The EMBO Journal vol. 1 0 no. 1 pp. 101 - 107, 1 991

Mapping of the high affinity FcE receptor binding site tothe third constant region domain of IgE

Ahuva Nissim, Marie-Helene Jouvin1 andZelig Eshhar

Department of Chemical Immunology, The Weizmann Institute ofScience, Rehovot, 76100, Israel and 'Molecular Allergy andImmunology Section, NIAID, National Institute of Health, Rockville,MD 20852, USA

Communicated by B.Geiger

Identification of the precise region(s) on the IgE moleculethat take part in the binding of IgE to its high affinityreceptor (FccRI) may lead to the design of IgE analoguesable to block the allergic response. To localize theFcERI-binding domain of mouse IgE, we attempted toconfer on human IgE, which normally does not bind tothe rodent receptor, the ability to bind to the rat FcURI.Employing exon shuffling, we have expressed chimerice5-heavy chain genes composed of a mouse (4-hydroxy-3-nitrophenyl)acetic acid (NP)-binding VH domain, andhuman CE in which various domains were replaced bytheir murine counterparts. This has enabled us to testthe FcERI-binding of each mouse IgE domain whilemaintaining the overall conformation of the molecule. Allof the chimeric IgE molecules which contain the murineCE3, bound equally to both the rodent and humanreceptor, as well as to monoclonal antibodies recogniz-ing a site on IgE which is identical or very close to theFctRI binding site. Deletion of the second constant regiondomain did not impair either the binding capacity of themutated IgE or its ability to mediate mast cell degrada-tion. These results assign the third epsilon domain of IgEas the principal region involved in the interaction withthe FcERI.Key words: chimeric antibodies/exon shuffling/FcERIbinding/IgE-FceRI interaction

IntroductionThe interaction between immunoglobulin E (IgE) and its highaffinity receptor (FccRI) on mast cells and basophils is a keystep in the allergic response. Bridging of receptor bound IgEby a specific antigen triggers mast cell degranulation andrelease of substances mediating type I immediate hyper-sensitivity. Identification of the precise site on the IgEmolecule which binds to the FcERI is therefore an essentialprerequisite for the understanding of the molecularmechanism of this interaction, and may allow the design ofIgE analogues able to inhibit the allergic response.

Early studies (Stanworth et al., 1968; Ishizaka andIshizaka, 1975), have shown that the receptor binding sitemay be explicitly assigned to the constant region of IgE asisolated Fcc fragment was able to bind to mast cells withan affinity similar to that of the intact molecule. Nevertheless,

fragmentation of the FcE into smaller pieces destroyed itsreceptor binding capacity (Ishizaka et al., 1970). In addition,it has been shown that maintenance of the native conforma-tion of IgE is important in its binding to the FcsRI (Ishizakaand Ishizaka, 1975; Rousseaux-Prevost et al., 1984).Circular dichroism measurements of reduced or heat in-activated IgE implicate the C-3 and Cc4 as the principaldomains involved in the receptor binding (Dorrington andBennich, 1973, 1978). More recent studies (Perez-Monfortand Metzger, 1982) demonstrated that a region in the cleftbetween the second and third C region of rodent IgE wasprotected from proteolysis when bound to the FciRI. Manystudies have attempted to use synthetic peptide analoguesof defined regions of IgE or antibodies against such peptidesto inhibit the IgE-FcERI interaction (Hamburger, 1975;Bennich et al., 1977; Burt et al., 1987; Burt and Stanworth,1987; Robertson et al., 1988). The inhibition obtained inmost of the studies was either not reproducible (Hamburger,1975; Bennich et al., 1977) or resulted in only partial orinefficient inhibition (Burt et al., 1987; Burt and Stanworth,1987). Several anti-IgE monoclonal antibodies (mAb), havebeen raised against the native IgE molecule and are able toblock the binding of IgE to mast cells effectively (Baniyashet al., 1986, 1988). However, because of the possibility ofsteric hindrance, they fail to provide definitive informationregarding specific amino acids involved in the interactionwith the FcERI.The most successful approach for elucidating the precise

