Proc. Nati. Acad. Sci. USAVol. 81, pp. 1351-1355, March 1984Biochemistry

Biologically active synthetic fragments of epidermal growth factor:Localization of a major receptor-binding region

(phosphorylation/DNA synthesis/receptor clustering/antigenic determinants/peptide synthesis)

A. KOMORIYA*t, M. HORTSCHt§, C. MEYERS*¶, M. SMITH*II, H. KANETYt, AND J. SCHLESSINGERt*Laboratory of Chemoprevention, National Cancer Institute, National Institutes of Health, Bethesda, MD 20205; and *The Department of ChemicalImmunology, The Weizmann Institute of Science, Rehovot 76100, Israel

Communicated by Christian B. Anfinsen, November 14, 1983

ABSTRACT A primary receptor-binding region of mouseepidermal growth factor (EGF) was identified by comparingthe relative affinities of selected synthetic fragments with over-lapping sequences in the EGF receptor-binding assay, usinghuman foreskin fibroblasts. Only synthetic peptides contain-ing the amino acid residues 20-31 in the mouse EGF sequenceshowed the ability to compete with 1251-labeled EGF in bindingto EGF receptors. The affinities of the cyclic EGF fragment[Ala2°JEGF-(14-31) and the linear [(S-acetamidomethyl)-Cys20,31]-EGF-(20-31) were approximately 1/104 of the affini-ty of EGF. Despite their reduced receptor affinities, these twopeptides exhibited the in vitro biological activities of nativeEGF, while fragments from other regions of the EGF moleculewere devoid of these biological properties. The peptides in-duced DNA synthesis in human foreskin fibroblasts as mea-sured by [3H]thymidine incorporation into DNA. They alsoinduced EGF receptor clustering and activated the EGF-sensi-tive kinase, enhancing the autophosphorylation of EGF recep-tors in a dose-related manner. Moreover, a major antigenicdeterminant of EGF for rabbit anti-EGF antibodies was iden-tified within this same localized region of the EGF molecule bycompetition experiments utilizing the synthetic EGF frag-ments. The predominant EGF antigenic determinant(s) wasalso found within the fragment [(S-acetamidomethyl)Cys20,311-EGF-(20-31). The accessibility of the residues in positions 20-31 for antibody recognition is consistent with the conclusionthat these residues constitute or contain a major receptor-binding region for EGF.

Epidermal growth factor (EGF) is a single-chain polypeptideof 53 residues with three disulfide bonds which define threelooped regions from residues 1-20, 14-31, and 32-53 (1). It isa potent stimulator of cellular proliferation and inhibitor ofgastric acid secretion (2). EGF has received a renewed inter-est in recent years for the finding that tyrosine-specific ki-nase activity is associated with the EGF receptor (3). EGFalso enhances the action of transforming growth factors insoft agar assays (4, 5), and the recently isolated and charac-terized sarcoma growth factor binds to EGF receptor (6).Recent studies on the mechanisms of the mitogenic action

of EGF indicate that, upon binding to its diffusely distribut-ed receptor, rapid clustering of the EGF receptors is fol-lowed by their internalization. Concurrently, EGF inducesrapid responses such as the uptake of ions (7), enhancedphosphorylation of various membrane proteins (including theautophosphorylation of the EGF receptor) (8, 9), changes incell morphology (10), and the reorganization of cytoskeletalproteins (11). How these early cellular responses to EGF re-late to the mitogenic process and the way in which EGF acti-vates its receptors to initiate these responses are unknown.While the isolation and characterization ofEGF were report-

ed a number of years ago (2, 12), very little is known aboutthe structure-function relationships of EGF. To date, onlythe importance of the COOH-terminal sequence of EGF-(47-53) has been studied in some detail (2, 13-15). Derivatives ofEGF and urogastrone lacking either one or two residuesfrom the COOH terminus possess the same activity as theintact molecule both in vivo and in vitro. Further deletion ofthe COOH-terminal five or six residues leads to a markedreduction in both receptor affinity and mitogenic activity invitro (13, 16). Despite the reduced potency of the derivativedevoid of the COOH-terminal hexapeptide, EGF-(1-47) iscapable of inducing a full biological response. Therefore, theresidues critical to the intrinsic activity of the molecule arecontained within the 1-47 sequence.We report here further structure-function studies ofEGF,

