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

人们早就知道肉毒梭菌能引起人类食源性骨病、肠道和创伤性毒血症，以及野生动物和家畜肉毒杆菌中毒的爆发。肉毒杆菌神经毒素（BoNTs）是自然界已知的最有效的毒素，通过抑制胆碱能突触的神经递质释放，引起潜在的致命瘫痪。肉毒杆菌菌株产生的BoNT与由辅助无毒蛋白组成的毒素复合物（L-TC）一样大，这在食源性肉毒中毒中发挥了重要作用，不仅保护BoNT免受胃肠道低pH值和蛋白酶的影响，而且还帮助BoNT在肠粘膜层转运。不幸的是，即使是肉毒杆菌毒素复合物的亚基结构图像也从未被捕获，因为它的大尺寸与多种无毒成分相关，从300kDa到900kDA不等，这是由平衡沉降分析确定的，取决于BoNT。本研究首次通过透射电镜（TEM）观察到一系列血清型D型肉毒毒素复合物，这些复合物由150 kDa BoNT、130 kDa无毒非血凝素（NTNHA）和3种血凝素亚组分（70 kDa HA-70、30 kDa HA-30和17 kDa HA-17）根据序列特异性结合顺序组装而成。成熟L-TC的TEM图像显示其呈椭球状，呈“三臂”状。体切片由一个BoNT、一个NTNHA和三个HA-70分子组成。臂部分由HA-33和HA-17分子复合物组成。我们首次确定了由两个HA-33和单个HA-17分子组成的配合物的x射线晶体结构。基于TEM图像和本工作的生化结果，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、三个HA-70、六个HA-30和三个HA-17分子）。关于肉毒杆菌毒素复合物的亚基结构，历史上只有我们基于生化结果想象构建的示意图模型。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，这将得到非常仔细执行的实验结果的合理支持。我们认为，由于使用了D血清型独特菌株4947 (D-4947)，该结构得以成功制造。与其他血清型染色不同，D-4947毒素复合物仅由完整的组分组成，包括bont在内的亚基组分中没有任何缺口，这意味着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，我们可以通过体外重组毒素复合物，使用分离的每个亚基，来证明包括单个亚基结合顺序的组装途径。这个新颖而不寻常的模型将合理地解释，在食源性肉毒杆菌中毒中，无毒成分构成了BoNT的“运载工具”，它可以安全地保护BoNT不受消化道的影响，并有助于BoNT内化到血液中。换句话说，BoNT被无毒蛋白包裹，随后HA-30蛋白在最前面附着在肠细胞上，导致BoNT在生理条件下从复合物释放到细胞内部。据我们所知，这种独特的系统在细菌产生的蛋白质毒素中只有肉毒杆菌毒素是已知的。因此，我们可以表明肉毒杆菌无毒复合物可能用作治疗和功能蛋白制剂的新型药物递送系统。我们相信该模型将是完成整个肉毒毒素复合物晶体学分析的第一步，这将在不久的将来进行。少

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