the role of the reactive disulfide bond in the interaction of cholera-toxin functional regions

Eur. J. Biochem. 93. 621 --627 (1979)

The Role of the Reactive Disulfide Bond in the Interaction of Cholera-Toxin Functional Regions

Maurizio TOMASI, Angela BATTISTINI, Antonio ARACO, L. Ciorgio RODA, and Giuliano D’AGNOLO

Laboratorio di Biologia Cellulare e Immunologia, Istituto superiore di saniti, Romd, and Laboratorio di Farmacologia, Universiti di Ancona

(Received August 28, 1978)

The chemical reactivity of disulfide bonds towards reducing agents, in the absence of denaturing conditions, in cholera toxin has been studied. Treatment of the toxin with dithiothreitol or other mercaptans gave selective reduction of one of the six disulfide bonds of the protein. This reactive disulfide links two distinct functional regions of the toxin, fragment CI, which activates adenylate cyclase, and fragment y /35 , which recognizes the cell surface receptors. Upon reduction, the two fragments remain bound together and the secondary structure of the protein is retained. The two functional regions have been separated and purified only by methods based on charge differences. When mixed together, purified a and purified y/35 fragments spontaneously and rapidly re-form the disulfide bond. However, reduction of the disulfide bond is an absolute requirement for freeing the catalytic site of the CI functional region. Thus, while other non-covalent binding regions are involved in maintaining cholera toxin molecular structure, the reactive disulfide bond may play a role in the mechanism of cell intoxication.

Cholera toxin is a protein extremely potent in stimulating the membrane-bound enzyme, adenylate cyclase, in a variety of mammalian cells [1,2]. It has been shown that the toxin molecule is composed of two protomeric species A and B [3-81. Protonier A contains two non-identical polypeptide chains CI and y, linked through a single disulfide bond. The remainder of the molecule consists of five identical polypeptide chains [9], each containing a single intra-chain disulfide bond, forming a stable aggregate, protomer B. Thus, the toxin has a molecular formula ay/35. The A protomer is required for the activation of adenylate cyclase [lo], while protomer B is responsible for the binding of the toxin to the cell [ll,12], interacting specifically with the hydrophilic moiety of ganglioside GMI [13].

Protomer A and B are very tightly bound as shown by their slow dissociation in sodium dodecyl sulfate or urea solutions. Dissociation conditions in acid pH, with or without unfolding agents, separate cholera toxin into two fractions, protomer A and the dis- aggregated /3 polypeptide chains [3,6,7]. Although the lipid environment of the cell membrane might

Ahhreviufiun. Ganglioside GI, galactosyl-~’-acetylgalactosaniI-

Enzymr. Adenylate cyclase (EC 4.6.1.1). ayl-(N-acetylneuraminyl)-galactosylglucosylceramide.

mimic the properties of detergents, no direct evidence of the dissociation in vivo of protomers A and B has been described. It seems more probable, as suggested by Gill [8], that one of the commited steps in the intoxication process is the cleavage of one of the six disulfide bonds of cholera toxin. Exposure of native toxin to dithiothreitol, in the absence of denaturing agents, splits selectively the reactive disulfide bond joining the CI and y polypeptide chains. This partial reduction results in the formation of two fragments, possessing functional properties, the a chain and a complex of the y polypeptide chain and protomer B, y/35 [3 - 8,141. The a fragment stimulates adenylate cyclase in cell-free systems in an NAD-dependent reaction [15,16], while the yps complex comprises the functional region responsible for recognition of the cell surface receptors [14]. In addition, it has been shown that protomer A, in the presence of a large excess of dithiothreitol, catalyzes the hydrolysis of NAD to ADP-ribose and nicotinamide [I71 and func- tions as acceptor protein for ADP-ribosylation [18]. Although the process of adenylate cyclase activation is still poorly understood, this evidence suggests that, after the initial interaction of the toxin binding region with ganglioside GMI, the cyclase stimulation requires a partial reduction of cholera toxin and the subsequent

622 Disulfide Bond in Interaction of Cholera-Toxin Functional Regions

exposure to the solvent of the NAD-binding region Reduction of Cholera Toxin of the ct chain.

