The inter-heavy chain disulfide bonds of IgG4 are in equilibrium with intra-chain disulfide bonds
The inter-heavy chain disulfide bonds of IgG4 are in equilibrium with intra-chain disulfide bonds
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DOI：
10.1016/s0161-5890(01)00050-5
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发表时间：
2001-01-01
影响因子：
3.6
通讯作者：
Aalberse, RC
中科院分区：
文献类型：
--
作者：
Schuurman, J;Perdok, GJ;Aalberse, RC
与其他免疫球蛋白G（IgG）亚类不同，据报道血浆中的IgG 4抗体在功能上是单价的。在先前的论文中，我们表明，循环IgG 4抗体的表观单价是由血浆IgG 4的不对称性引起的-大部分具有两个抗原结合位点，导致双特异性。我们假设双特异性抗体的产生是由分泌后机制引起的，涉及IgG 4半分子（即一条重链和一条轻链）的交换。该假设基于观察到的IgG 4重链间二硫键的不稳定性。为了研究这种不稳定性，我们构建了IgG 4突变体，并在非还原条件下通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析了重链之间的共价相互作用。参与重链间键形成的铰链半胱氨酸之一Cys 226突变为丝氨酸，导致更稳定而不是更不稳定的重链间键联。此外，我们证实了将IgG 4铰链序列Cys-Pro-Ser-Cys突变为IgG 1铰链序列Cys-Pro-Pro-Cys也显著稳定了重链之间的共价相互作用。这两个观察结果表明了对观察到的重链间二硫键不稳定性的解释：形成替代的链内胱氨酸。显然，该链内胱氨酸不能在Cys 226被Set取代的突变体中形成，并且由于脯氨酸的扭转自由度受限，不能容易地在具有IgG 1铰链序列（Cys-Pro-Pro-Cys）的突变体中形成。因此，我们假设IgG 4亚群中缺乏共价重链相互作用反映了链间和链内胱氨酸之间的平衡。基于已发表的IgG 4相关铰链缺失的IgG 1骨髓瘤蛋白Meg的结构，我们提出了两种形式的IgG 4和半分子交换反应的模型，这可能导致形成双特异性IgG 4抗体。(C)2001爱思唯尔科技有限公司版权所有。
Unlike other immunoglobulin G (IgG) subclasses, IgG4 antibodies in plasma have been reported to be functionally monovalent. In a previous paper, we showed that the apparent monovalency of circulating IgG4 antibodies is caused by asymmetry of plasma IgG4 - a large fraction has two antigen-binding sites resulting in bispecificity. We postulated that the generation of bispecific antibodies was caused by a post-secretion mechanism, involving the exchange of IgG4 half-molecules (i.e. one heavy and one light chain). This hypothesis was based on the observed instability of the inter-heavy chain disulfide bonds of IgG4. To investigate this instability, we constructed IgG4 mutants and analyzed the covalent interaction between the heavy chains by sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions. The mutation to serine of one of the hinge cysteines involved in the inter-heavy chain bond formation, Cys226, resulted in a more stable rather than a more labile inter-heavy chain linkage. Moreover, we confirmed that mutating the IgG4 hinge sequence Cys-Pro-Ser-Cys to the IgG1 hinge sequence Cys-Pro-Pro-Cys also markedly stabilizes the covalent interaction between the heavy-chains. These two observations suggested an explanation for the observed instability of the inter-heavy chain disulfide bonds: the formation of an alternative, intra-chain cystine. Obviously, this intra-chain cystine cannot be formed in the mutant where Cys226 is replaced by Set, and cannot easily be formed in the mutant with the IgG1 hinge sequence (Cys-Pro-Pro-Cys) due to the restricted torsional freedom of prolines. We, therefore, postulate that the lack of a covalent heavy-chain interaction in a subpopulation of IgG4 reflects an equilibrium between inter- and intra-chain cystines. Based upon the published structure of the IgG4-related hinge-deleted IgG1 myeloma protein Meg, we propose a model for the two forms of IgG4 and for the half-molecule exchange reaction, which might result in the formation of bispecific IgG4 antibodies. (C) 2001 Elsevier Science Ltd. All rights reserved.