Naunyn-Schmiedeberg's Arch. Pharmacol. 276, 271--288 (1973) 9 by Springer-Verlag 1973
Original Papers On the Structure of Tetanus Toxin B e r n a r d Bizzini, Andr6 Turpin, a n d Marcel R a y n a u d Service Immunochimie, Garehes, Institut Pasteur, Garehes Received June 22, 1972 Summary. Previous results from the literature pertaining to the molecular state of tetanus toxin are reported. By use of disc electrophoresis, gel filtration and ultracentrifugation, it is shown in this paper that tetanus toxin is likely to be constituted of subunits. Furthermore, it is postulated, starting from various lines of evidence, that the toxin should be recovered from the purification schedule as a dimer. The constituting monomeric units of the dimer are held together most probably by strong nomcovMent interactions. Each monomer is proposed to consist again of two subunits of different sizes linked by one S-S bridge. The results which further this view are discussed together with the questions arising from this mode]. Key words: Tetanus Toxin -- Structure. M a n y d a t a f o u n d in t h e l i t e r a t u r e concerning t h e a c t u a l molecular s t a t e of t e t a n u s t o x i n are c o n t r a d i c t o r y . P i l l e m e r a n d Moore (1948) a n d Pifiemer et al. (1948) r e p o r t e d a s e d i m e n t a t i o n coefficient of 4.5 S for a freshly p r e p a r e d , biologically active toxin, t h a t t h e y claimed to be monomerie. T h e also o b s e r v e d t h a t t h e toxin, on s t a n d i n g in isotonic n e u t r a l solution a t 0 ~C, was c o n v e r t e d into a flocculating, a t o x i c p r o d u c t , whose s e d i m e n t a t i o n coefficient was near 7 S. S u b s e q u e n t l y , L a r g i e r a n d J o u b e r t (1956) confirmed t h e o b s e r v a t i o n s of P i l l e m e r et al., b y using a t o x i n p r e p a r a t i o n of higher p u r i t y . These a u t h o r s a s c e r t a i n e d a s e d i m e n t a t i o n coefficient of 3.9 S a n d c a l c u l a t e d a molecular weight of a b o u t 68 000. T h e toxoid, t h a t was f o r m e d spontaneously on storage h a d a s e d i m e n t a t i o n coefficient of 7.6 S, corresponding to a molecular weight of 144500. Using a s a m p l e of t o x i n purified from b a c t e r i a l e x t r a c t s , R a y n a u d et al. (1960) o b s e r v e d s e d i m e n t a t i o n coefficients ranging from 6.0 to 7.1 S. Since t h e l a t t e r t o x i n e x h i b i t e d a t o x i c i t y of t h e same order of m a g n i t u d e as t h e 4.5 S crystalline t o x i n of Pillemer, t h e question arose as to w h e t h e r t h e t o x i n could exist u n d e r several a g g r e g a t i o n states, g a y n a u d et al. (1960) were able to o b t a i n d e p o l y m e r i z e d forms from t h e 7 S toxin, w i t h s e d i m e n t a t i o n coefficients a r o u n d 4 or 2 S, b y t h e a c t i o n of reducing or a l k y l a t i n g agents.
272
B. Bizzini et al. :
More recently, D a w s o n a n d M a u r i t z e n (1967), a n d M u r p h y a n d Miller (1967) r e p o r t e d values of 7.0 S a n d 6.4 S, respectively, for t h e p u r e toxin. M u r p h y a n d Miller suggested t h a t t h e m a t e r i a l h a d to be t h e biologically active monomer. Mangalo et al. (1968), using a t o x i n twice as active as Pillemer's, e x h i b i t i n g a specific t o x i c i t y of 1 . 6 6 x 1 0 S M L D / m g N, d e t e r m i n e d a molecular weight of a b o u t /48000, with a s e d i m e n t a t i o n coefficient of 7.60--7.88 S. This l a s t value for the molecular weight was s u b s e q u e n t l y confirmed b y M u r p h y et al. (1968) who f o u n d 141000 b y gel filtration. Besides of Pillemer et al. a n d L a r g i e r et al., no one has succeded in isolating a 4.5 S t e t a n u s toxin, w h e t h e r t h e t o x i n was purified from culture filtrates or from b a c t e r i a l extracts. However, it should be recalled that Peetom and van der Veer (1966) observed that tetanus toxin could be degraded by freezing at --20~ The resulting toxin, while having lost most of its toxicity, showed not only a modification of the antigenic structure but also a reduced sedimentation coefficient of 3.3 S. Latham et al. (1965) has previously reported the isolation from the toxoid by Sephadex G-100 gel filtration of a component (Fraction III) that gave a reaction of partial identity with the toxoid. This component, that exhibited a decreased molecular weight in respect to the native toxin, was considered by these authors as a haptenlike substance, which is toxoid-speeific in vitro but only feebly immunogenic in vivo. In an attempt to reveal the existence of subunits in tetanus toxin, Dawson and Nichol (1969) determined the sedimentation coefficient and molecular weight of the toxin before and after the action of the reducing agent, sodium sulphite. For the native toxin, a value of 176000 was obtained with a sedimentation coefficient of 6.47 S. The toxin contained a smaller eonstituent, whose concentration was estimated to 50/0. The action of sulphite brought about a reduction of the molecular weight to 152000--158000, showing an asymmetrical peak. The sedimentation coefficient