FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1993581461> ?p ?o ?g. }

• W1993581461 endingPage "17002" @default.
• W1993581461 startingPage "16993" @default.
• W1993581461 abstract "Hyperexcitability disorders of cholinergically innervated muscles are treatable with botulinum neurotoxin (BoNT) A. The seven serotypes (A–G) potently block neurotransmission by binding to presynaptic receptors, undergoing endocytosis, transferring to the cytosol, and inactivating proteins essential for vesicle fusion. Although BoNT/A and BoNT/E cleave SNAP-25, albeit at distinct sites, BoNT/E blocks neurotransmission faster and more potently. To identify the domains responsible for these characteristics, the C-terminal heavy chain portions of BoNT/A and BoNT/E were exchanged to create chimeras AE and EA. After high yield expression in Escherichia coli, these single chain chimeras were purified by two-step chromatography and activated by conversion to disulfide-linked dichains. In vitro, each entered neurons, cleaved SNAP-25, and blocked neuromuscular transmission while causing flaccid paralysis in vivo. Acidification-dependent translocation of the light chain to the cytosol occurred more rapidly for BoNT/E and EA than for BoNT/A and AE because the latter pair remained susceptible for longer to inhibitors of the vesicular proton pump, and BoNT/A proved less sensitive. The receptor-binding and protease domains do not seem to be responsible for the speeds of intoxication; rather the N-terminal halves of their heavy chains are implicated, with dissimilar rates of cytosolic transfer of the light chains being due to differences in pH sensitivity. AE produced the most persistent muscle weakening and therefore has therapeutic potential. Thus, proof of principle is provided for tailoring the pharmacological properties of these toxins by protein engineering. Hyperexcitability disorders of cholinergically innervated muscles are treatable with botulinum neurotoxin (BoNT) A. The seven serotypes (A–G) potently block neurotransmission by binding to presynaptic receptors, undergoing endocytosis, transferring to the cytosol, and inactivating proteins essential for vesicle fusion. Although BoNT/A and BoNT/E cleave SNAP-25, albeit at distinct sites, BoNT/E blocks neurotransmission faster and more potently. To identify the domains responsible for these characteristics, the C-terminal heavy chain portions of BoNT/A and BoNT/E were exchanged to create chimeras AE and EA. After high yield expression in Escherichia coli, these single chain chimeras were purified by two-step chromatography and activated by conversion to disulfide-linked dichains. In vitro, each entered neurons, cleaved SNAP-25, and blocked neuromuscular transmission while causing flaccid paralysis in vivo. Acidification-dependent translocation of the light chain to the cytosol occurred more rapidly for BoNT/E and EA than for BoNT/A and AE because the latter pair remained susceptible for longer to inhibitors of the vesicular proton pump, and BoNT/A proved less sensitive. The receptor-binding and protease domains do not seem to be responsible for the speeds of intoxication; rather the N-terminal halves of their heavy chains are implicated, with dissimilar rates of cytosolic transfer of the light chains being due to differences in pH sensitivity. AE produced the most persistent muscle weakening and therefore has therapeutic potential. Thus, proof of principle is provided for tailoring the pharmacological properties of these toxins by protein engineering. Seven serotypes (A–G) of botulinum neurotoxin (BoNT), 2The abbreviations used are: BoNT, botulinum neurotoxin; DC, dichain; HC, C-terminal half; HC, heavy chain; LC, light chain; SC, single chain; HN, N-terminal half; BafA1, bafilomycin A1; ConA, concanamycin A; IMAC, immobilized metal affinity chromatography; BisTris, 2-[bis(2-hydroxyethyl)-amino]-2-(hydroxymethyl)propane-1,3-diol; GFP, green fluorescent protein; DTT, dithiothreitol; DAS, digit abduction score; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; CGNs, cerebellar granule neurons; SCGNs, superior cervical ganglion neurons. 