using synthetic fragments prepared by solid-phase methods(17). In designing the synthetic fragments, we took advan-tage of the high conformational stability of the EGF mole-cule owing to the presence of three disulfide bonds. Thethree cyclic EGF fragments prepared were therefore definedby the three disulfide bonds of EGF, and a short peptide(residues 20-31) was designed according to an insight pro-vided by a computer-modeled tertiary structure of EGF (un-published data). Determination of the relative receptor affin-ities of the three cyclic peptides, combined with those of se-lected synthetic peptides with overlapping sequences,allowed us to identify a major receptor-binding region ofEGF. The EGF-specific nature of the cellular responses tothe active synthetic peptides is indicated by their ability toinduce various "EGF-like" early and delayed responses.

MATERIALS AND METHODSEGF was purified from submaxillary glands of adult malemice by the method of Savage and Cohen (14). EGF wasradiolabeled with 1251 by using the Chloramine-T method(18) to a specific activity of 100,000 cpm/ng of protein. [-32P]ATP (5000 Ci/mmol; 1 Ci = 37 GBq) was purchasedfrom New England Nuclear, and [methyl-3H]thymidine (47Ci/mmol) was purchased from Amersham. Pansorbin waspurchased from Calbiochem-Behring. Anti-EGF serum wasraised in rabbits and gamma globulin fraction was obtainedby ammonium sulfate precipitation. The monoclonal anti-body TL5-IgG was prepared and purified as described (19).TL5-IgG was labeled with the sulfonyl chloride derivative

Abbreviations: EGF, epidermal growth factor; HFF, human fore-skin fibroblasts; Acm, S-acetamidomethyl.tPresent address: The Department of Biology, The Johns HopkinsUniversity, 3400 N. Charles Street, Baltimore, MD 21218.§Present address: European Molecular Biology Laboratory, Post-fach 10.2209, D-600, Heidelberg, F.R.G.Present address: SmithKline and French Laboratories, Departmentof Immunology, 709 Swedeland Road, Swedeland, PA 19401.1Present address: Clinical Hematology Branch; National Institute ofArthritis, Diabetes, and Digestive and Kidney Diseases, NationalInstitutes of Health, Bethesda, MD 20205.

1351

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "adv ertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

1352 Biochemistry: Komoriya et al.

(RB200 Sc) of rhodamine lissamine-B (Research Organics,Cleveland, OH), according to the method of Brandtzaeg(20).

Cells. Human foreskin fibroblasts (HFF) were from pri-mary cultures obtained from M. Gabbay. Only cells frompassages 9-14 were used. Human epidermoid carcinomacells (A-431) were kindly provided by G. Todaro. The cellswere grown in Dulbecco's modified Eagle's medium(GIBCO) with 10% heat-inactivated fetal calf serum andequilibrated with 10% CO2 in air.

Synthesis and Purification of EGF Fragments. EGF frag-ments were synthesized according to the Merrifield solid-phase method (17), using a Beckman model 990B peptidesynthesizer. Anhydrous hydrogen fluoride treatment of pep-tide resins for 1 hr at 0C in the presence of 10% (vol/vol)anisole and 1% ethanedithiol effected the cleavage from res-in and the deprotection of peptides. Further deprotection offormyltryptophan in fragment (43-51) was accomplishedwith 0.1 M piperidine treatment in ice for 30 min (21). Thefree disulfhydryl peptides were cyclized in dilute aqueoussolution at pH 6.8-7.0 by oxidation with potassium ferricya-nide (22). The peptides were purified by gel filtration chro-matography on Sephadex G-10 and G-25 (fine) eluted with 1M acetic acid, followed by ion-exchange chromatography onDEAE-cellulose or CM-cellulose as appropriate. Purity ofthe synthetic peptides was monitored by high-performanceliquid chromatography on Waters Associates ,BondapakC18 analytical columns (0.39 x 30 cm), in water/acetonitrilegradients of 10-40% containing 0.1% CF3COOH. Where fur-ther purification was necessary, reverse-phase liquid chro-matography on ,uBondapak C18 columns (0.78 x 30 cm) wasperformed with the same elution system. The peptide con-centrations and amino acid compositions were determinedafter acid hydrolysis in constant-boiling HCl containing traceamounts of phenol at 110°C for 24 hr.Radioimmunoassays. The selected concentrations of the