decreased to 5.60--5.80 S. These workers calculated that the sedimenting material consisted of 200/o of a 4 S constituent, with a molecular weight of 88000 and 800/0 of a constituent with a molecular weight of 176000, the average molecular weight of the combination being 158000. Bizzini et al. (1970) d e m o n s t r a t e d t h e presence of 6 free s u l f h y d r y l a n d 2 disulfide groups in t h e t o x i n molecule. These results agreed with those of M u r p h y et al. (1968), who stressed t h e difficulty to a c t i v a t e one of the six SI-I groups. W h i l e we f o u n d leucine as t h e only N - t e r m i n a l group of t h e toxin, t h e former a u t h o r s failed to d e m o n s t r a t e any. H o l m e s a n d I~yan (unpublished results) r e p o r t e d glycine to be t h e only N - t e r m i n a l residue. I f t h e S-S bridges in t h e t o x i n are a s s u m e d to be i n t e r e h a i n bridges, t h e n the t o x i n could c o n t a i n two or t h r e e subunits. I n p r e l i m i n a r y studies we o b s e r v e d dissociation of t h e t o x i n b y filtration on S e p h a d e x G-200 in t h e presence of 6 M g u a n i d i n e of the reduced, e a r b o x y m e t h y l a t e d t o x i n d e r i v a t i v e . H o w e v e r , u p o n filtering a s i m i l a r l y t r e a t e d t o x i n t h r o u g h S e p h a d e x G-200 in 8 M urea, M u r p h y et al. (unpublished results) o b t a i n e d only one p e a k a n d concluded t h a t t h e t o x i n m i g h t consist of one single p o l y p e p t i d e chain.
On the Structure of Tetanus Toxin
273
I n t h e p r e s e n t w o r k we r e i n v e s t i g a t e d t h e p r o b l e m o f t h e m o l e c u l a r s t a t e o f t e t a n u s t o x i n . W e s h a l l p r e s e n t n e w e v i d e n c e s t h a t t h e t o x i n is constituted by the assembly of subunits. Methods and Materials
Preparation o/~he Toxin. Tetanus toxin was isolated a n d purified from bacterial extracts according to the method described previously (Bizzini et al., 1969). The preparation yielded 3200 Lf/mg N a n d 2.0 X 10 s LD/mg N. I t was homogeneous upon disc electrophoresis. Chemicals. Tris (hydroxymethyl-aminomethane) was purchased from KochLight Laboratories. Guanidine hydrochloride (GUA) was a n AG product from Carlo E r b a ; it was twice recrystallized from 95O/o ethanol. Iodoacetic acid, a product from Serlabo, was twice recrystallized from petroleum ether a n d stored a t -- 25~ Urea l%P (Prolabo) was once recrystallized from hot water as sodium lauryl sulphate (SDS) from Koch-Light. Dithiothreitol (DTT) was obtained from Calbiochem; 2-Mercaptoethanol (2-MeOH) was from Mann l~esearch Laboratories, used without previous distillation.
Bu//ers. Tris-EDTA buffer was prepared according to U l l m a n n et al. (1968). Denaturing agents were added to the buffers, when stated, in the following concentrations: GUA 6 M; urea 8 M; SDS 1~ Ultraeentri/ugation. B o t h sedimentation velocity a n d equilibrium experiments were carried out with a Spinco (Model E) ultracentrifuge at 20 ~= 0.1~ The sedimentation velocity runs were made a t rotor speed of 59 780 rev/min using Schlieren optics. The a p p a r e n t s values were corrected to 20~ and also for the viscosity of the buffers b y reference to water. The partial specific volume of the toxin was calculated from the previously determined amino acid composition (Bizzini et al., 1970) according to Cohn a n d Edsall (1943) a n d was found to be equal to 0.74 ml/g in water a n d assumed to be the same in buffer. W h e n 6 M GUA was present the partial specific volume was t a k e n as 0.73 ml/g according to Wallenfels et al. (1963), who assumed a 0.01 ml/g reduction of the partial specific volume in the presence of the denaturant. Molecular weight determinations were conducted according to Yphantis (1960), or to U l l m a n n et al. (1968) when GUA was present. Gel filtration was carried out on Sephadex G-200 or Bio-Gel A 5 M in Pharmaeia columns, type K 25/45, fitted with two adaptors. Columns were prepared according to the recommendations of the manufacturer, except when 8 M urea or 6 M GUA were added to the buffers, in which case the technique of Olesen a n d Pedersen (1968) was followed. After packing of the columns, they were calibrated b y running substances of known molecular weights : bovine-y-globulin (BGG) ; bovine serum albumin (BSA); ovalbumin (OVA); myoglobin (MYO) and cytochrome C (CYT C) (Calibration Kit; l~ann Research). W h e n gel filtration was to be performed through Bio-Gel A 5 1~I, Davison's technique (1968) was followed. I n each case exclusion a n d inclusion volumes were specified b y eofiltration of a mixture of dextran blue and DNP-alanine. Disc Gel Eleetrophoresis. Disc electrophoresis in 6.0 or 7.5 ~ polyacrylamide gel was carried out according to the technique of Davis (1964) with the glycine-Tris buffer system which gives a final p H near 9.5. The upper gel was omitted. Bromophenol blue was used as the tracking dye. I n a few cases, 5 M urea was added. Electrophoresis was conducted with a constant current of 5 mA per tube. The gels were then stained with 1~ Amido Black in 70/0 acetic acid.
