Cytotoxic Effects of a Chimeric Protein Consisting of Tetanus Toxin

THE JOURNAL OF BIOLWICAL CHEMISTRY Vol. 269, No. 42, Issue of October 21, pp. 26165-26171, 1994 Printed in U S A .

Cytotoxic Effects of a Chimeric Protein Consisting of Tetanus Toxin Light Chain and Anthrax Toxin Lethal Factor in Non-neuronal Cells*

(Received for publication, March 24, 1994, and in revised form, June 22, 1994)

Naveen AroraS, Lura C. Williamson$, Stephen H. LepplaS, and Jane L. Halpernnll From the SLaboratoy of Microbial Ecology, NIDR and the $Laboratory of Developmental Neurobiology, NICHD, National Institutes of Health, and the Wivision of Bacterial Products, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892

The light chain of tetanus toxin is a zinc endoprotease two chains are covalently associated by a single disulfide bond. that inhibits neurotransmitter release by selective pro- The heavy chain is required for binding to neuronal cells (7) teolysis of the synaptic vesicle-associated protein syn- and translocation of the light chain across membranes (8, 9). aptobrevidvesicle-associated membrane protein. Cel- The light chain is responsible for inhibition of exocytosis (10- lubrevin is a homologue of synaptobrevin that is found 12). This inhibition has been shown to be due to an endopro- in most cell types and is also a substrate for tetanus tease activity of the light chain (13-15). LC contains the con- toxin. The lack of receptors for tetanus toxin on most sensus sequence H- (amino acids 233-237) that is found

non-neuronal difficult. To characterize tetanus molysin (16, 17). In this group of endoproteases, the 2 histi- toxin effects in non-neuronal cells, a fusion protein con- dines are involved in zinc binding, and the glutamic acid plays sisting of the 254 amino-terminal amino acids of lethal a catalytic role. binds zinc atom per molecule (13, 18) factor (LF) of anthrax toxin and tetanus toxin light chain (LC) was prepared. This protein (LF-LC) inhibited which is coordinated by the 2 histidine residues (7), and inhibi- evoked glycine release from primary spinal cord neu- tors of zinc endoproteases such as captopril and phosphorami- rons at concentrations between l.o and ng/ml. L ~ - L ~ don inhibit LC action (13, 19). Recombinant light chain in was cytotoxic to RAW 264.7, ANA-^ cells (mouse mat- which cysteine or valine are substituted for the 2 histidine rophage cell lines), and Chinese hamster ovary cells in a residues in the zinc-binding domain has no activity in perme- dose-dependent manner. These effects required the abilized adrenal chromafin cells (20). presence of protective antigen, the receptor Ending The protease activity of LC is highly specific for synaptobre- component of anthrax toxin. In contrast, LF-LC was not vin (also referred to as vesicle-associated membrane protein), a cytotoxic to RBL-2H3, Vero, or mouse hybridoma cell 13-kDa protein which is a component of synaptic vesicle mem- lines. Mutagenesis of conserved amino acids (Hism7 and branes (21, 22). Recently, synaptobrevin has been shown to GluZs4) in the zinc-binding motif of LC resulted in fusion function as a receptor for the soluble N-ethylmaleimide-sensi- proteins having no biological activity. LF-LC did not in- tive fusion protein (NSF) attachment proteins (SNAP) (23). hibit regulated secretion of serotonin in RBL-2H3 cells SNAPS, SNAP receptors, and NSF can be purified as a 20 S O r Constitutive Secretion in ZUlY nOn-neUrOna1 Cell lines complex that may be required for intracellular membrane fu- as measured in several different assays. we suggest that sion (23, 24). The identification of synaptobrevin as a SNAP the cytotoxic effects Of LF-LC result *Om inhibition Of a receptor suggests that it is important for targeting transport specific intracellular membrane fusion event mediated to the correct acceptor membrane. by cellubrevin. Although synaptobrevin is expressed exclusively in the nerv-

ous system, a homologue of this protein has been identified that is present in non-neuronal cells (25, 26). Cellubrevin is a syn-

Tetanus toxin is a protein neurotoxin produced by Clostrid- aptobrevin homologue that is found in all tissues (25). Cellubre- iurn tetani that inhibits the exocytosis Of neurotransmitter (for vin is enriched in clathrin-coated vesicles and is also cleaved by recent reviews, see Refs. 1 and 2). Tetanus toxin is synthesized tetanus toxin; however, no specific function has been identified as a single Polypeptide chain of 150 containing 1315 amino for this protein (27). Its localization and identification as a acids (3, 4) that is cleaved by clostridial Proteases ( 5 ) between homologue of synaptobrevin has led to the hypothesis that it is residues 457 and 458 to generate two fragments, a heaV chain involved in the targeting and fusion of vesicles in the constitu- (HC)’ of -100 kDa and a light chain (LC) of -50 kDa (6). The tive secretion pathway. Tetanus toxin has been reported to

inhibit secretion in certain non-neuronal cell types such as * The costs of publication of this article were defrayed in part by the primary human macrophages and a mouse macrophage cell

Papent of Page charges. This article must therefore be hereby marked line (28-30). In these studies, both the constitutive and stirnu- “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. lated release of lysozyme was inhibited by tetanus toxin. The

