THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 13, Issue of May 5, pp. 9053-9058,1992 Printed in U.S.A.

Identification and Partial Characterization of a Low Affinity Metal- binding Site in the Light Chain of Tetanus Toxin*

(Received for publication, November 1, 1991)

J. Fraser Wright$, Martine Pernollet, Angeline Reboul, Catherine Aude, and Maurice G . Colombj From the Dhpartement de Biologie Mokcu@ire et Structurale, Laboratoire d’zmmunochimie, Unite Znstitut National de la Sante et de la Recherche Medicale 238, Centre d’Etudes Nuclpaires de Grenoble, 85 x, 38041 Grenoble Cedex, France

Tetanus toxin was shown to contain a metal-binding site for zinc and copper. Equilibrium dialysis binding experiments using ‘j6Zn indicated an association con- stant of 9-15 PM, with one zinc-binding siteltoxin mol- ecule. The zinc-binding site was localized to the toxin light chain as determined by binding of “Zn to the light chain but not to the heavy chain after separation by sodium dodecyl sulfate-polyacrylamide gel electropho- resis and transfer to Immobilon membranes. Copper was an efficient inhibitor of “Zn binding to tetanus toxin and caused two peptide bond cleavages in the toxin light chain in the presence of ascorbate. These metal-catalyzed oxidative cleavages were inhibited by the presence of zinc. Partial characterization of metal- catalyzed oxidative modifications of a peptide based on a putative metal-binding site (HELIH) in the toxin light chain was used to map the metal-binding site in the protein.

Tetanus toxin is a rather well defined 150-kDa protein comprising two disulfide-linked polypeptidic chains (light chain (L) 50 kDa, heavy chain (H) 100 kDa). Its complete amino acid sequence has been established recently (Eisel et al., 1986; Fainveather and Lyness, 1986) and shows conserved zones in common with other clostridial neurotoxins such as botulinum toxins A and B, particularly a HELIHXXH se- quence within a domain of predicted a-helical secondary structure of the light chain (Binz et al., 1990). Screening data bases showed that the HELIH motif of the above sequence was a signature for a family of zinc-dependent metallopepti- dases (Jongeneel et al., 1989).

Our current interest in tetanus toxin is based on its anti- genic properties in human: we use tetanus toxin as a model protein to analyze molecular details of its intracellular pro- teolytic processing in antigen-presenting cells. The presence of a putative zinc peptidase site in the toxin sequence prompted us to investigate in more detail the following com- puter findings as: 1) the zinc binding area in the light chain of the toxin is a predicted T epitope, and 2) autoproteolysis is likely to be one of the multifacet aspects of antigen proc- essing in antigen-presenting cells. As a matter of fact, a recent

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

$ Recipient of an INSERM Poste Vert, a Franco-Canadian scien- tific exchange fellowship, and a C. T. E. Commissariat a 1’Energie Atonique (Saclay, France)EA fellowship. Present address: Protein Chemistry Section, National Reference Laboratory, Canadian Red Cross Society, 1800 Alta Vista Drive, Ottawa, Ontario K1G 455, Canada.

To whom correspondence should be addressed.

report on neonatal tetanus despite protective serum antitoxin concentration (Maselle et aL, 1991) underlines the interest of refined studies of the T antigenicity of tetanus toxin.

This paper reports the first experimental evidence for zinc binding to tetanus toxin and the competitive binding of cop- per. Precision on the location of the metal-binding site is given by conventional methods as well as by metal-catalyzed oxidative cleavage of the toxin or of synthetic peptides copying the metal binding sequence.

MATERIALS AND METHODS

Chemicals-The chloride salts of Fez+, Zn2+, Cu2+, Co2+, Cd2+, and Ca2+ were from Sigma and those of Mn2+, Ni2+, and MgZf were from Merck. (65)ZnCl2 was from Amersham Corp. EDTA, dithiothreitol, ascorbate, MOPS’, and other laboratory chemicals were from Sigma.

