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Identification of a small epitope in domain Ib of Pseudomonas aeruginosa exotoxin A that elicits enzyme-neutralizing antibodies
FEMS Microbiology Immunology 89 (1992) 267-272 @ 1992 Federation of European Microbiological Societies 0920-8534/92/$05.00 Published by Elsevier
267
FEMSIM 00215
Identification of a small epitope in domain Ib of Pseudomonas aeruginosa exotoxin A that elicits enzyme-neutralizing antibodies K a r i n e R u t a u l t ~, D o m i n i q u e Coin ~, M a r i e - J e a n n e V a c h e r o n ~, Micheline G u i n a n d ~, J e a n W a l l a c h b a n d G e o r g e s Michel ~' " Laboratoire de Biochimie Microbienne, and h Laboratoire de Biochirnie Analytique, Unit:ersit~ Claude Bernard Lyon I, Villeurbanne, France
Received 12 March 1992 Accepted 24 March 1992
Key words: Pseudomonas aeruginosa; Exotoxin A; Peptide-thyroglobulin conjugate
1. SUMMARY A peptide corresponding to amino acids 392404 of the amino acid sequence of Pseudomonas aeruginosa exotoxin A (the last 13 amino acids of domain Ib) was synthesized and coupled to thyroglobulin. The conjugate induced an antiserum in rabbits with high antibody titer against native toxin as measured by ELISA, and this antiserum was highly efficient in inhibiting the ADP-ribosyltransferase activity of exotoxin A. These data corroborate the potential importance of amino acids 400-404 in the enzymatic mechanism of exotoxin A.
2. I N T R O D U C T I O N Exotoxin A (ETA) is the most toxic of the extracellular enzymes produced by Pseudomonas Correspondence to: M. Guinand, Laboratoire de Biochimie Microbienne, Universit~ Claude Bernard Lyon I, 43 Boulevard du 11 Novembre 1918, F-69622 Villeurbanne, France.
aeruginosa. It has been isolated, purified and extensively characterized as described in several reviews [1-3]. It is a single-chain protein with a 613-amino acid sequence and four disulfide bridges. It contains three distinct domains: Ia-Ib, II and III (Fig. 1). The active site is located in the carboxy-terminal domain III but the last five residues of the adjacent domain lb are essential for a full expression of enzymatic activity [4]. The other two domains Ia and II are involved in eucaryotic cell binding and in translocation respectively [5]. Toxicity is considered due to an inhibition of protein synthesis in eucaryotic cells, by transfer of ADP-ribose from NAD + to elongation factor 2 (EF2) [6,7]. Diphtheria toxin (DT) has a remarkably similar enzymic property [8]. Several approaches have been used to define functional epitopes ofP. aeruginosa ETA. Synthetic peptides or mutants containing different deletions in the ETA structural gene [9] as well as monoclonal antibodies [10,11] have proven to be very useful in this respect. Antibodies against a subtiIisin-generated fragment of ETA (S-fragment), containing the last 13 residues of domain
268
Ia
I[
I ExotoxinA
NH
252
lh
II1
364 404
613
2-I
S- Fragment
Tridecapeptide
392{ 613 Nil 2-- { [~"~'~i~{~;~'~:'}~i'~i':'~i~i~ - - COOil
t
I R-N-Y-P-T-G-A-E-F-L-G-D-G [
Fig. 1. Location of the S-fragment and of the tridecapeptide within the ETA molecule. The three domains of the molecule are indicated as follows: domains la and Ib are white, domain II is hatched, domain III is grey. The S-fragment derived from subtilisin cleavage contains residues 392-613. The tridecapeptide (white, residues 392-404) is the C-terminal portion of domain lb.
lb and the entire domain III (Fig. 1), neutralize the ADP-ribosyltransferase activity of ETA [12], but antibodies against the sole domain III fail to neutralize this activity [13]. The N-terminal tridecapeptide of the S-fragment was therefore proposed to be a functional epitope [12]. In order to test this assumption, the corresponding tridecapeptide R N Y P T G A E F L G D G was synthesized and conjugated to a carrier protein for rabbit immunization. Antiserum against this sequence was obtained, and its inhibitory effect on ADPribosylation activity of ETA and of D T was studied.
3. M A T E R I A L S AND M E T H O D S
3.1. Toxins ETA used in this study was prepared fromP.
established by UV analysis of a resin sample d e p r o t e c t e d by 20% p i p e r i d i n e / d i m e t h y l formamide. The - S H group of Cys was protected by the triphenylmethyl group and the guanidino function of Arg by the 4-methoxy2,3,6-trimethylbenzene sulfonyl group. Threonine was introduced as Fmoc-Thr-l-oxo-2-hydroxy-dihydrobenzotriazine ester and all other Fmocamino acids as Pfp esters. After the linkage of the Gly residue, all Fmoc-amino acids were coupled using a 3.5 molar excess in presence of 1-hydroxybenzotriazole with a coupling time of 30 rain for the next six amino-acids and 60 min for subsequent residues. Each Fmoc deprotection stage involved t r e a t m e n t with 20% p i p e r i d i n e / dimethyl-formamide for 10 rain. Cleavage of the peptide from the resin was achieved by a 6 h t r e a t m e n t with trifluoracetic acid-anisoleethanedithiol (95:3:2), followed by successive washings of the resin with ether.
aeruginosa strain 103, as previously described [12]. DT was a gift from Prof. J.P. Reboud (University Lyon I).
