Vol. 254, No. 21, Issue of November 10, pp. 10728-10733, 1979 Printed m U.S.A.

Structure of Tetanus Toxin DEMONSTRATION AND SEPARATION OF A SPECIFIC ENZYME CONVERTING INTRACELLULAR TETANUS TOXIN TO THE EXTRACELLULAR FORM*

(Received for publication, June 27, 1979)

Torsten B. Helting,$ Sieglinde Parschat, and Hermann Engelhardt

From the Kesenrch Laboratories, Behringwerke AG, 3550 Mnrburg/Lahn, Gernaany

Protease activity has been demonstrated in culture supernatants of Clostridium tetani at various stages of fermentation. Gel chromatography of the concentrated filtrates revealed the presence of three enzymatically active fractions eluting at separate positions off the column. The smallest protease was found to “nick” the single chain intracellular tetanus toxin, producing the extracellular, two-chain structure of the molecule. As little as 3 ng of active protease were sufficient to cleave 50 pg of intracellular tetanus toxin, suggesting that this enzyme is responsible for the observed structural change of the toxin molecule during its release into the culture medium. By comparison, the second protease, eluting at an intermediate position, exhibited only mar- ginal activity towards intracellular toxin. The third, largest, enzyme was not active under the conditions of the assay. However, the latter protease effectively hy- drolyzed low molecular weight histidyl peptides, and it is concluded that this enzyme is similar to the one described by Miller, P. A. Gray, C. T., and Eaton, M. D. (1960) J. Bacterial. 79, 95-102.

The properties of the partially purified enzymes, in- cluding their differential behavior towards a number of protease inhibitors, are reported.

Recent work focused on the structure of tetanus toxin has helped to establish the existence of two molecular forms of this protein (l-6). Apparently, the toxin molecule is elabo- rated by Clostridium tetani as a single polypeptide chain with a molecular weight of about 140,000. This single chain mole- cule has been termed intracellular toxin and may be obtained by interrupting the fermentation prior to lysis, followed by extraction of the harvested bacteria with salt solutions. Alter- natively, an extracellular form of tetanus toxin may be re- covered from the filtrates of autolyzed cultures. Craven and Dawson (1) showed that extracellular toxin was separable into two polypeptide chains (heavy chain and light chain), which were linked to each other by a disulfide bond. Matsuda and Yoneda confirmed and extended these findings, reporting the conversion of the intracellular tetanus toxin to the extracel- lular form by digestion with trypsin (2). The two fragments, termed a- and ,&fragment, were apparently similar to the light chain and heavy chain polypeptide, respectively.

New data on the NHa-terminal structure of tetanus toxin seem to confirm the present concepts regarding its polypeptide chain composition (6). Thus, proline was the sole amino acid

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

$ Address after January 1, 1980: Sandoz Research Institute, Brun- ner Strasse 59, 1235 Vienna, Austria.

detected in NH&erminal position of the intracellular toxin, whereas the extracellular species contained NHa-terminal leu- tine in addition to proline. Furthermore, isolated light chain was found to exhibit proline as its NHz-terminal amino acid, whereas leucine occupied the equivalent position of the heavy chain polypeptide. These data suggest that the light chain fragment constitutes the NH?-terminal region of the original protein (6).

Since treatment of the intracellular toxin with trypsin re- sulted in the conversion to a structure very similar to that seen in toxin preparations derived from the culture filtrate, it is conceivable that a proteolytic enzyme of C. tetani is involved in this alteration of the molecular structure. Previously, pro- tease activity in cultures of C. tetani has been demonstrated (7-10) and there is some indication that the formation of a histidyl peptidase occurs concomitantly with tetanus toxin (8). In the present communication, three separate proteases were demonstrated in the bacterial culture filtrate. Only one of these was highly active in generating the heavy chain-light chain framework of tetanus toxin. A second proteolytic en- zyme exhibited substantial activity towards general protease substrates but produced only a marginal quantity of “nicked” tetanus toxin from the intracellular species. Finally, a protease lacking the capacity to convert the toxin to its extracellular form, but displaying properties consistent with the previously described histidyl peptidase (8), was separated from the other two enzymes.

