Journal of Protein Chemistry, Vol. 19, No. 8, 2000

6990277-8033/00/1100-0699$18.00/0 © 2000 Plenum Publishing Corporation

Recombinant Derivatives of Clostridial Neurotoxins asDelivery Vehicles for Proteins and Small Organic Molecules

Marina V. Zdanovskaia,1 Georgyi Los,1 and Alexey G. Zdanovsky1,2

Received December 6, 2000

Clostridial neurotoxins are the most powerful toxins known. Nevertheless, derivatives of these tox-ins may find broad applications both in science and medicine because of their unique abilities torecognize neurons and deliver small and large molecules into them. In this paper we describe theconstruction of two types of such derivatives. Proteins belonging to the first class were designed toallow direct conjugation with one or few molecules of interest. Proteins belonging to the secondclass contain biotin residue and therefore could be easily connected to streptavidin loaded with mul-tiple molecules of interest. Only C-terminal regions of neurotoxin heavy chains were incorporatedin the structure of recombinant proteins. Nevertheless, recombinant proteins were found to be ableto recognize specific neuronal receptors and target model molecules to rat synaptosomes and humanneuroblastoma cells.

KEY WORDS: Expression; clostridial neurotoxins; targeting; neurons.

1. INTRODUCTION

Tetanus toxin (TeNT)3 produced by Clostridium tetaniand seven serologically distinct botulinum neurotoxins(BoNT/A, /B, /C, /D, /E, /F, /G) produced by C. botu-linum, C. butyricum, C. argentinense,and C. barati arecausative agents of such life-threatening diseases as bot-ulism and tetanus. These homologous toxins are pro-duced by host bacteria cells in the form of singlepolypeptide chains of about 1300 amino acid residues inlength (Mr ≈ 150 kDa) (Binz et al., 1990; East et al.,1992; Eisel et al.,1986; Hauser et al.,1990; Poulet et al.,1992; Thompson et al.,1990, 1993). A single moleculeof each toxin possess three functional domains: receptor-recognizing, transport, and catalytic. As a result of lim-ited proteolysis conducted in the presence of reducingagents, a single polypeptide chain of each neurotoxin canbe converted in two chains: light (Mr ≈ 50 kDa) andheavy (Mr ≈ 100 kDa). Light chains correspond to thecatalytic domain. Catalytic domains of these toxins are

Zn2+ metalloproteases that recognize and selectivelycleave proteins involved in targeting of presynaptic vesi-cles and fusion of these vesicles with the neuronalplasma membrane (Schiavo et al., 1992a, b, 1993a, b).By this means neurotoxins block neurotransmitter re-lease into the synaptic cleft, resulting in muscle paraly-sis and eventually death in humans and animals due toasphyxiation.

Heavy chains of clostridial neurotoxins are respon-sible for recognition of specific receptors on the cell sur-face and for the transport of corresponding light chainsinto cytosol of neuronal cells. The opposite clinicalsymptoms of tetanus and botulism, spastic versus flaccidparalysis, are due to the different sites of action of thesetoxins. TeNT utilizes an existing intraaxonal transportmechanism to travel from the site of entry in the body tothe spinal cord where it is released from the postsynapticdendrites. The release is followed by an uptake into thepresynaptic terminals of the inhibitory interneurons, wherethe catalytic domain penetrates the cytosol and eventually

1 Promega Corporation, 2800 Woods Hollow Road, Madison, Wiscon-sin 53711-5399.

2 To whom correspondence should be addressed at e-mail: azdanovs@promega.com

3 Abbreviations: BoNT/A, /B, /C, /E, botulinum neurotoxins ofserotypes A, B, C, and E, respectively, TeNT tetanus toxin; RAP, re-ceptor-associated protein; SNAP25, synaptosomal-associated proteinof 25 kDa; SynB, synaptobrevin 2.

impairs the neuronal circuit that ensures balanced volun-tary muscle contraction (Schwab et al.,1979). In contrast,BoNTs act peripherally at the neuromuscular junction,blocking neurotransmitter release in the same neuron it en-ters. It was demonstrated that the C-terminal part of TeNTknown as tetanus fragment C (Mr ≈ 50kDa) possessesstructure required not only for neuronal uptake, but alsofor axonal transport of this protein (Bizzini et al., 1981;Buttner-Ennever et al.,1981; Figueiredo et al.,1997). Inaddition, it was recently shown that fragment C is capableof delivering DNA into the cytosol of neurons (Knightet al.,1999). Therefore it might possess structures capableof penetrating cell membrane. Because of these properties,fragment C of tetanus toxin as well as corresponding frag-ments of botulinum neurotoxins might find a broad appli-cation in science and in medicine. Indeed, these proteinscould be used as tags for visualization of cholinergic nerveterminals and as vehicles for targeted delivery of large andsmall molecules into neurons. The ability to produce theseproteins by recombinant methods would make productionof such proteins more robust and safe and thus would re-move the main obstacle preventing their regular use.