site on the IgE molecule that interacts with the Fc-RI utilizedrecombinant DNA technology. Cloned CE gene segmentsof both human (Flanagan and Rabbitts, 1982; Nishida et al.,1982) and mouse IgEs (Ishida et al., 1982; Liu et al., 1982)were expressed either in bacteria (Liu et al., 1984; Kentenet al., 1984; Coleman et al., 1985) or myeloma cells,yielding functional molecules. In a recent study, a 76 aminoacid monomeric recombinant peptide (rE2'-3') spanning theCE2-CE3 junction of human IgE was reported to bind to thehuman FcERI with an affinity similar to native IgE (Helmet al., 1988). A smaller octapeptide, containing sequencesincluded in the CE3 part of Helm's rE2'-3' fragment, wasrecently reported to specifically inhibit histamine release byhuman peripheral basophils (Nio et al., 1990).One of the aims in elucidating the receptor binding site

is to design an IgE analogue which will serve for effectiveblockage of allergic responses. Thus, we have focused ourefforts on murine IgE which allows the testing of the feas-ibility of such an approach using in vivo model systems. Tothis end we have constructed and expressed recombinantmurine IgE, and by deletions, truncations and site directedmutagenesis compiled evidence suggesting that the CE3 isthe principal domain responsible for the receptor binding ofmurine IgE (Schwarzbaum et al., 1989). However, becausesome of the effects observed in our previous study could,in fact, result from mutations distal to the actual binding site

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A.Nissim, M.-H.Jouvin and Z.Eshhar

which induced gross conformational changes in the molecule,we have attempted to employ a complementary approachdesigned to minimize such changes. Therefore, rather thanto destroy the murine receptor binding site, we attemptedto introduce it into a molecule which allows the conserva-tion of its overall conformation. To achieve this, we tookadvantage of the fact that in spite of the large degree ofsequence homology between mouse and human IgE (Liu,1986), the latter does not bind to the rodent receptor, whileboth murine and human IgE bind equally well to the humanFcERI (Conrad et al., 1983). In this study we describe oursuccess in conferring upon human IgE the ability to bindto the rodent FcERI by replacing the human CE3 with itsmurine counterpart. This allows us to unequivocally assignthe receptor binding site to the murine C-3 domain.

a

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Hind III

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Results

Chimeric human-mouse IgEIn order to study the role played by the various Cc domainsof murine IgE in the binding to the Fc-RI, we took advantageof the fact that human IgE does not bind to the rodentreceptor, and replaced various domains in the humanmolecule with their murine homologues. The principalexpression vector we constructed (Hu-pSVC-, Figure la)was similar to one described previously (Neuberger et al.,1985), and contains a murine anti-NP VH segment and theentire human CE region gene. To construct it we replacedthe murine Ce of the Mu-pSVCE (PSV2VH-CE,Schwarzbaum et al., 1989), with the gene encoding humanCc (Flanagan and Rabbitts, 1982). This plasmid contains the

5 A- BomH I

lbo I

I1730I17751860

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Puu 11 1800

Mouse CE Human Cc

bCHM3

CHM2

CHM2M3

CSPD

PCDD

ii Ce NCa2 hlC77I NCe4

| NCd NCH3 NCe4

NHCel 1 ..LJ.EW.L|H16 s.a from MCe2

HCd [1 | HCe4

S1 a. from MCe2HCd

HCeI I

S

Fig. 1. Schematic diagram of (a) the Mu-pSVCE and Hu-pSVCE plasmids which were used to construct the murine-human chimeric rIgE and (b)deletion mutants. The plasmids contain the rearranged VDJ genomic segment encoding VH of an anti-NIP mAb and mouse (Mu-pSVCE) or human(Hu-pSVCE) CE gene. The mutants represent combinations of human exons (open boxes) and murine exons (dotted boxes). CSPD and PCDDrepresent partially (leaving 16 residues) or completely CE2 deletion mutants. S- indicates cysteine forming the interheavy chain disulfide bond.

