Engineering Botulinum Neurotoxin to Extend Therapeutic Intervention

Engineering botulinum neurotoxin to extend therapeutic intervention

Sheng Chen and Joseph T. Barbieri1

Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Road, Room 256, Milwaukee, WI 53226

Edited by R. John Collier, Harvard Medical School, Boston, MA, and approved April 22, 2009 (received for review March 20, 2009) Clostridium botulinum neurotoxins (BoNTs) are effective therapeu- glabellar facial lines, and axillary hyperhidrosis (11). BoNT/A tics for a variety of neurological disorders, such as strabismus, efficacy in dystonia and other disorders related to involuntary blepharospam, hemificial spasm, and cervical dystonia, because of skeletal muscle activity, coupled with a satisfactory safety pro- the toxin’s tropism for neurons and specific cleavage of neuronal file, has prompted empirical/off-label use in a variety of secre- soluble N-ethylmaleimide-sensitive fusion protein-attachment pro- tions and pain and cosmetic disorders (12). tein receptors (SNARE) proteins. Modifying BoNT to bind nonneu- The clinical use of BoNTs is limited to targeting inflictions ronal cells has been attempted to extend therapeutic applications. affecting neuromuscular activity (11, 12). Elucidation of the However, prerequisite to develop nonneuronal therapies requires structure-function relationship of BoNTs has enabled the design the retargeting the catalytic activity of BoNTs to nonneuronal of therapies that retarget BoNT to unique neurons and non- SNARE isoforms. Here, we reported the engineering of a BoNT neuronal cells. Replacement of BoNT HCR domain with nerve derivative that cleaves SNAP23, a nonneuronal SNARE protein. growth factor, lectin from Erythrina cristagalli, or epidermal SNAP23 mediates vesicle-plasma membrane fusion processes, in- growth factors enable retargeting of BoNT/A to neuronal or cluding secretion of airway mucus, antibody, insulin, gastric acids, nonneuronal cells such as nociceptive afferents and airway and ions. This mutated BoNT/E light chain LC/E(K224D) showed epithelium cells (13–15). However, the selective cleavage of extended substrate specificity to cleave SNAP23, and the natural neuronal-specific SNARE proteins by BoNT has limited devel- substrate, SNAP25, but not SNAP29 or SNAP47. Upon direct protein opment of therapies in these nonneuronal systems. Prerequisite delivery into cultured human epithelial cells, LC/E(K224D) cleaved to develop therapies requires the retargeting of the catalytic endogenous SNAP23, which inhibited secretion of mucin and IL-8. activity of the BoNTs to nonneuronal SNARE isoforms. Here, These studies show the feasibility of genetically modifying LCs to we extend the substrate specificity of BoNT/E by engineering a target a nonneuronal SNARE protein that extends therapeutic catalytic derivative that cleaves the nonneuronal SNARE pro- potential for treatment of human hypersecretion diseases. tein, SNAP23, as a platform to develop therapies for nonneuronal human secretory diseases (16, 17). SNAP23 ͉ SNAP25 ͉ SNARE proteins Results Clostridium botulinum neurotoxins (BoNTs) are the most potent Previous studies identified residues 167–186 as the minimal, protein toxins for humans (1). BoNTs elicit neuronal-specific optimal peptide of SNAP25, a 206 amino acid protein, for LC/E flaccid paralysis by targeting neurons and cleaving neuron- in vitro cleavage (18). SNAP25 (167–186) comprises 2 subsites specific soluble N-ethylmaleimide-sensitive fusion protein- that include a substrate binding ‘‘B’’ region and an active site attachment protein receptors (SNARE) proteins. BoNTs are ‘‘AS’’ region (Fig. 1A). LC/E recognizes the P3 residue to organized