Eur. J. Biochem. 209. 823-828 (1992) PEBS 1992

Identification of functionally active fragments of staphylococcal enterotoxin B Valcry Yu. ALAKHOV', Eugenii Yu. KLINSKY Mikhail I. KOLOSOV ', Ingrid MAURER-FOGY', Eli7aveta Yu. MOSKALEVA', Peter G. SVESHNIKOV', Lyubov P. POZDNYAKOVA', Olga B. SHEMCHUKOVA' and Eugcnii S. SEVERIN' Research Center of Molecular Diagnostics and Therapy, Moscow, Russia Department of Protein Chemistry, Bendcr, Vienna, Austria (Received September 9, 1991/February 24, 1992) - EJB 91 1201

It has been found that staphylococcal enterotoxin B contains a proteolysis-sensitive sequence in the cysteine loop formed by two half-cystines located in the middle of the toxin polypeptide chain. Fragments of the enterotoxin formed as a result of its digestion in this region have been isolated, their N-terminal scyuences have been determined and sites of proteolysis have been identified. It has been demonstrated that the N-terminal fragment of staphylococcal enterotoxin B is capable of activating T cell proliferation in the culture of human mononuclear cells practically to the same degree as the intact enterotoxin. The toxin's C-terminal fragment possesses an ability to activate calmodulindependent enzymes and is probably the toxicogenic part of the enterotoxin.

Staphylococcal enterotoxins (S. enterotoxins) represent a group of proteins with molecular masses ranging between 27 - 30 kDa which are secreted by Staphylococcus aureus and cause food poisoning [l]. At present, sevcn S. enterotoxins are known, namely A, B. C1, C2, C3, D and E [2]. Their amino acid sequences have been determined [3] and it was shown that all are single-chain polypeptides containing one disulfide bond formed by two half-cystines located in the middle of the polypeptide chain which form the so-called cysteine loop. S. enterotoxins are known to be most potent T cell mitogens [4]. T cell activation accompanied by induction of interleukin 2 and interferon [5]is conditioned by high-affinity interaction of S. enterotoxins with class 11 main histocompatibility complex (MHC) molecules [6] and subsequent presentation of the complex formed to a variable region of the subunit of the T cell receptor (V,TCR) [7]. From the character of receptor interactions and the nature of T cell proliferative response, the mechanism of S. enterotoxins' action on cells of the immune system is completely identical to that of MIS antigens [8, 91; on this basis, the foregoing proteins were united in the family of the so-called 'superantigens'. Despite the fact that interaction of S. enterotoxins with immunocompetent cclls has been studied in sufficient detail, there are no precise data explaining the interrelationship between immunomodulatory properties and the possible role of these toxins in pathogenesis of staphylococcal infections. There are also no data about the structural and functional organization of S. cnterotoxins and the regions of their moelcules responsible for receptor interactions and toxicogenic functions. Correspondence to V. Yu. Alakhov, Research Center of Molecular Diagnostics and Thcrapy, Simpheropotsky blvd. 8, Moscow, Russia 113149 Fax: +70951132633. Abbreviutions. S. enterotoxins, staphylococcal enterotoxins; MHC, main histocompatibility complex; V,TCK, variable part o f psubunit of T cell receptor; NaCI/Pi,phosphate-buffered saline; BacM. bacteriomodulin. Enzjwe. Adenylatc cyclase (EC 4.6.1.I).

The present work is devoted to the study of the domain structure of S. enterotoxin B. We have found that it can be easily digested in the cysteine loop by contaminating proteases that are present in the toxin preparation. The fragments formed were isolated and their N-terminal amino acid sequences were determined, which allowed these fragments to be identified in the S . enterotoxin B structure. Studying the functional properties of the fragments obtained also allowed us to identify a region in the S. enterotoxin B structure that is responsible for its mitogenic activity, as well as to demonstrate the presence in S. enterotoxin B of a Ca2+-indcpendent peptide analog of calmodulin, previously discovered by us in S. enterotoxins A [lo].


