Palmer M, Harris R, Freytag C, Kehoe M, Tranum-Jensen J, Bhakdi S
Institute of Medical Microbiology, University of Mainz, Augustusplatz, D-55101 Mainz, Germany.
EMBO J. 1998 Mar 16;17(6):1598-605. doi: 10.1093/emboj/17.6.1598.
Streptolysin O (SLO) is a bacterial exotoxin that binds to cell membranes containing cholesterol and then oligomerizes to form large pores. Along with rings, arc-shaped oligomers form on membranes. It has been suggested that each arc represents an incompletely assembled oligomer and constitutes a functional pore, faced on the opposite side by a free edge of the lipid membrane. We sought functional evidence in support of this idea by using an oligomerization-deficient, non-lytic mutant of SLO. This protein, which was created by chemical modification of a single mutant cysteine (T250C) with N-(iodoacetaminoethyl)-1-naphthylamine-5-sulfonic acid, formed hybrid oligomers with active SLO on membranes. However, incorporation of the modified T250C mutant inhibited subsequent oligomerization, so that the hybrid oligomers were reduced in size. These appeared as typical arc lesions in the electron microscope. They formed pores that permitted passage of NaCl and calcein but restricted permeation of large dextran molecules. The data indicate that the SLO pore is formed gradually during oligomerization, implying that pores lined by protein on one side and an edge of free lipid on the other may be created in the plasma membrane. Intentional manipulation of the pore size may extend the utility of SLO as a tool in cell biological experiments.
链球菌溶血素O（SLO）是一种细菌外毒素，它能与含有胆固醇的细胞膜结合，然后寡聚化形成大孔。在细胞膜上，除了环状结构外，还会形成弧形寡聚体。有人提出，每个弧形代表一个未完全组装的寡聚体，构成一个功能性孔，脂质膜的自由边缘在其对面。我们通过使用SLO的寡聚化缺陷型非裂解突变体来寻找支持这一观点的功能证据。这种蛋白质是通过用N-（碘乙酰氨基乙基）-1-萘胺-5-磺酸对单个突变半胱氨酸（T250C）进行化学修饰而产生的，它能在细胞膜上与活性SLO形成杂合寡聚体。然而，掺入修饰后的T250C突变体抑制了随后的寡聚化，因此杂合寡聚体的尺寸减小。在电子显微镜下，这些呈现为典型的弧形损伤。它们形成的孔允许NaCl和钙黄绿素通过，但限制了大的葡聚糖分子的渗透。数据表明，SLO孔在寡聚化过程中逐渐形成，这意味着在质膜中可能会形成一侧由蛋白质内衬而另一侧由自由脂质边缘构成的孔。对孔径的有意操控可能会扩展SLO作为细胞生物学实验工具的用途。
