37 and 42 (anti-Electrophorus nAChR) and mAb 6 (anti-Torpedo nAChR) react preferentially with Torpedo sequences. With both mouse and Torpedo-derived proteins, mAb 198 binds equally well to long and short constructs, mAbs 37 and 42 react better with longer constructs (this difference is more marked with mAb 42, see Figs. 2 , 3 , and 6), while mAb 6 binds equally well t o long and short constructs of mouse a chain but reacts preferentially with the shorter Torpedo construct (as does a-BTX).

Although part of the binding site for the two groups of mAbs is clearly carried on residues 61-76, we cannot as yet distinguish between two possible interpretations, namely (i) that this peptide represents the whole antigenic site for all anti-MIR antibodies which recognize different epitopes within the peptide but whose binding is influenced by the disparate conformations assumed by different fusion proteins and (ii) that part of the epitope(s) for different anti-MIR mAbs is provided by residues contained in other parts of the a chain sequence. This analysis is complicated for two rea- sons. Comparison of the binding of mAbs to fusion proteins

of different length from the same species might be misleading because of the possibility that these artificially produced hybrid proteins fold up in unusual ways. At least in the case of mAb 6 and a-BTX, a clear effect related to the conforma- tion of different fusion proteins can be seen when Torpedo constructs are tested (Fig. 11, as both probes bind more strongly to the shorter construct. Second, direct comparison of the affinities of the mAbs for intact nAChR and the synthetic peptide is difficult, as the peptide is of mouse origin whereas the only intact nAChR readily available for compar- ison is that from Torpedo. However, future measurement of the relative affinities of the mAbs for intact Torpedo nAChR and the corresponding Torpedo peptide might be expected to give some indication of the conformational dependence of the MIR.

As anti-MIR antibodies bind only to the a chain, it is relevant to compare peptide M1 with the corresponding seg- ments of the other subunits. In mouse nAChR, the P, ^7, and 6 chains share 38, 50, and 38% of residues, respectively, with M l ( 2 1 ) ; i n Torpedo nAChR, the homology is 63,50, and 44%

(22). In either case, no more than 2 consecutive residues are shared by the different subunits explaining the observed lack of cross-reactivity.

The present work demonstrates that all or part of the binding site for monoclonal anti-MIR antibodies is present on peptide 61-76 of the a chain. Since some of the mAbs used in this study were among those originally used to demonstrate that a large proportion of polyclonal antibodies, produced either by immunization of rats with Torpedo (3) or Electro- phorus (4) nAChRs or naturally in myasthenic patients (23) are directed against the MIR, it would be reasonable t o expect that such polyclonal antibodies would also recognize peptide M1. However, such sera have low titers of antibodies to mammalian nAChR, rats immunized with Torpedo nAChR having titers of anti-rat nAChR of the order of 0.2-20 nM5 and myasthenic patients having titers of anti-human nAChR of the order of 1-100 nM (24). Because of these low titers, it is necessary to test such antisera at high concentrations (1/

100-1/200 dilutions) and, in our ELISA system, such concen- trations, even of control sera, produce high and, in the case of non-inbred animals, extremely variable background levels of binding. In spite of this, we can detect significant binding of polyclonal antibodies from the sera of 4/16 rats immunized with Torpedo receptor.‘ We are currently testing other detec- tion systems to assess the binding of polyclonal antibodies to peptide M1.

Recently, two other groups have investigated the binding of polyclonal anti-receptor antibodies to synthetic peptides chosen from the sequence of the a chain of Torpedo nAChR (25, 26). Mulac-Jericevic et al. (25) suggest that polyclonal anti-nAChR antibodies from 5 mice and 1 rabbit (all outbred animals) are directed against several regions of the first 210 residues of the a chain (peptides 1-14, 25-36, 41-53, 63-75, 102-114, 128-138, 172-182, 188-198). The apparent discrep- ancy with our results might result from differences in the test systems used (immunoabsorbents instead of ELISA) or the actual peptides used (56-71 and 67-82 in their study compared with 61-76 in ours). A further possibility is that the MIR (defined in the inbred rat but shown to be present in myas- thenic patients) is not immunodominant in the outbred mice and rabbit used in this study. Ralston et al. (26) investigated the binding of polyclonal rat anti-nAChR antibodies t o a range of peptides accounting for 57% of the a chain sequence.

Using iodinated peptide, they found only weak binding of

’

T. Barkas and J-M. Gabriel, unpublished results.

5920 Main Immunogenic Region of Acetylcholine Receptor anti-nAChR antibodies to region 73-90. Again, methodology

differences may play a role; for example we know that chlor- amine-T oxidation of peptide M1 destroys its antigenicity.6 These authors also tested the binding of anti-nAChR to peptides immobilized directly on nitrocellulose, diazophenol- thiol paper, and Biodyne membranes with negative results. It is possible that binding of the peptide to the membrane might mask the binding site as already described for the binding of a-BTX to peptide 185-199 by the same authors (26). How- ever, in both approaches no peptide corresponding directly to M1 was tested (peptides 44-60, 66-83, and 73-90 in the radioimmunoassay; in addition 52-70 was tested on mem- brane-bound peptides). We believe that experiments of this sort show the superiority of the fusion protein approach compared with that of the overlapping (or nonoverlapping) peptide approach in the initial stages of binding site localiza- tion.

Acknowledgments-We would like to thank Dr. J. Schmidt for the chick muscle cDNA library and Dr. J. Boulter for Torpedo cDNA clone aT3. Artwork was provided by the Medical Photography Unit, Lausanne.

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