into 3 functional domains: an N-terminal zinc-metal- facilitate alignment of the P2 and P1Ј residues of SNAP25. The loprotease light chain (LC), a translocation domain (HCT), and S1Ј pocket of LC/E is formed by F191,T159, and T208 with a C-terminal receptor binding domain (HCR) (1, 2). BoNTs bind hydrophobic interactions between F191 of LC/E and the P1Ј luminal domains of synaptic vesicle proteins, upon the fusion of residue I181 of SNAP25 (19). The basic S2 pocket contains K224, synaptic vesicles with the plasma membrane (3–5). BoNTs are which recognizes the P2 residue, D179, through a predicted salt internalized into endosomes and upon acidification, the LC is bridge. Docking the P2 and P1Ј residues of SNAP25 into the translocated into the cytoplasm, where SNARE proteins are active site pockets of LC/E aligns the scissile bond for cleavage cleaved (1, 2). (18, 19). Mammalian neuronal exocytosis is driven by the formation of Binz and coworkers reported that BoNT/E did not cleave protein complexes between the vesicle SNARE, VAMP2, and human SNAP23 (8), which provided a framework for defining the plasma membrane SNAREs, SNAP25 and syntaxin 1a (6). SNAP isoform specificity of the BoNTs. Many of the residues There are 7 serotypes of BoNTs (termed A–G) that cleave that contributed to LC/E recognition of SNAP25 were con- specific residues on 1 of 3 SNARE proteins: serotypes B, D, F, served in human SNAP23, except T173/A179,D179/K185,M182/T188, and G cleave VAMP-2, serotypes A and E cleave SNAP25, and and E183/D189, respectively (Fig. 1B). T173 in SNAP25 played only serotype C cleaves SNAP25 and syntaxin 1a (1). Thus, neuronal a limited contribution for LC/E substrate recognition (19) and specificity is based upon BoNT binding to neurons and cleaving only main chain interactions of M182-D186 contributed to LC/E neuronal isoforms of the SNARE proteins. For example, substrate recognition. Thus, the T173/A179,M182/T188, and E183/ BoNT/A cleaves human SNAP25, but not the human nonneu- D189 differences between SNAP25 and SNAP23 did not appear ronal isoform SNAP23 (7, 8). The nonneuronal SNARE iso- to contribute to inability of LC/E to cleave SNAP23. In contrast, forms are involved in divergent cellular processes, including fusion reactions in cell growth, membrane repair, cytokinesis, and synaptic transmission (reviewed in 9). Author contributions: S.C. and J.T.B. designed research; S.C. performed research; S.C. and The reversible nature of muscle function after BoNT intoxi- J.T.B. analyzed data; and S.C. and J.T.B. wrote the paper. cation that replace toxin-affected nerves with new nerves (10) The authors declare no conflict of interest. has turned the BoNT from a deadly agent to therapies for This article is a PNAS Direct Submission. neuromuscular conditions. As early as 1989, BoNT/A was ap- 1To whom correspondence should be addressed. E-mail: [email protected]. proved by the FDA to treat strabismus, blepharospam, and This article contains supporting information online at www.pnas.org/cgi/content/full/ hemificial spasm and then for cervical dystonia, cosmetic use, 0903111106/DCSupplemental.

9180–9184 ͉ PNAS ͉ June 9, 2009 ͉ vol. 106 ͉ no. 23 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0903111106 Downloaded by guest on September 28, 2021 Fig. 2. Cleavage of SNAP23 by LC/E(K224D). (A) Five micromolar SNAP23 was incubated with indicated amounts of LC/E(K224D) and subjected to SDS/PAGE [stained gel is shown in insert, SNAP23 (152–211) is designated (SN23 (152– 211)] and the cleavage product SNAP23 (152–186) is designated *. % SNAP23 cleavage was determined by densitometry. (B) Kinetic constant for LC/E to cleave SNAP25 and LC/E(K224D) to cleave SNAP23.