Tetanus toxin was supplied by Bio-MCrieux, Lyon, France and was subjected, after DFP-treatment, to an additional gel-filtration puri- fication step using a Sephacryl S-300 HR column, as described by Villiers et al. (1991). The toxin was also purified in the laboratory from its protoxin monochain form from the washed bacterial pellet of cultured Clostridium tetuni, as described by Ozutsumi et al. (1985) with the following modifications: the toxin-containing fractions fol- lowing HPLC gel filtration using a TSKG3000 SWG preparative column (300 X 21.5 mm) (Waters) were combined and concentrated by ultracentrifugation on Amicon PM-10 dialysis membrane. The concentrate was diluted with water to give an ionic strength of 10 millisiemens and applied to a Pbarmacia Mono-& fast protein liquid chromatography column equilibrated with 100 mM NaCl, 10 mM phosphate, pH 7.0, and eluted with a gradient of NaCl to 500 mM. The toxin peak eluted at 20 millisiemens was concentrated using an Amicon apparatus and stored at -80 “C. Protoxin was processed into its two-chain form using trypsin (1:50 (w/w), 20 min, 30 “C).

Synthetic Peptides-One peptide (TT225-243:DPALLLMHELI- HVLHGLYG) was set up on zinc binding consensus sequence (Jon- geneel et al., 1989). The second one (TT233-248:ELIHVLHGL- YGMQVSS) was based on predictive epitopy, determined by M.-B. Villiers, according to Stille et al. (1987) and Rothbard and Taylor (1988). They were obtained by the solid-phase procedure of Merrifield et ul. (1964) using an Applied Biosystems 430A synthesizer.

Fast atom bombardment mass spectrometry (FAB-MS) of TT225- 243 and of purified oxidized products generated by treatment of this peptide with copper and ascorbate was performed using a model FISONS VG type ZAB2-Seq mass spectrometer.

NH2-terminal sequence was determined by a microsequencing method (Matsudaira, 1987), using a 470A Applied Biosystems gas- phase protein sequencer.

SDS-PAGE was performed according to Laemmli (1970). Samples for electrophoresis were prepared by boiling 5 mn in 1% SDS, 10% glycerol, 50 mM Tris, pH 6.8. For reduced samples 50 mM ditbiothre- itol was included.

“Zn binding to tetanus toxin was performed as described by Mazen et al. (1988): after reduction of TT, its H and L chains were separated by SDS-PAGE, transferred to Immobilon membranes, and the mem-

The abbreviations used are: MOPS, 4-morpholineethanesulfonic acid HPLC, high performance liquid Chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; FAB-MS, fast atom bombardment mass spectrometry.

9053

9054 Metal Binding by Tetanus Toxin branes were finally incubated with =Zn.

A second method in which the native conformation of the protein is maintained was also used to evaluate =Zn binding and for compe- tition studies: tetanus toxin (1 mg/ml) was equilibrated in 2 mM MOPS, 150 mM NaCI, pH 7.0, using 2-ml Sephadex G-50 minicol- umns equilibrated with the same buffer. To 20 pl of the protein solution was added 2 pl of MOPS, NaCl buffer containing approxi- mately 0.2 pCi of 65ZnC12 corresponding to a final zinc concentration of 2 p ~ . After incubation for 1 h at 20 "C, the protein solution was applied to a TSK G3000 SW analytical column (300 X 7.5 mm) (Waters), and the eluate was monitored for =Zn radioactivity and for protein (280 nm). Competition for 6sZn binding to the toxin by various divalent cations was performed using a 500-fold molar excess of these cations.

The quantitative parameters of =Zn binding were investigated by using also two different methods: first, by equilibrhm dialysis essen- tially as previously described for "Ca binding to C 1 (Thielens et al. 1990). Tetanus toxin in 2 mM MOPS, 150 mM NaCI, pH 7.0, was at a final concentration of 10 p ~ . Incubation lasted 42 h at 4 "C. The binding data were plotted according to Scatchard (1949).

