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• W2073113603 abstract "Botulinum neurotoxin (BoNT) types A and B selectively block exocytosis by cleavage of SNAP-25 and synaptobrevin, respectively; in humans, many months are required for full recovery from the resultant neuromuscular paralysis. To decipher the molecular basis for such prolonged poisoning, intoxication in adreno-chromaffin cells was monitored over 2 months. Exocytosis from BoNT/B-treated cells resumed after 56 days because of the appearance of intact synaptobrevin. However, inhibition continued in BoNT/A-treated cells, throughout the same interval, with a continued predominance of cleaved SNAP-25-(1–197) over the intact protein. When recovery from poisoning was attempted by transfection of the latter cells with the gene encoding full-length SNAP-25-(1–206), no restoration of exocytosis ensued even after 3 weeks. To ascertain if this failure was because of the persistence of the toxin's protease activity, the cells were transfected with BoNT/A-resistant SNAP-25 constructs; importantly, exocytosis was rescued. C-terminal truncation of the toxin-insensitive SNAP-25 revealed that residues 1–201, 1–202, 1–203 afforded a significant return of exocytosis, unlike shorter forms 1–197, −198, −199, or −200; accordingly, mutants M202A or L203A of full-length SNAP-25 rescued secretion. These findings give insights into the C-terminal functional domain of SNAP-25, demonstrate the longevity of BoNT/A protease, and provide the prospect of a therapy for botulism. Botulinum neurotoxin (BoNT) types A and B selectively block exocytosis by cleavage of SNAP-25 and synaptobrevin, respectively; in humans, many months are required for full recovery from the resultant neuromuscular paralysis. To decipher the molecular basis for such prolonged poisoning, intoxication in adreno-chromaffin cells was monitored over 2 months. Exocytosis from BoNT/B-treated cells resumed after 56 days because of the appearance of intact synaptobrevin. However, inhibition continued in BoNT/A-treated cells, throughout the same interval, with a continued predominance of cleaved SNAP-25-(1–197) over the intact protein. When recovery from poisoning was attempted by transfection of the latter cells with the gene encoding full-length SNAP-25-(1–206), no restoration of exocytosis ensued even after 3 weeks. To ascertain if this failure was because of the persistence of the toxin's protease activity, the cells were transfected with BoNT/A-resistant SNAP-25 constructs; importantly, exocytosis was rescued. C-terminal truncation of the toxin-insensitive SNAP-25 revealed that residues 1–201, 1–202, 1–203 afforded a significant return of exocytosis, unlike shorter forms 1–197, −198, −199, or −200; accordingly, mutants M202A or L203A of full-length SNAP-25 rescued secretion. These findings give insights into the C-terminal functional domain of SNAP-25, demonstrate the longevity of BoNT/A protease, and provide the prospect of a therapy for botulism. /B, /C, and /E, botulinum neurotoxins type A, B, C, and E, respectively chloramphenicol acetyltransferase cellubrevin Chinese hamster ovary dithiothreitol glutathioneS-transferase human growth hormone immunoglobulin type G light chain large dense core vesicle low ionic strength medium neuromuscular junction polyacrylamide gel electrophoresis radioimmunoassay synaptobrevin synaptosomal-associated protein with molecular mass of 25 kDa the products of SNAP-25 proteolysis by BoNT/A and BoNT/C, respectively solubleN-ethylmaleimide-sensitive factor attachment protein receptor tetanus toxin enzyme-linked immunosorbent assay Botulinum neurotoxin type A (BoNT/A)1 is being employed successfully for the therapy of dystonias and dysphonias because it potently and selectively inhibits acetylcholine release at the neuromuscular junction (NMJ), thereby resulting in paralysis that lasts for several months (Refs. 1Angaut-Petit D. Molgo J. Comella J.X. Faille L. Tabti N. Neuroscience. 