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Construction of Artificial Capsule by Using Novel Bio- Materials

利用新型生物材料构建人工胶囊

基本信息

批准号：
11470078
负责人：
HANDA Hiroshi
金额：
$ 7.87万
依托单位：
Tokyo Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

Adeno-associated virus (AAV) capsids are composed of three proteins, VP1, VP2 and VP3. Recombinant protein of VP2 and VP3 are prepared. The simian virus 40 capsid is composed of 72 pentamers of VP1 protein. Although the capsid is known to dissociate to pentamers in vitro following simultaneous treatment with reducing and chelating agents, the functional roles of disulfide linkage and calcium ion-mediated interactions are not clear. To elucidate the roles of these interactions, we introduced amino acid substitutions in VP1 at cysteine residues and at residues involved in calcium binding. We expressed the mutant proteins in a baculovirus system and analyzed both their assembly into virus-like particles (VLPs) in insect cells and the disassembly of those VLPs in vitro. We found that disulfide linkages at both Cys-9 and Cys-104 conferred resistance to proteinase K digestion on VLPs, although neither linkage was essential for the formation of VLPs in insect cells. In particular, reduction of the disulfide linkage at Cys-9 was found to be critical for VLP dissociation to VP1 pentamers in the absence of calcium ions, indicating that disulfide linkage at Cys-9 prevents VLP dissociation, probably by increasing the stability of calcium ion binding. We found that amino acid substitutions at carboxy-terminal calcium ion binding sites (Glu-329, Glu-330, and Asp-345) resulted in the frequent formation of unusual tubular particles as well as VLPs in insect cells, indicating that these residues affect the accuracy of capsid assembly. In addition, unexpectedly, amino acid substitutions at any of the calcium ion binding sites tested, especially at Glu-157, resulted in increased stability of VLPs in the absence of calcium ions in vitro. These results suggest that appropriate affinities of calcium ion binding are responsible for both assembly and disassembly of the capsid.

腺相关病毒（AAV）衣壳由三种蛋白质VP 1、VP 2和VP 3组成。制备了VP 2和VP 3的重组蛋白。猴病毒40衣壳由72个五聚体的VP 1蛋白组成。尽管已知衣壳在体外用还原剂和螯合剂同时处理后解离成五聚体，但二硫键和钙离子介导的相互作用的功能作用尚不清楚。为了阐明这些相互作用的作用，我们在VP 1中的半胱氨酸残基和参与钙结合的残基处引入了氨基酸取代。我们在杆状病毒系统中表达了突变蛋白，并分析了它们在昆虫细胞中组装成病毒样颗粒（VLP）以及这些VLP在体外的分解。我们发现，在Cys-9和Cys-104的二硫键赋予抗性蛋白酶K消化的VLP，虽然没有连接是必不可少的VLP在昆虫细胞中的形成。特别地，发现在Cys-9处的二硫键的还原对于在不存在钙离子的情况下VLP解离成VP 1五聚体是关键的，这表明在Cys-9处的二硫键可能通过增加钙离子结合的稳定性来防止VLP解离。我们发现羧基端钙离子结合位点（Glu-329、Glu-330和Asp-345）的氨基酸取代导致在昆虫细胞中频繁形成不寻常的管状颗粒以及VLP，表明这些残基影响衣壳组装的准确性。此外，出乎意料的是，在所测试的任何钙离子结合位点处，尤其是在Glu-157处的氨基酸取代导致VLP在体外不存在钙离子的情况下的稳定性增加。这些结果表明，适当的亲和力的钙离子结合负责组装和拆卸的衣壳。