The second method was by centrifugation-filtration, as described by Penefsky (1977). Incubations of toxin with various concentrations of ZnClp containing trace 'j5Zn were carried out for 1 h at 20 "C before passage of the mixture through miniature Sephadex G-50 columns. The binding data were plotted according to Scatchard (1949).

Copper-mediated cleavage of tetanus toxin was as follows: 100 pl of 10 p M toxin in 2 mM MOPS, 150 mM NaCI, pH 7.0, was made 10 p~ CuC12. Ascorbate was added to a final concentration of 100-200 p ~ . The mixture was incubated for various time periods, a t 37 "C, and subsequently fragments were analyzed by 12.5% SDS-PAGE under reducing conditions. SDS-PAGE lanes were transferred to ProBlott membranes and after Coomassie Blue staining, the protein bands were excised and subjected to NH2-terminal microsequencing as described above. Zinc and various peptides were tested for their ability to inhibit copper-mediated cleavage of tetanus toxin light chain. The modification of TT225-243 by copper and ascorbate was investigated by incubation of a solution of 100 p M peptide in the MOPS/NaCI buffer with 100 p M copper and ascorbate ranging in concentration from 100 p M to 100 mM for various time periods at 20 "C. The oxidized products were analyzed and purified using a HPLC C18 reverse-phase analytical column (Nova Pak (150 X 3.9 mm) (Waters)) employing a gradient from 5 to 80% acetonitrile in 0.1% trifluoroacetic acid. Controls were performed in which TT225- 243 was treated with CuC12 alone or with ascorbate alone.

RESULTS

Zinc Binding Parameters-The binding of =Zn to the light chain of tetanus toxin separated by SDS-PAGE and subse- quently transferred to an Immobilon membrane is shown in Fig. 1. Under reducing conditions the light chain but not the heavy chain of the toxin shows 65Zn binding. The monochain form of the toxin was also able to bind 'j5Zn under the same conditions.

When purified toxin was preincubated with a solution of "'Zn and subsequently chromatographed by gel filtration un- der physiological pH and salt concentration, the protein peak monitored at 280 nm coeluted with a peak of radioactivity, indicating binding of 'j5Zn to the toxin molecule. An equivalent amount of 65Zn alone in buffer solution did not pass through the column, suggesting that free zinc bound to the column matrix under these conditions. A similar approach has been described to investigate binding of fi4Cu by dopamine 8-mon- ooxygenase (Skotland and Flatmark, 1983). Accumulated free zinc could be removed from the column by flushing with 10 mM EDTA overnight. The binding of 'j5Zn by tetanus toxin measured by this technique was sensitive to pH, being maxi- mal at 7.0 and dropping off sharply below 6.5 (data not shown).

Using the same gel filtration assay, the ability of various divalent cations to displace or compete with 'j5Zn was meas- ured. Fig. 2 indicates that copper was more effective than nonradioactive zinc in its ability to prevent 65Zn binding to tetanus toxin. Nickel, and to a lesser extent cobalt, were able

T T - H Chain,

L Chain,

1 2 3 4

Kd

- 30

FIG. 1. Zinc binding to tetanus toxin. 50 pg (300 pmol) of tetanus toxin, in reduced or non-reduced conditions, were loaded on a 8% acrylamide SDS gel. After electrophoresis proteins were trans- ferred to an Immobilon membrane. The membrane was incubated with 1.94 pCi of @ZZn as described under "Materials and Methods." For the detection of 65Zn bound to unreduced ( l a n e 3) or reduced tetanus toxin ( l a n e 4 ) , an autoradiogram was obtained by exposure to Fuji x-Ray film at -70 "C for 5 days. After autoradiography, the Immobilon membrane was stained for proteins with Coomassie Blue ( l a n e 1, unreduced tetanus toxin; lane 2, reduced tetanus toxin). Phosphorylase b, bovine serum albumin, ovalbumin, and carbonic anhydrase were used as molecular weight markers.

