Enhancement of Diphtheria Toxin Potency by Replacement of the ...

Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 2000 International Society for Neurochemistry

Enhancement of Diphtheria Toxin Potency by Replacement of the Receptor Binding Domain with Tetanus Toxin C-Fragment: A Potential Vector for Delivering Heterologous Proteins to Neurons Jonathan W. Francis, Robert H. Brown, Jr., *Dayse Figueiredo, †Mary P. Remington, ‡Orlando Castillo, ‡Michael A. Schwarzschild, *†Paul S. Fishman, §John R. Murphy, and §Johanna C. vanderSpek Cecil B. Day Center for Neuromuscular Research and ‡Molecular Neurobiology Laboratory, Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown; §Department of Medicine, Boston University School of Medicine, Boston, Massachusetts; *Department of Neurology, University of Maryland School of Medicine; and †Research Service, Baltimore Veterans Affairs Medical Center, Baltimore, Maryland, U.S.A.

Abstract: This study describes the expression, purification, and characterization of a recombinant fusion toxin, DAB389T TC, composed of the catalytic and membrane translocation domains of diphtheria toxin (DAB389) linked to the receptor binding fragment of tetanus toxin (C-fragment). As determined by its ability to inhibit cellular protein synthesis in primary neuron cultures, DAB389TTC was ⬃1,000-fold more cytotoxic than native diphtheria toxin or the previously described fusion toxin, DAB389MSH. The cytotoxic effect of DAB389T TC on cultured cells was specific toward neuronal-type cells and was blocked by coincubation of the chimeric toxin with tetanus antitoxin. The toxicity of DAB389T TC, like that of diphtheria toxin, was dependent on passage through an acidic compartment and ADP-ribosyltransferase activity of the DAB389 catalytic fragment. These results suggest that a catalytically inactive form of DAB389T TC may be useful as a nonviral vehicle to deliver exogenous proteins to the cytosolic compartment of neurons. Key Words: Diphtheria toxin:tetanus toxin fusion protein—Vector delivery system—Neurons. J. Neurochem. 74, 2528 –2536 (2000).

erties of these proteins, but there is growing interest in using fragments from some of the toxins to facilitate the delivery of exogenous “passenger” biomolecules, i.e., peptides, proteins, and plasmid DNA, to various target cells (Bizzini et al., 1977; Stenmark et al., 1991; Madshus et al., 1992; Donnelly et al., 1993; Fominaya and Wels, 1996; Goletz et al., 1997; Knight et al., 1999). This interest is grounded in the observation that many bacterial protein toxins have a modular structure/function organization that reflects a clear partitioning of different functions to distinct domains within the polypeptide. For example, botulinum toxin, tetanus toxin (TTx), DTx, and Pseudomonas exotoxin A are all synthesized in situ as single polypeptide chains (Montecucco et al., 1994). Each of the toxins must then undergo a proteolytic “nicking” step, which generates the active toxophore structure consisting of two fragments joined by a single interfragment disulfide bond. One fragment of the toxin serves (a) to bind the toxin to its specific receptor on the surface of a target cell, (b) to promote internalization of the toxin into endosomal vesicles, and (c) to facilitate translocation of the toxin’s catalytic fragment through the endo-

Bacterial protein toxins are the most deadly natural substances (Middlebrook, 1989). Despite their lethality, bacterial toxins are gaining increasing attention as therapeutic agents in human disease. Intramuscular injections of botulinum neurotoxin (BOTOX) are used to treat certain neuromuscular disorders involving local muscle spasticity or dystonia. Most recently, a diphtheria toxin (DTx)–interleukin 2 (IL-2) fusion protein (ONTAC) has been approved by the Food and Drug Administration for treatment of refractory cutaneous T cell lymphoma. The current use of bacterial protein toxins as biopharmaceuticals has exploited the exquisite cytotoxic prop-

Received November 11, 1999; revised manuscript received January 18, 2000; accepted January 19, 2000. Address correspondence and reprint requests to Dr. J. W. Francis at Cecil B. Day Center for Neuromuscular Research, Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Building 149, 13th Street, Room 6627, Charlestown, MA 02129, U.S.A. E-mail: [email protected] Abbreviations used: DTx, diphtheria toxin; IL-2, interleukin-2; IPTG, isopropyl ␤-D-thiogalactopyranoside; MSH, melanocyte-stimulating hormone; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SOD-1, superoxide dismutase; TTC, carboxyl 451amino acid fragment (C-fragment) of tetanus toxin heavy chain; TTx, tetanus toxin.