MATERIALS AND METHODS

Chemicals-Sephadex G-100 gel was purchased from Pharmacia, Uppsala, Sweden. Ultrogel AcA 44 gel was ordered from LKB, Bromma, Sweden. Soybean trypsin inhibitor, benzamidine, and PMSF’ were delivered by Sigma, St. Louis, MO. TPCK-trypsin was obtained from Worthington, Freehold, N. J. Azocoll was a product of Calbiochem-Behring Corp., San Diego, Calif. I’rotease inhibitors de- rived from pooled human sera (tu-2.macroglobulin, cu-I-antitrypsin, and inter-a-trypsin inhibitor) were kindly supplied by Mr. Haupt, Behringwerke AG. Equine tetanus antitoxin (different lots) were stock preparations of Behringwerke AG.

Tetanus Toxin-The extracellular form of the toxin was derived from culture filtrates and purified as described previously (5). Intra- cellular tetanus toxin was prepared after interrupting the fermenta- tion process at 40 h. The live cells from 2-liter cultures were harvested by centrifugation, washed twice in 0.15 M NaCl, and suspended in %(I of the original volume in a buffer containing 1 M NaCl and 0.1 M

sodium citrate (3). The buffer was supplemented with benzamidine (2 g/liter) to minimize any proteolytic activity present. After incu- bating at 4°C for 5 days, the cells and debris were separated from the extracted protein by centrifugation. The supernatant was dialyzed against several changes of 0.01 M phosphate buffer and applied to a column (2 x 10 cm) of DEAE-cellulose, eluted with a gradient mixture

’ The abbreviations used are: PMSF, phenylmethanesulfonyl fluo- ride; IgG, immunoglobulin G fraction, produced by gel chromatogra- phy of whole serum on Sephadex G-150; SIX, sodium dodecyl sulfate.

10728

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Tetanus Toxin Converting Protease 10729

(500 ml of 0.2 M phosphate buffer into an equal volume of 0.01 M phosphate). The fractions containing the toxin constituted the major peak. This material was pooled, concentrated, and further purified by gel chromatography, on a column (2 X 100 cm) of Ultrogel AcA 44, eluted with 0.1 M Tris buffer, pH 8.0, containing 1 M NaCl. Tetanus toxin prepared in this fashion yielded a single polypeptide band on SDS-gel electrophoresis and contained only traces of nicked toxin as revealed by gel electrophoresis under reducing conditions.

Demonstration of Proteolytic Activity in Cultures of C. tetani- Samples (50 ml) of actively growing cultures were taken every 24 h and separated by centrifugation into a cell fraction and a supernatant fraction.The cells were washed with 0.15 M NaCl and resuspended in 2 ml of 0.1 M Tris-HCl buffer, pH 8.0. The supernatant fraction was also concentrated to 2 ml by ultrafiltration prior to enzymatic analysis. Proteolytic activity was determined as described below.

Separation and Partial Purification of Bacterial Proteases- After preliminary experiments (ll), the following procedure was adopted to separate the three proteases. The culture filtrate (6000 ml) was concentrated to 100 ml by ultrafiltration and applied in two portions to a column (5 X 100 cm) of Sephadex G-100, eluted with 0.1 M Tris-HCl buffer, pH 8.0, 1 M NaCl. Aliquots (0.05 ml) of each fraction were diluted to 1 ml with the same buffer and incubated for 6 h with Azocoll (5 mg/tube). Appropriate pools were formed and purified by a second chromatographic run on a column (2.5 x 250 cm) of Ultrogel AcA 44, eluted as above.