1990; 37: 799-808Crossref PubMed Scopus (114) Google Scholar and 2Sloop R.R. Cole B.A. Escutin R.O. Neurology. 1997; 49: 189-194Crossref PubMed Scopus (160) Google Scholar; and reviewed in Ref. 3Cava T.J. Eur. J. Neurol. 1995; 2: 57-60Google Scholar). Seven serotypes of BoNTs (A to G) are produced by Clostridium botulinum, whereas only a single form of tetanus toxin (TeTX) is synthesized by Clostridium tetani; the latter blocks transmitter release at central inhibitory nerve terminals (4Pellizzari R. Rossetto O. Schiavo G. Montecucco C. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 1999; 354: 259-268Crossref PubMed Scopus (221) Google Scholar). These homologous, but immunologically distinct, proteins share many properties; each active neurotoxin consists of a heavy chain and a light chain (LC) linked by a disulfide bond and non-covalent interactions. The heavy chain is required for high affinity binding to specific neuronal ecto-acceptors, subsequent internalization, and translocation of the LC into the cytosol, where the latter blocks synaptic vesicle exocytosis (reviewed in Ref. 4Pellizzari R. Rossetto O. Schiavo G. Montecucco C. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 1999; 354: 259-268Crossref PubMed Scopus (221) Google Scholar). The LCs of BoNTs and TeTX are Zn2+-dependent endoproteinases that exhibit remarkable substrate selectivities, targeting single bonds (except for BoNT/C, see below) in one of three SNARE (soluble N -ethylmaleimide-sensitive factor attachment protein receptor) proteins proposed to constitute the core components of the vesicle docking/fusion complex that mediates the regulated exocytosis of neurotransmitters (5Söllner T. Bennett M.K. Whiteheart S.W. Scheller R.H. Rothman J.E. Cell. 1993; 75: 409-418Abstract Full Text PDF PubMed Scopus (1577) Google Scholar). Synaptosomal-associated protein with a molecular mass of 25 kDa (SNAP-25) is proteolyzed by BoNT/A, /C, and /E at separate sites near the C terminus: Gln197-Arg198, Arg198-Ala199, and Arg180-Ile181 (6Schiavo G. Santucci A. DasGupta B.R. Mehta P.P. Jontes J. Benfenati F. Wilson M.C. Montecucco C. FEBS Lett. 1993; 335: 99-103Crossref PubMed Scopus (377) Google Scholar, 7Vaidyanathan V.V. Yoshino K. Jahnz M. Dörries C. Bade S. Nauenburg S. Niemann H. Binz T. J. Neurochem. 1999; 72: 327-337Crossref PubMed Scopus (184) Google Scholar, 8Binz T. Blasi J. Yamasaki S. Baumeister A. Link E. Südhof T.C. Jahn R. Niemann H. J. Biol. Chem. 1994; 269: 1617-1620Abstract Full Text PDF PubMed Google Scholar), respectively. Syntaxin 1A/B is also cleaved by BoNT/C and synaptobrevin (Sbr) by BoNT/B, /D, /F, /G, and TeTX (reviewed in Ref. 4Pellizzari R. Rossetto O. Schiavo G. Montecucco C. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 1999; 354: 259-268Crossref PubMed Scopus (221) Google Scholar). Unlike syntaxin 1A/B and Sbr, which each possess a transmembrane anchor (9Niemann H. Blasi J. Jahn R. Trends Cell Biol. 1994; 4: 179-185Abstract Full Text PDF PubMed Scopus (291) Google Scholar), SNAP-25 is attached to the membrane through thioester-linked palmitate modifications of one or more of its four centrally located cysteines (10Lane S.R. Liu Y.C. J. Neurochem. 