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CHIMERIC DIPHTHERIA:TETANUS TOXIN somal membrane into the cell cytosol. The second fragment is an enzyme that catalyzes a toxic biochemical reaction within the cytosol of the intoxicated cell. Of the bacterial toxins, the clostridial toxins, botulinum toxin, and TTx are distinctive in their high degree of specificity for neurons (Halpern and Neale, 1995). Bizzini et al. (1977) suggested ⬎20 years ago that an atoxic piece of TTx might be used as a vector to target the delivery of other proteins to neurons. TTx has a well-documented capacity for neuronal binding and internalization (Lee et al., 1979; Rogers and Snyder, 1981). In particular, when administered systemically or intramuscularly to animals, the toxin is taken up selectively by motor neurons in the brainstem and spinal cord (Habermann and Dimpfel, 1973). The carboxyl 451amino acid fragment (C-fragment) of TTx heavy chain, called TTC, retains the neuronal binding and uptake properties of the holotoxin without the toxic domains (Bizzini et al., 1977; Dumas et al., 1979; Morris et al., 1980; Weller et al., 1986). Linking TTC to other large proteins by either chemical conjugation or genetic fusion enhances their uptake by neurons in vitro and in vivo (Bizzini et al., 1980; Beaude et al., 1990; Fishman et al., 1990; Dobrenis et al., 1992; Francis et al., 1995; Coen et al., 1997). In aggregate, these studies strongly indicate that TTC possesses considerable potential as a nonviral vector for delivering therapeutic proteins to neurons. We have previously expressed a recombinant “hybrid” protein composed of TTC joined to Cu/Zn superoxide dismutase (SOD-1) (Francis et al., 1995). This bifunctional hybrid protein, called SOD:Tet451, possesses the neuronal binding and internalization properties of TTC as well as the enzymatic activity of SOD-1. Intravenous administration of SOD:Tet451 to rats following transient middle cerebral artery occlusion substantially reduces cerebral infarction in this model of focal brain ischemia/ reperfusion (Francis et al., 1997). Although the mechanism of action behind SOD:Tet451’s neuroprotective activity in vivo has yet to be fully delineated, the SOD-1 passenger protein most likely remains associated with membranous vesicles following internalization. Passenger proteins, e.g., SOD-1, directly linked to TTC would not a priori be expected to escape from intracellular vesicles after internalization because TTC does not contain the membrane translocation domain of TTx. That the same restricted intracellular distribution would probably befall other passenger proteins directly linked to TTC suggests that such fusion constructs would probably not be very useful for delivering passenger proteins that need access to other subcellular compartments to carry out their particular function(s). This led us to ask whether incorporation of a membrane translocation domain into our TTC fusion constructs would facilitate delivery of passenger proteins to the cytosol following their internalization into neuronal cell endosomes/vesicles. To address this question, we have begun to evaluate the membrane translocation apparatus of DTx. The catalytic fragment of DTx is well known to undergo membrane translocation to the cyto-

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solic compartment of an intoxicated cell, where it shuts down cellular protein synthesis by inactivating the translation elongation factor EF-2. Both TTx and DTx have unique transmembrane domains, but we decided to use that of DTx because DTx is the most thoroughly characterized of the membrane-translocating toxins (Pappenheimer, 1977; London, 1992). Moreover, it has already been demonstrated that the recombinant fusion of selected passenger peptides or proteins to DTx facilitates passenger protein delivery to the cytosol of various nonneuronal cultured cells (Stenmark et al., 1991; Madshus et al., 1992; Wiedlocha et al., 1992; Klingenberg and Olsnes, 1996). In the present report we have examined the biological activity of a recombinant DTx:TTC fusion toxin, DAB389TTC, in cultured neuronal cells to determine if the chimeric toxin retains the characteristic functional properties of its component TTC and DTx moieties. Because this fusion toxin combines the catalytic and membrane translocation domains of DTx with the atoxic cell targeting domain of TTx (TTC), one would predict that DAB389TTC would possess both the neuronal specificity of TTC and the characteristic ability of DTx to inhibit potently cellular protein synthesis. The observed profile of DAB389TTC cytotoxicity suggests that a nontoxic analogue of this reagent may be a useful vehicle for delivering exogenous passenger proteins to the cytosolic compartment of neurons. EXPERIMENTAL PROCEDURES Materials All DNA-modifying enzymes were purchased from wellknown commercial sources. A codon-engineered cDNA clone for TTC was obtained from Dr. Neil Fairweather (Imperial College, London, U.K.). Recombinant TTC was purchased from Boehringer Mannheim (Indianapolis, IN, U.S.A.). DTx and rabbit anti-TTC antisera were purchased from Calbiochem (La Jolla, CA, U.S.A.), and mouse monoclonal antibody against the catalytic domain of DTx was purchased from Accurate Chemical and Scientific (Westbury, NY, U.S.A.). Additional immunochemical reagents were purchased from Vector Laboratories (Burlingame, CA, U.S.A.). Chloroquine and monensin were from Sigma (St. Louis, MO, U.S.A.).

