©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Diphtheria Toxin Binds to the Epidermal Growth Factor (EGF)-like Domain of Human Heparin-binding EGF-like Growth Factor/Diphtheria Toxin Receptor and Inhibits Specifically Its Mitogenic Activity (*)

(Received for publication, August 22, 1994; and in revised form, October 21, 1994)

Toshihide Mitamura (§) Shigeki Higashiyama (1) Naoyuki Taniguchi (1) Michael Klagsbrun (2) Eisuke Mekada (¶)

From the  (1)Division of Cell Biology, Institute of Life Science, Kurume University, Kurume, Fukuoka 830, Japan, the Department of Biochemistry, Osaka University Medical School, Suita, Osaka 565, Japan, the (2)Department of Surgery, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The membrane anchored form of human heparin-binding epidermal growth factor-like growth factor (HB-EGF) acts as the diphtheria toxin (DT) receptor. Transfection of human HB-EGF cDNA into mouse LC cells, L cells stably expressing DRAP27, conferred sensitivity to DT, but transfection of mouse HB-EGF cDNA did not. To define the essential regions of HB-EGF that serve as the functional DT receptor, we examined the sensitivity to DT and DT binding of cells expressing several human/mouse HB-EGF chimeras. It was found that DT binds to the EGF-like domain of the human HB-EGF. However, mouse HB-EGF does not serve as a functional DT receptor due to non-conserved amino acid substitutions in this domain. In addition, CRM197, a non-toxic mutant of DT, inhibited strongly the mitogenic activity of the secreted form of human HB-EGF, but not of mouse HB-EGF and other EGF receptor-binding growth factors. These results confirmed further that DT interacts with the EGF-like domain of HB-EGF and that this interaction is specific for human HB-EGF.


INTRODUCTION

Diphtheria toxin (DT)^1 is a cytotoxic protein (M(r) = 58,342) that inhibits cellular protein synthesis in eukaryotes by inactivating elongation factor 2 through ADP-ribosylation(1, 2) . The toxin binds to a specific receptor on the cell surface(3, 4) , then is internalized by receptor-mediated endocytosis (5) and finally, the enzymatically active A fragment is translocated to the cytosol (6, 7) where the A fragment inactivates elongation factor 2.

The DT sensitivity is determined primarily by the presence or absence of specific receptors for DT(8, 9, 10) . DT receptor (DTR), originally identified in monkey Vero cells(3, 4, 11) , has been shown to be identical to the membrane-anchored form of heparin-binding EGF-like growth factor (HB-EGF)(12, 13) , first identified in the conditioned medium of macrophages and macrophage-like U937 cells(14, 15) . Analysis of the nucleotide sequence of human HB-EGF (hHB-EGF) cDNA predicts a membrane-anchored precursor protein composed of putative signal peptide, pro sequence, heparin-binding, EGF-like, transmembrane, and cytoplasmic domains(14) . The HB-EGF precursor can be cleaved to yield a mature biologically active growth factor containing 75-86 amino acids(16) . Many cell types can release a spontaneously soluble form and/or substantial amounts of the membrane-anchored form of HB-EGF which serves as DTR(13) . HB-EGF/DTR forms a complex with membrane protein DRAP27(13). DRAP27 has been shown to be identical to CD9(17) , and it up-regulates the number of functional HB-EGF/DTR and DT sensitivity in the presence of HB-EGF/DTR (13, 17, 18) .

HB-EGF is expressed in multiple tissues of human, monkey, mouse, and rat species and with a very similar tissue distribution(19) . However, while cells from human and monkey are highly sensitive to DT, those from mouse and rat are resistant(20, 21, 22, 23) . Given these observations, the question arises as to why mouse (e.g. L cells) and rat cells are resistant to DT despite expressing a considerable amount of endogenous HB-EGF(19) . Transfection of hHB-EGF cDNA into mouse L cells confers sensitivity to DT(12, 13) , suggesting that mouse L cells have the machinery needed for DT sensitivity but that for some reason mouse HB-EGF (mHB-EGF) is not a functional DTR. Since the HB-EGF precursor shows about 80% amino acid sequence identity between human and mouse (19) , one possibility is that mHB-EGF may not serve as a functional receptor for DT because of amino acid substitutions.