E(K224D) did not cleave SNAP23(I187D) (Fig. 3B) supported that LC/E(K224D) cleaved human SNAP23 between residues BIOCHEMISTRY 186R-I187. SNAP25 isoforms include SNAP25a, SNAP25b, SNAP23a, SNAP23b, SNAP29, and SNAP47 (22, 23). SNAP23 and SNAP25 mediate synaptic membrane fusion in nonneuronal and neuronal cells, respectively, whereas SNAP29 and SNAP47 have Fig. 1. K185 of human SNAP23 contributes to the substrate recognition by not been implicated in membrane fusion events. SNAP29 was BoNT/E. (A) Substrate recognition by LC/E. Two subsites in SNAP25 contribute shown to inhibit SNARE disassembly and was implicated in to substrate binding ‘‘B’’ (Km) and catalysis ‘‘AS’’ (kcat), where the P3, P2, and synaptic transmission (24). Whereas the function of SNAP47 is P1Ј residues contribute to recognition by LC/E. (B) Sequence alignment of not clear, SNAP47 can substitute for SNAP25 in SNARE human SNAP25 (SN25) and human SNAP23 (SN23). (C)(Upper) modeled complex formation and proteoliposome fusion. The substrate complex structure of LC/E-SNAP25 predict the recognition of P site residues of 224 SNAP25 by LC/E. (Lower) modeled complex structure of LC/E(K224D)-SNAP23 specificity of LC/E(K D) on SNAP25 isoforms including predict the recognition of P site residues of SNAP23 by LC/E(K224D). Models SNAP23a, SNAP25b, SNAP29, and SNAP47 were tested. were generated by SWISS-MODEL, using LC/E crystal structure (PDB:3d3x), and SNAP23b and SNAP25a were not tested because the a-b iso- images were generated in PyMol. forms of SNAP23 and SNAP25 were identical at the LC/E

the P2 residue of SNAP25, D179, is recognized by the basic S2 pocket of LC/E via the basic residue, K224, which contributes to LC/E substrate recognition (Fig. 1C, upper panel). This allowed the hypothesis that ‘‘the salt bridge between K224 of LC/E and D179 of SNAP25 contributes the ability of LC/E to cleave SNAP25 and that charge repulsion between K224 of LC/E and the P2 residues of SNAP23, K185, contributes to the inability of LC/E to cleave SNAP23.’’ To test this hypothesis, a point mutation, K224D, was introduced into LC/E and tested for the ability to cleave human SNAP23 (Fig. 1C, lower panel). 224 LC/E(K D) cleaved human SNAP23 with a Km of approxi- Ϫ1 mately 3 ␮M and kcat of approximately 17 S (Fig. 2), within 2-fold of the Km and 5-fold of the kcat of LC/E for the cleavage of human SNAP25. The specific activity for the cleavage of SNAP23 by LC/E(K224D) was similar to the cleavage of VAMP-2 by the B serotype of BoNT and approximately 10-fold faster for the cleavage of VAMP-2 by tetanus toxin (20, 21). The site that LC/E(K224D) cleaved SNAP23 was identified by MALDI-TOF MS where a major peak with an m/z value of 2,812.5 was Fig. 3. Site of SNAP23 cleavage by LC/E(K224D) (A) Five micromolar SNAP23 ␮ 224 identified in a reaction mixture that contained SNAP23 and was incubated with 2 M of LC/E(K D) and subjected to MALDI-TOF mass 224 spectrometry. Intensity (100%) on the y axis was set to the 2,812.5 band and LC/E(K D) (Fig. 3A), corresponding to the C-terminal 25 the x axis represents mass-to-charge units, m/z.(B) SNAP23(I187D) was incu- amino acids of human SNAP23, IKRITDKADTNRDRIDI- bated with the indicated amounts of LC/E(K224D) and subjected to SDS/PAGE. 224 ANARAKKLIDS. This indicated that LC/E(K D) cleaved The Coomassie-stained gel is shown with the migrations of LC/E(K224D and SNAP23 between 186R-I187. The determination that LC/ SNAP23(I187D) indicated on the left.