Plasmid constructs A fusion gene encoding the first 388 amino acids of DTx linked to TTC was constructed by slight modification of the previously described plasmid, pET-JV127. Derived from the prokaryotic expression vector pET11d (Novagen, Madison, WI, U.S.A.), pET-JV127 encodes for a DTx–IL-2 fusion toxin, DAB389IL-2 (vanderSpek et al., 1993). The present DTx:TTC fusion protein, designated DAB389TTC, was assembled by replacing the IL-2 cDNA in pET-JV127 with a 1.4-kb semisynthetic cDNA for TTC obtained from plasmid pMAL:TetC (Fig. 1) (Makoff et al., 1989; Figueiredo et al., 1995). The amino terminus of TTC was fused to the carboxyl terminus of the DAB389 moiety because the normal position of the respective receptor binding domains for both DTx and TTx is at the toxin’s carboxyl terminus. To allow ligation of the 5⬘ end of the TTC cDNA to the 3⬘ terminus of the DAB389 coding sequence in pET-JV127, a PCR protocol was first used to introduce a

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FIG. 1. Recombinant construction of the DAB389T TC fusion gene.

new SphI restriction site just upstream from the beginning of the TTC coding sequence. This PCR used a partially degenerate forward primer that encoded for an SphI site at the 5⬘ end of the primer as well as a BamHI site immediately upstream from the SphI site to allow subcloning of the PCR product back into plasmid pMALc:TetC. The reverse primer was positioned over a unique SacII restriction site in the TTC cDNA located ⬃0.4 kb downstream from the start of the coding sequence. The resulting 0.4-kb PCR product was then restricted with BamHI/SacII and inserted by fragment exchange back into pMAL:TetC. The sequence of the PCR product was subsequently checked by the

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dideoxy chain termination method (Sanger et al., 1977). Finally, the IL-2 cDNA in pET-JV127 was replaced with the modified TTC cDNA from pMAL:TetC using SphI/HindIII fragment exchange, creating plasmid pET11d:DAB389TTC. A similar fusion construct containing the E149S mutation in the DTx catalytic domain [DA(E149S)B389TTC] was assembled in parallel using the plasmid pET-JV127(E149S) (vanderSpek et al., 1996).

Fusion protein purification The DAB389TTC and DA(E149S)B389TTC fusion proteins were expressed in Escherichia coli strain BL21(DE3) (Nova-

CHIMERIC DIPHTHERIA:TETANUS TOXIN gen). Liquid broth cultures (1.5 L) in NZCYM media (Sigma) were propagated to an OD600 of 0.5– 0.6 in a shaking incubator at 37°C, at which point isopropyl ␤-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM to induce target protein expression. Bacteria were harvested 4 h later by centrifugation at 5,000 g for 10 min and then stored at ⫺20°C until further use. Fusion proteins were purified from total soluble bacterial protein using ammonium sulfate precipitation followed by ion exchange chromatography. After resuspending the bacterial pellet in 50 ml of cold 0.1 M potassium phosphate buffer (pH 8.0) [containing 2 mM EDTA, 20 mM ␤-mercaptoethanol, 0.5% Triton X-100, and Complete Antiprotease Cocktail (Boehringer Mannheim)], bacteria were lysed by lysozyme treatment followed by sonication. The total lysate was centrifuged at 20,000 g for 20 min to obtain the soluble protein fraction (supernatant), to which 25 g of solid ammonium sulfate was slowly added with stirring. The precipitated protein was collected by centrifugation at 20,000 g for 20 min and resolubilized in 20 ml of 0.05 M Tris-HCl (pH 8.4) containing 2 mM EDTA, 20 mM ␤-mercaptoethanol, and Complete Antiprotease Cocktail without EDTA. The sample was then dialyzed overnight at 4°C against 0.05 M Tris-HCl (pH 8.4) containing 0.2 mM phenylmethylsulfonyl fluoride. DEAE-anion exchange chromatography was carried out by passing the dialyzed ammonium sulfate cut over a 6-ml bed volume of DEAE Sepharose Fast Flow (Amersham Pharmacia) at a flow rate of 0.6 ml/min. The column was then washed with 100 ml of 0.02 M Tris-HCl (pH 8.4) containing 100 mM NaCl and Complete Antiprotease Cocktail without EDTA. Sample remaining bound to the column was subsequently eluted using a step gradient of 125, 150, and 175 mM NaCl in 0.02 M Tris-HCl, pH 8.4. After the eluted fractions were analyzed by Coomassie Blue staining of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels electrophoresed under reducing conditions, fractions were pooled, and buffer was exchanged against phosphate-buffered saline using a Centricon Plus-20 (Amicon; 50,000 molecular weight cutoff). Sample purity was assessed by densitometric analysis of dried gels using a flatbed scanner along with Adobe Photoshop version 4.0 and NIH Image software. Sample protein concentrations were determined by Pierce Coomassie Blue Assay. The antigenic identity of DAB389TTC and DA(E149S)B389TTC fusion proteins was confirmed by immunoblot analysis using rabbit anti-TTC antisera (1:100,000 dilution) and mouse anti-DTx antibody (1:1,000 dilution). Purified sample protein was aliquoted and stored at ⫺20°C until further use. A 1.5-L bacterial culture typically yielded ⬃20 mg of purified target protein.

