Dual Beneficial Effect of Interloop Disulfide Bond for Single Domain Antibody Fragments
Dual Beneficial Effect of Interloop Disulfide Bond for Single Domain Antibody Fragments
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DOI：
10.1074/jbc.m111.242818
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发表时间：
2012-01-13
影响因子：
4.8
通讯作者：
Saerens, Dirk
中科院分区：
文献类型：
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作者：
Govaert, Jochen;Pellis, Mireille;Saerens, Dirk
骆驼体内功能性重链抗体(HCAbs)的抗原结合片段由单一结构域组成，称为HCAbs重链可变区(VHH)。VHH在骨架区域-2上有显着的氨基酸替换，以产生一个抗原结合区域，该区域在没有轻链伙伴的情况下发挥作用。这些取代为VHH提供了更亲水、更易溶的特性，但降低了结构域的内在稳定性。在这里，我们研究了单倍体VHH的另一个标志的功能作用，即第一和第三个抗原结合环之间的额外二硫键。在用所有20个氨基酸取代形成环间半胱氨酸后，我们选择并鉴定了几个保留抗原结合能力的VHH。虽然VHH结构域可以在没有环间二硫键的情况下发挥作用，但我们证明了它的存在构成了净优势。首先，二硫键稳定了结构域，并通过骨架区域-2标志氨基酸抵消了不稳定。其次，二硫键使长的第三个抗原结合环变硬，导致更强的抗原相互作用。这种双重有益的效应解释了体内抗体成熟过程有利于具有环间二硫键的VHH结构域。
The antigen-binding fragment of functional heavy chain antibodies (HCAbs) in camelids comprises a single domain, named the variable domain of heavy chain of HCAbs (VHH). The VHH harbors remarkable amino acid substitutions in the framework region-2 to generate an antigen-binding domain that functions in the absence of a light chain partner. The substitutions provide a more hydrophilic, hence more soluble, character to the VHH but decrease the intrinsic stability of the domain. Here we investigate the functional role of an additional hallmark of dromedary VHHs, i.e. the extra disulfide bond between the first and third antigen-binding loops. After substituting the cysteines forming this interloop cystine by all 20 amino acids, we selected and characterized several VHHs that retain antigen binding capacity. Although VHH domains can function in the absence of an interloop disulfide bond, we demonstrate that its presence constitutes a net advantage. First, the disulfide bond stabilizes the domain and counteracts the destabilization by the framework region-2 hallmark amino acids. Second, the disulfide bond rigidifies the long third antigen-binding loop, leading to a stronger antigen interaction. This dual beneficial effect explains the in vivo antibody maturation process favoring VHH domains with an interloop disulfide bond.