3.2. Synthesis of the peptide The pentadecapeptide RNYPTGAEF L G D G C G was synthesized by the solid-phase method (Ultrosyn A resin, 0.1 m e q / g ) with 9-fluorenylmethoxycarbonyl (Fmoc) derivatives. The first amino acid (GIy) was coupled as Fmocglycine-pentafluorophenyl ester (Pfp) (5 molar excess). The degree of attachment of Gly (93%) was
3.3. Conjugation of the peptide to thyroglobulin The pentadecapeptide was conjugated to thyroglobulin (TGB) using m-maleimidobenzoyl Nhydroxysuccinimide ester (MBS) as hetero-bifunctional cross-linker according to the procedure of Schmidt and Schmidt [14]. TGB (10 mg) was dissolved in 1.5 ml of phosphate-buffered saline (PBS) and mixed with 1 ml of dimethylformamide containing 5 mg of MBS. After 2 h with stirring at room temperature, the conjugate was
269 separated from unreacted cross-linker by dialysis against 0.1 M phosphate buffer, pH 6. The pentadecapeptide (1.9 rag) was combined with the TGB-MBS conjugate and stirred for 15 h at room temperature.
as a standard [15]. Peptide sequencing was performed in a protein microsequencer (Applied Biosystems, model 407 A).
4. RESULTS
3.4. Peptide- TGB conjugate antiserum New Zealand white rabbits were immunized intramuscularly (5 X 0.2 ml) and subcutaneously (5 x 0.2 ml) on days 0, 28 and 42 with the peptide-TGB conjugate (150/xg, protein weight) dissolved in 1 ml PBS supplemented with 1 ml of Freund's complete (first injection) or incomplete (following injections) adjuvant solutions (Difco). Rabbits were bled 57 days after the first injection and the serum was decomplemented (56°C, 30 min), filter-sterilized (0.45 ixm) and stored at - 2 0 ° C . Antisera against exotoxin A and S-fragment have been previously obtained [12]. 3.5. Enzyme-linked immunosorbent assay (ELISA) The ELISA was performed essentially as described [12]. The microtiter plates (Micro Elisa Dynatech) were coated with 100 /xl of the antigens at 5 /xg/ml (ETA, DT, TGB protein and conjugate) or at 10 /xg/ml (free peptide). All assays were performed in duplicate. ELISA titers were calculated by multiplying the reciprocal of a serum dilution within the linear portion of the dilution curve (A490, 0.4 to 1) by the corresponding A490 value. 3.6. Neutralization of ADP-ribosyltransferase actiuity ETA and DT were extemporaneously activated at 25°C for 15 min with 4 M urea and 1% dithiothreitol. Neutralization of enzymatic activity with antisera was evaluated by incubating 10 Izl of activated ETA (160 ng) or activated DT (117 ng) with 10/xl of antiserum at final dilutions of 1:2 to 1:200, at 37°C for 30 min. The enzymatic assays were then performed in duplicate as described previously [12]. Normal rabbit serum was used as a negative control. 3. 7. Other procedures Protein concentration was determined by the Bradford procedure with bovine serum albumin
4.1. Synthesis of the peptide and conjugation to TGB The selected pentadecapeptide RNYPTGAEFLGDGCG, prepared by the solid-phase method, corresponds to residues 392-404 of ETA with an additional Cys residue at the C-terminal end for coupling to a protein carrier by a heterobifunctional cross-linking agent. A Gly residue was also added to avoid peptide racemisation during the synthesis. The crude peptide (88 rag) was purified by HPLC on a C18 reversed-phase column with a discontinuous gradient of acetonitrile in 0.1% trifluoracetic acid. The pure peptide was eluted between 20 and 23% of acetonitrile (data not shown). It was lyophilized immediately to prevent disulfide bond formation. Total identity between the selected and the synthesized peptide was demonstrated by peptide sequencing. The conjugation to TGB was then performed and the resuiting peptide-TGB conjugate (3.6 rag, protein wt) was separated from unreacted free peptide by gel filtration on Sephadex G25 in 0.1 M ammonium hydrogen carbonate buffer, pH 7.4. It was assessed by ELISA with dilutions of anti-(S-fragment) antiserum and was recognized by this antiserum indicating that it included the N-terminal tridecapeptide of the S-fragment, as expected. 4.2. Immunochemical characterization of the conjugate The conjugate was used as immunogen in rabbits and the antiserum collected after 57 days was assayed for reactivity by ELISA. It was able to induce antibodies that cross-reacted with native ETA. Using ETA as coating antigen, the titers of the anti-(peptide-TGB) and anti-ETA antisera were 55 x 103 and 90 x 103 respectively (Fig. 2). They differ by a factor of 1.7. Using the protein carrier or the synthetic peptide as coating antigens, it was shown that the peptide-TGB conju-
270
gate also elicited antibodies against both of them with titers of 48 × 103 and 64 × 103 respectively. The anti-(peptide-TGB) antiserum was also tested with D T as coating antigen. As seen in Fig. 2 it did not cross-react with DT.