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The defined medium consisted of Dulbecco’s modified Eagle’s medium/F12 medium (GIBCO) containing 0.45% glucose, B-27 supplement (GIBCO), and antibiotics. Cells were plated at a density of 1.5 ⫻ 106 cells/2 ml per well in six-well plates (Costar) precoated with 0.2% polyethylenimine.

Cytotoxicity assay Because the cytotoxicity of DTx is known to be mediated by its ability to inhibit cellular protein synthesis, we chose to examine the effect of our fusion toxins on cell viability through the use of a [14C]leucine incorporation assay. [14C]Leucine incorporation into HUT 102/6TG, N18-RE105, HEPG2, and Swiss 3T3 cells was measured using previously established methods (vanderSpek et al., 1996). For the N18-RE-105, HEPG2, and Swiss 3T3 lines, 1 ⫻ 105 cells in 0.1 ml of complete medium were seeded into each well of a flat-bottom, 96-well microtiter plate. Assays conducted with HUT 102/6TG cells used 5 ⫻ 104 cells per well. In general, test proteins were serially diluted in medium such that addition of 0.1 ml to each well resulted in a final concentration ranging from 100 nM to 100 fM. Cultures were then incubated for 18 h at 37°C in a 5% CO2/95% air atmosphere. The medium was subsequently removed and replaced with 0.2 ml of leucine-free minimal essential medium (GIBCO) containing 1 ␮Ci/ml [14C]leucine (0.250 mCi/mmol; DuPont NEN), 2 mM glutamine, and antibiotics. After 2 h, the medium was again removed, and the cells were lysed by treatment with 60 ␮l of 0.4 M KOH per well for 10 min at room temperature. Proteins were precipitated by addition of 140 ␮l of 10% trichloroacetic acid per well followed by another 10-min incubation at room temperature. Insoluble protein was collected on glass fiber filters using a PhD cell harvester (Cambridge Technology, Watertown, MA, U.S.A.). Sample radioactivity was determined according to standard methods. [14C]Leucine incorporation into the primary neuron cultures was similarly determined by adjusting for the increased volumes required for the larger culture wells. Cells were used for the cytotoxicity assays after 4 –5 days in vitro when the neurons were incubated with test proteins for 18 h in a 1-ml volume of fresh medium. Cells were subsequently pulsed for 2 h with 1 ␮Ci of [14C]leucine in a total volume of 1 ml of minimal essential medium. After lysing the cells with 0.25 ml of KOH, proteins were precipitated by addition of 0.6 ml of 10% trichloroacetic acid. Insoluble protein was collected on glass fiber filters, and radioactivity was measured as above. Calculation of IC50 values was performed by determining the concentration of fusion toxin required to cause 50% inhibition of [14C]leucine incorporation following incubation of the cells with the toxin for 18 h.

Cell culture N18-RE-105 neuronal hybrid cells were cultured as previously described with minor modifications (Malouf et al., 1984). Cells were maintained in Dulbecco’s modified Eagle’s medium (GIBCO) supplemented with 10% fetal bovine serum (GIBCO), 1⫻ HAT (hypoxanthine/aminopterin/thymidine) media supplement (Sigma), and antibiotic/antimycotic (Sigma). HUT 102/6TG, HEPG2, and Swiss 3T3 cells were maintained in RPMI 1640 medium (GIBCO) supplemented with 10% fetal bovine serum (Hyclone), 2 mM glutamine, 50 IU/ml penicillin, and 50 ␮g/ml streptomycin (Gazdar et al., 1980; Waldmann, 1987). Primary dissociated cultures from embryonic day 17–18 rat striatum were prepared as described with the exception that cells were placed in serum-free, defined medium 4 h after plating to curb proliferation of glial cells (Konradi et al., 1994).