No information about the reactivity of the active disulfide bridge of cholera toxin has been obtained until now because of the insolubility of protomer A, and its constituent the ct polypeptide chain, in the usual buffers. Recently, we have described conditions for optimum solubility of the CI polypeptide chain in the absence of denaturing agents [14]. This paper describes the role of the active disulfide bond joining the a chain to the yf l5 complex as well as the isolation and purification of cholera toxin functional regions.

MATERIALS AND METHODS

Materials

Blue dextran, Sephadex, Sepharose 4B and DEAE- Sephadex gels were obtained from Pharmacia (Upp- sala, Sweden). The polyacrylamide-agarose gels, Ultro- gel, were purchased from LKB (Bromma, Sweden), while BioGel P-100 was obtained from BioRad (Rich- mond, Cal., U.S.A.). Dithiothreitol was purchased from Calbiochem (Sand Diego, Cal., U.S.A.). Iodo- [l-14C]acetamide was obtained from The Radiochemi- cal Centre (Amersham, England). Collodion bags and a filtration apparatus were obtained from Sar- torius Membranfilter (Gottingen, F.R.G.). The pre- blended liquid scintillation solution, Instagel, was obtained from Packard Instruments (Milano, Italy). Cholera toxin was obtained from Schwarz-Mann Bio- chemicals (Orangeburg, N.Y., U.S.A.). The ganglioside GM1 was the generous gift of Prof. Tettamanti (Istituto di Chimica Biologica, Universita di Milano, Italy).

Cholera Toxin

Cholera toxin was prepared from the cell-free supernatant obtained from cultures of Vibrio cholerae 569 B, serotype Inaba, grown on syncase medium [l]. In large-scale preparations, significant concentration of the toxin was achieved by treatment of the supernatant with calcium phosphate gel, prepared as described by Colowick [19]. Sufficient calcium phosphate was used to give a ratio of 25 ml of gel suspension per liter of culture filtrate. After adjusting the pH to 6.5, the suspension was stirred gently for 2 h. If necessary the pH was re-adjusted and the suspension was left overnight at 4 "C without stirring. Most of the supernatant was removed by suction and dis- carded. The toxin was recovered from the precipitate by elution with 0.1 M sodium citrate, pH 8.2 (1/40 of the volume of the initial cell-free supernatant). Cholera toxin was further purified by means of published procedures [20].

For reduction, unless otherwise specified, aliquots of a toxin solution (1 mg/ml) in 0.1 M Tris-HC1 buffer, pH 8.2, 0.002 M EDTA, 0.01 M dithiothreitol, were made 20% (v/v) in ethylene glycol. The incubation tubes were flushed with nitrogen, stoppered and placed at 37 "C for 20 min.

For reduction with sodium borohydride, the protein was dissolved in deoxygenated 0.1 M Tris-HC1 buffer, pH 8.5, 0.002 M EDTA, at a concentration of 0.3 mg/ml. An equal volume of variable amounts (0.2- 1.0 M) of NaBH4 in deoxygenated 0.1 M Tris- HC1 buffer, pH 8.5, was added. The solutions were stirred, kept under a nitrogen atmosphere, and incubated at 0 "C, 20 "C and 37 "C. Samples were with- drawn periodically (0 - 3 h) for sulfhydryl determinations and polyacrylamide gel electrophoresis analyses.

Electrophoresis

Polyacrylamide gel electrophoresis, with and without sodium dodecyl sulfate, was done according to the method of Davis [21] as modified by Laemmli [22]. The gels were stained with a solution of 0.1 % Coo- massie blue R250. Elution of protein from unstained gels was performed by mincing l-mm gel slices in 0.5 ml of 0.1 M Tris-HC1 buffer, pH 8.2, 0.002 M EDTA, 20% (v/v) ethylene glycol and keeping the mixture overnight at 4 "C.