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~----
75
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4.3. Neutralization of exotoxin A and diphtheria toxin ADP-ribosyltransferase actiHty Anti-(peptide-TGB) and anti-ETA antisera were assayed in parallel for their inhibitory effects on ADP-ribosyltransferase activity of E T A or DT. Inhibition was calculated relative to the activity of E T A in the presence of a non-immune antiserum. As seen in Fig. 3, both antisera inhibited 50% of E T A ADP-ribosyltransferase activity with dilutions of 1 : 50 and 1 : 90 respectively, differing only by a factor of 1.8. However, at the lowest dilution, anti-exotoxin A antiserum was able to completely neutralize the ADP-ribosyltransferase activity of ETA, whereas anti(peptide-TGB) antiserum was only able to neutralize 90% of this activity. This difference is presumably due to the presence of antibodies in
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Antiserum dilution Fig. 3. Neutralization of ADP-ribosyltransferase activity. The reaction was performed with 160 ng of E T A and various dilutions of anti-ETA ([23) or anti-(peptide-TGB) ( I I ) antiserum. The inhibition was calculated relative to the activity of E T A in the presence of a non-immune antiserum.
the anti-ETA antiserum, affecting ADP-ribosyltransferase activity in several parts of the E T A molecule. Whatever the dilutions were, both antisera were completely unable to inhibit ADP-ribosyltransferase activity of DT.
5. D I S C U S S I O N ...............
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Antiserum reciprocal dilution
90 X 103 Fig. 2. T i t r a t i o n o f t h e a n t i s e r a by t h e E L I - S A m e t h o d , T h e titration of the anti-(peptide-TGB) (m) or anti-ETA (~) a n t i s e r u m w a s w i t h 500 /xg o f c o a t e d E T A p e r well. T h e
titration of the anti-(peptide-TGB) antiserum (e) was with 500 /zg of coated DT per well. The titers were calculated by multiplying the reciprocal of the antiserum dilution within the linear portion of the dilution curve by the corresponding A490 value.
Genetically engineered E T A deletion mutants, monoclonal antibodies and synthetic peptides were used by several groups to identify functional epitopes in E T A [9-11]. Recently, eight mono~ clonal antibodies have been characterized [11]. They reacted with at least six independant epitopes: two in domain II (residues 264-308) three in domain Ib (residues 365-399 and 381-399) and one in domain III (residues 408-613). Two antibodies, recognizing domain II and domain lb respectively, neutralized the cytotoxic effect of ETA. Neither of these cytotoxicity neutralizing antibodies inhibited ADP-ribosylating activity. The third approach to identify functional E T A epitopes was to use synthetic peptides, as in previous studies of cholera toxin [16], diphtheria toxin [17-19] and pertussis toxin [20,21]. With
271
antibodies directed against peptides accessible on the surface of E T A crystalline structure, three E T A epitopes were identified: two within domain I (residues 1-30 and 233-242) and one within the last 33 amino acids of domain III [9]. When synthetic peptide antisera were assayed for their effect on enzyme activity, the C-terminal peptide antiserum caused a slight inhibition in activity but no inhibition was observed by any of the other peptide antisera. A sequential epitope 422-432 has been defined using a series of synthetic peptides [22]. This epitope includes His 426 which has previously been shown to be essential for ADPribosyltransferase activity [23]. Our work focused on the identification of a new determinant. Limited proteolysis of E T A by subtilisin generates an S-fragment with a potential epitope in its N-terminal sequence [12]. In the present study, a tridecapeptide corresponding to this sequence (residues 392-404 of ETA) has been synthesized and conjugated to a carrier-protein (TGB). The peptide-TGB conjugate, appears to produce E T A cross-reactive antibodies which were shown to almost completely abolish the ADP-ribosylation activity of ETA. This result has to be emphasized since, as indicated above, none of the monoclonal antibodies isolated by Ogata et al. neutralized the ADP-ribosylation activity of E T A even though two of these antibodies were directed against residues 381-399 [11]. Thus, the 1/ast five residues of domain Ib (residues 400-404) are demonstrated to be very important for the function of the epitope 392-404. Those residues have already been shown essential to the full expression of E T A ADP-ribosyltransferase activity [4]. It is not possible that the anti-(peptide-TGB) antiserum inhibits E T A enzyme activity by interfering with the NAD + binding site, since several pieces of evidence substantiate the role of the domain III cleft in NAD + binding [2,3]. Moreover E T A and D T have virtually the same enzymatic activity and 23% of identical amino acids in their enzymatic domains with the same residues His, Glu and Trp being involved in the catalytic center [24,25]. Now, the anti-(peptide-TGB) antiserum inhibits E T A activity but does not inhibit D T activity. The sequence we have identified
does not appear then to have a correlate in D T and it is possible that the anti-(peptide-TGB) antiserum inhibits enzyme activity by interfering either specifically or non-specifically with the acceptor substrate (EF 2) binding. The peptide sequence may also have a role in imparting chemical stability to the enzymatic fragment and antibody binding may interfere with this supposed function. Given the evidence that the peptide may represent a functionally important structural feature of ETA, a site-specific mutagenic alteration of the sequence could provide additional corroborating evidence for a potential enzymatic role.
