An Inhibitory Antibody Blocks the First Step in the Dithiol/Disulfide Relay Mechanism of the Enzyme QSOX1
An Inhibitory Antibody Blocks the First Step in the Dithiol/Disulfide Relay Mechanism of the Enzyme QSOX1
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DOI：
10.1016/j.jmb.2013.07.011
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发表时间：
2013-11-15
影响因子：
5.6
通讯作者：
Fass, Deborah
中科院分区：
文献类型：
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作者：
Grossman, Iris;Alon, Assaf;Fass, Deborah
Quiescin巯基氧化酶1（QSOX 1）是二硫键形成的催化剂，其经历成纤维细胞的调节分泌，并且在腺癌和其他癌症中过度产生。我们最近已经表明，QSOX 1是所需的特定层粘连蛋白异构体掺入到培养的成纤维细胞的细胞外基质（ECM），因此，肿瘤细胞粘附和渗透的ECM。层粘连蛋白在整联蛋白介导的细胞存活和运动中的已知作用表明，控制QSOX 1活性可能提供一种对抗转移性疾病的新方法。基于这个动机，我们开发了一种抑制人QSOX 1活性的单克隆抗体。在这里，我们提出了这种抗体的生物化学和结构表征，并证明它是一种紧密结合的抑制剂，阻断酶中的氧化还原活性位点之一，但不是从头二硫化物催化产生的位点。因此，巯基氧化酶活性被阻止而不直接结合巯基氧化酶结构域，证实了最初基于诱变和蛋白质解剖推测的结构域间QSOX 1电子转移机制的模型。此外，我们开发了抗体的单链变体，并表明它是一种有效的QSOX 1抑制剂。QSOX 1抑制性抗体将是研究ECM组成和结构在细胞迁移中的作用的有价值的工具，并且重组版本可以进一步开发用于基于肿瘤微环境操纵的潜在治疗应用。(C)2013爱思唯尔有限公司保留所有权利。
Quiescin sulfhydryl oxidase 1 (QSOX1) is a catalyst of disulfide bond formation that undergoes regulated secretion from fibroblasts and is over-produced in adenocarcinomas and other cancers. We have recently shown that QSOX1 is required for incorporation of particular laminin isoforms into the extracellular matrix (ECM) of cultured fibroblasts and, as a consequence, for tumor cell adhesion to and penetration of the ECM. The known role of laminins in integrin-mediated cell survival and motility suggests that controlling QSOX1 activity may provide a novel means of combating metastatic disease. With this motivation, we developed a monoclonal antibody that inhibits the activity of human QSOX1. Here, we present the biochemical and structural characterization of this antibody and demonstrate that it is a tight-binding inhibitor that blocks one of the redox-active sites in the enzyme, but not the site at which de novo disulfides are generated catalytically. Sulfhydryl oxidase activity is thus prevented without direct binding of the sulfhydryl oxidase domain, confirming the model for the interdomain QSOX1 electron transfer mechanism originally surmised based on mutagenesis and protein dissection. In addition, we developed a single-chain variant of the antibody and show that it is a potent QSOX1 inhibitor. The QSOX1 inhibitory antibody will be a valuable tool in studying the role of ECM composition and architecture in cell migration, and the recombinant version may be further developed for potential therapeutic applications based on manipulation of the tumor microenvironment. (C) 2013 Elsevier Ltd. All rights reserved.