Determination of Proteolytic Actiuity-Column effluents were monitored by incubating each fraction with Azocoll and measuring the release of dye from the insoluble carrier calorimetrically (12). For convenience, some kinetic experiments and most inhibition assays were also performed using this substrate. General protease activity was also determined by following the liquefaction of fibrin in agar plates (13). Conversion of the intracellular form of tetanus toxin to the nicked species was demonstrated by SDS-gel electrophoresis under reducing conditions following incubation with various enzyme pools and using the intracellular toxin as the substrate. The disap- pearance of the toxin band at 140,000 daltons and the appearance of the composite polypeptide chains at approximately 93,000 and 48,000 daltons, respectively (5), indicated the presence of converting enzyme activity.

1 o-o 150

AGE OF CULTURE (HOURS)

FIG. 1. Formation of proteases by C. tetani as a function of fermentation. Every 24 h, samples (50 ml) were withdrawn from the culture and filtered. The culture filtrate was concentrated to 2 ml and aliquots (0.4 ml) were incubated with Azocoll at pH 8.0. After 3 h at 37”C, the tubes were read at 520 nm. C---O, samples taken from nonstirred l-liter cultures; H, samples from a lo-liter fermenter. In the latter case, the growth rate is always slightly below that observed in still cultures and maximal release of protease activity is obtained after 120 h (instead of 96 h) into the fermentation.

(-1

A B C

+Me +Me+Me

(+I FIG. 2. SDS-gel electrophoretic analysis after digestion of

intracellular tetanus toxin with the crude culture filtrate. A, intracellular toxin after incubation with the culture filtrate (sample taken at 120 h and concentrated 25-fold) overnight; B, the correspond- ing culture filtrate without added intracellular tetanus toxin; and C, intracellular tetanus toxin used as substrate. In each incubation the concentration of the components was kept constant. Preliminary treatment of the material with mercaptoethanol prior to gel electro- phoresis is indicated by the symbol +Me. The presence of extracel- lular tetanus toxin in the enzyme preparation interferes with the interpretation of the gel patterns. However, the disappearance of the single chain polypeptide due to intracellular toxin after digestion with the culture filtrate is indicative of the presence of converting enzyme activity (I$ Gel A +Me and C +Me).

Histidyl peptidase activity was demonstrated by incubating the enzyme preparation with glycyl-L-histidine (8) followed by high volt- age paper electrophoresis of the digest.

Other Analytical Methods-Gel electrophoresis in SDS buffer was performed by the procedure of Weber and Osborn (14). Estimation of the molecular size was achieved by determining the elution position of the enzyme on Ultrogel AcA 44. The column had been calibrated by a number of globular proteins and the approximate molecular size of each protease was computed as described by Andrews et al. (15).

Antisera-Rabbit antisera against fractions of the culture super- natant from C. tetani were produced by three subcutaneous injections of the partially purified enzyme. Prior to injection, the protease with an activity equivalent to 0.5 pg of trypsin was mixed with an equal volume of Freund’s complete adjuvant. In order to eliminate traces of tetanus toxin contaminating the preparations, two methods were employed. Preliminary treatment of each fraction with formaldehyde (0.1% HCHO, pH 6.5, 96 h at room temperature), or with rabbit antibodies against Fragment B of tetanus toxin (16) rendered all materials nontoxic. In some cases, the immunoglobulin fraction was separated from serum protease inhibitors by gel chromatography on Sephadex G-150.

RESULTS

Demonstration of Protease Activity-Fig. 1 shows the re- lease of proteolytic activity into the extracellular medium of

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Tetanus Toxin Converting Protease

TT t

a

160

P-3R d

t t

P\ / ‘\ I -e-m;----oJ . t, ,

-,. ‘\

40 60 80 100 120 140 60 80 & @?-

100 120 I-”

140 16b

FRACTION NUMBER

TABLE I Isolation of C. tetani proteases by gel chromatography

The proteases were prepared by gel chromatography on Sephadex G-100 followed by rechromatography of each peak on Ultrogel AcA 44. After concentration by ultrafiltration, the activity was measured with the Azocoll assay (see “Materials and Methods”) and expressed as trvnsin eauivalents at nH 8.0.