ACKNOWLEDGEMENTS This work was supported by the Centre National de la Recherche Scientifique (Unitd Mixte de Recherches 24) and by a grant of the Association Fran~aise de Lutte contre la Mucoviscidose. We thank L. Denoroy (Service Central d'Analyses du Centre National de la Recherche Scientifique, Solaize, France) for the peptide sequencing and B. Danve (Pasteur Mdrieux, Marcy l'Etoile, France) for providing us with antisera.
REFERENCES [1] Pastan, I. and FitzGerald, D. (1989) Pseudomonas exotoxin: chimeric toxins. J. Biol. Chem. 264, 15157-15160. [2] Wick, M.J., Franck, D.W., Storey, D.G. and Iglewski, B.H. (1990) Structure, function and regulation of Pseudomonas aeruginosa exotoxin A. Annu. Rev.. Microbiol. 44, 335-363. [3] Wick, M.J., Hamood, A.N. and Iglewski, B.H. (1990) Analysis of the structure-function relationship of Pseudornonas aeruginosa exotoxin A. Mol. Microbiol. 4, 527535. [4} Siegall, C.B., Cbaudbary, V.K., FitzGerald, D.J. and Pastan, I. (1989) Functional analysis of domains ii, Ib and III of Pseudomonas exotoxin. J. Biol. Chem. 264, 1425614261. [5] Hwang, J., FitzGerald, D.J., Adhya, S. and Pastan, I. (1987) Functional domains of Pseudomonas exotoxin identified by deletion analysis of the gene expressed in E. coll. Cell 48, 129-136. [6] Iglewski, B.H. and Kabat, D. (1975) NAD-dependent inhibition of protein synthesis by Pseudomonas aeruginosa toxin. Proc. Natl. Acad. Sci. USA 72, 2284-2288.
272 [7] Iglewski, B.H., Liu, P.V, and Kabat, P. (1977) Mechanism of action of Pseudomonas aeruginosa exotoxin A: Adenosine diphosphate-ribosylation of mammalian elongation factor 2 in vitro and in vivo. Infect. Immun. 15, 138 144. [8] Collier, R.J. and Mekalanos, J.J. (1980) ADP-ribosylating exotoxins. In: Multifunctional Proteins (Bisswanger, H. and Schmincke-Ott, E., Ed.), pp. 261-291 John Wiley & Sons, New York. [9] Olson, J.C., Hamood, A.N., Vincent, T.S., Beachey, E.H. and Iglewski, B.H. (1990) Identification of functional epitopes of Pseudornonas aeruginosa exotoxin A using synthetic peptides and subclone products. Mol. Immunol. 27, 981-993. [10] Chia, J.K.S., Pollack, M., Avigan, D. and Steinbach. S. (19861 Functionally distinct monoclonal antibodies reactive with enzymatically active and binding domains of Pseudomonas aeruginosa toxin A. Infect. Immun. 52, 756-762. [11] Ogata, M., Pastan, 1. and FitzGerald, D. (19911 Analysis of Pseudomonas exotoxin activation and conformational changes by using monoclonal antibodies as probes. Infect. Immun. 59, 407-414. [12] Bourdenet, S., Vacheron, M.J., Guinand, M., Michel. G. and Arminjon, F. (1990) Biochemical and immunochemical studies of proteolytic fragments of exotoxin A from Pseudomonas aeruginosa. Eur. J. Biochem. 192, 379-385. [13] Hwang, J. and Chen, M. (1989) Structure and function relationship of Pseudomonas exotoxin A. J. Biol. Chem. 264,2379-2384. [14] Schmidt, W. and Schmidt, M.A. (19891 Mapping of linear B-cell epitopes of the $2 subunit of pertussis toxin. Infect. Immun. 57, 438-445. [15] Bradford, M.M. (19761 A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. [16] Jacob, C.O., Sela, M. and Arnon, R. (1983) Antibodies against synthetic peptides of the B subunit of cholera toxin: cross-reaction and neutralization of the toxin. Proc. Natl. Acad. Sci. USA 80, 7611-7615. [17] Audibert, F. (1987) Utilization of diphtheria peptides for
the study of adjuvants and of optimized presentation of protective epitopes in synthetic vaccines. In: Synthetic Vaccines, Volume II (Arnon, R., Ed), pp. 1 14. CRC Press, Florida. [18] Audibert, F., Jolivet, M., Chedid, L., Alouf, J.E., Baqnet, P., Rivaille, P. and Siffert, O. (1981) Active antitoxic immunization by a diphtheria toxin synthetic o[igopeptide. Nature 289, 593-594. [19] Audibert, F., Jolivet, M., Chedid, L., Arnon. R. and Sela. M. (1982) Successful immunization with a totally synthetic diphtheria vaccine. Proc. Natl. Acad. Sci. USA 79. 5042-5046. [21/] Askel6f, P., Rodmalm, K, Wrangsell, G., Larsson, U., Svenson, S.B., Cowell, J.L., Unden, A. and Bartfai, T. (19901 Protective immunogenecity of two synthetic peptides selected from the amino sequence of Bordetella pertussis toxin subunit S1. Proc. Natl. Acad. Sci. USA 87, 1347-1351. [21] Presentini, R., Perin, F., Ancilli, G., Nucci, D., Bartoloni, A., Rappuoli, R. and Antoni, G. (1989) Studies of the antigenic structure of two cross-reacting proteins, pertussis and cholera toxins, using synthetic peptides. Mol. lmmunol. 26, 95-100. [22] McGowan, J.L., Kessler, S.P., Anderson, D.C. and Galloway, D.R. (19911 Immunochemical analysis of Pseudomonas aeruginosa exotoxin A. Analysis of the His ~2~' determinant. J, Biol. Chem. 266, 49l 1-4916. [23] Wosniak, D.J., Hsu, L.Y. and Galloway, D.R. (19881 His 42~' of the Pseudomonas aeruginosa exotoxin A is required for ADP-ribosylation of elongation factor II. Proc. Natl. Acad. Sci. USA. 85, 8880-8884. [24] Carrol, S.F. and Collier, R.J. (19881 Amino acid sequence homology between the enzymic domains of diphtheria toxin and Pseudornonas aeruginosa exotoxin A. Mol. Microbiol. 2, 293-296. [25] Domenighini, M., Montecucco, C., Ripka, W.C. and Rappuoli, R. (1991) Computer modelling of the NAD binding site of ADP-ribosylating toxins active-site structure and mechanism of NAD binding. Mol. Microbiol. 5, 23-31.