WZn)

cu Zn

Mi

co ca

Mn

F.3

Cd

Ma

65 Zlnc bound (x103cpm),

FIG. 2. Inhibition of e6Zn binding to tetanus toxin by diva- lent cations. Tetanus toxin (20 pl, 10 p ~ ) was incubated with =ZnCl2 alone (final concentration 0.2 p ~ ) or with 6sZn in the presence of a 500-fold molar excess of the chloride salts of various divalent cations for 1 h at 20 "C. Following incubation the mixture was applied to a TSKG-3000SW analytical gel filtration column (Waters) a t a flow rate of 1 ml/l min. Radioactivity associated with the protein peak was determined.

to weakly reduce 65Zn binding to the toxin molecule. Equilibrium dialysis was used to determine the parameters

of 'j5Zn binding to tetanus toxin. Fig. 3 shows the combined results of two independent experiments. The dissociation constant was calculated to be 15 p~ and the number of sites was 0.81 molecule 6sZn/mo1ecule of tetanus toxin. Equilibrium dialysis was carried out at 4 "C due to a tendency of the toxin molecule to stick to the dialysis membranes when incubated a t 20 or 37 "C for extended periods. The results were sup- ported by an independent binding assay performed at ambient temperature in which toxin-bound 6sZn and unbound 65Zn were separated using small (200 pl) gel filtration columns (Penefsky, 1977). The results of these experiments (not shown) indicate a K d of 9 p~ and 0.86 'j5Zn-binding sites/ toxin molecule.

Targeted Metal-catalyzed Oxidative Modification-When TT was incubated with copper and dithiothreitol or ascorbate

Metal Binding by Tetanus Toxin 9055

0 0.02 0.04 BOUND/FREE ,. 0.06

z?nom*/mol p o m w p m a zn

FIG. 3. Scatchard analysis of zinc binding to tetanus toxin. Binding of f"'ZnC12 (from 5 to 110 p ~ ) to tetanus toxin (10 p M ) was measured hy equilibrium dialysis. Data are plotted according to Scatchard (1949), and the straizht l i n e is derived by linear regression analysis.

1 2 3 4 kcla ".". ~

1 7 0 116

8 5 H chaln -

L chain - 5 5

PI 7' 3 9

p28-

p25 - - 27

peptide cleavages of the toxin light chain under oxidative conditions, consistent with the scheme below.

One cleavage site is located close to the middle of the light chain, and gives rise to fragments p 25 and p 28. The second site is close to the carboxyl terminus of the light chain and gives rise to fragment p 47. The 5 kDa COOH-terminal sequence of the light chain has not been identified.

It is noteworthy that the two copper-mediated oxidative cleavages of TT are inhibited in the presence of zinc excess (1 mM) (Fig. 4, lane 3 ) .

Mapping of the Zinc Binding Rpgion Using Synthetic Pep- tides-Peptide TT225-243 containing the putative metal binding motif HELIH was effectively demonstrated to bind "Zn by gel filtration, under conditions in which free ""Zn binds to the column matrix (data not shown). This peptide was found to be an effective inhibitor of the copperlascorhate- induced polypeptide cleavage of the toxin light chain (Fig. 5, lane 3), giving 50% inhibition a t a concentration of 20 pM. Peptide TT233-248, which does not bind ""Zn in our condi- tions, and oxidized peptide '"225-243 were found to be much weaker inhibitors of the oxidative cleavage (Fig. 5, hnes 4 and

r 28 II 25 I r e : : 7 SCHEME 1. - 2 0

1 4

H chaln -

FIG. 4. Effect of zinc on the metal-catalyzed oxidation of tetanus toxin. Tetanus toxin (10 p ~ ) was incubated for 6 h as described under "Materials and Methods," a t 37 "C in 2 mM MOPS, 150 mM NaCI, pH 7.0, alone (control, lone I ), with 10 p~ CuCI2 and 200 p M sodium ascorbate (lane 2 ) or with 1 mM ZnC12, 10 p M cucI2, and 200 p~ sodium ascorhate ( l o n e 3 ) . Lane 4 corresponds to molec- ular weight standards. After incuhation, samples were reduced and analyzed by SDS-PAGE in a 12.5% SDS-polyacrylamide gel.