RESULTS Expression of the DAB389TTC and DA(E149S)B389TTC fusion proteins in E. coli BL21(DE3) host cells was initially examined by Coomassie Blue staining of SDS-PAGE gels. Compared with total cell lysates obtained from parallel cultures of DAB389TTC and DA(E149S)B389TTC bacteria that were not treated with IPTG, the respective lysates obtained from IPTG-induced cultures each revealed a prominent band with an estimated molecular weight of 93,000 (Fig. 2A, arrow). This electrophoretic mobility is in good accord with the size predicted for each of these 840-amino acid polypeptides. The DA(E149S)B389TTC fusion protein appeared to J. Neurochem., Vol. 74, No. 6, 2000

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FIG. 2. SDS-PAGE and western blot analysis of the DAB389T TC and DA(E149S)B389T TC fusion proteins. A: SDS-PAGE analysis of DAB389T TC and DA(E149S)B389T TC expression in E. coli host strain BL21(DE3). Each lane was loaded with 15 ␮g of sample protein: lane 1, DAB389T TC total lysate, no IPTG; lane 2, DAB389T TC total lysate, 4-h induction; lane 3, DA(E149S)B389T TC total lysate, no IPTG; lane 4, DA(E149S)B389T TC total lysate, 4-h induction; lane 5, DAB389T TC soluble fraction, no IPTG; lane 6, DAB389T TC soluble fraction, 4-h induction; lane 7, DAB389T TC insoluble fraction, no IPTG; and lane 8, DAB389T TC insoluble fraction, 4-h induction. B: SDSPAGE analysis of DAB389T TC and DA(E149S)B389T TC protein purification using ammonium sulfate precipitation and DEAE anion-exchange chromatography. Lanes 1–3 were loaded with 15 ␮g of protein, whereas lanes 4 –9 were loaded with 10 ␮g of protein: lane 1, DAB389T TC total lysate, 4-h induction; lane 2, DAB389T TC soluble fraction; lane 3, DAB389T TC, 40% ammonium sulfate cut; lane 4, DAB389T TC, DEAE elution at 125 mM NaCl; lane 5, DAB389T TC, DEAE elution at 150 mM NaCl; lane 6, DA(E149S)B389T TC soluble fraction, 4-h induction; lane 7, DA(E149S)B389T TC, 40% ammonium sulfate cut; lane 8, DA(E149S)B389T TC, DEAE elution at 125 mM NaCl; and lane 9, DA(E149S)B389T TC, DEAE elution at 150 mM NaCl. C: Western blot analysis of DAB389T TC using anti-DTx and anti-T TC antibodies. Lanes 1–3 show anti-DTx immunoreactivity; lanes 4 – 6 show anti-T TC immunoreactivity. Lanes 1 and 4, DAB389T TC soluble fraction, no IPTG (2 ␮g of protein); lanes 2 and 5, DAB389T TC soluble fraction, 4-h induction (2 ␮g of protein); and lanes 3 and 6, DAB389T TC, DEAE elution at 125 mM NaCl (125 and 250 ng of protein).

be present in a substantially greater amount than DAB389TTC. SDS-PAGE analysis of soluble and insoluble protein fractions prepared from total lysates indicated that the DAB389TTC and DA(E149S)B389TTC target proteins were present in both fractions (data for E149S mutation not shown). The fusion proteins were subsequently purified from soluble bacterial protein using ammonium sulfate precipitation and ion exchange chromatography (Fig. 2B). Analysis of the purified target proteins by SDS-PAGE/Coomassie Blue staining indicated that the samples were ⬃85–90% pure. Western blot analysis of purified DAB389TTC protein with anti-TTC and antiDTx A fragment antibodies confirmed the antigenic identity of the fusion protein; a major immunoreactive band corresponding to a molecular weight of 93,000 was recognized by both antibodies (Fig. 2C). The presence of this immunopositive band in the soluble protein fraction obtained from a control culture indicates that there is leaky expression of DAB389TTC in the absence of IPTG. We studied the functional properties of the DAB389TTC fusion protein through its ability to inhibit protein synthesis in N18-RE-105 neuronal hybrid cells and cultured embryonic rat striatal neurons. The N18RE-105 cell line was chosen because, unlike most neuroblastoma cell lines, N18-RE-105 cells have a surface ganglioside composition similar to normal brain tissue and thus bind high amounts of TTx (Rogers and Snyder, 1981; Staub et al., 1986). This cell line has also been previously used to study the binding and internalization J. Neurochem., Vol. 74, No. 6, 2000