peptides and 125I-labeled EGF (125I-EGF) (100,000 cpm/ng)were prepared in phosphate-buffered saline containing 1%Triton X-100 and 0.1% NaDodSO4. Aliquots (200 ,ud) of1:3000 dilutions of rabbit anti-EGF antibody were incubatedwith 50 IlI of 125I-EGF (or 50,000 cpm) and 50 ,.l of eachpeptide solution overnight at 4°C. A suspension of Pansorbin(50 u1l of 10%, wt/vol) was added to each sample and themixture was incubated for an additional 2 hr. Then the sam-ples were centrifuged and washed three times with phos-phate-buffered saline, and the radioactivity was determined.

Visualization of Fluorescently Labeled EGF Receptors. TheA-431 cells were plated at low density on glass coverslips.Cells were washed twice with phosphate-buffered saline.Coverslips were then incubated at 4°C for 30 min and at 37°Cfor an additional 40 min in 60 ,ul of Dulbecco's modified Ea-gle's medium (40 mM Hepes, 0.1% bovine serum albumin)containing either EGF or synthetic peptides with rhodamine-labeled monoclonal antibody against EGF receptor, TL5-IgG. Cells were washed three times with cold phosphate-buffered saline and fixed with 3% (vol/vol) formaldehyde.Rhodamine fluorescence was observed with a Zeiss IM35inverted microscope. Photographs were taken on Kodak Tri-X films.

Phosphorylation Reaction. The levels of enhancement ofEGF receptor autophosphorylation by the synthetic peptideswere determined as follows: shed membrane proteins (30 ,ug)from A-431 cells prepared as described by Cohen et al. (23)were incubated with either EGF or synthetic peptides at25°C for 25 min and at 0°C for 5 min. Then an aliquot of [y-32P]ATP (10 ,ul, 10 nM) was added and the mixture was incu-bated an additional 10 min at 0°C. Final incubation volumewas 50 Al containing 20 mM Hepes at pH 7.4, 3 mM MnC12,0.2% Triton X-100, and 0.01% bovine serum albumin. Elec-trophoresis sample buffer was added to terminate reactions

and samples were boiled for 5 min. Then, the samples wereelectrophoresed on NaDodSO4/5-15% polyacrylamide gra-dient gels. After the electrophoresis, gels were dried andautoradiography of the gels was done. The extent of the EGFreceptor autophosphorylation was quantified by scanningpercent transmission of the autoradiogram, using a BeckmanDU-8 spectrophotometer.

Radioreceptor binding competition assays were carriedout according to previously described procedures (24) ex-cept for the incubation temperature of 40C.

Stimulation of DNA synthesis by the synthetic peptideswas determined by using HFF cells as previously described(24).

RESULTSBinding Activity of Various Synthetic Peptides to EGF Re-

ceptor and to Anti-EGF Antibodies. Synthetic peptides cov-ering the sequence ofmouse EGF from residue Asn-1 to Glu-51 were tested for their ability to inhibit the binding of 125I1EGF to EGF receptor of HFF. The following syntheticpeptides were used in the radioreceptor assay: [Ala14]EGF-(1-20), [Ala20]EGF-(14-31), [(Acm)Cys20'31]EGF-(20-31)(Acm, S-acetamidomethyl), EGF-(32-48), and EGF-(43-51).Neither the NH2-terminal fragment [Ala14]EGF-(1-20) northe COOH-terminal fragments EGF-(32-48) and EGF-(43-51) showed any ability to compete with 125I-EGF in the re-ceptor binding assay (see Fig. 1) at concentrations reaching100 gM. Only the synthetic peptides containing the middleregion of the EGF molecule, namely cyclic [Ala20]EGF-(14-31) and linear [(Acm)Cys20'31]EGF-(20-31), were able toblock 60% and 40% of the binding of 125I-EGF to EGF recep-tor, respectively. In another experiment the same peptidesblocked 90% of the binding of 1251-EGF to EGF receptor ofrat pituitary cell line 6H3 (unpublished data). In terms of theconcentration required for 50% inhibition of 125I-EGF bind-ing to the EGF receptors, the affinity of EGF is 3 x 104times that of these peptides, while in terms of the free energyof ligand-receptor complex formation, the peptide retainedabout 50% of the free energy of association for EGF andEGF receptor (-6 kcal/mol as compared to -12 kcal/molfor EGF**).