S. enterotoxin €3 was obtained as previously described [ l l , 121. The procedure of purification included chromatography on Bio-Rex 70 (Bio-Radj, CM-cellulose (Whatman) and reversed-phase HPLC on wide-pore carrier Ultrapore C8 (Beckman). When chromatography on BIO-Rex 70 was performed at room temperature, a preparation of S . enterotoxin B digested in the cysteine loop was obtained. This cysteine loop was reduced with 100 mM dithiothreitol for 2 h at room temperature. Gel electrophoresis was performed according to Laemmli [17]. 12.5% SDSjPAGE was used with the following protein markers: x-lactalbumin (14.4 kDaj, trypsin inhibitor (20.1 kDa), carbonic anhydrase (30 kDa) (Pharmacia). Gels were stained with Coomassie brilliant blue R-250. S. enterotoxin B fragments were separated by means of reversed-phasc HPLC on wide-pore carrier Bakerbond WPCls in 0.1 %n trifluoroacetic acid. Elution was performed using a 0 - 75% linear gradient of acetonitrilc. The N-terminal amino acid sequences of S. enterotoxin B and its fragments were determined by automated Edman

824 degradation using an Applied Biosysterns protcin sequencer (type 477A). Human mononuclear cells were isolated according to a previous method [13] and cultured in 96-well tissue-culture clusters (Costar Corp.) at a concentration of 1 x lo6 or 2 x lo6 cellsjml in RPMI 1640 medium containing 10% fetal bovine serum, 100 Ulml penicillin and 100 pglml streptomycin. The cells were cultured with S. cnterotoxin B and its fragment during 72 h at 37°C and 56% COz. Then incorporation of [3H]thymidine (25 Cijmmol, Amersham) into cellular DNA was determined; [3H]thymidine was used at a final concentration of 1 pCi/ml. The activity of adenylate cyclase kindly provided by Dr E. Taffelshtein, Irkutsk Antiplague lnstitute of Siberia and Far East, Russia) was determined at 30°C in the medium (50 pl) containing 20 mM TrisiHCl pH 7.5, 10 mM MgCI2, 0.1 mM GTP, 1 mM CAMP, 0.1 mM [ U - ~ ~ P I A T(1P x lo6 cpm, Amersham), 0.5 mM isobutylmethylxanthine, 20 mM crcatine phosphate and 0.5 mg/ml creatine kinase. The reaction was stopped after 20 min and the [32P]cAMP formed was assayed by the method described [14]. To obtain hybridomas producing monoclonal antibodies to S. enterotoxin B, BALBIc mice were twice immunized in hind footpads with heat-inactivated (30 min boiling in Na/Pi). S. enterotoxin B (50 pg/mouse) with an interval of 14 days. Hybridomas were obtained by fusing lymphocytes from popliteal lymph nodes with myeloma cell line X63-AgS-653 in the presence of poly(ethy1cne glycol) 4000 (Merck) [I 51. Cloning was performed twice by the limiting dilution technique in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 5% macrophase-conditioned medium, antibiotics and 2 mM L-glutamine. Screening of hybridomas was routinely carried out by ELISA using 96-well micro-titre plates (Costar Corp.) coated with S. enterotoxin B. Twice cloned hybridomas were injected intraperitoneally (lo6 cells in 0.5 ml NaCI/Pi) in pristaneprimed BALB/c mice. Monoclonal antibodies were purified from ascitic fluids by means of ammonium sulfate precipitation with subsequent immunoaffinity chromatography on S. enterotoxin-B - Sepharose obtained by a method described previously [I 61. RESULTS AND DISCUSSION

S. enterotoxin B fragmentation and separation of fragments S. enterotoxins are generally stable to the action of proteases but in the cysteine loop, located in the central part of the polypeptide chain, there are regions that are sensitive to proteolysis. Thus, in particular, treatment of native S. enterotoxin B with trypsin leads to its digestion at Lys97Thr-98. S. enterotoxin C1 also undergoes limited proteolysis under the action of trypsin and is digested in the region located in the cysteine loop [18]. S. enterotoxin A is rather resistant to the action of trypsin but papain digests this toxin with formation of peptidcs that can be separated only after reduction of the S-S bond [19]. This indicates that in this case sites of proteolysis are also located in the cysteine loop. We have established that during its isolation S. enterotoxin B undergoes limited proteolysis. As can be seen in Fig. 1, in the absence of reducing agent, SDS electrophoresis reveals only one protein band in the S.renterotoxin B preparation corresponding to a molecular mass of 28 kDa. The reduction of S. enterotoxin B with dithiothreitol leads to the appearance of thrcc additional bands with molecular masses of 17,16 and

Fig. 1. Analysis of S. enterotoxin B preparation by means of electrophoresis in 12.5% SDS/PAGE. The positions of protein markers are shown (see Materials and Methods). (a) S. enterotoxin B rcduccd with 100 mM dithiothrcitol for 2 h at room ternpcrature; (b) S. enterotoxin B, not treated with the reducing agent.