Protease Yield Approximate molecular weight

nghl P-l 11.8 P-2 40.5 P-3 5.5

P-1R 7.3 65,000 P-2R 11.2 40,ooo P-3R 3.2 270

C. tetuni cultures as fermentation proceeds. The overall pro- tease activity peaked concomitant with the attainment of maximal cell mass, but measurable activity was demonstrable at all stages during fermentation. Incubation of intracellular toxin with the crude protease from the culture supernatant also resulted in the conversion to the extracellular form; however, the interpretation of the stained gels was compli- cated by several protein bands which may be attributed to contaminants present in the culture filtrate (Fig. 2). In con- trast, no converting enzyme activity was detected after re- moving the culture supernatant and incubating the harvested bacterial cells with intracellular toxin. Therefore, the culture filtrate was selected as the starting material for the purifica- tion of the protease activity.

Purification-of C. tetani Proteases-Fig. 3 shows the elu- tion pattern obtained after ultrafiltration of the culture filtrate and gel chromatography on a column (5 X 100 cm) of Sepha- dex G-100. Three Azocoll-positive peaks, eluting behind teta- nus toxin and denoting P-l, P-2, and P-3, respectively, were separated by the gel column. The fractions corresponding to each peak were pooled and subjected to rechromatography on long columns (2.5 x 250 cm) of Ultrogel AcA 44 (Fig. 3). These semipurified materials (P-lR, P-2R, and P-3R) were used for most studies reported here. The yields of each protease ex- pressed as trypsin equivalents at pH 8.0 are summarized in Table I.

Substrate Specificity-Protease P-1R did not effect the conversion of intracellular tetanus toxin (Fig. 4) but caused

FIG. 3. Preparation and analysis of proteases from C. tetani. a, gel chromatography of the culture filtrate from C. tetani. The fdtrate derived from 3000 ml of autolyzed culture was concen- trated to 50 ml and applied to a column (5 X 100 cm) of Sephadex G-100, eluted as described under “Materials and Meth- ods.” 0---Q ultraviolet absorbance pattern (280 mn); H, proteolytic activity (AzocolI assay, 520 nm). Tetanus toxin (TT) constitutes the major protein peak. The three proteases from two sep- arate gel runs were appropriately pooled, concentrated, and rechromatographed on Ultrogel AcA 44 columns. b, rechro- matography of protease P-l, yielding P- IR; c, rechromatography of P-2, yielding P-2R, and d, rechromatography of P-3, yielding P-3R.

a

(+I

FIG. 4. Preparation and analysis of proteases from C. tetani. a, digestion of intracellular tetanus toxin with the semipurified pro- teases from the culture filtrate (cf Fig. 3, b to d). A, intracellular tetanus toxin used as substrate; B, P-1R enzyme (100 ng) incubated overnight with intracellular tetanus toxin (50 cg) in a total volume of 0.25 ml at pH 8.0; C, same as B with a reduced amount (20 ng) of P- 1R enzyme; D, P-2R enzyme (100 ng) incubated with intracellular toxin as in B, E, same as D with reduced amount (20 ng) of P-2R enzyme; F, P-3R enzyme (15 ng) incubated with intracellular tetanus toxin as in B; G, same as F with reduced amount (3 ng) of P-3R enzyme. The symbol +Me indicates that the samples had been reduced with mercaptoethanol prior to the electrophoretic analysis. b, controls with the same amount of enzyme in the absence of added substrate. Only protease P-2R at the higher concentration (D) showed the presence of contaminating polypeptides, which, however, did not interfere significantly with the results.