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B. Bizzini et al. :
Disc eleetrophoresis was also conducted in the presence of SDS according to Weber and Osborn (1969). Maleylation, Reduction and Carboxymethylation o/ the Toxin. Maleylation was effeeted according to Butler et al. (1969) in 0.1 M Phosphate buffer pH 8.0. Maleic anhydride was added in an amount thrice in excess on the number of free amino groups present in the toxin. The maleylated toxin was then reduced in the presence of 6 M GUA by addition of solid DTT to 0.25 M final concentration. The reduced maleylated toxin was alkylated by adding iodoacetate three times in excess on a molar basis (Butler et al., 1969). Reduction and Carboxymethylation o/the Native Toxin. These were accomplished in the presence of 6 M GUA either according to Crestfield et al. (1963) with 2-MeOH or according to Butler et al. (1969) with DTT. For alkylation, iodoacetate was used in an excess three times on a molar basis. Recovery of the reduced and carboxymethylated products was performed by dialysis against COaHNH4, pH 8.6, and freeze-dried. Whereas the derivatives of tile native toxin are quite insoluble in the absence of GUA, the derivatives of the maleylated toxin remain soluble in ordinary buffers. However, the insoluble reduced alkylated products can be dissolved in the presence of 1~ SDS. Determination o/the Reactivity o] Free S H Groups. Titration of SH groups was carried out with 5,5'-dithionitrobenzoicacid (DTNB) according to Ellman (1959), as modified by Habeeb (1966), but in addition to 2~ SDS, 8 M urea or 6 M GUA were employed, as specified in certain cases. Amino Acid Analysis. The amino acid analyses were performed in a Spinco Automatic Analyzer, Model 120, under conditions essentially as specified in another paper (Raynaud et al., 1965). Determination of the Stokes Radius. It was done according to Page and Godin (1970).
Results On disc electrophoresis, n a t i v e t o x i n formed b u t one band, whereas a m a r k e d heterogeneity was i n d u c e d b y its maleylation. After r e d u c t i o n a n d alkylation, t e t a n u s t o x i n resulted i n a n insoluble derivative. However, a soluble c o m p o u n d could be o b t a i n e d when the t o x i n was m a l e y l a t e d prior to its r e d u c t i o n a n d alkylation. This reduced a n d c a r b o x y m e t h y l a t e d m a l e y l - t o x i n showed the presence of two b a n d s on disc electrophoresis (Fig. 1). Disc eleetrophoreses were Mso accomplished according to W e b e r a n d Osborn (1969) i n the presence of sodium dodecylsuplhate (SDS) w i t h or w i t h o u t the reducing a g e n t 2-Me0H, which p e r m i t t e d us to e v a l u a t e the molecular weights of the dissociated moieties after calibration of the gels b y r u n n i n g substances with k n o w n molecular weights. I n the absence of a reducing agent, n a t i v e t o x i n gave i n the presence of 1 ~ SDS only one band, for which a molecular weight of a p p r o x i m a t e l y 140000 could be estimated. U n d e r the same conditions, m a l e y l - t o x i n separated into two sharp bands, as well as two m i n o r bands, the firs~ m a j o r c o n s t i t u e n t formed of aggregates, the second one e x h i b i t i n g a molecular weight of 140 000. Molecular weights of 102 000 a n d 73 000 were
On the Structure of Tetanus Toxin
275
Fig. 1. Patterns given on 7.50/0 ordinary polyacrylamide gel from left to right by: native toxin, maleyl-toxin and reduced and carboxymethylated maleyl-toxin
a
b
c
d
Fig. 2 a--d. SDS gel patterns shown in the absence of 2-MeOtt by tetanus toxin and its derivatives: From left to right, a native toxin, b reduced and carboxymethylated toxin, c maleyl-toxin, d reduced and carboxymethylated maleyl-toxin
e s t i m a t e d for t h e m i n o r constituents. I n contrast, r e d u c e d c a r b o x y m e t h y l a t e d t o x i n dissolved in 1 ~ SDS gave rise to t h r e e sharp b a n d s w i t h e s t i m a t e d m o l e c u l a r weights of 141000; 108000 a n d 69000, respecti-
