INFECTION AND IMMUNITy, Sept. 1976, p. 680-686Copyright © 1976 American Society for Microbiology

Vol. 14, No. 3Printed in U.S.A.

Molecular Forms of Neurotoxins in Proteolytic Clostridiumbotulinum Type B CulturesBIBHUTI R. DASGUPTA* AND H. SUGIYAMA

Food Research Institute* and Department ofBacteriology, University of Wisconsin,Madison, Wisconsin 53706

Received for publication 23 February 1976

A modified purification method was used to isolate the neurotoxin of proteo-lytic Clostridium botulinum type B strain Lamanna. The preparation was foundto be a mixture of two protein forms. They were of molecular weight 152,000 andcould not be separated by ion-exchange chromatography or electrophoresis inpolyacrylamide gel. One was a single polypeptide chain, and the other was adichain molecule (nicked toxin) held together by an interchain disulfide bond(s).Trypsinization increased the toxicity of the toxin preparation and converted thesingle-chain molecules into dichain forms that were indistinguishable from theendogenously generated nicked toxin. A protease of the type B culture, withsubstrate specificity similar to that of trypsin, did not change detectably themolecular form of unnicked type E toxin, although toxicity was increased.Higher toxicity was obtained when unnicked type E was trypsinized; the result-ing preparation contained only nicked toxin molecules.

Neurotoxins of Clostridium botulinum aresynthesized as molecules having low or possiblyno toxicity. Their characteristically high spe-cific toxicity is attained when these "native"toxins are activated. With proteolytic cultures(types A and B), activation occurs endoge-nously during toxin production from the actionof protease(s) produced by the organisms (10,11); in cases of nonproteolytic strains (types B,E, and F), the full activation is obtained whenthe toxin is trypsinized experimentally (8, 10).Trypsin (EC 3.4.4.4) does not increase the toxic-ity of type A toxin isolated from cultures incu-bated for maximum toxicity, but gives highactivation of type E toxin (10). Toxin of proteo-lytic type B strain Lamanna is intermediate inthat its trypsinization results in a low but de-tectable toxicity increase (10).The fully activated botulinum toxin types

studied have a common "nicked" protein struc-ture that is comparable to that described fordiphtheria toxin (2, 13). Nicked botulinum tox-ins as defined here are the toxic molecules ofmolecular weight 145,000 to 167,000 which arecomposed of a heavy (H) and a light (L) chainheld together by a disulfide bond(s). H is ap-proximately twice the molecular weight ofL (1,5, 12, 14). The untrypsinized type E toxin hasthe molecular weight of the activated form, butis a single-chain unnicked protein which, dur-ing its activation by trypsin, is nicked to be-come a two-chain protein. This dichain mole-cule is comparable to the endogenously acti-vated toxin of proteolytic cultures. The H and L

chains of nicked toxins can be separated bypolyacrylamide sodium dodecyl sulfate electro-phoresis only after their disulfides are reduced(5).The work reported here relates the molecular

structure of the toxin purified from the proteo-lytic type B Lamanna strain to its low activata-bility with trypsin. Possible other molecularchanges of native toxin during its endogenousactivation are considered.

MATERIALS AND METHODS

Buffers were prepared by titrating acidic andbasic components of the same molarity; pH valuesare those determined at 23°C. Citrate buffer compo-nents were citric acid and trisodium citrate; phos-phate buffer was made with di- and monosodiumphosphate and acetate buffer was made with aceticacid and sodium acetate; and Tris-hydrochloride wastris(hydroxymethyl)aminomethane and HCl. Seph-adex materials (Pharmacia Fine Chemicals) were of40- to 120-,um beads, and chromatographic stepswere by gravity flow at room temperature exceptwhere noted. Protein concentrations were deter-mined as absorbances at 278 nm (A278). Trypsin,freed of chymotryptic activity with L-tosylamido-2-phenylethylchloromethyl ketone, and soybean tryp-sin inhibitor were from Worthington BiochemicalCorp., Freehold, N.J. Toxicity was assayed as 50%lethal dose (LD50) for mice (7).Toxin preparations were trypsinized by incuba-

tion at 37°C with enzyme dissolved in 0.01 or 0.02 Mphosphate buffer (pH 6.0). After incubation, a 0.1-mlaliquot was mixed with soybean trypsin inhibitor ata trypsin/inhibitor ratio of 1:1.5 (wt/wt). A secondaliquot, added to a solution made of 20 ul of 10%