If indeed these residues constitute a primary receptor-binding region of EGF, then the residues 20-31 should beexposed and accessible for other intermolecular interac-tions. We examined the accessibility and conformation ofthe middle loop region by using antibodies against nativeEGF (i.e., identification of the antigenic determinant of EGFin this region). Fig. 2 shows the ability of the fragment[(Acm)Cys20'31]-EGF-(20-31) to compete with 125I-EGF fordirect binding to rabbit anti-EGF antibodies. Overnight incu-bation with the peptide, chosen to ensure attainment of equi-librium, caused 80% inhibition of the binding of 125I-EGF toanti-EGF antibodies. This indicates that the residues 20-31constitute major antigenic determinants for mouse EGF.Hence the location of the middle loop residues in native EGFtertiary structure is available for intermolecular interactions.Observed accessibility of the regions to antibody binding isconsistent with the view that residues 14-31 or a shorter se-

**The free energy of EGF and EGF receptor complex formation canbe estimated from the reported apparent equilibrium dissociationconstant of the complex, 1-1.5 x 1O-' M (25). Assuming no coop-erativity involved in the ligand-receptor complex formation, thefree energy of peptide and EGF receptor complex formation canbe determined from the apparent equilibrium dissociation con-stant for peptide estimated from radioreceptor competitive bind-ing assays using the expression Kj = ID50/(1 + L/Kd), in whichKi, ID50, L, and Kd are equilibrium dissociation constant for fig-and, concentration of ligand at 50%o inhibition, concentration offree radiolabeled ligand, and dissociation constant for radiola-beled ligand, respectively.

Proc. NatL Acad Sci. USA 81 (1984)

Proc. NatL. Acad Sci USA 81 (1984) 1353

0.D 50-

25

50 100Peptide, AM

FIG. 1. Inhibition of 125I-EGF binding to HFF by synthetic EGFfragments. HFF cells were incubated at 40C with 1 nM 125I-EGFwith increasing concentration of peptides: o, [Ala IEGF-(1-20); *,[Ala20]EGF-(14-31); *, [(Acm)Cys20'31]EGF-(20-31); o, EGF-(32-48); and A, EGF-(43-51).

quence of 20-31 constitute a primary receptor-binding re-gion for EGF. The NH2- and COOH-terminal synthetic frag-ments were not recognized by anti-EGF antibodies (data notshown).

Synthetic Peptides Activate the EGF-Sensitive Kinase andStimulate DNA Synthesis. Possible segregation of residues re-sponsible for receptor binding and biological activity was ex-amined. EGF activates a tyrosine-specific cyclic-nucleotide-independent protein kinase, which phosphorylates the re-ceptor molecule and other cellular proteins (3, 8, 26). EitherEGF or the various synthetic- peptides were added to mem-branes prepared from A-431 bells. Fig. 3 shows that besidesEGF only the two peptides that block the binding of EGF toits membrane receptor, namely peptide [Ala20]EGF-(14-31)and the shorter peptide R(Acm)Cys20'31]EGF-(20-31), inducethe phosphorylation of various membrane proteins. As withEGF, the main phosphorylated protein is the 170,000-daltonEGF receptor. Further, the enhancement of the phosphoryl-ation of this receptor by the peptides is dose related (see Fig.4)._EGF is a potent mitogen of fibroblasts. Hence, we exam-

0

-6.0.0vjos

.-I

Peptide, M

FIG. 2. Competitive binding of "'I5-EGF to rabbit anti-EGF se-

rum with either unlabeled EGF (-) or [(Acm)Cys20'31]EGF-(20-31)(----). Error bars represent SEM.