In this study we created derivatives of tetanus toxinand botulinum neurotoxin serotypes B and A specificallydesigned for use as delivery vehicles. A previously de-scribed method (Zdanovsky and Zdanovskaia, 2000) forproduction of clostridial proteins in E. coli was found tobe effective in the case of these derivatives also. Deriv-atives of tetanus toxin and botulinum neurotoxinserotypes A and B were accumulated in E. coli cells inthe form of inclusion bodies. Using a specially designedrenaturation procedure, we were able to recover solubleproteins that possessed the ability to recognize specificneuronal receptors and were able to target model com-pounds to human neuroblastoma cells.

2. MATERIALS AND METHODS

2.1. Bacterial Cells and Plasmids

Escherichia coliJM109 cells were used to propagateplasmids. E. coli strain BL21(λDE3) containing plasmidpACYC-IleArg10 (Zdanovsky and Zdanovskaia, 2000)was used for expression of recombinant proteins.

pGEM-T® Vector (Promega) was used to clonesequence amplified by PCR from E. coli chromosomeand encoding biotin ligase, BirA. PinPoint™ Vector(Promega) was used as source for the sequence encodingpeptide biotinated by BirA protein from E. coli.PlasmidspETBoNT/A-H1, pETBoNT/E-H10, pETBoNT/B-H18,pETTeNT-H4Km, pETBoNT/A-L22Km, and pET-

BoNT/B-L10 were constructed earlier (Zdanovsky andZdanovskaia, 2000) and encode heavy chains of botu-linum neurotoxin serotypes A, E, and B, heavy chain oftetanus toxin, and light chains of botulinum neurotoxinserotypes A and B, respectively.

2.2. Mammalian Cells

Human neuroblastoma cell line SH-SY5Y as wellas human adenocarcinoma cell line HeLa were obtainedfrom ATCC. Human neuroblastoma cells were main-tained in ATCC medium: a 1:1 mixture of Eagle’s min-imum essential medium with nonessential acids andHam’s F12K medium (90%) and fetal bovine serum(10%). For differentiation, cells were incubated for 3 daysin medium containing 1 µM of retinoic acid (Sigma).HeLa cells were maintained in RPMI medium containing5% of fetal bovine serum and mixture of antibiotics(penicillin, 100 u/ml; streptomycin, 100 u/ml; ampho-tericin B, 0.25 µg/ml).

2.3. DNA-Modifying Enzymes

Restriction enzymes BamH I, EcoR I, Hpa II, NcoI, Nde I, Nhe I, SnaB I, SpeI, and Xho I as well as T4DNA polymerase were obtained from Promega. RapidDNA Ligation Kit and Expand™ High Fidelity PCRSystem were supplied by Boehringer Mannheim.

2.4. Oligonucleotides

Oligonucleotides used for polymerase chain reac-tion as well as oligonucleotides used as adaptor forcloning are listed in Table I. All these oligonucleotideswere synthesized at Promega.

2.5. Nucleic Acids

Total DNAs from E. coli were purified according tothe procedure described earlier.

700 Zdanovskaia, Los, and Zdanovsky

Table I. Oligonucleotides Used

Name Sequence

CysLys-N 5′CATGAGCGCTTGCAAGGGCysLys-C 5′CATGCCCTTGCAAGCGCTBirA-3′ 5′GGGAATTCTTTTCTGCACTACGCAGGGATBirA-5′ 5′-AAAAAACCATGGGAATGAAGGATAACAC

CGTGCCACTGA

2.6. Polymerase Chain Reaction

PCR was performed using E. coli genome DNA;primers BirA-3′ and BirA-5′ (Table I) and Expand™High Fidelity PCR System were supplied by BoehringerMannheim.

2.7. Construction of Plasmids

pGEM-BirA5 was constructed by cloning a frag-ment amplified by PCR from E. coli chromosome andencoding biotin ligase (BirA) into the vector pGEM-T®.

pETBirA was constructed by substitution of thesmall Nco I–EcoR I fragment in the plasmid pET-BoNT/A-L22Km with the small Nco I–EcoRI fragmentfrom plasmid pGEM-BirA5.

pET-CysLysBoNT/E-H4 was constructed by inser-tion into the Nco I site of plasmid pETBoNT/E-H10 ofthe adaptor formed by oligonucleotides CysLys-N andCysLys-C (Table I).

pETBoNT/B-CH11 was constructed by connectingthe big Nhe I–Xho I fragment of plasmid pET-CysLys-BoNT/E-H4 with the small SpeI–XhoI fragment of plas-mid pETBoNT/B-H18.

pETbioBoNT/B-L11 was constructed by connect-ing the big Nde I–BamH I fragment of plasmid pET-BoNT/B-L10 with the small Nde I–BamH I fragment ofplasmid PinPoint.

pETbioBoNT/B-CH8 was constructed by connect-ing the small fragment of plasmid pETBoNT/B-H18generated by subsequent treatment of plasmid DNA withSpeI, T4 DNA-polymerase, and EcoRI with the big frag-ment of plasmid pETbioBoNT/B-L11 generated by sub-sequent treatment of plasmid DNA with SpeI, T4 DNApolymerase, and EcoR I.