680

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NEUROTOXIN OF C. BOTULINUM TYPE B 681

dodecyl sulfate and 0.1 ml of 0.1 M phospb(pH 7.0) containing 0.1% dodecyl sulfate amercaptoethanol, was immediately placecing water bath and heated for 5 min.

Polyacrylamide gel electrophoresis wilsulfate (PAGE) was that used previouslytive concentrations of unnicked toxin,chains, in a solution were estimated, aseparation by PAGE, from densitometrimade of the stained gels at 560 nm withlinear transport scanner (model 24108) rucm/min and with recorder chart speed ofmin. That the areas in the traced peakEprotein concentrations was shown by a tesseparate gels were charged with 100, 80,or 10 ,l. of a type B toxin preparation arphoresed for 6 h. For each molecular specieof areas against sample volume showed aline relationship (Fig. 1). The H chainchromogenic than the L chain, so that, vprecision of the procedure, H/LAN. ratios1.6:1 represented equimolar amounts of thteins.Type E neurotoxin preparation (7) sh

the unnicked toxin band (molecular weighin PAGE. Toxicity after trypsinization w107 LD50 per 1.0 A278.The protease (TLE) with substrate

similar to that of trypsin was an essentiallneous preparation that was derived frormanna culture strain used as the source

12001

soo0

400

20 40 60 s0Sample Volume (,1)

FIG. 1. Relationship between protein abands separated by PAGE and the ared

densitometric peaks of stained bands. Sadifferent volumes of a type B toxin (A278reduced with mercaptoethanol; HandL chua 1:1 molar ratio in all samples.

iate buffer toxin (6). Before use, it was reduced for 1 h at 370CLnd 3 ,ul of with 5 mM dithiothreitol solution made in 0.05 MI in a boil- acetate buffer (pH 6.0) containing 2 mM CaCl2.

Type B neurotoxin was purified from 8-liter cul-th dodecyl ture lots grown in carboys of 9-liter capacity. Cul-(5). Rela- ture medium for producing progenitor toxin (3),H and L without added NaCl, was inoculated with 2 ml ofLfter their culture stock that had been stored at - 20°C. Incuba-ic tracings tion for toxin production was 96 h at 37°C or 168 h ata Gilford 30°C. Toxin purification was by a procedure modi-nning at 2 fied from the one used previously (4).:5.01 mm/ Crude toxic precipitate was obtained by dissolv-B reflected ing 313 g of (NH4)2SO4 per liter of incubated culture;t in which and holding it for 48 h at 40C. The precipitate was60, 40, 20, collected by centrifugation and extracted twice withid electro- 100 ml of 0.05 M citrate buffer (pH 5.5). The precipi-'s, the plot tate, obtained by dissolving 351 g of (NH,)2SO4 pera straight- liter of clarified (by centrifugation) pool of the ex-was more tracts, was dissolved in 40 ml of citrate buffer. Afterwithin the dialyzing against the same buffer, a 20-ml sampleof 1.3:1 to (A278 = 25.6 and A260 = 37 to A278 = 80 and A260 =

Le two pro- 124) was loaded on a diethylaminoethyl (DEAE)-Sephadex A-50 column (2.2 by 45 cm). Essentially all

owed only the applied toxicity was recovered in the first pro-Lt, 147,000) tein peak that was eluted in washing the column{as 1.71 x with the citrate buffer. The A26d/A278 ratio of 0.5 to

0.55 in the toxic fractions indicated removal of mostspecificity of the nucleic acids.y homoge- Disodium ethylenediaminetetraacetate (EDTA)m the La- and (NH4)2SO4 were dissolved in the pool of the toxicof type B fractions to 5 mM and 351 g/liter, respectively. The

precipitate, collected after 48 h at 40C, was dissolvedin 5.0 ml of 0. 15 M Tris-hydrochloride buffer (pH 7.4)containing 5 mM EDTA. No more than 1.6 ml of the

