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B细胞标志物CD20的分离与鉴定

Isolation and characterization of the B-cell marker CD20.

作者信息

Ernst James A, Li Hong, Kim Hok Seon, Nakamura Gerald R, Yansura Daniel G, Vandlen Richard L

机构信息

Department of Protein Chemistry, MS 63, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA.

出版信息

Biochemistry. 2005 Nov 22;44(46):15150-8. doi: 10.1021/bi0511078.

Abstract

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.

摘要

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。包括治疗性抗体利妥昔单抗在内的许多抗体与CD20的结合已被证明严重依赖于第三和第四螺旋跨膜区域之间环结构的构象。在这项工作中,在大肠杆菌中表达的人和鼠CD20蛋白显示定位于细胞膜,并在非变性去污剂溶液中纯化。通过圆二色光谱法测量,纯化的人和鼠CD20蛋白具有大量的螺旋结构。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。CD20的还原和烷基化消除了利妥昔单抗的结合,数据与所提出的抗体表位在半胱氨酸残基167和183之间形成的二硫键结合环内一致。还原的CD20再氧化后,部分恢复了二硫键依赖性抗体结合。抗体结合不受拟为CD20细胞内结构域中的半胱氨酸突变的影响。完整的利妥昔单抗及其Fab片段与分离纯化的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细胞以一种允许观察到显著亲和力效应的方式展示CD20,可能是通过将CD20单体交联到脂筏中,这限制了受体在膜中的扩散。这种交联可能在将CD20分配到脂筏中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细胞清除活性中发挥作用。

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