267
FEMSIM 00215
Identification of a small epitope in domain Ib of Pseudomonas aeruginosa exotoxin A that elicits enzyme-neutralizing antibodies K a r i n e R u t a u l t ~, D o m i n i q u e Coin ~, M a r i e - J e a n n e V a c h e r o n ~, Micheline G u i n a n d ~, J e a n W a l l a c h b a n d G e o r g e s Michel ~' " Laboratoire de Biochimie Microbienne, and h Laboratoire de Biochirnie Analytique, Unit:ersit~ Claude Bernard Lyon I, Villeurbanne, France
Received 12 March 1992 Accepted 24 March 1992
Key words: Pseudomonas aeruginosa; Exotoxin A; Peptide-thyroglobulin conjugate
1. SUMMARY A peptide corresponding to amino acids 392404 of the amino acid sequence of Pseudomonas aeruginosa exotoxin A (the last 13 amino acids of domain Ib) was synthesized and coupled to thyroglobulin. The conjugate induced an antiserum in rabbits with high antibody titer against native toxin as measured by ELISA, and this antiserum was highly efficient in inhibiting the ADP-ribosyltransferase activity of exotoxin A. These data corroborate the potential importance of amino acids 400-404 in the enzymatic mechanism of exotoxin A.
2. I N T R O D U C T I O N Exotoxin A (ETA) is the most toxic of the extracellular enzymes produced by Pseudomonas Correspondence to: M. Guinand, Laboratoire de Biochimie Microbienne, Universit~ Claude Bernard Lyon I, 43 Boulevard du 11 Novembre 1918, F-69622 Villeurbanne, France.
aeruginosa. It has been isolated, purified and extensively characterized as described in several reviews [1-3]. It is a single-chain protein with a 613-amino acid sequence and four disulfide bridges. It contains three distinct domains: Ia-Ib, II and III (Fig. 1). The active site is located in the carboxy-terminal domain III but the last five residues of the adjacent domain lb are essential for a full expression of enzymatic activity [4]. The other two domains Ia and II are involved in eucaryotic cell binding and in translocation respectively [5]. Toxicity is considered due to an inhibition of protein synthesis in eucaryotic cells, by transfer of ADP-ribose from NAD + to elongation factor 2 (EF2) [6,7]. Diphtheria toxin (DT) has a remarkably similar enzymic property [8]. Several approaches have been used to define functional epitopes ofP. aeruginosa ETA. Synthetic peptides or mutants containing different deletions in the ETA structural gene [9] as well as monoclonal antibodies [10,11] have proven to be very useful in this respect. Antibodies against a subtiIisin-generated fragment of ETA (S-fragment), containing the last 13 residues of domain
268
Ia
I[
I ExotoxinA
NH
252
lh
II1
364 404
613
2-I
S- Fragment
Tridecapeptide
392{ 613 Nil 2-- { [~"~'~i~{~;~'~:'}~i'~i':'~i~i~ - - COOil
t
I R-N-Y-P-T-G-A-E-F-L-G-D-G [
Fig. 1. Location of the S-fragment and of the tridecapeptide within the ETA molecule. The three domains of the molecule are indicated as follows: domains la and Ib are white, domain II is hatched, domain III is grey. The S-fragment derived from subtilisin cleavage contains residues 392-613. The tridecapeptide (white, residues 392-404) is the C-terminal portion of domain lb.
lb and the entire domain III (Fig. 1), neutralize the ADP-ribosyltransferase activity of ETA [12], but antibodies against the sole domain III fail to neutralize this activity [13]. The N-terminal tridecapeptide of the S-fragment was therefore proposed to be a functional epitope [12]. In order to test this assumption, the corresponding tridecapeptide R N Y P T G A E F L G D G was synthesized and conjugated to a carrier protein for rabbit immunization. Antiserum against this sequence was obtained, and its inhibitory effect on ADPribosylation activity of ETA and of D T was studied.