276
B. Bizzini et
a
b
al. :
c
d
Fig. 3 a--d. SDS gel patterns shown in the presence of 2-MeOH by tetanus toxin and its derivatives. From left to right: anative toxin, b reduced and carboxymethylated toxin, c maleyl-toxin, d reduced and earboxymethylated maleyl-toxin
vely. l~edueed carboxymethylated maleyl-toxin also gave three distinct bands with their molecular weights at 160000, 105000 and 70000 (Fig. 2). I n the presence of 1 ~ SDS and 2-Me0H, native toxin was split into three strong bands exhibiting molecular weights of 141000, 104000 and 66000, respectively. Reduced carboxymethylated toxin yielded three bands, for which molecular weights of 141000, 105000 and 65000 were evaluated. As to reduced carboxymethylated maleyl-toxin, it separated into two bands with estimated molecular weights of 105000 and 69000 (Fig. 3). On the basis of the disc electrophoresis experiments mentioned, we have tried to isolate the subunits by gel filtration under dissociating conditions. The pattern of gel filtration on Sephadex G-200 of tetanus toxin in the presence of 6 M GUA is shown in Fig. 4. I t can be seen that the toxin was essentially eluated as a symmetrical peak indicating a molecular weight near 150000. When the gel filtration was performed with the reduced and carboxymethylated toxin in the presence of 8 M urea, no dissociation occurred and the same molecular weight could be calculated as for the native toxin in the presence of 6 M GUA (Fig. 5).
On The Structure of Tetanus Toxin 1.0" D,O. 280nm
277
BGG
1
/\ r,
0.5.
i i
_..., "
\
5'0
7'0
\ g0
mt
Fig. 4. Sephadex G-200 filtration of tetanus toxin in Tris-EDTA buffer containing 6 M Guanidine D,O. 280 nm
BGG
!\
0.5.
!i
I
/
i
\ I
\
"\
jc~-_' 5O
7~
16o
m[ Fig. 5. Sephadex G-200 filtration of the reduced and carboxymethylated toxin in Tris-EDTA in 8 M urea
I n contrast, t h e gel filtration of t h e r e d u c e d a n d e a r b o x y m e t h y l a t e d t o x i n in t h e presence of 6 M G U A showed the existence of two constit u e n t s , C 1 a n d C~ (Fig. 6). l ~ e e h r o m a t o g r a p h y of each c o n s t i t u e n t t h r o u g h the same gel allowed us to p u r i f y C 1 a n d C 2 a n d to e s t i m a t e t h e i r m o l e c u l a r weights as 110 000 a n d 40000 r e s p e c t i v e l y (Fig. 7).
278
B. Bizzini et
al. :
BGG
I
D.0. 28Ohm 1.0-
i
i
9
i
I IBsA
iii II II
0.5
ii
/ 0
I/
" io
/
,,
J \/\. V
7b
\.~. ,
gb
13o
m[
"~.
Fig. 6. First Sephadex G-200 filtration of tile reduced and carboxymethylated toxin in Tris-EDTA containing 6 M Guanidine
a.o. 280 nm
D.O. BGG
BSA
/\ j\
0.5-
I /
0,5-
\
\
OVA
/
"k
F\
"\
#-
5o
7b
mt
io
9b
Fig. 7. Second Sephadex G-200 filtration of each of the constituents separated in the first filtration (Fig. 6)
The amino acid compositions of both C1 and C 2 are reported in Table 1. The composition of C] and C~ is relatively similar. This fact m a y account for the difficulty in separating t h e m from one another b y other techniques t h a n the gel filtration in 6 M GUA.
On the Structure of Tetanus Toxin
279
Table 1. Comparative amino acid composition of the reduced and carboxymethylated toxin (CM-T6) and of the constituents C1 and C2 Amino acid
Lysine ttistidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleueine Leucine Tyrosine Phenylalanine Total
Carboxymethyl toxin (CM-T6)
Constituent C1
Constituent C2
g/lO0 g protein
N
g/lO0 g protein
N'
g/lOOg protein
N"
10.38 1.49 4.01 18.30 5.55 6.80 10.81 4.41 3.21 3.16 1.20 4.78 2.32 11.69 10.50 10.10 6.59
106 14 35 206 70 97 110 57 64 53 10 61 23 134 120 83 60
10.78 1.88 4.43 18.40 5.55 6.72 10.68 3.54 3.32 3.15 0.83 4.42 1.89 10.52 10.52 8.85 5.83
81 13 28 152 51 70 80 34 49 39 5 42 14 89 89 54 39
10.79 1.79 4.96 19.45 5.75 6.88 11.32 4.95 3.60 2.96 1.59 4.46 3.29 9.38 9.74 10.25 8.86
30 5 11 58 19 26 31 17 19 13 4 15 9 29 30 23 21
115.30
112,15
120.02
N -- Assumed number of residues for 150000 g. N' = Assumed number of residues for 110000 g. N" -Assumed number of residues for 40000 g.
