TMT spectrometry unveils key M6a interactors

The membrane glycoprotein M6a -together with proteolipid protein (PLP), DM20 and M6b- belongs to the tetraspan PLP family. M6a is a neuronal surface protein that promotes neuronal stem cell differentiation, migration, neurite and axonal outgrowth, filopodia/spine induction and synapse formation in primary neuronal cultures and non-neuronal cell lines. M6a has two extracellular loops (EC1 and EC2). four transmembrane domains, one intracellular loop and the N and C terminus facing the cell cytoplasm. However, the complete mechanism of action or proteins associated with it through its extracellular loops remained unknown. In previous work we provided strong evidence that M6a´s extracellular loops widely contribute to its function. To asses this question, we designed and purified a chimera protein (M6a-loops as a bait protein) which was subjected to co-immunoprecipitation (Co-Ip) with rat hippocampi homogenates followed by TMT-MS. The TMT-MS and data analysis was done by the Proteomics Core Facility from the European Molecular Biology Laboratory (EMBL, Heidelberg, Germany).

细胞膜糖蛋白M6a，与蛋白脂质蛋白（PLP）、DM20及M6b共同构成四跨膜蛋白家族。M6a是一种神经元表面蛋白，能够促进神经元干细胞的分化、迁移、突起和轴突生长、丝状伪足/棘突诱导以及原代神经元培养和非神经元细胞系中的突触形成。M6a具有两个细胞外环（EC1和EC2）、四个跨膜结构域、一个细胞内环以及面向细胞质浆的N端和C端。然而，M6a通过其细胞外环所发挥的完整作用机制或与之相关的蛋白仍属未知。在先前的研究中，我们提供了强有力的证据表明M6a的细胞外环广泛参与了其功能的实现。为了评估这一问题，我们设计并纯化了嵌合蛋白（以M6a环作为诱饵蛋白），随后使用大鼠海马组织匀浆进行共免疫沉淀（Co-Ip）实验，并紧接着进行了TMT-MS分析。TMT-MS及数据分析工作由欧洲分子生物学实验室（EMBL，海德堡，德国）的蛋白质组学核心设施完成。