Disulfide Bond Requirements for Active Wnt Ligands
Disulfide Bond Requirements for Active Wnt Ligands
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DOI：
10.1074/jbc.m114.575027
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发表时间：
2014-06-27
影响因子：
4.8
通讯作者：
He, Xi
中科院分区：
文献类型：
--
作者：
MacDonald, Bryan T.;Hien, Annie;He, Xi
分泌型Wnt脂蛋白是富含半胱氨酸和脂质修饰的形态发生素，与卷曲蛋白（FZD）受体和低密度脂蛋白受体相关蛋白6（LRP 6）结合。Wnt通过突出的拇指和食指结构域接合FZD，所述拇指和食指结构域各自由通过二硫键固定的成对β链组装并抓住FZD胞外域的两侧。Wnt二硫键的重要性已被假定但未表征。我们系统地分析了原型Wnt 3a中的半胱氨酸和相关的二硫键。我们的数据表明，Wnt 3a的任何单个半胱氨酸的突变通过异位分子间二硫键形成导致共价Wnt寡聚体，并减少/消除Wnt信号传导。尽管鞘脂激活蛋白样结构域的氨基部分和食指基部中的个体半胱氨酸突变具有更好的耐受性并允许残留的Wnt 3a分泌/活性，但氨基末端、拇指和食指尖端中的那些半胱氨酸突变与分泌和/或活性不相容。基于二硫键模式的一些选择的双半胱氨酸突变体恢复Wnt分泌/活性。此外，食指指尖的双半胱氨酸突变导致Wnt 3a具有正常分泌，但最小的FZD结合和显性阴性性质。我们的研究结果实验验证了Wnt晶体结构的预测，并突出了saposin样和cavine样结构域的关键但不同的作用，包括拇指和食指在Wnt折叠/分泌和FZD结合。最后，我们修改了现有的表达载体的19个表位标记的人WNT蛋白的标签提供的异位半胱氨酸的去除，从而产生标记的WNT配体活性的典型和非典型的信号。
Secreted Wnt lipoproteins are cysteine-rich and lipid-modified morphogens that bind to the Frizzled (FZD) receptor and LDL receptor-related protein 6 (LRP6). Wnt engages FZD through protruding thumb and index finger domains, which are each assembled from paired beta strands secured by disulfide bonds and grasp two sides of the FZD ectodomain. The importance of Wnt disulfide bonds has been assumed but uncharacterized. We systematically analyzed cysteines and associated disulfide bonds in the prototypic Wnt3a. Our data show that mutation of any individual cysteine of Wnt3a results in covalent Wnt oligomers through ectopic intermolecular disulfide bond formation and diminishes/abolishes Wnt signaling. Although individual cysteine mutations in the amino part of the saposin-like domain and in the base of the index finger are better tolerated and permit residual Wnt3a secretion/activity, those in the amino terminus, the thumb, and at the tip of the index finger are incompatible with secretion and/or activity. A few select double cysteine mutants based on the disulfide bond pattern restore Wnt secretion/activity. Further, a double cysteine mutation at the index finger tip results in a Wnt3a with normal secretion but minimal FZD binding and dominant negative properties. Our results experimentally validate predictions from the Wnt crystal structure and highlight critical but different roles of the saposin-like and cytokine-like domains, including the thumb and the index finger in Wnt folding/secretion and FZD binding. Finally, we modified existing expression vectors for 19 epitope-tagged human WNT proteins by removal of a tag-supplied ectopic cysteine, thereby generating tagged WNT ligands active in canonical and non-canonical signaling.