11 To whom correspondence and reprint requests should be addressed: inhibition of lysozyme secretion from macrophages may be due Bldg. 29, Rm. 103, 8800 Rockville Pike, Bethesda, MD 20892. Tel.: to cleavage of cellubrevin by tetanus toxin. 301-496-9695; F a : 301-402-2776. Although studies with macrophages indicate that tetanus light chain oftetanus toen; LF, lethal factor; PA, protective anti&; E6 toxin can inhibit events Other than neuroexoc~osis,

cell types has made studies of tetanus toxin action in in a class of zinc-binding endoproteases represented by ther-

The abbreviations used are: HC, heavy chain of tetanus toxin. LC

edema factor; LF-PE, a fusion protein consisting of the amino-terminal 254 amino acids of LF and residues 398-613 of Pseudomonas exotoxin 2-yl)-2,5-diphenyltetrazolium bromide; HBSS, Hepes balanced salt so- A LF-LC, a fusion protein consisting of the amino-terminal 254 amino lution; NSF, N-ethylmaleimide-sensitive fusion protein; SNAP, soluble acids of LF and residues 1-477 of tetanus toxin; PCR, polymerase chain NSF attachment protein; dATPaS, deoxyadenosine 5’-O-(l-thiotriphos- reaction; CHO, Chinese hamster ovary; MTT, 3-(4,5-dimethylthiazol- phate).

26165

26166 Tetanus B x i n Light Chain Anthrax Toxin Fusion Proteins

studies of the action of this toxin have been possible only in the few cell types possessing tetanus toxin receptors (l ,2). In order to study tetanus toxin action in non-neuronal cells, we con- structed a fusion protein that makes use of the ability of the anthrax toxin components to deliver heterologous proteins to the cytosol. Three distinct proteins (31-35), lethal factor (LF), edema factor (EF), and protective antigen (PA), comprise anthrax toxin. Each protein of the anthrax toxin complex is non- toxic individually. PA binds to cell surface receptors (35,36) and is cleaved by a protease (37,381, probably furin (39,40), resulting in a 63-kDa protein that remains bound to the receptor and a 20-kDa fragment that is released. Removal of the 20-kDa fragment from PA exposes a high affinity binding site for LF and EF (35, 41). The complex of PA and LF or EF is then inte~al ized into vesicles that undergo acidi~cation, resulting in the translocation of LF or EF into the cytosol. EF is a cal- modulin-dependent adenylate cyclase which increases CAMP concentrations in the cytosol (35,42). Based upon mutagenesis and protease inhibitor studies, LF appears to be a zinc-binding protease (43); however, no cellular substrate has been identified. Injection of lethal toxin (LF plus PA) results in pulmonary edema and death in mice and rats. Some macrophage cell lines are rapidly lysed when treated with lethal toxin, while other cell types are not affected. The interaction of lethal toxin with macrophages has been shown to be important for its systemic effects in mice (44). Mice depleted of macrophages by silica injections are resistant to subsequent challenge with lethal toxin. Treatment of cultured macrophages with sublytic concentrations of lethal toxin resulted in the production of inter- leukin 1, suggesting that symptoms associated with anthrax may result from the release of cytokines.

Anthrax toxin been shown to be a useful system for deliver- ing proteins to the cytosol. A protein consisting of LF fused to the ADP-ribosylation domain of Pseudomonas exotoxin A is a potent cytotoxin in the presence of PA (45). This fusion protein retained ADP-ribosylation activity and inhibited protein synthesis in CHO cells. It has since been demonstrated that the first 254 residues of LF, which constitute the PA-binding domain, are sufficient for delivery of heterologous proteins to the cytosol (46). In the present study, a protein consisting of the 254 amino-termin~l residues of LF fused to the amino terminus of the light chain of tetanus toxin {LF-LC) was prepared, and its effects were studied in various cell lines. LF-LC mimicked the action of tetanus toxin on neuronal cells and had marked cytotoxic effects on certain types of cells. This activity appears to be due to the protease activity of the tetanus toxin light chain.

EXPERIMENTAL PROCEDURES Reagents-Restriction endonucleases and DNA modifying enzymes

were purchased from Life Technologies, Inc., Boehringer Mannheim, or New England Biolabs. Escherichia coli DH5a competent cells were from Life Technologies, Inc. Low melting point agarose (Sea Plaque) was obtained from FMC COT. Glutathione Sepharose 4B was purchased from Pharmacia Biotech Inc. Oligonucleotides were synthesized on a PCR Mate (Applied Biosystems) and purified using oligonucleotide purification cartridges obtained from Applied Biosystems. P5S1dATPaS, ~-[3,4,5-~H]leucine (60.0 Ciimmol), and 5-[1,2-3Hlserotonin (15-30 Cit mmol) were purchased from DuPont NEN, and [3Hlglycine (12.2 CiJ mmol) was from Amersham. Tetanus toxin was purified as described earlier (47). The fusion protein LF"254.TR~PE"98"'3 (LF-PE) was prepared as described earlier (46). This protein contains amino acid residues 1-254 of LF fused to domain 111 (containing the ADP-ribosyltrans- ferase domain) of Pseudomonas exotoxin A. The polymerase chain reaction was performed in a thermocycler (Perkin Elmer Cetus) using AmpliTaq DNA polymerase from Perkin Elmer Cetus and dNTPs from U. S. Bi~hemical Corp. The vector pGEX-KG was obtained from the American Type Culture Collection. This vector will express the gene of interest as a fusion protein with glutathione S-transferase. A thrombin cleavage site is downstream from the multiple cloning site. This vector is derived from pGEX-2T (Pharmacia Biotech Inc.) and has the coding

sequence for 5 glycines inserted between the thrombin cleavage site and the multiple cloning site (48). This glycine linker has been shown to facilitate thrombin cleavage of glutathione S-transferase from the de- sired protein product.

