Isolation and Characterization of the B-Cell Marker CD20

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• 作者：James A. Ernst ; Hong Li ; Hok Seon Kim ; Gerald R. Nakamura ; Daniel G. Yansura ; and Richard L. Vandlen
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：November 22, 2005
• 年：2005
• 卷：44
• 期：46
• 页码：15150 - 15158
• 全文大小：368K
• 年卷期：v.44,no.46(November 22, 2005)
• ISSN：1520-4995

文摘

The integral membrane protein CD20 has been identified as an important therapeutic target inthe treatment of non-Hodgkin's lymphoma (NHL). CD20 binding of many antibodies including thetherapeutic antibody, rituximab, has been shown to be critically dependent upon the conformation of aloop structure between the third and fourth helical transmembrane regions. In this work, human and murineCD20 proteins expressed in Escherichia coli are shown to be localized with the cell membrane and arepurified in nondenaturing detergent solutions. The purified human and murine CD20 proteins have asubstantial helical structure as measured by circular dichroism spectroscopy. Only small changes in thesecondary structure are observed following the reduction of CD20, with the addition of SDS, or afterheating. 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 proposedantibody 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 ofCD20. The affinities of intact rituximab and its Fab fragment to the isolated and purified CD20 are similarto the observed affinity of rituximab Fab for CD20 on the surface of B cells. However, the intact rituximabantibody shows much higher affinity for CD20 on B cells. This suggests that B cells display CD20 insuch a way that allows for marked avidity effects to be observed, perhaps through cross-linking of CD20monomers into lipid rafts, which limits receptor diffusion in the membrane. Such cross-linking may playa role in partitioning CD20 into lipid rafts and in enhancing antibody-dependent B-cell depletion activitiesof rituximab and other therapeutic anti-CD20 antibodies.