11 kDa, it can be concluded that. in this case, S. entcrotoxin B is digested in at least two sites; this is supported by the number of fragments formed, the sites of proteolysis being located in the cysteine loop. The formation of the S. entcrotoxin B fragments shown in Fig. 1 is evidently caused by the action of contaminating proteases that are isolated with the toxin. This result also confirms the fact that the region of the polypeptide chain that possesses increased sensitivity to proteolysis is limited by two half-cystines in the molecule of S. enterotoxin B. It should be noted that this region of S. enterotoxins is characterized by the highest degree of structural similarity. The second half-cystine is located in a part of the sequence that exhibits 100% similarity in all S. enterotoxins ~31. Huang et al. [20] formulated a hypothesis that this part of the molecule is responsible for the emetic activity of S. cntcrotoxin. However, the data stated above suggest that this region contains two functional domains of S. enterotoxins which act separately on their interaction with biological targets. In this case, a high degree of conservatism of this region may be conditioned by the necessity of its recognition by thc enzyme systems mediating the processing of S. enterotoxins. To elucidate the role of the fragments foiined as a result of S. enterotoxin B digestion and their biological effects, it seemed reasonable to isolate these fragments, identify them in the structure of S. enterotoxin B and try to detect their activities. The fragments formed after reduction of S. enterotoxin B were separated by HPLC on wide-pore reversed-phase carrier Bakerbond WPCI8 (Fig. 2a). The analysis of the fractions obtained by SDS electrophoresis (Fig. 2b) revealed that fraction 1 contained an 11-kDa peptide, fraction 2 full-size S. enterotoxin B, and fraction 3 a mixture of 1QkDa and 17-kDa fragments with more of the 16-kDa fragment. Identification of S. enterotoxin B fragments in the amino acid sequence

Table 1 lists the N-terminal amino acid sequences of the peptides obtaicnd. The 11-kDa peptide in fraction 1 has a sequence corresponding to the N-terminal sequence of S. enterotoxin B. This preparation also contains, as an admixture, a peptide with a sequence beginning from the second amino acid residue of S. enterotoxin B. This means that partial dcgradation of S. entcrotoxin B results not only in formation of the

825 ed at Asn100-Asp101 and Gln106-Thr107, which leads to formation of the N-terminal fragment with a molecular mass of about 11 kDa and two C-terminal peptides with molecular masses of 16 and 17 kDa. The fact that only one N-terminal fragment of S. enterotoxin B was discovered can be explained in two ways: either fragments 1-100 and 1-106 cannot be separated by the methods used, or fragment 101 -106 is unstable in the N-terminal part of S. enterotoxin B and fragment 1 - 106 degrades before fragment 1 - 100. It should be noted that S. enterotoxin B is digested with formation of fragments that have C-terminal Gln and Asn. It can be assumed that this is conditioned by the action of staphylococcal glutamine protease [21] that may be present in the preparation of S. enterotoxin B as an endogenous contaminant. This finding is also supported by cleavage of N-terminal Gln occurring during formation of the 11-kDa peptide. Thus, from the point of view of structural organization, the molecule of S. enterotoxin B may be represented as two domains formed by N- and C-terminal regions of the toxin connected by the cysteine loop that contains a proteolysissensitive region. It should be noted that this type of structural organization is characteristic of many protein toxins, both of bacterial (diphtheria toxin [22]) and plant (ricin [23]) origin. In all the foregoing cases, one of the domains, termed B (binding) subunit, provides for the toxin interaction with the receptor,

11-kDa, 16-kDa and 17-kDa peptides, but is accompanied by the cleavage of N-terminal Glu. The data depicted in Fig. 2a and the results of determination of the N-terminal amino acid sequence of fraction 2 indicate that this preparation contains homogeneous full-size S. enterotoxin B. Fraction 3 contains a mixture of the 16-kDa and 17-kDa peptides (Fig. 2b). Substantial difference in the content of these peptides in the preparation (72% and 28%, respectively) allowed us to determine their N-terminal amino acid sequences (Table 1). By comparison of these sequences with the primary structure of S. enterotoxin B (Fig. 3), we were able to localize the 11-kDa, 16-kDa and 17-kDa peptides in the polypeptide chain of S. enterotoxin B and identify the sites of proteolysis. As can bc sccn in Fig. 1, S. enterotoxin B is digest-

a I




















........... . .. .