3. M A T E R I A L S AND M E T H O D S
3.1. Toxins ETA used in this study was prepared fromP.
established by UV analysis of a resin sample d e p r o t e c t e d by 20% p i p e r i d i n e / d i m e t h y l formamide. The - S H group of Cys was protected by the triphenylmethyl group and the guanidino function of Arg by the 4-methoxy2,3,6-trimethylbenzene sulfonyl group. Threonine was introduced as Fmoc-Thr-l-oxo-2-hydroxy-dihydrobenzotriazine ester and all other Fmocamino acids as Pfp esters. After the linkage of the Gly residue, all Fmoc-amino acids were coupled using a 3.5 molar excess in presence of 1-hydroxybenzotriazole with a coupling time of 30 rain for the next six amino-acids and 60 min for subsequent residues. Each Fmoc deprotection stage involved t r e a t m e n t with 20% p i p e r i d i n e / dimethyl-formamide for 10 rain. Cleavage of the peptide from the resin was achieved by a 6 h t r e a t m e n t with trifluoracetic acid-anisoleethanedithiol (95:3:2), followed by successive washings of the resin with ether.
aeruginosa strain 103, as previously described [12]. DT was a gift from Prof. J.P. Reboud (University Lyon I).
3.2. Synthesis of the peptide The pentadecapeptide RNYPTGAEF L G D G C G was synthesized by the solid-phase method (Ultrosyn A resin, 0.1 m e q / g ) with 9-fluorenylmethoxycarbonyl (Fmoc) derivatives. The first amino acid (GIy) was coupled as Fmocglycine-pentafluorophenyl ester (Pfp) (5 molar excess). The degree of attachment of Gly (93%) was
3.3. Conjugation of the peptide to thyroglobulin The pentadecapeptide was conjugated to thyroglobulin (TGB) using m-maleimidobenzoyl Nhydroxysuccinimide ester (MBS) as hetero-bifunctional cross-linker according to the procedure of Schmidt and Schmidt [14]. TGB (10 mg) was dissolved in 1.5 ml of phosphate-buffered saline (PBS) and mixed with 1 ml of dimethylformamide containing 5 mg of MBS. After 2 h with stirring at room temperature, the conjugate was
269 separated from unreacted cross-linker by dialysis against 0.1 M phosphate buffer, pH 6. The pentadecapeptide (1.9 rag) was combined with the TGB-MBS conjugate and stirred for 15 h at room temperature.
as a standard [15]. Peptide sequencing was performed in a protein microsequencer (Applied Biosystems, model 407 A).
4. RESULTS
3.4. Peptide- TGB conjugate antiserum New Zealand white rabbits were immunized intramuscularly (5 X 0.2 ml) and subcutaneously (5 x 0.2 ml) on days 0, 28 and 42 with the peptide-TGB conjugate (150/xg, protein weight) dissolved in 1 ml PBS supplemented with 1 ml of Freund's complete (first injection) or incomplete (following injections) adjuvant solutions (Difco). Rabbits were bled 57 days after the first injection and the serum was decomplemented (56°C, 30 min), filter-sterilized (0.45 ixm) and stored at - 2 0 ° C . Antisera against exotoxin A and S-fragment have been previously obtained [12]. 3.5. Enzyme-linked immunosorbent assay (ELISA) The ELISA was performed essentially as described [12]. The microtiter plates (Micro Elisa Dynatech) were coated with 100 /xl of the antigens at 5 /xg/ml (ETA, DT, TGB protein and conjugate) or at 10 /xg/ml (free peptide). All assays were performed in duplicate. ELISA titers were calculated by multiplying the reciprocal of a serum dilution within the linear portion of the dilution curve (A490, 0.4 to 1) by the corresponding A490 value. 3.6. Neutralization of ADP-ribosyltransferase actiuity ETA and DT were extemporaneously activated at 25°C for 15 min with 4 M urea and 1% dithiothreitol. Neutralization of enzymatic activity with antisera was evaluated by incubating 10 Izl of activated ETA (160 ng) or activated DT (117 ng) with 10/xl of antiserum at final dilutions of 1:2 to 1:200, at 37°C for 30 min. The enzymatic assays were then performed in duplicate as described previously [12]. Normal rabbit serum was used as a negative control. 3. 7. Other procedures Protein concentration was determined by the Bradford procedure with bovine serum albumin
4.1. Synthesis of the peptide and conjugation to TGB The selected pentadecapeptide RNYPTGAEFLGDGCG, prepared by the solid-phase method, corresponds to residues 392-404 of ETA with an additional Cys residue at the C-terminal end for coupling to a protein carrier by a heterobifunctional cross-linking agent. A Gly residue was also added to avoid peptide racemisation during the synthesis. The crude peptide (88 rag) was purified by HPLC on a C18 reversed-phase column with a discontinuous gradient of acetonitrile in 0.1% trifluoracetic acid. The pure peptide was eluted between 20 and 23% of acetonitrile (data not shown). It was lyophilized immediately to prevent disulfide bond formation. Total identity between the selected and the synthesized peptide was demonstrated by peptide sequencing. The conjugation to TGB was then performed and the resuiting peptide-TGB conjugate (3.6 rag, protein wt) was separated from unreacted free peptide by gel filtration on Sephadex G25 in 0.1 M ammonium hydrogen carbonate buffer, pH 7.4. It was assessed by ELISA with dilutions of anti-(S-fragment) antiserum and was recognized by this antiserum indicating that it included the N-terminal tridecapeptide of the S-fragment, as expected. 4.2. Immunochemical characterization of the conjugate The conjugate was used as immunogen in rabbits and the antiserum collected after 57 days was assayed for reactivity by ELISA. It was able to induce antibodies that cross-reacted with native ETA. Using ETA as coating antigen, the titers of the anti-(peptide-TGB) and anti-ETA antisera were 55 x 103 and 90 x 103 respectively (Fig. 2). They differ by a factor of 1.7. Using the protein carrier or the synthetic peptide as coating antigens, it was shown that the peptide-TGB conju-
270
gate also elicited antibodies against both of them with titers of 48 × 103 and 64 × 103 respectively. The anti-(peptide-TGB) antiserum was also tested with D T as coating antigen. As seen in Fig. 2 it did not cross-react with DT.