Chemical Procedures

S-Carboxymethylation of the - SH groups in reduced cholera toxin was carried out with iodo- [l-'4C]acetamide. Reduced protein (2- 10 pg) was incubated at 25 "C in 0.1 M Tris-HC1 buffer, pH 8.2, 0.002 M EDTA, 20 % (v/v) ethylene glycol, with various concentrations (0.2- 1.0 mM) of iodoacetamide, in a final volume of 0.2 ml. After 3 h, unreacted iodoacetamide was inactivated by the addition of 1 pmol of dithiothreitol. Following the addition of 0.5 mg of bovine serum albumin, the reactions were terminated with 0.4 ml of 5 % (v/v) trichloroacetic acid. The precipitates were recovered by centrifuga- tion, washed three times with 0.2 ml of 5 % trichloroacetic acid, dissolved in 0.1 ml of 1.0 M NaOH and counted in a Packard Tri-Carb liquid scintillation spectrometer, after mixing with 10 ml of Instagel solution. The amount of radioactivity bound indicates the number of cysteine residues of the protein.

The number of sulfhydryl groups per molecule. of protein was also determined according to the Ellman procedure 1231.

Protein was determined by the method of Munkres and Richards [24] or Lowry et a]. [25]. Amino acid analyses were performed according to Spackman et al.

M. Tomasi, A. Battistini, A. Araco, L. G. Roda, and G. D'Agnolo 623

[26] using a Beckman Spinco model 120C automatic analyser.

Blue dextran has been covalently coupled to Sepharose 4B by the cyanogen bromide procedure of Ryan and Vestling [27].

Ultracentrifigal Studies

Sedimentation velocity runs were made with the use of a Beckman E analytical ultracentrifuge equipped with schlieren optics. Samples, at a concentration of 1 .O - 1.5 mg/ml of protein were run in 0.1 M Tris-HC1 buffer, pH 8.2, 0.002 M EDTA, 20% (v/v) ethylene glycol, at 20 "C and 60000 rev./min. The buffer for reduced cholera toxin contained also 0.01 M dithiothreitol. Aliquots of the same solutions were used for diffusion experiments using a synthetic boundary cell in the ultracentrifuge at 20 "C and 12000 rev./min, under conditions where little or no sedimentation occurred. The diffusion coefficients were determined by the graphical method described by Shachman [28].

Optical Methods

Circular dichroism spectra were measured with a Cary 60 spectropolarimeter equipped with a model 6002 circular dichroism attachment. The spectra were recorded at 24 "C with protein concentrations ranging from 0.3 to 1.2 mg/ml and cells having 0.1 - 1.0-cm pathlengths. The results are given as mean residue ellipticity, based on a mean residue weight of 112. Protein concentrations were calculated from the absor- bance at 280 nm [l], obtained with a Cary 17 spectro- photometer.

Purification of Cholera Toxin Functional Regions

All operations were carried out at 4 "C. The functional regions of cholera toxin were isolated by DEAE- Sephadex A-50 column chromatography. The column was equilibrated with 0.02 M Tris-HC1 buffer, pH 8.2, 0.005 M 2-mercaptoethanol, 0.002 M EDTA, 20 % (v/v) ethylene glycol. The toxin was reduced in the same buffer and the column load was 2 mg of reduced cholera toxin per ml bed volume. The column was washed with 2 column volumes of equilibrating buffer and was then eluted with a linear gradient of 10 column volumes of LiC1 from 0.0 to 0.2 M, containing 0.02 M Tris-HC1, pH 8.2,0.005 M 2-mercaptoethanol, 0.002 M EDTA, 20 % (v/v) ethylene glycol. Two discrete peaks were found corresponding to the y P 5 complex and the CI polypeptide chain. The y p 5 complex was unretarded in the equilibrating buffer and the a chain was eluted at about 0.1 M LiCI. Routinely, the CI chain contami- nated with small amounts of yP5 complex (Fig.lb),

was re-chromatographed in order to obtain a better purification.

In subsequent studies the two functional regions were separated in pure form by a more time-consuming method : preparative disc gel electrophoresis. Prepara- tive disc gel electrophoresis on 7.5 % polyacrylamide at pH 8.9 was performed in a Canalco preparative disc gel apparatus. The separating and stacking gels were the same as those of the anlytical acrylamide system. 4 ml of separating gel (7.5 % acrylamide, 2.1 % N,N'- methylenbisacrylamide) and 0.6 ml of stacking gel were used for the electrophoresis of about 3 mg of protein. The toxin was reduced in 0.02 M Tris-HC1, pH 8.2, 0.002 M EDTA, 20% (v/v) ethylene glycol, 0.01 M dithiothreitol, for 1 h at 25 "C. The reduced sample, 1 - 2 ml containing 2.5 - 3 mg of protein and bromophenol blue as tracking dye was layered above. Electrophoresis was begun at 5 mA and continued until the elution of non-reduced toxin; the current was then increased to 10mA. The eluting buffer was the same of the reduction reaction and was pumped at a flow rate of 2.7 ml/h. Fractions were analyzed by standard analytical polyacrylamide disc gel electrophoresis. Those fractions which showed a single band upon staining with Coomassie blue were pooled and concentrated without stirring in a model SM 26314 Sartorius membrane filter apparatus and stored at 4 "C for further characterization.

