The Y54(L)W mutation of anti-leukotriene C4 single-chain antibody increases affinity to leukotriene E4
The Y54(L)W mutation of anti-leukotriene C4 single-chain antibody increases affinity to leukotriene E4
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抗白三烯 C4 单链抗体的 Y54(L)W 突变增加了对白三烯 E4 的亲和力
DOI：
10.1093/jb/mvw055
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发表时间：
2016
期刊：
影响因子：
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通讯作者：
Yoshitaka Takahashi.
中科院分区：
文献类型：
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作者：
Yuki Kawakami;Mai Kinoshita;Yoshiko Mori;Shuji Okochi;Shiori Hirano;Ichika Shimoda;Keita Kanzaki;Toshiko Suzuki-Yamamoto;Masumi Kimoto;Mitsuaki Sugahara;Tetsuya Hori;Hiromichi Saino;Masashi Miyano;Shozo Yamamoto;Yoshitaka Takahashi.
确定了与 LTC4 复合的抗白三烯 (LT) C4 单克隆抗体 (mAbLTC) 的 X 射线晶体结构，然而，仅晶体学研究不足以完全了解抗原结合位点的结构。为了阐明 mAbLTC 轻链中 Tyr-54 和 Asn-58 的各自贡献（两者均与 LTC4 的谷氨酸形成氢键），我们使用抗 LTC4 单链可变片段 (scFvLTC) 检查了残基的取代是否影响抗原结合亲和力和特异性。在Tyr-54(L)突变体中，Y54(L)W对LTE4的亲和力显着增加，与LTD4相当。使用在大肠杆菌和毕赤酵母中表达的Y54(L)W突变体获得了基本上相同的结果。结构模型表明抗体中的取代色氨酸和LTE4中的半胱氨酸残基之间形成了新的氢键。 Y54(L)R、Y54(L)E和Y54(L)L对LTC4的亲和力显着降低，而其他测试的Tyr-54(L)突变体以及Asn-58(L)突变体在LT结合方面没有表现出显着变化。结果可以深入了解抗体特异性 LT 识别的分子基础。
The X-ray crystal structure of an anti-leukotriene (LT) C4monoclonal antibody (mAbLTC) in complex with LTC4was determined, however, crystallographic studies alone are not enough to fully understand the structures of the antigen-binding site. To elucidate the individual contribution of Tyr-54 and Asn-58 in the light chain of mAbLTC, both of which formed a hydrogen bond with glutamic acid of LTC4, we examined whether substitution of the residues affects the antigen binding affinity and specificity using an anti-LTC4single chain variable fragment (scFvLTC). Among the Tyr-54(L) mutants, Y54(L)W showed a dramatic increase in the affinity to LTE4which was comparable to that to LTD4. Essentially the same results were obtained using the Y54(L)W mutant expressed inEscherichia coliandPichia pastoris.The structural modeling suggested the formation of a novel hydrogen bond between the substituted tryptophan in the antibody and the cysteine residue in LTE4. The affinity of Y54(L)R, Y54(L)E and Y54(L)L to LTC4was markedly reduced, whereas other tested Tyr-54(L) mutants as well as Asn-58(L) mutants did not show significant change in LT binding. The results may provide an insight into the molecular basis of specific LT recognition by the antibody.