Mutational analysis of sclerostin shows importance of the flexible loop and the cystine-knot for Wnt-signaling inhibition.
Mutational analysis of sclerostin shows importance of the flexible loop and the cystine-knot for Wnt-signaling inhibition.
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DOI：
10.1371/journal.pone.0081710
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Mueller TD
中科院分区：
文献类型：
--
作者：
Boschert V;van Dinther M;Weidauer S;van Pee K;Muth EM;Ten Dijke P;Mueller TD
含胱氨酸结的蛋白质硬骨素是骨骼生长的重要负调节因子，因此是一个有前景的治疗靶点。它通过抑制Wnt（无翅型和int1）信号通路来发挥其生物学作用，该通路通过促进间充质干细胞向成骨细胞分化而参与骨形成。硬骨素的核心结构由三个环组成，其中第一环和第三环（指1和指2）形成一个结构化的β -折叠，第二环无结构且高度灵活。生化数据表明，灵活环对于硬骨素与低密度脂蛋白相关蛋白家族（LRP）的Wnt共受体结合很重要，它通过与Wnt共受体LRP5或 - 6相互作用来抑制Wnt信号。为了进一步研究Wnt抑制的结构要求，我们在硬骨素核心结构域的所有三个环内进行了广泛的突变研究，包括单突变和多突变以及重要区域的截断。通过这种方法，我们可以证实第二环的重要性，特别是氨基酸Asn92和Ile94对于与LRP6结合的重要性。基于在一个患有骨硬化症的土耳其家族中发现的硬骨素变体，我们生成了一个硬骨素突变体，其中半胱氨酸84和142被替换，从而去除了胱氨酸结的第三个二硫键。该突变体与LRP6的结合亲和力降低，并且对Wnt1的抑制活性也大大降低，这表明灵活环之外的元件对于硬骨素抑制Wnt也很重要。此外，我们研究了突变对两种不同Wnt蛋白（Wnt3a和Wnt1）抑制的影响。我们可以检测到对这些蛋白抑制的明显差异，这表明硬骨素拮抗Wnt1和Wnt3a的机制存在根本差异。
The cystine-knot containing protein Sclerostin is an important negative regulator of bone growth and therefore represents a promising therapeutic target. It exerts its biological task by inhibiting the Wnt (wingless and int1) signaling pathway, which participates in bone formation by promoting the differentiation of mesenchymal stem cells to osteoblasts. The core structure of Sclerostin consists of three loops with the first and third loop (Finger 1 and Finger 2) forming a structured β-sheet and the second loop being unstructured and highly flexible. Biochemical data showed that the flexible loop is important for binding of Sclerostin to Wnt co-receptors of the low-density lipoprotein related-protein family (LRP), by interacting with the Wnt co-receptors LRP5 or -6 it inhibits Wnt signaling. To further examine the structural requirements for Wnt inhibition, we performed an extensive mutational study within all three loops of the Sclerostin core domain involving single and multiple mutations as well as truncation of important regions. By this approach we could confirm the importance of the second loop and especially of amino acids Asn92 and Ile94 for binding to LRP6. Based on a Sclerostin variant found in a Turkish family suffering from Sclerosteosis we generated a Sclerostin mutant with cysteines 84 and 142 exchanged thereby removing the third disulfide bond of the cystine-knot. This mutant binds to LRP6 with reduced binding affinity and also exhibits a strongly reduced inhibitory activity against Wnt1 thereby showing that also elements outside the flexible loop are important for inhibition of Wnt by Sclerostin. Additionally, we examined the effect of the mutations on the inhibition of two different Wnt proteins, Wnt3a and Wnt1. We could detect clear differences in the inhibition of these proteins, suggesting that the mechanism by which Sclerostin antagonizes Wnt1 and Wnt3a is fundamentally different.
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