2The abbreviations used are: BoNT, botulinum neurotoxin; DC, dichain; HC, C-terminal half; HC, heavy chain; LC, light chain; SC, single chain; HN, N-terminal half; BafA1, bafilomycin A1; ConA, concanamycin A; IMAC, immobilized metal affinity chromatography; BisTris, 2-[bis(2-hydroxyethyl)-amino]-2-(hydroxymethyl)propane-1,3-diol; GFP, green fluorescent protein; DTT, dithiothreitol; DAS, digit abduction score; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; CGNs, cerebellar granule neurons; SCGNs, superior cervical ganglion neurons. dichain (DC) proteins (∼150 kDa) from Clostridium botulinum, selectively bind and enter certain nerve terminals. They potently block the exocytosis of neurotransmitters by cleaving proteins that are essential for the fusion of synaptic vesicles with the presynaptic membrane: SNAP-25 (synaptosome-associated protein of 25 kDa; BoNT/A, BoNT/E, and BoNT/C1), vesicle-associated membrane protein (BoNT/B, BoNT/D, BoNT/F, and BoNT/G), and syntaxin (a second substrate of BoNT/C1) (reviewed in Refs. 1Dolly J.O. Lawrence G. Ward A.B. Barnes M.P. Clinical Uses of Botulinum Toxins. University Press, Cambridge, Cambridge, UK2007: 9-25Crossref Scopus (3) Google Scholar and 2Dolly J.O. de Paiva A. Foran P. Lawrence G. Daniels-Holgate P. Ashton A.C. Semin. Neurosci. 1994; 6: 149-158Crossref Scopus (55) Google Scholar). Potent and preferential blockade by the toxins of cholinergic synaptic transmission in the peripheral nervous system underlies the clinical symptoms of botulism, which include dry mouth, ptosis, difficulty in swallowing, and respiratory failure (3Arnon S.S. Brin M.F. Hallet M. Jankovic J Scientific and Therapeutic Aspects of Botulinum Toxin. Lippincott Williams & Wilkins, Philadelphia2002: 145-150Google Scholar). However, these same properties have proven most advantageous for the successful treatment, particularly with the BoNT/A complex, of a wide variety of neurogenic hyperactivity disorders such as dystonias, dysphonias, spasticity, overactive bladder, hyperhydrosis, hypersalivation, and certain types of headache (4Ward A.B. Barnes M.P. Clinical Uses of Botulinum Toxins. University Press, Cambridge, Cambridge, UK2007: 1-384Crossref Scopus (0) Google Scholar). BoNT/A gains access to overactive nerve endings by binding with high affinity, via the C-terminal half (HC) of its heavy chain (HC; ∼100 kDa), to a lumenal domain of SV2A/SV2B/SV2C (synaptic vesicle protein 2), which is exposed at the cell surface after exocytotic fusion of synaptic vesicles (5Dong M. Yeh F. Tepp W.H. Dean C. Johnson E.A. Janz R. Chapman E.R. Science. 2006; 312: 592-596Crossref PubMed Scopus (617) Google Scholar, 6Mahrhold S. Rummel A. Bigalke H. Davletov B. Binz T. FEBS Lett. 2006; 580: 2011-2014Crossref PubMed Scopus (275) Google Scholar). An intravesicular region of synaptotagmin (I and II), another synaptic vesicle protein, acts as an avid receptor for BoNT/B and BoNT/G (7Nishiki T. Tokuyama Y. Kamata Y. Nemoto Y. Yoshida A. Sato K. Sekiguchi M. Takahashi M. Kozaki S. FEBS Lett. 1996; 378: 253-257Crossref PubMed Scopus (215) Google Scholar, 8Dong M. Richards D.A. Goodnough M.C. Tepp W.H. Johnson E.A. Chapman E.R. J. Cell Biol. 2003; 162: 1293-1303Crossref PubMed Scopus (264) Google Scholar, 9Rummel A. Karnath T. Henke T. Bigalke H. Binz T. J. Biol. Chem. 2004; 279: 30865-30870Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). BoNTs also interact with gangliosides with lower affinity, and the latter stabilize binding of the toxins to their respective protein receptors (10Rummel A. Eichner T. Weil T. Karnath T. Gutcaits A. Mahrhold S. Sandhoff K. Proia R.L. Acharya K.R. Bigalke H. Binz T. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 359-364Crossref PubMed Scopus (160) Google Scholar, 11Montecucco C. Rossetto O. Schiavo G. Trends Microbiol. 2004; 12: 442-446Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The distinct protein receptors (2Dolly J.O. de Paiva A. Foran P. Lawrence G. Daniels-Holgate P. Ashton A.C. Semin. Neurosci. 1994; 6: 149-158Crossref Scopus (55) Google Scholar) for the other BoNT serotypes remain to be identified. As synaptic vesicle proteins are recovered from the plasmalemma, BoNTs are carried into the lumen of recycling vesicles, and acidification triggers the translocation of their light chain (LC; ∼50 kDa) into the presynaptic cytosol. A basis for a disulfide link between the LC and HC being required for uptake (12de Paiva A. Poulain B. Lawrence G.W. Shone C.C. Tauc L. Dolly J.O. J. Biol. Chem. 1993; 268: 20838-20844Abstract Full Text PDF PubMed Google Scholar) has been demonstrated; a membrane-spanning channel formed by the HC mediates translocation of the LC, which is released upon reduction in the cytosol (13Schmid M.F. Robinson J.P. DasGupta B.R. Nature. 