In this study, we have used serial human/mouse HB-EGF chimeras to study the relationship between HB-EGF primary structure and its function as a DTR. The results of these studies demonstrate that DT binds to the EGF-like domain of hHB-EGF but not of mHB-EGF due to amino acid substitutions. We also show that binding of non-toxic mutant DT, CRM197, to hHB-EGF, but not mHB-EGF, results in the inhibition of the mitogenic activity.


EXPERIMENTAL PROCEDURES

Materials

DT and CRM197 were prepared as described previously(24) . A recombinant mature hHB-EGF corresponding to amino acids 73-147 of the HB-EGF precursor (25) was obtained from Dr. Judy Abraham (Scios-Nova, Mountain View, CA). Mature mHB-EGF was prepared from conditioned medium of LC cells expressing mHB-EGF and partially purified by heparin-Sepharose (Pharmacia Biotech, Ltd., Tokyo, Japan) affinity chromatography. Recombinant beta-cellulin was kindly provided by Dr. Reiko Sasada (Takeda Chemical Industries, Ltd., Osaka, Japan). Amphiregulin was a kind gift from Dr. Gregory Plowman (Bristol-Myers Squibb, Seattle, WA). Transforming growth factor alpha (TGF-alpha) was purchased from Otsuka Pharmaceutical Co., Ltd. (Osaka, Japan).

Cell Lines and Cell Cultures

LC cells, the stable transfectants of mouse L cells expressing DRAP27(17) , were used as the recipient cells for transfection experiments. EP170.7 cells, the stable transfectants of 32D cells expressing human EGFR, were used for the mitogenic activity assay(26) . LC cells were maintained in Dulbecco's modified Eagle's medium supplemented with 100 units/ml penicillin G, 100 µg/ml streptomycin, and 10% fetal bovine serum (ICN Biomedical, Inc., Costa Mesa, CA). EP170.7 cells were maintained in RPMI 1640 supplemented with 100 units/ml penicillin G, 100 µg/ml streptomycin, 10% fetal bovine serum (ICN Biomedical, Inc., Costa Mesa, CA), and 5% WEHI-3 cells conditioned medium(27) . WEHI-3 cell-conditioned medium as the source of IL-3 was prepared as described(28) .

Plasmid Constructions and Transfection

pUC119-21 was constructed from pUC119 (TAKARA SHUZO, Ltd., Kyoto, Japan). A DraII site and two PvuII sites of pUC119 were destroyed by blunting with T4 DNA polymerase and by inserting the HpaI linker, respectively. Then the multicloning sites were replaced with the synthetic linker containing the EcoRI-BstXI-HincII-NotI-HindIII sites in this order. A 740-base pair MluI-DraIII fragment of hHB-EGF cDNA subcloned into pCMV5(13) , and a 650-base pair AvaI-DraII fragment of mHB-EGF cDNA subcloned in pBluescript SK(-) (Stratagene, La Jolla, CA.) (19) were blunted by using the T4 DNA polymerase, and the blunted fragments were inserted into a HincII site of pUC119-21. The resulting plasmids containing hHB-EGF and mHB-EGF cDNA were designated as pTHG-1 and pTMHG, respectively. DraII and SmaI sites were introduced into the coding region of mHB-EGF cDNA of pTMHG by site-directed mutagenesis with an in vitro mutagenesis kit (Amersham, Ltd., Tokyo, Japan), and the resulting plasmid was designated as pTMHG-1. The mutations introduced were synonymous, thus no amino acid substitutions occurred. Chimera plasmids were constructed by exchanging fragments using the corresponding restriction enzyme sites present in pTHG-1 and either pTMHG or pTMHG-1. A plasmid encoding HDelta(63-105) was constructed as follow. pTHG-1 was digested with PvuII and DraII, then ligated to the synthetic adapters (5`-CTGCTACCCCTAGGAGGCGGCCGGGACCGGAAAGTCAGG-3`, and 5`-GTCCCTGACTTTCCGGTCCCGGCCGCCTCCTAGGGGTAGCAG-3`). The amino acids corresponding to the restriction enzyme sites and the structures of the chimeric HB-EGF molecule are illustrated in Fig. 1. After constructing the various chimera plasmids, BstXI-NotI fragment of each plasmid was inserted into the corresponding restriction enzyme sites in the expression vector, pRc/CMV (Invitrogen Corp., San Diego, CA). Construction of all chimera plasmids was confirmed by the DNA sequencing. Resulting plasmids were transfected transiently as described previously (13) by the calcium phosphate method(29) . Transfected cells were cultured for 48 h and then used for further studies.