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Mapping of the FcERI binding site on IgE

Ig enhancer (- 1 kb upstream from the leader exon (E,Figure IA) and upon its transfection into the lambda lightchain expressing J588L myeloma, relatively large amounts(-3-10 tg/ml) of chimeric human IgE with anti-NIPspecificity could be obtained from the supernatants of stabletransfectants; even higher levels were obtained in ascites fluidof mice injected with the transfectomas. The proteinproduced by this transfectoma, was extensively characterizedand found to be identical to the native human IgE moleculein size (Figure 2) and in its ability to bind to the humanFCERI (see below) and low affinity receptor, FcERII (datanot shown). Immunologically, this molecule reacted withpolyclonal and monoclonal anti-human IgE specific anti-bodies (S.Schwarzbaum, A.Hasner and Z.Eshhar, un-published).To construct the chimeric IgE molecules containing the

various murine constant region domains within the humanCE, we swapped the human and murine exons, usingconvenient restriction sites as detailed in Materials andmethods. A schematic diagram of the different human-mouse chimeric IgE mutants generated by the exon shufflingapproach is depicted in Figure lb. CHM2, CHM3 andCHM2M3 are chimeric human CE molecules containing themurine CE2, Cc3 and Ce2+Cc3 respectively. In addition,in order to study the involvement of the CE2 in the FcERIbinding of IgE, we made two deletion mutants, one in whichthe entire CE2 is missing and another in which only 16 aminoacids from the carboxy terminus of the murine CE2 arepresent (Figure lb). Thus, the PCDD mutant contains humanCEc, murine CE3 and human CE4. The CSPD mutantcontains, in addition, 16 amino acids (from Val314-Pro329)of the murine CE2. Because of the localization of the twointerheavy chain disulfide bonds at the CE2 (Cys241 andCys328), we preserved Cys328 in both PCDD and CSPDdeletion mutants to ensure proper dimerization.

All the mutant chimeric genes were expressed in the J588Lmyeloma as secreted anti-NIP IgE antibodies reactive withanti-human IgE. The molecular weight of the resulting rIgEmolecules was studied by immunoblot analysis (Figure 2).All the nondeleted chimeric molecules migrate with the sameapparent molecular weight of 190 kd under non-reducing

conditions (data not shown), and upon reduction displayeda heavy chain band with apparent molecular weight of

- 70 kd. Whereas the human IgE reacted only with anti-human IgE antibodies (Figure 2 lane 2) and mouse IgE onlywith anti-mouse (Figure 2 lane 1), all human-mousechimeras reacted with both anti-human IgE and anti-mouseIgE antibodies (lanes 3-5, Figure 2). The different stain-ing intensities of the chimeric molecules by the anti-mouseIgE reflect the varying content of the relevant antigenicdeterminants in these molecules as seen by the polyclonalantibodies. The antimouse IgE antibodies also contain anti-lambda light chain antibodies (Schwarzbaum et al., 1989)and therefore react to the same extent with the mouse lambdalight chain in all the preparations. The CE2-deleted mutantsreacted under reducing conditions with both anti-human andmouse IgE antibodies and migrated faster (Figure 3A lanes3,4) than the chimeric molecule (lane 2) or the native SPEIgE which always displays somewhat higher molecularweight than the recombinant IgE, most likely due todifferences in glycosylation. The immunoblot analysis of thedeleted mutants electrophoresed under non-reducing condi-tions (Figure 3B lane 3,4), revealed in addition to theexpected shorter H2L2 molecules, a major 93-96 kd band,probably corresponding to heavy-light monomer, a band inthe range of 150-160 kd, and bands of mol. wt > 200 kd(in the case of PCDD). These additional bands most likelyrepresent abnormal pairing, reflecting the possible instabilityof the interchain disulfide bond due to the deletion of oneof the two cysteines involved in the heavy chain dimerization.

_o IW, ap, --

Fig. 2. Immunoblot analysis of the human-mouse IgE chimeras withanti-murine and anti-human IgE antibodies. Affinity purified IgEpreparations were electrophoresed under reducing conditions on 10%acrylamide gels, electroblotted to nitrocellulose and hybridized with'25I-labeled rabbit anti-mouse 1gE or peroxidase labeled goat anti-human IgE antibodies. Note that the anti-mouse IgE antibody alsoreacts with the X chain. The recombinant IgEs were loaded in thefollowing order: murine (lane 1), human (lane 2), CHM3 (lane 3),CHM2 (lane 4), and CHM2M3 (lane 5).