The same p a t t e r n of gel filtration is o b t a i n e d when n a t i v e t o x i n is filtered t h r o u g h Sephadex G-200 in 6 M G U A buffer i n the presence of 0.1 M 2-MeOtt (Fig.8). I t is to be noted, however, t h a t the molecular weights of both c o n s t i t u e n t s (148 000 a n d 69 000 respectively) are signific a n t l y higher t h a n after r e d u c t i o n a n d e a r b o x y m e t h y l a t i o n . Thus, free SH groups m i g h t still i n t e r a c t u n d e r these conditions. I n 1 M formic acid, gel filtration on Sephadex G-200 gave a symmetrieal peak w i t h o u t modification of the molecular weight. I f the t o x i n was s u b m i t t e d previously to performie acid oxidation according to Moore (1963), the t o x i n eluted from the gel i n formic acid b y two peaks with molecular weights of 106000 a n d 21000, respectively (Fig. 9). Gel filtration t h r o u g h Bio-Gel A 5 M in 8 M urea according to D a v i s o n did result i n only a slight dissociation (Fig. 10). The results of the u l t r a e e n t r i f u g a t i o n runs performed with the native, or the maleylated, or the reduced a n d e a r b o x y m e t h y l a t e d maleylt o x i n in o r d i n a r y buffers are s u m m a r i z e d i n Table 2. 19
Naunyn-Schmiedcberg's Arch. Pharmacol., Vol. 276
280
B. Bizzini
et al. :
BGG
I
D,O.
i~,
280 n m
il ji I i i IJBSA i i
1.0.
0.5-
I
/
/
\
i
\ gb
2'0
ml Fig.8. Sephadex G-200 filtration of tetanus toxin in Tris-EDTA containing 6 M Guanidine and 0.1 M 2-Mereaptoethanol
BSA
BGG
D.O.
280 nm
i\
1.0
MYO
i \
i 0.5
I I
i i
/ m
\
\
\.
/
\
\
\
I
5O
m[ Fig. 9. Sephadex G-200 filtration of tetanus toxin oxidized by performie acid in 1 M formic acid
On the Structure of Tetanus Toxin
281
BGG BSA
ao. 280nm
i
1.5
I
~.
ih
II ~.o
I II i i ; I
o.~
BGG
I I I i I
i! il il ii
o.s \
-'~
4'0
ii i!
i!
/ 0
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i
6'0
\
~'J
~
9i3
ml
0
~'"" ......
5'0
7'0
1150
Fig. 10. Bio-Gel A 5 M filtration in Tris-EDTA containing 8 M urea and 1 M 2-Mercaptoethanoh 1 left: reduced and carboxymethylated toxin, 2 right: native toxin
Table 2. Results of the ultracentrifug~tion runs in ordinary buffers Nature of the toxin
Buffer
Native
0.07 M Phosphate pI-I 8.2
Maleylated
Tris-EDTA NaC1 10H 7.6
Reduced and c~rboxymethylated Tris-EDTA maleyl-toxin NaC1 p i t 7.6
Concert- Sedimentation tration coefficient ~ 6.85 -4- 0.05 0.5
0.75
Molecular weight
141375 • 5375
2.50 S 2 = 2.80 S 1 --
S 1 = 1.60 S 2 = 2.02
Ig is t o be seen a f t e r m a l e y l a t i o n or a f t e r r e d u c t i o n a n d a l k y l a t i o n o f t h e m a l e y l - t o x i n , t h a t t h e s e d i m e n t a t i o n coefficients a r e l o w e r e d d o w n to a b o u t 2 - - 3 , a t t h e s a m e t i m e as t w o e n t i t i e s a r e v i s u a l i z e d (Fig. 11). W h e n t h e n l t r a e e n t r i f u g a g i o n studies w e r e c a r r i e d o u t in t h e p r e s e n c e o f 6 M G U A , s o m e w h a t differing r e s u l t s w e r e o b t a i n e d , as s h o w n i n T a b l e 3. 1.9"
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Fig. 11. Ultracentrifugation pattern of the mMeylated toxin in Tris-EDTA buffer pH 7.6. Photograph taken at 266 min. Upper curve: concentration = 1~ Lower curve: concentration = 0.5~ Table 3. Results of the ultracentrifugation runs under denaturing conditions Nature of the toxin
Buffer
Sedimentation coefficient
Native
Tris-EDTA ~- 6 M S0.5~ -- 0.66 GUA pH 7.6
Native
Tris-EDTA pH 7.6 S0.vs~ = 0.55 ~- 6M GUA -~ 0.3 M 2-MeOH
39900 •
Reduced and alkylated
Tris-EDTA pH 7.6 So.75~ -- 0.85 ~- 6 M GUA
52706 • 1156
Treated withDTNB, Tris-EDTA pH 7.6 St.o~ = 0.61 without reduction @ 6 M GUA
Molecular weight 145800 4- 5450 968
73573 4- 1253
U n d e r these conditions, the molecular weight of t e t a n u s t o x i n averaged a b o u t 146000. I t s e d i m e n t e d as a single peak. W h e n t o x i n was sedim e n t e d i n 6 M G U A i n the presence of a reducing agent, the molecular weight was calculated to be a b o u t 40000. The s e d i m e n t a t i o n p a t t e r n i n d i c a t e d a homogeneous m a t e r i a l Thus, when the free SH groups on the t o x i n molecule are blocked i n 6 M G U A b y D T N B a n d i n the absence of a r e d u c i n g agent, a reduced molecular weight can be calculated (Table 3).