Figure 1: Structures of human HB-EGF, mouse HB-EGF, and human/mouse HB-EGF chimeras. A, alignment of amino acid sequences of human and mouse HB-EGF. The amino acid residues are numbered on each lane. Single and double underlines indicate the predicted signal sequence and the transmembrane domain, respectively(14) . A basic amino acid cluster which serves as a heparin-binding domain (HBD) is represented by a bold underline. EGF-like domains are indicated by shaded boxes. Restriction enzyme sites used for constructing chimera plasmids are indicated by the upper lines. B, schematic structures of the HB-EGF chimeras and the summary for the results of DT sensitivity and DT binding assays are shown. Shaded and open boxes indicate the regions of human HB-EGF and mouse HB-EGF, respectively. ED were determined from the data of Fig. 2. Specific binding of DT was determined by the incubation with 100 ng/ml I-DT as described under ``Materials and Methods.'' The values represent the average of two independent experiments, and variations from the means are shown in parentheses.




Figure 2: DT sensitivity of LC cells transiently expressing hHB-EGF, mHB-EGF, and the human/mouse chimeras. LC cells were transfected with various plasmids and cultured for 2 days. Cells were incubated with various amounts of DT for 2 h, followed by incubation with 1 µCi/ml [^3H]leucine for 1 h. Radioactivity incorporated in protein was determined. The values represent the average of two independent experiments, and bars indicate variations from the means. Some points omitted bars to avoid the complication of lines, but variations from the means were less than 6%. A, LC cells expressing hHB-EGF (bullet), and mHB-EGF (), and LC cells transfected with the vector, pRc/CMV (). B, LC cells expressing H(1-50) (bullet), H(1-68) (), H(1-84) (), H(1-106) (circle), H(1-136) (up triangle), and H(1-186) (box). C, LC cells expressing H(186-208) (bullet), H(136-208) (), H(106-208) (), H(84-208) (circle), H(68-208) (up triangle), and H(50-208) (box). D, LC cells expressing H(106-136) (bullet), H(136-186) (), H(106-186) (), and HDelta(63-105) (circle).



DT Sensitivity Assays

DT sensitivity was assayed by measuring the inhibition of protein synthesis by DT as described previously(30) . The values represent the average of two independent experiments. In this study, ED was defined as the effective dose of DT which reduces protein synthesis to 75% of the control values.

DT Binding Assays

Binding of I-labeled DT to cell was measured as described(13) . Nonspecific binding of I-DT was assessed in the presence of a 1,000-fold excess of unlabeled DT. Specific binding was determined by subtracting the nonspecific binding from the total binding obtained with I-DT alone. The values represent the average of two independent experiments.

Inhibition of Growth Factor Activity by CRM197

EP170.7 cells (26) were incubated with one of the EGF family growth factors or IL-3 (WEHI-3 cell-conditioned medium) and various concentrations of CRM197 for 48 h at 37 °C, followed by incubation with [^3H]thymidine for 4 h, and radioactivity incorporated into DNA was measured(13) . The values represent the average of two independent experiments.


RESULTS

To show that mHB-EGF cannot serve as the DTR, the plasmid containing mHB-EGF cDNA, cloned from a macrophage library(19) , was introduced into recipient cells. LC cells, which are the stable transfectants of mouse L cells expressing DRAP27, were used as the recipient cells through this study. Although DRAP27 itself does not confer any DT sensitivity without HB-EGF/DTR, it up-regulates the number of functional HB-EGF/DTR and DT sensitivity in the presence of HB-EGF(13, 17, 18) . LC cells were transiently transfected with mHB-EGF cDNA. Immunoprecipitation of surface-biotinylated HB-EGF with anti-HB-EGF antibody showed the expression of substantial amounts of mHB-EGF protein on the cell surface (data not shown), but cells did not show any DT sensitivity (Fig. 2A). In contrast to mHB-EGF, transfection with hHB-EGF cDNA made these cells sensitive (Fig. 2A), confirming previous reports(13) . The rate of protein synthesis of LC cells was not diminished below 50% in the range of DT concentrations studied because the transfection efficiency was about 50% throughout the experiments, and the remaining cells were left untransfected. These results indicate that mHB-EGF does not have the optimal amino acid sequence for being a functional DTR.