1997; 69: 1864-1869Crossref PubMed Scopus (104) Google Scholar). BoNT/A- or /E-truncated SNAP-25 remain membrane-bound, but release is inhibited because of the destabilization of the ternary complex even though assembly and disassembly can still occur (11Hayashi T. McMahon H.J. Yamasaki S. Binz T. Hata Y. Südhof T.C. Niemann H. EMBO J. 1994; 13: 5051-5061Crossref PubMed Scopus (661) Google Scholar, 12Pellegrini L.L. O'Connor V. Lottspeich F. Betz H. EMBO J. 1995; 14: 4705-4713Crossref PubMed Scopus (127) Google Scholar). The precise basis of their blockade of neuroexocytosis has yet to be elucidated. While the mechanism of BoNT-induced inhibition has received much attention, the molecular processes involved in recovery of the NMJ from poisoning, which can take up to 4 months depending on the serotype used, remains poorly understood. In humans, the extent of recovery from the neuromuscular paralysis caused by BoNT/A or /B was found to be ∼6 and ∼66%, respectively, at 49 days post-injection (2Sloop R.R. Cole B.A. Escutin R.O. Neurology. 1997; 49: 189-194Crossref PubMed Scopus (160) Google Scholar). Recently, the sequence of events involved in the protracted resumption of neurotransmission in BoNT/A poisoned motor endplates has been revealed by monitoring synaptic function in individual, identified nerve endings of living animals (13de Paiva A. Meunier F.A. Molgo J. Aoki K.R. Dolly J.O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3200-3205Crossref PubMed Scopus (551) Google Scholar). This showed that the transient appearance of functional nerve sprouts mediates a partial return of neuromuscular function, with full recovery relying on the originally poisoned nerve terminals reacquiring synaptic activity. However, it is still unclear how the lifetime of the SNAREs and/or persistence of toxin activity influences the time course of the recovery. To gain insights into the molecular basis of the extended but varied recovery periods, this process was studied in adreno-chromaffin cells. Evoked exocytosis from the latter shares many characteristics with synaptic vesicle-mediated neurotransmitter release from neurons; in particular, SNAP-25, Sbr, and syntaxin 1A/B are present and have all been shown to be required for release of their large dense core vesicles (LDCVs) (14Lawrence G.W. Foran P. Dolly J.O. Eur. J. Biochem. 1996; 236: 877-886Crossref PubMed Scopus (38) Google Scholar, 15Foran P. Lawrence G.W. Shone C.C. Foster K.A. Dolly J.O. Biochemistry. 1996; 35: 2630-2636Crossref PubMed Scopus (233) Google Scholar) because cleavage of these targets by BoNT/A, /B, or /C blocks exocytosis. Previous studies performed on chromaffin cells suggested that persistence of BoNT/A or TeTX activity was responsible for prolonged inhibition of catecholamine release (16Bartels F. Bergel H. Bigalke H. Frevert J. Halpern J. Middlebrook J. J. Biol. Chem. 1994; 269: 8122-8127Abstract Full Text PDF PubMed Google Scholar). Thus, it was pertinent to compare the time course of recovery of exocytosis in chromaffin cells to the different durations of paralysis of NMJ in humans poisoned with BoNT/A and B (2Sloop R.R. Cole B.A. Escutin R.O. Neurology. 1997; 49: 189-194Crossref PubMed Scopus (160) Google Scholar). The changing amounts of the respective BoNT-truncated and full-length SNARE could be quantified throughout the periods of intoxication in these cells, a feat that cannot be easily achieved at the NMJ. Further, this cell model allowed rescue of BoNT/A poisoning to be attempted through the introduction of vectors encoding various forms of SNAP-25. Failure to recover exocytosis in this way with the wild-type molecule, up to 3 weeks post-intoxication, suggested continued activity of the toxin. This was shown conclusively by the successful rescue with SNAP-25 mutants that were engineered to be highly resistant to proteolysis. This important achievement facilitated experimentation which established that substituting residues in SNAP-25 at positions 197, 198, 202, or 203 does not affect its role in regulated secretion, whereas truncating SNAP-25 past residue 201 inhibits the process. Highly purified BoNT/A was isolated as described previously (17Shone C.C. Tranter H.S. Curr. Top. Microbiol. Immunol. 