On the Structure of Tetanus Toxin
283
Fig. 12. Ultracentrifugation pattern of the reduced and earboxy methylated toxin in Tris-EDTA buffer pH 7.6, containing 8 M urea. Photograph on the left was taken 23 rain after full speed was attained. Photograph on the right was taken at 329 min
Finally, when the sedimentation of the reduced and alkylated toxin was performed in the presence of 8 IV[ urea, no complete dissociation took place as in 6 M GUA. At the beginning of the run two peaks, one composite (1-L 2), the other homogeneous (3) were visualized. As sedimentation was proceeding, the composite peak was further resolved into its constitutive components (1) @ (2) (Fig. 12). All three peaks (1), (2) and (3) appear in the proportions of 50.6~ 42.6~ and 6.8~ with relative sedimentation coefficients of 0.64, 0.98 and 3.00 respectively. Discussion
I t appears from the results of the disc electrophoresis experiments t h a t tetanus toxin is likely to be constituted of subunits. Thus, in the presence of SDS, both the reduced toxin and the reduced and alkylated toxin gave three bands with estimated molecular weights of 140000, 105000 and 68000--70000. The value of 140000 might represent the weight of the unaltered toxin. The same splitting of tetanus toxin was observed when the native toxin was electrophoresed in the presence of SDS and of a reducing agent, 2-MeOIt. In agreement with the disc eleetrophoresis results, the gel filtration experiments also strongly further the view the toxin to be built of subunits. The gel filtration experiments, while pointing to the existence of smaller molecular entities after reduction of the S-S bridges of the toxin, did never result in the quantitative isolation of the constituting subunits. Again in these experiments, a part of the toxin after reduction was separated with unchanged molecular weight (148 000), whereas some of it was isolated as a smaller compound (69000--72000). When the reduced
284
B. Bizzini et
al. :
and alkylated toxin was investigated under conditions promoting the breakage of hydrogen bonds, a reduction in the molecular weight was invariably verified. From Sephadex G-200, in the presence of 6 M GUA, the reduced and carboxymethylated toxin was thus elutcd as two peaks with respective molecular weights of 110000 and 40 000. On the other hand, the incapacity to dissociate the reduced and alkylated toxin reported by Murphy et al. (unpublished results) in 8 M urea is substantiated by our own experiments. However, when 6 M GUA was substituted for 8 M urea, partial dissociation was observed, as is evidenced by the isolation of smaller entities. But, whatever the conditions, no complete dissociation was ever reached as on disc electrophoresis. Particularly interesting was the observation made on toxin, which had been previously oxidized by performic acid. Gel filtration through Sephadex G-200 in the presence of formic acid revealed two peaks with molecular weights of 106000 and 21000 respectively. By the light of the results reported above, the value of 106000 assumes a significant meaning. This value, that is also found on disc electrophoresis experiments, strongly indicated that a large portion of the toxin molecule should be constituted of subunits linked by non-covalent bonds. This moiety is put to light, when the other part, which is likely to be linked to it by S-S bridges, is released from the toxin molecule by reduction of such bridges. When we examine the results of the ultracentrifugation studies the view the toxin to be built of subunits is still further supported. Under non dissociating conditions, the splitting of the toxin into lighter molecular entities is demonstrated by the ultracentrifugation patterns of both the maleylated and the reduced alkylated maleyl-toxins (Fig. 11 and Table 2). Habceb (1967) investigated the conformational changes brought about when proteins were chemically modified by maleylation. The chemical modification associated with the introduction of new groups, without any breakage of primary chemicals bonds, is assumed to alter the intramolecular forces in such a way as to induce a "reorganization" of the conformation. Furthermore, the blockage of free amino groups with maleyl-groups effects a sharp change in the overall net charge of the molecule. These assumptions are substantiated by the following modifications of the properties of native toxin by maleylation: 1. loss of the toxicity and of the immunochemical reactivity; 2. small increase in the reactivity of free SH groups to DTNB. For instance, in the presence of 8 M urea or of 2~ SDS one more SH group is titrated ;
On the Structure of Tetanus Toxin
285
3. augmentation of the Stokes' radius from 4.74 for the native toxin to 5.309 after introduction of 54 maleyl groups per mole toxin. I t could thus be postulated t h a t the modification induced in the molecule b y maleylation might reflect the depolymerization of aggregates of toxin and the rupturing of hydrogen bonds linking the subunits which are eventually held together by non-covalent bonds or released b y reduction and carboxymethylation. Our results seem to confirm this postulate, since the sedimentation coefficients are lowered down to 1.75--2.50 after maleylation; at the same time two entities are visualized. In addition, the fact that the sedimentation coefficients determined for the reduced and alkylated maleyl-toxin are not significantly different from those for the maleylated toxin, enhances the view, that maleylation brings about the dissociation of moieties of the