Henson S E, Smith D, Boackle S A, Holers V M, Karp D R
The Harold C. Simmons Arthritis Research Center and the Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, TX 75390-8884, USA.
J Immunol Methods. 2001 Dec 1;258(1-2):97-109. doi: 10.1016/s0022-1759(01)00471-9.
CD21 (complement receptor 2, CR2) binds the terminal proteolytic fragments of the third component of complement (C3) that have been covalently attached to immune complexes or other targets during the activation of complement. We used the technique of in vivo biotinylation to create a recombinant multivalent ligand for CD21. A sequence coding for a biotinylation signal peptide was added to the 3' end of the human C3dg cDNA. The modified C3dg was expressed in Escherichia coli and biotinylated intracellularly by the bacterial biotin holoenzyme synthetase (BirA) enzyme. Monomeric C3dg was unable to bind to CD21 as determined by flow cytometry, while biotinylated recombinant C3dg (rC3dg) complexed with fluorochrome-conjugated streptavidin bound tightly. Binding was observed using CD21 positive B cells but not seen on pre-B cells that do not express this complement receptor. Two assays were used to assess the functional capacity of the recombinant C3dg. First, multimeric C3dg caused the phosphorylation of the mitogen-activated kinase, p38, in mature B lymphoma cells. Second, C3dg greatly enhanced the activation of primary B cells in combination with a sub-stimulatory concentration of anti-IgM monoclonal antibody. These results illustrate the utility of the technique of in vivo biotinylation to generate ligands for cell surface receptors that require multimerization for high avidity binding and function.
CD21（补体受体2，CR2）可结合补体第三成分（C3）的末端蛋白水解片段，这些片段在补体激活过程中已共价连接至免疫复合物或其他靶标。我们利用体内生物素化技术制备了一种用于CD21的重组多价配体。将编码生物素化信号肽的序列添加到人C3dg cDNA的3'末端。修饰后的C3dg在大肠杆菌中表达，并通过细菌生物素全酶合成酶（BirA）在细胞内进行生物素化。通过流式细胞术测定，单体C3dg无法与CD21结合，而与荧光染料偶联的链霉亲和素复合的生物素化重组C3dg（rC3dg）则紧密结合。在表达CD21的B细胞上观察到了结合，但在不表达这种补体受体的前B细胞上未观察到。使用两种测定法评估重组C3dg的功能能力。首先，多聚体C3dg可导致成熟B淋巴瘤细胞中的丝裂原活化激酶p38磷酸化。其次，C3dg与亚刺激浓度的抗IgM单克隆抗体联合使用时，可大大增强原代B细胞的活化。这些结果说明了体内生物素化技术在生成用于细胞表面受体的配体方面的实用性，这些受体需要多聚化才能实现高亲和力结合和功能。