Plasmid Construction-The fusion protein LF-LC (Fig. 1) was con- structed using the polymerase chain reaction (PCR) as described previously (451. LF"B4 was amplified from the plasmid pLF7 using primers having the sequences 5"TCTAGATCTAGAAGC~C~TCATGGTGA TGTAGG-3' (primer 1) and 5'-GATC""I'AAGTTCACGCGTGGATA- GATTTA'I""C1YTG-3' (primer 2). A DNA fragment coding for the amino-terminal 477 amino acids of tetanus toxin (LC plus 20 amino

5 ' - A C ~ ~ T A C ~ G T A T G C C ~ T A A C C A T A A A T A A ~ A G - 3 ' (primer acids of HC) was amplified from total C. tetani DNA using the primers

3) and 5'-AAGCTTAAGCTTTCAGTTAAATC'ITCAWM"l"3' (primer 4). The LF"264 PCR product was digested with XbaI and MluI restriction enzymes and the tetanus toxin light chain PCR product was digested sequentially with MluI and HindIII. The digested PCR products and the vector pGEX-KG which had been digested sequentially with XbaI and HindIII and dephosphorylated using calfintestinal phos- phatase were electrophoresed on low melting point agarose, purified, and ligated overnight at 16 "C. After transformation into competent E. coli DH5a cells, transformants were screened by restriction enzyme digestion. Selected plasmids were transformed into E. coli strain SG12036, kindly provided by Susan Gottesman, NCI, NIH. This strain has mutations in the gal, lon, and sul A genes (49). Fusion proteins identical with LF-LC, but having mutations in the zinc-binding domain f,m3HELIHm7) were made by a two-step PCR technique. To substitute an alanine for histidine 237, the 5' portion of the gene was amplified using primers 3 and 6 (5'-TAGTACmTATAAGTTCGTGCA1YTAATAG-3'),

(5'-CTAlTAATGCACGAACTTATA=GTACTA-3'). F'rimers 5 and 6 and the 3' portion of the gene was amplified using primers 4 and 5

are overlapping oligonucleotides that result in a change in residue 237 from histidine to alanine. "he two overlapping fragments were then spliced together in a second PCR amplification using primers 3 and 4. Alanine was also substituted for glutamic acid 234 and histidine 233 using the same technique. The mutated LC genes and LF"'& were ligated into pGEX-KG as described above. The accuracy of each con- struct was confirmed by DNA sequencing using Sequenase version 1.0 from U. S. Biochemical Corp.

Expression and Purification of Fusion Proteins-Cultures were grown in super broth to an A,, of 0.6-0.8 at 37 "C with shaking. Iso- propyl-1-thio-(3-D-galactopyranoside (1.0 mM final concentration) was added, and the incub~tion continued for an additional 4 h. The bacteria were harvested by centrifugation at 4 "C and resuspended in 100 m sodium phosphate, pH 7.4,150 m~ NaCl, 1% Triton X-100,2 mM EDTA, 5 pg/ml leupeptin, 10 pg/ml aprotinin, 10 pg/ml 4-(2-aminoethyl)ben- zenesulfonyl fluoride hydrochloride and 750 pg/ml benzamidine hydrochloride. Lysates of soluble proteins were prepared by sonication of the cells (2 x 1 min, 75% maximum power) a t 0 "C in a Branson Sonifier. The lysates were centrifuged at 20,000 x g for 30 min at 4 "C, and the pellet was discarded. Supernatants containing the soluble proteins were passed through a 0.45-pm filter and loaded onto a glutathione Sepharose 4B column. ARer washing the column with 100 mM sodium phosphate, pH 7.4, 150 mM NaCl, the fusion proteins were eluted with 50 mM Tris, pH 8.0, 10 mM glutathione, 0.5 m~ EDTA, and were con- centrated in Centriprep-100 units (Amicon). Purified fusion proteins were incubated with thrombin (4 pg/ml) for 1 h in 50 mM Tris, pH 8.0, 3 mM CaCl,, 150 mM NaCl to separate glutathione S-transferase from the remainder of the fusion protein. Free glutathione S-transferase was removed by passing the digested fusion protein through a glutathione Sepharose 4B column. The purity of LF-LC was estimated from sodium dodecyl sulfate gel electrophoresis as >70%, and its concentration in all experiments has been corrected to account for degradation products.