14 0











Fig. 2. Separation of fragmcnts forming upon reduction of S. enterotoxin B by reversed-phase HPLC (a) and analysis of the fragments obtained by means of electrophoresis in 12.5% SDS/PAGE (b).

Fig. 3. Comparison of the primary structure of S. enterotoxin B with N-tcrminal scquences of the peptides obtained.

Table 1. N-terminal amino acid sequences of S. enterotoxin B and its fragments. The fraction numbers correspond to those in Fig. 2a.

Y r act ion number

Amino acid scqucnce 1















15 %




















Ser Glu

Gln Ser

Pro Ciln

Asp Pro

Pro Asp

Lys Pro

Pro Lys

Asp Pro

Glu Asp

Leu Glu

Hi5 Leu

Lys His

Scr Lys

Ser Ser

Lys Ser

18 100

































826 while the second one, A (activatory) subunit, is responsible for its toxicogenic function. A and B subunits connected by a proteolysis-sensitive sequence in the cysteine loop are translated as a single polypeptide chain which is processed either in host cells (e.g. ricin), or in target cells (e.g.diphtheria toxin).

Hence, it can be concluded that the regions responsible for the mitogenic effect of S. enterotoxin B and therefore, for its interaction with class I1 MHC molecules and V,TCR are located in the N-terminal part of the toxin. These data differ from the results obtained for S. enterotoxins A [6]. In this work it was demonstrated that digestion of' carboxyinethylated S. enterotoxins A by cyanogen bromide The functional properties of S. enterotoxin B fragments at Metl07-ThrlOX located near the cysteine loop led to a The receptor interactions of S. enterotoxin B are con- considerable decrease in the mitogenic effect of the toxin. This ditioned by its high-affinity binding with class I1 MHC mol- divergence of the results can be explained in at least two ways. ecules and subsequent presentation to V, TCR. These process- First, it is probable that the receptor-recognizing regions of S. es result in activation of T cell proliferation. As can be seen enterotoxins A and S. enterotoxin B are located in different in Fig. 4, native S. enterotoxin Band S. enterotoxin B digested parts of the toxin and, in the case of S. enterotoxins A, the in the cysteine loop display practically the same mitogenic cysteine loop is involved in the interaction. Second, in thc activities with respect to human mononuclear cells. The N- above-mentioned work, the composition of the products terminal 11-kDa peptide also activates T cell proliferation, obtained after treatment of carboxymethylated S. enterothough to a lesser degree. At the same time, the C-terminal toxins A with cyanogen bromide was not analyzed. Therefore, 16-kDa peptide practically does not affect T cell proliferation. it cannot be excluded that digestion of S. enterotoxins A A small mitogenic effect observed under the action of high with cyanogen bromide leads to abnormal cleavage of the Nconcentrations of this fragment is evidently conditioned by terminal part of the toxin or some other modification which the presence of an admixture of S. enterotoxin B (no more leads to its inactivation. At the same time, the fact that the Nthan 1%) in the 16-kDa peptide preparation which is terminal part of S. enterotoxins A is involved in the interaction undetectable by SDS electrophoresis and sequencing analysis. with class I1 MHC molecules or V,TCR is supported by results of a study [24] in which it was shown that a synthetic Nterminal fragment of S. enterotoxins A (residues 1-28) and antibodies to this peptide blocked the ability of S. enterotoxins A to activate T cell proliferation. We have obtained a panel of monoclonal antibodies to S. enterotoxin B among which only one type, designated mAbS5, possesses an ability to neutralize the mitogenic effect of S. enterotoxin B with respect to human mononuclear cells (Table 2). Fig. 5 shows the results of immunoblotting that illustrate the specificity of mAb-S5 with respect to S. enterotoxin B fragments. The data presented clearly show that these antibodies interact with the 1 I-kDa N-terminal fragment of S . enterotoxin B. However, the data do not establish exactly what stage of receptor interactions of S. enterotoxin B is blocked by mAb-S5: binding with class I1 MHC molecules or presentation to V,TCR. Nevertheless, the above-discussed results confirm our previous finding that the N-terminal part of S. enterotoxin B is responsible for its mitogenic effect. Previously we discovered that limited proteolysis of S. I I I enterotoxin A resulted in formation of a fragment termed O - 01 -1 t -10 -9 lg [Peptide] (M] bacteriomodulin (BacM) that was capable of Ca2'-independent activation of calmodulin-dependent enzymes [25]. The Fig. 4. Dependence of the rate of [3H)thymidineincorporation into DNA of human mononuclear cells on concentrations of native (7) and reduced same peptide was discovered by us in the lysate ofproliferating human lymphoblastoid cells pretreated with S. enterotoxin A (0) S. enterotoxin B, 11-kDa (A)and 16-kDa (@) peptides.

