Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters

Methods that do not require animal sacrifice to detect botulinum neurotoxins (BoNTs) are critical for BoNT antagonist discovery and the advancement of quantitative assays for biodefense and pharmaceutical applications. Here we describe the development and optimization of fluorogenic reporters that detect the proteolytic activity of BoNT/A, B, D, E, F, and G serotypes in real time with femtomolar to picomolar sensitivity. Notably, the reporters can detect femtomolar concentrations of BoNT/A in 4 h and BoNT/E in 20 h, sensitivity that equals that of animal-based methods. The reporters can be used to determine the specific activity of BoNT preparations with intra- and inter-assay coefficients of variation of approximately 10%. Finally, we find that the greater sensitivity of our reporters compared with those used in other commercially available assays makes the former attractive candidates for high-throughput screening of BoNT antagonists

Novel long-chain neurotoxins from Bungarus candidus distinguish the two binding sites in muscle-type nicotinic acetylcholine receptors

αδ-Bungarotoxins, a novel group of long-chain α-neurotoxins, manifest different affinity to two agonist/competitive antagonist binding sites of muscle-type nicotinic acetylcholine receptors (nAChRs), being more active at the interface of α–δ subunits. Three isoforms (αδ-BgTx-1–3) were identified in Malayan Krait (Bungarus candidus) from Thailand by genomic DNA analysis; two of them (αδ-BgTx-1 and 2) were isolated from its venom. The toxins comprise 73 amino acid residues and 5 disulfide bridges, being homologous to α-bungarotoxin (α-BgTx), a classical blocker of muscle-type and neuronal α7, α8, and α9α10 nAChRs. The toxicity of αδ-BgTx-1 (LD50 = 0.17–0.28 µg/g mouse, i.p. injection) is essentially as high as that of α-BgTx. In the chick biventer cervicis nerve–muscle preparation, αδ-BgTx-1 completely abolished acetylcholine response, but in contrast with the block by α-BgTx, acetylcholine response was fully reversible by washing. αδ-BgTxs, similar to α-BgTx, bind with high affinity to α7 and muscle-type nAChRs. However, the major difference of αδ-BgTxs from α-BgTx and other naturally occurring α-neurotoxins is that αδ-BgTxs discriminate the two binding sites in the Torpedo californica and mouse muscle nAChRs showing up to two orders of magnitude higher affinity for the α–δ site as compared with α–ε or α–γ binding site interfaces. Molecular modeling and analysis of the literature provided possible explanations for these differences in binding mode; one of the probable reasons being the lower content of positively charged residues in αδ-BgTxs. Thus, αδ-BgTxs are new tools for studies on nAChRs

The Effects of Biogeography on Ant Diversity and Activity on the Boston Harbor Islands, Massachusetts, U.S.A

Many studies have examined how island biogeography affects diversity on the scale of island systems. In this study, we address how diversity varies over very short periods of time on individual islands. To do this, we compile an inventory of the ants living in the Boston Harbor Islands National Recreation Area, Boston, Massachusetts, USA using data from a five-year All Taxa Biodiversity Inventory of the region's arthropods. Consistent with the classical theory of island biogeography, species richness increased with island size, decreased with island isolation, and remained relatively constant over time. Additionally, our inventory finds that almost half of the known Massachusetts ant fauna can be collected in the BHI, and identifies four new species records for Massachusetts, including one new to the United States, Myrmica scabrinodis. We find that the number of species actually active on islands depended greatly on the timescale under consideration. The species that could be detected during any given week of sampling could by no means account for total island species richness, even when correcting for sampling effort. Though we consistently collected the same number of species over any given week of sampling, the identities of those species varied greatly between weeks. This variation does not result from local immigration and extinction of species, nor from seasonally-driven changes in the abundance of individual species, but rather from weekly changes in the distribution and activity of foraging ants. This variation can be upwards of 50% of ant species per week. This suggests that numerous ant species on the BHI share the same physical space at different times. This temporal partitioning could well explain such unexpectedly high ant diversity in an isolated, urban site

Residues at the Subunit Interfaces of the Nicotinic Acetylcholine Receptor That Contribute to α-Conotoxin M1 Binding

International audienceThe two binding sites in the pentameric nicotinic acetylcholine receptor of subunit composition alpha2 beta gamma delta are formed by nonequivalent alpha-gamma and alpha-delta subunit interfaces, which produce site selectivity in the binding of agonists and antagonists. We show by sedimentation analysis that 125I-alpha-conotoxin M1 binds with high affinity to the alpha-delta subunit dimers, but not to alpha-gamma dimers, nor to alpha, gamma, and delta monomers, a finding consistent with alpha-conotoxin M1 selectivity for the alpha delta interface in the intact receptor measured by competition against alpha-bungarotoxin binding. We also extend previous identification of alpha-conotoxin M1 determinants in the gamma and delta subunits to the alpha subunit interface by mutagenesis of conserved residues in the alpha subunit. Most mutations of the alpha subunit affect affinity similarly at the two sites, but Tyr93Phe, Val188Lys, Tyr190Thr, Tyr198Thr, and Asp152Asn affect affinity in a site-selective manner. Mutant cycle analysis reveals only weak or no interactions between mutant alpha and non-alpha subunits, indicating that side chains of the alpha subunit do not interact with those of the gamma or delta subunits in stabilizing alpha-conotoxin M1. The overall findings suggest different binding configurations of alpha-conotoxin M1 at the alpha-delta and alpha-gamma binding interfaces