To define which part of substitutions are needed to confer DT sensitivity, plasmids encoding various human/mouse HB-EGF chimeras were constructed. Since the amino acid substitutions between human and mouse HB-EGF are distributed throughout the entire region of the HB-EGF open reading frame (Fig. 1A), serial chimeras were constructed, covering the entire structure (Fig. 1B). The various chimera plasmids were transiently transfected into LC cells and assayed for DT sensitivity and DT binding. Among the chimeric HB-EGF constructs containing human sequence in the N-terminal region, only the expression of H(1-186) conferred DT sensitivity at the same level as hHB-EGF; others, H(1-50), H(1-68), H(1-84), H(1-106) and H(1-136), did not (Fig. 2B). Among the chimeric HB-EGF constructs containing mouse sequence in the N-terminal region, the expression of H(50-208), H(68-208), H(84-208), and H(106-208) conferred full DT sensitivity at the same level as hHB-EGF. Cells expressing H(136-208) showed moderate sensitivity (about 100 times less sensitive than cells expressing hHB-EGF), and cells expressing H(186-208) did not show any sensitivity at all (Fig. 2C). These results suggested that the human sequence between Asp and Tyr was sufficient to confer full DT sensitivity to LC cells but that the homologous region in mHB-EGF was not. The importance of the human sequence between Asp to Tyr for DT sensitivity was confirmed by three other chimeric HB-EGFs, H(106-136), H(136-186), and H(106-186). Of these, only the expression of H(106-186) conferred full DT sensitivity (Fig. 2D). The expression of H(136-186) slightly increased DT sensitivity, similar to H(136-208), but the expression of H(106-136) had no effect at all. These experiments showed directly that the human sequence from Asp to Tyr is essential for the expression of complete DTR activity. Furthermore, to define the essential region for DT sensitivity more precisely, we constructed plasmid encoding HDelta(63-105) which is hHB-EGF with a deletion from Asp to Arg. LC cells expressing HDelta(63-105) showed full DT sensitivity (Fig. 2D), indicating that the region between Asp to Arg is not necessary for DT sensitivity. These results were not due to different transfection efficiencies or different expression levels of chimeric HB-EGF molecules on the cell surface as could be shown by immunofluorescence staining or immunoprecipitation of surface biotinylated HB-EGF with anti-HB-EGF antibody (data not shown).

To examine whether the inability of mHB-EGF to promote DT sensitivity was due to a deficiency in DT binding, the DT binding activity of several LC cells expressing transiently the native and chimeric forms of HB-EGF was examined (Fig. 3, summarized in Fig. 1B). DT binding correlated positively with DT sensitivity in that cells with higher sensitivity to DT showed greater DT binding. These results suggest that the inability of mHB-EGF to promote DT sensitivity stems from its inability to bind DT due to the amino acid substitutions. Furthermore, as shown in the Fig. 3inset, Scatchard plot analysis demonstrated that the binding constant for DT of LC cells expressing H(106-186) (K(a) 3.1 times 10^8M) was almost same as that obtained with native hHB-EGF (K(a) 3.6 times 10^8M), indicating that this chimeric HB-EGF sequence possesses full binding activity. In addition, LC cells expressing HDelta(63-105) also showed the similar binding constant for DT (K(a) 4.7 times 10^8M) and further confirms that region Asp to Arg is not necessary for the binding of DT.


