A Novel Subunit Structure of Clostridium botulinum Serotype D Toxin Complex with Three Extended Arms

Abstract

The botulinum neurotoxins (BoNTs) are the most potent toxins known in nature, causing the lethal disease known as botulism in humans and animals. The BoNTs act by inhibiting neurotransmitter release from cholinergic synapses. Clostridium botulinum strains produce large BoNTs toxin complexes, which include auxiliary non-toxic proteins that appear not only to protect BoNTs from the hostile environment of the digestive tract but also to assist BoNT translocation across the intestinal mucosal layer. In this study, we visualize for the first time a series of botulinum serotype D toxin complexes using negative stain transmission electron microscopy (TEM). The complexes consist of the 150-kDa BoNT, 130-kDa non-toxic non-hemagglutinin (NTNHA), and three kinds of hemagglutinin (HA) subcomponents: 70-kDa HA-70, 33-kDa HA-33, and 17-kDa HA-17. These components assemble sequentially to form the complex. A novel TEM image of the mature L-TC revealed an ellipsoidal-shaped structure with “three arms” attached. The “body” section was comprised of a single BoNT, a single NTNHA and three HA-70 molecules. The arm section consisted of a complex of HA-33 and HA-17 molecules. We determined the x-ray crystal structure of the complex formed by two HA-33 plus one HA-17. On the basis of the TEM image and biochemical results, we propose a novel 14-mer subunit model for the botulinum toxin complex. This unique model suggests how non-toxic components make up a “delivery vehicle” for BoNT. The botulinum neurotoxins (BoNTs) are the most potent toxins known in nature, causing the lethal disease known as botulism in humans and animals. The BoNTs act by inhibiting neurotransmitter release from cholinergic synapses. Clostridium botulinum strains produce large BoNTs toxin complexes, which include auxiliary non-toxic proteins that appear not only to protect BoNTs from the hostile environment of the digestive tract but also to assist BoNT translocation across the intestinal mucosal layer. In this study, we visualize for the first time a series of botulinum serotype D toxin complexes using negative stain transmission electron microscopy (TEM). The complexes consist of the 150-kDa BoNT, 130-kDa non-toxic non-hemagglutinin (NTNHA), and three kinds of hemagglutinin (HA) subcomponents: 70-kDa HA-70, 33-kDa HA-33, and 17-kDa HA-17. These components assemble sequentially to form the complex. A novel TEM image of the mature L-TC revealed an ellipsoidal-shaped structure with “three arms” attached. The “body” section was comprised of a single BoNT, a single NTNHA and three HA-70 molecules. The arm section consisted of a complex of HA-33 and HA-17 molecules. We determined the x-ray crystal structure of the complex formed by two HA-33 plus one HA-17. On the basis of the TEM image and biochemical results, we propose a novel 14-mer subunit model for the botulinum toxin complex. This unique model suggests how non-toxic components make up a “delivery vehicle” for BoNT. Different strains of Clostridium botulinum produce seven distinct serotypes of neurotoxins (BoNTs), 2The abbreviations used are: BoNTbotulinum neurotoxinHAhemagglutininNTNHAnon-toxic non-hemagglutininTCtoxin complexTEMtransmission electron microscopy 2The abbreviations used are: BoNTbotulinum neurotoxinHAhemagglutininNTNHAnon-toxic non-hemagglutininTCtoxin complexTEMtransmission electron microscopy classified A through G. BoNT has attracted much interest in recent years due to extensive research on its biochemistry, determination of its crystal structure, and investigations into the pharmacology and applications of BoNTs as therapeutic agents for the treatment of human disease (1Schiavo G. Matteoli M. Montecucco C. Physiol. Rev. 2000; 80: 717-766Crossref PubMed Scopus (1047) Google Scholar, 2Bigalke H. Schoer L.E. Aktories K. Just I. Bacterial