Fig. 3. Immunoblot analysis of the deletion mutants. A, Belectrophoresis under reducing and non-reducing conditions, using a

10% and 5% acrylamide gel respectively. The samples were loaded as

follows: SPE-7- native murine IgE (lane 1), CHM3 (lane 2), CSPD(lane 3), PCDD (lane 4). The presents of 93-96 kd, 150-160 kd and>200 kd bands in the non-reduced deletion mutant preparationsreflects different molecular species due to lack of one of the interchaindisulfide bonds.

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Binding to FceRIThe ability of the different chimeric and mutated IgEmolecules to bind to FC-RI was evaluated by severalindependent assays. The RBL-2H3 rat mast cell line,expressing high levels of surface receptors, served as thesource for the rodent mast cells, while as a source for thehuman receptor we used COS cells transfected with the genesof the at and oy chains of the human FcERI (Kuster et al.,1990; Blank et al., 1989). Figure 4A describes the resultsof several separate competitive inhibition experimentscomparing the ability of different amounts of purifiedchimeric IgE to block the binding of radiolabeled murineIgE to RBL cells. As shown in Figure 4A, the recombinantmurine IgE could effectively block the binding of nativemouse IgE. On the other hand, the chimeric IgE containingthe entire human Cc could not bind to the rodent receptoreven at very high concentrations. As expected, both humanand mouse recombinant molecules and all the chimeras couldbind to the human receptor, thereby inhibiting the bindingof radiolabeled human IgE (Figure 5). Exchanging the 2ndconstant domain of the human IgE with its murine counter-part (CHM2) did not endow the human IgE with the abilityto bind to the rodent receptor. However, exchanging the thirddomain (CHM3), or both CE2 and Ce3 domains (CHM2M3),conferred on the human IgE full reactivity towards the rodentFccRI (Figure 4A).

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01- .,

z0H-mIz

In addition, as an independent test of Fc-RI binding, wefound that the chimeric human-mouse IgE moleculesretained the effector function of IgE, i.e. the ability tomediate mast cell degranulation. As can be seen inFigure 6A, mouse IgE as well as the chimeric moleculescontaining the murine CE3 could bind to RBL, and upon theaddition of polyvalent antigen (NIP-BSA) caused degranula-tion and release of the granular enzyme, 3 hexosaminidase.Once again, human IgE and the chimeric molecule contain-ing the murine Cc2 were not active in this functional assay.Because of sequence homology between the human and

mouse C-2 (36%), the studies described above do notexclude the possibility that amino acids from a homologousportion of the human CE2 play a part in the binding of theCHM3 chimera to the rodent FccRI. To test this possibility,the biological activity of the C-2 deletion mutants werestudied. As shown in the blocking and degranulation experi-ments (Figures 4B and 6B), both the partially deleted CSPDand the fully deleted PCDD mutants could bind to RBL cells.In both assays the apparent specific activity of the PCDDand CPSD mutants was about 8- to 10-fold lower than thatof CHM3. However, because of the heterogeneity in themolecular composition of these deletion mutants, it wasimportant to determine the proportion of the active molecularspecies in the preparations. Indeed, by reacting 1251-affinity-purified preparations with large excess of RBL-2H3 cellsit was found that only 15% of PCDD and 45% of CPSDcould actually bind to the mast cells (data not shown). Usingthese correction factors the actual specificity was determinedto be only 3- to 4-fold lower than CHM3. Interestingly, thesetwo Cc2 deleted IgE molecules could bind (Table I) as wellas mouse IgE to the 84. lc mAb, an anti-IgE antibody whichwas found to recognize an epitope which is identical orclosely associated with the receptor binding site (Baniyashet al., 1988) and has been shown to recognize an epitopeon CE3 (Schwarzbaum et al., 1989).

