The first step of glycosylphosphatidylinositol (GPI) anchor biosynthesis in all eukaryotes is the addition of N-acetylglucosamine (GlcNAc) to phosphatidylinositol (PI) which is catalysed by a UDP-GlcNAc : PI α1-6 GlcNAc-transferase. This enzyme has been shown to be a complex of at least seven subunits in mammalian cells and a similar complex of homologous subunits has been postulated in yeast. Homologs of most of these mammalian and yeast subunits were identified in the <Trypanosoma brucei> predicted protein database. The putative catalytic subunit of the <T. brucei> complex, TbGPI3, was epitope tagged with three consecutive c-Myc sequences at its C-terminus. Immunoprecipitation of TbGPI3-3Myc followed by native polyacrylamide gel electrophoresis and anti-Myc Western blot showed that it is present in a ~240 kDa complex. Label-free quantitative proteomics were performed to compare anti-Myc pull-downs from lysates of TbGPI-3Myc expressing and wild type cell lines. TbGPI3-3Myc was the most highly enriched protein in the TbGPI3-3Myc lysate pull-down and partner proteins TbGPI15, TbGPI9, TbGPI2, TbGPI1 and TbERI1 were also identified with significant enrichment. Our proteomics data also suggest that an Arv1-like protein (TbArv1) is a subunit of the <T. brucei> complex. Yeast and mammalian Arv1 have been previously implicated in GPI biosynthesis, but here we present the first experimental evidence for physical association of Arv1 with GPI biosynthetic machinery. A putative E2-ligase has also been tentatively identified as part of the <T. brucei> UDP-GlcNAc : PI α1-6 GlcNAc-transferase complex.

中文翻译：

---

蛋白质组学鉴定的<Trypanosoma brucei>的糖基磷脂酰肌醇生物合成途径的UDP-GlcNAc：PIα1-6GlcNAc-转移酶亚基。

在所有真核生物中糖基磷脂酰肌醇（GPI）锚定生物合成的第一步是将N-乙酰氨基葡糖（GlcNAc）添加到磷脂酰肌醇（PI）中，后者由UDP-GlcNAc：PIα1-6GlcNAc-转移酶催化。该酶在哺乳动物细胞中已显示为至少七个亚基的复合物，并且已在酵母中假定了类似的同源亚基的复合物。在<Trypanosoma brucei>预测蛋白数据库中鉴定了大多数这些哺乳动物和酵母亚基的同源物。<T的推定催化亚基。布鲁斯复合体TbGPI3在其C末端被三个连续的c-Myc序列标记为抗原决定簇。TbGPI3-3Myc的免疫沉淀，天然聚丙烯酰胺凝胶电泳和抗Myc Western印迹表明，它存在于〜240 kDa的复合物中。进行了无标记的定量蛋白质组学，以比较来自表达TbGPI-3Myc和野生型细胞系裂解物的抗Myc下拉。TbGPI3-3Myc是TbGPI3-3Myc裂解物下拉物中最富集的蛋白质，伴侣蛋白TbGPI15，TbGPI9，TbGPI2，TbGPI1和TbERI1也被发现具有明显的富集。我们的蛋白质组学数据还表明，类似Arv1的蛋白（TbArv1）是<T的一个亚基。brucei>复杂。酵母和哺乳动物Arv1以前已经参与了GPI生物合成，但是在这里，我们提供了Arv1与GPI生物合成机制之间物理联系的第一个实验证据。暂时还假定推定的E2-连接酶是<T的一部分。Brucei> UDP-GlcNAc：PIα1-6GlcNAc-转移酶复合物。