for 6 h at 37 "C, conditions which are known to cause metal- catalyzed site-specific oxidative modifications in proteins (Stadtman, 1990), a series of discrete bands were generated. Toxin alone (Fig. 4, lane 1 ) or TT in the absence of ascorbate or Cu (data not shown) was not cleaved, revealing that the purified toxin was free of extrinsic contaminating proteasic activity. Oxidative cleavage of the tetanus toxin, as shown by SDS-PAGE under reducing conditions (Fig. 4, lane Z), gen- erated fragments of 47 (p 47), 28 (p 28), and 25 kDa (p 25), corresponding to a decrease of the light chain of about lo%, as estimated by densitometry of the gel colored by Coomassie Blue. NH,-terminal microsequencing of fragments after transfer to ProBlott membranes indicated that p 47 and p 28 contained the NH2-terminal sequence of the toxin light chain, while the NH, terminus of p 25 was blocked. These results are consistent with generation of two copper-mediated poly-

1 2 3 4 5 6 kD.

p20 - p25 -

170 116

05

55

39

27

20

14

FIG. 5. Effect of native and oxidized TT22R-243 peptides and of native TT233-247 peptide on t h e metal-catalyzed oxidative cleavage of tetanus toxin. Tetanus toxin 0 0 pvJ wax incuhated for f h as descrihetl under "Materials and Methods." at 37 "C in 2 mM MOPS, 150 mM Na('I, pH 7 .0 . alone (control. low 1 1 , with 10 pM CuCI? and 200 p~ sodium ascorhate (Inn@ 2 ) , and with 10 p M cuCl2 and 200 p M sodium ascorhate in the presence of 50 p M "T22.5-243 peptide ( lane.?) , 50 pM oxidized lT225-243 peptide (Ian@ 4 ) , or FiO p M 'IT2.13-248 peptidr ( h n @ 5 ) . fanp 6 corrrsponds to molecular weight standards. After incuhation, samples were reduced and analyzed hy SDS-PAGE in a 12.5"h SDS-polyacrylamide gel.

9056 Metal Binding by Tetanus Toxin

5), giving 50% inhibition at 200 and 500 WM, respectively. Peptide 1'1'225-243 was treated with copper and ascorbate

under conditions which led to polypeptide cleavage in the intact toxin. Assessed by reverse-phase HPLC chromatogra- phy, no modification of the peptide was observed at concen- trations of ascorbate (50-500 WM) equivalent to those which induced toxin light chain cleavage. However, concentrations of ascorbate approximately 100-fold higher (5-50 mM) caused a time-dependent modification of the peptide, giving rise to two products TT225-243A and TT225-243B (Fig. 6A). Mass spectrometry analysis indicated molecular masses of 2142.2 for unmodified TT225-243, 2158.6 for TT225-243A, and 2158.6 for TT225-243B, suggesting the addition of a single atom of oxygen in each case (Fig. 6, B and C). NH2-terminal microsequencing suggested modifications of amino acids His-

232, Glu-233, and His-236 in TT225-243A and His-232 and Glu-233 in TT225-243B, as shown by the absence of these residues.

