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Structure and function of bacterial phospholipase C

细菌磷脂酶C的结构和功能

基本信息

批准号：
10670275
负责人：
NAGAHAMA Masahiro
金额：
$ 2.05万
依托单位：
Tokushima Bunri University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

项目摘要

N-domain(1 -250residues ; CP_<1-250>) of alpha-toxin and Bacillus cereus phospholipase C (BcPLC) possessed phospholipase C(PLC) activity of about 2% of wild-type alpha-toxin (WT) activity, but not hemolytic activity. PLC and hemolytic activities of C.bifermentans PLC(CbPLC) were about 10% and 1% of these activities of WT, respectively. These activities of the hybrid protein consisting of CP_<1-250> and the C-domain of CbPLC(CP_<1-250>-CB_<251-372>) were lower than those of WT and the activities of the hybrid protein consisting of the N-domain of CbPLC and the C-domain of alpha-toxin(CB_<1-250>-CP_<251-370>) were similar to those of WT.On the other hand, PLC activity of the hybrid protein consisting of BcPLC and the C-domain of alpha-toxin(BC-CP_<251-370>) or CbPLC(BC- CB_<251-372>) was not significantly different from that of BcPLC.However, BC-CP_<251-370> significantly hemolyzed rabbit erythrocytes, but BC-CB_<251-372> did not. WT, CB_<1-250>-CP_<251-370> and BC-CP_<251-370> strongly bound to the red cells, CbPLC and BC-CB_<251-372> bound faintly, and CP_<1-250> and BcPLC not at all. Incubation of CP_<1-250> with CP_<251-370> completely complemented hemolytic and PLC activities. Furthermore, CP_<251-370> significantly conferred hemolytic activity on BcPLC, but inhibited PLC activity of BcPLC.CP_<251-370> significantly stimulated PLC activity of CB_<1-250>, but induced no effect on hemolytic activity. CP_<251-370> significantly inhibited PLC activity of BcPLC.The C-domain mutants with acrylodan attached to residues at position 263 and 365 exhibited a marked blue shift, indicative of movement of the fluorophore to a hydrophobic environment. These observations suggest that interaction of the C-domain of alpha-toxin with fatty acyl residues of phosphatidylcholine plays an important role in biological activities of N-domain of alpha-toxin.

α-毒素的N-结构域(1 -250残基；CP_<1-250>)和蜡状芽孢杆菌磷脂酶C(BcPLC)具有野生型α-毒素(WT)活性的约2％的磷脂酶C(PLC)活性，但没有溶血活性。 C.bifermentans PLC (CbPLC) 的 PLC 和溶血活性分别约为 WT 的 10% 和 1%。由CP_<1-250>和CbPLC的C结构域(CP_<1-250>-CB_<251-372>)组成的杂合蛋白的这些活性低于WT和由CbPLC的N-结构域和α-毒素的C结构域组成的杂合蛋白(CB_<1-250>-CP_<251-370>)的活性。另一方面，由BcPLC和α毒素C结构域(BC-CP_<251-370>)或CbPLC(BC-CB_<251-372>)组成的杂化蛋白的PLC活性与BcPLC没有显着差异。然而，BC-CP_<251-370>显着溶血兔红细胞，但 BC-CB_<251-372> 没有。 WT、CB_<1-250>-CP_<251-370>和BC-CP_<251-370>与红细胞强烈结合，CbPLC和BC-CB_<251-372>微弱结合，而CP_<1-250>和BcPLC根本不结合。 CP_<1-250>与CP_<251-370>的孵育完全补充了溶血和PLC活性。此外，CP_<251-370>显着赋予BcPLC溶血活性，但抑制BcPLC的PLC活性。CP_<251-370>显着刺激CB_<1-250>的PLC活性，但对溶血活性没有影响。 CP_<251-370>显着抑制BcPLC的PLC活性。在263和365位残基上连接有acrylodan的C结构域突变体表现出明显的蓝移，表明荧光团移动至疏水环境。这些观察结果表明，α-毒素的C-结构域与磷脂酰胆碱的脂肪酰基残基的相互作用在α-毒素的N-结构域的生物活性中起着重要作用。