~ e u r ~ n a ~ Cell Culture-Gultures of fetal mouse spinal cord neurons with their associated dorsal root ganglia were prepared by a previously published method (50) and grown in 35-mm dishes for 2-3 weeks. Neu- rotransmitter release was measured as described by Williamson et al. (51). Briefly, cultures were incubated for 30 min at 35 "C with radioac- tive glycine (2 $i/ml) to label intracellular pools. The cultures were then incubated in Hepes balanced salt solution (HBSS) without calcium and containing 0.5 mM EGTA and then incubated sequentially for 4.5 min at 35 "C in the following solutions: HBSS without calcium and with 0.5 mM EGTA; HBSS containing 56 mM KC1, 83 m~ NaCl, and no calcium; HBSS containing 56 mix KCl, 83 mM NaC1, and 2.0 mM CaCl,; and HBSS with no calcium and with 0.5 mM EGTA. The radioactivity released into each of the incubation solutions and remaining in the cells was determined. Calcium-dependent release was defined as the amount of material released hy exposure to 56 mM potassium in the presence of

Tetanus Toxin Light Chain Anthrax Toxin Fusion Proteins 26167

Lethal factor Tetanus toxin

254 776 457 1315 PA bind catalytic I Light chain I Heavy chain I

FIG. 1. Structure of lethal factor, tetanqs toxin light chain, and the fusion proteins. The lethal factor light chain fusion proteins contain 477 amino 233 237 acids of tetanus toxin, which corresponds to the light chain plus 20 amino acids of 254 HELIH 477 the heavy chain. The fusion protein also contains 14 residues of the vector LF-LC pGEX-KG at the amino terminus and the amino acids TR between LF1-254 and the LC sequence as a result of the cloning scheme.

HEFGH

HALIH LF-LC E234A k/ HELIA v LF-LC H237A

calcium, minus that released in the absence of calcium, and was calculated as a percentage of the total radioactivity present at the start of the release assay.

Measurement of Viable Cell Number-FLAW 264.7 macrophages obtained from the American Type Culture Collection were grown in Dul- becco’s modified Eagle’s medium (DMEM) with high glucose (Biofluids) containing 10% fetal bovine serum, 2 m glutamine, 10 m Hepes, and 50 pg/ml gentamicin. CHO cells were grown in a-minimal essential medium supplemented with 5% fetal bovine serum, 10 mM Hepes, and 50 pg/ml gentamicin. Cells were plated in 48-well plates (50-100 x lo4 celldwell) and incubated overnight. The cells were rinsed one time, and fresh medium containing the indicated additions was added. At the specified time, 3-(4,5-dimethylthiazol-2-yl~-2,5-diphenyltetrazolium bromide (M’R’) was added to the cells a t a final concentration of 0.5 mg/ml for 1 h. The medium was removed, and the cells dissolved in a solution of 0.5% SDS, 40 mM HCl, and 90% isopropyl alcohol. Aliquots from each well were transferred to a 96-well plate, and the absorbance at 540 or 570 nm read on a microplate reader.

Measurement of Serotonin Release-The rat basophil cell line RBL- 2H3 was provided by Dr. Robert Hohman (Oncor, Rockville, MD). Cells were maintained in Eagle’s minimal essential medium supplemented with 15% fetal bovine serum and 2 mM glutamine. To measure serotonin release, cells were plated into 24-well dishes (2 x lo5 cells/well) and incubated for 48 h with the indicated additions. Radiolabeled serotonin (0.5 pCUwel1) was added for the last 16 h of this incubation. Serotonin release was stimulated by the addition of a mouse monoclonal anti-

described (52). dinitrophenyl IgE antibody and dinitrophenyl-bovine serum albumin as

Measurement ofprotein Secretion-Bulk protein secretion was measured as described by Oda and Wu (53). Cell monolayers in 48-well plates (50-100 x lo4 celldwell) were incubated with the indicated proteins for 24 h. Cells were then labeled for 20 min with [3Hlleucine (20 pCi/ml) in leucine-free medium, rinsed, and incubated for 0.5 or 1 h in DMEM containing 1% fetal bovine serum. The medium was removed from the cells and centrifuged to remove nonadherent cells. Cells were dissolved in 0.2% Triton X-100, and the amount of trichloroacetic acid-precipitable material in the medium and cells was measured using a filter assay and liquid scintillation counting. The amount of secreted protein was calculated as a percent of total labeled protein by dividing the secreted protein by the sum of secreted plus intracellular protein.

Measurement of Lysozyme Secretion-FLAW 264.7 macrophages in 48-well plates were incubated with various additions as described above. At the designated time, medium was removed from the cells and assayed directly to measure secreted lysozyme levels. Cells were dissolved in 0.01% Triton X-100 to measure intracellular lysozyme levels. Lysozyme levels were measured with a spectrophotometric assay (54) in which samples were added to a suspension of Micrococcus lysodeikticus (0.3 mg/ml) in 0.067 M potassium phosphate, pH 6.25, and the initial rate of lysis was monitored at 450 nm.

Secretion of Monoclonal Antibody 18.1.7-The mouse hybridoma cell line 18.1.7 is derived from the fusion of the myeloma cell line P3X63 and

spleen cells from a mouse immunized with tetanus toxoid (55). Mono- clonal antibody 18.1.7 binds to the HC of tetanus toxin and inhibits neurotoxin binding to gangliosides and neuronal cells. Hybridoma cells were plated into 48-well dishes (lo4 cells/dish) and incubated with the indicated additions for 48 h. After a 1-h incubation with MTT, the contents of each well were transferred to an Eppendorf tube and centrifuged at 200 x g. The medium was assayed for levels of secreted monoclonal antibody using an enzyme-linked immunosorbent assay method, and the cells were solubilized as described to determine the number of viable cells.