~

1 2 3 4 5 6 7 8

FIG. 3. Enhancement of membrane protein phosphorylation byEGF and synthetic peptides. The figure is autoradiography of Na-DodSO4/5-15% polyacrylamide gradient gel electrophoretic analy-sis of shed membrane vesicle preparation using A-431 cells incubat-ed with [Y_32P]ATP and EGF (33 nM, lane 1), [Ala14]EGF-(1-20)(350 MM, lane 2), [Ala20]EGF-(14-31) (350 ,M, lane 3), [(Acm)-Cys20,31]EGF-(20-31) (125 AM, lane 4), buffer alone (lane 5),[(Acm)Cys31]-EGF-(25-42) (100 MM, lane 6), EGF-(32-48) (210 AM,lane 7), or EGF-(43-51) (210 ,M, lane 8). Arrow by lane 1 indicatesthe position for 170,000-dalton EGF receptor.

ined the capacity of the synthetic peptides to induce DNAsynthesis in HFF (see Fig. 5). Again, only the synthetic pep-

tides containing residues 20-31, [Ala20]-EGF-(14-31) and[(Acm)Cys20'31]EGF-(20-31), enhanced the [methyl-3H]thy-midine incorporation into HFF. The [Ala20]EGF-(14-31) in-duced more than an 8-fold increase in [methyl-3H]thymidineuptake in HFF, reaching the level achieved by the addi-tion of EGF (5 nM). The shorter linear synthetic peptide

FIG. 4. Dose-related enhancement of the EGF receptor auto-phosphorylation by [Ala']EGF-(14-31). Shed membrane vesiclesprepared from A-431 cells were incubated with [Ala2']EGF-(14-31)at various concentrations. The levels of autophosphorylation werequantified by scanning percent transmission of autoradiograph filmsof the gels (traces A, D, E, and F). The results are plotted at the top.

Biochemistry: Komoriya et aL

1354 Biochemistry: Komoriya et al.

800

.2

ao0 Q.

u 0 400C di-0

.

E 200

25 50

Peptide, ,uM75 100

FIG. 5. Enhancement of [methyl-3H]thymidine incorporation inquiescent HFF by synthetic peptides [Ala'4]EGF-(1-20) (o),[Ala24]EGF-(14-31) (e), EGF-(32-48) (o), and EGF-(43-51) (A).Broken line indicates the level of [methyl-3H]thymidine uptake ob-served for 5 nM EGF stimulation.

[(Acm)Cys20'31]EGF-(20-31) was equally as active as cyclic[Ala20]EGF-(14-31) (data not shown). The concentrationsof these two peptides used to achieve maximal uptake of[methyl-3H]thymidine were 75 AM and 100 ,uM, respective-ly.

Synthetic Peptides Induce the Clustering of EGF Receptor.To confirm that the effects of the synthetic peptides are me-diated by the EGF receptor, we examined the effects of thepeptides on the distribution of the EGF receptor. Fig. 6 illus-trates the immunofluorescence visualization of the receptorsin A-431 cells. We made use of a monoclonal antibodyagainst EGF receptors, TL5-IgG, which neither interfereswith binding of EGF to the receptor nor induces receptor

clustering by itself (19). Hence, the fluorescently labeledTL5-IgG allowed us to visualize the EGF receptors and ob-serve the effects of either EGF or the synthetic peptides onthe cellular distribution of this molecule. As previously re-ported (19), TL5-IgG alone failed to induce the receptor clus-tering at 37TC (Fig. 6A). However, the addition of eitherEGF (10 nM; Fig. 6B) or the synthetic peptides [Ala20]EGF-(14-31) (480 AM; Fig. 6C) and [(Acm)Cys20'31]EGF-(20-31)(190 ,4M; Fig. 6D) induced receptor clustering indicated bythe appearance of the fluorescent patches. The other NH2-and COOH-terminal synthetic peptides [Ala14]EGF-(1-20),EGF-(32-48), and EGF-(43-51) failed to induce the receptorclustering. Therefore, the induced cellular responses by thepeptides containing residues 20-31 are receptor-mediatedevents.