*c

solution was applied on a Sephadex G-200 column/ Hchain (2.5 by 95 cm) equilibrated with the same buffer.pToxin The toxin, which was recovered in the second pro-

tein peak, was concentrated with (NH4)2SO4 (351g/liter) and rechromatographed on Sephadex G-200

L chain without EDTA in the buffer.All subsequent steps were performed at 4°C. The

cooled toxic pool was applied to a DEAE-SephadexA-50 column (1.5 by 25 cm) equilibrated with theTris-hydrochloride buffer. The major protein peakeluted with a NaCl gradient (150 ml of Tris-hydro-chloride buffer + equivolume buffer with 0.15 MNaCl) contained most of the toxin; a small secondpeak that developed in some runs was discarded.The toxic pool was dialyzed against 0.02 M phos-phate buffer (pH 5.95) and then chromatographed ona sulfoethyl (SE)-Sephadex C-50 column to recoverneurotoxin in a sharp, symmetrical peak (Fig. 2).The pool of toxic fractions was stored at 40C in thepresence of 5mM EDTA and 3 mM NaN3. To processan 8-liter culture lot required sequentially fourDEAE-Sephadex A-50, four Sephadex G-200, two

z . repeat Sephadex G-200, one DEAE-Sephadex A-50,100 and one SE-Sephadex C-50 columns.

mounts in RESULTSas of Are The type B toxin purification proceduretmples are3 = 0.146) yielded uniform toxin preparations more con-ains are in sistently than the one used previously (4).

About 5 to 7% of the starting toxicity was re-

t

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682 DASGUPTA AND SUGIYAMA

Tube No.

FIG. 2. Final chromatographic step in purifica-tion oftypeB toxin. A SE-Sephadex C-50 column (1.5by 25 cm) equilibrated with 0.02 Mphosphate buffer(pH 5.95) was loaded with 56 ml ofdialyzed solutionof A278 = 0.38. Gradient elution (150 ml of the pH5.95 buffer + 150 ml ofsame buffer containing 0.15MNaCI) started at arrow. Fractions (4.8 ml/tube) 30to 42 pooled as purified toxin.

covered such that 7 to 9 mg of purified toxin(A260/A278 = 0.48) was obtained from 8-litercultures incubated at 370C for 96 h. Specifictoxicity of the preparations (trypsinized)ranged from 4.7 x 107 to 6.2 x 107 mouse LD5011.0 A278, or 1.14 x 108 LD50/mg of toxin based on

E °m of 18.5 (1). The molecular weights of threeseparately purified type B neurotoxin prepara-tions determined by seven PAGE experiments(5) appeared to be similar. This value of 152,000was lower than the previously reported valuesof 165,000, 167,000, and 170,000 (1, 4, 12). Themolecular weight of the H chain was essen-

tially twice that of the L chain.PAGE analyses of toxin preparations not

treated with a disulfide reducing agent showedessentially only the expected single band (pro-teins of molecular weight 152,000), but traces oftwo other bands could occasionally be detected(Fig. 3A). That these faint, fast-moving bandsprobably represent the H and L chains, whichare normally held together as nicked toxin, wasindicated by results of electrophoresing untryp-sinized but reduced toxin. The test resolved thesame three bands, except that the two faster-moving ones were now much more prominent;the H and L chains, made separable by thereductive treatment, moved coincidently withthe original trace bands so that the latter couldnot be distinguished from the known H and Lbands (Fig. 3A,B).The tests showed the toxin preparations to be

essentially a mixture of nicked toxin and un-nicked protein of molecular weight 152,000

