Ruff-Jamison S, Glenney J R
Lucille P. Markey Cancer Center, Department of Biochemistry, University of Kentucky College of Medicine, Lexington 40536-0084.
J Immunol. 1993 Apr 15;150(8 Pt 1):3389-96.
Sequence analysis of a panel of antibodies to phosphotyrosine revealed predominant H and L chain V regions in the immune response and a unique D segment in the Py20 mAb, which exhibits a high affinity for phosphotyrosine. In order to determine the influence of somatic diversity on the high affinity binding of Py20, H and L chain V regions were expressed in Escherichia coli as an Fv dimer. Whereas the H or L chain V regions of Py20 alone were unable to bind phosphotyrosine, the Fv binds phosphotyrosine with an affinity comparable with the intact IgG as determined by fluorescence quenching experiments (1.55 x 10(-7) M vs 1.25 x 10(-7) M, respectively). Substitution of the Py20 V regions with other IgG V regions that differed greatly in sequence abolished binding. A high affinity Py20-combining site was dependent on the presence of the unique D-D segment. Replacement of the Py20 D-D region with a single homologous D region resulted in a decrease in affinity (5.9 x 10(-7) M). Substitution of this D-D region for the D region of another anti-phosphotyrosine antibody that is known to bind phosphotyrosine weakly (1 x 10(-3) M) conferred high affinity binding. Removal of three tyrosines from the first of the two D regions was accompanied by a fivefold reduction in affinity for phosphotyrosine. In addition, changing the VK and VH junctional amino acids resulted in a complete loss of binding. Therefore, the formation of the high affinity Py20 combining site requires both a H and L chain that are similar in sequence to those of Py20 including the unique D region and the junctional amino acids.
对一组抗磷酸酪氨酸抗体的序列分析显示，免疫反应中主要的重链和轻链V区以及Py20单克隆抗体中一个独特的D片段，该抗体对磷酸酪氨酸具有高亲和力。为了确定体细胞多样性对Py20高亲和力结合的影响，重链和轻链V区在大肠杆菌中作为Fv二聚体表达。单独的Py20重链或轻链V区不能结合磷酸酪氨酸，而通过荧光猝灭实验测定，Fv与完整IgG具有相当的亲和力结合磷酸酪氨酸（分别为1.55×10⁻⁷M和1.25×10⁻⁷M）。用序列差异很大的其他IgG V区替换Py20 V区则消除了结合。高亲和力的Py20结合位点依赖于独特的D-D片段的存在。用单个同源D区替换Py20 D-D区导致亲和力降低（5.9×10⁻⁷M）。用另一种已知对磷酸酪氨酸结合较弱（1×10⁻³M）的抗磷酸酪氨酸抗体的D区替换该D-D区赋予了高亲和力结合。从两个D区中的第一个去除三个酪氨酸伴随着对磷酸酪氨酸亲和力降低五倍。此外，改变VK和VH连接氨基酸导致结合完全丧失。因此，高亲和力Py20结合位点的形成需要重链和轻链在序列上与Py20相似，包括独特的D区和连接氨基酸。
