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

脑膜中存在的糖基磷脂酰肌醇（GPI）锚定蛋白在神经网络系统的构成中起着至关重要的作用。一些报道认为gpi锚定蛋白的糖基化在胚胎发育过程中可能发生改变，但由于纯化和分析困难，仅报道了少数gpi锚定蛋白的碳水化合物功能和结构。在本项目中，我们通过SDS-PAGE和LC/Ms^n分离，研究了大鼠脑中gpi锚定蛋白的位点特异性糖基化。用SDS-PAGE对经PIPLC处理的大鼠脑膜释放的gpi连接蛋白进行分离。在20-25 kDa和45-85 kDa波段分别鉴定为Thy-1和LAMP、OBCAM、NTM、kilon的混合物。首先，用1%的SDS从凝胶中提取Thy-1，用LC/MS^n对Thy-1进行胰蛋白酶消化，进行位点特异性糖基化分析。结果表明，Asn23和98附着于高甘露糖型、杂化型和复合型低聚糖上，Asn74附着于杂化型低聚糖上。同样，LAMP、OBCAM、NTM和kilon的位点特异性糖基化分析是通过在45-85 kDa的波段提取蛋白质，然后采用LC/MS^n进行的。我们发现在四种蛋白质中，第一结构域的糖基化位点被高甘露糖型低聚糖占据，而第三结构域的糖基化位点则附着在带有Le^x基序的低聚糖上。我们计划将该方法应用于其他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.