1995; 195: 143-160Crossref PubMed Scopus (44) Google Scholar). BoNT/B and /E were supplied by Drs. C. C. Shone (Centre for Applied Microbiology and Research, UK) and B. DasGupta (Madison, WI) and activated prior to use (18Lawrence G.W. Foran P. Mohammed N. DasGupta B.R. Dolly J.O. Biochemistry. 1997; 36: 3061-3067Crossref PubMed Scopus (48) Google Scholar). Antiserum specific for SNAP-25-(1–197) was a gift from Drs. T. A. N. Ekong and D. Sesardic (National Institute for Biological Standards and Control, UK). Mouse cDNA encoding SNAP-25b and its BoNT/A-truncated version were provided by Dr. L. Zhou of this laboratory. Urografin (Schering Healthcare, Germany), radioimmunoassay (RIA) kit (Nichols Institute, CA, USA), Quick-ChangeTM (Stratagene), calcium phosphate reagents (Life Technologies, Inc., UK), SuperfectTM(QIAGEN, Inc.), pGEX-2T and enhanced chemiluminesence (ECL) reagents (Amersham Pharmacia Biotech, UK), and pcDNA1.1/Amp (Invitrogen) were purchased. Chromaffin cells were prepared from bovine adrenal glands and maintained as primary cultures, as described previously (14Lawrence G.W. Foran P. Dolly J.O. Eur. J. Biochem. 1996; 236: 877-886Crossref PubMed Scopus (38) Google Scholar). Cells required for transfection were further enriched using urografin density-gradient centrifugation (19Wilson S.P. J. Neurosci. Methods. 1987; 19: 163-171Crossref PubMed Scopus (72) Google Scholar). Within 2–3 days after preparation, the cells were incubated with a low ionic strength medium (LISM) at 37 °C for 24 h in the absence or presence of 6.6 nm BoNT/A or 66 nm BoNT/B; after washing, they were maintained at 37 °C (14Lawrence G.W. Foran P. Dolly J.O. Eur. J. Biochem. 1996; 236: 877-886Crossref PubMed Scopus (38) Google Scholar). At various intervals after treatment with or without BoNT/A or /B, the cells were washed briefly with Locke's solution (19Wilson S.P. J. Neurosci. Methods. 1987; 19: 163-171Crossref PubMed Scopus (72) Google Scholar) and incubated in quadruplicate at 22 °C with this buffer in the absence or presence of 2 mm Ba2+. After 15 min, aliquots of the medium bathing the cells were removed and assayed for catecholamine content using a fluorometric procedure (14Lawrence G.W. Foran P. Dolly J.O. Eur. J. Biochem. 1996; 236: 877-886Crossref PubMed Scopus (38) Google Scholar). Values for basal release of catecholamine were subtracted from the amounts recorded in the presence of Ba2+ to determine the evoked components, which were expressed as a percentage of the total catecholamine content within the cells. The mouse SNAP-25b gene was ligated into either pGEX-2T or pcDNA1.1/Amp, using the BamHI and EcoRI restriction sites. Site-specific point mutations or stop codons were introduced into these constructs, using Pfu-DNA polymerase either by conventional polymerase chain reaction strategies or using the Quick-Change site-directed mutagenesis methodology. When required, unidirectional ligations were performed using the BamHI andEcoRI restriction sites. These mutations or deletions in the SNAP-25 gene were verified by sequencing. Full-length variants of glutathioneS-transferase (GST)-SNAP-25 were expressed inEscherichia coli and purified by affinity chromatography (20Smith D.B. Johnson K.S. Gene ( Amst. ). 1988; 67: 31-40Crossref PubMed Scopus (5046) Google Scholar). Each of the recombinant proteins was coated onto 96-well plates, rinsed, blocked with 2% (w/v) bovine serum albumin and exposed to various concentrations of DTT-reduced BoNT/A at 37 °C for 4 h. The wells were then aspirated, washed, and probed with anti-SNAP-25(C terminus)-IgG (generated against the last 12 residues of SNAP-25) (14Lawrence G.W. Foran P. Dolly J.O. Eur. J. Biochem. 