Tsumoto K, Ogasahara K, Ueda Y, Watanabe K, Yutani K, Kumagai I
Department of Chemistry and Biotechnology, Faculty of Engineering, University of Tokyo, Japan.
J Biol Chem. 1995 Aug 4;270(31):18551-7. doi: 10.1074/jbc.270.31.18551.
It has been shown that Tyr residues are unusually localized in the regions of antibodies responsible for contact with antigens (Padlan, E. A. (1990) Proteins Struct. Funct. Genet. 7, 112-124). In order to clarify the role of these Tyr residues in antigen binding, the interaction between hen egg white lysozyme (HEL) and its monoclonal antibody HyHEL10, whose structure has been well studied in complex with its antigen, was investigated. Four Tyr residues in the VH chain (HTyr-33, HTyr-50, HTyr-53, and HTyr-58) were replaced with Ala, Leu, Phe, or Trp, and the interactions between these mutant Fv fragments and HEL were studied by inhibition assay of the enzymatic activity of HEL and isothermal titration calorimetry. Twelve mutant Fv fragments could be expressed, but two mutants (HY50W and HY58W) could not be obtained in the Escherichia coli expression system, and a further two mutants (HY33A and HY50A) could not be purified by affinity chromatography. It was shown by inhibition assay that Tyr residues at each mutated site made positive contributions to the interaction to different degrees. Thermodynamic studies showed that the role of Tyr residues in antigen binding was to obtain enthalpic energy. The roles of Tyr residues in antibody HyHEL10 for the association with antigen, HEL, can be summarized as follows: 1) formation of hydrogen bonds by the hydroxyl group, 2), creating more favorable interactions through the aromatic ring and decreasing the entropic loss upon binding, and 3) allowing hydrophobic interaction through the side chain. The four Tyr residues studied here were found to play significant roles in the association in various ways.
已表明酪氨酸（Tyr）残基异常定位于抗体与抗原接触的区域（帕德兰，E. A.（1990年）《蛋白质结构、功能与遗传学》7，112 - 124）。为了阐明这些酪氨酸残基在抗原结合中的作用，研究了鸡蛋清溶菌酶（HEL）与其单克隆抗体HyHEL10之间的相互作用，该抗体与抗原形成复合物的结构已得到充分研究。重链可变区（VH链）中的四个酪氨酸残基（HTyr - 33、HTyr - 50、HTyr - 53和HTyr - 58）被替换为丙氨酸（Ala）、亮氨酸（Leu）、苯丙氨酸（Phe）或色氨酸（Trp），并通过HEL酶活性抑制测定和等温滴定量热法研究了这些突变Fv片段与HEL之间的相互作用。可以表达12个突变Fv片段，但在大肠杆菌表达系统中无法获得两个突变体（HY50W和HY58W），另外两个突变体（HY33A和HY50A）无法通过亲和层析纯化。抑制测定表明，每个突变位点的酪氨酸残基对相互作用都有不同程度的正向贡献。热力学研究表明，酪氨酸残基在抗原结合中的作用是获得焓能。抗体HyHEL10中酪氨酸残基与抗原HEL结合的作用可总结如下：1）通过羟基形成氢键；2）通过芳香环产生更有利的相互作用并减少结合时的熵损失；3）通过侧链允许疏水相互作用。研究发现，这里研究的四个酪氨酸残基在结合过程中以各种方式发挥着重要作用。