(Fig. 3A,B; Fig. 4A,B). The unnicked proteinwas identified as unnicked toxin by the corre-lated, progressive disappearance of its bandwith increases of H and L chains during tryp-sinization of toxin samples. Since this changein electrophoretic pattern could be shown onlywhen the trypsinized sample was reduced (Fig.3C,D; Fig. 4C), trypsin must have cleaved abond of a protein, but the cleavage productsremained together until the connecting S-Slinkage(s) was broken. The behavior was thatexpected of unnicked toxin. The toxin purifiedfrom the proteolytic type B culture was, there-fore, a mixture ofnicked and unnicked toxin.The ratio of nicked (sum of H and L chains

obtained by PAGE ofreduced but untrypsinizedpreparations) to unnicked toxins in the purifiedtoxin preparations varied with the incubationsused for toxin production. In samples purifiedfrom cultures incubated at 37°C for 96 h, theratio ranged from 1:0.4 to 1:0.6; in samples de-rived from cultures incubated at 30°C for 168 h,the ratio ranged from 1:1.6 to 1:1.8. Trypsiniz-ing these toxin preparations for maximum tox-icity increased specific toxicity only two- tothreefold.Type B toxin samples from the two differ-

ently incubated cultures were trypsinized atratios of substrate to enzyme of 20:1, 14.7:1,9.8:1, 4.9:1, and 3.25:1 (wt/wt) and incubationtimes of 0, 10, 20, 40, and 60 min. Comparisonsof protein concentration in the bands resolvedby PAGE showed that L and H chains increasedin a fixed (1:1) molar ratio (Fig. 3B-D; Fig.4B,C).

Possible differences in action of TLE andtrypsin on unnicked type E toxin were studied.Toxin (A278 = 0.113) was incubated separatelywith an equal volume of TLE (A278 = 0.487) ortrypsin (final concentration, 28 ,ug/ml), both in0.05 M acetate buffer (pH 6.0) with 2 mM CaCl2.Control was toxin mixed with buffer. After 1 hat 37°C, the mixtures were analyzed for toxicityand by PAGE. The same tests were made witha portion of the TLE-treated sample that wasfurther incubated for 1 h with trypsin at a finalconcentration of 28 Ag/ml.

Toxicities, corrected for dilutions, of toxintreated with TLE, trypsin, and TLE plus tryp-sin were, respectively, 4, 29, and 8 times (seereference 7 for explanation on such low, high,and intermediate values) higher than the 5.6 x103 LD50/ml ofthe control. All samples analyzedby PAGE without reduction gave only the bandrepresentative of molecules of 147,000 molecu-lar weight. Reductive treatment of the controland the TLE-treated samples did not changethe electrophoretic pattern. When toxin wasreduced after being treated with trypsin alone

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VOL. 14, 1976 NEUROTOXIN OF C. BOTULINUM TYPE B 683

A

.....

.. L

B

P~~~~~~~~~~~~~~~~........... .........

..~~~~~.7.7%+v.;

-,~~~~~~~~~'X

J it

FIG. 3. Densitometric tracings ofbands resolved by PAGE oftype B toxin purifi'ed from a culture incubatedfor 96 h at 37C.A.7.of toxin before trypsinization = 0142. Sample on gel is 100 pi. (A) Unreduced; (B)reduced; (C) reduced after trypsinization for 10 min at a toxin/enzyme ratio of 4.9:1 (wt/wt); (D) same as Cexcept trypsinized for 60 min. Ratios ofH and L peak areas in B, C, and D are, respectively, 1.2:1, 1.3:1, and1.3:1. Direction of migration is left to right (anode).

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684 DASGUPTA AND SUGIYAMA

A

B

C

FIG. 4. Densitometric tracings ofbands resolved by PAGE oftype B toxin purified from a culture incubatedfor 168 h at 30°C. A278 of toxin before trypsinization = 0.125. Sample on gel is 100 pl. (A) Unreduced; (B)reduced; (C) reduced after trypsinization for 60 min at a toxin/enzyme ratio of4.9:1 (wtlwt). Ratio ofHandLpeaks in B and C are both 1.6:1. Direction of migration is left to right (anode).

or with TLE and then trypsin, the originalband was replaced wth those of proteins of mo-lecular weight 102,000 (H chain) and 50,000 (Lchain). These PAGE results are identical tothose already published (Fig. 1 of reference 5).