DiscussionThe main finding of the present work is that the entirebinding site of IgE which binds with high affinity to the

100

.1 1 10

0

100 c

IgE (,ug/ml)Fig. 4. Competitive inhibition of IgE binding to rodent mast cells bythe chimeric (A) and mutated (B) rIgE molecules. RBL-2H3 rat mastcells were preincubated with varying concentrations of purifiedchimeric rigE followed by addition of 75ng 1251-labeled SPE-7 (nativemurine IgE). Percentage inhibition was calculated by the degree ofradioactivity bound using non-inhibited sample as 0% inhibition andsample incubated with 50 Ag cold IgE as 100% inhibition. The actualamount of IgE used in the experiments displayed in B were calculatedto account for the active binding fraction in each preparation, asdetermined from the level of binding plateau obtained in the presenceof saturating amounts of mast cells. Results described in A representthe average of three separate experiments.

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80

60

40

20

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-0--(- Munne-rigE---- Human-rigE

CHM3* CHM2

I& CHM2M3

1 10

Relative Dose1 00

Fig. 5. The recombinant human-mouse chimeric IgE inhibit thebinding of human IgE to human FcERI. The inhibition of binding ofhuman [1251]IgE (PS myeloma protein) to COS cells transientlyexpressing the human FcERI a and y transgenes was determined. Theresults are expressed as the degree of binding obtained when 60 ng ofthe human [1 2I]IgE was added to the transfected cells in the presenceof equivalent amounts (relative dose) of the various chimeric rIgEmolecules. Determination was performed in triplicates.

Mapping of the FccRI binding site on IgE

specialized FcE receptor on mast cells can be assigned to

the third constant region domain. Until now, the second andthird constant region domains and their interface have allbeen implicated as possible FceRI binding sites. Althoughinitial studies suggested the C-3 and Cc4 as the interactionsite (Dorrington and Bennich, 1973, 1978), later studiesfound that the C-4 can be replaced by C-y3 without impairingthe receptor-binding affinity or kinetics (Baird et al., 1990).

100 .

0

z0

-i

z

(9

0

10 100

IgE (ng/ml)Fig. 6. Degranuation of mast cells mediated by the chimeric rIgE (A)and the deletion mutants (B). RBL-2H3 cells were preincubated withdifferent rIgE preparations and washed before the addition ofNIP-BSA. Percentage degranulation was calculated from the release ofthe granular enzyme ,B-hexosaminidase. A and B represent two

separate experiments; each point is the average of three determinations.The actual amount of IgE in the experiments described in B was

determined as explained for Figure 4B.

Table I. Inhibition of rosette formation by anti-IgE mAbs

IgE Preparation Concentration Rosette forming cells

-84. lc +84. Ic

(jsg/ml) (%)

Murine-rIgE 3.0 100 0Human-rIgE 4.3 0 0

CHM3 5.0 100 0CHM2 6.0 0 0CHM2M3 3.0 100 0CSPD 6.0 100 0

PCDD 4.0 100 0

RBL-2H3 mast cells were incubated with the indicated amounts of therIgE in the presence and absence of the anti-IgE mAb 84. ic whichrecognizes an epitope on murine IgE which is closely associated withthe FCERI binding site. The number of rosette forming cells was

determined following the addition of NIP-SRBC. Under theexperimental conditions employed all RBL-2H3 cells gave specificrosettes, all of which were inhibited by the anti-IgE mAb.

Thus, suggests that the role played by Ce4 is in the stabiliza-tion of the active conformation of 1gE required for IgEbinding. In our previous studies (Schwarzbaum et al., 1989),using deletion mutants of murine recombinant IgE, we coulddemonstrate that deletion of a 45 amino acid fragment fromthe carboxy end of CE3 completely eliminated the FcERIbinding activity as well as the binding of anti-IgE specificmAbs which had been found to be related to the IgE- FcERIinteraction site. Although these data favored the CE3 as thereceptor binding site, we could not rule out the possibilitythat the effect seen using the deletion approach could in factresult from conformational changes distal to the site ofmutation.