pETbioTeNT-CH4 was generated by connecting thefragment of plasmid pETTeNT-H4Km generated by sub-sequent treatment of plasmid DNA with Hpa II and T4DNA polymerase with the big fragment of plasmid pET-bioBoNT/B-L11 generated by subsequent treatment ofplasmid DNA with SpeI, EcoR I, and T4 DNA poly-merase.

pETbioTeNT-CH9 was generated from the plasmidpETbioTeNT-CH4 by subsequent treatment of plasmidDNA with BamH I, T4 DNA polymerase, and poly-nucleotide ligase from bacteriophage T4.

pETBoNT/E-H32 was generated from plasmidpET-CysLysBoNT/E-H4 by subsequent treatment ofplasmid DNA with BamH I, NheI, T4 DNA polymerase,and polynucleotide ligase from bacteriophage T4.

pETBoNT/A-CH5 was constructed by connectingthe small SnaB I–Xho I fragment of plasmid pETBoNT/

A-H1 with the big fragment of plasmid pETBoNT/E-H32generated by subsequent treatment of plasmid DNA withBamH I, T4 DNA polymerase, and Xho I.

2.8. Expression and Purification of RecombinantProteins

When cell cultures were at an absorbency of 0.4–0.5(600 nm), protein expression was induced by the additionof IPTG. Cells were harvested 90 min later. Derivatives ofBoNT/A, BoNT/B, and TeNT were recovered by a three-step denaturation/renaturation procedure: (1) dissolving ofinclusion bodies in buffer containing 6 M guanidine hy-drochloride, 100 mM Tris-HCl, pH 8.0, 2 mM EDTA, and65 mM dithioerythrol, (2) renaturation in 100 mM Tris-HCl, 2 mM EDTA, 500 mM L-arginine, and 0.9 mM ox-idized glutathione, pH 7.0 for 24–36 hr (renaturation ofbotulinum neurotoxin derivatives was conducted in thepresence of trisialoganglioside T16), and (3) dialysisagainst buffer containing 20 mM Tris-HCl, pH 7.5, and100 mM urea. Soluble protein then was concentratedusing an Ultrafree-15 Centrifugal Filtration Device fromMillipore and was used for further manipulations.

2.9. Biotinylation of Recombinant Proteins In Vitro

Renatured bioBoNT/B-CH and bioTeNT-CH wereadded to reaction mixture containing 10 mM Tris-HCl,10 mM MgCl2, 150 mM KCl, pH 7.4, 100 µM ATP, 200µM biotin, and 0.06 mg/ml of biotin ligase at a concen-tration 0.4 mg/ml and the mixture was incubated for 1 hrat 37°C. After completion of the reaction biotin ligasewas removed by affinity chromatography on Ni-Sepharose, and ATP and biotin were removed by dialysisagainst buffer containing 10 mM Tris-HCl, pH 8.0, and1 mM EDTA. Proteins were then concentrated using anUltrafree-MC Centrifugal Filtration Device from Millipore.

2.10. Preparation of Synaptosomes

Rat cerebrocortical synaptosomes were prepared bydiscontinuous Percoll density gradient centrifugation ofan S1 fraction essentially as described (Dunkley et al.,1988; Harrison et al., 1988). Briefly, two adult maleSprague-Dawley rats (180–200 g) were killed by decap-itation. The brains were rapidly removed and dissectedin cold Krebs–Ringer buffer (containing 125 mM NaCl,3 mM KCl, 1.2 mM MgSO4, 1.2 mM CaCl2, 22 mMNaHCO3, 1 mM NaH2PO4, and 10 mM glucose). Beforeuse, buffer was gassed with a mixture of 95% O2 and 5%CO2 for 20 min and pH was adjusted to 7.4). Stedie-Riggs

Heavy Chains of Clostridial Neurotoxins as Delivery Vehicles 701

slices (0.5 mm thick) from the cerebral cortices of tworats were homogenized in 32 ml of chilled isotonic su-crose buffer (containing 0.32 M sucrose, 0.1 mM EDTA,1.0 mM dithiotreitol, and 5 mM HEPES, pH 7.4) at 4°C.The crude homogenate was centrifuged at 1000 × g for10 min (4°C). Eight ml of the supernatant (S1 fraction)was layered onto each of four discontinuous Percoll gra-dients, comprised of 10 ml each of 20%, 10%, and 6%Percoll (v/v) in isotonic sucrose buffer. The gradientswere centrifuged at 32,000 × g for 5 min (4°C). Synapto-somes were harvested at the interface between the 10%and 20% layers, pooled together, diluted in 20 ml ofisotonic sucrose buffer, and centrifuged at 15,000 × g for15 min (4°C). The loose pellet was resuspended in iso-tonic sucrose buffer, placed on ice, and used within 5 min.

