Kimiko Hasegawa

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

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.

HA-33 facilitates transport of the serotype D botulinum toxin across a rat intestinal epithelial cell monolayer.

A large size botulinum toxin complex (L-TC) is composed of a single neurotoxin (BoNT), a single nontoxic nonhaemagglutinin (NTNHA) and a haemagglutinin (HA) complex. The HA complex is comprised of three HA-70 molecules and three arm structures of HA-33/HA-17 that consist of two HA-33 and a single HA-17. In addition to the mature L-TC, smaller TCs are present in cultures: M-TC (BoNT/NTNHA), M-TC/HA-70 and immature L-TCs with fewer HA-33/HA-17 arms than mature L-TC. Because L-TC displays higher oral toxicity than pure BoNT, it was presumed that nontoxic proteins are critical for food poisoning. In this study, the absorption of TCs across intestinal epithelial cells was assessed by examining the cell binding and monolayer transport of serotype D toxins in the rat intestinal epithelial cell line IEC-6. All TCs, including pure BoNT, displayed binding and transport, with mature L-TC showing the greatest potency. Inhibition experiments using antibodies revealed that BoNT, HA-70 and HA-33 could be responsible for the binding and transport. The findings here indicate that all TCs can transport across the cell layer via a sialic acid-dependent process. Nonetheless, binding and transport markedly increased with number of HA-33/HA-17 arms in the TC. We therefore conclude that the HA-33/HA-17 arm is not necessarily required for, but facilitates, transport of botulinum toxin complexes.

Clostridium botulinum serotype D neurotoxin and toxin complex bind to bovine aortic endothelial cells via sialic acid

Botulinum neurotoxin (BoNT) is produced as a large toxin complex (L-TC) associated with nontoxic nonhemagglutinin (NTNHA) and three hemagglutinin subcomponents (HA-70, -33 and -17). The binding properties of BoNT to neurons and L-TC to intestinal epithelial cells are well documented, while those to other tissues are largely unknown. Here, to obtain novel insights into the pathogenesis of foodborne botulism, we examine whether botulinum toxins bind to vascular endothelial cells. BoNT and 750 kDa L-TC (a complex of BoNT, NTNHA and HAs) of Clostridium botulinum serotype D were incubated with bovine aortic endothelial cells (BAECs), and binding to the cells was assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot. Both BoNT and L-TC bound to BAECs, with L-TC showing stronger binding. Binding of BoNT and L-TC to BAECs was significantly inhibited by N-acetyl neuraminic acid in the cell culture medium or by treatment of the cells with neuraminidase. However, galactose, lactose or N-acetyl galactosamine did not significantly inhibit toxin binding to the cells. This is the first report demonstrating that BoNT and L-TC bind to BAECs via sialic acid, and this mechanism may be important in the trafficking pathway of BoNT in foodborne botulism.

Sialic acid-dependent binding and transcytosis of serotype D botulinum neurotoxin and toxin complex in rat intestinal epithelial cells.

A large toxin complex (L-TC) produced by Clostridium botulinum is composed of neurotoxin (BoNT), non-toxic non-hemagglutinin (NTNHA) and hemagglutinin subcomponents (HA-70, -33 and -17). In animal botulism, BoNT or L-TC is internalized by intestinal epithelial cells. Previous studies showed that L-TC binds to intestinal cells via sugar chains on the cell surface, but the role of toxin binding to sugar chains in the toxin absorption from intestine is unclear. To clarify whether the toxin binding to sugar chains on intestinal cell surface leads to its transcytosis across the cells, we examined binding and permeation of BoNT and L-TC of C. botulinum serotype D strain 4947 to the rat intestinal epithelial cell line IEC-6 in semi-permeable filters in Transwell systems. Both BoNT and L-TC bound to and permeated the cell monolayers, with L-TC showing greater binding and permeation. In addition, both binding and permeation of toxins were potently inhibited by N-acetyl neuraminic acid in the cell culture medium or by treatment of the cells with neuraminidase. However, neither galactose, lactose nor N-acetyl galactosamine inhibited binding or permeation of toxins. These results support the idea that permeation of both BoNT and L-TC through the intestinal cell layer depends on prior binding to sialic acid on the cell surface. This is the first report demonstrating that the binding of botulinum toxins to cell surface sialic acid leads to their transcytosis through intestinal epithelial cells.

Small-angle X-ray scattering reveals structural dynamics of the botulinum neurotoxin associating protein, nontoxic nonhemagglutinin

In cell culture supernatants, the botulinum neurotoxin (BoNT) exists as part of a toxin complex (TC) in which nontoxic nonhemagglutinin (NTNHA) and/or hemagglutinins (HAs) are assembled onto the BoNT. A series of investigations indicated that formation of the TC is vital for delivery of the toxin to nerve cells through the digestive tract. In the assembly process, BoNT binds to NTNHA yielding M-TC, and it then matures into L-TC by further association with the HAs via NTNHA in the M-TC. Here, we report a crystal structure of the NTNHA from Clostridium botulinum serotype D strain 4947. Additionally, we performed small-angle X-ray scattering (SAXS) analysis of the NTNHA and the M-TC to elucidate the solution structure. The crystal structure of D-4947 NTNHA revealed that BoNT and NTNHA share a closely related structure consisting of three domains. The SAXS image indicated that, even though the N-terminal two-thirds of the NTNHA molecule had an apparently similar conformation in both the crystal and solution structures, the C-terminal third of the molecule showed a more extended structure in the SAXS image than that seen in the crystallographic image. The discrepancy between the crystal and solution structures implies a high flexibility of the C-terminal third domain of NTNHA, which is involved in binding to BoNT. Structural dynamics of the NTNHA molecule revealed by SAXS may explain its binding to BoNT to form the BoNT/NTNHA complex.

