Localization of a highly immunogenic region on the acetylcholine receptor α-subunit

Vol. 135, No. 1, 1986 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

February 26, 1986 Pages 82-89

LCEALI~~OFAHI~YI~ CRFGIcNcNmEArxIYumLINF. RE- U-SUBUNIT

MiryC.Souroujon, Drorit Neumann, Sergio Pizzi@ella, Anat Safran and Sara Fuchs

DeparlmEa-ltofchanicalIIIpnmology, TlxWeizmam Instituteof Scieme,

Re?mvot 76100, Israel

Received December 17, 1985

AntLbodiestosyn~ticpfptideswere ~+oyeciinorder toraqdmainsonthE? a-submit of thzac&ylcholine recqtortowhi~severalmoncclonalamibo&ia are directed. Five peptides correspomling to residues l-20, 126-143, 169-181, MO-340 ard 351-368 of the receptor Ck&mnitwere synthesized and antibodies against t&n were elicited ?heanti-pq3tickantibodieswereqAoym3alcng withthe monoclonal antibodiestoidentifyfra~tsof S. aurpysV8protease digested-a-skunk in immoblottingeqxrinxmts. Our resultsckmnstrate thatahiwy ~cregionof~OL-~tislocatedonacarboxy-ter- minal14kLhportionofthe~-submit. Thisregionalsosears to Lmdfrgo anti- g3i-ric &ages chringmscle~elopnmt. AmxxclonalantSoodydlrectxl a~thecholinergicbindFngsiteoft2-heacetylcholiner~~r react&with an18M>aseqmtofthechxkunitwhichbound Mmqaroto~ aswellas antibodies directed against pqtide 169-181. B 1986 Academic Press, Inc.

lhanicotinic ace~lcholinereceptor (AQ-R) is acanplex&nmltic%atennWant

protein, and consists of four sxhunits a,B,Y,&, present in a stoichiomtry of

2:l:l:l respectively. This protein mcoqases avarietyof fmctionaldmains

suchastkeligandbindingsite, anion&armel, phoqhorylationsites, glyco-

sylationsitfx and Mnunogmic regionshhichmaybe involved in the innmm

responseto~andin myadkmia gravis (see l-5 for reviews). In order to

analyze~dcwa~in~A(7NI~todel~~~~r~~toPICNi

inmyasUEMawahavepreviouslypreparedalibraryof Inonoclonalant&odies

(mAtx) elicited against Torpedo KhR (6-8). ThesemAkeweeqllcIyedas

probes to study the function, metabolic ti pathogxkity of the AUiR (8-10).

~~theantigenic~~~to~~themcAhsare~~isfeasi-

ble, byexrployingantibodig agahstsyn~cpq%idesco~gti

select&regionson~AG-Bmolecule. ?hus, itmaybepossible topiqmint

t&region responsible for a certain function. Thisqroa~has~qlayed

inourlaboratmyin ordertolocalize~ligandbindLngsiteofthere3eptor

0006-291X/86 $1.50 Copyright 0 1986 by Academic Press, Inc. All rights oJ’ reproduction in any form reserved. 82


(11) - 0th~ groupbaveutilizedantikdiesto syn~icpeptidfzs for analyz-

tig the tr ansnanbrane orientation of the ACZIR molecule (12-14), and its main

WC region (15,16).

Inthis~~have~loyedantibodlestosynthetic~~desoftheAChR

cl-~tinor~rtolocalizetheantigenic~~~~towardswhichsome

m=Abs are directed. Our results danonshate that tbre majority of xAke are

directedtowardsa14ki3acarbo~-termina 1 region of the a-submit, indicating

thataNc$ily hmmmqnic&nnainresidesthere. We also &mm&rate that a

~ZRJ (5.5) direc&d against the receptor ligand bindirq site (7), binds an 18

kDA fragnentoftia-subunitwhichalsorexc+zes antibodies againstpeptik

169-181.

-Durificatian: AChR purification frcm m was performad as previously ckscrihd (17).

-of A library of m&s has been elicited against Torpedo FLNi in two different hybridizations as previously reported (6,7)*.

): SynthesisofpqtickscorrespmdMg resides l-20 and 126-43 of the KhR u-submit was described elsewhere (11,:). Pqtidescoreqondiq to residues 169-181, 330-340 and 351-368 of Torpedo AChR a-subunit were synthesized similarly by Um Merrifield solid phase procedure (19) and their amino acid ratios correqorded well witi that expeckd. The

pqticks were conjugated to bovine serumalhmh (BSA; Signa)using l-ethyl-3-3'-dh&hylam.incprcpylcarhdiimide (Siqa) as the coupling reagent (20) -

Q: AChRwasresolved to its submits inpolycryl~degelelectsophoresis (PKE), containhg lithim ckodecyl sulfate (UX;21). Digestion of tha a-suhmitwithS. aura V8 protease was performed as previously described (11). Electr@oretic trans- fer of tha resolved AU-B subunits or tha proteolytic frawts of th a-tsukamit, aswell astoxlnwerl~Yandt~werlay~reperformedas described (11,22). For antibody overlay the stripe were imubated with a 1:lOO dilution of tha different. antibodies in whi6l-1 was used at 1:75 dilutions).

qum&ing buffer (except for IX& 5.5 Allantipqtideantiserakereaksorbedona

calm of S-aureus V8 protease prior to use. AfterSwashesin#xxphate buffered saline (Pm), and ore waq2p PBS contahing 0.5% tri@l x-100, the stripswereimxhatedforlhr~ I-1abeledProteinAor I-labeled-goat ~ti~-hnmoglobuUns(5xlO cpn/ml). lhestripswerethenwasbedas above, and autoradiogrw.