the hydrolysis of glycyl-L-histidine as shown by paper electro- phoresis after incubating the enzyme with the dipeptide over- night (Fig. 5). In contrast, proteases P-2R or P-3R exhibited no significant activity towards glycyl+histidine. However,

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Tetanus Toxin Converting Protease 10731

(+I

FIG. 5. High voltage paper electrophoresis of digests after incubating glycyk-histidine (2 gmol) with proteases P-lR, P- 2R, and P-3R (cf Fig. 3, 6 to d). Each enzyme was adjusted to a final concentration of 0.2 Zig/ml (trypsin equivalents, Azocoll assay) and kept overnight at room temperature in a total volume of 200 ~1. Aliquots (10 ~1) were spotted on Whatman No. 3MM paper and subjected to paper electrophoresis on a Gilson model D high voltage electrophorator (3500 V, 30 min). The dried papers were stained with ninhydrin. A, glycyl-L-histidine standard; B, D, and F, glycyl-n-histi- dine incubated in the absence of proteolytic enzymes; C, glycyl-n- histidine + enzyme P-lR, E, glycyl-L-histidine + enzyme P-2R; G, glycyl+histidine + enzyme P-3R, H, L-glycine standard; and Z, stan- dard mixture of L-glycine and L-histidine. The analysis was performed in pyridine/acetate buffer at pH 6.4.

the latter enzymes were found to modify the chain structure of tetanus toxin. Under the conditions of the assay, protease P-2R only partially degraded intracellular toxin (Fig. 4). By comparison, protease P-3R was by far the most active con- verting enzyme. As little as about 3 ng of protease P-3R (expressed in terms of trypsin equivalents) adequately cleaved 50 Zrg of intracellular toxin, producing the composite poly- peptide chains. At higher concentration, extensive degrada- tion of the toxin occurred (Fig. 4~). The partially purified enzymes did not interfere with the interpretation of the gel patterns.

All enzymes exhibited activity towards Azocoll or fibrin (13). Their approximate molecular sizes, based on gel chro- matography data, are listed in Table I.

Znnhibition Studies-Fig. 6 summarizes data from repre- sentative experiments carried out to determine the sensitivity of the three enzymes to several inhibitors. Protease P-1R was only marginally affected by general inhibitors of serine pro- teases, such as benzamidine or PMSF. Both P-2R and P-3R, respectively, showed greater susceptibility; thus, at 2 mg/ml

TABLE II

Effect of serum proteinase inhibitors on C. tetani proteases Each enzyme was subjected to a preliminary incubation with buffer

or with the inhibitor indicated (final concentration, 500 pg/ml). After 10 min, Azocoll (5 mg) was added to each tube and the incubation continued for 60 min. The protease activity is expressed as percentage of t,he color vield without added inhibitor.

InhibItor added Protease activity

% P-IR None

cY-2-Macroglobulin a-l-Antitrypsin Inter-a-trypsin inhibitor

104 106 104

P-2R None 100 n-2-Macroglobulin 98 o-l-Antitrypsin 67 Inter-a-trypsin inhibitor 96

P-3R None 100 n-2-Macroglobulin 31 a-1-Antitrypsin 122 Inter-a-trypsin inhibitor 112

6 8

INHIBITOR CONCENTRATION (mghl) ETHANOL CONCENTRATION I I V/V ’

FIG. 6. Inhibition of C. tetuni proteases by (a) benzamidine, the effect of solvent alone was also investigated. As is seen, P-3R was (b) PMSF, or (c) ethanol. Each protease was adjusted to a final not affected by ethanol itself. However, P-1R and in particular, P-2R concentration of 0.2 Zig/ml (trypsin equivalents) and the inhibitor was were strongly impeded by ethanol. Thus, the contribution of PMSF added in the final concentration indicated. After 10 min at 37”C, to the inhibition of these two enzymes is limited, and in the case of P- Azocoll (5 mg) was added and the incubation continued for 60 min. 2R, possibly negligible. O----0, enzyme P-IR; H, enzyme P- The activity is expressed as per cent color yield compared to controls 2R, and X-X, enzyme P-3R. without added inhibitor. Since PMSF was added dissolved in ethanol,

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10732 Tetanus Toxin Converting Protease

TABLE III Inhibition patterns of C. tetani proteases

+, indicates significant inhibition at the maximal concentration of inhibitor tested. -, indicates absence of inhibition within the concentration range tested. +, indicates a marginal inhibitory effect.