1993; 364: 827-830Crossref PubMed Scopus (84) Google Scholar, 14Fischer A. Montal M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 10447-10452Crossref PubMed Scopus (138) Google Scholar). Notably, BoNT/E enters cultured neurons more quickly than BoNT/A (15Keller J.E. Cai F. Neale E.A. Biochemistry. 2004; 43: 526-532Crossref PubMed Scopus (149) Google Scholar), has a higher potency, and acts faster to inhibit transmission at the neuromuscular junction (16Simpson L.L. J. Pharmacol. Exp. Ther. 1980; 212: 16-21PubMed Google Scholar, 17Simpson L.L. DasGupta B.R. J. Pharmacol. Exp. Ther. 1983; 224: 135-140PubMed Google Scholar, 18Lawrence G. Wang J. Chion C.K. Aoki K.R. Dolly J.O. J. Pharmacol. Exp. Ther. 2007; 320: 410-418Crossref PubMed Scopus (28) Google Scholar). Furthermore, BoNT/E totally inhibits vesicle fusion more akin to the effects of vesicle-associated membrane protein- or syntaxin-cleaving BoNTs than the reduction in fusion rate induced by BoNT/A (19Xu T. Binz T. Niemann H. Neher E. Nat. Neurosci. 1998; 1: 192-200Crossref PubMed Scopus (289) Google Scholar, 20Sakaba T. Stein A. Jahn R. Neher E. Science. 2005; 309: 491-494Crossref PubMed Scopus (121) Google Scholar). Moreover, the blockade of transmitter release by BoNT/E is relatively unaffected by treatments that elevate cytosolic [Ca2+] and antagonize inhibition by BoNT/A (20Sakaba T. Stein A. Jahn R. Neher E. Science. 2005; 309: 491-494Crossref PubMed Scopus (121) Google Scholar, 21Gerona R.R. Larsen E.C. Kowalchyk J.A. Martin T.F. J. Biol. Chem. 2000; 275: 6328-6336Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 22Cull-Candy S.G. Lundh H. Thesleff S. J. Physiol. (Lond.). 1976; 260: 177-203Crossref Scopus (159) Google Scholar, 23Meunier F.A. Lisk G. Sesardic D. Dolly J.O. Mol. Cell. Neurosci. 2003; 22: 454-466Crossref PubMed Scopus (131) Google Scholar, 24Lawrence G.W. Foran P. Mohammed N. DasGupta B.R. Dolly J.O. Biochemistry. 1997; 36: 3061-3067Crossref PubMed Scopus (48) Google Scholar). All these properties would be highly desirable for incorporation into new and improved therapeutic versions of BoNT, although BoNT/E produces transient muscle weakness compared with the long duration of action of BoNT/A (25Eleopra R. Tugnoli V. Rossetto O. De Grandis D. Montecucco C. Neurosci. Lett. 1998; 256: 135-138Crossref PubMed Scopus (164) Google Scholar). Thus, the feasibility of such an approach was assessed. Herein, chimeric BoNTs were constructed by recombinant substitution of the HC domains in BoNT/E and BoNT/A with their counterpart region from the other serotype and expressed as His6-tagged single chain (SC) proteins in Escherichia coli. The resultant chimeras could be converted to their disulfide-linked DC form by controlled proteolysis, which also removed the tag. The EA chimera, containing the LC and the N-terminal half (HN) of the HC of BoNT/E fused to the HC domain of BoNT/A, was shown to enter cultured neurons almost as rapidly as BoNT/E and blocked neuromuscular transmission with equipotency, although paralysis was transient (as for BoNT/E). Its speedy internalization into neurons could be attributed to an elevated sensitivity to pH of the BoNT/E LC-HN portion that facilitates expedient cytosolic transfer of the proteolytic LC rather than to the receptor utilized. On the other hand, chimera AE (the LC and HN domain of BoNT/A fused to the HC domain of BoNT/E) was less potent than EA or BoNT/E and much slower to enter neurons, but produced prolonged muscle weakness in mouse, matching that induced by clinical preparations of type A toxin-hemagglutinin complex. As AE should utilize a different receptor than BoNT/A for neuronal entry, it could be a useful alternative for clinical application in the minority of patients who are primary non-responders to the BoNT/A complex. Materials—The following materials were obtained as indicated; the pET29a expression vector, Novagen; E. coli BL21(DE3), Stratagene; restriction enzymes, New England Biolabs; TALON Superflow resin, Clontech; and UNO S1 and Q1 columns, Bradford protein assay kit, and Prescision Plus™ all blue protein standards, Bio-Rad. AccuPrime™ Pfx DNA polymerase, precast gels, and reagents for SDS-PAGE were supplied by Invitrogen. PD-10 desalting columns were provided by GE Healthcare. Substrates for ECL detection of horseradish peroxidase were purchased from Millipore. Antibodies were from Sigma (rabbit anti-SNAP-25, anti-HPC-1, and anti-IgG conjugated to either horseradish peroxidase or alkaline phosphatase), Sternberger Monoclonals (SMI-81), and Novagen (anti-His6). Custom antibodies specific for LC/A, LC/E, HC/E, or BoNT/A were prepared by Zymed Laboratories Inc. Purified natural BoNTs were purchased from List Laboratories (BoNT/A, DC form) and Metabiologics (BoNT/E, SC form). The latter was proteolytically nicked to the DC (>95%) with TrypZean (8 μg/mg of BoNT; Sigma) for 40 min at 27 °C. Bafilomycin A1 (BafA1) was supplied by LC Laboratories (Woburn, MA). Concanamycin A (ConA) and all other reagents were from Sigma. Mice were obtained from Charles River Laboratories (C57) and Harlan UK (female Tyler's Ordinary); Sprague-Dawley rats were bred in an approved animal unit at Dublin City University. All experimental procedures involving animals were approved by an Institutional Ethics Committee and licensed by the Irish Government, Department of Health and Children. Generation of Constructs for BoNT AE and EA Chimeras: Their Expression, Purification, and Characterization—The cloning and expression of BoNTs were performed in accordance with European Union regulations, registered in Ireland with the Environmental Protection Agency, and notified to the Health and Safety Authority. Genes encoding either BoNT/A or BoNT/E SCs were codon optimized for expression in E. coli and synthesized, and the sequences were verified. 3K. R. Aoki, unpublished data. For constructing chimera AE (see Fig. 1), a fragment encoding the LC plus the translocation domain (HN) of BoNT/A (LC-HN/A) and that encoding the binding domain of BoNT/E (HC/E) were amplified by standard PCR using a high fidelity polymerase. The products were cloned into the pET29a vector using suitable restrictions sites to generate the AE chimera (Fig. 1). Nucleotides encoding a nine-residue linker were inserted between the translocation and binding domains; also, modification of the original vector sequence introduced trypsin cleavage sites between HC/E and the C-terminal six histidines (His6) (see Fig. 1). For constructing chimera EA, a fragment encoding the binding domain of BoNT/A (HC/A) was likewise generated, and nucleotides were added to encode a trypsin cleavage site at its C terminus. This fragment, as well as a separate DNA sequence encoding the LC plus the translocation domain of BoNT/E (LC-HN/E), was amplified by PCR and cloned into pET29a to create an expression vector containing the EA insert (see Fig. 1). After DNA sequence verification, each new SC gene was transformed into E. coli BL21, and expression was elicited by autoinduction (26Studier F.W. Protein Expression Purif. 2005; 41: 207-234Crossref PubMed Scopus (4107) Google Scholar); optimal incubation periods and temperatures were determined empirically. Cells were then pelleted, washed, and lysed using lysozyme and several freeze/thaw cycles; insoluble material was removed by centrifugation. The AE SC was trapped by immobilized metal affinity chromatography (IMAC) on TALON resin, eluted with 500 mm imidazole, and transferred into 50 mm Tris-HCl buffer (pH 8.1) using Sephadex G-25. For further purification, the sample was loaded onto a UNO Q1 column, and after washing with 30 mm NaCl, a stepwise gradient up to 1 m NaCl in 50 mm Tris-HCl buffer was applied. Fractions containing the eluted SC toxin were pooled and either stored at –80 °C or proteolytically nicked by TrypZean (1 μg/mg of BoNT) for 1 h at 25 °C before the addition of trypsin inhibitor (10 μg/mg of toxin) and storage of the DC (as for the SC). The expression and affinity purification of the chimera EA SC were performed similarly. The pooled eluate from IMAC was gel-filtered into 0.02 m sodium phosphate buffer (pH 6.5) and further purified by loading onto a UNO S1 column, followed by washing with 150 mm NaCl and elution with a stepwise gradient up to 1 m NaCl in 0.02 m sodium phosphate buffer. After transfer of the eluted toxin into 50 mm HEPES and 145 mm NaCl (pH 7.4) on Sephadex G-25, purified SC toxin was either stored at –80 °C or nicked