Figure 3: Binding of I-DT to LC cells transiently expressing hHB-EGF, mHB-EGF, and human/mouse chimeras. LC cells were transfected with various plasmids and cultured for 2 days. Cells were incubated with various concentration of I-DT for 9 h at 4 °C, and the cell-associated radioactivity was determined. The data are expressed as specific binding. Nonspecific binding of I-DT for LC cells expressing hHB-EGF or H(106-186), H(136-186), and H(106-136) was less than 5%, 10-20%, and 30-40%, respectively. The values represent the average of two independent experiments, and bars indicate variations from the means. The symbols used are hHB-EGF (circle), mHB-EGF (up triangle), H(106-136) (bullet), H(136-186) (), or H(106-186) (). Inset, Scatchard plots of DT binding for hHB-EGF (circle) and H(106-186) ().



So far our studies have indicated that Asp-Tyr in HB-EGF is the critical region for the binding of DT and subsequent DT sensitivity. However, the critical region may be narrower. We have demonstrated previously that recombinant mature hHB-EGF, which is composed of Arg-Ser, binds DT with an affinity similar to that of the transmembrane HB-EGF/DTR(13) . This would suggest that the amino acid residues in Leu-Tyr are not necessary for DT binding and that the critical region of HB-EGF that mediates DT sensitivity is HB-EGF106-147, which is essentially the EGF-like domain of HB-EGF.

HB-EGF is mitogenic via the binding of its EGF-like domain to the EGFR (14) . The binding of DT to the EGF-like domain of hHB-EGF suggests that DT might be an inhibitor of the mitogenic activity of HB-EGF. Thus, the possibility that CRM197, a non-toxic mutant form of DT (24) with similar or higher binding affinity to DTR(31) , inhibits HB-EGF mitogenic activity was examined. EP170.7 cells (26) require IL-3 or EGFR ligands for growth. EP170.7 cells cultured with the human mature form of HB-EGF (HB-EGF73-147) in the absence of IL-3 were stimulated to incorporate [^3H]thymidine into DNA. Addition of CRM197 completely inhibited DNA synthesis in EP170.7 cells cultured with mature hHB-EGF but not with mature mHB-EGF (Fig. 4A). In addition, CRM197 did not inhibit DNA synthesis in EP170.7 cells cultured with IL-3, which ruled out the possibility that CRM197 itself is directly toxic for the growth of EP170.7 cells. Since CRM197 inhibited the mitogenic activity of hHB-EGF, but not that of mHB-EGF, it appears that the inhibition is due to interactions with specific amino acids in the EGF-like domain of hHB-EGF substituted for in mHB-EGF.


Figure 4: Effect of CRM197 on the mitogenic activities of human and mouse HB-EGFs and other EGF family growth factors. EP170.7 cells were incubated with either one of several EGF family growth factors (10 ng) or IL-3 (WEHI-3 cell-conditioned medium), and various concentration of CRM197 for 48 h, followed by incubation with [^3H]thymidine for 4 h. Radioactivity incorporated into DNA was measured. The values represent the average of two independent experiments, and bars indicate variations from the means. A, the symbols used are hHB-EGF (bullet), mHB-EGF (), or IL-3 (). B, the symbols used are EGF(bullet), TGF-alpha (), amphiregulin (), or beta-cellulin (circle).



The EGF homologous growth factors, TGF-alpha, EGF, beta-cellulin, and amphiregulin also are mitogenic via the EGFR. As shown in Fig. 4B, CRM197 did not inhibit any of these EGF-like growth factors, demonstrating that CRM197 specifically inhibits the mitogenic activity of HB-EGF.


DISCUSSION

DTR has been shown to be identical to the membraneanchored form of HB-EGF(12, 13) . HB-EGF/DTR is expressed in multiple tissues of many species including primate and murine(19) . Yet it has been known for a long time that murine cells are much less sensitive to DT than are primate cells. For example mouse L cells are about 100,000 times less sensitive to DT than are monkey Vero cells(9) . Several lines of evidence indicate that DT sensitivity is determined primarily by the number of DT-specific receptors, i.e. the amount of HB-EGF on the cell surface(4, 8, 9, 10) . Nevertheless, expressing cell surface HB-EGF is insufficient to produce DT sensitivity in mouse cells. We have demonstrated this directly in transfection experiments in which expression of hHB-EGF confers DT sensitivity to mouse cells, but expression of mHB-EGF does not. In exploring the mechanism of the differential sensitivity of human and mouse HB-EGF/DTR to DT, we have found that the ability to bind DT and the consequent toxicity is inherent in the primary structure of HB-EGF/DTR, particularly in the EGF-like domain. Non-conserved amino acid substitutions in mHB-EGF results in loss of DT sensitivity.

