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

研究结果如下：对中间溶素(ILY)及其相关毒素进行了分子模拟，获得了鉴定ILY细胞膜结合部位的有价值的分子信息。在此基础上，合成了多种具有ILY细胞膜结合区(ILY4D)部分一级结构的多肽，并对其竞争抑制作用进行了测试。此外，利用ILY及其近亲的嵌合体确定了人特异性结合所需的最小结构，发现该结构位于ILY4D的后半部分，除十一肽区域外，其余56个残基。然而，由于ILY4D的膜识别需要ILY4D的三级结构，其部分结构的短肽和ILY4D的C端56肽不能很好地应用于细胞膜结合模块。另一方面，尽管分子大小是大的…在此基础上，成功地制备了两个具有独立间隔区大小的ILY4D重组体和一个无毒的ILY突变体(ILY-SS)，该突变体具有完整的ILY4D结构，并在每个分子的N端有一个锚基(Cys)，作为细胞膜结合模块。将ILY4D模块与抗癌胚抗原(CEA)单抗的(Fab‘)2片段偶联，证实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，认为这些分子可用于肿瘤的靶向细胞免疫治疗，ILY-SS组件也可用于细胞膜修饰技术。此外，我们还构建了在N端分别与这两种模块融合的表达载体系统。使用这些模块的导弹细胞免疫治疗和用于基因治疗的药物输送系统的开发有望在今后进行。较少

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