A hybrid toxin containing fragment A from diphtheria toxin linked to the B promoter of cholera toxin

443

Biochimica et Biophysica A cta, 626 ( 1 9 8 0 ) 4 4 3 - - 4 5 0 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 38570

A H Y B R I D TOXIN CONTAINING F R A G M E N T A FROM D I P H T H E R I A TOXIN LINKED TO THE B P R O T O M E R OF C H O L E R A TOXIN

J O S E F W. M A N N H A L T E R a, D. G A R Y G I L L I L A N D a a n d R. J O H N C O L L I E R a,b

a Department o f Microbiology and b Molecular Biology Institute, University o f California, Los Angeles, CA 90024 (U.S.A.) (Received June 9th, 1980)

Key words: Diphtheria toxin; Cholera toxin; Hybrid protein; Protein synthesis inhibition

Summary We have constructed and characterized a hybrid toxin containing the A chain of diphtheria toxin linked via a disulfide bridge to the B protomer of cholera toxin. Cholera toxin B protomer, previously derivatized with 4--5 cystaminyl uroups per pentameric protomer, was reacted with reduced diphtheria toxin chain A to give the desired hybrid, containing an average of 2 molecules of diphtheria toxin chain A per cholera toxin B protomer. A concentration of 0.3 nM hybrid inhibited protein synthesis by 50% in 24 h in several cultured cell lines; thus the hybrid was a b o u t 10-fold more toxic than of a (diphtheria toxin chain A)-SS-(concanavalin A) conjugate described previously. Evidence was obtained that toxicity of the hybrid was dependent on the functional contributions of both the diphtheria toxin chain A and cholera toxin B protomer moieties.

Introduction Many toxic proteins contain two functionally distinct and physically separable moieties, devoted to intracellular action (A moiety) and binding to cell surfaces (B moiety) [1--3]. Both moieties are generally needed for significant toxicity on intact cells. In diphtheria toxin the A and B moieties are contained within a single polypeptide chain (Mr 60 000), which may be converted to A and B fragments (Mr 21 145 and 39 000, respectively) by mild proteolysis and reduction [1]. The A fragment catalyzes ADP-ribosylation of elongation factor 2 and thereby causes an inhibition of protein synthesis. The A chain alone is not toxic for whole cells, however, because it does n o t gain access to its intracellular target (elongaA b b r e v i a t i o n s : CT-B, t h e

B Protomer of c h o l e r a t o x i n ; D T - A , the A c h a i n of diphtheria toxin.

444

tion factor 2) with significant efficiency. Hence, the B chain is required for toxicity. The B chain is known to bind the toxin to specific cell-surface receptors and may also act in other ways to facilitate transfer of the A moiety across the plasma membrane. Cholera toxin is a multi-chain protein containing the A and B moieties on different subunits [2]. The A chain (Mr 29 000), like the diphtheria toxin mole= cule, is normally processed by limited proteolysis and reduction, to yield A~ and A2 chains (Mr 23 000 and 6000, respectively) [2,4]. The A, moiety catalyzes ADP-ribosylation of a 42 kdalton regulatory subunit of adenylate cyclase, which results in an acceleration of cyclic AMP production within the cell [5,6]. The A2 moiety apparently serves to attach the entire A subunit non-covalently to the B protomer. The latter, consisting of 5 B chains (each of Mr 11 500) [2], binds to ganglioside GM~ exposed on the cell surface, as first demonstrated by Van Heyningen and coworkers [7--9]. As in the case of diphtheria toxin, the catalytic center on the AI chain must penetrate at least to the inner face of the plasma membrane, but is unable to do so unless it is linked t o t h e B protomer. Here we report the construction and characterization of a hybrid protein containing the A moiety of diphtheria toxin linked to the B moiety of cholera toxin. The hybrid shows toxic activity dependent on the functional contributions of the two parts. Materials and Methods Fragment A (DT-A) was prepared from diphtheria toxin (Connaught Laboratories) as described [10] and was heated to 80°C for 10 min to inactivate any residual traces of toxin. S-Carboxymethylated DT-A was prepared according to the method of Waxdal et al. [11]. Enzymic activity of DT-A was assayed by ADP-ribosylation of wheat germ elongation factor 2 with '4C-labeled NAD as substrate [12]. CT-B was the A25B fraction, prepared as described [13]. 3T3 cells were maintained in Dulbecco-modified Eagle medium, supplemented with 10% fetal calf serum and 20 #g/ml of gentamycin or kanamycin. CHO-Klc and CH-RE1.22c cells were maintained in Ham's nutrient mixture F-12 supplemented with 10% fetal calf serum and 20 /~g of gentamicin per ml. For toxicity assays, cells (5 • 104 cells in 1 ml) were seeded into flint glass vials (17 X 52 mm, ICN). After incubating 24 h at 37°C, the medium was changed, samples were added in a volume of 10 #1, and the cells were incubated for an additional 24-h period. Cells were then pulsed for 2 h with ~4C-labeled amino acids, and incorporation of radioactivity into trichloroacetic acid precipitable material was determined as described [14]. Synthesis of (DT-A)-SS-(CT-B) conjugate. All solutions contained 0.02% NaN3 and 1 #g phenylmethanesulfonyl fluoride per ml. After dialysis against distilled water at 4°C, CT-B was brought to 0.5 M in cystamine dihydrochloride (Aldrich), and the pH was adjusted to 4.7. A 700-fold molar excess of 1-ethyl3-(3-dimethylaminopropyl)-carbodiimide (Sigma} was added, and the mixture was incubated at room temperature for 12 min while maintaining the pH at 4.7. The sample was then chromatographed on Sephadex G-25 (Pharmacia) equilibrated with 10 mM sodium phosphate buffer, pH 7.0. Reduced DT-A, prepared as described [10], was mixed with an equimolar amount of cysta-

