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Dynamics of membrane-associated complement regulatory proteins in red blood cells.

机译：膜相关补体调节蛋白在红细胞中的动力学。

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Complement activation involves a series of solution-phase and membrane-associated reactions that culminate in opsonizing noxious targets and, for Neisseria species, lysing the bacteria. Normal human cells are protected from autologous complement-mediated damage by the action of regulatory proteins on their plasma membranes. Although the dynamics of these regulatory proteins are likely to have a critical role in regulatory protein function, there is little to no information in the literature concerning the lateral diffusion and membrane domain localization of these molecules. In this thesis, single particle tracking (SPT) was used as the primary approach to examine the lateral diffusion of all three membrane-associated complement regulatory proteins in human red blood cells (RBC): complement receptor 1 (CR1, CD35), decay-accelerating factor (DAF, CD55), and CD59. CR1 is a single-span transmembrane protein, while both DAF and CD59 are glycosylphosphatidylinositol (GPI)-linked proteins.; In both normal and complement-activated RBC, the majority of CR1 molecules showed confined motion, while a minor population exhibited Brownian movement. The confinement of CR1 corresponded to the observation of receptor clustering on the membrane surface, and was due to CR1 interaction with a membrane skeleton-linked Fas-associated phosphatase-1 (FAP-1). CR1 confinement and clustering are likely to be important in the capture and transport of immune complexes by RBC through the circulation. The function of the mobile fraction of CR1 remains unclear.; Most of the DAF molecules exhibited Brownian lateral diffusion in normal RBC. Biochemical studies showed that activation of complement in the fluid phase caused the complement fragment Cab to deposit preferentially onto glycophorin A (GPA) on the RBC membrane surface, and SPT revealed that DAF, Cab, and GPA were all laterally immobilized in the membranes of complement-treated cells. Laser optical tweezers experiments showed that the major fraction of GPA molecules were not associated with the membrane skeleton in the native membrane, but that Cab deposition induced a physical association between GPA and the membrane skeleton. These results are consistent with a model in which complement activation stimulates the formation of a skeleton-linked DAF-Cab-GPA complex on the RBC surface. The accumulation of many such complexes over the life of the RBC may play a role in the removal of senescent RBC from the circulation.; In normal RBC, CD59 was transiently confined in local membrane domains (transient confinements). Within these domains, lateral diffusion of the molecule was significantly hindered. In the presence of C5b-8, CD59 molecules were mostly immobilized. C5b-8 motion was confined as well. Experiments using cholesterol-reduced RBC showed that the CD59 transient confinement and the C5b-8 membrane insertion were sensitive to changes in membrane cholesterol content. These results are consistent with a model in which terminal complement activation induces molecular interaction between CD59 and C5b-8, and in which the lateral mobility of CD59 and the membrane insertion of C5b-8 depend on membrane cholesterol content.
机译：补体激活涉及一系列溶液相和膜相关反应，最终导致调理有害靶标，对于奈瑟菌属菌种，裂解细菌。正常人细胞通过调节蛋白在其质膜上的作用而免受自身补体介导的损害。尽管这些调节蛋白的动力学可能在调节蛋白功能中起关键作用，但是在文献中几乎没有关于这些分子的横向扩散和膜结构域定位的信息。在本文中，单颗粒跟踪（SPT）被用作检查所有三种与膜相关的补体调节蛋白在人红细胞（RBC）中的横向扩散的主要方法：补体受体1（CR1，CD35），衰变-加速因子（DAF，CD55）和CD59。 CR1是单跨膜蛋白，而DAF和CD59都是糖基磷脂酰肌醇（GPI）连接的蛋白。在正常和补体激活的RBC中，大多数CR1分子均显示受限运动，而少数群体则表现出布朗运动。 CR1的限制对应于在膜表面上受体簇的观察，并且归因于CR1与膜骨架连接的Fas相关磷酸酶1（FAP-1）的相互作用。在RBC通过循环捕获和转运免疫复合物中，CR1的限制和聚集可能很重要。 CR1的移动部分的功能尚不清楚。大多数DAF分子在正常RBC中表现出布朗横向扩散。生化研究表明，补体在液相中的活化导致补体片段Cab优先沉积在RBC膜表面的糖蛋白A（GPA）上，SPT显示DAF，Cab和GPA都侧向固定在补体膜中处理的细胞。激光镊子实验表明，GPA分子的主要部分与天然膜中的膜骨架无关，但Cab沉积诱导了GPA与膜骨架之间的物理缔合。这些结果与其中补体激活刺激在RBC表面上形成骨架连接的DAF-Cab-GPA复合物的模型一致。在RBC的整个生命周期中，许多此类复合物的积累可能在循环中去除衰老的RBC中起作用。在正常的RBC中，CD59被暂时限制在局部膜结构域中（暂时限制）。在这些区域内，分子的横向扩散受到显着阻碍。在C5b-8存在下，CD59分子大部分被固定。 C5b-8运动也受到限制。使用降低胆固醇的RBC进行的实验表明，CD59瞬态限制和C5b-8膜插入对膜胆固醇含量的变化敏感。这些结果与其中末端补体活化诱导CD59和C5b-8之间的分子相互作用，以及其中CD59的横向迁移率和C5b-8的膜插入取决于膜胆固醇含量的模型相一致。

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