Inhibitor

Equine antitoxm:

Enzyme Benzami- dine (8 mg/

Id)”

Soybean u-2-MaCK- Inter-a- PMSF (2 IJrea (2 M) Ethanol (tetanus) (diphthe- trypsin in- globulm a-l-Ant]-

mg/ml) (4%) (50 floccu- ria) (50 hibitor (0.5 (O.Zg’

trypsin (0.5 trypsin in-

lation floccula- w/ml) mg/ml) hibitor (0.5

tion units/ w/d units/ml)

ml)

I’-1R + + + + P-2R + - + + I’-3R - + + -

~- ~~ ~~ ” Values in parentheses represent maximal concentration tested.

1.5 .

1.0

z z z

0”

a 0 0.5 ac .

0

q I I I I I I I 5 6 7 8 9 10 11

P”

FIG. 7. Dependence of the enzymatic activity on pH. Each enzyme was diluted to 0.2 pg/ml (trypsin equivalents) with the appropriate buffer solutions and incubated with Azocoll (5 mg) for 60 min at 37°C. Triplicate tubes were read at 520 nm. The buffers used were: 0.1 M acetate (pH 5 to 6) or 0.1 M Tris/acetate (pH 7 to 11). M, enzyme P-IR; M, enzyme P-2R, and X-X, enzyme P-3R.

of benzamidine, nearly half of the P-2R activity was sup- pressed.

When tested against proteinase inhibitors derived from pooled human sera, only P-3R was significantly impeded by a-2-macroglobulin at 500 pg/ml inhibitor concentration (Table II). This enzyme was also partially inhibited by inter-a-trypsin inhibitor. Furthermore, slight inhibition of P-2R by u-l-anti- trypsin was noted (Table II). Normal rabbit sera had no effect on the proteolytic activity of P-1R. However, the activity of protease P-2R was moderately affected by the serum. In keeping with the sensitivity of enzyme P-3R against a-2- macroglobulin, this protease was strongly inhibited by normal rabbit serum.

Equine tetanus antitoxin effectively inhibited all three pro- teases. Furthermore, the capacity of the antiserum to block enzymatic activity towards Azocoll was independent of prior absorption of the serum with highly purified tetanus toxin. Nonspecific horse serum did not interfere with the enzymatic activity of the proteases. A summary of the inhibition patterns is given in Table III.

Dependence on pH-Proteases P-1R and P-2R showed

TABLE IV Inhibition of C. tetaniproteases by rabbit antisera

To test for inhibition, each protease was adjusted to a concentration of 0.2 pg/ml (final concentration, expressed as trypsin equivalents) and mixed with 300 ~1 of whole serum or the corresponding amount of the IgG fraction. After 10 min at room temperature, Azocoll (5 mg) was added and the tubes were kept at 37°C for 1 h. Sera inhibiting the color development by 50% or more as compared to controls without antibodies, are indicated by a + sign. A combination showing no significant inhibition is indicated by a - sign.

Antiserum following immuniza- Controls (preimmune tion with” SCi)

EIlZYtae P-1R P-2R P-3R Whole se- IgG frac-

nun tionb

P-IR + - - - -

P-2R + + - -t -

P-3R - - + + -

n Except when tested against enzyme P-lR, all antisera were pu- rified by gel chromatography on Sephadex G-150 to remove serum inhibitors.

h Under the conditions of the assay, IgG fractions from preimmune sera were all completely devoid of inhibitory activity against any protease elaborated by C. tetani.

similar pH-profiles with a broad maximum in the range of pH 8.0 to 9.0. The third protease exhibited maximal activity below pH 7.0 and was distinguished from the other two enzymes in this assay (Fig. 7).