Protein Toxins, Handbook of Experimental Pharmacology. Springer-Verlag Ltd., Berlin2000: 407-443Google Scholar, 3Jankovic J. Hallett M. Therapy with Botulinum Toxin. Marcel Dekker, Inc., New York1994Google Scholar). After ingestion of BoNT, the BoNT is absorbed from intestinal epithelial cells into the bloodstream, after which it consequently reaches the neuromuscular junctions. BoNT enters nerve cells via receptor-mediated endocytosis, where it cleaves specific sites on target proteins, inhibiting release of neurotransmitters from peripheral cholinergic synapses through its zinc protease activity (4Schiavo G. Rossetto O. Catsicas S. Polverino de Laureto P. DasGupta B.R. Benfenati F. Montecucco C. J. Biol. Chem. 1993; 268: 23784-23787Abstract Full Text PDF PubMed Google Scholar, 5Montecucco C. Schiavo G. Mol. Microbiol. 1994; 13: 1-8Crossref PubMed Scopus (488) Google Scholar, 6Dolly J.O. Black J. Williams R.S. Melling J. Nature. 1984; 307: 457-460Crossref PubMed Scopus (256) Google Scholar). This process causes muscular paralysis in humans and animals, leading to the disease botulism. botulinum neurotoxin hemagglutinin non-toxic non-hemagglutinin toxin complex transmission electron microscopy botulinum neurotoxin hemagglutinin non-toxic non-hemagglutinin toxin complex transmission electron microscopy Toxins with serotypes A–D and G are encoded by two gene clusters in close proximity to each other; cluster 1 contains the bont and ntnha genes, and cluster 2 contains three genes: ha-70, ha-33, and ha-17 (7Hauser D. Eklund M.W. Boquet P. Popoff M.R. Mol. Gen. Genet. 1994; 243: 631-640Crossref PubMed Scopus (70) Google Scholar, 8Minton N.P. Curr. Top. Microbiol. Immunol. 1995; 195: 161-194PubMed Google Scholar). Therefore, botulinum TC consists of five components: BoNT, non-toxic non-hemagglutinin (NTNHA) and three hemagglutinin subcomponents (HA-70, HA-33, and HA-17). All serotypes of BoNT associate non-covalently with auxiliary non-toxic proteins, thereby forming large toxin complexes (TCs). Serotype A–D strains produce the M-TC (BoNT·NTNHA complex) and L-TC (BoNT·NTNHA·HAs complex) in the culture medium, while serotype E and F strains produce only M-TC. The major biological function of the non-toxic components appears to be protection of the BoNT against the hostile environment of the digestive tract (9Sakaguchi G. Kozaki S. Ohishi I. Alouf J.E. Fehrenbach F.J. Freer J.H. Jelijaszewicz J. Bacterial Protein Toxins. Academic Press Inc., London1984: 435-443Google Scholar, 10Ohishi I. Sugii S. Sakaguchi G. Infect. Immun. 1977; 16: 107-109Crossref PubMed Google Scholar). Additionally, several lines of evidence have shown that the non-toxic proteins play a role in the uptake and transcytosis of L-TC into the intestinal epithelium (11Nishikawa A. Uotsu N. Arimitsu H. Lee J.C. Miura Y. Fujinaga Y. Nakada H. Watanabe T. Ohyama T. Sakano Y. Oguma K. Biochem. Biophys. Res. Commun. 2004; 319: 327-333Crossref PubMed Scopus (53) Google Scholar, 12Uotsu N. Nishikawa A. Watanabe T. Ohyama T. Tonozuka T. Sakano Y. Oguma K. Biochim. Biophys. Acta. 2006; 1763: 120-128Crossref PubMed Scopus (32) Google Scholar, 13Fujinaga Y. Inoue K. Watanabe S. Yokota K. Hirai Y. Nagamachi E. Oguma K. Microbiology. 1997; 143: 3841-3847Crossref PubMed Scopus (130) Google Scholar, 14Fujinaga Y. Inoue K. Nomura T. Sasaki J. Marvaud J.C. Popoff M.R. Kozaki S. Oguma K. FEBS Lett. 2000; 467: 179-183Crossref PubMed Scopus (101) Google Scholar, 15Fujinaga Y. Inoue K. Watarai S. Sakaguchi Y. Arimitsu H. Lee J.C. Jin Y. Matsumura T. Kabumoto Y. Watanabe T. Ohyama T. Nishikawa A. Oguma K. Microbiology. 2004; 150: 1529-1538Crossref PubMed Scopus (62) Google Scholar). Recently, we have also demonstrated, using a cultured Caco-2 cell layer, that serotype D HA-33 plays a critical role in the permeation of TC species into the intestinal epithelium (16Niwa K. Koyama K. Inoue S. Suzuki T. Hasegawa K. Watanabe T. Ikeda T. Ohyama T. FEMS Immunol. Med. Microbiol. 