Komekado Hideyuki, Yamamoto Hideki, Chiba Tsutomu, Kikuchi Akira
Department of Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
Genes Cells. 2007 Apr;12(4):521-34. doi: 10.1111/j.1365-2443.2007.01068.x.
Wnt-3a is a representative ligand that activates the beta-catenin-dependent pathway in Wnt signaling and is modified with glycans and palmitate. In this study, we analyzed the relationship between glycosylation and lipidation of Wnt-3a. Secretion of a Wnt-3a mutant that lacks glycosylation (Wnt-3a NQ) was impaired. Wnt-3a C77A, which lacks palmitoylation at Cys77, was secreted with similar efficiency to wild-type Wnt-3a (Wnt-3a WT), but did not induce the internalization of low-density lipoprotein receptor-related protein 6 (LRP6). Furthermore, removal of palmitate from Wnt-3a suppressed the ability to bind to its receptors Frizzled8 and LRP6. Wnt-3a C77A was glycosylated to an extent similar to Wnt-3a WT, while Wnt-3a NQ was not modified with palmitate. Expression of porcupine, which is a putative acyltransferase, enhanced palmitoylation of Wnt-3a WT greatly, but that of Wnt-3a NQ slightly. While Wnt-3a WT was present in both the endoplasmic reticulum (ER) and Golgi, Wnt-3a NQ was located to the ER only. Furthermore, Wnt-3a was not palmitoylated but was glycosylated in the cells treated with Brefeldin A, which inhibits transport of vesicles from the ER to the Golgi. These results indicate that glycosylation of Wnt-3a precedes palmitoylation and that both modifications are necessary for secretion of an active Wnt-3a.
Wnt-3a是一种代表性配体，可激活Wnt信号通路中依赖β-连环蛋白的途径，并被聚糖和棕榈酸修饰。在本研究中，我们分析了Wnt-3a的糖基化与脂酰化之间的关系。缺乏糖基化的Wnt-3a突变体（Wnt-3a NQ）的分泌受损。在半胱氨酸77处缺乏棕榈酰化的Wnt-3a C77A以与野生型Wnt-3a（Wnt-3a WT）相似的效率分泌，但不诱导低密度脂蛋白受体相关蛋白6（LRP6）的内化。此外，从Wnt-3a中去除棕榈酸会抑制其与受体卷曲蛋白8（Frizzled8）和LRP6结合的能力。Wnt-3a C77A的糖基化程度与Wnt-3a WT相似，而Wnt-3a NQ未被棕榈酸修饰。假定的酰基转移酶豪猪蛋白的表达极大地增强了Wnt-3a WT的棕榈酰化，但对Wnt-3a NQ的增强作用较小。虽然Wnt-3a WT在内质网（ER）和高尔基体中均有存在，但Wnt-3a NQ仅定位于内质网。此外，在经布雷菲德菌素A处理的细胞中，Wnt-3a未被棕榈酰化但被糖基化，布雷菲德菌素A可抑制囊泡从内质网向高尔基体的转运。这些结果表明，Wnt-3a的糖基化先于棕榈酰化，并且两种修饰对于活性Wnt-3a的分泌都是必需的。
