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Development of Ganglioside Probes for Live Imaging of Lipid Rafts
Naoko Komura,Hiromune Ando,Kenichi Suzuki,Rahul Chadda,Yasuhiro Ikeda,Hideharu Ishida,Akihiro Kusumi,Makoto Kiso 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1
Lipid rafts have been assumed to work as a platform where protein receptors, cholesterols and gangliosides (representatively GM3 and GM1) assemble and interact for efficient signal transduction. According to the observation of these raft molecules by single molecule tracking technique, raft components were frequently but very transiently recruited to the cluster of GPI-anchored proteins formed upon binding of extracellular signaling molecules [1]. However, behaviors and functions of gangliosides in living cell membranes have never been extensively investigated because of the lack of their fluorescent probes that behave as equivalents of native gangliosides. To address this issue, we intend to develope novel fluorescent GM3 and GM1 probes for single molecule tracking, in which glycan parts are site-specifically labeled with various fluorescent dyes and evaluate the functionality of the fluorescent gangliosides. We designed the replacement of the C9 hydroxyl groups of sialic acid of GM3 and GM1 with amino groups to introduce fluorescent dyes. The synthesis of glycan parts of GM3 and GM1 were successfully achieved by using a Neu-Gal unit having a trifluoroacetamide at the C9 position. Next, the glycan parts were glycosidated with the Glc-Cer acceptor developed by our research group [2], yielding the skeletons of GM3 and GM1, respectively, which was followed by the conversion of trifluoroacetamide groups into amino groups. Finally the amino GM3 and GM1 were conjugated with fluorescent dyes through amide linkages, producing the targeted fluorescent gangliosides GM3 and GM1. The synthesized gangliosides were subjected to biophysical evaluations; DRM analysis and single molecule observation of colocalization with raft molecules (GPI-anchored protein, CD59 and epidermal growth factor receptor). Results obtained in these evaluations demonstrated great influence of the loaded position and polarity of fluorescent dye on the raftphilicity of gangliosides. Furthermore, the Kd value of the fluorescent GM1 probe for cholera toxin B subunit (CTXB) was comparable to that of native GM1, indicating that dye did not interfere the binding to CTXB. These results strongly suggest that the fluorescent gangliosides can be used to identify behaviors and functions of gangliosides in raft domains.
Synthesis of SSEA-1, SSEA-3 and SSEA-4 for developing bio-compatible-nanomaterials
Rita Pal,Naoko Komura,Akihiro Imamura,Hiromune Ando,Hideharu Ishida,Makoto Kiso 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1
Stage-specific embryonic antigens1 (SSEA’s) have been identified as glycosphingolipids which play diverse roles in embryonic development, such as regulation of cell growth, recognition and differentiation. It’s well-documented that human ES and iPS cell express predominantly glycosphingolipids of the globo- series2 such as SSEA-3 and SSEA-4, which are also hallmarks of human embryonal carcinoma cells. While, SSEA-1 represents a marker for murine pluripotent stem cells, in which it plays an important role in adhesion and migration of the cells in the preimplantation embryo. We are intended to integrate their biological functions into biocompatible nanomaterials for developing ES and iPS cell culturing system. To conjugate with nanomaterials these molecules have been suitably functionalized at the terminal position. In the present study, a convergent chemical synthesis of SSEA-1 SSEA-3 and SSEA-4 will be discussed.