In the present study we undertook a different strategy toovercome the ambiguity resulting from the mutationalapproach in which a loss of activity following a change ina site has been used to indicate the involvement of this sitein the binding activity. This alternative, more directapproach, involves the construction of the binding site inanalogous but non-binding molecule(s). The system weapplied makes use of the well defined species specificity ofIgE and the fact that human IgE does not bind to the rodentreceptor. Because of the substantial homology in sequence(Ce1, 40%; CE2, 36%; C-3, 47%; C-4. 51%; Liu, 1986)and overall tertiary and quaternary structure between themouse and the human IgE molecules, we expected it to bean excellent system for studying the contribution of differentmurine CE fragments to the FceRI binding site. Thus, byexchanging murine domains with the homologous humanones, it should be possible to pinpoint the exact regionresponsible for the binding of mouse IgE to its FcERI.Employing this rationale, we expressed chimeric human IgEmolecules containing both, or either one, of the murine CE2and CE3 domains (Figure 1 B). In various independent setsof experiments, using either direct or indirect binding to theFccRI, the data obtained (Figure 4,6) clearly demonstratethat introducing the murine CE3 into the human IgE wasnecessary to endow the human molecule with the ability tobind to the rodent receptor with high affinity. Such domainswapping did not impair the ability of the chimeric moleculesto bind to the human FceRI (Figure 5). These data howeverdo not imply that the third constant region domain containsall the necessary elements and is sufficient by itself to accountfor the high affinity interaction with the receptor. In fact,several studies have implicated the CE2 - CE3 interface asthe site of IgE- FceR interaction (Peretz-Monfort et al.,1982; Holowka and Baird, 1983; Baird and Holowka, 1985).Most recently, a recombinant peptide spanning 76 aminoacids at the Ce2-CE3 junction of human IgE (containingthe Gln30' -Arg376 sequence) has been shown to be anefficient inhibitor of human IgE binding to mast cells in vivoand in vitro (Helm et al., 1988, 1989). The CE2 and the CE3parts of this peptide need to be joined since fragments con-taining the separate domains were found to be inactive. Theseresults were challenged in a recent report by Nio et al., whodescribed a synthetic octapeptide (Pro35 - Lys352) fromwithin the human CE3 that was sufficient to block IgEmediated histamine release from human basophils (Nioet al., 1990). In the light of these findings, and the homologybetween mouse and human IgE, the fact that the CHM3chimeric molecule containing the human CE 1 and Ce2,mouse CE3 and human Ce4, binds to both mouse and humanFcERI (Figures 4A,5,6A), could be interpreted as suggestingthat a part of the human C-2 is supplementing the murine

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CE3 in constituting the FcERI binding site. However, bothCE2 deletion mutants generated in the present study, whichlack either most (CSPD) or all (PCDD) of the human CE2but contain the entire murine CE3 (Figure lb), appearedto be almost as active as the other chimeric IgE moleculesin their FccRI-binding capacity either using the indirectinhibition method (Figure 4B) or direct degranulation (Figure6B) and rosette (Table I) assays. We believe that this stronglysuggests that the murine CE3 is both necessary and suffi-cient as the rodent receptor binding site on IgE. In all ourexperiments, the CE2-deleted molecules displayed apparentspecific activities about 8- to 10-fold lower than the nativeor the chimeric molecules. This can be attributed to a dropin affinity due to the CE2 deletion or due to heterogeneityof the CSPD and PCDD preparations. Indeed, comparisonof the electrophoretic pattern of the deleted and intactmolecules under non-reducing conditions (Figure 3), revealsseveral molecular species. Thus, the decrease in specificactivity may be due to instability of the CE2 deleted mutants(resulting from a missing cysteine residue which takes partin the interheavy-chain disulfide bond). If only the intactmolecule (H2L2, which is seen to comprise - 60% (forCSPD) and 20% (for PCDD)) is capable of binding theFcERI, then the actual specific activity of the deletedmolecules is only 3- to 4-fold lower than that of the non-mutated molecules. In fact, binding experiments with excessmast cells revealed similar values (15% for PCDD, 45%for CSPD, data not shown) and were used to determine theactual amount of the IgE used in the experiments displayedin Figures 4B and 6B.