RESULTS

Reduction of Cholera Toxin

Treatment of cholera toxin with reducing agents in the absence of denaturing agents gave partial disulfide reduction and resulted in the formation of two fragments (Fig. 1).

Substrate quantities of reduced cholera toxin were incubated with i~do[l-~"C]acetamide. The S-['"CI- carboxymethylated toxin was isolated from the reaction mixture by trichloroacetic acid precipitation. When the toxin was reduced and S-carboxymethylated at room temperature a maximum of 2 mol of S-car- boxymethyl residue bound/mol of reduced toxin was found to occur. The arrangement of the cysteines in the two fragments was determined, after reduction and S-carboxymethylation, by separating the fragments from each other and from excess reagent by polyacrylamide disc gel electrophoresis. Protein bands were extracted from unstained gels and counted. One reactive cysteine was present on each of the fragments obtained after reduction of the toxin. Thus a single disulfide bond connecting the two fragments is reduced under the conditions employed.

Molecular weight determinations, and analyses by acrylamide gel electrophoresis with sodium dodecyl sulfate, have demonstrated that the slower migrating


Fig. 1 . Polyacrylumide disc gel electrophoresis of' cholera toxin junc- tional regions. The samples were subjected to electrophoresis on 7.5 % polyacrylamide gels in a Tris/glycine, pH 8.4, buffer system according to the method of Davis 1211. (a) Cholera toxin reduced as described under Materials and Methods (20 pg); (b) a chain from DEAE-Sephadex (20 pg); (c) y P 5 from DEAE-Sephadex (20 pg); (d) OL from the preparative disc gel electrophoresis (20 pg); (e) y p ~ from the preparative disc gel electrophoresis (20 kg)

fragment, shown in Fig. 1, was an association of the p and y chains attached to form the y p ~ complex, while the faster-migrating fragment was identical to the CI chain, as already reported [14]. Amino acid analyses of the fragments, isolated by preparative disc gel electrophoresis, confirmed such a polypeptide chain composition, and were in agreement with data from other laboratories [4,7]. Further evidence on the identity of the fragments was obtained by studies on their biological properties. The complex y/$ retained the ability of cholera toxin to form insoluble com- plexes with ganglioside G M ~ [5,14] and it is thus responsible for the recognition of the cell surface receptors. The CI chain retained the ability of cholera toxin to activate adenylate cyclase [14,16] and it is thus responsible for the cell intoxication process.

The reduction reaction was found to be very slow below pH 8 and very fast above pH 9, but accompanied by a rapid deamidation of the toxin, resulting in functional fragments heterogeneous in charge [20]. Apart from the common environmental parameters, like pH, reducing agents and ionic strength, reduction is strongly influenced by temperature. As shown in Fig. 2, the sharp transition points to good cooperativity of the process. The biphasic temperature profile reflects local conformational transitions or changes in the

1oc

75 - 7J m

7 3 5c

E3

P .E x

25

25.0 325 Temperature ("C)

).O

Fig. 2. Temperature-dependent reduction of cholera toxin. Cholera toxin, 0.24 nmol, was incubated in 0.1 M Tris-HCl buffer, pH 8.2, 0.002 M EDTA, 0.01 M dithiothreitol, 20% (v/v) ethylene glycol in a final volume of 0.1 ml, at the indicated temperatures. After 10 min, the samples were quickly cooled in ice and analyzed by polyacrylamide disc gel electrophoresis as described under Mate- rials and Methods. Results are expressed as percentage of reduced cholera toxin relative to experiments in which reduction was complete, as shown by sulfhydryl titration

loo 1 I

Time (min)