of another TTC fusion protein, SOD:Tet451 (Francis et al., 1995). Because N18-RE-105 cells have certain characteristics that are not typical of mammalian nerve cells, however, we further evaluated the activity of DAB389TTC in primary dissociated cultures of rat striatal neurons. Striatal neurons are not the natural target cells of TTx in vivo, although the high density of TTx binding sites in rat striatum (Rogers and Snyder, 1981) indicated that these cells would nonetheless be useful for evaluating the interaction of the chimeric toxin with primary neurons in vitro. Following overnight treatment of cultured striatal neurons or N18-RE-105 cells with various concentrations of DAB389TTC, the chimeric toxin was shown to be a potent inhibitor of cellular protein synthesis. The inhibition of cellular [14C]leucine incorporation by DAB389TTC was dose-dependent and resulted in an IC50 of 4 pM in the primary neuron cultures and 2 nM in cultures of N18RE-105 cells (Fig. 3). The cytotoxic effect of DAB389TTC on cultured cells appeared to be specific for neuronal-type cells given that the chimeric toxin at a concentration of 100 nM produced only a modest 23% inhibition of protein synthesis in HUT 102/6TG cells, a human T lymphocyte cell line known to have a large number of IL-2 receptors (Fig. 3) (Gazdar et al., 1980; Waldmann, 1987). DAB389TTC was also not cytotoxic to HEPG2 (human hepatic carcinoma) or Swiss 3T3 cell lines (mouse fibroblast) (data not shown). When HUT 102/6TG cells were treated overnight with the DTx:IL-2

CHIMERIC DIPHTHERIA:TETANUS TOXIN

FIG. 3. Dose–response relationship of DAB389T TC cytotoxicity in primary neurons, N18-RE-105 cells, and HUT 102/6TG cells. Cultured cells were incubated overnight with various concentrations of the chimeric toxin before pulse labeling the next day with [14C]leucine. For primary neurons and N18-RE-105 cells, data are mean ⫾ SEM (bars) values of three separate experiments having two to four replicate cultures per experiment. For HUT 102/6TG cells, data are mean ⫾ SEM (bars) values of quadruplicate cultures. Data presented for untreated control cells were derived from primary neurons. Mean values for untreated N18RE-105 cells and HUT 102/6TG cells similarly had SEs of ⱕ5% (data not shown).

fusion toxin, DAB389IL-2, however, incorporation of [14C]leucine was markedly reduced (IC50 ⫽ 10 pM; data not shown). We further studied the targeting specificity of DAB389TTC by comparing its cytotoxic potency in primary neurons with that of native DTx and another DTx fusion protein having a different cell targeting domain, DAB389melanocyte-stimulating hormone (MSH) (Murphy et al., 1986). As shown in Fig. 4, the potency of DAB389TTC for inhibiting [14C]leucine incorporation in cultured striatal neurons was generally 1,000-fold greater

FIG. 4. Comparative cytotoxicity of DAB389T TC, DTx, and DAB389MSH in primary neuron cultures. Cells were treated overnight with various concentrations of each of the toxins before radiolabeling with [14C]leucine. Data are mean ⫾ SEM (bars) values of triplicate cultures.

