Synthesis And Application Of The Ganglioside-Magnetosome Complex And Lectin-Magnetosome Complex

Magnetosomes are synthesized by magnetotactic bacteria, which have drawn great interest as potential carriers to couple with diverse bioactive macromolecules, anticancer drugs and enzymes due to their unique features such as paramagnetism, nano-scale, narrow size distribution and membrane-bounded. However, it was rarely not applied in the glycobiology research.Gangliosides, a family of glycosphingolipids with one or more sialic acids, act as distinguishing surface markers and serve as specific determinants in cellular recognition and cell-to-cell communication. Exogenous gangliosides, are widely performed to study transport and metabolism of their endogenous counterparts, as well as their biological functions. However, gangliosides usually aggregate into micelles in the aqueous media, resulted in the restrained absorption into cells. Therefore, it is very important to find a kind of carrier to couple with gangliosides, which can enhance the absorption of gangliosides into cells. Lectins are a kind of proteins, which can recogenize some specific carbohydrates. The carriers coupled with lectins have the great potential application in separation and enrichment of glycoproteins.Firstly, we extracted gangliosides from dog blood and pig brain. 43.26 mg GM1 and 53.26 mg GM3 were obtained. The purity of GM1 and GM3 were 96.7% and 98.2%, which were determined by Thin-Layer Chromatography(TLC) and Dot-blot. Then, 213.8 mg magnetosomes which contain the whole magetosome membranes were extracted from Magnetospirillum gryphiswaldense MSR-1.Secondly, we established a strategy to remove the proteins on the membrane of magnetosome by sodium dodecyl sulfate(SDS) and efficiently immobilized the gangliosides, GM1 and GM3, onto the magnetosome by ultrasonic treatment. Complex of magnetosome-GM1 presented the greatly enhanced cell incorporation, while complex of magnetosome-GM3 showed significant inhibition on phosphorylation of epithelial growth factor receptor(EGFR) stimulated by EGF. Moreover, cell incorporation of GM1 and inhibition on activation of EGFR by GM3 were further enhanced with exposure to the magnetic field.Fianlly, the magnetosomes were modified with phosphatidylethanolamine(PE), coupled with Bis(sulfoduccinimidyl) suberate sodium salt(BS3), and ligated with lectin, sequentially. Lectin-PE-magnetosome complexes were prepared and used for the separation of glycoproteins. The results showed that the proteins on magnetosomes surface were removed, and modified PE-magnetosomes showed the better dispersion than native magnetosomes. Moreover, 294.64 μg of wheat germ agglutinin(WGA) were coupled to one milligram of PE-magnetosomes, 1.6 times than that coupled to one milligram of magnetosomes. WGA-PE-magnetosomes could specifically separate the ovalbumin(OVA) from the mixture of OVA and Concanavalin A(Con A). Taken in all, the magetosomes showed the great potential application in separation and enrichment of glycoproteins in the glycomics research.