toxin. In the presence of the denaturing agent, 6 M GUA, although only approximate values can be calculated because of the m a n y assumptions which are to be made as to the molecular parameters under these conditions, the decrease in the molecular weight of the reduced or of the reduced and alkylated toxins is quite significant. I n effect, while a molecular weight of 146000 was calculated for the native toxin in the presence of 6 M GUA, values ranging from 40000 to 50000 were determined for the reduced toxin or for the reduced and alkylated toxin. I n contrast, when the blockage of free SH groups was effeeted with D T N B without any breakage of S-S bridges, a molecular weight of 73 000 was obtained in the presence of 6 M GUA. I n an a t t e m p t to reconcile these seemingly contradictory results, we were prompted to postulate for tetanus toxin the following structure (Fig. 13). According to this scheme, tetanus toxin is isolated from the purification procedure as a dimer. Each molecule of monomer in the dimer would h sJ I S
~
21ooo
|
monomer "/3 000
I non covatent interactions
dimer o,-
.
.
.
.
.
.
.
.
.
.
.
.
.
146 000 S'
"~"
S2 000
I
s
I Fig. 13. Tentative representation of the structure of tetanus toxin
286
B. Bizzini et al. :
be built by the linkage of two polypeptide chains with respective molecular weights of 52000 and 21000 by means of one S-S bridge. I n the dimer, the two molecules of monomer would be held together by very strong non-covalent interactions. In support to the postulated structure we may allege the following facts : I. when the non-covalent interactions are ruled out by blockage of free SH groups by DTNB, a molecular weight of 73 000 is determined by ultracentrifugation in the presence of 6 M GUA; 2. when the toxin is reduced and alkylated a molecular weight of 105000 and 40000 is obtained by gel filtration. This might indicate that under these conditions (6 M GUA) the two heavy chains (polypeptide chains with molecular weight 52 000) remain attached and that the two light chains (polypeptide chains with molecular weight 21000), after being released, reassociate by non-covalent interactions. 3. when the toxin is reduced and alkylated three bands were separated by disc electrophoresis. The band with molecular weight 140000 should correspond to the unmodified toxin. While the band with molecular weight 105000 should represent the association of two heavy chains, the band with molecular weight 65 000 might be constituted by the linking of three light chains. This originates from the dissociation of 3 moles of dimer according to :
3 moles dimer --~ 3 moles (heavy chain ~- heavy chain) ~- 2 moles (light chain ~ light chain Jr light chain). 4. finally, when the toxin was oxidized with performic acid, two constituents with molecular weights 106000 and 21000 were separated. The constituent 106000 could be accounted for by the assembly of two heavy chains and the constituent 21000 by the release of the light chains as such without further recombination. I f the proposed representation seems tentatively to be supported b y the results of our present investigations, it still is vexed b y previous results of our own or from the literature. Murphy et al. (1968) found tetanus toxin not to possess any N-terminal amino acid residue. Bizzini et al. (1970) demonstrated leucine as the single N-terminal residue, while }Iolmes and R y a n (unpublished results) revealed glycine as the single ~-terminal residue. This situation might arise from the fact t h a t the conditions under which the determination of N-terminal amino acid residues is carried out are not suitable to unmask N-terminal residues in tetanus toxin. The incapacity to quantitatively isolate the subunits, even under strong dissociating conditions, as is evidenced by the disc electrophoresis and gel filtration experiments
On the Structure of Tetanus Toxin
287
could a t least p a r t l y a c c o u n t for this view. F u r t h e r i n v e s t i g a t i o n s in t h i s direction will be needed, before a n y definite conclusion m a y be d r a w n . L a t h a m et al. (1965) r e p o r t e d t h e isolation f r o m t e t a n u s t o x o i d b y gel filtration of a h a p t e n - l i k e substance, t o x o i d specific i n vitro a n d feebly i m m u n o g e n i c in vivo. This h a p t e n - l i k e s u b s t a n c e e x h i b i t e d a smaller molecular weight t h a n t h e n a t i v e toxin. P e e t o m a n d v a n d e r Veer (1969) b r o u g h t a b o u t t h e d e g r a d a t i o n of the t o x i n b y freezing a n d thawing. The t h u s o b t a i n e d d e g r a d e d t o x i n was still e x h i b i t i n g a r e a c t i o n of p a r t i a l i d e n t i t y with t h e toxoid. These a u t h o r s a s s u m e d t h e d e g r a d e d t o x i n to c o n t a i n only one of t h e two antigenic d e t e r m i n a n t groups (at least) p r e s e n t on t h e t o x i n molecule. A t t h e same time, t h e s e d i m e n t a t i o n coefficient of t h e d e g r a d e d t o x i n was lowered down to 3.3. F u r t h e r i n v e s t i g a t i o n s are being carried o u t to cheek our m o d e l a n d to e l u c i d a t e t h e n a t u r e of the i n t e r a c t i o n s which are p r e d i c t e d from it. Aclcnowledgments. The authors are indebted to Dr. 1~. Mangalo for some of the preliminary ultracentrifugal analyses and to Mr. L. Sagaert for most of the determinations with the ultracentrifuge which have been used in this work. They wish to thank Miss M. Pissavy for a very competent technical assistance.