We have concluded that the critical area for DT binding resides in the residues Asp to Ser as found in the human sequence, but not in the mouse sequence. This is the EGF-like domain of HB-EGF. The strongest evidence is that (i) a chimeric HB-EGF encompassing human, but not mouse, amino acids Asp to Tyr is fully active in mediating DT toxicity, (ii) that a deletion mutant encompassing human Asp to Arg is also fully active, and (iii) that as previously shown, secreted HB-EGF, encompassing Arg to Ser is sufficient for full DT binding activity(13) .

The critical amino acids within Asp to Ser needed for DT sensitivity have not been refined any further. Altogether there are 10 substitutions between human and mouse HB-EGF, 8 non-conserved residues in the region Asp to Pro, and, in addition, human Glu mouse His and human Ser mouse Thr. That the chimera H(106-136), which contains human residues 106-136, and the chimera H(1-136), which contains human residues 1-136, are mostly inactive in DT binding and sensitivity implicates the importance of the downstream human Glu and human Ser residues in DTR activity. The relative inactivity of the chimera H(136-186), which contains human residues 136-186, and the chimera H(136-208), which contains human residues 136-208, suggests that the human residues upstream of Pro are important for DTR activity. Further studies involving single amino acid substitutions might provide additional insight into exactly which amino acid residues in the EGF-like domain are the critical ones for DT sensitivity.

We have also demonstrated that the non-toxic DT mutant CRM197 inhibits specifically the mitogenic activity of hHB-EGF but not the mitogenic activity of mHB-EGF. Studies of the structure-function relationships for EGF-EGFR interactions indicate that several amino acid residues located in anti-parallel beta-sheets of EGF as well as those highly conserved amino acid residues in the other EGFR ligands contribute to the EGF-EGFR interaction without drastic alteration of the overall structure(32, 33, 34) . This suggests that EGF binds to EGFR at the entire region of the molecule. Present studies suggest that the interaction of hHB-EGF with DT may also occur at the entire region of the EGF-like domain. Thus, it is reasonable to speculate that inhibition of mitogenic activity of hHB-EGF by CRM197 is due to the masking of or competition for binding sites for EGFR on the hHB-EGF molecule.

Moreover, CRM197 does not inhibit other EGFR-binding growth factors. Therefore, although these EGF family members are also produced as membrane-anchored precursors and bind to EGFR in a manner similar to HB-EGF, HB-EGF is probably the only EGF family member that can function as an efficient receptor for DT among these growth factors. Thus, CRM197 may be useful as a specific inhibitor for hHB-EGF. Alteration of CRM197 structure, based on knowledge of the three-dimensional structure of DT, may be useful for producing highly potent inhibitors of HB-EGF, stronger than CRM197, or compounds of altered specificity that would inhibit other EGF family members.


FOOTNOTES

*
This work was supported by grants from the Ministry of Education, Science and Culture of Japan and Ciba-Geigy (Japan) Foundation and National Institutes of Health Grants CA37392 and GM47397 (to M. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of the Japan Society for the Promotion of Science Postdoctoral Fellowship.

To whom correspondence should be addressed: Div. of Cell Biology, Institute of Life Science, Kurume University, Kurume, Fukuoka 830, Japan. Tel.: 81-942-376317; Fax: 81-942-313320.

(^1)
The abbreviations used are: DT, diphtheria toxin; DTR, DT receptor; EGF, epidermal growth factor; EGFR, EGF receptor; HB-EGF, heparin-binding EGF-like growth factor; hHB-EGF, human HB-EGF; mHB-EGF, mouse HB-EGF; TGF-alpha, transforming growth factor alpha; IL-3, interleukin-3.


ACKNOWLEDGEMENTS

We thank Kuniaki Nakamura for help with the indirect immunofluorescence staining and Fumie Nakano for help with the plasmid preparations. We would like to thank Dr. Judy Abraham for providing recombinant mature human HB-EGF, Dr. Reiko Sasada for recombinant beta-cellulin, and Dr. Gregory Plowman for amphiregulin.


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