by TrypZean (8 μg/mg of toxin) for 1 h at 25 °C before storage. The expression, purification, and nicking of the chimeras were monitored by SDS-PAGE on precast BisTris gels after heating in lithium dodecyl sulfate sample buffer to 80 °C for 5 min. Gels were stained with Coomassie Blue or electrotransferred to polyvinylidene difluoride membranes, probed by Western blotting with primary antibodies (as specified in the figure legends) followed by horseradish peroxidase-conjugated anti-species secondary antibodies, and visualized using an ECL protocol. The protein concentrations of SC and DC samples were determined using the Bradford assay. Note that, except where stated otherwise, all functional assays were performed on the DC. Measurements of the Toxins' Protease Activities—A model substrate was created for assay of SNAP-25 cleavage by BoNTs and chimeras (27Williams D. Steward L.E. Gilmore M.A. Okawa Y. Webber J.A. Aoki K.R. Neurotox. Res. 2006; 9: 239Google Scholar). DNA encoding green fluorescent protein (GFP) was fused to nucleotides encoding the 73 C-terminal residues of mouse SNAP-25 and a His6 tag. The GFP-SNAP-25-(134–206)-His6 fusion protein was expressed in E. coli and purified by IMAC. Toxins were diluted to 100 or 200 nm in HBS-20 (20 mm HEPES, 100 mm NaCl (pH 7.4), 10 μg/ml bovine serum albumin, 5 mm dithiothreitol (DTT), and 10 μm ZnCl2) and incubated for 30 min at 37 °C (to activate the protease) before serial dilution (typically ranging from 0.003 to 100 nm) in HBS-20 and mixing with GFP-SNAP-25-(134–206)-His6 (13.5 μm final concentration). After an additional 30 min at 37 °C, cleavage reactions were stopped by the addition of 8 m guanidine HCl, loaded onto 50 μl of TALON resin, and incubated for 15 min in the dark at room temperature. After washing, the amount of GFP-peptide fragment in fractions not bound to the resin was determined using a Synergy HT fluorometer (λex = 485/20 nm, λem = 528/20 nm) and quantified with Gen5 (both from BioTek Instruments). Data were plotted as fluorescence intensity against the log of toxin concentration and fitted with sigmoidal curves, and the amount of toxin required to cleave 10% of the substrate was interpolated. Assessment of the Neuromuscular Paralytic Activity and Lethality of BoNTs—Mouse hemidiaphragms were bathed at 35 °C in Krebs-Ringer buffer (containing 0.1% (w/v) bovine serum albumin and continuously aerated with 95% O2 and 5% CO2). Muscle contractions were elicited by electrical stimulation (50-ms square pulses of 5–10 V at 0.2 Hz) of the nerve and measured using FORT25 isometric force transducers (World Precision Instruments) as described previously (18Lawrence G. Wang J. Chion C.K. Aoki K.R. Dolly J.O. J. Pharmacol. Exp. Ther. 2007; 320: 410-418Crossref PubMed Scopus (28) Google Scholar). BoNTs were added directly to the Krebs-Ringer buffer, and the time taken for a 90% reduction in muscle tension was recorded. BoNT-induced neuromuscular paralysis in vivo was monitored by the digit abduction score (DAS) assay (28Aoki K.R. Toxicon. 2001; 39: 1815-1820Crossref PubMed Scopus (171) Google Scholar). The maximal tolerated dose was determined for each neurotoxin; this is the largest dose that could be injected into the gastrocnemius muscle without producing systemic symptoms (e.g. complete immobilization of the injected leg, lower abdomen contraction, and subsequent general paralysis). Mice received single 5-μl injections into the right gastrocnemius muscle of a maximal tolerated dose of toxin diluted in sterile normal saline (0.9% (w/v) NaCl containing 0.5% (w/v) bovine serum albumin). Mice were monitored on a daily basis, and the DAS was determined using a five-point scale (0 = normal to 4 = maximal reduction in digit abduction) as described (28Aoki K.R. Toxicon. 2001; 39: 1815-1820Crossref PubMed Scopus (171) Google Scholar). Toxin lethalities were determined by intraperitoneal injection into mice as described previously (29Maisey E.A. Wadsworth J.D. Poulain B. Shone C.C. Melling J. Gibbs P. Tauc L. Dolly J.O. Eur. J. Biochem. 