Isolation and characterization of the B-cell marker CD20
Isolation and characterization of the B-cell marker CD20
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DOI：
10.1021/bi0511078
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发表时间：
2005-11-22
期刊：
影响因子：
2.9
通讯作者：
Vandlen, RL
中科院分区：
文献类型：
--
作者：
Ernst, JA;Li, H;Vandlen, RL
整合膜蛋白CD20已被确定为治疗非霍奇金淋巴瘤（NHL）的重要治疗靶点。许多抗体（包括治疗性抗体利妥昔单抗）的 CD20 结合已被证明严重依赖于第三和第四螺旋跨膜区之间的环结构的构象。在这项工作中，在大肠杆菌中表达的人和鼠 CD20 蛋白被证明位于细胞膜上，并在非变性洗涤剂溶液中纯化。通过圆二色光谱测量，纯化的人和鼠 CD20 蛋白具有显着的螺旋结构。在 CD20 还原、添加 SDS 或加热后，仅观察到二级结构的微小变化。利妥昔单抗抗体显示以纳摩尔亲和力与纯化的人 CD20 结合。利妥昔单抗结合通过 CD20 的还原和烷基化而被消除，数据与所提出的抗体表位一致，该表位位于半胱氨酸残基 167 和 183 之间形成的二硫键环内。在还原的 CD20 重新氧化后，二硫键依赖性抗体结合部分恢复。抗体结合不受 CD20 胞内结构域中半胱氨酸突变的影响。完整的利妥昔单抗及其 Fab 片段对分离纯化的 CD20 的亲和力与观察到的利妥昔单抗 Fab 对 B 细胞表面 CD20 的亲和力相似。然而，完整的利妥昔单抗抗体对 B 细胞上的 CD20 显示出更高的亲和力。这表明 B 细胞展示 CD20 的方式可以观察到明显的亲和力效应，可能是通过 CD20 单体交联成脂筏，从而限制了受体在膜中的扩散。这种交联可能在将 CD20 分隔成脂筏以及增强利妥昔单抗和其他治疗性抗 CD20 抗体的抗体依赖性 B 细胞消耗活性中发挥作用。
The integral membrane protein CD20 has been identified as an important therapeutic target in the treatment of non-Hodgkin's lymphoma (NHL). CD20 binding of many antibodies including the therapeutic antibody, rituximab, has been shown to be critically dependent upon the conformation of a loop structure between the third and fourth helical transmembrane regions. In this work, human and murine CD20 proteins expressed in Escherichia coli are shown to be localized with the cell membrane and are purified in nondenaturing detergent solutions. The purified human and murine CD20 proteins have a substantial helical structure as measured by circular dichroism spectroscopy. Only small changes in the secondary structure are observed following the reduction of CD20, with the addition of SDS, or after heating. The rituximab antibody is shown to bind to purified human CD20 with nanomolar affinity. Rituximab binding is abolished by reduction and alkylation of CD20, with data consistent with the proposed antibody epitope being within the disulfide-bonded loop formed between cysteine residues 167 and 183. Disulfide-bond-dependent antibody binding is partially recovered following reoxidation of reduced CD20. Antibody binding is unaffected by mutations of cysteines proposed to be in the intracellular domain of CD20. The affinities of intact rituximab and its Fab fragment to the isolated and purified CD20 are similar to the observed affinity of rituximab Fab for CD20 on the surface of B cells. However, the intact rituximab antibody shows much higher affinity for CD20 on B cells. This suggests that B cells display CD20 in such a way that allows for marked avidity effects to be observed, perhaps through cross-linking of CD20 monomers into lipid rafts, which limits receptor diffusion in the membrane. Such cross-linking may play a role in partitioning CD20 into lipid rafts and in enhancing antibody-dependent B-cell depletion activities of rituximab and other therapeutic anti-CD20 antibodies.