Chen and Barbieri PNAS ͉ June 9, 2009 ͉ vol. 106 ͉ no. 23 ͉ 9181 Downloaded by guest on September 28, 2021 Fig. 4. Sequence alignment and substrate specificity of LC/E(K224D) and Wt-LC/E on SNAP25 isoforms. (A) Alignment of SNAP23a,b, SNAP25a,b, SNAP29, and SNAP47 (ClustalW2) in the regions corresponding to SNARE proteins that interact with the binding region and active sites region of LC/E. Indicated are conserved residues (*) and similar residues (:, .) among the SNAP25 isoforms. Cleavage site of SNAP25 by LC/E (arrow) and P site resides are indicated. Linear velocity assays of LC/E(K224D) (B) and Wt-LC/E (C) with the Fig. 5. Recombinant LC/E(K224D) cleaves SNAP23 and inhibits mucin and IL-8 indicated isoforms of SNAP25. Five micromolar SNAP25 isoform was incubated secretion in TGF-␣ stimulated HeLa cells. HeLa cells were treated with digito- with the indicated amounts of LC, subjected to SDS/PAGE and gels were nin and then incubated with His-LC/E(K224D) or His-Wt-LC/E (3-Xflag tagged stained with Coomassie. The amount of SNAP25 isoform cleavage was deter- proteins). After an overnight incubation, cells were washed and then incu- mined by densitometry. bated with serum free MEM media supplemented with 20 ng/mL TNF-␣ for 36 h when cell supernatants were collected and cell lysates were prepared. (A) 224 Cell lysates were subjected to SDS/PAGE and LC/E expression and SNAP23 substrate recognition region (Fig. 4A). LC/E(K D) showed cleavage was measured by Western blot, using ␣-3Xflag antibody and similar activity on both SNAP23 and SNAP25 (Fig. 4B), but did ␣-SNAP23 antibody, respectively; * indicates migration of the SNAP23 not cleave SNAP29 and SNAP47. Wt-LC/E cleaved SNAP25 cleavage product. Culture supernatants were assayed IL-8 (B) and mucin (Fig. 4C), but not the other SNAP25 isoforms. The specificity of (C) secretion, using an ELISA, using 1.0 as a reference for cells treated with another LC/E K224 mutation (K224A) on SNAP23 and SNAP25 recombinant LC/E. was also characterized. LC/E (K224A) cleaved SNAP23 and SNAP25 with similar efficiencies, but at a slower rate than ␣ LC/E(K224D). HeLa cells by analyzing TNF- -mediated mucin and IL-8 se- Next, the ability of LC/E(K224D) to cleave endogenous cretion. Control HeLa cells secreted mucin and IL-8 upon ␣ 224 SNAP23 in HeLa cells was tested. Whereas a role for SNAP23 addition of TNF- , whereas LC/E(K D)-transfected HeLa in constitutive exocytosis is not apparent (25), SNAP23 contrib- cells showed reduced mucin and IL-8 secretion (Fig. S1 B and C). utes to regulated exocytosis (26). Transfection of approximately The inhibition was specific, because Wt-LC/E-transfected HeLa 60% of a HeLa cell population with LC/E(K224D) resulted in the cells showed the same amount of mucin and IL-8 secretion as cleavage of approximately 45% of the SNAP23, whereas control cells and did not cleave endogenous SNAP23 (Fig. S1). SNAP23 cleavage was not detected when HeLa cells were To test the feasibility of using LC/E(K224D) as a protein transfected with Wt-LC/E or a no plasmid control (Fig. S1). This therapy, recombinant LC/E(K224D) was delivered into HeLa indicated that LC/E(K224D), but not wt-LC/E, cleaved endoge- cells, using digitonin. Recombinant LC/E(K224D) cleaved en- nous SNAP23 in cultured cells. The effect of SNAP23 cleavage dogenous SNAP23 (Fig. 5A), which inhibited TNF-␣-mediated on HeLa cell secretion was tested in LC/E(K224D)-transfected mucin and IL-8 secretion (Fig. 5B and C). Digitonin treatment