2.11. Interaction of the Fragments of bioBoNT/B-CH and bioTeNT-CH with Synaptosomes

Ten µl of synaptosomes (10 µg of protein) was in-cubated with or without fragments of clostridial neuro-toxins in Krebs–Ringer bicarbonate buffer at 4°C or37°C in 2.0-ml tubes. Total volume of the mixture was45 µl. Concentrations of the fragments as well as in-cubation times are indicated in the figure legends. Insome experiments bovine serum albumin (BSA) wasadded to the incubation mixture (final concentration upto 100 µg/ml). To stop reaction, 1.7 ml of Krebs–Ringerbuffer was added to each tube, and synaptosomes werecollected by centrifugation of the mixture at 12,000 × gfor 15 sec. Supernatants were discarded and pellets wereresuspended in the SDS–PAGE sample buffer and usedfor Western blot analysis.

2.12. Interaction of Derivatives of TeNT withEukaryotic Cells

Biotinilated or conjugated fluorescein isothio-cyanate fragments of TeNT (3 µg) as well as the strepta-vidin-conjugated fluorescent dye Alexa488 (3 µg) wereadded to eukaryotic cells grown at 37°C in 96-wellplates. After incubation proteins not bound to cells wereremoved by three subsequent washes with fresh media.The cells then were examined by phase contrast and flu-orescence using an Axiovert 25 CFL inverted micro-scope (Carl Zeiss, Inc.).

2.13. SDS–PAGE

Proteins were separated using 4–20% gradient poly-acrylamide Tris-glycine gels from Invitrogen and either

were stained with Coomassie blue or were used for West-ern blot analysis.

2.14. Western Blot Analysis of Proteins

Electrophoretic transfer of proteins to the nitrocellu-lose membrane (0.2 µm, Schleicher & Schuell, Germany)or ImmobiloneP (Millipore) was carried out in 25 mMTris base/188 mM glycine (pH 8.3), 20% (v/v) methanolfor 2.5 hr with a constant current of 60 mA (at 4°C) inXcell II Blot Module from Invitrogen. The membraneswere rinsed with TBST buffer (10 mM Tris-HCl, 150 mMNaCl, pH 7.6, containing 0.05% Tween 20) and incubatedin blocking solution (1% BSA in TBST buffer) for 30 min.The membranes were washed with 50 ml of TBST bufferand incubated in TBST for 45 min with either horserad-ish-conjugated streptavidin or streptavidin-alkaline phos-phatase (Promega). Then membranes were washed (50 ml,5 min, three times) with TBST buffer and proteins werevisualized either by the enhanced chemiluminescence(ECL) system (Pharmacia-Amersham) or Western Blue®

stabilized substrate for alkaline phosphatase (Promega)according to manufacturer’s instructions. All procedureswere conducted at room temperature.

2.15. Protein Measurement

Protein was measured by the standard protocol ofthe Pierce BCA Protein Assay (Pierce Chemical Co.),using BSA as a standard.

2.16. Other Materials

Tetanus toxin C fragment–fluorescein isothio-cyanate conjugate was obtained from List BiologicalLaboratories, Inc. Streptavidin-Alexa Fluor™ 488 con-jugate was purchased from Molecular Probes. Mono-sialogangliosides GM1, GM2, and GM3, asialogangliosidesGM1 and GM2, disialogangliosides D1a, D1b, and D2, aswell as trisialoganglioside T1b were obtained fromSigma. Protein markers used were Mark12 wide-rangeprotein standard (myosin, 200 kDa; β-galactosidase,116.3 kDa; phosphorylase B, 97.4 kDa; bovine serumalbumin, 66.2 kDa; glutamate dehydrogenate, 55 kDa;lactate dehydrogenase, 36.5 kDa; carbonic anhydrase,31 kDa; soybean trypsin inhibitor, 21.5 kDa; lysozyme,14.4 kDa; and aprotinin, 6 kDa) and SeeBlue Pre-Stained Standard (myosin, 250 kDa; bovine serum albu-min, 98 kDa; alcohol dehydrogenase, 50 kDa; carbonicanhydrase, 36 kDa; myoglobin, 30 kDa; and lysozyme,16 kDa) from Invitrogen as well as midrange molecular

702 Zdanovskaia, Los, and Zdanovsky

weight markers from Promega (phosphorylase B, 97.4kDa; bovine serum albumin, 66.2 kDa; glutamate de-hydrogenate, 55 kDa; ovalbumin, 42.7 kDa; aldolase,40 kDa; carbonic anhydrase, 31 kDa; soybean trypsininhibitor, 21.5 kDa; and lysozyme, 14.4 kDa).