RESULTS

A DNA fragment corresponding to the 477 amino-terminal amino acids of tetanus toxin was produced using the polymerase chain reaction and ligated in the correct reading frame to a fragment encoding the amino-terminal 254 amino acids of LF. The structure of the protein encoded by this gene fusion is shown schematically in Fig. 1. This gene fusion was inserted into pGEX-KG and the resulting plasmid (pNA67) transformed into E. coli SG12036 for expression. After purification on glutathione Sepharose 4B and cleavage with thrombin, LF-LC migrated as a band of -80 kDa when analyzed by SDS-poly- acrylamide gel electrophoresis. Approximately 400 pg of LF-LC could be purified from a 1-liter culture.

Effect of LF-LC on Neuronal Cells-The effect of LF-LC on neurosecretion was measured in primary mouse spinal cord neurons. In preliminary experiments, the presence of PA receptors on these cells was demonstrated by incubating them with LF-PE (100 ng/ml) in the presence or absence of PA for 48 h and measuring the rate of protein synthesis. LF-PE alone had no effect on protein synthesis, while LF-PE plus 1.0 pg/ml PA inhibited protein synthesis by 80% (data not shown). Spinal cord neurons were then incubated with LF-LC in the presence or absence of PA (1 pg/ml) for 16 h, and glycine release was stimulated as described under “Experimental Procedures” (Fig. 2). In the absence of PA, LF-LC (100 ng/ml) had no effect on evoked glycine release (data not shown). However, in the presence of 1 pg/ml PA, LF-LC was a highly potent inhibitor of glycine release with an EC,, of approximately 10 ng/ml (0.1 nM). The EC,, of tetanus toxin is between 0.1 and 0.01 nM in this assay (51). The action of LF-LC plus PA on spinal cord neurons was similar to that of tetanus toxin in that neurosecretion appeared to be the only function affected. Cells that had been treated with LF-LC plus PA appeared morphologically normal, and uptake of L3H1glycine was not altered (data not shown).

26168 Tetanus Toxin Light Chain Anthrax Toxin Fusion Proteins

0 1 10 100 LF-LC (nglml)

FIG. 2. Inhibition of evoked glycine release by LF-LC plus PA.

LF-LC plus 1 pg/ml PA for 16 h. The cells were then incubated with Spinal cord cultures were incubated with varying concentrations of

L3Hlglycine for 30 min and washed, and evoked release was measured as described under “Experimental Procedures.” Incubation of cells with LF-LC in the absence of PA had no effect on evoked release. Error burs represent the S.D. of triplicate determinations. This experiment was repeated three times with similar results.

These data suggest that this fusion protein can be efficiently delivered to the cytosol of neuronal ceIls by a pathway that includes internalization after binding to the PA receptor.

Cytotoxic Effects of LF-LC on Non-neuronal Cells-Several different cell lines were treated with LF-LC to determine its effects on non-neuronal cells. Cells were incubated with 10 ng/ml LF-LC in the presence or absence of PA (1 pg/ml) for 48 h, and the number of viable cells was quantitated with MTT (Table I). In the presence of PA, LF-LC had cytotoxic effects in two macrophage cell lines tested, RAW 264.7 and ANA-1 cells, and in CHO cells. LF-LC was not cytotoxic in the absence of PA. In three other cell lines that were tested, RBL-2H3 (rat ba- sophils), Vero (African green monkey kidney), and 18.1.7 (a hybridoma cell line secreting an anti-tetanus toxin HC antibody), LF-LC had no effect on cell viability. The presence of FA receptors on each of these cell lines was verified by demonstrat- ing that LF-PE in combination with PA inhibited protein synthesis (data not shown).

Visual inspection of cells treated with LF-LC and PA suggested that the cytotoxic effects of this toxin occur slowly relative to the effects of LF-PE and PA. The time course of LF-LC action was measured by determining the total amount of protein on dishes of control and PA plus LF-LC-treated RAW 264.7 and CHO cells at different time points during a 48-h incubation (Fig. 3). In control RAW 264.7 cells (Fig. 3 A ) , the amount of proteidwell increased by -500%, while the amount of protein in wells treated with 10 ng/ml LF-LC decreased by approximately 30%. The amount of protein in wells of RAW 264.7 cells treated with 1 nglml LF-LC increased by approximately 300%, indicating that their rate of growth was inhibited relative to control cells. Similar effects were seen in CHO cells (Fig. 3B).

The effect of LF-LC on RAW 264.7 and CHO cells was dependent on the concentration of LF-LC (Fig. 4) and the dura- tion of toxin treatment (Fig. 5). LF-LC was approximately 10- fold more potent in RAW 264.7 cells (EC,, = 1.0 ng/ml) than CHO cells (EC,, = 10 ng/ml). To measure the time dependence of LF-LC effects, RAW 264.7 cells were incubated with LF-LC plus PA for different periods of time from 0.5 to 8 h. The cells were then washed to remove unbound toxin, and cytotoxicity was measured after a 48-h incubation (Fig. 5). The number of viable cells was reduced by approximately 10% following a

TABLE I Growth inhibition by LF-LC in cultured cell lines

Cells were incubated with 10 ng/ml LF-LC plus 1.0 pg/ml PA for 48 h. The number of viable cells was measured with MTT as described under “Experimental Procedures.” Each value is expressed as a percent of the value obtained for untreated cells and is representative of at least three experiments. Variability between experiments was less than 10%.