9182 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0903111106 Chen and Barbieri Downloaded by guest on September 28, 2021 also delivered Wt-LC/E into HeLa cells, but Wt-LC/E-treated BoNT has contributed to the understanding vesicle fusion and HeLa cells, did not show detectable inhibition of mucin and IL-8 neurotransmitter release mechanisms in neuronal cells. The secretion and did not cleave endogenous SNAP23 (Fig. 5). This ability of a BoNT derivative to cleave nonneurological SNAREs supports a role for SNAP23 in regulated exocytosis pathways in may provide a useful tool to investigate intracellular vesicular epithelial cells and indicates the utility of LC/E(K224D) as a trafficking and the mechanism of membrane fusion in nonneu- research tool to study SNAP23-regulated exocytosis (26). ronal systems. Although BoNT/A could be considered the logical serotype to Discussion be engineered for various applications because of its wide clinical Understanding of substrate specificity of botulinum neurotoxins applications, analyses of the mechanisms of SNAP25 recognition has enabled the engineering of a light chain derivative of indicate that LC/A requires a longer substrate for optimal BoNT/E with extended substrate specificity, providing a proof of SNAP25 recognition with a greater number of residue interac- principle to extend the clinical potential of BoNT therapy tions than LC/E (19). The less-complex SNAP25-LC/E interac- beyond neurological applications. Whereas airway mucus pro- tions make BoNT serotype E amenable for engineering to tects the epithelial lining by entrapping and clearing foreign modify substrate recognition. In addition, alignment of human debris, bacteria, and viruses from the airway by ciliary move- SNAP25 and SNAP23 showed that these proteins had a high ment, a process termed mucociliary clearance (16, 17), excessive level of homology at the P3 and P1Ј sites that are involved in airway mucus secretion, mucus hypersecretion, may cause mucus SNAP25 recognition by LC/E. Thus, BoNT/E is a useful plat- accumulation that is associated with human clinical conditions form to engineer mutations that effect SNARE protein recog- such as asthma and chronic obstructive pulmonary disease where nition. The successful delivery of LC/E(K224D) into cells to mucus accumulation contributes to respiratory diseases. Mucus inhibit IL-8 and mucin secretion supports a role for LC/E(K224D) secretion is a regulated process coordinated by several mole- as a research tool and also shows the potential for therapy to cules, including SNARE proteins, myristoylated alanine-rich C regulate human hypersecretion diseases such as asthma and kinase substrate (MARCKS), and Munc proteins, which coor- inflammatory diseases. The therapeutic specificity of LC/ dinate the docking of mucin containing vesicles with the secre- E(K224D) would be based upon the receptor binding component, tory cell plasma membrane for exocytosis (16, 17). Targeting as described for toxin chimeras, such as diphtheria toxin A SNAP23 by a substrate modified BoNT may reduce the secretion fragment-IL2 (29) and Exotoxin A fragment-IgG variable region processes of hypersecretion syndromes. A SNAP23-specific fragment (30). In conclusion, the current study shows proof of BoNT may also be targeted for other therapeutic applications principle for altered substrate specificity to extend the applica- BIOCHEMISTRY that include diabetes and inflammatory and immune disorders tion of BoNTs beyond neurological inflictions. which include a hypersecretory component (27, 28). Alignment and biochemical analyses allow prediction of the Methods mechanism for the catalytic activity of Wt-LC/E and LC/ Molecular Modeling. Molecular modeling was performed using SWISS-MODEL. 