3. RESULTS AND DISCUSSION

3.1. Construction of Genes Encoding C-TerminalFragments of Clostridial Neurotoxins and TheirExpression in E. coli

In developing a targeting system for the delivery ofsmall and large molecules into neuronal cells we tookinto consideration two different scenarios. In some casesthe delivery of only few molecules of interest into neu-ronal cells may be required to achieve the desired effect.In these cases the molecules could be directly connectedto the targeting protein. In other situations a massive de-livery of molecules of interest into the neurons may benecessary. In this case direct connection of the moleculeswith the targeting protein will not be advisable becausemodification of the targeting protein at multiple sitesmight interfere with its functions. Instead, moleculescould be loaded onto a carrier protein that could belinked through a single linker to the targeting proteinwithout affecting its function. We decided to use C-ter-minal regions of clostridial neurotoxins as targeting pro-teins because of their previously described ability torecognize neuronal cells and deliver even large DNAmolecules into these cells. Using previously cloned frag-ments of clostridial DNA encoding heavy chains oftetanus toxin and botulinum neurotoxins type A and B,we assembled two types of constructions (for details seeSection 2). The first type of construction (plasmids pET-BoNT/A-CH5 and pETBoNT/B-CH11) encodes C-ter-minal fragments of BoNT/A and /B heavy chains thatcontain cysteine and lysine residues incorporated into itsN-terminal sequence. Located on a flexible and exposedsection of the protein, these residues may be used fordirect chemical conjugation of these proteins to the mol-ecules of interest. The second type of construction (plas-mids pETbioBoNT/B-CH8 and pETbioTeNT-CH9)encodes C-terminal fragments of BoNT/B and TeNT thatare fused at the N-terminus with the sequence that is rec-ognized by E. coli biotin ligase. Biotin ligase causesmodification of only one residue in the whole sequence.As a result proteins containing a single biotin moiety inthe area that is not important for receptor recognition andtransport may be easily connected with streptavidin thatis loaded with multiple molecules of interest.

Previously we found that amplification in the E.coli of genes for tRNAs recognizing ATA and AGAcodons rarely used in E. coli resulted in a substantial in-crease in the production of fragments of clostridial pro-teins in E. coli.Data presented in Fig. 1 demonstrate thatusing the same approach, we were able to produce de-rivatives of C-terminal fragments of BoNT/A, BoNT/B,and TeNT to the level of up to 10% of total cell protein.

3.2. Purification of BoNT/A, BoNT/B, and TeNTDerivatives

We determined that derivatives of C-terminal frag-ments of BoNT/A, BoNT/B, and TeNT were accumu-lated in E. coli cells in the form of inclusion bodies. Torecover proteins in the soluble form we used a denatura-tion/renaturation procedure that includes (1) dissolvinginclusion bodies in 6 M guanidinium hydrochloride, (2)a renaturation step that occurs in the buffer with 100-foldlower concentration of guanidinium hydrochloride, and(3) the dialysis step. This procedure was successfullyused before to recover enzymatically active light chainsof clostridial neurotoxins produced in E. coli (Zdanovskyand Zdanovskaia, 2000). Nevertheless, when used for re-covery of C-terminal fragments of clostridial neurotox-ins this procedure gave soluble product only in the caseof the TeNT derivative (bioTeNT-CH). Earlier it wasfound that C-terminal areas of botulinum neurotoxins, aswell as tetanus toxin, interact with gangliosides (Kita-mura et al., 1980; Kozaki et al., 1987; Morris et al.,1980; Rogers and Snyder, 1981). We speculated that ad-dition of gangliosides to the renaturation solution might

Heavy Chains of Clostridial Neurotoxins as Delivery Vehicles 703

Fig. 1. Expression of derivatives of clostridial neurotoxins in E. coli.Total cell extracts of E. coli BL21(DE3) (odd lanes) and BL21(DE3,pACYC-IleArg10) (even lanes) containing plasmids pETBoNT/E-H10(lanes 1 and 2), pETCysLysBoNT/E-H4 (lanes 3 and 4), pETBoNT/A-CH5 (lanes 5 and 6), pETBoNT/B-H18 (lanes 7 and 8),pETBoNT/B-CH11 (lanes 9 and 10), pETbioBoNT/B-CH8 (lanes 11and 12), and pETbioTeNT-CH9 (lanes 13 and 14) were separated on4–20% SDS–PAGE and proteins were visualized with Coomassie blue.Lane M1 correspond to Mark 12 wide-range protein standard fromNovex and lane C corresponds to total cell extract of E. coliBL21(DE3) that does not have any plasmid.

stimulate formation of the correct structure of the recep-tor-recognizing domain of clostridial neurotoxins andeventually might improve the yield of renatured recombi-nant proteins. We tested nine different gangliosides (seeSection 2). As shown in Fig. 2, both asialogangliosidesGM1 and GM2 failed to improve renaturation of BoNT/A-CH, BoNT/B-CH, and bioBoNT/B-CH (proteins encodedby plasmids pETBoNT/A-CH5, pETBoNT/B-CH11, andpETbioBoNT/B-CH8, respectively). Among the mono-sialogangliosides, GM1 was the best in helping to recoversoluble protein and the use of GM3 resulted in a loweryield of renatured protein than the use of GM2. Amongdisialogangliosides, D2 had some positive effect on re-naturation of BoNT/B derivatives but did not improve re-naturation of BoNT/A-CH, and D1a had a moderately

positive effect on recovery of soluble BoNT/A-CH, witha much stronger effect on recovery of BoNT/B deriva-tives. Disialoganglioside D1b as well as trisialoganglio-side T1b were the most potent in terms of efficientrecovery of both BoNT/A and BoNT/B derivatives in thesoluble form. These data seem to be in an agreementwith the previously reported data on affinity of botu-linum neurotoxins to di- and trisialogangliosides with thepreference for gangliosides of the 1b structure (Holm-gren et al.,1980; Kitamura et al.,1999; Rogers and Sny-der, 1981). Sialic acid residue was proposed previouslyand recently shown to be a component of ganglioside in-volved in interaction with neurotoxin (Swaminathan andEswaramoorthy, 2000) and was also found to be impor-tant for ganglioside-stimulated refolding of hybridproteins.