445

minyl-(CT-B) and dialyzed for 36 h against 10 mM phosphate buffer, pH 7.0. The conjugate was purified on phosphocellulose as described in the legend to Fig. 1. Results We constructed a conjugate containing fragment A from diphtheria toxin (DT-A) linked to the B protomer from cholera toxin (CT-B) and measured its capacity to inhibit protein synthesis in cultured cells. We chose to incorporate a disulfide bridge into the linkage between DT-A and CT-B, since a disulfide bridge links the enzymically active moiety to the remainder of the molecule in several toxins, including diphtheria toxin, and may be necessary for the expression of toxicity. CT-B, which contained A2 moiety attached, was derivatized with cystamine in the presence of a water-soluble carbodiimide, according to the method of Gilliland et al. [15]. The modified protein, containing an average of 4 to 5 cystaminyl groups per pentameric CT-B, was then reacted with reduced DT-A under conditions facilitating disulfide interchange. Purification of the conjugate was achieved by ion-exchange and molecular exclusion chromatography (Fig. 1). The lower molecular weight peak in panel B of Fig. 1 apparently corresponds to a mixture of fragmented CT-B protomers, some of which contain attached DT-A. The low specific toxicity of the material in this peak presumably results from reduced affinity of fragmented CT-B conjugates for cell surfaces. The final product, (DT-A)-SS-(CT-B), contained traces of unconjugated CT-B, b u t no free DT-A or its dimer. The ratio of DT-A to CT-B in the product was approximately 2. Formation and purification of the conjugate were monitored by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (Fig. 2). After the crosslinking reaction a new band was observed corresponding in molecular weight (32 000) to that predicted for a particle containing the cholera toxin B chain m o n o m e r linked to DT-A. This band was absent if the SH group of DT-A had been blocked by carboxymethylation or if 2-mercaptoethanol was included in the gel buffer. After the purification, t w o bands (Mr 11 000 and 32 000) were observed, as expected from the structure of the conjugate. Toxic activity was assayed by measuring inhibition of protein synthesis in cultured human or animal cells and is expressed in terms of the concentration of conjugate or toxin required to inhibit protein synthesis by 50% in a 24-h incubation (IDs0(24)). We used 3T3 cells (Swiss) for most toxicity assays to rule out the possibility that the conjugate 's toxic effect might be due to traces of diphtheria toxin. These and other cells of murine origin are highly insensitive to diphtheria toxin due either to a lack of receptors or a deficiency in toxin transport [16,17]. Such cells are highly sensitive to exotoxin A from Pseudom o n a s aeruginosa, however. This toxin, like diphtheria toxin, catalyzes the ADP-ribosylation of elongation factor 2, but its entry is mediated by a different cell surface receptor. The conjugate gave an IDs0(24) value of a b o u t 0.3 nM on 3T3 cells and was at least 3 to 4 orders of magnitude more toxic than diphtheria toxin (Fig. 3A). DT-A, CT-B, or cystaminyl-CT-B showed essentially no toxicity; the same was true of S-carboxymethylated DT-A mixed with cystaminyl-CT-B. Reduction of