224 E(K D) on SNAP25 isoforms. The low overall homology The structure of LC/E-SNAP25 (146–202) complex was obtained as described within the active site regions of SNAP29 and SNAP47 to (18), using the crystal structure of LC/E (PDB:3d3x). The structure of LC/ SNAP25 and the lack of an isoleucine at the P1Ј site explain the E(K224D)-SNAP23 was modeled using LC/E-SNAP25 complex structure as tem- inability of Wt-LC/E and LC/E(K224D) to cleave SNAP29 and plate, using PyMol. Data presented are the average of experiments performed SNAP47. In contrast, the overall homology between SNAP23 at least 3 times. and SNAP25 is high, except at the P2, P2Ј and P3Ј residues with the most dramatic change at the P2 residue where SNAP25 Plasmid Construction and Protein Expression. BoNT LC/E expression vector was contains an aspartate and SNAP23 contains a lysine. Thus, 1 constructed by amplifying DNA encoding LC/E (1–400) of C. botulinum serotype E Belugaand subcloned into pET-15b. For transfection experiments, LC/E reason for the inability of Wt-LC/E to cleave SNAP23 may be (1–400) was also subcloned into pEGFP vector to generate an EGFP-LC/E because of electrostatic repulsion of the P2 residue lysine within (1–400) fusion protein expressed under the CMV promoter. Expression vectors 224 SNAP23 by K of LC/E. This may destabilize the S2 pocket and for SNAP23 (152–211), SNAP29 (202–258), and SNAP47 (406–464), the protein affect alignment of the P1Ј residue into the S1Ј pocket. The equivalents of SNAP25 (145–206), were constructed by amplifying the cDNA ability of LC/E(K224D) to cleave SNAP23 may be because of the template: human SNAP23 (ATCC, 2900640), SNAP29(ATCC, 10700609), and introduction a salt bridge between the P2 residue Lys of SNAP23 SNAP47(ATCC, 10468826) by PCR and subcloning into pGEX-2T. Site-directed and the mutated S2 pocket residue D224. LC/E(K224D) also mutagenesis was performed using QuikChange (Stratagene). Protein expres- retained the ability to cleave SNAP25, although at a rate that was sion and puriﬁcation were performed as previously described (18). approximately 10-fold slower than Wt-LC/E. This suggests that the repulsion between the P2 residue aspartate of SNAP25 and Cleavage of SNARE Proteins by LC/E and LC/E(K224D). Linear velocity reaction. 224 Reactions contained (10 ␮L): 5 ␮M human SNARE proteins, 10 mM Tris-HCl (pH the mutated S2 pocket residue D was not sufficient to inhibit 224 sessile bond cleavage by LC/E(K224D). Because LC/E(K224D) 7.6) with 20 mM NaCl, and the indicated amounts of LC/E and LC/E(K D). Reactions were incubated for 10 min at 37°C, subjected to SDS/PAGE, and gels cleaved SNAP25 and SNAP23, but at a reduced rate relative to were stained with Coomassie. The amount of SNARE protein cleavage was LC/E(K224D), both charge and size of the R-group at residue 224 determined by densitometry. Kinetic parameters. Km and kcat determinations contribute to optimal scissile bond cleavage. Overall, the bio- were made for Wt-LC/E and LC/E derivatives using SNAP25 isoforms. LC con- chemical properties of LC/E and LC/E-K224 derivatives are centrations were adjusted to cleave Ͻ10% substrate at several concentrations consistent with P2 residue-S2 pocket residue interactions con- of substrate (1.5ϳ18 ␮M SNARE protein). Reactions were carried out at 37°C tributing to the efficiency of sessile bond cleavage. Whereas the for 10 min, subjected to SDS/PAGE, and the amount of cleaved product was ability of native LC/E to bind SNAP23 has not been determined, calculated by densitometry. Reaction velocity versus substrate concentration kinetic values for LC/E and SNAP25 and LC/E(K224D) and was ﬁt to the Michaelis-Menten equation, using Lineweaver-Burk plots, using SNAP23 are within 2-fold, indicating similar binding affinities. SigmaPlot IX.