Similarly to BoNTs, tetanus toxin also interactswith gangliosides. Nevertheless, with one exception (ad-dition of asialoganglioside GM1 caused degradation ofbioTeNT-CH) gangliosides did not affect recovery ofbioTeNT-CH (Fig. 3), suggesting that the C-fragmentof TeNT is more prone to form the correct 3D structurethan the corresponding sequences of BoNTs. Because re-covery of botulinum neurotoxin derivatives was consis-tently better in the presence of trisialoganglioside T1b itwas used for the large-scale purification of these proteins.

Analysis of yields of recovered proteins revealedthat bioTeNT-CH and bioBoNT/B-CH were recoveredmore efficiently than BoNT/A-CH or BoNT/B-CH. Also,

704 Zdanovskaia, Los, and Zdanovsky

Fig. 2. Effect of gangliosides on renaturation of derivatives ofbotulinum neurotoxins. Inclusion bodies recovered from E. coli BL21(DE3, pACYC-IleArg10) containing plasmids pETBoNT/A-CH5 (A)or pETBoNT/B-CH11 (B) were renatured either without (lane 1) or inthe presence of gangliosides: asialoganglioside GM1 (lane 2),monosialoganglioside GM1 (lane 3), asialoganglioside GM2 (lane 4),monosialoganglioside GM2 (lane 5), monosialoganglioside GM3 (lane6), disialogangliosides D1a (lane 7), D1b (lane 8) and D2 (lane 9) aswell as trisialoganglioside T1b (lane 10). After completion ofrenaturation and dialysis soluble proteins were separated on 4–20%SDS-PAGE and proteins were visualized with Coomassie Blue. LaneM corresponds to mid-range molecular weight markers from Promega.

Fig. 3. Effect of gangliosides on renaturation of derivatives of tetanustoxins. Inclusion bodies recovered from E. coli BL21 (DE3, pACYC-IleArg10, pETbioTeNT-CH9) were renatured either without (lane 1)or in the presence of gangliosides: asialoganglioside GM1 (lane 2),monosialogangliosides GM1 (lane 3), GM2 (lane 4) and GM3 (lane 5),disialogangliosides D1a (lane 6), as well as trisialoganglioside T1b (lane7). After completion of renaturation and dialysis soluble proteins wereseparated on 4–20% SDS-PAGE and proteins were visualized withCoomassie Blue. Lane M corresponds to mid-range molecular weightmarkers from Promega.

the first two proteins were found to be more stabile, asthough peptide located on the N-termini of these proteinswould have some stabilizing effect. Preparations of theseproteins recovered after the renaturation procedure con-tained more full-size protein than preparations ofBoNT/A-CH and BoNT/B-CH. Therefore for furthertesting we used bioTeNT-CH and bioBoNT/B-CH.

3.3. Biotinylation of bioTeNT-CH and bioBoNT/B-CH

Our attempts to purify bioTeNT-CH and bioBoNT/B-CH using monomeric avidin covalently bound to arigid methacrylate matrix (SoftLink™ Soft ReleaseAvidin Resin, Promega) revealed that the majority of re-natured protein did not bind to the resin, suggesting thatthe majority of protein molecules did not have a biotinmoiety on them. Therefore we developed a procedure forbiotinylation of these proteins in vitro. For this purposewe have constructed a plasmid, pETBirA, which encodesbiotin ligase from E. coli (see Section 2). Protein en-coded by this plasmid was recovered from the solublefraction of E. coli cells using affinity chromatographyon Ni-Sepharose and in combination with biotin andATP was used for modification of bioTeNT-CH andbioBoNT/B-CH. Results of the in vitro biotinylation ex-periment are shown in Figure 4. In this experiment rena-tured bioBoNT/B-CH was incubated with biotin ligase,ATP, and biotin or without these compounds. After com-pletion of incubation subsequent twofold dilutions ofeach sample were separated by SDS–PAGE and wereused for Western blotting probed with streptavidine-alkaline phosphatase conjugate. In the case of proteintreated with biotin ligase we could detect bioBoNT/B-CH in samples fourfold more dilute than in the case ofprotein not treated with biotin ligase. Therefore at least75% of renatured bioBoNT/B-CH originally did not con-tain biotin. Similar results were obtained in the case ofbioTeNT-CH (data not presented).