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E l u t i o n v o l u m e , ml Fig. 1. P u r i f i c a t i o n o f ( D T - A ) - S S - ( C T - B ) c o n j u g a t e . A. I o n e x c h a n g e c h r o m a t o g r a p h y o f t h e c o n j u g a t i o n p r o d u c t s . C r u d e c o n j u g a t e ( 8 - - 9 m g p r o t e i n in 5 m l ) w a s a p p l i e d to p h o s p h o c e l l u l o s e ( W h a t m a n P-11; 4 . 5 m l p a c k e d gel) e q u i l i b r a t e d w i t h 10 m M s o d i u m p h o s p h a t e b u f f e r , p H 7.0. T h e r e s i n w a s w a s h e d w i t h b u f f e r a n d t h e p r o t e i n w a s e l u t e d w i t h 0 . 2 M s o d i u m p h o s p h a t e b u f f e r , p i t 8.0. - - , a b s o r b a n c e at 2 8 0 n m ; b o t h p e a k s s h o w e d A D P - r i b o s y l t r a n s f e r a s e a c t i v i t y ( d a t a n o t s h o w n ) . B. Gel c h r o m a t o g r a p h y o n S e p h a c r y l S - 2 0 0 . F r a c t i o n s e l u t e d f r o m t h e p h o s p h o c e l l u l o s e c o l u m n w e r e p o o l e d , c o n c e n t r a t e d , a n d passed t h r o u g h a S e p h a c r y l S - 2 0 0 c o l u m n ( P h a r m a c i a , 1 1 6 c m X 1.2 c m , f l o w r a t e 12 m l / h ) e q u i l i b r a t e d w i t h 50 m M s o d i u m p h o s p h a t e b u f f e r , p H 8.0, c o n t a i n i n g 1 0 p g g e n t a m i c i n p e r ml. - - , a b s o r b a n c e at 2 8 0 nm; ...... , A D P - r i b o s y l t r a n s f e r a s e a c t i v i t y ; v e r t i c a l bars, p r o t e i n s y n t h e s i s o f 3 T 3 cells a f t e r a 24-h incub a t i o n w i t h e q u i v a l e n t c o n c e n t r a t i o n s o f t h e f r a c t i o n s i n d i c a t e d ( a p p r o x . 3 nM c o n j u g a t e ) ; m e a s u x e d as p e r c e n t o f r a d i o a c t i v e a m i n o a c i d s i n c o r p o r a t e d in u n t r e a t e d c o n t r o l c u l t u r e s .

the conjugate 's disulfide linkage abolished its toxicity (data not shown). The conjugate was as toxic for several diphtheria toxin sensitive cell lines {CHO; Vero; V79) as for 3T3 cells. However, diphtheria toxin was about 100 times more toxic than the conjugate on these lines. To confirm that the observed toxicity was due to inactivation of elongation factor 2, the conjugate was tested on CH-RE1.22c cells [14]. These cells, which are mutants of CHO-Klc cells, contain an altered form of elongation factor 2, incapable of acting as substrate of ADP-ribosyl transfer. The cells are therefore insensitive to both diphtheria toxin and exotoxin A from Ps. aeruginosa [13]. As shown in Fig. 3B, the conjugate did not inhibit protein synthesis in the m u t a n t cells. Several controls were performed to ensure that the activity of the CT-B moiety was required for toxicity of the conjugate. As shown in Table I, toxic-