?he~tspecificityofthemonoclonal~tibodleserplayedinthis~

wasdetelmhed~~ ixmmmoblotting method (Fig. 1). Allthesem=Absboradto

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a b c d ef g

Fia.1: subunit specificity of nrAbs. ToqxdoACnRwaselectr~resedina l~plyauylamide gel, blotted ~~nitrocellulose filters ardcwerlayedwith ths differsIt mzAk5 follow& by I-labeled-GaMIg. Overlaywitha, mAb 1.34; b, mcAb 5.14; c, rxAb 1.39; d, mSb 1.22; e, mAb 1.26; f, mAb 5.5 and g, rdib 5.46.

thecr-sukunitkereas someofthanboundto additionalsuhmits aswell. Thus,

mmoclonal antibodies 1.34, 5.14 and 5.5 reacted predcminatly with the

a-subunit (Fig. la,b,f), mzi#x 1.39 and 5.46 cross reacted with the c1 and

6 -Wts (Fig. lc,g) and m=Abs 1.22 and 1.26 cross reacted with the a and B

subnits (Fig. ld,e) . It should be noted that the imnmoblotting conditions

qzplied, enabled to determine the suhmit specificity of mzAks which previously

failed to react with the isolated subunits in radiohmnmoassay in solution

(e.g. 5.5 and 5.46 (8).).

ForprecisemaFpingoftlreantigenic~~~tamrdswN~themcAbs

are directed w a&plied blot analysis of S. sure V8-proteolytic fragnmts of

the a-subunit. ?Ire V8-proteolytic framts were overlayed with the m=Ahs, and

withantibodiestosyntheticpeptidescorrespandingto~ frcmtheAChR

a--t. All the anti-pE@ick imtibodiesglployedcross-reactedwith~

AChR and bound specifically to its a-subunit (11, 18 and data not Shown).

Blot analysis of seven xmdbe is ShowIl in Fig. 2. Of Chese, IIKAX 1.22,

5.14, 1.34, 1.39 and. 5.46, all reacted with W same proteolytic fravt of

14 kDa (Fig. 2a-e). 0-1 the other hand, m3it.1 5.5 (Fig. 2f) andm=Ab 1.26 (Fig.

2g) bou& to an 18 kDa fragnmt and a 22 kDa fraqxznt respectively. As can be

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a b c d e f 9 a b c defgh

-2: Binding of IIKACS to proteolyzed a-submit. ?I& ehxtr~b, proteolyzed a-dxmitwasl~~otted.antonitrccellulose filter andoverlaydwith e%a TfcA?ze foll&by I-labele&-GaMIg. CNerlay with: a, nrAb 1.22; b, nkdb 5.14; c, nrAb 1.34; d, mAb 1.39; e, IKNJ 5.46; f, nrAb 5.5 and. g, ITC& 1.26.

-3: BbdingofmcAbs andantipeptide antibodies toproteolyzed a-submit. ~~~ipeptide~t~~2~readedwithproteolyzeda-~tfol- 1oWd.W I-labeled-G&lIa or I-labeled-protein A as follows: a, n&b 1.26; b; II&&I 5.14; c, anti-peptiae 351-368ktibodies; d, anti--da l-20 antibaiies; e, anti-peptide 126-143 anrtibodig; f, mcAb 5.5; g, a-ECX andh, anti-pqtide 169-181 antibodies.

sem in Fig. 3 WE 14 kEa fragnent which contajns m antigenic sites of these

five m&s (Fig. Za-e and Fig. 3b) is also r-z& by antibodies prepared

againsta~~ticpEptidewfiichcorrespondstoresi~ 351-368 oftha

a-subunit (Fig. 3~). Antibodies to mtide l-20 and to pqtide 126-143 of the

a-subunit did not bind to this 14 kDa fragwnt, nor did

1251-labeled-a-bungarotoxin (a-EYIX) (Fig. 3d,e,g). Thissuggeststhattbe14

kJk fragnmtrqx-esmts asegnmt frcxnthecarboxyterminalpx-tionofthe

a-subunit.

