Structural and Functional analyses of GPI-anchored proteins by nano-LC/MS

存在于脑细胞膜上的糖基磷脂酰肌醇（GPI）锚定蛋白在神经网络系统的构成中起着重要的作用。一些研究表明GPI锚定蛋白的糖基化在胚胎发育过程中会发生改变，但由于纯化和分析的困难，目前只有少数GPI锚定蛋白的糖基化功能和结构被报道。本研究采用SDS-PAGE和LC/MS^n分离技术研究了大鼠脑内GPI锚定蛋白的位点特异性糖基化。20-25 kDa和45-85 kDa的条带分别被鉴定为Thy-1和LAMP、OBCAM、NTM、kilon的混合物。首先，用1%SDS从凝胶中提取Thy-1，并将Thy-1的胰蛋白酶消化物进行LC/MS^n以进行位点特异性糖基化分析。Asn 23和98分别为高甘露糖型、杂合型和复合型寡糖，Asn 74为岩藻糖基复合型寡糖。同样，通过从45-85 kDa处的条带中提取蛋白质，然后进行LC/MS^n，进行LAMP、OBCAM、NTM和kilon的位点特异性糖基化分析。我们证明了四个蛋白质中第一结构域的糖基化位点被高甘露糖型寡糖占据，第三结构域的糖基化位点被带有Le^x基序的寡糖所占据，我们计划将这种方法应用于其他GPI结合蛋白的位点特异性糖基化分析。

Glycosylphosphatidylinositol (GPI)-anchored proteins existing in brain membrane play an essential role in constitution of nerve network system. Some reports suggest that glycosylation of GPI-anchored proteins could alter during the embryo development, however, the carbohydrate function and structure of only a few GPI-anchored proteins are reported due to the difficulty of their purification and analysis. In this project we studied site-specific glycosylation of GPI-anchored proteins in rat brain by the separation with SDS-PAGE followed LC/Ms^n.GPI-linked proteins, which were released by PIPLC treatment from the rat brain membrane, were fractionated and separated by SDS-PAGE. The bands at 20-25 kDa and 45-85 kDa were identified as Thy-1 and a mixture of LAMP, OBCAM, NTM, kilon, respectively. First, Thy-1 was extracted from the gel with 1% SDS, and the tryptic digest of Thy-1 was subjected to LC/MS^n for site-specific glycosylation analysis. It was confirmed that Asn23 and 98 are occupied with high-mannose type, hybrid and complex type oligosaccharides, and Asn74 was attached to fucosylcomplex type oligosaccharides. Likewise, site-specific glycosylation analysis of LAMP, OBCAM, NTM, and kilon were carried out by extraction of the proteins from the band at 45-85 kDa followed by LC/MS^n. We demonstrated that the glycosylation sites in the first-domain are occupied with high-mannose type oligosaccharide among four proteins, and those in the third-domain are attached to oligosaccharides bearing Le^x motif.We are planning to apply this method to the site-specific glycosylation analysis of other GPI-binding proteins.