1988; 177: 683-691Crossref PubMed Scopus (62) Google Scholar). The lowest amount of toxin that killed 50% of mice within 4 days was taken as one minimal lethal dose (mLD50) and expressed as the number of mLD50 units/mg of toxin. Isolation and Culturing of Mouse Spinal Cord, Rat Cerebellar Granule, and Superior Cervical Ganglionic Neurons: Exposure to BoNTs and Assay of SNARE Cleavage—All neuronal cultures were maintained at 37 °C in a 5% CO2 atmosphere. Neurons were prepared from mouse spinal cords removed at gestation day 13, dissociated with trypsin, and seeded at a density of 106 cells/well on rat tail collagen-coated 24-well plates in Dulbecco's modified Eagle's medium supplemented with 5% heat-inactivated horse serum and other factors (15Keller J.E. Cai F. Neale E.A. Biochemistry. 2004; 43: 526-532Crossref PubMed Scopus (149) Google Scholar). At 21 days in vitro, cultures were exposed for 20 min to each toxin in stimulation buffer (15Keller J.E. Cai F. Neale E.A. Biochemistry. 2004; 43: 526-532Crossref PubMed Scopus (149) Google Scholar), washed twice with toxin-free medium, and incubated for the periods specified in the figure legends before harvesting for analysis. In some experiments, an endosomal acidification inhibitor, ConA, was added (250 nm final concentration) at various times after toxin removal as described (15Keller J.E. Cai F. Neale E.A. Biochemistry. 2004; 43: 526-532Crossref PubMed Scopus (149) Google Scholar); the cells were harvested after an additional 20 h of culturing. Rat cerebellar granule neurons (CGNs) were prepared and maintained following established methodology (30Foran P.G. Mohammed N. Lisk G.O. Nagwaney S. Lawrence G.W. Johnson E. Smith L. Aoki K.R. Dolly J.O. J. Biol. Chem. 2003; 278: 1363-1371Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). At 7 days in vitro, the cells were exposed for 24 h at 37 °C to a series of toxin concentrations. In some cases, cells were incubated for 5 min with 500 pm BoNTs in HEPES-buffered solution containing 70 mm K+ (stimulation) with adjustment of the NaCl concentration (30Foran P.G. Mohammed N. Lisk G.O. Nagwaney S. Lawrence G.W. Johnson E. Smith L. Aoki K.R. Dolly J.O. J. Biol. Chem. 2003; 278: 1363-1371Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar); after being washed twice and incubated in medium at 37 °C for the periods specified in the figure legends, the cells were harvested. The isolation and culture of neurons from the superior cervical ganglia of 1–3-day-old rat pups were performed as described (31Mahanthappa N.K. Patterson P.H. Press MIT Cambridge MA Banker G. Goslin K. Culturing Nerve Cells. 2nd Ed. 1998: 289-316Google Scholar). The neurons were dispersed in L15 medium supplemented with vitamins, plated on rat tail collagen-coated 48-well plates, and maintained for 8–10 days before experimentation. The neurons were exposed to 10 nm toxin for the periods indicated in the figure legends before harvesting. At the end of each experiment, all cell types were solubilized in lithium dodecyl sulfate sample buffer and heated to 80 °C for 5 min before SDS-PAGE and Western blotting; SNAP-25 was visualized with a monoclonal antibody (SMI-81, which recognizes intact and BoNT/A- or BoNT/E-cleaved products) and quantified as described (30Foran P.G. Mohammed N. Lisk G.O. Nagwaney S. Lawrence G.W. Johnson E. Smith L. Aoki K.R. Dolly J.O. J. Biol. Chem. 2003; 278: 1363-1371Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 32Meng J. Wang J. Lawrence G. Dolly J.O. J. Cell Sci. 2007; 120: 2864-2874Crossref PubMed Scopus (188) Google Scholar). Statistical Analysis—Data were calculated and graphs generated using GraphPad Prism 4.0. p values were calculated by Student's t test. Generation of BoNT Chimeras by Genetic Recombination of Functional Domains from BoNT/A and BoNT/E—Based on sequence alignments and published crystal structures of BoNT/A and BoNT/B (33Lacy D.B. Tepp W. Cohen A.C. DasGupta B.R. Stevens R.C. Nat. Struct. Biol. 1998; 5: 898-902Crossref PubMed Scopus (670) Google Scholar, 34Swaminathan S. Eswaramoorthy S. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 1024-1026Crossref PubMed Scopus (18) Google Scholar), a portion of the gene deduced to encode the protease and translocation domains of BoNT/A was linked to that for the C-terminal receptor-binding moiety of BoNT/E plus 19 additional amino acids, including a His6 tag, to generate chimera AE (LC-HN/A-HC/E-His6) (Fig. 1). Moreover, AE possesses a linker of nine exogenous residues between the HN/A and HC/E domains to increase flexibility between these functional moieties (Fig. 1). This chimeric toxin was expressed in E. coli using Studier's autoinduction medium (26Studier F.W. Protein Expression Purif. 