1996; 236: 877-886Crossref PubMed Scopus (38) Google Scholar). Unbound IgG was removed by rapid washing, and the bound IgGs were detected indirectly using anti-species-specific IgGs-conjugated to alkaline phosphatase; appearance of the colored product was quantified upon addition of p-nitrophenyl phosphate. TheA 405 nm values recorded from toxin-treated wells were expressed as percentages of those for toxin-free control and plotted against the toxin concentration used. Standard curves relating amounts of intact SNAP-25 remaining in wells were made, using defined mixtures of full-length and BoNT/A-truncated GST-SNAP-25. TheA 405 nm readings observed were expressed as percentages of that recorded for the 100% intact protein sample and plotted against the amounts of intact GST-SNAP-25 coated. This was used to extrapolate the actual amount of intact SNAP-25 remaining in each well; generally, ∼70% of the maximal A 405 nm signal represented 50% intact SNAP-25 in wells. CHO cells, which lack SNAP-25, were transfected with pcDNA1.1/Amp-SNAP-25s using SuperfectTM and 24 h later were incubated in LISM with and without 6.6 nmBoNT/A, as described for chromaffin cells. Membranes were isolated, subjected to immunoblotting with the following IgGs (14Lawrence G.W. Foran P. Dolly J.O. Eur. J. Biochem. 1996; 236: 877-886Crossref PubMed Scopus (38) Google Scholar, 15Foran P. Lawrence G.W. Shone C.C. Foster K.A. Dolly J.O. Biochemistry. 1996; 35: 2630-2636Crossref PubMed Scopus (233) Google Scholar); anti-SNAP-25(full-length)-IgG (an antibody raised against recombinant GST-SNAP-25), anti-SNAP-25A (an antiserum solely reactive with SNAP-251–197), anti-SNAP-25(C terminus)-IgG and anti-Sbr/Cbr-IgG (raised against residues 33–94 of human Sbr-2). Bound antibodies were detected indirectly using anti-species-specific IgGs conjugated to horseradish peroxidase and visualized by the ECL detection system. At the indicated times after incubation in LISM with or without 6.6 nm BoNT/A, the cells were transfected with the hGH construct together with pcDNA1.1/Amp-chloramphenicol acetyltransferase (CAT), as a control, or the appropriate SNAP-25 gene (wild-type or mutants) using the calcium phosphate precipitation method (21Holz R.W. Senter R.A. Uhler M.D. Methods Enzymol. 1995; 257: 221-231Crossref PubMed Scopus (9) Google Scholar). Four to six days after transfection, hGH secretion from intact chromaffin cells was stimulated (as described for catecholamine release) and quantified using a RIA. In some experiments, transfected cells were permeabilized with 20 μm digitonin in a permeabilization buffer (18Lawrence G.W. Foran P. Mohammed N. DasGupta B.R. Dolly J.O. Biochemistry. 1997; 36: 3061-3067Crossref PubMed Scopus (48) Google Scholar) and exposed for 15 min to reduced BoNT/E. Release over the subsequent 15 min was evoked by the addition of 20 μm free Ca2+ and was then assessed. Aliquots were removed and assayed for hGH content; Ca2+-evoked hGH secretion was calculated as outlined above for Ba2+-evoked hGH release. To investigate the important question of how BoNTs exert their inhibitory actions for prolonged periods, neuroendocrine chromaffin cells were treated for 24 h with BoNT/A (6.6 nm) or B (66 nm), using a LISM to overcome the absence of high affinity acceptors (14Lawrence G.W. Foran P. Dolly J.O. Eur. J. Biochem. 1996; 236: 877-886Crossref PubMed Scopus (38) Google Scholar). Evoked secretion and cell complement of SNAREs were assessed at 5, 19, 40, and 56 days post-intoxication (Fig. 1). Ba2+ was employed instead of other commonly used stimuli (e.g. 55 mm K+ or nicotine which induce Ca2+ entry) because it evokes much more catecholamine release (up to 50% of the total cell complement compared with only ∼20% by the latter stimuli) while still exhibiting the same requirements for SNAP-25 and Sbr/Cbr (14Lawrence G.W. Foran P. Dolly J.O. Eur. J. Biochem. 