Immunochemical Distinction between the Three Pro- teases-Table IV shows the effect on the enzymatic activity of the three proteases after preliminary incubation with anti- bodies raised in rabbits against each enzyme preparation. Each enzyme was immunogenic and elicited antibodies affect- ing the catalytic center, in agreement with the data obtained with polyvalent equine antitoxin (Table III). In addition to inhibiting the homologous protease, anti P-1R serum also interfered with the activity of protease P-2R. Enzyme P-3R was strongly impeded by preimmune sera, presumably due to the presence of serum inhibitors (cf Table II). However, after preparing the corresponding immunoglobulin fraction, this enzyme was affected by the homologous serum only.

DISCUSSION

The thorough study of Miller et al. (8) indicated a close association between the formation of a histidyl peptidase by C. tetani, and the appearance of the toxin in culture filtrates. However, the relationship between this proteolytic enzyme and the structural features of tetanus toxin has remained unclear, although recent work on the polypeptide chain com- position of the toxin has suggested an important role for a bacterial protease in transforming intracellular toxin to its extracellular counterpart (2, 6, 11).

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Tetanus Toxin Converting Protease 10733

From the data presented in this communication, it appears highly likely that the histidyl peptidase activity demonstrated by Miller et al. (8) is very similar to the P-1R protease eluting immediately after tetanus toxin on gel chromatography. This enzyme catalyzed the hydrolysis of glycyl-L-histidine but did not, under the reaction conditions employed, induce the char- acteristic modification of the intracellular toxin. However, with general protease substrates, two further enzymes were detected and separated from P-1R by gel chromatography. The two additional enzymes failed to hydrolyze low molecular weight histidyl peptides in the standard assay but converted intracellular tetanus toxin to the extracellular form. Whereas only slight converting activity was associated with P-2R, enzyme P-3R was highly efficient in this capacity. Thus, three enzymes with differential proteolytic activities may now be distinguished in culture filtrates of C. tetalzi. The yield of each enzyme (Table I) was considerably below that of the tetanus toxin released by the bacteria, but sufficient protease P-3R is clearly available for conversion of the toxin to the extracellular form during cultivation.

Since the intracellular form of tetanus toxin may be isolated as an intact polypeptide chain with a molecular weight of about 140,000 (1-4, 6, ll), one might tentatively conclude that the toxin is excreted into the culture medium in the single chain form and is subsequently hydrolyzed by the extracellu- lar proteases. Furthermore, the finding that bacteria isolated from the growing cultures by centrifugation and subsequently incubated with intracellular tetanus toxin were unable to effect hydrolytic cleavage of the polypeptide chain, suggests the absence of significant activity of the relevant proteases on the surface of the bacterial membrane. However, at least traces of the extracellular toxin was present in all preparations derived by extraction of early cultures with NaCl/citrate buffer. In a few such “intracellular” toxin preparations, the quantity of nicked protein amounted to 50% of the total amount of tetanus toxin isolated, although Azocoll analyses routinely carried out prior to purification confirmed the ab- sence of detectable proteolytic activity. Therefore, under cer- tain culture conditions, at least, the cleavage of the poly- peptide chain of tetanus toxin may possibly occur prior to the release of the molecule into the medium. Thus, the site where proteolytic cleavage occurs remains unsettled and this prob- lem will require further attention.

