Expression and characterization of recombinant soluble human CD3 molecules: presentation of antigenic epitopes defined on the native TCR-CD3 complex.
Expression and characterization of recombinant soluble human CD3 molecules: presentation of antigenic epitopes defined on the native TCR-CD3 complex.
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重组可溶性人 CD3 分子的表达和表征：天然 TCR-CD3 复合物上定义的抗原表位的呈现。
DOI：
10.1093/intimm/14.4.389
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发表时间：
2002
影响因子：
4.4
通讯作者：
Ledbetter,JeffreyA
中科院分区：
文献类型：
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作者：
Law,Che-Leung;Hayden-Ledbetter,Martha;Buckwalter,Sonya;McNeill,Lisa;Nguyen,Hieu;Habecker,Phil;Thorne,BarbaraA;Dua,Raj;Ledbetter,JeffreyA
TCR-CD3 复合物由克隆型二硫键连接的 TCRαβ 或 TCRδγ 异二聚体以及不变的 CD3δ、ε、γ 和 δ 链组成。我们生成了表达与人 IgG1 Fc 融合的 CD3δ、ε 或 γ 亚基胞外结构域的质粒构建体。由单个 CD3δ、ε 或 γ 亚基组成的重组融合蛋白与抗 CD3 mAb（包括 G19-4、BC3、OKT3 和 64.1）反应较差。 CD3ϵ-Ig 与 CD3δ-Ig (CD3ϵδ-Ig) 或 CD3γ-Ig (CD3ϵγ-Ig) 共表达导致融合蛋白与 G19-4 的结合大大增加。对纯化的 CD3ϵδ-Ig 融合蛋白进行短暂的酸处理显着改善了其与 BC3、OKT3 和 64.1 的结合。表面等离子共振分析显示，CD3εδ-Ig 和抗 CD3 mAb 的解离常数范围为 10-8 至 10-9M。基于这些结果，表达了编码 CD3δ 链的单链 (sc) 构建体，该链通过柔性接头连接到 CD3ϵ 链，随后是人 IgG1 Fc。 sc CD3δϵ–scIg 与抗 CD3 mAb 反应，无需酸处理。此外，抗 CD3 mAb 与 CD3εδ-Ig 的结合亲和力高于 CD3εγ-Ig，表明 CD3εδ 和 CD3εγ 亚基之间存在潜在的结构差异。总之，我们报告了可溶性重组 CD3 蛋白的表达，证明了 T 细胞表面表达的天然 CD3 复合物的结构特征。这些CD3融合蛋白可用于进一步分析TCR-CD3复合物的结构，并鉴定可通过破坏CD3和TCR亚基之间的相互作用来干扰TCR-CD3介导的信号转导的分子。
The TCR–CD3 complex consists of the clonotypic disulfide‐linked TCRαβ or TCRδγ heterodimers, and the invariant CD3δ, ϵ, γ and ζ chains. We generated plasmid constructs expressing the extracellular domains of the CD3δ, ϵ or γ subunits fused to human IgG1 Fc. Recombinant fusion proteins consisting of individual CD3δ, ϵ or γ subunits reacted poorly with anti‐CD3 mAb including G19‐4, BC3, OKT3 and 64.1. Co‐expression of the CD3ϵ–Ig with either the CD3δ–Ig (CD3ϵδ–Ig) or the CD3γ–Ig (CD3ϵγ–Ig) resulted in fusion proteins with much increased binding to G19‐4. A brief acid treatment of the purified CD3ϵδ–Ig fusion protein substantially improved its binding to BC3, OKT3 and 64.1. Surface plasmon resonance analysis revealed that the dissociation constants for CD3ϵδ–Ig and anti‐CD3 mAb ranged from 10–8to 10–9M. Based on these results, a single‐chain (sc) construct encoding the CD3δ chain linked to the CD3ϵ chain with a flexible linker followed by human IgG1 Fc was expressed. The sc CD3δϵ–scIg reacted with anti‐CD3 mAb without requiring acid treatment. Moreover, anti‐CD3 mAb bound CD3ϵδ–Ig at a higher affinity than CD3ϵγ–Ig, suggesting potential structural differences between the CD3ϵδ and CD3ϵγ subunits. In summary, we report the expression of soluble recombinant CD3 proteins that demonstrate structural characteristics of the native CD3 complex expressed on the T cell surface. These CD3 fusion proteins can be used to further analyze the structure of the TCR–CD3 complex, and to identify molecules that can interfere with TCR–CD3‐mediated signal transduction by disrupting the interaction between CD3 and TCR subunits.