2007; 49: 346-352Crossref PubMed Scopus (37) Google Scholar). It is therefore likely that the non-toxic components play a role in the trans-epithelial transport of BoNT across the intestinal barrier. However, few studies have addressed the structure of the TC, because of the large sizes of the complexes consisting of multiple non-toxic components. These complexes vary from 300 kDa up to 900 kDa, depending upon the BoNT serotypes. C. botulinum serotype D strain 4947 (D-4947) produces TC species, which consist only of intact components, without any nicking. We previously proposed an assembly pathway, including the binding order of individual subunits, through in vitro reconstitution of the D-4947 L-TC (17Kouguchi H. Watanabe T. Sagane Y. Sunagawa H. Ohyama T. J. Biol. Chem. 2002; 277: 2650-2656Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar): the M-TC is formed first by assembly of a single BoNT and a single NTNHA molecule, and is subsequently converted to the complete L-TC by forming complexes with HA-70 and HA-33·HA-17 molecules. Additionally, we found hemagglutination-negative TC species in which HA-33·HA-17 molecules were missing from the mature L-TC (18Mutoh S. Kouguchi H. Sagane Y. Suzuki T. Hasegawa K. Watanabe T. Ohyama T. Biochemistry. 2003; 42: 10991-10997Crossref PubMed Scopus (32) Google Scholar, 19Mutoh S. Suzuki T. Hasegawa K. Nakazawa Y. Kouguchi H. Sagane Y. Niwa K. Watanabe T. Ohyama T. Microbiology. 2005; 151: 3847-3858Crossref PubMed Scopus (14) Google Scholar). These TC species are considered to be intermediates in the 650-kDa L-TC assembly pathway (20Kouguchi H. Suzuki T. Hasegawa K. Mutoh S. Watanabe T. Niwa K. Yoneyama T. Katoh Y. Ohyama T. J. Microbiol. Method. 2006; 67: 416-423Crossref PubMed Scopus (19) Google Scholar). Regarding the subunit structure of the botulinum TC, historically there has been only one model, our schematic one that was constructed based on biochemical results (18Mutoh S. Kouguchi H. Sagane Y. Suzuki T. Hasegawa K. Watanabe T. Ohyama T. Biochemistry. 2003; 42: 10991-10997Crossref PubMed Scopus (32) Google Scholar, 21Suzuki T. Watanabe T. Mutoh S. Hasegawa K. Kouguchi H. Sagane Y. Fujinaga Y. Oguma K. Ohyama T. Microbiology. 2005; 151: 1475-1483Crossref PubMed Scopus (23) Google Scholar). In this work, we propose for the first time a more detailed structure of a botulinum serotype D TC, and we describe a potential assembly process that specifies the binding order of individual subunits. This novel and unusual 14-mer model may explain how non-toxic components comprise a delivery vehicle for BoNT in food-borne botulism, how BoNT is protected in the digestive environment, and how BoNT is internalized into the bloodstream. Production and Purification of Botulinum D-4947 TC Species—C. botulinum D-4947 was cultured using a dialysis method as previously described (22Hasegawa K. Watanabe T. Sato H. Sagane Y. Mutoh S. Suzuki T. Yamano A. Kouguchi H. Takeshi K. Kamaguchi A. Fujinaga Y. Oguma K. Ohyama T. Protein J. 2004; 23: 371-378Crossref PubMed Scopus (24) Google Scholar). The TC species were purified from the culture supernatants by SP-Toyopearl 650 S cation-exchange column chromatography as described previously (18Mutoh S. Kouguchi H. Sagane Y. Suzuki T. Hasegawa K. Watanabe T. Ohyama T. Biochemistry. 