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Habeeb, A. F. S. A. : Quantitation of conformational changes on chemical modification of proteins: Use of suceinylated protehls as a model. Arch. Bioehem. Biophys. 121, 652--664 (1967). Habeeb, A. F. S.A., Schrohenloher, R. E., Bennett, J. C.: Studies of maleyated chains of human IgM. J. Immunol. 105, 846--855 (1970). Largier, J. F., Joubert, F. J. : Investigation of the physical properties of tetanus toxoids. Biochim. biophys. Acta (Amst.) 20, 407--408 (1956). Latham, W. C., Jenness, C. P., Timperi, R. J.K., Michelsen, C. B. H., Zipilivan, F.M., EdsaI1, G., Ley, H. L., Jr. : Purification and characterization of tetanus toxoid and toxin. I. Fractionation of tetanus toxoid by gel filtration. J. Immunol. 95, 487--493 (1965). Mangalo, R., Bizzini, B., Turpin, A., Raynaud, M. : The molecular weight of tetanus toxin. Biochim. biophys. Acta (Amst.) 168, 583--584 (1968). Moore, S. : On the determination of cystine as cysteie acid. J. biol. Chem. 238, 235--237 (1963). Murphy, S. G., Miller, K. D. :Tetanus toxin and antigenic derivatives. I. Purification of the biologically active monomer. J. Bact. 94, 580--585 (1967). Murphy, S. G., Plummet, T. H., Miller, K. D. : Physical and chemical characterization of tetanus toxin. Fed. Proc. 27, 268 (1968). Olesen, H., Pedersen, P. 0. : Gel filtration of albumin on sephadex G-200 in urea. Acta chem. seand. 22, 1386--1394 (1968). Page, M., Godin, C. : Determination of the stokes radius of native and succinylated glutamate dehydrogenase on Sepharose 4B. J. Chromatogr. 50, 66--71 (1970). Peetom, F., Van der Veer, M. : The antigenic structure of tetanus toxin and toxoid and its relationship with tetanus immunology. In: Principles on tetanus. Proceedings of the International conference on tetanus, Bern, 1966, pp. 237--244. Bern: It. Huber 1967. Pillemer, L., Moore, D. H. : The spontaneous conversion of crystalline tetanal toxin to a flocculating atoxic dimer. J. biol. Chem. 173, 427--428 (1948). Pillemer, L., Wittler, R. G., Burrel, J. I., Grossberg, D. B. : The immunochemistry of toxins and toxoids. IV. The crystallization and characterization of tetanal toxin. J. exp. Med. 88, 205--221 (1948). Raynaud, M., Bizzini, B., Relyveld, E . H . : Composition en aminoacides de la toxine dipht6rique purifi6e. Bull. Soe. Chim. biol. (Paris) 47, 261--266 (1965). Raynaud, M., Turpin, A., Bizzini, B. : Existence de la toxine t6tanique sous plusieurs 6tats d'agr6gation. Ann. Inst. Pasteur 99, 167--172 (1960). Ullmann, A., Goldberg, M. E., Perrin, D., Monod, J. : On the determination of molecular weight of proteins and protein subunits in the presence of 6 M guanidine hydroehloride. Biochemistry 7, 261--265 (1968). Wallenfels, K., Sund, H., Weber, K. : Die Untereinheiten der fl-Galaktosidase aus E. coll. Biochem. Z. 338, 714--727 (1963). Weber, K., Osborn, M.: The reliability of molecular weight determinations by dodecyl sulphate polyacrylamide gel eleetrophoresis. J. biol. Chem. 244, 4406--4412 (1969). Yphantis, P. A. : Rapid determination of molecular weights of peptides and proteins. Ann. :N. u Aead. Sci. 88, 586--601 (1960). B. Bizzini
Service d'Immunochimie, Garches Institut Pasteur 92380 Garches/France