Alignment of SNAP25 and SNAP23 within the LC/E binding 224 region (Fig. 1) is nearly identical with 7 of 8 residues identical LC/E and LC/E(K D) Activity in Human Cultured Epithelial Cells. Cell transfection. HeLa cells were cultured in 6-well plates in MEM supplemented with 10% and the nonidentical pair being T:A; a conserved substitution newborn calf serum. Subconﬂuent cells were transfected with 0.5 or 1.0 ␮g pair, which also supports similar binding affinities of LC/E for indicated plasmid using Lipofectamine LTX (GIBCO/BRL). Protein delivery. SNAP25 and SNAP23. Protein delivery was performed as described (31) with modiﬁcation. HeLa cells SNARE proteins are key proteins in membrane fusion and were permeabilized with 1 mL/well of permeabilization buffer containing 30 trafficking within neuronal secretory pathways (9). The use of ␮M digitonin for 7 min and then incubated in permeabilization buffer with

Chen and Barbieri PNAS ͉ June 9, 2009 ͉ vol. 106 ͉ no. 23 ͉ 9183 Downloaded by guest on September 28, 2021 and without the indicated LC. Protein Secretion Assays. After an overnight and quenched with 100 ␮Lof1MH2SO4.A450 was expressed as fraction relative incubation, transfected and protein-delivered cells were incubated in 2 mL to secreted mucin or IL-8 in control supernatants. Cleavage of SNAP23. Lysates serum-free MEM containing 20 ng/mL TNF-␣. After 36 h, 1.5 mL supernatant from cells incubated with TNF-␣ for 36 h were assayed for LC/E and LC/E was collected, centrifuged at 13,000 ϫ g for 1 min, and assayed for secreted (K224D)-mediated a cleavage of endogenous SNAP23, using ␣-SNAP23 mouse mucin and IL-8, using an ELISA. Supernatants (150 ␮L) were mixed with 50 ␮L IgG (Abcam) by Western blot analysis (18). of 0.2 M Na2CO3 (pH 9.6) and added to 96-well plates and incubated overnight at 4°C. Plates were washed and locked with 50 mM Na2CO3 (pH 9.6) buffer ACKNOWLEDGMENTS. This work was sponsored by the National Institutes of ␮ containing 1% (wt/vol) BSA. Plates were washed and incubated with 100 L Health (NIH)/National Institute of Allergy and Infectious Diseases Regional Center ␣-mucin IgG (1/200 dilution, Abcam) or ␣-IL-8 IgG (1/200 dilution, Abcam) for of Excellence for Bio-defense and Emerging Infectious Diseases Research (RCE) 1 h at RT. Plates were washed 3 and incubated with ␣-mouse horseradish Program. The authors wish to acknowledge membership within and support peroxidase-conjugated antibody (1:10,000 dilution, Pierce) for1hatRT.Plates from the Region V ‘Great Lakes’ RCE (NIH award 1-U54-AI-057153). We thank the were washed and developed with 100 ␮L Ultra-TMB (Pierce) for 20 min at RT members of the Barbieri laboratory for helpful discussion.

1. Montecucco C, Schiavo G (1994) Mechanism of action of tetanus and botulinum 17. Rogers DF (2007) Physiology of airway mucus secretion and pathophysiology of hy- neurotoxins. Mol Microbiol 13:1–8. persecretion. Respir Care 52:1134–1146, discussion 1146–1139. 2. Poulain B, Humeau Y (2003) [Mode of action of botulinum neurotoxin: pathological, 18. Chen S, Barbieri JT (2007) Multiple pocket recognition of SNAP25 by botulinum cellular and molecular aspect]. Ann Readapt Med Phys 46:265–275. neurotoxin serotype E. J Biol Chem 282:25540–25547. 3. Dong M, et al. (2003) Synaptotagmins I and II mediate entry of botulinum neurotoxin 19. Chen S, Barbieri JT (2006) Unique substrate recognition by botulinum neurotoxins B into cells. J Cell Biol 162:1293–1303. serotypes A and E. J Biol Chem 281:10906–10911. 4. Dong M, et al. (2006) SV2 is the protein receptor for botulinum neurotoxin A. Science 20. Sikorra S, Henke T, Galli T, Binz T (2008) Substrate recognition mechanism of VAMP/ 312:592–596. synaptobrevin-cleaving clostridial neurotoxins. J Biol Chem 283:21145–21152. 