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Fig. 2. S D S - p o l y a c r y l a m i d e gel e l e c t z o p h o r e s i s of ( D T - A ) - S S - ( C T - B ) c o n j u g a t e . ( A ) CT-B; since t h e B p e n t a m e z ring dissociates in b u f f e r s c o n t a i n i n g SDS, o n l y t h e B m o n o m e r (M r 11 0 0 0 ) is seen; t h e A2 m o i e t y (M r 6 0 0 0 ) d o e s n o t s t a i n w i t h C o o m a s s i e Blue, b u t c a n be m o n i t o r e d r u n n i n g w i t h t h e f r o n t in 1 2 5 i . l a b e l e d p r e p a r a t i o n s . (B) D T - A m o n o m e r (M r 21 0 0 0 ) a n d d i m e z (M r 4 2 0 0 0 ) . ( C ) C r u d e c o n j u g a t e ; besides D T - A m o n o m e r a n d d i m e r a n d B-chain m o n o m e r , an a d d i t i o n a l b a n d (M r 32 0 0 0 ) is s e e n corres p o n d i n g to ( D T - A ) - S S - ( C T - B c h a i n m o n o m e r ) . (D) P u r i f i e d ( D T - A ) - S S - ( C T - B ) c o n j u g a t e ; o n l y b a n d s corr e s p o n d i n g t o t h e ( D T - A ) - S S - ( C T - B c h a i n m o n o m e r ) a n d CT-B m o n o m e r are seen. (E) ( D T - A ) - S S - ( C T - B ) conjugate treated with dithiothreitol. Fig. 3. A. E f f e c t of ( D T - A ) - S S - ( C T - B ) c o n j u g a t e o n p r o t e i n s y n t h e s i s in 3 T 3 cells. T h e p r e p a r a t i o n s t e s t e d w e r e , c o n j u g a t e (o o), d i p h t h e r i a t o x i n (n a), D T - A (~ D), Ps. aeruginosa e x o t o x i n A (o . . . . . . o), CT-B (" • ) , c y s t a m i n y l - ( C T - B ) (~ A), a n d c a r b o x y m e t h y l a t e d D T - A m i x e d w i t h e y s t a m i n e - d e r i v a t i z e d CT-B (~ . . . . - - ~ ) . B. E f f e c t of c o n j u g a t e o n C H O - K l c a n d C H - R E 1 . 2 2 c cells. Closed s y m b o l s C H O - K l e cells; o p e n s y m b o l s C H - R E 1 . 2 2 e cells. S a m p l e s t e s t e d w e r e ( D T - A ) - S S - ( C T - B ) conjugate (e o, © o), d i p h t h e r i a t o x i n (A. . . . - - A ~. . . . - ~ ) , a n d Ps. aeruginosa e x o t o x i n A (u m, D . D). T o x i c i t y assays w e r e p e r f o r m e d as d e s c r i b e d a b o v e e x c e p t t h a t H a m ' s F-12 m e d i u m w a s u s e d . All assays w e r e p e r f o r m e d in d u p l i c a t e or t r i p l i c a t e .

ity was abolished by incubation of the conjugate with anti-(CT-B) antibodies or ganglioside GM1. A mixture of crude gangliosides was a weaker inhibitor of conjugate activity than purified ganglioside GM1, but was strongly antagonistic at high concentrations. Addition of excess CT-B also blocked the action of the conjugate on cells. None of the antagonists alone significantly altered the toxicity of e x o t o x i n A on 3T3 cells (data n o t shown). We also tested the sensitivity of a ganglioside GMl-deficient transformed line of 3T3 cells to the conjugate [18]. This line (KBalb/C 3T3) was significantly less sensitive to the toxin than the parental line (Balb/C 3T3), which contained higher levels of ganglioside GM~ (IDs0(24) = 7 nM and 0.25 nM, respectively). After incubation of the t w o

448

TABLE I E F F E C T OF A N T A G O N I S T S OF C H O L E R A T O X I N B I N D I N G ON T H E T O X I C I T Y OF T H E ( D T - A ) SS-(CT-B) C O N J U G A T E Toxicity assays were performed w i t h 3 nM c o n j u g a t e o n S w i s s 3 T 3 c e l l s as d e s c r i b e d in t h e l e g e n d t o Fig. 3A. T h e a n t a g o n i s t s a l o n e w e r e n o n - t o x i c f o r t h e s e c e l l s at t h e c o n c e n t r a t i o n s i n d i c a t e d a n d h a d n o e f f e c t o n t h e t o x i c i t y o f 1 nM e x o t o x i n A. W h e r e g a n g l i o s i d e s w e r e u s e d ( G M , S u p e l c o ; c r u d e g a n g l i o s i d e s , S i g m a ) , t h e y w e r e p r e i n c u b a t e d w i t h t h e p r o t e i n f o r 2 h at r o o m t e m p e r a t u r e a n d t h e m i x t u r e w a s a d d e d i n a v o l u m e o f 10 pl t o t h e cells. A l l o t h e r a n t a g o n i s t s (10 #1) w e r e a d d e d s i m u l t a n e o u s l y with the conjugate or exotoxin. Antagonist

Concentration (nM)