2003; 42: 10991-10997Crossref PubMed Scopus (32) Google Scholar). Each TC species fraction was collected separately, and the molecular mass was estimated by gel filtration. The M-TC·HA-70 and 540-, 610-, and 650-kDa L-TC species were then purified to homogeneity, as determined by SDS- and native-PAGE. The TC species termed M-TC, M-TC·HA-70, 540-kDa, 610-kDa, and 650-kDa L-TC were thus isolated for TEM and other experiments. Isolation of the HA-33·HA-17 complex from 650-kDa L-TC—Isolation of the HA-33·HA-17 complex from a 250-mg pellet resulting from ammonium sulfate precipitation of the 650-kDa L-TC was performed as described (17Kouguchi H. Watanabe T. Sagane Y. Sunagawa H. Ohyama T. J. Biol. Chem. 2002; 277: 2650-2656Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). PAGE and Densitometric Analyses—PAGE under non-denaturing conditions (native-PAGE) was carried out at pH 8.8 using a 5–12.5% polyacrylamide linear gradient gel. SDS-PAGE was performed using a 13.6% polyacrylamide gel in the presence of 1% 2-mercaptoethanol. The Coomassie Brilliant Blue R-250 (CBB) staining intensities of the components were analyzed with ImageJ 1.30v. The protein concentration was determined using a BCA protein assay kit (Pierce, IL) with bovine serum albumin as the standard. Hemagglutination Assay and Titration of TC Species with the HA-33·HA-17 Complex—A hemagglutination assay was performed by a microtitration method (18Mutoh S. Kouguchi H. Sagane Y. Suzuki T. Hasegawa K. Watanabe T. Ohyama T. Biochemistry. 2003; 42: 10991-10997Crossref PubMed Scopus (32) Google Scholar). To determine the binding capacities of the TC species, with isolated HA-33·HA-17 as titrant, the titration experiment was performed by adding HA-33·HA-17 to the TC species. The titration mixtures containing 0.7 μm TC species and several concentrations of HA-33·HA-17 were incubated for 1 h at room temperature, followed by a hemagglutination assay and native-PAGE. Analytical Ultracentrifugation—The purified HA-33·HA-17 complex was dialyzed against 50 mm acetate buffer (pH 5.0) containing 0.15 m NaCl and adjusted to an appropriate protein concentration. Sedimentation equilibrium experiments were carried out using an Optima XL-I analytical ultracentrifuge (Beckman Coulter Inc.) with a 4-hole An60Ti rotor and two-channel charcoal-filled Epon cells at a rotor speed of 17,000 rpm. The data were analyzed using the software Ultrascan 6.01. Mass Spectrometric Analyses—Nano-liquid chromatography/electrospray ionization time-of-flight mass spectrometry (nanoLC/ESI-TOF-MS) was carried out with a nanoLC system consisting of a Famos auto sampler and a Switchos automated switching valve (LC Packings), and a micrOTOF-focus orthogonal acceleration TOF instrument (Bruker Daltonics, Bremen, Germany) interfaced with a nano-ESI HA-33·HA-17 was in mm acetate buffer (pH 5.0) with a protein concentration of of was a mm mm After the to the nano-ESI the was with a linear gradient of All experiments were carried out in ionization time-of-flight mass spectrometry was carried out with an mass (Bruker in linear at an acceleration of the and were used to and HA-33·HA-17 was in mm (pH at a protein concentration of of the TC in 50 mm acetate buffer (pH 5.0) were on The were with were with the and the at a of and an of of HA-33·HA-17 of the HA-33·HA-17 the protein was by with an and by using with a molecular of followed by through an μm of conditions was to HA-33·HA-17 for x-ray and 2 were used as for and H. K. M. S. M. Y. Sasaki T. D Biol. 2003; PubMed Scopus Google at and The protein concentrations from to The of to were and x-ray data were collected at on a using from a with The used for this experiment up to To a data x-ray data were collected from at using a on the at the data collected using the were up to are in of data and for D-4947 HA-33·HA-17 complex in 50 mm (pH m m (pH m cell of of of from in a and x-ray data were and using the data was carried out using the D. Biol. 1994; PubMed Scopus Google Scholar). was by molecular using A. D Biol. 2000; PubMed Scopus Google Scholar). The model for molecular was the structure of the from serotype which the to the D-4947 HA-33 and were carried out to the using the K. and on Protein 1994; to the of A. 42: Scopus Google which were then used for of the The was using of model were carried out using the Protein and D Biol. 1997; PubMed Scopus Google Scholar). The of the model was using for and After the model a was to the structure using A. Biol. PubMed Scopus Google