DISCUSSIONThe present studies establish that the synthetic peptides[Alal']EGF-(14-31) and [(Acm)Cys20'31]EGF-(20-31) inducea series of cellular responses like those to EGF. Judgingfrom the ability of these peptides to compete with 125I-EGFin binding to the receptors and by their retention of half ofthe free energy (estimated from apparent Kd of ligand**) as-sociated with EGF-receptor complex formation, residues20-31 define a primary receptor-binding region of EGF.Rather low affinities of these peptides to the receptors canbe explained by the expected low value for the conforma-tional equilibrium constant (Kc; see Appendix) for the pep-tides, as was determined from the levels of anti-EGF anti-body recognition toward these peptides.The concentration difference between EGF and the pep-

tide [(Acm)Cys20'31]-EGF-(20-31) required for competitivebinding of 125I-EGF to antibody at the half-maximal competi-tion level was about five orders of magnitude (see Fig. 2).Hence, one can estimate a conformational equilibrium con-

FIG. 6. Induction of EGF receptor clustering in A-431 cells by synthetic peptides. Patches of fluorescence indicate the clustering of EGFreceptors induced by 10 nM EGF (B), 430 ,uM [Ala20]EGF-(14-31) (C), or 190 ,uM [(Acm)Cys20'31]EGF-(20-31) (D). (A) Blank with onlyrhodamine-labeled anti-EGF receptor monoclonal antibody (TL5) added.

I 2

_ ~~ ~ ~ =Qm 0

Proc. NatL Acad Sci. USA 81 (1984)

1.

Proc. Natl. Acad ScL USA 81 (1984) 1355

stant for [(Acm)Cys20'31]EGF-(20-31) in native conforma-tion to be in the order of 10-5 (see Appendix). The affinity ofthe peptide to EGF receptors would then be expected to belower by the same magnitude (105) when compared withEGF. We observed a concentration difference of 3 x 104_fold. A similar degree of reduced affinity is reported for theNH2-terminal hexapeptide of glucagon. This hexapeptidebinds to glucagon receptors and stimulates adenylate cyclaseactivity at 105-fold higher concentrations than required forglucagon (27). Hence, if the cooperativity needed for stabi-lizing native conformation of a peptide containing receptorbinding residues is not present, one would expect the recep-tor affinity and biological activity to be affected by the re-duced conformational equilibrium to native conformation,Kc. The data suggest, therefore, that the primary functionalroles of the NH2- and COOH-terminal regions of the EGFare to provide conformational stability for the middle region(residues 20-31). The reduced /3-sheet conformation and re-ceptor affinity observed for EGF-(1-48) (28) support theproposed functional role of COOH-terminal residues. It isnoteworthy that, unlike EGF, the receptor-binding region ofinsulin seems to be defined by residues brought together inspace by its tertiary structure (29). Also, in contrast to ourobservations with EGF fragments, micromolar concentra-tions of insulin B-chain fragments induce biological respons-es that are not receptor mediated (30). The observed proper-ties of [Ala20]EGF-(14-31) and [(Acm)Cys20 31]EGF-(20-31)indicate that the receptor-binding residues and the receptor-activating residues reside within the sequence 20-31 region.The present data, however, support the view that the bindingof a small peptide to the receptor is by itself sufficient toinduce both the early and delayed EGF cellular responses.

APPENDIX

Kc [1]Pr Pn,

in which Pr and Pn are peptide fragments in random and na-tive conformation. Kc is a conformational equilibrium con-stant for peptide and is defined as [Pn]/[Pr] (31).Antibody (Ab), which has been raised against native pro-

tein, binds to a peptide fragment in native conformation as:

Ka 2Ab + Pn AbPn, [2]

in which the peptide contains antigenic determinant(s). Ka isthe association constant for the peptide and antibody and isdefined as [AbPn]/[Ab][Pnj. The value of Ka can be estimat-ed to be 109 M-1 from the apparent equilibrium dissociationconstant of 10-9 M determined from radioimmunoassays.Hence,

Kc = [Pn]/[PrI = [AbPn]/Ka[Ab][PrI. [3]

Since Pr >> Pn, Pr Pt, in which Pt is the total peptideconcentration. Also, at 50% inhibition of radiolabeled EGFbinding to antibody by peptide, [AbPn] = [Ab]. The expres-sion for Kc is simplified as:

K, [AbPn]/Ka[Ab][Pt] 1/Ka[Pt].

For the present study,

K__ 1/(109 M-1)(10-4 M) _ 10-5.We acknowledge the assistance of Dr. Nancy Acton in peptide

synthesis. We thank Richard Feldmann for assistance in EGF struc-ture model bulding. We thank S. Gill for expert technical assistanceand Y. Yarden and M. Burmeister for fruitful discussions. We thankalso S. Dalton and T. Wilson for manuscript preparation. M.H. is arecipient of a long-term Minerva Fellowship.

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