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15

70

99

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lines with 50 ~M ganglioside GM, for 24 h the ganglioside-deficient strain showed increased sensitivity to the conjugate (ID50(24) = 2.5 nM). These results are consistent with other data indicating that exogenously added ganglioside GM1 can become incorporated into ganglioside-deficient cells and increase the binding of cholera toxin [18--21]. Discussion Previous studies have demonstrated significant toxicity of conjugates containing DT-A linked to the lectin concanavalin A [15] or to a monomeric subunit of Wistaria floribunda lectin [22]. Similar results were reported for conjugates containing the A subunit of ricin toxin, linked to concanavalin A [23] or to the B subunit of human chorionic gonadotropin [24]. However, none of the conjugates reported to date have been as toxic as whole toxin for sensitive cell lines. This result is not entirely unexpected in that none of the binding moieties described above normally serves as a functional attachment vehicle for toxin A chains. Here we describe a toxic hybrid containing a toxin A chain linked through a disulfide linkage to a heterologous toxin B moiety, the cholera B protomer. Toxic activity was found to be dependent on functional contributions of both protein moieties and on their physical linkage to one another. Although (DTA)-SS-(CT-B) was less toxic than whole diphtheria toxin for sensitive cell lines, it was at least 5- to 10-fold more toxic than other DT-A conjugates reported [22], including a (DT-A)-SS-(concanavalin A) conjugate previously prepared in

449 this laboratory [15]. These results suggest that CT-B may be more efficient in promoting entry of DT-A into cells than concanavalin A. Inhibition of protein synthesis by toxins, or by conjugates such as (DT-A)SS-(CT-B), is the end result of a complex series of events initiated by binding to the mammalian cell surface. Although binding of cholera toxin and diphtheria toxin to certain cell types has been well-characterized, analysis of subsequent steps in entry of these proteins into the cytoplasm of cells has been difficult, and these steps remain poorly defined. Perhaps the major problem has been that toxins such as diphtheria toxin apparently enter the cytoplasm by a relatively inefficient process. Although a single molecule is sufficient to kill a cell when introduced directly into the cytoplasm [25], a much larger number of toxin molecules must normally bind to cells in culture for a single 'productive' entry to occur. It is therefore difficult to delineate those processes which are relevant to membrane traversal from those which are abortive or nonproductive. Thus, the difference in toxicity between the t w o conjugates might also be attributed to (i) a larger number of cell surface receptors for CT-B than concanavalin A, or (ii) more efficient internalization of ligand by CT-B receptors than concanavalin A receptors. Whether or n o t entry of DT-A mediated by CT-B is b y the same mechanism as the normal route of entry of the catalytic moiety of cholera toxin, CT-A1 is n o t known. Regardless of the m o d e of entry of the (DT-A)-SS-(CT-B) conjugate, the results presented here suggest that attachment of CT-B to ganglioside GM1 on cell surfaces can mediate entry of a heterologous, attached polypeptide. The (DT-A)-SS-(CT-B) conjugate m a y therefore be useful as a selective agent in isolating mutant cells with altered gangliosides or deficient in internalizing macromolecules b o u n d to the cell surface. Conjugates analogous to (DT-A)SS-(CT-B) may also have applications in probing entry and modes of action of other celI-surface ligands than employ gangliosides as cell-surface receptors, such as interferon and certain polypeptide hormones [26,27]. Finally, conjugation to CT-B may serve as a general m e t h o d of introducing heterologous proteins into mammalian cells. Acknowledgements We thank Drs. T. and J. Moehring and Dr. S. Aaronson for providing tissue culture cell lines. This work was supported b y Grant AI-07877 from the National Institute of Allergy and Infectious Disease. One of the authors (D.G.G.) was supported b y Grant GM-07185 from the National Institute of General Medical Sciences. References 1 Collier, R . J . ( 1 9 7 5 ) B a c t e r i o l . R e v . 3 9 , 5 4 - - 8 5 2 Gill, D.M. ( 1 9 7 7 ) A d v . C y c l i c N u c l e o t i d e R e s . 8, 8 5 - - 1 1 8 3 0 l s n e s , S. a n d Pihl, A. ( 1 9 7 7 ) in T h e S p e c i f i c i t y a n d A c t i o n o f A n i m a l , B a c t e r i a l a n d P l a n t T o x i n s , R e c e p t o r s a n d R e c o g n i t i o n , Series B, ( C u a t r e c a s a s , P., e d . ) , Vol. 1, p p . 1 3 1 - - 1 7 3 , C h a p m a n a n d Hall, London 4 M e k a l a n o s , J . J . , Collier, R . J . a n d R o m i g , W . R . ( 1 9 7 9 ) J. Biol. C h e m . 2 5 4 , 5 8 5 5 - - 5 8 6 1 5 Gill, D.M. a n d M e r e n , R . ( 1 9 7 8 ) P r o c . N a t l . A c a d . Sci. U . S . A . 7 5 , 3 0 5 0 - - 3 0 5 4 6 Casscl, D. a n d P f e u f f e r , T. ( 1 9 7 8 ) P r o c . N a t l . A c a d . Sci. U . S . A . 75, 2 6 6 9 - - 2 6 7 3

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