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than that observed with native DTx or DAB389MSH. A similar rank order of cytotoxic potency was observed in treated N18-RE-105 cells. In this cell line, both DTx and DAB389MSH failed to produce any significant inhibition of protein synthesis up to a concentration of 100 nM (the highest concentration tested in our experiments; data not shown). To ascertain that DAB389TTC cytotoxicity in cultured striatal neurons was mediated by the TTC cell targeting domain of the chimeric toxin, we first compared the cytotoxicity of DAB389TTC alone with that of DAB389TTC and tetanus antitoxin together. Simultaneous incubation of cultured striatal neurons with 100 pM DAB389TTC and 75 units of tetanus antitoxin almost completely prevented the inhibition of [14C]leucine incorporation observed after treatment with DAB389TTC by itself (data not shown). The binding profile of the chimeric toxin was further assessed by testing whether a large molar excess of purified recombinant TTC could block the ability of DAB389TTC to inhibit protein synthesis in primary neurons. Coincubation of 10 pM chimeric toxin with a 10,000-fold higher concentration of recombinant TTC only partially attenuated the decrement in protein synthesis seen with the chimeric toxin alone (62% vs. 42% of control, respectively). Following receptor-mediated endocytosis of DTx into cellular endosomes, delivery of the DTx catalytic domain through the endosomal membrane into the cytosol is dependent on acidification of endocytic vesicles. Agents that interfere with endosomal acidification, such as chloroquine, monensin, and bafilomycin A, are well known to diminish the cytotoxicity of DTx and DTx fusion proteins (Draper and Simon, 1980; Sandvig and Olsnes, 1980; Papini et al., 1993; Fisher et al., 1996). To determine if inhibition of protein synthesis in DAB389TTCtreated cells is likewise dependent on endosomal acidification, N18-RE-105 neuronal hybrid cells were coincubated with the chimeric toxin and either chloroquine or monensin. As shown in Fig. 5, both chloroquine and monensin substantially reversed the cytotoxic effect of DAB389TTC in N18-RE-105 cells. Finally, to demonstrate that inhibition of protein synthesis associated with DAB389TTC treatment of neuronal cells is mediated through the ADP-ribosylation activity of the DTx catalytic domain, we compared the cytotoxicity of DAB389TTC with that of an ADP-ribosyltransferase-defective mutant, DA(E149S)B389TTC (Barbieri and Collier, 1987; Wilson et al., 1990). Consistent with previous results obtained in other cells treated with DTx and other DTx-related fusion proteins bearing this same mutation (Fisher et al., 1996; vanderSpek et al., 1996; Lemichez et al., 1997), DA(E149S)B389TTC was seen to be almost 1,000-fold less potent than DAB389TTC in inhibiting [14C]leucine incorporation into cultured striatal neurons (Fig. 6). The reduction in cytotoxicity associated with the E149S mutant was also observed in the N18-RE-105 cell line. Whereas the IC50 for DAB389TTC inhibition of protein synthesis in N18-RE-105 cells was J. Neurochem., Vol. 74, No. 6, 2000

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J. W. FRANCIS ET AL. were treated with a large excess of TTC along with DAB389TTC, the binding of the chimeric toxin to cultured neurons appeared to be poorly saturable. These observations are in excellent agreement with previous studies of TTC and TTx binding that showed (a) an inhibitory effect of anti-tetanus antibodies on TTx binding to intact cultured neuronal cells (Staub et al., 1986; Walton et al., 1988; Sandberg et al., 1989) and (b) that the binding of TTC and TTx to various preparations of primary neural cells and tissues was nonsaturable under physiological conditions (Critchley et al., 1986; Pierce et al., 1986; Staub et al., 1986; Walton et al., 1988; Halpern and Loftus, 1993). The poor ability of excess TTC to inhibit DAB389TTC cytotoxicity in intact primary neurons at 37°C may also reflect a constant turnover of TTx receptors, as was demonstrated previously in N18-RE-105 cells (Staub et al., 1986). Our results thus strongly support the conclusion that the TTC domain of DAB389TTC remains fully functional as a neuronal cell targeting ligand following its recombinant fusion to the DAB389 moiety. It is interesting that our study revealed that the potency of DAB389TTC for inhibiting [14C]leucine incorporation in cultured primary neurons (IC50 ⫽ 4 pM) was ⬃100 – 1,000-fold greater than might be expected given previously reported affinity constants for TTC and TTx binding to the TTx receptor, generally in the range of 0.5–5 nM (Goldberg et al., 1981; Rogers and Snyder, 1981; Pierce et al., 1986; Staub et al., 1986). This finding very likely reflects a powerful amplification step within the mechanism of DAB389TTC intoxication that occurs downstream of toxin receptor binding. The level of signal amplification provided by the DAB389 catalytic do-

FIG. 5. Inhibition of DAB389T TC cytotoxicity in N18-RE-105 cells by cotreatment with lysosomotropic agents. A: Cells were incubated overnight with 10 nM chimeric toxin, 10 ␮M chloroquine, or both. *p ⬍ 0.05, significantly different from control; **p ⬍ 0.05, significantly different from control but not chloroquine alone. B: Cells were treated overnight with 1 nM chimeric toxin, 1 ␮M monensin, or both. Data are mean ⫾ SEM (bars) values of three or four replicate cultures. *p ⬍ 0.05, significantly different from control.

