Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Shimogamo Hangi-cho, Sakyo-ku, Kyoto, 606-8522, Japan.
Graduate School of Biomedical Sciences, Tokushima University, 3-18-15 Kuramoto, Tokushima, 770-8504, Japan.
Protein J. 2022 Apr;41(2):293-303. doi: 10.1007/s10930-022-10053-w. Epub 2022 Apr 30.
Somatic hypermutation (SHM) is one of the driving forces that increases antibody (Ab) affinity. We studied the effects of SHM on thermostability and affinity using three single-chain Fv fragments (scFvs) of anti-(4-hydroxy-3-nitrophenyl)acetyl Abs, namely 9TG, 9T7, and E11. 9TG has a germline structure that lacks SHM and is an ancestor of 9T7 with 11 mutations. E11, which has 21 mutations, is a mature Ab and has its own ancestor. The thermostabilities and antigen-Ab interactions were analyzed by circular dichroism (CD), differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC). Far-UV CD spectra showed that all scFvs were folded into a structure referred to as immunoglobulin-fold and were unfolded by heating at different melting temperatures. Comparison of thermodynamic parameters obtained from DSC and ITC revealed that the magnitude of stabilization free energy at 37 °C was in the order, 9TG > 9T7 > E11, while that of the free energy of interaction with antigen was 9TG < 9T7 < E11, suggesting that Abs make a trade-off between stability and affinity during affinity maturation.
体细胞超突变（SHM）是增加抗体（Ab）亲和力的驱动力之一。我们使用三种抗-（4-羟基-3-硝基苯乙酰基）Ab 的单链 Fv 片段（scFv）研究了 SHM 对热稳定性和亲和力的影响，即 9TG、9T7 和 E11。9TG 具有缺乏 SHM 的种系结构，是具有 11 个突变的 9T7 的祖先。E11 具有 21 个突变，是成熟的 Ab，有自己的祖先。通过圆二色性（CD）、差示扫描量热法（DSC）和等温滴定量热法（ITC）分析热稳定性和抗原-Ab 相互作用。远紫外 CD 光谱表明，所有 scFv 均折叠成一种称为免疫球蛋白折叠的结构，并在不同的熔点下通过加热而展开。从 DSC 和 ITC 获得的热力学参数的比较表明，在 37°C 时稳定自由能的大小顺序为 9TG>9T7>E11，而与抗原相互作用的自由能顺序为 9TG<9T7<E11，这表明 Abs 在亲和力成熟过程中在稳定性和亲和力之间进行了权衡。
