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

通过 Nano-LC/MS 对 GPI 锚定蛋白进行结构和功能分析

• 批准号: 15590052
• 负责人: KAWASAKI Nana
• 金额: $ 1.98万
• 依托单位: National Institute of Health Sciences
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590052/
• 关键词: LC; MS; glycosylation; GPI-anchored protein; Thy-1; LAMP; OBCAM; NTM; kilon; 電気泳動; 糖鎖構造; 2次元電気泳動

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.

糖基磷脂酰肌醇(GPI)锚定蛋白存在于脑膜上，在神经网络系统的构成中起着重要作用。一些报道认为GPI锚定蛋白的糖基化可能会在胚胎发育过程中发生变化，但由于纯化和分析的困难，仅有少数GPI锚定蛋白的碳水化合物功能和结构的报道。在本项目中，我们研究了大鼠脑内GPI锚定蛋白的位点特异性糖基化作用，采用LC/MS法分离GPI锚定蛋白。通过PIPLC处理从大鼠脑膜中释放的GPI连接蛋白用SDS-PAGE进行分级和分离。20-25 kDa和45-85 kDa的条带分别被鉴定为Thy-1和LAMP、OBCAM、NTM、KYON的混合物。首先，用1%的十二烷基硫酸钠从凝胶中提取Thy-1，用LC/MS^n对Thy-1的胰酶切进行位点特异性糖基化分析。Asn23和98以高甘露糖型、杂交型和复合型寡糖为主，Asn74以岩藻糖基复合型寡糖为主。同样，对LAMP、OBCAM、NTM和KEON进行了位点特异性糖基化分析，从45-85 kDa的条带中提取蛋白质，然后进行LC/MS^n。我们证明，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，而在第三结构域的糖基化位点与带有Le^x基序的低聚糖结合。

项目成果

Profiling analisis of oligosaccharides in antibody pharmaceuticals by capillary electrophoresis.

通过毛细管电泳对抗体药物中的寡糖进行轮廓分析。

• 发表时间: 2005
• 期刊: J.Chromatogr.A 1050
• 作者: Satoru Kamada;Chie Nomura;Mitsuhiro Kinoshita;Saori Nishiura;Rika Ishikawa;Kazuaki Kakehi;Nana Kawasaki;Takao Hayakawa
• 通讯作者: Takao Hayakawa

Glycomic/ glycoproteomic analysis by LC/MS : Analysis of glycan structural alternation in the cells

通过 LC/MS 进行糖组/糖蛋白质组分析：分析细胞中的聚糖结构变化

• 发表时间: 2005
• 期刊: Proteomics 5
• 作者: Noritaka Hahii;Nana Kawasaki;Satsuki Itoh;Masashi Hyuga;Toru Kawanishi;Takao Hayakawa
• 通讯作者: Takao Hayakawa

Sawada Kinetic analysis of peptide digestion of chicken egg white ovomucoid and allergenic potential pepsin fragments

Sawada 鸡蛋清卵类粘蛋白肽消化和潜在过敏性胃蛋白酶片段的动力学分析

• 发表时间: 2005
• 期刊: Int.Arch.Allergy Immunol. 136
• 作者: Kayoko Takahi;Reiko Teshima;Haruyo Okunuki;Satsuki Itoh;Nana Kawasaki;Toru Kawanishi;Takao Hayakawa;Yuichi Kohno;Atsuo Urisu;Jun-ichi
• 通讯作者: Jun-ichi

Analysis of site-specific glycosylation in recombinant human follistatin expressed in Chinese hamster ovary cells

中国仓鼠卵巢细胞表达的重组人卵泡抑素位点特异性糖基化分析

• 发表时间: 2004
• 期刊: Biologicals 32
• 作者: Masashi Hyuga;Satsuki Itoh;Nana Kawasaki;Miyako Ohta;Akiko Ishii;Sumiko Hyuga;Takao Hayakawa
• 通讯作者: Takao Hayakawa

Glycosylation analysis of glycoproteins by LC/MS/MS. Analysis of glycosylation site and of site-specific heterogeneity

通过 LC/MS/MS 对糖蛋白进行糖基化分析。

• 发表时间: 2004
• 期刊: J. Electrophoresis 48
• 作者: Satsuki Itho;Akira Harazono;Nana Kawasaki;Noritaka Hashii;Yukari Matsuishi;Toru Kawanishi;Takao Hayakawa
• 通讯作者: Takao Hayakawa