3.4. Analysis of Targeting Properties of bioBoNT/B-CH and bioTeNT-CH

To ascertain whether renatured and biotinilatedbioBoNT/B-CH and bioTeNT-CH retained biologicalactivity, both proteins were tested for binding to rat brainsynaptosomes. In this experiment initially solublebioBoNT/B-CH and bioTeNT-CH were mixed togetherwith synaptosomes. After 15 min of incubation synapto-somes were separated from soluble proteins and spectraof proteins associated with synaptosomes and capable of

interacting with streptavidin were analyzed by Westernblot. As shown in Fig. 5, this procedure has allowed us torecover biotinylated fragments of both botulinum neuro-toxin and tetanus toxin from synaptosome-containingfractions. Binding of bioBoNT/B-CH and bioTeNT-CHwith synaptosomes could be a result of nonspecific in-teraction with synaptosomal membrane (e.g., absorption)or highly specific interaction with receptors. In our ex-periments, addition of BSA to the reaction mixtures inconcentrations 100-fold higher (10 mg/ml) than concen-trations of recombinant proteins used for binding had noeffect on the binding of either bioBoNT/B-CH or bio-TeNT-CH (data not shown). Additionally, preparationsof bioBoNT/B-CH and bioTeNT-CH used for coincuba-tion with synaptosomes in addition to full-size proteinsalso contained several fragments. Nevertheless, not allfragments present in the original preparations werefound in the synaptosome-containing pellet. In the caseof bioBoNT/B-CH only full-size protein was detected in

Heavy Chains of Clostridial Neurotoxins as Delivery Vehicles 705

Fig. 4. In vitro biotinylation of bioBoNT/B-CH. bioBoNT/B-CH wasincubated alone (A) or in the presence of biotin, ATP and biotin-ligase(B) for 1h at 37ºC in the reaction buffer (see Materials and Methods).After completion of the incubation subsequent two-fold dilutions ofeach mixture (lanes 1–11) were separated on 4–20% SDS-PAGE,transferred on ImmobiloneP membrane and biotin-containing proteinswere visualized with streptavidin-alkaline phosphatase conjugate. LaneM corresponds to SeeBlue Pre-Stained Standard from Invitrogen.

the synaptosome-containing pellet. In the case of bio-TeNT-CH, in addition to the full-size recombinant pro-tein we found that the synaptosome-containing pelletalso carried fragments that contained biotin and thereforecorrespond to the N-terminal region of the recombinantprotein. Finding of such fragments in association withsynaptosomes is in agreement with the previously re-ported data that the N-terminal half of tetanus toxin frag-ment C is able to interact with the cell surface (Herreroset al., 2000). Thus, data suggest that the interaction wehave observed between recombinant proteins and synap-tosomes was specific. To further examine the question ofspecificity we tested the interaction of bioTeNT-CH withdifferentiated and nondifferentiated human neuroblas-toma cells as well as with HeLa cells. Data presented inFig. 6 demonstrate that we were able to label differenti-ated neuroblastoma cells using a complex formed by bio-TeNT-CH and streptavidin conjugated with fluorescentdye Alexa488. The signal generated was relatively weak,requiring several hours of incubation for complete de-velopment. Nevertheless, the intensity and the pattern ofthe signal obtained with complex formed by bioTeNT-CH and streptavidin conjugated with fluorescent dyeAlexa488 were very similar to those generated when weused for labeling C-fragment from native TeNT that wasconjugated with fluorescein isothiocyanate. Additionally,we did not observe fluorescence associated with cells

when nondifferenciated neuroblastoma or HeLa cellswere incubated with both bioTeNT and Alexa488-labeled streptavidin or when differentiated neuroblas-toma cells were incubated with Alexa488-labeledstreptavidin alone. Therefore, one may conclude that theobserved targeting of streptavidin and associated fluro-phore molecules to differentiated neuroblastoma cells wasspecific and was mediated by the recombinant protein.

In summary, we demonstrated that receptor-recog-nizing domains of clostridial neurotoxins could be effi-ciently produced in E. coli. Also, using recombinantlyproduced receptor-recognizing domains of clostridialneurotoxins, we developed a new system that could beused for targeted delivery of small and large moleculesto neuronal tissue.

706 Zdanovskaia, Los, and Zdanovsky

Fig. 5. Interaction of bioTeNT-CH with rat cerebrocortical synap-tosomes. Rat synaptosomes were incubated alone (lane, 1 and 5) or inthe presence of either bioBoNT/B-CH (lanes 3 and 4) or bioTeNT-CH(lanes 7 and 8). After 15 min of incubation at 37°C synaptosomes wereisolated by centrifugation (lanes 4 and 8). Proteins were separated on4–20% SDS–PAGE, transferred on a nitrocellulose membrane, andprobed with streptavidin–horseradish peroxidase conjugate as describedin Section 2. Lanes 2 and 6 correspond to original preparations ofbioBoNT/B-CH and bioTeNT-CH, respectively.

