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Fluorescing Labeled Polysaccharide and Carbohydrate Chips for Studying Protein-Carbohydrate Interactions

Author: HanZhangRun
Tutor: YuGuangLi
School: Ocean University of China
Course: Medicinal Chemistry
Keywords: carbohydrate micro arrays fluorescent labeling retention rate carbohydrate protein interaction
CLC: Q504
Type: Master's thesis
Year: 2011
Downloads: 231
Quote: 2
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Abstract


Carbohydrates and their conjugates are involved in various biological events, including viral and bacterial infection, the immune response, differentiation and development, and the progression of tumor cell metastasis. Glycan arrays are a new technology that has enabled the high-sensitivity and rapid analysis carbohydrate-protein interaction and contribute to significant advances in glycomics. Glycan arrays use a minute amount of materials and can be used for high-throughput profiling and quantitative analysis and provide information for the development of carbohydrate-based vaccines and new drug discover. Polysaccharide chips is one part of Carbohydrates arrays,the fabrication of polysaccharide chips will accelerate progress of the study on glycome and drug discover.Labeling regent choose the fluorescent chromophore isothiocyanatofluorescein and its derivatives (Amino Fluorescent, AF).Aminofluorescein were covalently linked to the carboxyl groups of polysaccharide using 1-ethyl-3-(1,3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide as catalytic reagent; Isothiocyanatofluorescein reacts with polysaccharide in methyl sulphoxide at elevated temperatures t catalyzed by dibtityltin dilaurate. Polysaccharide was used to selectively insert primary amines by reductive animation followed by labeling FITC. Successfully labeled 34 different structures of the polysaccharide with degree of substitution rang 0.4%-2%。After purified those labeled polysaccharide and the appropriate sugar, fluorescence intensity quantitative, The fluorescent labeled polysaccharides were printed on the nitrocellulose coated glass chips with a microarrayer。Nitrocellulose membrane can hold those polysaccharides by Spatial network, and adsorption。after washing, some sugar float on the surface of nitrocellulose membrane just like cell surface glycoprotein, Then fluorescent mark of polysaccharide can be used to as probe indicates the polysaccharide retained on the chip, The preserved rate of polysaccharide on nitrocellulose membrane were calculated after washing,this result is very eventful in Carbohydrate and protein interactions.Interaction with Monoclonal Anti-Chondroitin Sulfate antibody (clone CS-56) was further studied, the experimental results showed that the preserving rate apparently different. The interaction of GAGs with CS-56 was scientifically compared according to their preserving rate on nitrocellulose glycoship, and after quantitatively calculation, the binding ability to CS-56 is:chondroitin sulfate C> D> A, otherwise, chondroitin sulfate B, heparin and heparan sulfate did not show any interaction with CS-56.In summary, study polysaccharides fluorescent labeling different polysaccharides systematically, Successfully labeled 34 different polysaccharide using different methods。the fluorescent labeling polysaccharides successfully applied to carbohydrate micro array, using the fluorescence intensity Changes as the probe retention rate of nitrocellulose membrane, This method solve the problem polysaccharide micro array can not be quantified before and after washing。The blinding experimental with Anti-Chondroitin Sulfate antibody and Connective Tissue growth factor Verified the fluorescence labeled polysaccharide chip carbohydrate-protein interaction, found out some polysaccharides can bind to CTGF.Those polysaccharide micro array will became one powerful tool for study glycome and drug discover。

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