In a separate series of experiments we have recently found(A.Nissim and Z.Eshhar, submitted for publication) that theCSPD and PCDD mutants maintained their binding capacityto the human high affinity FcERI. These and the datadescribed in the present study, suggest that the CE2 mostlikely does not participate in the IgE-FcERI interaction. Webelieve that in the native IgE molecule the Cc2 plays animportant role in the stabilization of the conformation of theFcERI binding site mainly through the two interheavy chaindisulfide bonds. In fact, the 3- to 4-fold drop in the bindingactivity of the CE2- deletion mutants (which lack one of theseS - S bonds) is most likely a direct result of such instability.Our interpretation does not agree with the conclusion drawnby the study of Helm et al. who reported that for efficientbinding of their monomeric polypeptide to human mast cells,a portion of - 30 residues of C-2 has to be joined to a 46amino acids stretch from the CE3 (Helm et al., 1988). Thisapparent controversy is most probably due to the basicdifference in structure between the two preparations. In thepresent study we kept the conformation of the molecule asclose as possible to its native form. On the other hand, therE2'-3' recombinant monomeric polypeptide described byHelm et al. does not maintain the native conformation ofIgE. Therefore it is likely that in this polypeptide the actualFcERI binding site is within the CE3 sequence and that theCE2 portion is required to impose the proper conformationupon the monomeric peptide. In fact, attempts to crystallizeIgE for structural determination has been as yet unsuccessful.Thus the spatial structure of IgE and exact delineation ofits FcERI binding site has to await the X-ray analysis of FcE.Nevertheless, information from studies such as thosereported here, using the same approach to introduce shorterfragments from the murine CE3 into the human IgE molecule

will be necessary for the mapping of the extent and preciselocation of the FcE receptor binding site of IgE.

Materials and methodsPlasmids and vectorsThe Mu-pSVCE and the Hu-pSVCE expression vectors used to express themurine and human IgE molecules in the J588L myeloma are schematicallydepicted in Figure la. They were constructed by inserting the genomic DNAof cloned murine and human e-chain constant region, kindly obtained fromDr T.Honjo (Ishida et al., 1982; Nishida et al., 1982), into the PSV2-VH6plasmid containing the rearranged VH gene of an anti-NP antibody, asdetailed in our previous study (Schwarzbaum et al., 1989).

Exon shufflingTo construct chimeric human IgE molecules which contain various murineCE domains, we deleted defined gene segments from the Hu-pSVCE andexchanged them with various fragments of the murine CE using convenientrestriction enzyme sites. In choosing such sites we preferred those in intronicsequences, thus keeping the splicing signals unharmed to allow in-frameexpression of the designed domains. Schematic representation of the differentchimeric human mouse E-heavy chains generated in this study is describedin Figure lb.To simplify the genetic manipulations, we generated the PGM 14 plasmid

by subcloning the Sail390-PvuII1800 fragment of the human CE into thePGM3 vector (Promega). To construct the CHM3 we deleted the humangene segment spanning Ncol1350-bp1002 using ExolIl (Erase-a-BaseTM,Promega) and replaced it by the murine Aoc 315-860fragment containingthe third CE exon of mouse IgE. The BgII650-Hpa 1730 from this PGM14was then inserted into the Hu-pSVCE instead of Bg1II650-Ncol1350. Theresulting plasmid contained the human CE1, CE2, mouse CE3 and humanCc4 exons. To construct the CHM2 containing the murine CE2 exon in ahuman background, the human AvaI fragments containing the human CE2and CE3 from PGM14 were replaced by the AvaI880_1775 fragmentcontaining the murine CE2 and CE3 exons. From this PGM 14-AvaI plasmida fragment containing the mouse CE3 was removed by Aoc I digest(Aoc 11315 1530 ) and replaced by the Bgl 1960-1470 fragment containingthe human CE3. Finally, the SalI390-NcoII350 fragment from this chimericPGM14 was subcloned instead of the corresponding SalI-NcoI fragmentof the Hu-pSVCE. The CHM2M3 chimeric gene was generated by removingfrom CHM3 the Sa1I390-BstXI1405 fragment containing the human CE2 andreplacing it by the same SaIl -BstXI fragment of PGM 14-Aval containingthe murine CE2.