Fig. 3. Reduction of cholera toxin. Cholera toxin, 2.4 nmol, was incubated in 0.1 M Tris-HCI buffer, pH 8.2, 0.002 M EDTA, 0.01 M dithiothreitol, 20% (v/v) ethylene glycol, in a final volume of 0.2 ml, at 25 "C. At the times indicated, the samples were quickly cooled in ice and subjected to polyacrylamide disc gel electrophoresis. After staining the gels were analyzed by densitometric scanning. Results are expressed as percentage of protein bands relative to experiments in which incubation with dithiothreitol were omitted. Unreduced cholera toxin (0----0); ct functional region (0-0); y 8 5 functional region (-0)

solvation of groups in the vicinity of the active disulfide bond.

When the protein was incubated with various concentrations of dithiothreitol, 2-mercaptoethanol or reduced glutathione, it was apparent that all of these mercaptans caused reduction of the toxin. The kinetics of reduction of cholera toxin at a dithiothreitol con-

M. Tomasi, A. Battistini, A. Araco, L. G. Roda, and G. D'Agnolo 625

centration of 0.01 M is shown in Fig.3. Comparison of the rates of reduction, under the same conditions with equivalent amounts of the sulfhydryl compounds, showed that the reaction with dithiothreitol was only slightly faster than the reactions with 2-mercaptoethanol or glutathione. Therefore the toxin has little specificity towards these mercaptans. However specific reduction of cholera toxin could not be obtained with other reducing agents such as sodium borohydride. Concentrations of sodium borohydride up to 1.0 M were unable to reduce the disulfide bridge under the conditions used, as shown by the sulfhydryl content titrated at regular time intervals. Similarly increasing the temperature from 0 "C to 37 "C showed a negative sulfhydryl reaction during the entire incubation period. Analytical polyacrylamide electrophoresis showed that treatment of the protein with sodium borohydride gave partial loss of amide groups, resulting in the microheterogeneity previously encountered P O I .

Properties of Reduced Cholera Toxin

The hydrodynamic properties of native and reduced cholera toxin were investigated by measurements of sedimentation and diffusion coefficients. The sedimentation coefficients were 5.48 S and 4.63 S for the native and the reduced toxin respectively. Diffusion coefficients determined using the same solutions, by the method of Shachman [28] were 6.14 x cm2 sp l and 5.40 x cm2 s-l for the native and the reduced toxin. Calculations of the apparent molecular weights from the sedimentation and diffusion coefficients using the Svedberg equation gave, within the experi- mental error, the same value for the two proteins, i.e. 80000 for the native toxin and 76000 for the reduced one. The lower values for the sedimentation and diffusion coefficients found with the reduced protein can be attributed to a different shape, resulting from a change in hydration and consequently in the molecular volume of the toxin. At no point in our experiments did we obtain any evidence of low-molecular-weight material. This was to be expected if the reduction of the disulfide bridge brings about the dissociation of the functional regions a and y p 5 . Thus the CI chain and the yp5 complex still bind to each other after reduction.

It is of interest to note that the secondary structure of the protein is relatively unperturbed by the difference in volume between native and reduced cholera toxin. The far-ultraviolet circular dichroism spectra of the two proteins were found to have no significant differences and to be indistinguishable from those previously reported [20]. The spectra in the near- ultraviolet region are significantly different (Fig. 4). When the protein was reduced with 2-mercaptoethanol, there was a loss of the positive maximum at

40

- 20

Fig.4. Circular dichroism of native and reduced cholera toxin. Measurements were made at 24 "C in 0.1 M Tris-HCI buffer, pH 8.2, containing 0.01 M 2-mercaptoethanol in the case of reduced toxin, The protein concentration was 1.2 mg/ml. Native toxin (-); reduced toxin (~ - ~ )

310 nm, probably a contribution in the native toxin of tryptophan(s) shielded from the solvent. In addition, it is worth noting that at the wavelengths showing similar maxima and minima, these were less pro- nounced for the reduced protein. This change in circular dichroism behaviour results both from the loss of the disulfide bond contribution, which is very large and smooth at these wavelengths [29], and from the contributions of the side chains of aromatic amino acids, mostly tyrosine having a higher degree of free- dom after reduction, as shown by the loss of structure at 277 and 287 nm.