DISCUSSION

The binding of metal ions to proteins has been established as important in both structural and functional aspects of protein chemistry. Zinc (reviewed by Vallee and Auld, 1990) as well as copper (Scott et al., 1988; Calabrese et al., 1989) have been shown to be essential components of the active sites of a variety of enzymes. Our demonstration of the binding of one zinc atom/toxin light chain suggests that these metals bind to a HELIH motif located within this chain, a site analogous to that established as forming part of the zinc

D . O d A

4

! I

TT 225-243 A 00.010- 11 225-243 B - lo.-- 2 a d o J "

1 0 10 20 30

0 40

T- (-1 lB loo 1 2158.6

FIG. 6. FAB-MS of peptide TT225-243 and metal-catalyzed oxidized forms. TT225-243 was treated with 100 ~ L M CuClz and 50 mM sodium ascorbate as described under "Materials and Methods," and the prod- ucts were separated by reverse-phase HPLC (C18 Nova Pak column) (panel A ) . FAB-MS of the two modified prod- ucts were identical and are shown in panel B. Panel C corresponds to FAB- MS of unmodified TT225-243 peptide.

I 1 80 -

60 -

40 -

9) u

3 5 Q

d 2142.6

9) .- - - m 20 -

2100.6 2042.5

2113.7

0

2181.4

loo ] DPALLLMHELWVLHGLYG "4'6

20 -

656.4 1214.6 o i , , I I , , " . 500 1000 1 goo 2000

MIZ

Metal Binding by Tetanus Toxin 9057

coordination site in thermolysin and a number of other zinc enzymes (Jongeneel et al., 1989). This motif as the site of metal binding in tetanus toxin is supported experimentally by binding of 'j5Zn to TT225-243, a synthetic peptide based on the toxin sequence and containing this motif.

The pH dependence of zinc binding to the toxin and the low affinity of this binding are suggestive of binding to histi- dinyl residues, consistent with a loss of zinc binding upon protonation of the histidine imidazole nitrogens at values of pH below 6.5.

Interestingly we have found that, in the qualitative zinc binding assay involving coelution of 65Zn with protein by gel filtration, the toxin light chain bound several times more 65Zn than a comparable molar quantity of intact toxin. This can be due to the rather drastic conditions used for the preparation of the TT light chains. However, it cannot be excluded that in whole tetanus toxin, the heavy chain down-regulates the binding of zinc to the light chain.

Our demonstration of zinc and copper binding by the light chain of tetanus toxin suggests that these metals may play a structural role in the toxin light chain as discussed above.

In the context of our studies of antigen processing and presentation using tetanus toxin as a model antigen, the ability of the toxin to bind metals with ensuing alterations of the toxin conformation is an important aspect for considera- tion.

According to Stille et al. (1987) and Rothbard and Taylor (1988), the amino acid sequence 233-248 in the tetanus toxin light chain is a potential T lymphocyte epitope. The amino acid sequence containing the HELIH motif appears to fulfill the requirements of a T-cell epitope as proposed in the am- phipathic helix hypothesis (Cornette et al., 1989) and of a region of high hydropathy contrast which typifies metal- binding sites in proteins (Yamashita et al., 1990). Thus, binding of a metal atom may be important for the conforma- tion of this putative T-cell epitope. More generally, the pres- ence of metal bound to tetanus toxin may play a role during the processing of the toxin by antigen processing cells and play on the B or T antigenic reactivity.

Preliminary data on the interaction of peptide 233-248 with antibodies raised in rabbit show that zinc favors the peptide- antibody interactions.'

Experimentally, the copper/zinc competition for binding to TT and the ability of copper to cleave about 10% of the light chain of tetanus toxin in the presence of ascorbate to give discrete fragments suggested to us that copper could be used as an oxidative probe of the metal-binding site in the toxin light chain. This is based upon the observation that metal- catalyzed oxidative modification of a protein occurs at or close to the metal-binding ligands of proteins (Marx and Chevion, 1985; Farber and Levine, 1986; Stadtman, 1990; Miller et al., 1990). Zinc competitively inhibits the copper-mediated oxi- dative cleavage of TT in agreement with previous experiments showing that zinc protects bacteria from copper-mediated oxidative damage (Korbashi et al., 1989). Comparable ion protection has been reported for Mg in iron-mediated oxida- tive cleavage of Escherichia coli glutamine synthetase (Jhon et al., 1991). The precision of metal-catalyzed oxidative mod- ification of biomolecules has also been used to establish specific metal-binding sites in peptides (Uchida and Kawak- ishi, 1990) and DNA (reviewed by Sigman, 1990).