Cell line Cell number

RAW % control

CHO <5 50

ANA- 1 30 RBL-2H3 100 Vero 100 18.1.7 100

50

40

- 9 30

d n 2 20

Y

c .-

10

0

IA

0 10 20 30 40 50 Time (h)

60 B 1

50

A 40 OI a. Y

.E 30 s n e

20

10

0 0 10 20 30 40 50

Time (h)

FIG. 3. Inhibition of cell growth by LF-LC. RAW 264.7 ( A ) and CHO cells ( B ) were plated at a density of lo5 celldm1 in 48-well plates. 24 h later, cells were washed, and fresh medium containing 1 pglml PA plus 0 (m), 1.0 ( + 1, or 10 ng/ml (A) LF-LC or 0.1 nglml LF-PE (0) was added. At each time point, cells were washed and dissolved in 0.01% Triton X-100. The amount of protein from each well was determined with bicinchoninic acid using the Pierce BCAprotein assay (Pierce) and bovine serum albumin as a standard. Error bars represent the S.D. of triplicate determinations.

30-min incubation with 1 ng/ml LF-LC and by approximately 60% after an 8-h exposure. At a concentration of 100 ng/ml LF-LC, cell number was reduced by 70% after a 30-min exposure and by 85% after an 8-h exposure.

Tetanus Toxin Light Chain Anthrax Toxin Fusion Proteins 26169

0 0.001 t 0.01 0.1 1 10

LF-LC (nglml)

100 1000

FIG. 4. Concentration dependence of LF-LC effects. RAW 264.7 (m, 0) and CHO (0,O) cells were incubated with varying concentrations of LF-LC in the presence (m, 0) or absence (0,O) of 1 pg/ml PA for 48 h. The number of viable cells was quantitated with “IT as described under “Experimental Procedures.” Error bars represent the S.D. of triplicate determinations.

120 I I

0 2 4 6 8 10 Time (h)

FIG. 5 . Time dependence of LF-LC effects in RAW 264.7 cells. Cells were incubated with 0.1 (m), 1.0 (O), 10 (01, or 100 (x) ng/ml plus 1 pg/ml PA for the indicated times. Medium containing LF-LC was removed and the cells were incubated in fresh medium without LF-LC. After a total incubation time of 48 h, the number of viable cells was quantitated with M’IT uptake. Error bars represent the S.D. of triplicate determinations.

Effect of LF-LC on Secretion in Non-neuronal Cells- Inhibition of exocytosis from neuronal cells due to synaptobrevin cleavage is the only characterized action of tetanus toxin. The synaptobrevin homologue cellubrevin which is also cleaved by tetanus toxin has been proposed to function in membrane fusion events in non-neuronal cells. We therefore studied whether LF-LC may be blocking secretion in several of the cell lines listed in Table I. Tetanus toxin has been reported to inhibit secretion of lysozyme from both human peripheral blood macrophages and cultured macrophage cell lines (28-30). The release of lysozyme was measured in RAW 264.7 cells which were incubated with or without LF-LC plus PA. At a concentration of 10 ng/ml LF-LC, there was no change in the level of secreted lysozyme after a 12-h incubation and a slight decrease after a 24-h incubation with LF-LC (data not shown).

RBL-2H3 cells undergo granule exocytosis in response to IgE receptor activation in a calcium-dependent manner (52). The exocytosis of serotonin was not inhibited in cells which had

TABLE I1 Effect of LF-LC on bulk protein secretion from CHO cells

tion of LF-LC + 1 pg/ml PA for 24 h. Bulk protein secretion was then Cultures of CHO cells were incubated with the indicated concentra-

measured as described under “Experimental Procedures.” Values represent the mean 2 S.D. of triplicate values. The average amount of radioactivity incorporated into trichloroacetic acid-precipitable material was 92,777 cpm in control cells, 106,917 cpm in cells treated with 0.1 ng/ml LF-LC, and 37,208 in cells treated with 10 ng/ml LF-LC. Secretion was measured at each concentration of LF-LC in at least three experiments with similar results.

Time Protein secreted

% total

0.40 2 0.15 0.49 -c 0.06 0.71 2 0.16

1.13 -c 0.09 1.09 2 0.24 1.36 -c 0.2

0.5 h Control 10 ng/nl LF-LC 1.0 ng/ml LF-LC

Control 10 ng/ml LF-LC 1.0 ng/ml LF-LC

1.0 h

been incubated with LF-LC plus PA for 48 h (data not shown). Stimulation of serotonin release with the calcium ionophore A23187 gave similar results. In other experiments, the constitutive secretion of a monoclonal antibody from the 18.1.7 hybridoma cell line was not inhibited by LF-LC plus PA (data not shown).

The bulk rate of protein secretion was measured in CHO cells to determine if any step in this process was inhibited by LF-LC. Control and treated cells were pulsed with [3Hlleucine for 20 min, and the amount of newly synthesized protein was determined at 0.5 and 1 h (Table 11). The percent of labeled protein secreted was not decreased in cells treated with LF-LC and PA. The total amount of newly synthesized protein was reduced by approximately 60% in LF-LC plus PA-treated cells, probably reflecting the decreased number of viable cells. There was no measurable protein secretion from RAW 264.7 cells as measured by this assay.