Purification of Cholera Toxin Functional Regions

The stability of the complex between the functional regions of reduced cholera toxin was also shown by attempts to separate CI and y p 5 by gel filtration. Re- duced toxin was subjected to gel filtration chromatography on columns equilibrated with the reducing buffer and kept under nitrogen. As shown by analytical polyacrylamide electrophoresis, the reduced toxin co- chromatographed with the native protein, on Sephadex G-75, BioGel P-100 and Ultrogel ACA 44, as a single peak comprising the c( and yps regions. No free a or y p s were separated under the conditions of these experiments. In a few instances yields of a were low (i.e. about 50 - 60 %), indicating a slow dissociation of the reduced toxin and the subsequent precipitation of the a chain.

The recent demonstration that reduced cholera toxin has a specific affinity for NAD [15- 181 suggested an additional attempt for the separation of the two regions on columns of blue-dextran - Sepharose. A variety of proteins, with strong affinity for NAD, bind to blue dextran affinity columns and can be quantita-


Fraction number

Fig. 5. Preparative polyacrylamide gel electrophoresis separation of CI and y / l s functional regions. Electrophoresis of reduced cholera toxin (3 mg) on 7.5 "/, polyacrylamide gel, pH 8.9, was conducted as described under Materials and Methods. Fractions were analyzed by standard polyacrylamide gel electrophoresis

tively eluted by the presence of low concentrations of the nucleotide in the equilibration buffer [27,30]. Reduced toxin (8 nmol) was applied to a blue-dextran-Sepharosecolumn (0.5 x 5 cm) equilibrated with 0.1 M Tris-HC1 buffer, pH 8.2, containing 0.01 M dithiothreitol, 0.002 M EDTA and 20 % (v/v) ethylene glycol. The column was washed with five column volumes of equilibrating buffer. A single homogeneous peak of non-reduced toxin, which is not absorbed by the resin, passed through the column unaffected. However, at least 90 % of the reduced toxin was bound to the affinity resin and was quantitatively eluted, with 0.001 M NAD, as a single peak containing the a and yp5 fragments.

The functional regions of reduced toxin can be separated only by methods based on charge differences. However separation was poor on DEAE- Sephadex (Fig. 1) where the a chain is not completely dissociated from y B 5 . When old, or commercial preparations, of the toxin were subjected to this procedure, routinely we found a better separation of the two fragments. Chronological studies on the partial reduction of the same batches clearly indicated that the reduction proceeded at a faster rate (approximately 5-fold) as compared to freshly prepared toxin. This behaviour, similar to that observed in experiments performed with sodium dodecyl sulfate, was the result of a partial denaturation of cholera toxin.

With native cholera toxin an electrophoretic procedure was necessary to fully discriminate u and y p ~ (Fig. 5). The two regions maintained their relative mobility when re-run on disc gel electrophoresis and appeared to be homogeneous as judged by the same technique (Fig. 1). The isolated y p ~ complex did precipitate with ganglioside G M I under the conditions described by van Heyningen [5], while the isolated a chain was found to stimulate adenylate cyclase in pigeon erythrocyte membrane as described [16]. Recombination experiments with equivalent amounts of a and yp5 showed the reformation of the disulfide

bond, joining the two regions, to be concentration- dependent. Almost total disulfide reformation can occur, as shown by analytical polyacrylamide gel electrophoresis and sulfhydryl titration, when the total concentration of the protein is above 1 mg/ml. Reoxidation was faster in the presence of 20"/, ethylene glycol, which avoids the aggregation of the u chain [14], and probably favours the correct pairing of the interacting regions by stabilizing electrostatic interactions [31- 331. In a typical experiment about 40-50Z of the fragments were capable of forming the toxin disulfide bond in 4- 5 h at room temperature.