Sugar-induced conformational change found in the HA-33/HA-17 trimer of the botulinum toxin complex

Large-sized botulinum toxin complex (L-TC) is formed by conjugation of neurotoxin, nontoxic nonhemagglutinin and hemagglutinin (HA) complex. The HA complex is formed by association of three HA-70 molecules and three HA-33/HA-17 trimers, comprised of a single HA-17 and two HA-33 proteins. The HA-33/HA-17 trimer isolated from serotype D L-TC has the ability to bind to and penetrate through the intestinal epithelial cell monolayer in a sialic acid-dependent manner, and thus it plays an important role in toxin delivery through the intestinal cell wall. In this study, we determined the solution structure of the HA-33/HA-17 trimer by using small-angle X-ray scattering (SAXS). The SAXS image of HA-33/HA-17 exhibited broadly similar appearance to the crystal image of the complex. On the other hand, in the presence of N-acetylneuraminic acid, glucose and galactose, the solution structure of the HA-33/HA-17 trimer was drastically altered compared to the structure in the absence of the sugars. Sugar-induced structural change of the HA-33/HA-17 trimer may contribute to cell binding and subsequent transport across the intestinal cell layer.

Molecular Characterization of GroES and GroEL Homologues from Clostridium botulinum

We report novel findings of significant amounts of 60- and 10-kDa proteins on SDS-PAGE in a culture supernatant of the Clostridium botulinum type D strain 4947 (D-4947). The N-terminal amino acid sequences of the purified proteins were closely related to those of other bacterial GroEL and GroES proteins, and both positively cross-reacted with Escherichia coli GroEL and GroES antibodies. Native GroEL homologue as an oligomeric complex is a weak ATPase whose activity is inhibited by the presence of GroES homologue. The 2634-bp groESL operon of D-4947 was isolated by PCR and sequenced. The sequence included two complete open reading frames (282 and 1629 bp), which were homologous to the groES and groEL gene family of bacterial proteins. Southern and Northern blot analyses indicate that the groESL operon is encoded on the genomic DNA of D-4947 as a single copy, and not on that of its specific toxin-converting phage.

Crystallization and preliminary X-ray analysis of the Clostridium botulinum type D nontoxic nonhaemagglutinin.

Clostridium botulinum produces botulinum neurotoxin (BoNT) as a large toxin complex assembled with nontoxic nonhaemagglutinin (NTNHA) and/or haemagglutinin components. Complex formation with NTNHA is considered to be critical in eliciting food poisoning because the complex shields the BoNT from the harsh conditions in the digestive tract. In the present study, NTNHA was expressed in Escherichia coli and crystallized. Diffraction data were collected to 3.9 Å resolution. The crystal belonged to the trigonal space group P321 or P3(1)21/P3(2)21, with unit-cell parameters a = b = 147.85, c = 229.74 Å. The structure of NTNHA will provide insight into the assembly mechanism that produces the unique BoNT-NTNHA complex.

Cyclopropane‐Based Peptidomimetics Mimicking Wide‐Ranging Secondary Structures of Peptides: Conformational Analysis and Their Use in Rational Ligand Optimization

Detailed conformational analyses of our previously reported cyclopropane-based peptidomimetics and conformational analysis-driven ligand optimization are described. Computational calculations and X-ray crystallography showed that the characteristic features of cyclopropane function effectively to constrain the molecular conformation in a three-dimensionally diverse manner. Subsequent principal component analysis revealed that the diversity covers the broad chemical space filled by peptide secondary structures in terms of both main-chain and side-chain conformations. Based on these analyses, a lead stereoisomer targeting melanocortin receptors was identified, and its potency and subtype selectivity were improved by further derivatization. The presented strategy is effective not only for designing non-peptidic ligands from a peptide ligand but also for the rational optimization of these ligands based on the plausible target-binding conformation without requiring the three- dimensional structural information of the target and its peptide ligands.

Building-block architecture of botulinum toxin complex: Conformational changes provide insights into the hemagglutination ability of the complex

Clostridium botulinum produces the botulinum neurotoxin (BoNT). Previously, we provided evidence for the “building-block” model of botulinum toxin complex (TC). In this model, a single BoNT is associated with a single nontoxic nonhemagglutinin (NTNHA), yielding M-TC; three HA-70 molecules are attached and form M-TC/HA-70, and one to three “arms” of the HA-33/HA-17 trimer (two HA-33 and one HA-17) further bind to M-TC/HA-70 via HA-17 and HA-70 binding, yielding one-, two-, and three-arm L-TC. Of all TCs, only the three-arm L-TC caused hemagglutination. In this study, we determined the solution structures for the botulinum TCs using small-angle X-ray scattering (SAXS). The mature three-arm L-TC exhibited the shape of a “bird spreading its wings”, in contrast to the model having three “arms”, as revealed by transmission electron microscopy. SAXS images indicated that one of the three arms of the HA-33/HA-17 trimer bound to both HA-70 and BoNT. Taken together, these findings regarding the conformational changes in the building-block architecture of TC may explain why only three-arm L-TC exhibited hemagglutination.