Scholar). The model was analyzed by M.W. J. 1993; Google to its The toxin fraction from the C. botulinum D-4947 culture was to an SP-Toyopearl 650 S column under conditions (pH After several for each five purified TC species were were termed M-TC (BoNT·NTNHA M-TC·HA-70 540-kDa, 610-kDa, and 650-kDa with the molecular determined by gel and The and a on SDS-PAGE to that of the 650-kDa but with staining intensities of the HA-33 and HA-17 were for the 650-kDa L-TC that to intermediates species in the pathway to 650-kDa L-TC The 650-kDa L-TC is the complete L-TC because complex was purified from the culture All two on under where the of non-toxic components NTNHA for from the BoNT, as shown in The of the non-toxic complex from the M-TC·HA-70, 540-kDa, 610-kDa, and 650-kDa L-TC revealed a with at to molecular TC species, for the 650-kDa hemagglutination we that the HA-33 and HA-17 molecules have been missing in the subunit of TC species other the complete the BoNT the purified D-4947 TC species were by negative stain the of TC species that were after of in the TEM the of subunit components are in The M-TC, which is of BoNT and appears as an The M-TC·HA-70 complex is an with the HA-70 on the M-TC up sequentially to form a complex. It is that the HA-70 appears to have This may be by the that the isolated HA-70 molecules distinct through the to not L-TC species revealed unique that to be We that were HA-33·HA-17 complexes that to the M-TC·HA-70 complex via the HA-70 molecule, as shown in The of with with the of the L-TC the of one two and three for and 650-kDa TC image with more was in the of This is therefore a novel the structure of the botulinum In an study, based on negative stain electron microscopy of the serotype A BoNT and of a non-toxic S. S. Infect. Immun. PubMed Google a model for the complex that the BoNT to the of the F. F. J. Biol. 1997; PubMed Scopus Google proposed a model for the serotype A of that was in with in the electron that were using a However, the of the components To our the of HA-33·HA-17 molecules in TC species consisted of one the by our schematic model T. Watanabe T. Mutoh S. Hasegawa K. Kouguchi H. Sagane Y. Fujinaga Y. Oguma K. Ohyama T. Microbiology. 2005; 151: 1475-1483Crossref PubMed Scopus (23) Google which was from experiments. These experiments to of HA-33·HA-17 molecules in the 650-kDa three in the L-TC and two in the This because gel an of 50 kDa for the HA-33·HA-17 that the complex is of one each of HA-33 and HA-17. results that the L-TC species may have for the of the of the 650-kDa L-TC and were because HA-33·HA-17 molecules to the and thus be the TC The HA-70 molecules also to a more upon binding of HA-33·HA-17 to form A that is to be a zinc an was in TEM of TC this a for the BoNT electron in a of the subunit structure of botulinum TC, which be by x-ray of complexes in the of the HA-33·HA-17 TEM and PAGE that the arm section consists of HA-33 and we to its molecular The HA-33·HA-17 which not be as a the was isolated from L-TC using m as a as previously described (17Kouguchi H. Watanabe T. Sagane Y. Sunagawa H. Ohyama T. J. Biol. Chem. 2002; 277: 2650-2656Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). using equilibrium a molecular of On the other of HA-33·HA-17 a mass of while a mass of the and the These were close to the by adding the of two HA-33 molecules and one HA-17 of the HA-33·HA-17 of the HA-33·HA-17 complex mm in were for x-ray using the We determined the x-ray crystal structure of the isolated HA-33·HA-17 complex from D-4947 L-TC at by molecular using the serotype HA-33 structure K. M. Oguma K. M. Microbiology. 2003; PubMed Scopus Google as the The cell were a and and each cell one complex in the The model was to an of and for HA-33 and for data and are in The that were the crystal structure of the HA-33·HA-17 complex as and The model two HA-33 molecules and one HA-17 in the This model is with the molecular of HA-33·HA-17 complex as determined by molecular Each HA-33 consists of a single of into a up of and in the crystal of serotypes A HA-33 J. F.J. J. Mol. Biol. 2005; PubMed Scopus Google and HA-33 K. M. Oguma K. M. Microbiology. 