DISCUSSION

When native cholera toxin was treated with dithiothreitol, or 2-mercaptoethanol, the disulfide bond linking the u and yp5 functional regions was selectively reduced while the other disulfide bonds, connecting half-cystine residues within the f i polypeptide chain remained entirely intact. This difference in the sus- ceptibility of the inter-chain and intra-chain disulfide bonds to reduction has been explained by Cecil and Wake [34]. They proposed that intra-chain disulfide bonds commonly form compact ring structures stabi- lized by hydrogen bonds (or other non-covalent bonds). In cholera toxin the five f l chains are probably attached to each other through identical binding sites that form a structure further protecting the disulfide bonds from thiol reagents. The inter-chain disulfide bond connecting the two functional regions is instead at a partially exposed position near the surface of the protein, as shown by the differential reactivity towards different reducing agents and the increased rate of reduction after denaturation. The sharp temperature- dependence and the narrow optimal conditions found for reduction further corroborate this assumption. Thus, the rate of reduction appears to be dependent on small conformational fluctuations of the protein binding domains, which provide some accessibility to the reducing agents.

Evidence for the presence of interacting surfaces capable of holding together the two fragments of cholera toxin after reduction comes from the ultracentrifuge and molecular sieving experiments. By using these techniques we have shown that reduced cholera toxin is present in solution in the form of a complex of the u and yp5 functional regions. However, in the absence of stabilizing agents, the reduced toxin slowly dissociates as shown by the aggregation of the a chain [14]. The dissociation-aggregation transition of the a chain can be avoided by addition of non- aqueous solvents such as ethylene glycol to the solution of reduced toxin. By lowering water activity, ethylene glycol tends to stabilize the intra-molecular hydrogen bonds and to enhance inter-molecular and intra-

M. Tomasi, A. Battistini, A. Araco, L. G. Roda, and G. D’Agnolo 621

molecular electrostatic interactions [31- 331. The importance of electrostatic interactions between different domains of the functional regions, maintaining the molecular organization of the reduced toxin, is demonstrated by the fact that a good separation is achieved only by electrophoretic methods (Fig. 5).

Evidence on the way these domains affect cholera toxin structure comes from the circular dichroism spectra. The binding domains are strong enough to ensure a similar internal folding of the polypeptide chains of the two regions. This assumption is corro- borated by the far-ultraviolet circular dichroism spectra of the native and the reduced toxin, whose identity suggests the same overall conformation for both proteins. However, reduction induces significant differences in the circular dichroism spectra of the two proteins in the range 250 - 300 nm (Fig. 4). The spec- trum of reduced toxin has features indicative of differences in the fine structure of the two proteins in the neighbourhood of some of their aromatic amino acids. On the other hand, these alterations of the toxin structure enables the protein to bind NAD, which is essential for adenylate cyclase activation [15,16].

The single reactive disulfide bond of cholera toxin is not necessary for maintaining the conformation of the protein required for toxic activity, since upon reduction the two functional regions remain bound together and the reduced protein possesses high catalytic activity [35].

In conclusion, the reported results, as well as the Rosen data [36] on the selective phosphorylation of the CI chain after reduction, allow us to formulate the following hypotheses : (a) the interaction between the LX and the y/?5 functional regions is, so to say, rigid and that the new properties and conformational changes, that appear after reduction, can be ascribed to the exposure of a contact surface previously difficult to reach by the solvent; (b) the above-mentioned properties could, on the contrary, be ascribed to changes, after reduction, of the protein tertiary structure, con- cerning regions even far off the contact area. Although the above-mentioned results do not permit an unequi- vocal choice between the two hypotheses, the absence of changes in the secondary structure make the first hypothesis more acceptable.

REFERENCES 1. Finkelstein, R. A. (1973) CRC Crit. Rev. Microhiol. 2,553 -623. 2. Sharp, G. W. G. (1973) Annu. Rev. Med. 24, 19-28.

3. Finkelstein, R. A,, Boesman, M., Neoh, S. H., LaRue, M. K. & Delaney, R. (1 974) J . Immunol. 113, 145 - 150.

4. Sattler, J., Wiegandt, H., Staerk, J., Kranz, T., Ronneberger, H. J., Schmidtberger, R. & Zilg, H. (1975) Eur. J . Biochem. 57,309- 316.

5. van Heyningen, S. (1976) J . Znfect. Dis. 133, S5-SI3. 6. Kurosky, A,, Markel, D. E., Touchstone, B. 8 Peterson, J. W.