Toxins selective for subunit interfaces as probes of nicotinic acetylcholine receptor structure

International audienceThe pentameric structure of the nicotinic acetylcholine receptor with two of the five subunit interfaces serving as ligand binding sites offers an opportunity to distinguish features on the surfaces of the subunits and their ligand specificity characteristics. This problem has been approached through the study of assembly of subunits and binding characteristics of selective peptide toxins. The receptor, with its circular order of homologous subunits (αγαδβ), assembles in only one arrangement, and through mutagenesis, the residues governing assembly can be ascertained. Selectivity of certain toxins is sufficient to readily distinguish between sites at the αγ and αδ interfaces. By interchanging residues on the γ and δ subunits, and ascertaining how they interact with the α-subunit, determinants forming the binding sites can be delineated. The α-conotoxins, which contain two disulfide loops and 12–14 amino acids, show a 10 000-fold preference for the αγ over the αγ subunit interface with αε falling between the two. The waglerins, as 22–24 amino acid peptides with a single core disulfide loop, show a 2000-fold preference for αε over the αγ and αδ interfaces. Finally, the 6700 Da short α-neurotoxin from N. mossambica mossambica shows a 10 000-fold preference for the αγ and αδ interfaces over αε. Selective mutagenesis enables one to also distinguish α-neurotoxin binding at the αγ and αδ subunits. This information, when coupled with homology modeling of domains and site-directed residue modification, reveals important elements of receptor structure and conformation.La structure pentamérique du récepteur nicotinique, dans laquelle deux des cinq interfaces entre sous-unités servent de sites de liaison pour l'acétylcholine, offre la possibilité de distinguer les caractéristiques des deux sites et leur spécificité pour les ligands. Nous avons abordé l'étude de l'assemblage des sous-unités par la liaison de toxines peptidiques sélectives. Le récepteur s'assemble d'une seule manière avec un ordre circulaire unique de sous-unité (αγδαβ), et il est possible d'identifier les résidus qui contrôlent ces interactions par mutagenèse dirigée. La sélectivité de certaines toxines est suffisante pour distinguer clairement les sites des interfaces αγ et αδ. En échangeant des résidus entre les sous-unités α et δ, et en analysant leurs associations avec la sous-unité α, on peut définir les déterminants des sites de liaison. Les conotoxines α formées de 12–14 acides aminés et contenant deux ponts disulfure se lient 10 000 lois mieux au site αγ, et de façon intermédiaire à αε. Les waglérines, formées de 20–24 acides aminés et possédant un seul pont disulfure se lient 2 000 fois mieux à αε qu'à αγ et αδ. Finalement, la neurotoxine α courte de 6 700 Da de Naja mossambica mossambica présente une préférence de 10 000 fois pour les interfaces αγ et αδ, par rapport à αε. La mutagenèse permet aussi de distinguer la liaison de l'α neurotoxine aux sites αγ et αδ. Ces informations, associées à la modélisation des domaines par homologie et la modification de résidus par mutagenè se dirigée, donnent d'importants éléments pour comprendre la structure et la conformation du récepteur

Residues at the Subunit Interfaces of the Nicotinic Acetylcholine Receptor That Contribute to α-Conotoxin M1 Binding

International audienceThe two binding sites in the pentameric nicotinic acetylcholine receptor of subunit composition alpha2 beta gamma delta are formed by nonequivalent alpha-gamma and alpha-delta subunit interfaces, which produce site selectivity in the binding of agonists and antagonists. We show by sedimentation analysis that 125I-alpha-conotoxin M1 binds with high affinity to the alpha-delta subunit dimers, but not to alpha-gamma dimers, nor to alpha, gamma, and delta monomers, a finding consistent with alpha-conotoxin M1 selectivity for the alpha delta interface in the intact receptor measured by competition against alpha-bungarotoxin binding. We also extend previous identification of alpha-conotoxin M1 determinants in the gamma and delta subunits to the alpha subunit interface by mutagenesis of conserved residues in the alpha subunit. Most mutations of the alpha subunit affect affinity similarly at the two sites, but Tyr93Phe, Val188Lys, Tyr190Thr, Tyr198Thr, and Asp152Asn affect affinity in a site-selective manner. Mutant cycle analysis reveals only weak or no interactions between mutant alpha and non-alpha subunits, indicating that side chains of the alpha subunit do not interact with those of the gamma or delta subunits in stabilizing alpha-conotoxin M1. The overall findings suggest different binding configurations of alpha-conotoxin M1 at the alpha-delta and alpha-gamma binding interfaces