2005; 41: 207-234Crossref PubMed Scopus (4107) Google Scholar) and isolated to ∼80% purity from lysed bacteria by IMAC on a Co2+-charged resin. A protein of ∼146 kDa was eluted by imidazole together with smaller amounts of lower molecular mass impurities as demonstrated by SDS-PAGE and Coomassie Blue staining (Fig. 2A); the major component co-migrated with natural BoNT/A and BoNT/E when lower quantities of proteins were run under nonreducing conditions (data not shown). All contaminants were removed by anion-exchange chromatography (Fig. 2B); the bound toxin was eluted by 70 mm NaCl (typically 10 mg of pure toxin (Fig. 3A) from a 1-liter culture). The resultant AE chimera gave a single band of ∼146 kDa upon SDS-PAGE in either the absence or presence of DTT, confirming that its expression is in the SC form (Fig. 3A). Western blotting demonstrated the presence in the 146-kDa SC of the BoNT/A LC and epitopes from the receptor-binding domain of BoNT/E and confirmed the absence of truncated forms (Fig. 3A). Controlled nicking with TrypZean gave near-complete conversion of the SC to a disulfide-linked DC as revealed by the appearance of the HC and LC upon SDS-PAGE in the presence of DTT (Fig. 3A); continued migration at ∼146 kDa in the absence of reducing agent indicates that the interchain disulfide was formed in virtually all of the DC (Fig. 3A). As expected, antibodies to HC/E recognized the HC but not LC, whereas anti-LC/A antibody labeled the latter (Fig. 3A); the observed lack of reactivity with an antibody against His6 highlighted that all of the C-terminal tag was removed during nicking (Fig. 3A).FIGURE 3Conversion of SC chimeras to DC forms by controlled nicking: simultaneous tag removal. The purified AE (A) and EA (B) chimeras were incubated with TrypZean (as described under “Experimental Procedures”). Aliquots were analyzed by SDS-PAGE in the absence or presence of 25 mm DTT (as indicated), followed by either Coomassie Blue staining or Western blotting with the antibodies specified. Arrows indicate the positions of the SC/DC, HC, and LC.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In a similar manner, DNA encoding the LC and HN of BoNT/E was fused to the sequence for the HC of BoNT/A to create a gene for chimera EA (Fig. 1), which was expressed in E. coli and affinity-purified as described for AE (Fig. 2C). Further purification was achieved by cation-exchange chromatography (Fig. 2D) on an UNO S1 column, with elution of the toxin by ≥220 mm NaCl (∼15 mg of pure EA from a 1-liter culture). Although the major protein band migrated upon SDS-PAGE at ∼145 kDa, a minor amount of larger, aggregated material (which disappeared upon reduction) was detected by Coomassie Blue staining and Western blotting (Fig. 3B). Complete conversion of the EA SC to DC with TrypZean was observed as described above for AE; likewise, the interchain disulfide was found to have been formed in the vast majority of the toxin. The presence of the requisite moieties (LC/E and HC/A) in EA and the successful removal of the His6 tag were confirmed by Western blotting (Fig. 3B). Staining of the nonreduced nicked toxin with IgGs specific for LC/E revealed a weak signal at ∼97 kDa, which is assumed to correspond to LC-HN resulting from a small degree of cleavage midway along the HC; accordingly, this disappeared upon reduction. Again, immunovisualization of LC and HC bands in the nicked sample, run without reductant, confirmed that the interchain disulfide failed to form only in a minor proportion of the toxin (Fig. 3B). Chimera EA Rapidly Enters Neurons, Cleaves SNAP-25, and B" @default.
• W1993581461 created "2016-06-24" @default.
• W1993581461 creator A5018691594 @default.
• W1993581461 creator A5019174550 @default.
• W1993581461 creator A5028787163 @default.
• W1993581461 creator A5033551724 @default.
• W1993581461 creator A5036088530 @default.
• W1993581461 creator A5041052962 @default.
• W1993581461 creator A5044500515 @default.
• W1993581461 creator A5048987262 @default.
• W1993581461 creator A5052827662 @default.
• W1993581461 creator A5060488022 @default.
• W1993581461 creator A5073552614 @default.
• W1993581461 creator A5081822429 @default.
• W1993581461 date "2008-06-01" @default.
• W1993581461 modified "2023-10-18" @default.