Site-directed Mutagenesis of the LC Zinc-binding Domain- The inhibition of secretion from neuronal cells and the effects of LF-LC on non-neuronal cells may be explained by the protease activity of tetanus toxin light chain or alternatively by an un- predicted toxic activity of the fusion protein. To determine if the effects reported here result from the known protease activity of tetanus toxin, mutations predicted to abolish protease activity were made in the gene encoding LF-LC. An alanine residue was substituted for histidine 233, histidine 237, or glutamic acid 234 of the light chain (Fig. 1). These residues are thought to be critical for zinc binding and protease activity. LF-LC H237A and LF-LC E234A were purified and tested for activity in spinal cord neurons (Table 111) and RAW 264.7 cells (Fig. 6). The LF-LC H233A mutant could not be purified. In the presence of PA, the LF-LC mutant proteins were not cytotoxic to RAW 264.7 cells and did not inhibit secretion from the spinal cord neurons. These results suggest that the effects of LF-LC on both neuronal and non-neuronal cells are due to the protease activity of tetanus toxin light chain moiety.

DISCUSSION

Tetanus toxin has recently been demonstrated to be a zinc endoprotease that inhibits exocytosis by cleaving the synaptic vesicle protein synaptobrevin (13-15). Cellubrevin, a homologue of synaptobrevin, is present in all eukaryotic cell types examined and is also a substrate for tetanus toxin in vitro (25). The specific function of cellubrevin and the consequences of its cleavage by tetanus toxin have not been determined, and the absence of receptors for tetanus toxin on most cell types has been an obstacle to addressing these questions. To investigate

26 170 Tetanus Ibxin Light Chain Anthrux lbxin Fusion Proteins TABLE rII

Egect of LF-LC mutants on evoked release from spinal cord cultures

LF-LC H237A plus 1 p&mI PA for 16 h and then with [~Hlglycine for 30 Cultures were incubated with 100 ngiml LF-LC, LF-LC E234A, or

min to allow uptake. Cultures were washed, and evoked release was measured as described under “Experimental Procedures.” Values represent the mean * S.D. of triplicate determinations,

Fusion protein Released PHlglycine

Control (none) % total 20 2 2.3

0.26 & 0.6 19.4 * 2.1 19.2 2 0.8

LF-LC LF-LC-E234A LF-LC-~237A

120

100

80

60

40

20

0 0.01 0.1 1 10 100 lo00

tosis by LF-LC occurred slowly relative to inhibition by tetanus toxin. Experiments are in progress to determine if the difference in time course can be a t t~buted to the rate of internalization of LF-LC uersus tetanus toxin or the rate of intracellular processing and activation.

Mutagenesis of specific amino acids important for zinc-binding and protease activity also supports the conclusion that LF-LC inhibited neuroexocytosis by proteolysis of synaptobrevin. The light chain of tetanus toxin includes the sequence HELIH (residues 233-237) which contains the consensus zinc- binding motif (HEXXH) of a class of metalloendoproteases 140, 41). X-ray c ~ s t a l l o ~ a p ~ c analysis of enzymes containing this sequence has established that the 2 histidine residues coordi- nate the zinc atom, and the glutamic acid is required for catal- ysis. Replacement of either His-237 or Glu-234 by alanine in LF-LC yielded a biolo~cally inactive molecule.

The major action of LF-LC in non-neuronal cells was a cytotoxic effect in several of the cell lines tested. In the presence of LF-LC, the growth of two macrophage cell lines and CHO cells was inhibited, and at higher concentrations the number of viable cells decreased during a 48-h incubation. RAW 264.7 cells were approximately 10-fold more sensitive to LF-LC than CHO cells. Since the number of PA receptors on these two cell lines is similar, the difference in sensitivity may reflect a dserence in the intracellular target. LF-LC did not need to be present continuously during the incubation in order to see maximal cytotoxicity. Comparable cytotoxic effects were seen regardless of whether the toxin was present for the entire experiment or removed after 4 h. These data suggest that sufficient LF-LC for

LF-LC (Rg/ml) maximal cytoto~city can be i n t e ~ a l i ~ e d within 4 h. FIG. 6. Inhibition of growtb by LF-LC mutants. RAW cells were

The cytotoxic effects of LF-LC were not seen in spinal cord

48 h. The number of viable cells was quantitated with M n . Error bars effects are due to a contaminant or nonspecific action of the represent the S.D. of triplicate determinations. fusion protein. Since the only identified activity of tetanus

incubated with LF-LC (e), LF..LC-E234A (x), or LF-LC-H237A (0) for neurons or other cell lines tested, making it unlikely that these

the actions of tetanus toxin in different cell types, we con- structed a fusion protein containing tetanus toxin light chain and the PA-binding domain from the lethal factor of anthrax toxin. Previously, the different protein components of anthrax toxin have been shown to be useful for mediating the uptake of heterologous proteins into cells (45, 46). Our present results provide an additional example of the ability of the anthrax toxin proteins to deliver a heterologous protein to the cytosol of a number of cell types. The presence of PA receptors on most eukaryotic cells (35, 36) makes this system useful for a wide number of cell types.