By qloying our anti~tide antibodies we also set forth to map the site on

the a-submit towards which mc?b 5.5 (specific for the ligand binding site of

AChR; 7) isdirected. As seen in Fig. 3 (f-h) mcAb 5.5, a-ETX and antibodies

to a synthetic pqtide correqonding to residues 169-181 of t&a-subunit, all

bound to W sama 18 kDa V8 fragnart of %e a-s&unit. This is in accordarxx

withourpreviousstudy (11) inwhichwehavepostiatedthatthis18kDa

ligandbimiing site, lies beyond residue Asp 152 of the a-subunit.

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In~sstudy~haveenployedantibodiesto~~icpeptidescorrespord-

ingtodefinadExqumms 0ftlxAchR u-subunit, inorder timaptiantigsllc

&terCmntstowhi~severalantiAChRnr=Abs aredirected. Tablelsunmarizes

the binding specificities of tk mAke and antipeptide antibodies with S,

aurecls V8 protease digested a-subunit. ItwasshownthatagfoupofmAbe

(m=Abs 1.34, 1.22, 5.14, 1.39 and 5.46) are all directed at one 14 kDa V8 pro-

teolytic fraQnent of the a-subunit. Itshouldbenotedhowever, thatalthouc$

thesemduls reactedwiththe sama fragnenttkyarenotdirectedagainstickm-

tical antigmicdete rminants as thy differ in thair subunit specificity (Fig.

l), as well as in other characteristics (8).

Blotting~rimentshavedanonstratedthattbe14E8a fragnentwhihbinds

manyofthemclAbsalsobindsantibodiestoasyntheticpepti~~~~to

residues 351-368, and is therefore derived frm the carboxy-terminal portion of

the a-subunit. Also, mAbs directed at this region as well as anti-peptide

351-368 antibodies, did not bind to trypsinat&. P&R (24) which is devoid of

its carboxy-terminal portion (25). Sincemkswouldbeexpe&edto represent

a statistical saupling of the antibody specificities obtained in anti-m

sera, this domain is probably a hi@-iLy inmmogmic region of the receptor.

Thiscor~lusionis StrengUxmedbyexpariments inourlabxatorywhichindicate

thatthemainresponseinra?,%its, two we&s following injection with ACbR, is

to this 14 kDa fragnent (data not shown). It mrs that tfie WC

TlYAEIE I: Imxmologicalcharacterizatimof

s. v8 proteasa digested a--t

Fragnmt

NW m3.b Anti-pptida oael-marIcers

22 1.26

18 5.5 169-181 a-m (11.22)

17 126-143 GUI A (23)

14 1.34,1.39,5.14 351-368

5.39.5.46

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regiontowhichmxtofournr=Abs aredirectedisdifferent franthemain i.mW

n~cregion(MIR)as~f~byTzartosandLindstram(26),~have~

theirMIRtotheNH2- terminal end of the o-subunit (16.27).

Unlikethe abovementioned fivemonoclonalantibodies~chbindthe1418a

fragxnt, we found that n-&b 1.26 reacted with a 22 kDa fragrrent (Fig. 2g).

This mcAb also reacted in a different manner than mcAbs 1.34, 1.22, 5.14 and

1.39 when tested with junctional and extrajunctional ACbR (28). All the latter

antibodies reacted preferentially, thou* not exclusively, with the extrajunc-

tional formof~receptor, whereasmAb1.26didnotdiscriminatebetween

junctional and extrajunctional receptors. In addition, antibodies against a

seqxme corresponding to residues 351-368 of the human and calf a-subunit

reactedprefermtially with the extrajunctional AChR from mouse muscle as corn-

paredtojunctionalAdXiR,inasimilar mxuker to that displayed by m=Abs 1.22,

1.34, 5.14 and 1.39 w et al., in preparation). +Ikese observations may

suggestt-hatthe14kDa fragnentcontains aregionwhi&urndergoes&velapnen-

talchangasintheprocess of synaptogslesis.

kkAb5.5whichisdirectedtowardstheligandbindingsite, boundto the

sama18kik fragm-&whi&was alsorecoSylizedbya-BTXandbyantibodiesto

stick 169-181 (Fig. 3 f,g,h). W results are in accordancewithprevious

findings from our laboratory that mcAb 5.5 is specifically directed towards the

actualbindingsite. As anti~tide l-20, 126-143 and 351-368 antibodies did

not bind to this 18 R&I toxin-binding fravt, we propose that it should map

at a sequmce present betwem residues 153 (the first S. V8 protease

cleavage point after AS-I 142) and 350.

hkawishtoUmnkDr.MatiFridkinfor fruitfuldi scussions aridhelpful advice in peptide synthesis. Thisworkwassqportedbygrantsfromthakscular I@trqhyAssociationofAmarica, TheLosAngeles Chapter ofthe&maWEnia ~avisFotndatian~theUnitedStates-IsraelBinatioMlScienceFoundatian (BSE) *

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Vol. 135, No. 1. 1986 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

24. Bartfeld, D. and Fubs, S. (1979) Biochm. Biqz&ys. Res. Commm. 8p, 512-519,

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Localization of a highly immunogenic region on the acetylcholine receptor α-subunit

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