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Construction of innovated drug delivery system for proteins and peptides by alteration of botulinum toxin complex

通过改变肉毒毒素复合物构建创新的蛋白质和肽药物递送系统

基本信息

批准号：
17300161
负责人：
OHYAMA Tohru
金额：
$ 10.09万
依托单位：
Tokyo University of Agriculture
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

项目摘要

Clostridium botulinum has long been known to cause food-bone illness, intestinal and wounded toxemias in humans, and outbreaks of botulism in wild and domestic animals. The botulinum neurotoxins (BoNTs) are the most potent toxins known in nature and elicit a potentially fatal paralysis, through the inhibition of neurotransmitter release from cholinergic synapses. C. botulinum strains produce BoNTs as large as toxin complex (L-TC) form consisted of auxiliary non-toxic proteins, which are well documented to play a critical role in food-borne botulism by not only protecting the BoNT from low pH and proteases in gastrointestinal tract but also by assisting BoNT translocation across the intestinal mucosal layer. Unfortunately, even the image of subunit structure of the botulinum toxin complex never been captured, due to its large sizes associated with multiple non-toxic components varying from 300kDa up to 900kDA as determined by equilibrium sedimentation analysis depending upon the BoNT t … More ypes. Here we the first visualized a series of the serotype D botulinum toxin complex forms by negative stain transmission electron microscopy (TEM) , which are assembled with 150 kDa BoNT, 130 kDa non-toxic non-hemagglutinin (NTNHA) , and three kinds of hemagglutinin subcomponents, 70 kDa HA-70, 30 kDa HA-30 and 17 kDa HA-17, according to the sequence-specific binding order. A novel TEM image of the mature L-TC reveals a ellipsoidal shaped appearance attached with "three arms". The body section was comprised of single BoNT, single NTNHA and three HA-70 molecules. The arm section was made up of the complex of HA-33 and HA-17 molecules. We for the first determined the X-ray crystal structure of the complex composed of two HA-33 and single HA-17 molecules. On the basis of the TEM image and biochemical results in this work, we propose 14-mer subunit structure model (comprise single BoNT, single NTNHA, three HA-70, six HA-30 and three HA-17 molecules) for botulinum toxin mature complex.Regarding the subunit structure of botulinum toxin complex, there has historically been only our schematic model imaginarily constructed based on biochemical results. In this work, we have achieved a major break-through since we are able to demonstrate the first subunit structure of a botulinum serotype D neurotoxin complex, which will be reasonably supported by the results derived from the experiments that were very carefully performed. We believe that this structure is successfully made due to use of serotype D unique strain 4947 (D-4947) . Unlike other serotype stains, D-4947 toxin complex consists only of intact components without any nicking in the subunit components including BoNTs, implying escape from further complicate the analysis of subunit structure due to the appearance of many fragments. Accordingly, we could demonstrate an assembly pathway including the binding order of individual subunits through in vitro reconstitution of the toxin complex, using isolated each subunit. This novel and unusual model will rationally explain that non-toxic components make up "delivery vehicle" for BoNT in food-borne botulism, which it can be safely protected from the digestive tract, and can help the BoNT to internalization into the bloodstream. In other word, the BoNT is armored with non-toxic proteins and subsequently HA-30 proteins at forefront catch on to the intestine cell, leading that the BoNT releases from the complex into the inner cell under the physiological conditions. To the best of our knowledge, such unique system has been known in only botulinum toxin among the protein toxins produced by bacteria. Thereby, we can show the insight into that botulinum non-toxic complex might be used as a novel drug delivery system for therapeutic and functional protein agents. We believe that model could be the first step to complete crystallographic analysis of overall botulinum toxin complex, which will be taken in near future. Less