We were unable to fully characterize the Cu2+-catalyzed cleavage of tetanus toxin light chain due to NHz-terminal blocking of the p 25 fragment and the inability to isolate and characterize the 5 kDa sequence COOH-terminal to the 47-

M.-B. Villiers, personal communication.

kDa light chain fragment. However, the apparent molecular weights of the polypeptides as well as the NHz-terminal sequence data for p 28 and p 47 allowed us to estimate the locations of the two sites. The cleavage site responsible for the generation of p 25 and p 28 is estimated to be within the light chain sequence leucine 230-methionine 260, consistent with the localization of the zinc-binding site within the HE- LIH sequence, while the second site is within the sequence aspartic acid 405-isoleucine 435.

Mass spectrometric analysis of peptide TT225-243 oxida- tion products gives evidence for major modification at the level of His-232 and -236. This last finding is consistent with the observation that peptides which contain the entire HE- LIH sequence, and at a lesser level peptide containing only ELIH are able to inhibit the oxidative cleavage. From the above observation, it is quite evident that the oxidized peptide is far less efficient as an inhibitor of tetanus toxin oxidative cleavage. Thus, in the absence of simple Cu binding test, the above observations strongly suggest that the copper-binding site is similar to the zinc-binding site,. As shown by Jongeneel et al. (1989), the homology of the HELIH motif of tetanus toxin light chain with the same motif found in established zinc metalloproteases such as thermolysin extends to regions flanking HELIH, supporting the hypothesis that zinc may play a role in a putative enzymatic active site in the tetanus toxin.

In preliminary experiments we have tested the ability of the protoxin and bichain toxin molecules and isolated light and heavy chains to cleave a limited number of established zinc metalloprotease substrates including endothelins (Vijay- araghavan et al., 1990), angiotensin, bradykinin, enkephalin, collagen, and gelatin. Although conclusive evidence for enzy- matic activity has not been established, we have observed a weak proteolytic activity directed against collagen and gelatin. The problems of finding the appropriate conditions to en- hance such activity and, more importantly, to eliminate the possibility of activity due to trace amounts of contaminating proteases remain to be resolved.

Although the molecular mechanism of tetanus toxin toxic- ity is largely unknown, the ability of minute amounts of the toxin to give rise to profound pathological consequences is consistent with the idea that tetanus toxin exerts its patho- logical effects by an enzymatic mechanism. Furthermore, the active component of the toxin molecule has been suggested to be the light chain which appears to act intracellularly, with the heavy chain serving to target the light chain to appropriate presynaptic neurons and enables the entry of the light chain into these cells (Mochida et al., 1989; Ahnert-Higler et al., 1989). The importance of histidine residues in the mechanism of toxicity of the closely related botulinum toxin, which shows a very high homology with tetanus toxin in the putative metal- binding HExxH region, has been suggested by loss of toxicity resulting from modification of histidine residues with diethyl pyrocarbonate (Dasgupta and Rasmussen, 1984). A similar loss of toxicity has been observed with DEPC-modified teta- nus toxin?

The possibility of a proteolytic activity associated with the tetanus toxin molecule, as suggested by the binding of zinc or copper atoms to the toxin light chain, must also be considered in its processing as an antigen. Although in the experimental conditions used no autoproteolytic activity of TT was ob- served, it cannot be excluded that in specific intracellular compartments of antigen processing cells the modification of normal antigen processing pathways due to such enzymatic activity present novel investigative approaches.

9058 Metal Binding by Tetanus Toxin Acknowledgments-We thank Anne-Marie Laharie, Isabelle Bally,

and Nathalie Scherrer for helpful technical assistance, Florence Du- bois for typing the manuscript, and H. Niemann, Jean Gagnon, GBrard Arlaud, and Marie-Bernadette Villiers for scientific discus- sion.

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