1996; 236: 877-886Crossref PubMed Scopus (38) Google Scholar). After 5 days, cells that had been treated with BoNT/A or /B showed extensive inhibition of Ba2+-evoked catecholamine release of 84 ± 0.7 and 90 ± 0.4% (means ± S.E.; n = 4), respectively, relative to that for toxin-free controls (Fig. 1). Full-length SNAP-25 was monitored using anti-SNAP-25(C terminus)-IgG, whereas intact Sbr and cellubrevin (Cbr; a BoNT-sensitive homologue (22McMahon H.T. Ushkaryov Y.A. Edelmann L. Link E. Binz T. Niemann H. Jahn R. Südhof T.C. Nature. 1993; 364: 346-349Crossref PubMed Scopus (400) Google Scholar)) were quantified using anti-Sbr/Cbr-IgG; both of these antibodies exhibit no reactivity toward the membrane-retained portion of their antigens upon proteolytic cleavage by BoNT/A or /B. The presence of BoNT/A-truncated SNAP-25 in the membrane was monitored using IgG specific for SNAP-25A; the latter does not recognize intact SNAP-25 but reacts strongly with the product of BoNT/A (18Lawrence G.W. Foran P. Mohammed N. DasGupta B.R. Dolly J.O. Biochemistry. 1997; 36: 3061-3067Crossref PubMed Scopus (48) Google Scholar, 23Ekong T.A.N. Feavers I.M. Sesardic D. Microbiology ( UK ). 1997; 143: 3337-3347Crossref PubMed Scopus (73) Google Scholar). Western blotting of the requisite membrane fraction of the cells showed that the BoNT-mediated inhibition of secretion noted at day 5 was accompanied by total or near-complete disappearance of intact SNAP-25 or Sbr/Cbr (Fig. 1), with the BoNT/A-truncated SNAP-25 being readily detected at this time (Fig. 1 A). At day 40 post-intoxication, only a trace of SNAP-25 C terminus immunoreactivity was observed in BoNT/A-poisoned cells relative to the amount in toxin-free control cells; accordingly, the level of evoked secretion was not significantly greater than that recorded on day 5 (Fig. 1 A). However, at day 56 post-intoxication, slightly larger amounts of intact SNAP-25 were noted in the poisoned cells, enough to cause a statistically significant level of recovery of exocytosis (from 16 to 34% of control). Throughout this time course, SNAP-25A persisted in the intoxicated cells (Fig. 1 A). Difficulties inherent to primary cell cultures precluded studies longer than 8 weeks post-intoxication, periods that would appear to be necessary to attain complete recovery from BoNT/A poisoning. In contrast, a much faster recovery from blockade by BoNT/B was observed. The high level of inhibition of evoked secretion in BoNT/B-poisoned cells, noted on day 5, of 90 ± 0.4% gradually subsided until only 10 ± 3.4% inhibition (means ± S.E.;n = 4) remained at day 56. This resumption of regulated exocytosis was coincident with reappearance of intact Sbr/Cbr to a level comparable with that observed in toxin-free cells (Fig. 1 B). Based on the above noted findings, it was hypothesized that expression of an excess of the full-length SNAP-25 in BoNT/A-poisoned cells might restore regulated exocytosis by competing with the SNAP-25Apresent. As a prerequisite to testing this possibility, expression of SNAP-25 in mammalian cells had to be demonstrated; this could be most readily achieved by transfecting pcDNA1.1/ Amp-SNAP-25 into CHO cells that lack endogenous SNAP-25. SDS-PAGE and Western blotting of a Triton X-100 solubilized extract of the cells showed that SNAP-25 was expressed and exhibited the same M r as the native species present in chromaffin cells (Fig. 2 A) when probed with anti-SNAP-25(full-length)-IgG; it was also detectable with anti-SNAP-25(C terminus)-IgG but not by IgG specific for SNAP-25A (Fig. 2 A). Notably, exposure of these transfected cells to BoNT/A resulted in the loss of labeling with anti-SNAP-25(C terminus)-IgG and the appearance of SNAP-25Areactivity (Fig. 2 A), establishing the susceptibility of the recombinant protein to the toxin. To evaluate the prospect of rescuing release through the introduction of SNAP-25 into BoNT/A-poisoned chromaffin cells, the protein was transiently co-expressed with hGH. The latter, which is normally absent from these cells, co-localizes with catecholamines in LDCVs; thus, evoked hGH secretion serves as an excellent reporter for LDCV exocytosis (21Holz R.W. Senter R.A. Uhler M.D. Methods Enzymol. 