Fig. 6. Interaction of bioTeNT-CH with human cells. Differentiatedhuman neuroblastoma cells (A, B, and C) and HeLa cells (D) wereincubated for 10 hr at 37°C in the presence of bioTeNT-CH and strep-tavindin–Alexa488 conjugate (A and D), tetanus toxin Cfragment–fluorescein isothiocyanate conjugate (B), orstreptavindin–Alexa488 conjugate (C). Each row of picturescorresponds to the image of the same area. Left column contains phaseand right column contains fluorescent microphotographs.

ACKNOWLEDGMENT

We thank Dr. Kari Kenefick (Promega Corporation)for a critical review of the manuscript.

REFERENCES

Binz, T., Kurazono, H., Popoff, M. R., Eklund, M. W., Sakaguchi, G.,Kozaki, S., Krieglstein, K., Henschen, A., Gill, D. M., and Nie-mann, H. (1990). Nucleic Acids Res.18, 5556.

Bizzini, B., Grob, P., and Akert, K. (1981). Brain Res.210, 291–299.Buttner-Ennever, J. A., Grob, P., Akert, K., and Bizzini, B. (1981).

Neurosci. Lett.26, 233–238.Dunkley, P. R., Heath, J. W., Harrison, S. M., Jarvie, P. E., Glenfield,

P. J., and Rostas, J. A. (1988). Brain Res.441, 59–71.East, A. K., Richardson, P. T., Allaway, D., Collins, M. D., Roberts,

T. A., and Thompson, D. E. (1992). FEMS Microbiol. Lett.75,225–230.

Eisel, U., Jarausch, W., Goretzki, K., Henschen, A., Engels, J., Weller,U., Hudel, M., Habermann, E., and Niemann, H. (1986). EMBOJ. 5, 2495–2502.

Figueiredo, D. M., Hallewell, R. A., Chen, L. L., Fairweather, N. F.,Dougan, G., Savitt, J. M., Parks, D. A., and Fishman, P. S. (1997).Exp. Neurol.145, 546–554.

Harrison, S. M., Jarvie, P. E., and Dunkley, P. R. (1988). Brain Res.441, 72–80.

Hauser, D., Eklund, M. W., Kurazono, H., Binz, T., Niemann, H., Gill,D. M., Boquet, P., and Popoff, M. R. (1990). Nucleic Acids Res.18, 4924.

Herreros, J., Lalli, G., and Schiavo, G. (2000). Biochem. J.347(1),199–204.

Holmgren, J., Elwing, H., Fredman, P., and Svennerholm, L. (1980).Eur. J. Biochem.106, 371–379.

Kitamura, M., Iwamori, M., and Nagai, Y. (1980). Biochim. Biophys.Acta628, 328–335.

Kitamura, M., Takamiya, K., Aizawa, S., and Furukawa, K. (1999).Biochim. Biophys. Acta1441, 1–3.

Knight, A., Carvajal, J., Schneider, H., Coutelle, C., Chamberlain, S.,and Fairweather, N. (1999). Eur. J. Biochem.259, 762–769.

Kozaki, S., Ogasawara, J., Shimote, Y., Kamata, Y., and Sakaguchi, G.(1987). Infect Immun.55, 3051–3056.

Morris, N. P., Consiglio, E., Kohn, L. D., Habig, W. H., Hardegree,M. C., and Helting, T. B. (1980). J. Biol. Chem.255, 6071–6076.

Poulet, S., Hauser, D., Quanz, M., Niemann, H., and Popoff, M. R.(1992). Biochem. Biophys. Res. Commun.183, 107–113.

Rogers, T. B. and Snyder, S. H. (1981). J. Biol. Chem. 256,2402–2407.

Schiavo, G., Benfenati, F., Poulain, B., Rossetto, O., Polverino de Lau-reto, P., DasGupta, B. R., and Montecucco, C. (1992a). Nature359, 832–835.

Schiavo, G., Poulain, B., Rossetto, O., Benfenati, F., Tauc, L., andMontecucco, C. (1992b). EMBO J.11, 3577–3583.

Schiavo, G., Rossetto, O., Catsicas, S., Polverino de Laureto, P., Das-Gupta, B. R., Benfenati, F., and Montecucco, C., (1993a). J. Biol.Chem.268, 23784–23787.

Schiavo, G., Santucci, A., Dasgupta, B. R., Mehta, P. P., Jones, J., Ben-fenati, F., Wilson, M. C., and Montecucco, C. (1993b). FEBSLett. 335, 99–103.

Schwab, M. E., Suda, K., and Thoenen, H. (1979). J. Cell Biol. 82,798–810.

Swaminathan, S. and Eswaramoorthy, S. (2000). Nature Struct. Biol.7,693–699.

Thompson, D. E., Brehm, J. K., Oultram, J. D., Swinfield, T. J., Shone,C. C., Atkinson, T., Melling, J., and Minton, N. P. (1990). Eur. J.Biochem.189, 73–81.

Thompson, D. E., Hutson, R. A., East, A. K., Allaway, D., Collins,M. D., and Richardson, P. T. (1993). FEMS Microbiol. Lett.108,175–182.

Zdanovsky, A. G. and Zdanovskia, M. V. (2000). Appl. Environ. Mi-crobiol. 66, 3166–3173.

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