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Mechanism of Immunoglobulin G4 Fab-arm Exchange
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2011-07-06 , DOI: 10.1021/ja203638y
Theo Rispens 1 , Pleuni Ooijevaar-de Heer 1 , Onno Bende 1 , Rob C. Aalberse 1
Affiliation  

Immunoglobulin G (IgG) antibodies are symmetrical molecules that may be regarded as covalent dimers of 2 half-molecules, each consisting of a light chain and a heavy chain. Human IgG4 is an unusually dynamic antibody, with half-molecule exchange ("Fab-arm exchange") resulting in asymmetrical, bispecific antibodies with two different antigen binding sites, which contributes to its anti-inflammatory activity. The mechanism of this process is unknown. To elucidate the elementary steps of this intermolecular antibody rearrangement, we developed a quantitative real-time FRET assay to monitor the kinetics of this process. We found that an intrinsic barrier is the relatively slow dissociation of the CH3 domains that noncovalently connect the heavy chains, which becomes rate determining in case disulfide bonds between the heavy chains are reduced or absent. Under redox conditions that mimic the previously estimated in vivo reaction rate, i.e., 1 mM of reduced glutathione, the overall rate is ca. 20 times lower because only a fraction of noncovalent isomers is present (with intra- rather than interheavy chain disulfide bonds), formed in a relatively fast pre-equilibrium from covalent isomers. Interestingly, Fab arms stabilize the covalent isomer: the amount of noncovalent isomers is ca. 3 times higher for Fc fragments of IgG4 (lacking Fab domains) compared to intact IgG4, and the observed rate of exchange is 3 times higher accordingly. Thus, kinetic data obtained from a sensitive and quantitative real-time FRET assay as described here yield accurate data about interdomain interactions such as those between Fab and/or Fc domains. The results imply that in vivo, the reaction is under control of local redox conditions.

中文翻译:

免疫球蛋白 G4 Fab-arm 交换机制

免疫球蛋白 G (IgG) 抗体是对称分子,可被视为 2 个半分子的共价二聚体,每个半分子由一条轻链和一条重链组成。人 IgG4 是一种异常动态的抗体,通过半分子交换(“Fab 臂交换”)产生具有两个不同抗原结合位点的不对称双特异性抗体,这有助于其抗炎活性。这个过程的机制是未知的。为了阐明这种分子间抗体重排的基本步骤,我们开发了一种定量实时 FRET 测定来监测该过程的动力学。我们发现一个内在障碍是非共价连接重链的 CH3 结构域相对缓慢的解离,如果重链之间的二硫键减少或不存在,这将成为速率决定因素。在模拟先前估计的体内反应速率的氧化还原条件下,即 1 mM 的还原型谷胱甘肽,总速率约为。低 20 倍,因为仅存在一小部分非共价异构体(具有重链内而不是重链间二硫键),由共价异构体在相对快速的预平衡中形成。有趣的是,Fab 臂稳定了共价异构体:非共价异构体的数量约为。与完整 IgG4 相比,IgG4 的 Fc 片段(缺少 Fab 结构域)高 3 倍,观察到的交换率相应地高 3 倍。因此,从此处描述的敏感和定量实时 FRET 测定中获得的动力学数据可产生有关域间相互作用的准确数据,例如 Fab 和/或 Fc 域之间的相互作用。结果表明,在体内,反应受局部氧化还原条件的控制。
更新日期:2011-07-06
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