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4.3. Neutralization of exotoxin A and diphtheria toxin ADP-ribosyltransferase actiHty Anti-(peptide-TGB) and anti-ETA antisera were assayed in parallel for their inhibitory effects on ADP-ribosyltransferase activity of E T A or DT. Inhibition was calculated relative to the activity of E T A in the presence of a non-immune antiserum. As seen in Fig. 3, both antisera inhibited 50% of E T A ADP-ribosyltransferase activity with dilutions of 1 : 50 and 1 : 90 respectively, differing only by a factor of 1.8. However, at the lowest dilution, anti-exotoxin A antiserum was able to completely neutralize the ADP-ribosyltransferase activity of ETA, whereas anti(peptide-TGB) antiserum was only able to neutralize 90% of this activity. This difference is presumably due to the presence of antibodies in
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the anti-ETA antiserum, affecting ADP-ribosyltransferase activity in several parts of the E T A molecule. Whatever the dilutions were, both antisera were completely unable to inhibit ADP-ribosyltransferase activity of DT.
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90 X 103 Fig. 2. T i t r a t i o n o f t h e a n t i s e r a by t h e E L I - S A m e t h o d , T h e titration of the anti-(peptide-TGB) (m) or anti-ETA (~) a n t i s e r u m w a s w i t h 500 /xg o f c o a t e d E T A p e r well. T h e
titration of the anti-(peptide-TGB) antiserum (e) was with 500 /zg of coated DT per well. The titers were calculated by multiplying the reciprocal of the antiserum dilution within the linear portion of the dilution curve by the corresponding A490 value.
Genetically engineered E T A deletion mutants, monoclonal antibodies and synthetic peptides were used by several groups to identify functional epitopes in E T A [9-11]. Recently, eight mono~ clonal antibodies have been characterized [11]. They reacted with at least six independant epitopes: two in domain II (residues 264-308) three in domain Ib (residues 365-399 and 381-399) and one in domain III (residues 408-613). Two antibodies, recognizing domain II and domain lb respectively, neutralized the cytotoxic effect of ETA. Neither of these cytotoxicity neutralizing antibodies inhibited ADP-ribosylating activity. The third approach to identify functional E T A epitopes was to use synthetic peptides, as in previous studies of cholera toxin [16], diphtheria toxin [17-19] and pertussis toxin [20,21]. With
271
antibodies directed against peptides accessible on the surface of E T A crystalline structure, three E T A epitopes were identified: two within domain I (residues 1-30 and 233-242) and one within the last 33 amino acids of domain III [9]. When synthetic peptide antisera were assayed for their effect on enzyme activity, the C-terminal peptide antiserum caused a slight inhibition in activity but no inhibition was observed by any of the other peptide antisera. A sequential epitope 422-432 has been defined using a series of synthetic peptides [22]. This epitope includes His 426 which has previously been shown to be essential for ADPribosyltransferase activity [23]. Our work focused on the identification of a new determinant. Limited proteolysis of E T A by subtilisin generates an S-fragment with a potential epitope in its N-terminal sequence [12]. In the present study, a tridecapeptide corresponding to this sequence (residues 392-404 of ETA) has been synthesized and conjugated to a carrier-protein (TGB). The peptide-TGB conjugate, appears to produce E T A cross-reactive antibodies which were shown to almost completely abolish the ADP-ribosylation activity of ETA. This result has to be emphasized since, as indicated above, none of the monoclonal antibodies isolated by Ogata et al. neutralized the ADP-ribosylation activity of E T A even though two of these antibodies were directed against residues 381-399 [11]. Thus, the 1/ast five residues of domain Ib (residues 400-404) are demonstrated to be very important for the function of the epitope 392-404. Those residues have already been shown essential to the full expression of E T A ADP-ribosyltransferase activity [4]. It is not possible that the anti-(peptide-TGB) antiserum inhibits E T A enzyme activity by interfering with the NAD + binding site, since several pieces of evidence substantiate the role of the domain III cleft in NAD + binding [2,3]. Moreover E T A and D T have virtually the same enzymatic activity and 23% of identical amino acids in their enzymatic domains with the same residues His, Glu and Trp being involved in the catalytic center [24,25]. Now, the anti-(peptide-TGB) antiserum inhibits E T A activity but does not inhibit D T activity. The sequence we have identified
does not appear then to have a correlate in D T and it is possible that the anti-(peptide-TGB) antiserum inhibits enzyme activity by interfering either specifically or non-specifically with the acceptor substrate (EF 2) binding. The peptide sequence may also have a role in imparting chemical stability to the enzymatic fragment and antibody binding may interfere with this supposed function. Given the evidence that the peptide may represent a functionally important structural feature of ETA, a site-specific mutagenic alteration of the sequence could provide additional corroborating evidence for a potential enzymatic role.