1995; 257: 221-231Crossref PubMed Scopus (9) Google Scholar). Control cells transfected with vectors encoding hGH and the non-toxic protein, CAT, exhibited an appreciable level of Ba2+-evoked hGH release, and this was inhibited extensively in cells pretreated with BoNT/A (Fig. 2 B). Notably, these two values approximate the respective levels of stimulated catecholamine secretion observed for non-transfected cells (Fig. 1 A). Transfection with a plasmid encoding wild-type SNAP-25 increased slightly the amount of hGH secreted from toxin-free cells; significantly, such overexpression of SNAP-25 failed to overcome the extent of inhibition because of BoNT/A poisoning (Fig. 2 B). These results suggested that persistence of BoNT/A protease for 5 days after intoxication maintained the blockade of release; to exclude other factors, SNAP-25 mutants insensitive to BoNT/A had to be constructed and tested. To examine the likelihood of the enzymic activity of the toxin being sustained within poisoned cells, suitable forms of full-length SNAP-25 that were not susceptible to BoNT/A but might be able to mediate exocytosis were constructed and characterized. Amino acids at positions 197 and/or 198 in wild-type SNAP-25, located on either side of the susceptible bond (i.e. the P1 or P′1 sites, nomenclature of Schechter and Berger (24Schechter I. Berger A. Biochem. Biophys. Res. Commun. 1967; 27: 157-162Crossref PubMed Scopus (4753) Google Scholar)), were altered because an investigation of SNAP-25 peptides had shown that the P′1 arginine residue is critical for cleavage by BoNT/A (25Schmidt J.J. Bostian K.A. J. Protein Chem. 1997; 16: 19-26Crossref PubMed Scopus (114) Google Scholar). Thus, the SNAP-25b gene was altered by PCR-based site-directed mutagenesis, and the mutant proteins were expressed as GST-linked products (see “Experimental Procedures”) so that their susceptibility to BoNT-mediated proteolysis could be assessed in vitro. Seven different SNAP-25 mutants were generated containing either single or double substitutions at the P1 and/or P′1 positions (listed in Fig. 3 A). Each GST-SNAP-25 variant was purified by affinity chromatography; SDS-PAGE and Western blotting confirmed that they exhibited the appropriate mobilities and immunoreactivities (not shown). The cleavage of recombinant SNAP-25 by BoNT/A was assessed using an ELISA which measured the amount of full-length substrate remaining. Notably, wild-type and SNAP-25 mutants all exhibited different susceptibilities to BoNT/A (Fig. 3 B); the EC50 values for the cleavage of each was determined from Fig. 3 B and displayed in Table I. Changes at the P′1, but not at the P1, position yielded proteins highly resistant to BoNT/A compared with the wild-type substrate; replacement of P′1 arginine with either alanine (R198A) or threonine (R198T) reduced degradation of SNAP-25 by ∼550- and ∼16,000-fold, respectively (Table I). In contrast, alteration of the P1 glutamine to alanine caused little change (Q197A; Table I). On the other hand, double-point mutations incorporating the innocuous P1 alanine replacement in addition to alterations at the P′1 site to alanine (Q197A/R198A) or lysine (Q197A/R198K) greatly decreased sensitivity to proteolysis by type A toxin, with the latter proving to be the most resistant (∼38,000-fold reduction in susceptibility). Other double-point mutations at the P1 and P′1 positions (i.e. the wild-type Gln197/Arg198 sequence to either KH, naturally occurring in Torpedo SNAP-25 (26Risinger C. Blomqvist