Exon deletionTwo deletion mutants of the chimeric human -mouse CHM3 gene lackingeither part or the entire human CE2 exon (CSPD and PCDD, Figure lb)were prepared. In the CSPD mutant most of the CE2 was deleted exceptfor a stretch encoding 16 amino acids at the carboxy terminus, keeping theCE2 -CE3 junction unchanged. The PCDD mutant was designed to retainonly one amino acid from the CE2 domain. Both mutants have cysteine328which is involved in the interchain disulfide bonding.To generate the CSPD mutant, a PstI1220 -SpeIS16r) fragment was isolated

from the PGM 14-Aval plasmid and subcloned into the CHM3 vector fromwhich the Sail -SpeI fragment was removed. As a result, a segment betweenhuman SalI390 to mouse PstI1220 consisting of most of CE2 was deleted.Mung bean nuclease was used to maintain the reading frame at the Sail -PstIjunction. To construct the PCDD we used the polymerase chain reaction(PCR) to regenerate a fragment between the bpl269-bpl676. The followingoligonucleotides were used as primers: (i) 5' GGGTCGACTGCCCAGG-TAGGTCTACA 3'; (ii) 5' CTATGGGGTCTTGGTGAT 3'. The firstprimer contains a Sall linker, the coding sequence for Cys328 whichaccounts for the interheavy chain disulfide bond and Pro329 which is thelast amino acid at the carboxy end of CE2 and the sequence of the 5' endof the CE2 -CE3 intron. The second primer contains the coding sequencefor the carboxy terminus of the mouse CE3. The PCR product amplifiedon the murine CE gene was digested by Sall and Spel and subcloned intoCHM3 from which the Sall-Spel fragment was deleted.

Transfection and ceOl cultureThe rat basophilic leukemia mast cell line RBL-2H3 (Barsumain et al., 1981),was grown in minimum Eagle's medium supplemented with L-glutamine,combined antibiotics and 10% fetal calf serum (FCS) (BioLabs, Jerusalem).J588L myeloma cells expressing the lambda-I light chain were grown inRPMI supplemented as above (Oi et al., 1983). The COS monkey kidney

106

Mapping of the FcERI binding site on IgE

fibroblast cell line were grown in DMEM plus supplements as above.Transfections of the chimeric and deleted e-genes into J588L cells and

of the a and y chain genes of the human FCeRI (kindly obtained fromDr J.P.Kinet) into COS cells was performed by electroporation as describedbefore (Potter et al., 1984). Transfected J588L cells were selected in mediumcontaining 1 Ag/ml mycophenolic acid, 250 jig/ml xanthine and 15 pgg/mlhypoxanthine (Sigma). COS cells transiently expressing the human FcERIwere used 48 h following transfection.

Immunoassays and binding assays for IgENative or mutated anti-NIP [(4-hydroxy-3-iodo-5-nitrophenyl)acetate] IgEwas affinity purified from culture supernatants of transfected cells onNIP-ovalbumin-Sepharose columns. IgE anti-NIP was detected by eitherenzyme-linked immunosorbent assay (ELISA) or radioimmunoassay aspreviously detailed (Schwarzbaum et al., 1989).

Binding of IgE to FceRI bearing cells was evaluated either directly orby inhibition of [125I]IgE binding to RBL-2H3 cells (using the SPE-7 mAbas a source of authentic murine IgE, Eshhar et al., 1980). These tests inaddition to the FcERI adsorption and the RBL-degranulation assays wereperformed as described (Schwarzbaum et al., 1989). The IgE specific rosetteassay was performed according to Rittenberg et al., 1969. Briefly, NIP-modified sheep red blood cells (NIP-SRBC), prepared as described wereadded to either RBL or FcERI-COS cells which were preincubated withdifferent concentrations of anti-NIP rigE for 1 h at room temperature. Tubeswere spun (100 x g, 5 min), and following an additional 2 h incubationmixed gently, and, the percentage of rosette forming cells was determinedby counting.

AcknowledgementsWe are grateful to Dr Shelley Schwarzbaum for helpful suggestions andreviewing the manuscript. We thank Dr T.Honjo for the gift of a mouseand human CE clones and Dr J.-P.Kinet for the human FcERI genes. Thisresearch was supported by grants from the NCRD-EEC and GIF. Z.E. isan incumbent of the Marshall and Renette Ezralow Chair in Chemical andCellular Immunology.

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Received on October 8, 1990

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