ACKNOWLEDGEMENTS This work was supported by the Centre National de la Recherche Scientifique (Unitd Mixte de Recherches 24) and by a grant of the Association Fran~aise de Lutte contre la Mucoviscidose. We thank L. Denoroy (Service Central d'Analyses du Centre National de la Recherche Scientifique, Solaize, France) for the peptide sequencing and B. Danve (Pasteur Mdrieux, Marcy l'Etoile, France) for providing us with antisera.
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272 [7] Iglewski, B.H., Liu, P.V, and Kabat, P. (1977) Mechanism of action of Pseudomonas aeruginosa exotoxin A: Adenosine diphosphate-ribosylation of mammalian elongation factor 2 in vitro and in vivo. Infect. Immun. 15, 138 144. [8] Collier, R.J. and Mekalanos, J.J. (1980) ADP-ribosylating exotoxins. In: Multifunctional Proteins (Bisswanger, H. and Schmincke-Ott, E., Ed.), pp. 261-291 John Wiley & Sons, New York. [9] Olson, J.C., Hamood, A.N., Vincent, T.S., Beachey, E.H. and Iglewski, B.H. (1990) Identification of functional epitopes of Pseudornonas aeruginosa exotoxin A using synthetic peptides and subclone products. Mol. Immunol. 27, 981-993. [10] Chia, J.K.S., Pollack, M., Avigan, D. and Steinbach. S. (19861 Functionally distinct monoclonal antibodies reactive with enzymatically active and binding domains of Pseudomonas aeruginosa toxin A. Infect. Immun. 52, 756-762. [11] Ogata, M., Pastan, 1. and FitzGerald, D. (19911 Analysis of Pseudomonas exotoxin activation and conformational changes by using monoclonal antibodies as probes. Infect. Immun. 59, 407-414. [12] Bourdenet, S., Vacheron, M.J., Guinand, M., Michel. G. and Arminjon, F. (1990) Biochemical and immunochemical studies of proteolytic fragments of exotoxin A from Pseudomonas aeruginosa. Eur. J. Biochem. 192, 379-385. [13] Hwang, J. and Chen, M. (1989) Structure and function relationship of Pseudomonas exotoxin A. J. Biol. Chem. 264,2379-2384. [14] Schmidt, W. and Schmidt, M.A. (19891 Mapping of linear B-cell epitopes of the $2 subunit of pertussis toxin. Infect. Immun. 57, 438-445. [15] Bradford, M.M. (19761 A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. [16] Jacob, C.O., Sela, M. and Arnon, R. (1983) Antibodies against synthetic peptides of the B subunit of cholera toxin: cross-reaction and neutralization of the toxin. Proc. Natl. Acad. Sci. USA 80, 7611-7615. [17] Audibert, F. (1987) Utilization of diphtheria peptides for
the study of adjuvants and of optimized presentation of protective epitopes in synthetic vaccines. In: Synthetic Vaccines, Volume II (Arnon, R., Ed), pp. 1 14. CRC Press, Florida. [18] Audibert, F., Jolivet, M., Chedid, L., Alouf, J.E., Baqnet, P., Rivaille, P. and Siffert, O. (1981) Active antitoxic immunization by a diphtheria toxin synthetic o[igopeptide. Nature 289, 593-594. [19] Audibert, F., Jolivet, M., Chedid, L., Arnon. R. and Sela. M. (1982) Successful immunization with a totally synthetic diphtheria vaccine. Proc. Natl. Acad. Sci. USA 79. 5042-5046. [21/] Askel6f, P., Rodmalm, K, Wrangsell, G., Larsson, U., Svenson, S.B., Cowell, J.L., Unden, A. and Bartfai, T. (19901 Protective immunogenecity of two synthetic peptides selected from the amino sequence of Bordetella pertussis toxin subunit S1. Proc. Natl. Acad. Sci. USA 87, 1347-1351. [21] Presentini, R., Perin, F., Ancilli, G., Nucci, D., Bartoloni, A., Rappuoli, R. and Antoni, G. (1989) Studies of the antigenic structure of two cross-reacting proteins, pertussis and cholera toxins, using synthetic peptides. Mol. lmmunol. 26, 95-100. [22] McGowan, J.L., Kessler, S.P., Anderson, D.C. and Galloway, D.R. (19911 Immunochemical analysis of Pseudomonas aeruginosa exotoxin A. Analysis of the His ~2~' determinant. J, Biol. Chem. 266, 49l 1-4916. [23] Wosniak, D.J., Hsu, L.Y. and Galloway, D.R. (19881 His 42~' of the Pseudomonas aeruginosa exotoxin A is required for ADP-ribosylation of elongation factor II. Proc. Natl. Acad. Sci. USA. 85, 8880-8884. [24] Carrol, S.F. and Collier, R.J. (19881 Amino acid sequence homology between the enzymic domains of diphtheria toxin and Pseudornonas aeruginosa exotoxin A. Mol. Microbiol. 2, 293-296. [25] Domenighini, M., Montecucco, C., Ripka, W.C. and Rappuoli, R. (1991) Computer modelling of the NAD binding site of ADP-ribosylating toxins active-site structure and mechanism of NAD binding. Mol. Microbiol. 5, 23-31.