Engineering of a functional interleukin-5 monomer: a paradigm for redesigning helical bundle cytokines with therapeutic potential in allergy and asthma.
Engineering of a functional interleukin-5 monomer: a paradigm for redesigning helical bundle cytokines with therapeutic potential in allergy and asthma.
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功能性白细胞介素 5 单体的工程设计：重新设计具有过敏和哮喘治疗潜力的螺旋束细胞因子的范例。
DOI：
10.1007/bf00204980
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Huston,DP
中科院分区：
文献类型：
--
作者：
Dickason,RR;English,JD;Huston,DP
白细胞介素 (IL) 5 特异性诱导嗜酸性粒细胞的分化，而嗜酸性粒细胞是过敏和哮喘发病机制的核心。从结构上看，IL-5 是细胞因子短链螺旋束亚家族的独特成员。与单分子折叠成单个螺旋束的其他亚家族成员相比，IL-5 通过由一对分子间二硫键共价连接的两个相同单体相互交叉形成一对螺旋束。尽管天然 IL-5 单体缺乏生物活性，但我们最近报道了 IL-5 插入突变体（称为 mono5）的工程改造，该突变体单分子折叠成单个螺旋束，并具有与天然 IL-5 相似的生物活性。在这里，我们通过早期酪氨酸磷酸化事件、Jak2 激活和丝裂原激活蛋白激酶激活的蛋白质印迹分析确定，mono5 和 IL-5 利用的信号转导途径没有差异。然而，利用构象依赖性中和性抗IL-5单克隆抗体的结合研究在mono5插入物附近定位了三级结构扰动。这种扰动使得能够定位与 IL-5 受体 α 链结合的 IL-5 三级结构的有限区域。荧光标记研究进一步揭示了mono5的半胱氨酸含有游离的巯基，从而证明IL-5的二硫键的作用是维持其他功能域的结构。在还原条件下，mono5 而非 IL-5 保留了构象表位，并且尽管 mono5 中不存在二硫键，但 mono5 和 IL-5 具有等效的热稳定性，这表明 mono5 所呈现的构象非常稳定。除了提供设计新型 IL-5 激动剂和拮抗剂的结构框架外，从 mono5 开发中获得的知识将使其他螺旋束蛋白能够重新设计，具有治疗潜力。
Interleukin (IL) 5 specifically induces the differentiation of eosinophils which are central to the pathogenesis of allergies and asthma. Structurally, IL-5 is a unique member of the short-chain helical bundle subfamily of cytokines. In contrast to other subfamily members which fold unimolecularly into a single helical bundle, IL-5 forms a pair of helical bundles by the inter-digitation of two identical monomers covalently linked by a pair of intermolecular disulfide bonds. Although a native IL-5 monomer lacks bioactivity, we recently reported the engineering of an insertional mutant of IL-5 (designated mono5) which folds unimolecularly into a single helical bundle and has biological activity similar to that of native IL-5. Here we demonstrate no differences in signal transduction pathways utilized by mono5 and IL-5, as determined by western blot analysis of early tyrosine phosphorylation events, Jak2 activation, and mitogen-activated protein kinase activation. However, binding studies utilizing conformationally dependent neutralizing anti-IL-5 monoclonal antibodies localized a tertiary structural perturbation near the insert of mono5. This perturbation enabled localization of a limited region of the tertiary structure of IL-5 that engages the IL-5 receptor α-chain. Fluorescent labeling studies further revealed that the cysteines of mono5 contained free sulfhydryl groups, thereby demonstrating that the role of the disulfide bonds of IL-5 is the structural maintenance of other functional domains. The retention of conformational epitopes by mono5, but not IL-5, under reducing conditions and the equivalent thermostability of mono5 and IL-5 despite the absence of a disulfide bond in mono5 indicated that the conformation assumed by mono5 is very stable. In addition to providing the structural framework for designing novel IL-5 agonists and antagonists, the knowledge gained from the development of mono5 will enable other helical bundle proteins to be redesigned with therapeutic potential.