A modification technique of cell membrane function using the membrane binding site of a cytolysin, intermedilysin

在授权期内，取得了如下成果：对中间溶素（intermedilysin，ILY）及其相关毒素进行了分子模拟，获得了ILY的分子信息，为确定ILY的细胞膜结合位点提供了有价值的分子信息。基于此信息，合成了具有ILY的细胞膜结合结构域（ILY 4D）的部分一级结构的各种肽，并测试了它们的竞争性抑制作用。此外，利用ILY及其相关基因的嵌合体确定了人特异性结合所需的最小结构，然后发现该结构位于ILY 4D的后半部分，除了十一肽区域外还有56个残基。然而，由于ILY 4D的三级结构对于ILY的膜识别是必需的，因此具有其部分结构的短肽和ILY 4D的C-末端56聚体肽对于应用于细胞膜结合模块并不令人满意。另一方面，虽然分子尺寸较大， 关于我们 除此之外，还成功地制备了两个具有不同间隔区大小的ILY 4D重组体和一个具有二硫键固定其构象的无毒ILY突变体（ILY-SS），它们具有完整的ILY 4D结构和每个分子的N端侧的锚残基（Cys）。将ILY 4D模块与抗CEA单克隆抗体（Fab '）_2片段偶联，证实偶联物包被的人红细胞能稳定、高效、特异地与CEA阳性癌细胞结合。因此，这些分子被认为是适用于导弹细胞免疫治疗癌症，ILY-SS模块也被认为是有用的细胞膜修饰技术。此外，还开发了在其N-末端与两种类型的模块融合的表达所需蛋白的载体系统。利用这些模块可以开发导弹细胞免疫治疗和基因治疗药物输送系统。少

During the granted period, the results were obtained as follows : Molecular modeling of intermedilysin(ILY) and its relative toxins was carried out and valuable molecular information of ILY to identify the cell membrane binding site of ILY was obtained. Based on this information, various peptides with partial primary structure of the cell membrane binding domain of ILY(ILY4D) were synthesized and tested for their competitive inhibitory action. Moreover, the minimum structure necessary for human-specific binding was determined using chimeras of ILY and its relatives, then the structure was found to locate in the latter part of ILY4D with 56 residues except for the undecapeptide region. However, since the tertiary structure of ILY4D was required for membrane recognition of ILY, short peptides with its partial structure and the C-terminal 56mer peptide of ILY4D were not satisfactory for the application to cell membrane binding modules. On the other hand, though the molecular size was larg … More er than such peptides, two recombinants of ILY4D with individual size of spacer and a non-toxic mutant of ILY with a disulfide bridge fixing its conformation (ILY-SS), possessing the complete ILY4D structure and an anchor residue (Cys) at the N-terminal side of each molecule were successfully prepared as cell membrane binding modules. ILY4D modules were conjugated with (Fab')_2 fragment of anti-carcinoembryonic antigen (CEA) monoclonal antibody and it was confirmed that human erythrocytes coated with the conjugates could stably, efficiently and specifically bind to CEA-positive cancer cells. Therefore, it was thought that these molecules were applicable to missile cellular immunotherapy against cancer, ILY-SS module was also suggested to be useful for cell membrane modification technique. Moreover, the vector systems expressing desirable protein fused with both types of module at their N-terminal were developed. Development of missile cellular immunotherapy and Drug delivery system for genetherapy using these modules is expected hereafter. Less