2005 Fiscal Year Final Research Report Summary
Structural and Functional analyses of GPI-anchored proteins by nano-LC/MS
| Project/Area Number |
15590052
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Physical pharmacy
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| Research Institution | National Institute of Health Sciences |
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Principal Investigator |
KAWASAKI Nana Natl.Inst.Health Sci., Biol.Chem.& Biologicals, Section Chief, 生物薬品部, 室長 (20186167)
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| Co-Investigator(Kenkyū-buntansha) |
HACHISUKA Akiko Natl.Inst.Health Sci, Biochem.& Immunochem, Senior Scientist, 機能生化学部, 主任研究官 (70184504)
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| Project Period (FY) |
2003 – 2005
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| Keywords | LC / MS / glycosylation / GPI-anchored protein / Thy-1 / LAMP / OBCAM / NTM / kilon |
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| Research Abstract |
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.
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Research Products
(33 results)
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[Journal Article] A non-sulfated from of the NHK-1 carbohydrate is specifically expressed in mouse kidney2005
Author(s)
Hideki Tagawa, Yasuhiko Kizuka, Tomoko Ikeda, Satsuki Itoh, Nana Kawasaki, Hidetaka Kurihara, Maristela Lika Onozaki, Akihiro Tojo, Tasuo Sakai, Toshisuke Kawasaki, Shogo Oka
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Journal Title
J. Biol. Chem. 280
Pages: 23876-23883
Description
「研究成果報告書概要(和文)」より
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[Journal Article] Mannan-binding protein blocks the activation of metalloproteases meprin a and b,2005
Author(s)
Makoto Hirano, Bruce Yong Ma, Nana Kawasaki, Kazumichi Okimura, Makoto Baba, Tomoaki Nakagawa, Keiko Miwa, Nobuko Kawasaki, Shogo Oka, Toshisuke Kawasaki
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Journal Title
J. Immunol. 175
Pages: 3177-3185
Description
「研究成果報告書概要(和文)」より
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[Journal Article] non-sulfated from of the NHK-1 carbohydrate is specifically expressed in mouse kidney2005
Author(s)
Hideki Tagawa, Yasuhiko Kizuka, Tomoko Ikeda, Satsuki Itoh, Nana Kawasaki, Hidetake Kurihara, Maristela Lika Onozaki, Akihiro Tojo, Tasuo Sakai, Toshisuke Kawasaki, Shogo Oka A
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Journal Title
J.Biol.Chem. 280
Pages: 23876-23883
Description
「研究成果報告書概要(欧文)」より
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