Assembly of complement components C5b-8 and C5b-9 on lipid bilayer membranes: visualization by freeze-etch electron microscopy.
Assembly of complement components C5b-8 and C5b-9 on lipid bilayer membranes: visualization by freeze-etch electron microscopy.
复制标题
补体成分 C5b-8 和 C5b-9 在脂质双层膜上的组装：通过冷冻蚀刻电子显微镜可视化。
DOI：
10.1021/bi00428a019
复制
发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Esser,AF
中科院分区：
文献类型：
--
作者：
McCloskey,MA;Dankert,JR;Esser,AF
加州大学尔湾分校生理与生物物理系，加州92717；佛罗里达大学比较与实验病理学系，佛罗里达盖恩斯维尔分校32610摘要：我们用冷冻刻蚀电镜观察了在合成磷脂双层上组装的补体C5b-8和C5b-9大分子复合物。这些复合物依次由纯化的人补体组分C5b-6、C7、C8和C9形成。C5b-8的配合物在囊泡的外表面（ES）观察到12 nm的颗粒，倾向于形成多分散的聚集体。聚集体有时具有规则的链状结构，包含不同数量的成对亚基。含有C5b-9配合物的囊泡蚀刻在外径= 27 nm的ES大环上。一个或两个旋钮通常附在环的周长上。膜的分裂导致C5b-9与外小叶的分裂。因此，C5b-9囊泡的原生质面（PF）存在直径为17 nm的圆孔，外质面（EF）存在大小相匹配的凸起的圆形残桩。C5b-9复合物通常位于最低脂质序列的区域。也就是说，在EF和ES的显微照片中，单个C5b-9配合物位于P/相波纹弯曲或到达死角的地方，并且C5b-9配合物的线性阵列在晶格中勾勒出类似斜位的结构；PF中的孔反映了这种分布。紧接在C5b-9环周围的膜常常下沉到比环本身大得多的区域。因此，在ES上的环和PF上的孔周围可以看到浅凹陷，而在EF上的树桩周围可以看到凸起的乳晕。这些乳晕可能是由脂质组成的，因为它们具有P/相的波纹外观特征。这些观察结果与C5b-9复合物能够在其直接环境中重组脂质组织的假设是一致的，这种作用可能参与了补体渗透细胞膜的机制。补体的膜攻击复合体（MAC） 1损害细胞膜功能的确切机制尚不清楚。自1972年迈耶（Mayer, 1972）提出孔隙或“甜甜圈”假说以来，该假说受到了广泛的欢迎。该模型的要点是，MAC，或C5b-9蛋白复合物，形成一个跨越双分子层的中央水孔，允许盐和水的快速跨膜交换。补体溶解红细胞的负染色电镜（EM）显示，在直径9-13纳米的暗圆形区域有染色物的积累，这是doughnu假说的早期支持。同一系统的冷冻蚀刻电镜也显示了外膜表面的甜甜圈状突起，以及从外单层疏水断裂面突出的蛋白质残根（lies等，1973；Bhakdi等，1974;Tranum-Jensen & Bhakdi, 1983）。最近对甜甜圈假说的支持来自于一项发现，即在某些条件下，例如高温或重金属或外源性蛋白酶的存在，纯化的补体成分C9形成由12-18个C9原体组成的管状结构（Podack & Tschopp, 1982; Tschopp etal ., 1984）。管的一端有一个环空，使得外部尺寸为= 21 x 15 x 15 nm （dispio & Hugh, 1985）。这种结构被称为poly (C9…
Department of Physiology and Biophysics, University of California, Irvine, California 92717, and Department of Comparative and Experimental Pathology, University of Florida, Gainesville, Florida 32610 Received April 14, 1988; Revised Manuscript Received August 16, 1988 abstract: We have visualized by freeze-etch electron microscopy the macromolecular complexes of complement, C5b-8 and C5b-9, respectively, assembled on synthetic phospholipid bilayers. These complexes were formed sequentially by using purified human complement components C5b-6 followed by C7, C8, and C9. Complexes of C5b-8 were observed on the external surface (ES) of vesicles as 12-nm particlesthat tended to form polydisperse aggregates. The aggregates were sometimes of a regular chainlike structure containing varying numbers of paired subunits. Etching of vesicles containing C5b-9 complexes revealed on the ES large rings of= 27-nm outer diameter. One or two knobs usually were attached to the perimeter of the rings. Splitting of the membrane resulted in partitioning of the C5b-9 with the outer leaflet. Thus, round holes of= 17-nm diameter were present in the protoplasmic face (PF), and raised circular stumps of a matching size were present on the exoplasmic face (EF) of C5b-9 vesicles. C5b-9 complexes were frequently localized in regions of the lowest lipid order. That is, in micrographs of the EF and ES, single C5b-9 complexes were located where the ripples of the P/phase bend or reach a dead end, and linear arrays of C5b-9 complexes outlined disclination-like structures in the lattice; the holes in the PF mirrored this distribution. Themembrane immediately surrounding C5b-9 rings was often sunk inwardly over an area much larger thanthat of the ring itself. Thus, shallow depressions were seen about the rings on the ES and around the holes in the PF, whereas raised areolas surrounded the stumps on the EF. These areolas presumably were composed of lipid because of their rippled appearance characteristic of the P/phase. These observations are consistent with the hypothesis that the C5b-9 complex is capable of restructuring lipid organization in its immediate environment and that such an effect may participate in the mechanism whereby complement permeabilizes cell membranes. e precise mechanism by which the membrane attack complex of complement (MAC) 1 impairs cell membrane function is not yet resolved. Since its formulation in 1972 the pore or “doughnut” hypothesis of Mayer (1972) has enjoyed wide popularity. The gist of this model is that the MAC, or C5b-9 protein complex, forms a central aqueous pore that spans the bilayer and allows rapid transmembrane exchange of salts and water. Early support for the doughnuthypothesis came from negative-stain electron micrographs (EM) of complement-lysed erythrocytes, which show accumulations of stain in darkcircular areas of 9-13-nm diameter. Freeze-etch EM of the same system also reveals doughnut-like projections on the external membrane surface and proteinaceous stubs projecting from the hydrophobic fracture face of the outer monolayer (lies et al., 1973; Bhakdi et al., 1974; Tranum-Jensen & Bhakdi, 1983).More recent support for the doughnut hypothesis comes from the finding that under certain conditions, eg, high temperature or the presence of heavy metals or exogenous proteases, purified complement component C9 forms a tubular structure composed of 12-18 C9 protomers (Podack & Tschopp, 1982; Tschopp et al., 1984). The tube has an annulus at one end such that the external dimensions are= 21 x 15 X 15 nm (DiScipio & Hugh, 1985). This structure, termed poly (C9 …