Singh B R, Giménez J A, DasGupta B R
Food Research Institute, University of Wisconsin, Madison 53706.
Biochim Biophys Acta. 1991 Mar 8;1077(1):119-26. doi: 10.1016/0167-4838(91)90533-6.
Production of botulinum-like neurotoxin by a non-Clostridium botulinum organism has profound implications in the epidemiology of the disease botulism. Molecular topography of the approximately 150 kDa neurotoxic protein produced by Clostridium butyricum (strain 5839) and its activation kinetics were examined and compared with a serologically related botulinum neurotoxin produced by C. botulinum type E to further characterize the butyricum neurotoxin. Botulinum neurotoxin was fully activated within 30 min of incubation with trypsin, whereas butyricum neurotoxin achieved maximum activation within 5 min of incubation. Molecular topography of the two neurotoxins was analyzed in terms of secondary structures and the surface accessibilities of the polypeptide domains containing aromatic amino acids. The secondary structure parameters of the butyricum neurotoxin (alpha-helix 22%, beta-sheet 41% and random coil 37%), as estimated from the far ultraviolet circular dichroic spectra, appeared similar to that of botulinum neurotoxin. (Singh, B.R. and DasGupta, B.R., (1989) Mol. Cell. Biochem. 86, 87). Second derivative ultraviolet spectral analysis revealed 37 and 41 Tyr residues exposed on the surface of butyricum and botulinum neurotoxins, respectively, suggesting a differential surface accessibility of polypeptide segments containing Tyr residues. Fluorescent Trp residues in both the botulinum type E and butyricum neurotoxins were in a relatively hydrophobic environment as indicated by the blue-shifted emission maxima (334 nm). About half of the fluorescent Trp residues of both proteins were accessible to acrylamide, a neutral fluorescence quencher, and appeared to be in a similar molecular environment. The ionic surface probe, I-, quenched the Trp fluorescence of botulinum significantly, but not that of butyricum neurotoxin. Thus, a considerable number of fluorescent Trp residues were apparently located on the surface of the botulinum, but not on that of the butyricum neurotoxin. Botulinum and butyricum neurotoxins, indistinguishable by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, migrated differently in the absence of sodium dodecyl sulfate suggesting difference(s) in their surface charge distribution. These results provide the first report of the secondary and tertiary structure parameters of the neurotoxin produced by a non-botulinum species and comparison of the molecular topography of the neurotoxin with the antigenically related botulinum neurotoxin type E.
一种非肉毒梭菌的生物体产生类肉毒杆菌神经毒素，这对肉毒中毒疾病的流行病学具有深远影响。对丁酸梭菌（菌株5839）产生的约150 kDa神经毒性蛋白的分子拓扑结构及其激活动力学进行了研究，并与E型肉毒梭菌产生的血清学相关肉毒杆菌神经毒素进行比较，以进一步表征丁酸梭菌神经毒素。肉毒杆菌神经毒素与胰蛋白酶孵育30分钟内完全激活，而丁酸梭菌神经毒素在孵育5分钟内达到最大激活。从二级结构以及含有芳香族氨基酸的多肽结构域的表面可及性方面分析了这两种神经毒素的分子拓扑结构。根据远紫外圆二色光谱估计，丁酸梭菌神经毒素的二级结构参数（α-螺旋22%，β-折叠41%，无规卷曲37%）与肉毒杆菌神经毒素相似。（辛格，B.R.和达斯古普塔，B.R.，（1989年）《分子与细胞生物化学》86卷，87页）。二阶导数紫外光谱分析显示，丁酸梭菌和肉毒杆菌神经毒素表面分别有37个和41个酪氨酸残基暴露，这表明含有酪氨酸残基的多肽片段表面可及性存在差异。E型肉毒杆菌和丁酸梭菌神经毒素中的荧光色氨酸残基处于相对疏水的环境中，这由蓝移的发射最大值（334纳米）表明。两种蛋白质约一半的荧光色氨酸残基可被丙烯酰胺（一种中性荧光猝灭剂）接近，且似乎处于相似的分子环境中。离子表面探针I-能显著猝灭肉毒杆菌的色氨酸荧光，但不能猝灭丁酸梭菌神经毒素的荧光。因此，相当数量的荧光色氨酸残基显然位于肉毒杆菌表面，而不在丁酸梭菌神经毒素表面。在十二烷基硫酸钠存在下通过聚丙烯酰胺凝胶电泳无法区分的肉毒杆菌和丁酸梭菌神经毒素，在没有十二烷基硫酸钠时迁移情况不同，这表明它们的表面电荷分布存在差异。这些结果首次报道了非肉毒杆菌物种产生的神经毒素的二级和三级结构参数，并将该神经毒素的分子拓扑结构与抗原相关的E型肉毒杆菌神经毒素进行了比较。