5. Rummel A, Karnath T, Henke T, Bigalke H, Binz T (2004) Synaptotagmins I and II act as 21. Chen S, Hall C, Barbieri JT (2008) Substrate recognition of VAMP-2 by botulinum nerve cell receptors for botulinum neurotoxin G. J Biol Chem 279:30865–30870. neurotoxin B and tetanus neurotoxin. J Biol Chem 283:21153–21159. 6. Brunger AT (2005) Structure and function of SNARE and SNARE-interacting proteins. 22. Oyler GA, et al. (1989) The identification of a novel synaptosomal-associated protein, Q Rev Biophys 38:1–47. 7. Sadoul K, et al. (1997) SNAP-23 is not cleaved by botulinum neurotoxin E and can SNAP-25, differentially expressed by neuronal subpopulations. J Cell Biol 109:3039– replace SNAP-25 in the process of insulin secretion. J Biol Chem 272:33023–33027. 3052. 8. Vaidyanathan VV, et al. (1999) Proteolysis of SNAP-25 isoforms by botulinum neuro- 23. Holt M, et al. (2006) Identification of SNAP-47, a novel Qbc-SNARE with ubiquitous toxin types A, C, and E: Domains and amino acid residues controlling the formation of expression. J Biol Chem 281:17076–17083. enzyme-substrate complexes and cleavage. J Neurochem 72:327–337. 24. Pan PY, et al. (2005) SNAP-29-mediated modulation of synaptic transmission in cul- 9. Jahn R, Scheller RH (2006) SNAREs–engines for membrane fusion. Nat Rev Mol Cell Biol tured hippocampal neurons. J Biol Chem 280:25769–25779. 7:631–643. 25. Okayama M, Arakawa T, Mizoguchi I, Tajima Y, Takuma T (2007) SNAP-23 is not 10. Mahajan ST, Brubaker L (2006) Botulinum toxin: From life-threatening disease to novel essential for constitutive exocytosis in HeLa cells. FEBS Lett 581:4583–4588. medical therapy. Am J Obstet Gynecol 196:7–15. 26. Abonyo BO, et al. (2004) Syntaxin 2 and SNAP-23 are required for regulated surfactant 11. Glogau RG (2002) Review of the use of botulinum toxin for hyperhidrosis and cosmetic secretion. Biochemistry 43:3499–3506. purposes. Clin J Pain 18:S191–197. 27. Martin-Martin B, Nabokina SM, Blasi J, Lazo PA, Mollinedo F (2000) Involvement of 12. Cheng CM, Chen JS, Patel RP (2006) Unlabeled uses of botulinum toxins: A review, part SNAP-23 and syntaxin 6 in human neutrophil exocytosis. Blood 96:2574–2583. 2. Am J Health Syst Pharm 63:225–232. 28. Pagan JK, et al. (2003) The t-SNARE syntaxin 4 is regulated during macrophage 13. Chaddock JA, et al. (2004) Retargeted clostridial endopeptidases: Inhibition of noci- activation to function in membrane traffic and cytokine secretion. Curr Biol 13:156– ceptive neurotransmitter release in vitro, and antinociceptive activity in in vivo models 160. of pain. Mov Disord 19 Suppl 8:S42–47. 29. Strom TB, et al. (1991) Immunotoxins and cytokine toxin fusion proteins. Ann N Y Acad 14. Duggan MJ, et al. (2002) Inhibition of release of neurotransmitters from rat dorsal root ganglia by a novel conjugate of a Clostridium botulinum toxin A endopeptidase Sci 636:233–250. fragment and Erythrina cristagalli lectin. J Biol Chem 277:34846–34852. 30. Du X, Ho M, Pastan I (2007) New immunotoxins targeting CD123, a stem cell antigen 15. Foster KA, et al. (2006) Re-engineering the target specificity of Clostridial neurotoxins on acute myeloid leukemia cells. J Immunother 30:607–613. - a route to novel therapeutics. Neurotox Res 9:101–107. 31. Washbourne P, et al. (2001) Cysteine residues of SNAP-25 are required for SNARE 16. Davis CW, Dickey BF (2008) Regulated airway goblet cell mucin secretion. Annu Rev disassembly and exocytosis, but not for membrane targeting. Biochem J 357:625– Physiol 70:487–512. 634.

9184 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0903111106 Chen and Barbieri Downloaded by guest on September 28, 2021