A.G. Lundell I. Lambertsson A. Nassel D. Pieribone V.A. Brodin L. Larhammar D. J. Protein Chem. 1993; 268: 24408-24414Google Scholar), or WW) caused far less resistance to BoNT/A (∼440- and ∼96-fold, respectively; Table I). It was hoped that, at least, some of these SNAP-25 variants could rescue evoked exocytosis from inhibition by BoNT/A.Table ISusceptibilities of wild-type and various SNAP-25 mutants to cleavage by BoNT/ASNAP-25 variantPosition of mutated residue(s)Minimum BoNT/A concentration necessary to proteolyse 50% of SNAP-25 in the standard assayaStandard curves, relating increasing amounts of intact GST-SNAP-25 per well to the increasing A 405 nm values recorded, were used to calculate the A 405 nm reading which was equivalent to 50% intact SNAP-25 remaining in wells. This absorbance value was used to determine the concentrations of BoNT/A required to proteolyse 50% of either the wild-type or mutant SNAP-25 from Fig. 3 B.Susceptibilities to proteolysis by BoNT/A (relative to wild-type)bObtained by dividing the minimal BoNT/A concentration giving 50% cleavage of SNAP-25 mutant by the corresponding values for wild-type; larger values indicate greater resistance to proteolysis.nmWild-typeNone0.00451.0(Gln197/Arg198)Q197AP10.00541.2R198AP1′2.5550R198TP1′7216000Q197A/R198AP1, P1′204400Q197A/R198KP1, P1′17038000Q197K/R198HP1, P1′2.0440Q197W/R198WP1, P1′0.4396a Standard curves, relating increasing amounts of intact GST-SNAP-25 per well to the increasing A 405 nm values recorded, were used to calculate the A 405 nm reading which was equivalent to 50% intact SNAP-25 remaining in wells. This absorbance value was used to determine the concentrations of BoNT/A required to proteolyse 50% of either the wild-type or mutant SNAP-25 from Fig. 3 B.b Obtained by dividing the minimal BoNT/A concentration giving 50% cleavage of SNAP-25 mutant by the corresponding values for wild-type; larger values indicate greater resistance to proteolysis. Open table in a new tab For the initial rescue experiments, R198T was selected for several reasons: (i) its high resistance to BoNT/A; (ii) a single substitution was deemed more likely than a double to be able to mediate exocytosis and cause rescue in toxin-treated cells, and (iii) in the unlikely event of it being cleaved intracellularly, the product would be detectable with anti-SNAP-25A-IgG because of the retention of Gln197 (which was altered in the double mutants). As a preliminary to the complicated rescue experiments, the expression of R198T and its lack of susceptibility to BoNT/A were first established in CHO cells. After transfection, as described above, R198T was found to be expressed at a level similar to that of wild-type SNAP-25, as judged from the labeling on blots using two antibodies reactive with full-length SNAP-25 or its C-terminal region (Fig. 2 A). Exposure of the transfected cells to BoNT/A did not lower the R198T reactivity with anti-SNAP-25(C terminus)-IgG, and furthermore, no product could be observed with SNAP-25A-IgG (Fig. 2 A). Having demonstrated that R198T mutant was expressed but not cleaved by BoNT/A in mammalian cells, plasmids encoding several such SNAP-25 mutants were introduced into chromaffin cells. Expression of these mutants in toxin-f" @default.
• W2073113603 created "2016-06-24" @default.
• W2073113603 creator A5005134724 @default.
• W2073113603 creator A5034426099 @default.
• W2073113603 creator A5041052962 @default.
• W2073113603 creator A5048987262 @default.
• W2073113603 creator A5080148077 @default.
• W2073113603 date "1999-12-01" @default.
• W2073113603 modified "2023-10-10" @default.
• W2073113603 title "Rescue of Exocytosis in Botulinum Toxin A-poisoned Chromaffin Cells by Expression of Cleavage-resistant SNAP-25" @default.
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