Table 2. Effect of monoclonal antibodies to S. enterotoxin B on the ability of 0.1 nM toxin to activate proliferation of human mononuclear cells Mean of five experiments is given with S.E.M. in parenthesis. Each experiment was performed in triplicate. mAbiSubisotype

S1 :IgG2a S5iIgC2a


l W 3 x Incorporation of ['Hlthymidine at mAb concn of 1.3 x 10'- M

1.3 x 10"-

12670 (607) 223 (22) 10135 (695)

10123 (913) 6865 (443) 13016 (849)


1.3~10'M ~

S. enterotoxin B

11016 (723) 10965 (821) 11625 (1015)

11 620 (963)

248 (37)


Fig.5. Rcsults of immunoblotting of reduced S. entcrotoxin B prcparation with mAb S5 (a) and protein staining (b). Electrophoresis waq carried out as in Fig. 1 .

terminal domain of the toxin, displays its activatory properties after removal of the N-terminal domain. The fact that BacM was detected both in S. enterotoxins A and S. enterotoxin B suggests that this activatory peptide is present in all other S. cnterotoxins. The appearance of a Ca2+-independent intracellular functional analog of calmodulin in a cell should inevitably dramatically influence Ca2 -dependent regulation of intracellular metabolism. Therefore, it can be assumed that BacM is related to the pathogenesis of S. enterotoxins and probably IS their toxicogenic fragment. It should be noted that thc activatory ability of BacM obtained from S. enterotoxin B is less pronounced than that of BacM from S. enterotoxins A, which correlates with toxicity of S. enterotoxins A and S. enterotoxin B. In the present work we have demonstrated that S. enterotoxin €3 consists of two functionally active domains connected by the cysteine loop that contains a proteolysis-sensitive sequence. The N-terminal domain is responsible for realization of the toxin’s mitogenic effect and probably contains sites involved in the interaction with class I1 MHC molecules and V,TCR. The C-terminal domain of S. enterotoxin B is a Ca2+-independent functional analog of calmodulin, BacM, and probably fulfils the toxicogenic function of S. enterotoxin B.








Fig. 6. Effect on the activity of calmodulin-dependent adenylate cyclase of Bacillus anfhraci~sof (a) 0.1 pM calrnodulin, (b) 0.1 pM S. entcrotoxin R, (c) 0.1 pM 11-kDa peptide, (d) 0.1 pM 16-kDa peptide, (e) basal activity of the enzyme. Data was averaged for lhrcc scparatc experiments.

[lo] to which this toxin displayed antiproliferative activity [ll]. We also established that the value of proliferative effect of S. enterotoxin A was directly proportional to intracellular penetration of the toxin 1261. Thus, it can be assumed that BacM is a toxicogenic fragment of S. enterotoxins A whose target is the system of calmodulin-dependent enzymes. In this work we have studied the effect of S. entei-otoxin B and its 11-kDa and 16-kDa fragments on the activity of calmodulin-dependent adenylate cyclase of Bacillus anthracis. These data are represented in Fig. 6. It can be seen that native S. enterotoxin B and its 11-kDa fragment do not produce any effect on the enzyme activity. At the same time the 16-kDa fragment activates adcnylate cyclase to a somewhat lesser extent than calmodulin. Thus, it can be concluded that S. enterotoxin B also contains BacM. This peptide, being a C-

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Identification of functionally active fragments of staphylococcal enterotoxin B.

It has been found that staphylococcal enterotoxin B contains a proteolysis-sensitive sequence in the cysteine loop formed by two half-cystines located...
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