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

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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