Thermodynamic consequences of grafting enhanced affinity toward the mutated antigen onto an antibody - The case of anti-lysozyme antibody, HyHEL-10
Thermodynamic consequences of grafting enhanced affinity toward the mutated antigen onto an antibody - The case of anti-lysozyme antibody, HyHEL-10
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DOI：
10.1074/jbc.275.17.12813
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发表时间：
2000-04-28
影响因子：
4.8
通讯作者：
Kumagai, I
中科院分区：
文献类型：
--
作者：
Nishimiya, Y;Tsumoto, K;Kumagai, I
为了从热力学角度探讨增强抗体对抗原亲和力的机制，研究了抗母鸡溶菌酶（HEL）抗体HyHEL-10，该抗体也能识别亲和力降低的突变抗原火鸡溶菌酶（TEL）。通过饱和诱变将对TEL具有高亲和力的四个残基（Tyr（53）、Ser（54）、Ser（56）和Tyr(58)）嫁接到HyHEL-10上，然后选择对TEL具有亲和力的残基。一些富集的克隆在58位点有一个Phe残基。热力学分析表明，与野生型相比，选择的克隆对TEL的亲和力增加了3倍以上，这是由于负焓变的增加。将HyHEL-10 HTyr（58）替换为Phe后，其负焓变增加，与选择的突变体相比，其对TEL的亲和力几乎相同，这表明其他位点的突变减少了熵损失，尽管对TEL的亲和力贡献不大。这些结果表明，抗体对抗原的亲和力通过某些有限突变的焓变增加而增强。由于突变引起的多余熵损失被其他能量中性突变所减少。
In order to address the mechanism of enhancement of the affinity of an antibody toward an antigen from a thermodynamic viewpoint, anti-hen lysozyme (HEL) antibody HyHEL-10, which also recognize the mutated antigen turkey lysozyme (TEL) with reduced affinity, was examined. Grafting high affinity toward TEL onto HyHEL-10 was performed by saturation mutagenesis into four residues (Tyr(53), Ser(54), Ser(56), and Tyr(58)) in complementarity-determining region 2 of the heavy chain (CDR-H2) followed by selection with affinity for TEL. Several clones enriched have a Phe residue at site 58. Thermodynamic analyses showed that the clones selected had experienced a greater than 3-fold affinity increase toward TEL in comparison with wild-type Fv, originating from an increase in negative enthalpy change. Substitution of HyHEL-10 HTyr(58) With Phe led to the increase in negative enthalpy change and to almost identical affinity for TEL in comparison with mutants selected, indicating that mutations at other sites decrease the entropy loss despite little contribution to the affinity for TEL. These results suggest that the affinity of an antibody toward the antigen is enhanced by the increase in enthalpy change by some limited mutation, and excess entropy loss due to the mutation is decreased by other energetically neutral mutations.