2 nM, the E149S mutant was essentially inactive at the highest concentration tested (100 nM). DISCUSSION In this report we have characterized the in vitro biological activity of a DTx–TTC fusion toxin, DAB389TTC, to determine whether the component TTC and DAB389 moieties remain functional following their recombinant linkage to each other. As determined indirectly by cytotoxic activity, the binding of DAB389TTC to cultured primary neurons was very characteristic of TTC. DAB389TTC cytotoxicity was thus seen to be specific to cultured neuronal cells and was almost completely inhibited when the cells were coincubated with tetanus antitoxin. Because this neurotoxicity was only partially blocked when the cells J. Neurochem., Vol. 74, No. 6, 2000

FIG. 6. Comparative cytotoxicity of DAB389T TC and DA(E149S)B389T TC in cultures of primary neurons or N18-RE-105 cells. Cells were incubated overnight with various concentrations of either fusion protein before pulse labeling the next day with [14C]leucine. Data are mean ⫾ SEM (bars) values of three or four replicate cultures. Data presented for untreated control cells were derived from primary neurons. The SEM for untreated N18-RE-105 cells was 6%.

CHIMERIC DIPHTHERIA:TETANUS TOXIN main may be so substantial that one molecule of catalytic domain translocated to the cytosol could kill the cell (Yamaizumi et al., 1978). Although we did not specifically examine the internalization step of DAB389TTC intoxication, the observed cytotoxicity of the chimeric toxin toward cultured neuronal cells argues that the fusion toxin must have been internalized. This interpretation is consistent with our previous characterization of another TTC fusion protein, SOD:Tet451, which showed that SOD:Tet451 was internalized into N18-RE-105 cells in a manner similar to that of TTx (Francis et al., 1995). Moreover, based on the comparative cytotoxic potency and mechanism of action described for DTx, the ability of DAB389TTC to inhibit cellular protein synthesis at low picomolar concentrations indicates that the chimeric toxin is fully competent not only for internalization but also for membrane translocation and catalytic activity. This conclusion is further supported by our investigation of the membrane translocation and catalytic steps of DAB389TTC intoxication. Thus, the ability of chloroquine or monensin to reduce the cytotoxicity of DAB389TTC in N18-RE-105 cells is consistent with previous findings demonstrating the importance of endosomal acidification for translocation of the DTx catalytic domain into the cytosol (Draper and Simon, 1980; Sandvig and Olsnes, 1980; Fisher et al., 1996). The attenuated capacity of the DA(E149S)B389TTC mutant to inhibit neuronal protein synthesis compared with DAB389TTC additionally suggests that the cytotoxicity of the chimeric toxin was mediated by ADP-ribosyltransferase activity (Barbieri and Collier, 1987; Wilson et al., 1990). Taken together, these findings establish that the recombinant fusion of TTC to DAB389 does not interfere with the membrane translocation activity of the DAB389 moiety within neurons. In summary, our analysis of DAB389TTC strongly indicates that the chimeric toxin retains in full the neuronal binding/internalization properties of TTC as well as the membrane translocation and catalytic functions of DTx. The potent cytotoxic effect of DAB389TTC on primary neurons in vitro argues that a nontoxic derivative of this reagent may be a useful vehicle for delivering exogenous proteins to the cytosolic compartment of neurons. If DAB389TTC is found to be cytotoxic to motor neurons in vivo, local injections of the chimeric toxin into muscle might also provide a longer-lasting therapy for many of the same neuromuscular disorders currently treated with botulinum toxin A. We are presently investigating the biological activity of DAB389TTC in vivo to see whether the chimeric toxin retains the characteristic axonal and transsynaptic transport properties of TTC. Acknowledgment: We gratefully acknowledge Dr. Neil Fairweather of Imperial College for providing us with a semisynthetic cDNA clone of TTC. We also thank Dr. George Oyler for his helpful discussion, Debra DiGregorio for her assistance with computer graphics, and Richard Milanich for his help with digital imaging. J.W.F. is supported by the Families of Spinal Muscular Atrophy and grant R01 NS38679-01 from the Na-

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tional Institutes of Health. R.H.B., Jr. is supported by the Amyotrophic Lateral Sclerosis Association, Muscular Dystrophy Association, grants 1P01NS31248-02 and 5F32HS10064 from the National Institutes of Health, the Pierre L. deBourgknecht ALS Research Foundation, and the Myrtle May MacLellan Fund. M.A.S. and O.C. are supported by grant DA07496 from the National Institutes of Health. P.S.F., D.F., and M.P.R. are supported by grant 1P01AG12992-01 from the National Institute on Aging and a Merit Review Award from the Department of Veterans’ Affairs. J.C.V. and J.R.M. are supported in part by grant CA-60934 from the National Cancer Institute to J.R.M.

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