Apart from separate elution positions on gel chromatogra- phy, the three proteases were also distinguished by their behavior towards various inhibitors (Tables II to IV). Only enzyme P-3R was markedly inhibited by a-2-macroglobulin and PMSF, but not by benzamidine. The inhibitory action of o-2-macroglobulin is presumably mediated by a conforma- tional change following interaction with the protease (17). Due to steric interference, the proteolytic activity of the trapped enzyme is impeded. It is conceivable that the sizes of proteases P-1R or P-2R were too great to permit a close association and entrapment by this inhibitor. Each enzyme was inhibited by the homologous antiserum derived in rabbits (Table IV). Interestingly, anti P-2R serum showed no inhibi- tory effect against protease P-lR, whereas the antiserum obtained after immunization with the latter enzyme also impeded the activity of P-2R. Thus, it cannot, at this stage, be ruled out that the two enzymes may express common antigenic determinants. An alternate explanation for this find-

ing would be given by assuming the presence of aggregated P- 2R molecules contaminating enzyme P-1R. Such aggregates may be highly immunogenic and induce anti P-2R antibodies upon immunization. The latter hypothesis is supported by the observation that culture filtrates which were kept for extended periods of time tended to lose some activity at the elution position of P-2R, although this enzyme, once isolated, was stable for months at 4°C.

All three enzymes were inhibited most effectively by hyper- immune sera produced against crude toxoid preparations. However, some lot-to-lot variation was observed and it is conceivable that toxoid preparations of slightly variable purity may influence the yield of specific antibody against the three proteases.

A possible role of the bacterial protease in the establishment of infection has previously been discussed (8), and the poten- tial value of anti-proteinase antibody to help control the spread of pathogens in the host tissue was implicated. Fur- thermore, the elaboration of proteases by the bacteria may be necessary for the conversion of tetanus toxin to the extracel- lular form. There is some indication that this conversion is required for the full expression of toxicity of the molecule (2, 18). Since only partial inhibition of protease P-3R was ob- tained with serum factors, it is clear that host tissue infected with C. tetani may contain active proteases of probable sig- nificance for the pathogenesis of tetanus during the initial stages of disease.

Achnowledgments-The expert technical assistance by Mrs. H. Dingeldein is gratefully acknowledged. We are indebted to Drs. M. C. Hardegree and D. J. Dungan for helpful discussions.

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3. Raynaud, M. (1951) Ann. Inst. Pasteur 80, 356-377 4. Murphy, S. G., Plummer, T. H., and Miller, K. D. (1968) Fed.

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Commun. 86,635-642 7. Amouraux, G. (1945) C. R. Seances Sot. Biol. Fil. 139,251-252 8. Miller, P. A., Gray, C. T., and Eaton, M. D. (1960) J. Bacterial.

79,955102 9. Hardegree, M. C., Palmer, A. E., and Duffin, N. (1971) J. Infect.

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(1979) Proceedings of the 5th International Conference on Tetanus, June 18-23, 1978, Ronneby, Sweden, in press

12. Jackson, R. L., and Matsueda, G. R. (1970) Methods Enzymol. 19, 591-599

13. Astrup, T., and Kok, P. (1970) Methods Enzymol. 19, 821-834 14. Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244,4406-4412 15. Andrews, P., Bray, R. C., Edwards, P., and Shooter, K. V. (1964)

Biochem. J. 93,627-632 16. Helting, T. B., Ronneberger, H.-J., Vollerthun, R., and Neubauer,

V. (1978) J. Biol. Chem. 253, 125-129 17. Laurels, C.-B., and Jeppsson, J.-O. (1975) in The Plasma Proteins

(Putnam, F. W. ed) 2nd Ed, Vol. 1, pp. 229-264, Academic Press, New York

18. Seki, T., Takaki, M., Hirabayashi, H., and Yamaguchi, H. (1954) Med. J. Osaka Uniu. 5, 271-289

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T B Helting, S Parschat and H Engelhardtconverting intracellular tetanus toxin to the extracellular form.

Structure of tetanus toxin. Demonstration and separation of a specific enzyme

1979, 254:10728-10733.J. Biol. Chem. 

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