Chung Hye-Shin, Lee Sunbae, Park Soon Jae
Department of Biological Sciences and Biotechnology, College of Life Science and Nano Technology, Hannam University, 1646, Yuseong-daero, Yuseong-gu, Daejeon, Korea.
Alteogen Inc., Yuseong-daero 62, Jeon-min Dong, Yuseong-gu, Daejeon, Korea.
PLoS One. 2016 Jul 15;11(7):e0159451. doi: 10.1371/journal.pone.0159451. eCollection 2016.
Here, we demonstrate that a metal ion binding motif could serve as an efficient and robust tool for site-specific conjugation strategy. Cysteine-containing metal binding motifs were constructed as single repeat or tandem repeat peptides and their metal binding characteristics were investigated. The tandem repeats of the Cysteine-Glycine-Histidine (CGH) metal ion binding motif exhibited concerted binding to Co(II) ions, suggesting that conformational transition of peptide was triggered by the sequential metal ion binding. Evaluation of the free thiol content after reduction by reducing reagent showed that metal-ion binding elicited strong retardation of cysteine oxidation in the order of Zn(II)>Ni(II)>Co(II). The CGH metal ion binding motif was then introduced to the C-terminus of antibody heavy chain and the metal ion-dependent characteristics of oxidation kinetics were investigated. As in the case of peptides, CGH-motif-introduced antibody exhibited strong dependence on metal ion binding to protect against oxidation. Zn(II)-saturated antibody with tandem repeat of CGH motif retains the cysteine reactivity as long as 22 hour even with saturating O2 condition. Metal-ion dependent fluorophore labeling clearly indicated that metal binding motifs could be employed as an efficient tool for site-specific conjugation. Whereas Trastuzumab without a metal ion binding site exhibited site-nonspecific dye conjugation, Zn(II) ion binding to antibody with a tandem repeat of CGH motif showed that fluorophores were site-specifically conjugated to the heavy chain of antibody. We believe that this strong metal ion dependence on oxidation protection and the resulting site-selective conjugation could be exploited further to develop a highly site-specific conjugation strategy for proteins that contain multiple intrinsic cysteine residues, including monoclonal antibodies.
在此，我们证明金属离子结合基序可作为一种高效且强大的工具用于位点特异性偶联策略。构建了含半胱氨酸的金属结合基序作为单重复或串联重复肽，并研究了它们的金属结合特性。半胱氨酸-甘氨酸-组氨酸（CGH）金属离子结合基序的串联重复序列表现出与Co(II)离子的协同结合，这表明肽的构象转变是由连续的金属离子结合引发的。用还原剂还原后对半胱氨酸游离含量的评估表明，金属离子结合以Zn(II)>Ni(II)>Co(II)的顺序强烈抑制半胱氨酸氧化。然后将CGH金属离子结合基序引入抗体重链的C末端，并研究了氧化动力学的金属离子依赖性特征。与肽的情况一样，引入CGH基序的抗体对金属离子结合表现出强烈的依赖性以防止氧化。具有CGH基序串联重复的Zn(II)饱和抗体即使在O2饱和条件下也能保持长达22小时的半胱氨酸反应性。金属离子依赖性荧光团标记清楚地表明金属结合基序可作为位点特异性偶联的有效工具。没有金属离子结合位点的曲妥珠单抗表现出非位点特异性染料偶联，而与具有CGH基序串联重复的抗体结合的Zn(II)离子表明荧光团位点特异性地偶联到抗体的重链上。我们相信，这种对氧化保护的强烈金属离子依赖性以及由此产生的位点选择性偶联可进一步用于开发针对包含多个内在半胱氨酸残基的蛋白质（包括单克隆抗体）的高度位点特异性偶联策略。
