@article{oai:nodai.repo.nii.ac.jp:00000495,
 author = {大山, 徹 and Ohyama, Tohru},
 issue = {4},
 month = {2016-04-20},
 note = {ボツリヌス神経毒素(BoNT)は,自然界最強の毒素であり,コリン作動性シナプスからの神経伝達物質放出の阻害によって,ヒトや動物のボツリヌス症として知られる致死的な疾病を引き起こす。ボツリヌス菌株は,BoNT の抗原性の違いにより,A から G 型の血清型に分類され,A,B,E および F 型はヒトに対して,一方 C および D 型は動物や鳥類のボツリヌス症の原因物質とされている。全ての血清型の BoNT には各々の無毒成分タンパク質が非共有結合的に会合して大きな毒素複合体(TC)を形成する。培養液中では,BoNT と非毒非血球凝集素(NTNHA)の複合体(M-TC)とさらに M-TC に 3 種の血球凝集素(HA;HA-70,HA-33 および HA-17)が会合したより大きな複合体(L-TC)が存在する。これらの TC には,構成成分のいくつかには特定の部位には分子内切断(nick)があるため,SDS-PAGE 上で多数のバンドが出現する。C および D 型のボツリヌス菌株から毒素の精製中に著者らは偶然にも無傷の TC 種を産生する特異的な D 型菌 4947 株(D-4947)を見出した。 本論文では,主に特異的 D-4947 の TC に関する主要な知見が述べられている。(1)C および D 型TC 構成成分(BoNT,NTNHA および HA-70)における菌体プロテアーゼあるいは自発的切断によるニック部位が特定された。(2)分離精製した各 TC 構成成分による L-TC の再構成に成功し,その形成機構を明らかにし,各構成成分遺伝子の発現が調べられた。(3)各種培養細胞系を用いて,C および D 型 L-TC の HA-33 が小腸内皮細胞透過において本質的な役割を果たしていた。(4)電顕観察および HA-33/HA-17 複合体の X 線結晶解析により,個々のサブユニットが会合する経路と 14 量体からなる L-TC 各サブユニットの立体的配置を初めて提唱した。(5)消化酵素耐性実験から,NTNHA が BoNT を消化から保護している決定的な証拠を提供した。また,NTNHA 分子は 1 個の亜鉛分子を含み,BoNT との構造的類似性が認められ,X 線小角散乱分析から NTNHA の水溶液中での動的構造の性質が示された。, Botulinum neurotoxins (BoNTs) are the most potent toxins known in nature, causing the lethal disease known as botulism in human and animals. The BoNTs act by inhibiting neurotransmitter release from cholinergic synapses. Accidental botulism often occurs through ingestion of Clostridium botulinum contaminated food. Different strains of C. botulinum produce seven distinct serotypes of BoNTs, classified A through G. Serotypes A, B, E and F in human botulism, whereas C and D appear to be causative toxins for animal and avian botulism. All serotypes of BoNT associate non-covalently with auxiliary nontoxic proteins, thereby forming large toxin complexes (TCs), M-TC (BoNT/NANHA) and L-TC (BoNT/NTNHA/HA-70/HA-33/HA-17) in the culture medium. The formation of TCs appear not only to protect BoNTs from the hostile environment of the digestive tract but also to assist neurotoxin translocation across the intestinal mucosal layer. However, BoNT, NTNHA and HA-70 components of the TC are nicked at specific sites by a bacterial protease, leading to the appearance of many fragments on SDS-PAGE. Fortunately, the author and collaborators serendipitously found unique serotype D strain 4947 (D-4947) which produces intact M-TC and L-TC without any nicking. In this manuscript, five topics on mainly D-4947 TC studies provided by the author and collaborators are described: (1) Specific nicking sites in the components of the serotype C and D TCs were characterized, including dichain structure in BoNT, spontaneous nicking in the NTNHA and endogenous cleavage in HA-70. (2) In vitro reconstitution of functional HAs of serotype C, and D-4947 L-TC assembly mechanism was achieved by the mixing of individual components, which was indistinguishable with native L-TC. Then the gene expressions of five individual D-4947 L-TC components were examined by quantitative reverse transcriptase PCR. (3) The HA-33 component of the serotypes C and D TCs played a critical role in the binding and transcytosis in intestinal epithelium, and other HA components protecting BoNT against gastrointestinal digestion, using various culture cells including Caco-2 cells, rat small intestinal epithelial cells, bovine aortic endothelial cells and equine erythrocytes. (4) A novel 14-mer subunit structure model of D-4947 L-TC was proposed on the basis of the negative stain transmission electron microscopy and X-ray crystal structure of the complex formed by two HA-33 plus one HA-17. (5) Definitive evidence was provided that both recombinant and native NTNHAs play a crucial role in protecting BoNT from proteolysis by digestive enzymes. Every single NTNHA contained a single Zn atom, of which small-angle X-ray scattering analysis of the NTNHA revealed the structural dynamics., E, 1, KJ00008376179, 綜説, Review},
 pages = {223--232},
 title = {ボツリヌス毒素複合体の構造と機能},
 volume = {57},
 year = {},
 yomi = {オオヤマ, トオル}
}