The addition of 254 amino acids from LF to LC did not appear to affect its function. With the exception of the zinc-binding domain, little is known about the function of different domains in LC. Kurazono et al. (56) reported that the removat of 10 amino acids from the amino terminus of LC resulted in a loss of inhibitory activity in Aplysia californica. It is not known if this region is important for maintaining LC in a active conforma- tion or if it has a more direct role in LC action. The protease activity of LC and intact tetanus toxin has not been compared quantitatively; however, in experiments using permeabilized bovine adrenal chromaffh cells, the light chain is a more efi- cient protease than holotoxin (10). We do not know if LF-LC is cleaved to release free LC after internalization.

The effects of LF-LC in cultured spinal cord neurons were similar to those of tetanus toxin, suggesting that the light chain was efficiently internalized and routed to the correct site of action in the cytosol. Evoked glycine release was inhibited in cells treated with LF-LC plus PA while microscopic appearance and amino acid uptake mechanisms were unaffected. In preliminary experiments, it was observed that inhibition of exocy-

toxin light chain is proteolysis of synaptobrevin and cellubrevin, a plausible hypothesis for our data is that the effects of LF-LC result from cleavage of cellubrevin. Cellubrevin is highly enriched in coated vesicle preparations and is thought to play a role in the targeting of vesicles in constitutive membrane traffic. Our data may be explained by a model in which specific vesicle fusion events involving cellubrevin are essential for cell growth and viability in macrophages and CHO cells. Proteoly- sis of cellubrevin by LF-LC would result in a disruption of cellular trafficking and inhibition of cell growth. The use of LF-LC mutants supports the conclusion that this protein is exerting its effects via proteolysis of cellubrevin. LF-LC mutants that were ineffective at inhibiting exocytosis in neuronal cells also had no effect on viability in RAW 264.7 cells.

The demonstration that LF-LC can interfere with normal cellular function in non-neuronal cells is in agreement with the results obtained by Eisel et al. (57) in transgenic mice. In these studies, mice that had been transfected with the gene for LC expressed this gene in Sertoli cells of the seminiferous epithe- lium. Sertoli cells expressing light chain contained large vacu- oles and had an abnormal distribution of actin filaments. The authors suggested that the effects of light chain are due to cellubrevin cleavage and a block in vesicular fusion processes.

Several interpretations may explain the lack of an effect of LF-LC in the other cell types studied. One possibility is that LF-LC is not efficiently routed to its site of action in the cytosol in resistant cells. However, each of the cell lines studied was sensitive to LF-PE, the fusion protein containing the enzymatic domain of Pseudomonas exotoxin A. LF-PE contains the identical region of LF as LF-LC, which suggests that the amino- terminal 254 residues of LF are able to interact with PA and translocate to the cytosol. We are currently measuring the ex-

Tetanus Toxin Light Chain A

tent of cellubrevin cleavage in cells sensitive and resistant to the cytotoxic effects of LF-LC to address this question more directly.

Another interpretation of the lack of effect of LF-LC on some cell lines is that in these cells, cellubrevin is not essential for cell viability. This would be similar to the effects of tetanus toxin in neurons. Tetanus toxin inhibits depolarization-evoked release of neurotransmitter in cultured spinal cord neurons; however, toxin-treated cultures remain viable with no apparent effect on other functions for a number of weeks. Removal of tetanus toxin results in a slow recovery of neuroexocytosis, indicating that the toxin has not interfered with cell viability (58). Our data would suggest that inhibition of cellubrevin function may not be cytotoxic in all cell types.

Although several experimental approaches were used to determine if LF-LC interfered with secretion in different cell lines, no clear effect was identified. In previous studies, tetanus toxin has been reported to inhibit the release of lysozyme from macrophages (28-30). In these studies, lysozyme release was measured after an 18-24-h incubation with tetanus toxin. In agreement with this, LF-LC also appeared to slightly inhibit lysozyme release from RAW cells after a 24-h incubation. Be- cause cytotoxic effects are also seen at 24 h, the inhibition of lysozyme secretion may represent a secondary effect rather than a specific action of LF-LC.

LF-LC also did not appear to inhibit the calcium-dependent secretion of serotonin from RBL-2H3 cells stimulated by IgE receptor cross-linking. Previously, tetanus toxin was shown to have no effect on the calcium-stimulated release of amylase from pancreatic acinar cells permeabilized with streptolysin 0 (59). The lack of effect of tetanus toxin in these cells indicates that there may be important differences in the mechanism of secretion in neuronal versus certain non-neuronal cell types, as well as differences in cellubrevin function between different cells.

Finally, LF-LC did not inhibit the bulk secretion of newly synthesized proteins in CHO cells. Newly synthesized proteins are secreted by a process that includes a series of budding and fusion events between sequential membrane-bound compart- ments. Our data indicate that LF-LC is probably acting at a step specific to one secretory process rather than inhibiting a universal step in constitutive secretion.

The family of proteins that is exemplified by synaptobrevid vesicle-associated membrane protein appear to be an important component in both constitutive and regulated exocytotic events. The role of these proteins in specific membrane fusion events has not yet been clarified. Use of the fusion protein described here should help in characterizing the importance of these proteins in different secretory pathways in eukaryotic cells.

Acknowledgments-We thank Scott Stibitz, Virginia Johnson, and Valery Gordon for helpful comments on the manuscript and Jerry Keith and William Habig for continued support and making this work possible.

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Documents

Cytotoxic Effects of a Chimeric Protein Consisting of Tetanus Toxin