2003; PubMed Scopus Google D-4947 HA-33 is also of two which consist of a by three and by a The two HA-33 molecules are by with sites by a On the other the HA-17 that a single of was not for two at the a that two HA-33 molecules. A with the structure of HA-33 that the were also found in the HA-17 structure, one was not in the due to electron Additionally, for were in the HA-17 molecule, that HA-17 may not shown in two HA-33 molecules form with each other at with extensive and in each in which the likely form On the other at the of HA-33 and make with the HA-17 molecule, forming an with one of HA-17 and as shown in is known the function of its most likely function is to two molecules of HA-33 and the HA-70 molecule, as by and to form the of HA-33·HA-17 in the performed in vitro reconstitution of the mature L-TC by adding of HA-33·HA-17 to form L-TC The hemagglutination activity of the 650-kDa L-TC was determined to be of while hemagglutination be for the other TC species. This that the of HA-33·HA-17 complexes is for hemagglutination To the molecular of TC species, we the hemagglutination activity of the M-TC·HA-70, and L-TC by binding of HA-33·HA-17 to the HA-33·HA-17 mass was TC species a to that of as shown in the of the the M-TC·HA-70, and TC species μm and 0.7 μm to hemagglutination TC species an on to that of the 650-kDa as in 1 not the of HA-33·HA-17 complexes was estimated as against M-TC·HA-70, and This to a model in which three HA-33·HA-17 complexes in the subunit structure of including the TEM are with the hemagglutination activity of the botulinum model suggests that HA-33 molecules be at the L-TC where play a role in the complex at the epithelial cell of the of HA-70 the of HA-70 in the L-TC has been we to the of HA-70 by of staining on SDS-PAGE on several of which were we that the of HA-70 was it consists of three subunits. This was by the of the BoNT by each molecular In of the of molecules in the this is of the of Botulinum that TC species from the culture appear to form subunits, which comprise The results described were to of the botulinum serotype D TC, to as to the of and the of each protein in botulinum D-4947 our proposed 14-mer model on the of of subunits, which an of the individual in the mature The complex a single BoNT, a single three HA-70, HA-33, and three HA-17. This model of the D-4947 BoNT structure was using the We serotype A DasGupta B.R. Biol. PubMed Scopus Google and BoNTs S. S. Biol. 2000; PubMed Scopus Google as and The and sizes of NTNHA and HA-70 molecules were from the TEM and molecular This model from our subunit model (18Mutoh S. Kouguchi H. Sagane Y. Suzuki T. Hasegawa K. Watanabe T. Ohyama T. Biochemistry. 2003; 42: 10991-10997Crossref PubMed Scopus (32) Google Scholar, 21Suzuki T. Watanabe T. Mutoh S. Hasegawa K. Kouguchi H. Sagane Y. Fujinaga Y. Oguma K. Ohyama T. Microbiology. 2005; 151: 1475-1483Crossref PubMed Scopus (23) Google which was based on biochemical The data described to propose a more model novel of this structure x-ray and a using the resulting The of NTNHA and HA-70 proteins have not been our model be to a few we that this model explain of botulinum TC as a “delivery in which subcomponents may the of BoNT into the via binding to intestinal In the model how BoNT is protected from protease is to the unique subunit structure of botulinum TC by TEM of the assembly of the botulinum TC with x-ray of the individual components. this model a first the complete structure of the botulinum toxin complex. We M. for Protein and E. and H. (Beckman Coulter Inc.) for of the HA-33·HA-17 and D. (Bruker K. for of the HA-33·HA-17 complex. We K. Y. S. D. Y. S. Y. S. and A. for in the botulinum TC species.