DISCUSSIONExcluding the trace amounts of H and L

chains that are apparently held together, bynoncovalent bonds after reduction of their in-terchain S-S bond(s), type B toxin purified froma proteolytic culture is a mixture of unnicked

and endogenously nicked proteins. The concen-tration ratios ofthe two forms oftoxin dependedon the incubations used to produce the toxin.The low but detectable toxicity increases

when these toxin preparations are trypsinizedcould result from the enzyme acting on un-nicked or nicked toxin or both. Data obtainedwith other botulinum toxin types suggest theaction to be on the unnicked form. There is notoxicity increase upon trypsinizing type A (un-published data) and F (K. H. Yang, Ph.D. the-sis, Univ. of Wisconsin, Madison, 1974) toxin

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NEUROTOXIN OF C. BOTULINUM TYPE B 685

preparations that are free of unnicked toxin. Incontrast, type E preparations containing onlyunnicked toxin show significant specific toxic-ity increases that are coincident with the nick-ing of the molecule by trypsin (5).Trypsinization of the single-polypeptide-

chain form of the neurotoxin may cleave morethen one bond, with at least one leading to theformation of the dichain molecule, but splittingof more than one bond may be associated withactivation. It is not yet known whether all ofthe toxicity increase upon trypsinization isfrom nicking, i.e., the peptide bond cleavagecreating the H and L subunits, but the maxi-mum threefold toxicity increase observed aftertrypsin treatment of the type B preparations issuggestive. These preparations have 1.8:1 to0.4:1 ratios of unnicked to endogenously nickedtoxin. Assuming that nicked toxins, formed en-dogenously or by trypsinization, are at thehighest attainable specific toxicity, it can becalculated that even with a 2:1 ratio of un-nicked to nicked toxin forms, trypsinizationwould not increase toxicity more than three-fold.The H chain of endogenously nicked type B

toxin has a molecular weight essentially twicethat of the L chain. Action of trypsin on theunnicked toxin form in the purified toxin prepa-rations generates nicked toxin whose constitu-ents are 2:1 in relative molecular sizes. How-ever, these observations do not prove identity ofthe nicked toxins that are formed endogenouslyand by trypsinization; cleavage of unnickedtoxin either one-third or two-thirds the distancefrom the C- to N-terminals would both givechains of the observed ratio.The probability is that the nicked toxins

being compared are similar molecules that re-sult from cleavage of the same or very close-bypeptide bond(s). If such were not the case, theendogenously formed H chain would have nearits center the bond susceptible to trypsin; itscleavage would create two pieces, each of sizecomparable to the L chain. The situation wouldbe such that trypsinization of thq purifiedtoxin, which is a mixture of unnicked and en-dogenously nicked molecules, would result inmore molecules of L-chain dimension than ofHchain. Seven separate experiments, of whichFig. 3 and 4 are examples, did not give such amolecular ratio; in all cases, L and H chainsincreased in the 1:1 molar ratio at which thesechains exist in endogenously nicked toxin.The conclusion does not rule out the possibil-

ity of minor differences. Trypsin could havecleaved also a bond(s) close to C- and/or N-terminals. Such an event would go unrecog-

nized if PAGE cannot detect the very smallfragment(s) and cannot distinguish the largepiece(s) from the endogenously generated H orL (or both) chain because of similarities in size.Aside from lower activations of type E and B

toxins (3, 7) by TLE than by trypsin, the molec-ular changes induced by the enzymes are strik-ingly different. Trypsin cleaved unnicked typeE toxin into H and L chains, but a molecularchange accompanying the toxicity increasefrom TLE action could not be detected. Thus,the bonds attacked by the two enzymes must bedifferent in spite ofthe closely similar substratespecificities of these enzymes (6).Treating the purified type B toxin samples

with TLE did not give a detectable toxicityincrease or evidence of conversion of unnickedmolecules to the nicked form (unpublisheddata). Since these toxin molecules were devel-oped in culture with TLE activity (3), unnickedtype E toxin was taken as a model substratethat was not previously exposed to TLE (7). Ifaction ofTLE on type E toxin is representativeof its effect on toxins of proteolytic C. botu-linum cultures, the presence of only nicked,completely activated toxin forms in type A andF cultures that have been incubated for maxi-mum toxicity must occur because these cul-tures have another enzyme(s) capable of carry-ing out the nicking to give the H and L chains.This inference, that complete natural activa-tion requires the action of at least two enzymes,is consistent with our previous hypothesis (7).