• W1993581461 title "Novel Chimeras of Botulinum Neurotoxins A and E Unveil Contributions from the Binding, Translocation, and Protease Domains to Their Functional Characteristics" @default.
• W1993581461 cites W1539759156 @default.
• W1993581461 cites W1543585694 @default.
• W1993581461 cites W1629865552 @default.
• W1993581461 cites W1966229750 @default.
• W1993581461 cites W1971272088 @default.
• W1993581461 cites W1979848038 @default.
• W1993581461 cites W1982932657 @default.
• W1993581461 cites W1987725158 @default.
• W1993581461 cites W1988075499 @default.
• W1993581461 cites W2000968769 @default.
• W1993581461 cites W2015905038 @default.
• W1993581461 cites W2020593055 @default.
• W1993581461 cites W2036059965 @default.
• W1993581461 cites W2041010702 @default.
• W1993581461 cites W2050781047 @default.
• W1993581461 cites W2058110544 @default.
• W1993581461 cites W2061853748 @default.
• W1993581461 cites W2069008538 @default.
• W1993581461 cites W2072108954 @default.
• W1993581461 cites W2075826540 @default.
• W1993581461 cites W2078359618 @default.
• W1993581461 cites W2079426657 @default.
• W1993581461 cites W2080250583 @default.
• W1993581461 cites W2083325186 @default.
• W1993581461 cites W2090106870 @default.
• W1993581461 cites W2091146475 @default.
• W1993581461 cites W2091467173 @default.
• W1993581461 cites W2093235237 @default.
• W1993581461 cites W2106246537 @default.
• W1993581461 cites W2128977928 @default.
• W1993581461 cites W2155781531 @default.
• W1993581461 cites W2162232417 @default.
• W1993581461 cites W2165882279 @default.
• W1993581461 cites W2170232101 @default.
• W1993581461 doi "https://doi.org/10.1074/jbc.m710442200" @default.
• W1993581461 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18400760" @default.
• W1993581461 hasPublicationYear "2008" @default.
• W1993581461 type Work @default.
• W1993581461 sameAs 1993581461 @default.
• W1993581461 citedByCount "101" @default.
• W1993581461 countsByYear W19935814612012 @default.
• W1993581461 countsByYear W19935814612013 @default.
• W1993581461 countsByYear W19935814612014 @default.
• W1993581461 countsByYear W19935814612015 @default.
• W1993581461 countsByYear W19935814612016 @default.
• W1993581461 countsByYear W19935814612017 @default.
• W1993581461 countsByYear W19935814612018 @default.
• W1993581461 countsByYear W19935814612019 @default.
• W1993581461 countsByYear W19935814612020 @default.
• W1993581461 countsByYear W19935814612021 @default.
• W1993581461 countsByYear W19935814612022 @default.
• W1993581461 countsByYear W19935814612023 @default.
• W1993581461 crossrefType "journal-article" @default.
• W1993581461 hasAuthorship W1993581461A5018691594 @default.
• W1993581461 hasAuthorship W1993581461A5019174550 @default.
• W1993581461 hasAuthorship W1993581461A5028787163 @default.
• W1993581461 hasAuthorship W1993581461A5033551724 @default.
• W1993581461 hasAuthorship W1993581461A5036088530 @default.
• W1993581461 hasAuthorship W1993581461A5041052962 @default.
• W1993581461 hasAuthorship W1993581461A5044500515 @default.
• W1993581461 hasAuthorship W1993581461A5048987262 @default.
• W1993581461 hasAuthorship W1993581461A5052827662 @default.
• W1993581461 hasAuthorship W1993581461A5060488022 @default.
• W1993581461 hasAuthorship W1993581461A5073552614 @default.
• W1993581461 hasAuthorship W1993581461A5081822429 @default.
• W1993581461 hasBestOaLocation W19935814611 @default.
• W1993581461 hasConcept C104317684 @default.
• W1993581461 hasConcept C138626823 @default.
• W1993581461 hasConcept C181199279 @default.
• W1993581461 hasConcept C185592680 @default.
• W1993581461 hasConcept C2776714187 @default.
• W1993581461 hasConcept C2777010668 @default.
• W1993581461 hasConcept C2777367657 @default.
• W1993581461 hasConcept C2994342964 @default.
• W1993581461 hasConcept C30278631 @default.
• W1993581461 hasConcept C55493867 @default.
• W1993581461 hasConcept C70721500 @default.
• W1993581461 hasConcept C86803240 @default.
• W1993581461 hasConcept C95444343 @default.
• W1993581461 hasConceptScore W1993581461C104317684 @default.
• W1993581461 hasConceptScore W1993581461C138626823 @default.

• next