Search Results (3)

Botulinum neurotoxins (BoNTs), which have been exploited as cosmetics and muscle-disorder treatment medicines for decades, are well known for their extreme neurotoxicity to humans. They pose a potential bioterrorism threat because they cause botulism, a flaccid muscular paralysis-associated disease that requires immediate antitoxin treatment and intensive care over a long period of time. In addition to the existing seven established BoNT serotypes (BoNT/A–G), a new mosaic toxin type termed BoNT/HA (aka type FA or H) was reported recently. Sequence analyses indicate that the receptor-binding domain (H_C) of BoNT/HA is ~84% identical to that of BoNT/A1. However, BoNT/HA responds differently to some potent BoNT/A-neutralizing antibodies (e.g., CR2) that target the H_C. Therefore, it raises a serious concern as to whether BoNT/HA poses a new threat to our biosecurity. In this study, we report the first high-resolution crystal structure of BoNT/HA-H_C at 1.8 Å. Sequence and structure analyses reveal that BoNT/HA and BoNT/A1 are different regarding their binding to cell-surface receptors including both polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Furthermore, the new structure also provides explanations for the ~540-fold decreased affinity of antibody CR2 towards BoNT/HA compared to BoNT/A1. Taken together, these new findings advance our understanding of the structure and function of this newly identified toxin at the molecular level, and pave the way for the future development of more effective countermeasures. Full article

Botulinum neurotoxins (BoNTs) exhibit extraordinary potency due to their exquisite neurospecificity, which is achieved by dual binding to complex polysialo-gangliosides and synaptic vesicle proteins. The luminal domain 4 (LD4) of the three synaptic vesicle glycoprotein 2 isoforms, SV2A‐C, identified as protein receptors for the most relevant serotype BoNT/A, binds within the 50 kDa cell binding domain H_C of BoNT/A. Here, we deciphered the BoNT/A‐SV2 interactions in more detail. In pull down assays, the binding of H_CA to SV2-LD4 isoforms decreases from SV2C >> SV2A > SV2B. A binding constant of 200 nM was determined for BoNT/A to rat SV2C-LD4 in GST pull down assay. A similar binding constant was determined by surface plasmon resonance for H_CA to rat SV2C and to human SV2C, the latter being slightly lower due to the substitution L563F in LD4. At pH 5, as measured in acidic synaptic vesicles, the binding constant of H_CA to hSV2C is increased more than 10-fold. Circular dichroism spectroscopy reveals that the quadrilateral helix of SV2C-LD4 already exists in solution prior to BoNT/A binding. Hence, the BoNT/A‐SV2C interaction is of different nature compared to BoNT/B‐Syt-II. In particular, the preexistence of the quadrilateral β-sheet helix of SV2 and its pH-dependent binding to BoNT/A via backbone–backbone interactions constitute major differences. Knowledge of the molecular details of BoNT/A‐SV2 interactions drives the development of high affinity peptides to counteract BoNT/A intoxications or to capture functional BoNT/A variants in innovative detection systems for botulism diagnostic. Full article

A new ganglioside transformed strain isolated from soil was identified as Cellulosimicrobium sp. 21. It produced a sialidase which transformed polysialo-gangliosides GD1 and GT1 into a monosialoterahexosylganglioside, i.e., ganglioside GM1. The sialidase had both NeuAc-α-2,3- and NeuAc-α-2,8-sialidase activity without producing asiolo-GM1. The optimum conditions were evaluated and it was found that the transformation was optimally performed at 30 °C and pH 7.0. The substrate should be added at the beginning of the reaction and the concentration of substrate was 3% (w/v). Under these optimum conditions, Cellulosimicrobium sp. 21 converted GD1 and GT1 into GM1 in inorganic medium in a 5 L bioreactor with the recovery rate of 69.3%. The product contained 50.3% GM1 and was purified on silica to give the product with 95% of GM1 with a recovery rate of 30.5%. Therefore, Cellulosimicrobium sp. 21 has potential to be applied in the production of GM1 in the pharmaceutical industry. Full article