肉毒梭菌长期以来一直被认为会引起食物性骨病、人类肠道和创伤性毒血症以及野生动物和家畜肉毒中毒的爆发。肉毒杆菌神经毒素（BoNT）是自然界中已知的最强的毒素，通过抑制胆碱能突触释放神经递质而引起潜在的致命性麻痹。C.肉毒杆菌菌株产生大至由辅助无毒蛋白质组成的毒素复合物（L-TC）形式的BoNT，所述辅助无毒蛋白质被充分证明在食源性肉毒杆菌中毒中发挥关键作用，不仅保护BoNT免受胃肠道中的低pH和蛋白酶的影响，而且还通过协助BoNT移位穿过肠粘膜层。不幸的是，甚至肉毒杆菌毒素复合物的亚基结构的图像也从未被捕获，这是由于其与多种无毒组分相关的大尺寸从300 kDa到900 kDA变化，如通过平衡沉降分析确定的，这取决于BoNT浓度。 ...更多信息类型本文首次通过负染透射电镜（TEM）观察到了一系列由150 kDa BoNT、130 kDa无毒非血凝素（NTNHA）和三种血凝素亚组分70 kDa HA-70、30 kDa HA-30和17 kDa HA-17按照序列特异性结合顺序组装而成的D型肉毒毒素复合物。成熟L-TC的一个新的TEM图像揭示了一个椭圆形的外观与“三个手臂”。主体部分由单个BoNT、单个NTNHA和三个HA-70分子组成。臂部分由HA-33和HA-17分子的复合物组成。我们首次测定了由两个HA-33和单个HA-17分子组成的复合物的X射线晶体结构。基于透射电镜图像和生化结果，我们提出了肉毒毒素成熟复合物的14-mer亚基结构模型（包括单个BoNT、单个NTNHA、3个HA-70、6个HA-30和3个HA-17分子）。在这项工作中，我们已经取得了重大突破，因为我们能够证明肉毒杆菌血清型D神经毒素复合物的第一个亚基结构，这将得到来自非常仔细进行的实验的结果的合理支持。我们认为，由于使用血清型D独特菌株4947（D-4947），成功制备了该结构。与其他血清型菌株不同，D-4947毒素复合物仅由完整组分组成，包括BoNT在内的亚基组分中没有任何切口，这意味着避免了由于许多片段的出现而使亚基结构的分析进一步复杂化。因此，我们可以证明组装途径，包括通过在体外重建的毒素复合物的各个亚基的结合顺序，使用分离的每个亚基。这一新颖而不寻常的模型将合理地解释无毒成分构成了食物中毒中BoNT的“运载工具”，它可以安全地免受消化道的影响，并可以帮助BoNT内化到血液中。换句话说，BoNT被无毒蛋白质装甲，随后HA-30蛋白质在最前沿捕获肠细胞，导致BoNT在生理条件下从复合物释放到内细胞中。据我们所知，在细菌产生的蛋白质毒素中，这种独特的系统仅在肉毒杆菌毒素中已知。因此，我们可以显示的洞察，肉毒杆菌无毒复合物可能被用作一种新的药物输送系统的治疗和功能蛋白剂。我们相信，该模型可能是第一步，以完成整个肉毒毒素复合物的晶体学分析，这将在不久的将来。少

项目成果

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专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

A novel electron microscopy structure of the toxin complex produced by the Clostridium botulinum serotpe D strain 4947

肉毒杆菌 Serotpe D 菌株 4947 产生的毒素复合物的新型电子显微镜结构

DOI：
发表时间：
2007
期刊：
影响因子：
0
作者：
Hasegawa;K.;et. al.
通讯作者：
et. al.

Characterization of trypsin like protease produced by Clostriduim botulinum type C strain Stockholm

C 型肉毒杆菌菌株斯德哥尔摩产生的胰蛋白酶样蛋白酶的表征

DOI：
发表时间：
期刊：
影响因子：
0
作者：
Saitoh;R.;Suzuki;T.;Yoneyama;T.;Mutoh;S.;Hasegawa;K.;Munehiro;M.;Niwa;K.;Watanabe;T.;Ohyama;T
通讯作者：
T

Current Topics in Food Science and Technology

食品科学与技术当前话题

DOI：
发表时间：
2005
期刊：
影响因子：
0
作者：
Saitoh;R.;Suzuki;T.;Yoneyama;T.;Mutoh;S.;Hasegawa;K.;Munehiro;M.;Niwa;K.;Watanabe;T.;Ohyama;T;Toshihiro Watanabe
通讯作者：
Toshihiro Watanabe

Cell internalization and traffic pathway of Clostridium botulinum type C neurotoxin in HT-29 cells