7. Lai, C. Y., Mendez, E. & Chang, D. (1976) J . Infect. Dis. 133,

8. Gill, D. M. (1976) Biochemistry, 15, 1242-1248. 9. Sigler, P. B., Druydn, M. E., Kiefer, 8. C. & Finkelstein, R. A.

(1977) Science (Wash.) 197, 1277- 1279. 10. van Heyningen, S. & King, C. A. (1975) Biochem. J. 146,

11. Holmgren, J., Lonnroth, I. & Svennerholm, L. (1973) Infect.

12. Cuatrecasas, P. (1973) Biochemistry, I?, 3547- 3558. 13. Sattler, J., Schwarzmann, G., Staerk, J., Ziegler, W. & Wie-

gandt, H. (1977) Hoppe-Seyler’s Z. Physiol. Chem. 358,

14. Tomasi, M., Battistini, A,, Ausiello, C., Roda, L. G. Sr

15. Gill, D. M. (1975)Proc. NatlAcad. Sci. U.S.A. 72,2064-2068. 16. Wodnar-Filipowicz, A. & Lai, C. Y. (1976) Arch. Biochem.

17. Moss, J., Manganiello, V. C. & Vaughn, M. (1976) Proc. Natl

18. Trepel, J. B., Chuang, D.-M. & Neff, N. H. (1977) Proc. Natl

19. Colowick, S . P. (1955) Methods Enzymol. I , 90-98. 20. Roda, L. G., Tomasi, M., Battistini, A,, Luzzi, I., Mastran-

tonio, P., Zampieri, A. & D’Agnolo, G. (1977) Biochim. Biophys. Acta, 492, 303 - 315.

(1976) J . Infect. Dis. 133, S14-S22.

S23 - S30.

269 - 271.

Immunity, 8, 208 -214.

159-163.

D’Agnolo, G. (1978) FEBS Lett. 94, 253 - 256.

Biuphys. 176,465-471.

Acad. Sci. U.S.A. 73, 4424-4427.

Acad. Sci. U.S.A. 74, 5440 - 5442.

21. Davis, B. J. (1964) Ann. N.Y . Acad. Sci. 121, 404-427. 22. Laemmli, U. K. (1970) Nature (Lond.) 227, 680-685. 23. Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77. 24. Munkres, K. D. & Richards, F. M. (1965) Arch. Biochem.

25. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.

26. Spackman, D. H., Stein, W. H. Sr Moore, S. (1958) Anal.

27. Ryan, L. D. s( Vestling, C. S. (1974) Arch. Biochem. Biophys.

28. Shachman, H. K. (1957) Methods Enzymol. 4, 32- 103. 29. Coleman, D. L,. & Blout, E. R. (1967) in Conformation of Bio-

polymers (Ramachandran, G. N., ed.) vol. 1, pp. 123-146, Academic Press, New York.

30. Thompson, S. T., Cass, H. K. & Stellwagen, E. (1975) Proc. Natl Acad. Sci. U.S .A 72, 669-672.

31. von Hippel, P. H. & Schleich, T. (1969) in Structure and Stability of Biological Macromolecules (Timasheff, S. N. & Fasman, G. D., eds) pp. 417-574, Marcel Dekker, New York.

32. Chauduri, S. R. & Yang, J. T. (1968) Biochemistry, 7, 1379- 1383.

33. Barteri, M. & Pispisa, B. (1973) Biopolymers, 12, 2309-2327. 34. Cecil, R. &Wake, R. G. (1962) Biochem. J . 82,401 -406. 35. Gill, D. M. (1976) J . Infect. Dis. 133, S55-S63. 36. Rosen, 0. M. (1976) Biochemistry, 15, 2902-2905.

Biophys. 109,446-479.

(1951) J . Biol. Chem. 193, 265-275.

Chem. 30,1190-1206.

160, 279 - 284.

M. Tomasi, A. Battistini, A. Araco, and G. D’Agnolo, Laboratorio di Biologia Cellulare e Immunologia, Istituto Superiore di Sanita, Viale Regina Elena 299, 1-00161 Roma, Italy

L. G. Roda, Laboratorio di Farmacologia, Istituto di Medicina Sperimentale e Clinica, Universiti di Ancona, Via delle Grotte di Posatora 2, 1-60100 Ancona, Italy

the role of the reactive disulfide bond in the interaction of cholera-toxin functional regions

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