Since the partially purified type B toxin prep-aration obtained from a 24-h culture, unlikethose purified from a 96- or 168-h culture, wasactivated by TLE (3), it is assumed at presentthat the nicked and unnicked toxins in purifiedtype B preparations have been endogenouslyacted upon by TLE.We can offer only speculation as to why un-

nicked toxin is present in a proteolytic culturethat has been incubated for as long as 7 days.The presence of nicked toxin shows that theculture has the mechanism for nicking nativetoxin, but complete conversion does not occur.One possibility would be that two kinds of toxicmolecules are synthesized; only one is suscepti-ble to endogenous "nicking enzyme," but bothare cleaved by trypsin. Alternatively, the rela-tively high concentration of an inhibitor oftrypsin that accumulates in type B cultures (9)might prevent nicking of toxin that is elabo-rated during the late stages of culture growth.

ACKNOWLEDGMENTSThis research was supported by the College of Agricul-

tural and Life Sciences, University of Wisconsin, Madison,and by Public Health Service grant 00712.

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INFECT. IMMUN.686 DASGUPTA AND SUGIYAMA

LITERATURE CITED

1. Beers, W. H., and E. Reich. 1969. Isolation and charac-terization of Clostridium botulinum Type B toxin. J.Biol. Chem. 244:4473-4479.

2. Collier, R. J. 1975. Diphtheria toxin: mode of action andstructure. Bacteriol. Rev. 39:54-85.

3. DasGupta, B. R. 1971. Activation of Clostridium botu-linum type B toxin by an endogenous enzyme. J.Bacteriol. 108:1051-1057.

4. DasGupta, B. R., D. A. Boroff, and K. Cheong. 1968.Isolation of chromatographically pure toxin of Clos-tridium botulinum type B. Biochem. Biophys. Res.Commun. 32:1057-1063.

5. DasGupta, B. R., and H. Sugiyama. 1972. A common

subunit structure in Clostridium botulinutn Type A,B and E toxins. Biochem. Biophys. Res. Commun.48:108-112.

6. DasGupta, B. R., and H. Sugiyama. 1972. Isolation andcharacterization of a protease from Clostridium botu-linum type B. Biochim. Biophys. Acta 268:719-729.

7. DasGupta, B. R., and H. Sugiyama. 1972. Role of a

protease in natural activation of Clostridium botu-

linum neurotoxin. Infect. Immun. 6:587-590.8. Eklund, M. W., F. T. Poysky, and D. I. Wieler. 1967.

Characteristics of Clostridium botulinum type F iso-lated from the Pacific coast of the United States.Appl. Microbiol. 15:1316-1323.

9. Hoyem, T., and A. Skulberg. 1962. Trypsin inhibitorsproduced by Clostridium botulinum cultures. Nature(London) 195:922-923.

10. lida, H. 1964. Experimental studies on toxin productionof Clostridium botulinum. Part I. Jpn. J. Bacteriol.19:458-461.

11. Inukai, Y. 1963. Activation of the toxin in the culture ofClostridium botulinum type A. Jpn. J. Vet. Res.11:87-93.

12. Kozaki, S., and G. Sakaguchi. 1975. Antigenicities offragments of Clostridium botulinum type B deriva-tive toxin. Infect. Immun. 11:932-936.

13. Sugiyama, H., B. R. DasGupta, and K. H. Yang. 1973.Disulfide-toxicity relationship of botulinal toxin typeA, E and F. Proc. Soc. Exp. Biol. Med. 143:589-591.

14. Yang, K. H., and H. Sugiyama. 1975. Purification andproperties of Clostridium botulinum type F toxin.Appl. Microbiol. 29:598-603.

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