| Project/Area Number |
13660323
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied veterinary science
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| Research Institution | Osaka Prefecture University |
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Principal Investigator |
KOZAKI Shunji Osaka Prefecture University, Graduate School of Agriculture and Biological Sciences, Professor, 農学生命科学研究科, 教授 (10109895)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2002: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2001: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Keywords | Clostridium botulinum / neurotoxin / receptor / site-directed mutagenesis / glycoprotein / ボツリヌス症 / 鳥類ボツリヌス症 / シナプトソーム |
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| Research Abstract |
Clostridium botulinum neurotoxin (BoNT) binds presynaptic neuronal cells and inhibits specifically neurotransmitter release. We have reported that BoNT/B recognizes a complex of synaptotagmin 2 (Stg2) and ganglioside GT1b as a type-specific receptor. We were cloning carboxyl-terminal heavy chain (Hc) gene and expressed the recombinant He in E. coli. The recombinant Hc inhibited ^<125>I-labeled BoNT/B binding to the Stg2/GT1b lipid vesicles and rat brain synaptosomes to the same extent, in comparison with unlabeled BoNT/B, suggesting that the recombinant Hc retains binding properties of holotoxin to the receptor. The blockade of neurotransmitter release by BoNT/B at the neuromuscular junction (NMJ) was rescued in the presence of recombinant Hc. We then constructed several site-directed mutants of Hc to investigate the role of individual residues in recognition of the receptor and GT1b. The binding capabilities of Hc mutants to the receptor and GTlb were examined by competition experiments with ^<125>I-labeled Hc. Three mutants, S1259A, W1261A and Y1262A, were found to drastically decrease the binding activities to the receptor and GT1b, although W1261 and Y1262 have been identified as contact residues between Hc domain and sialyllactose. Interestingly, K1260A hardly inhibited ^<125>I-labeled Hc binding to GT1b, whereas the binding of ^<125>I-labeled Hc to the receptor was effectively interfered by K1260A. From these results, it seems that the residues relating with ganglioside binding do not necessarily participate in formation of receptor recognition site. In the Hc derived from type C neurotoxins, the recombinant bound to the extract from chicken brain synaptosomes in the absence of ganglioside. We then purified further the binding substance by ion exchange and hydrophobic chromatography. Far-western analysis revealed that a acidic glycoprotein interacted with the Hc.
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