Establishment And Application Of Structure Analysis Methods Of Trace Amount Of Glycans In Animal Tissues

In the thesis, by using modern multiple extraction, separation and diverseanalysis techniques, a system method for structural analysis of ganglioside (GLS),N-glycans and glycosaminoglycan (GAGs) with small amount animal tissues wasestablished by using the liver, kidney and heart tissue of C57BL/6J mouse asmaterials. The method was firstly applied on studying the structural difference ofN-glycans from human cervical cancer cells (HeLa) and GAGs from kidneys of db/dbdiabetic mouse, respectively. Based on the method established, series endogenous andexogenous glycans were used as materials, the interaction of endogenous GLS andmarine neoglycolipids (NGLs) with (Neuraminidase, NA) of influenza A virus (H1N1)was firstly investigated by oligosaccharide chips method. Meanwhile, the interactionof endogenous GAGs and marine acidic polysaccharides with connective tissuegrowth factor (CTGF) was also firstly studied by polysaccharide chips method.The major conclusions were listed below:(1). A rapid analytical method of composition and structural characteristic of traceamount of GLS from animal tissues was established by high-performance thinlayer chromatography (HPTLC) and electrospray ionization mass spectrometry(ESI-MS) techniques. The method was firstly applied on the structural analysis ofGLS in the liver, kidney and heart tissue from C57BL/6J mice, respectively. It wasshown that the compositions of GLS from different tissues were significantlydifferent. The mice liver mainly contained GM1and GM2and the mice heartmainly contained GM1, GM2and GT1a. The content of GM3, GM2, GM1, GD1aand GT1a from mice kidney had little difference. The GLS content from liver washighest in the three tissues, and this maybe associate with it to be the major organof lipid metabolization.(2). A method of continuous release of trace amount of N-glycans and GAGssfrom the same tissue was established. The method was applied on N-glycans andGAGss extraction from the kidney of C57BL/6J mice. The result showed that thekidney mainly contained heparan sulfate (HS) and small amount of dermatan sulfate. Sixty kinds of glycans were detected according to the MALDI-TOF-MSprofiling, including high mannose types with relative high amount and mostN-glycans contained acetylneuraminic acid (NeuAc) and Fucose (Fuc). Theestablished N-glycan analysis method was firstly applied to the structure of HeLacells glycoprotein N-glycans profiling. The34types of N-glycans obtained fromHeLa cells included both high-mannose and complex (bi-, tri-, tetra-, andpenta-antennary) types, some of the latter containing bisecting and Lewisdeterminants, which were relevant to tumor metastasis in same way.(3). A microanalysis method of fine structural difference of trace amount of GAGsin animal tissues was established and applied on the disaccharides analysis ofGAGs from the kidney tissue of type2diabetic mice. By pre-columnderivatization of disaccharides with2-aminoacridone (2-AMAC) and followed byreverse-phase HPLC analysis, and compared with the normal C57BL/6J mouse,the N-sulfation degree of HS was obviously decreased in the kidney of db/dbdiabetes mice. It was notable that the marine-derived oligosaccharide drug (HS203)could enhance the sulfation degree of HS from the abnormal kidney tissue, thisresult suggest HS203could be a potential drug for treatment of diabeticnephropathy.(4). The interaction of6kinds of endogenous GLS and36kinds of NGLs preparedfrom marine red alga with H1N1neuraminidase (NA) was firstly analyzed byoligosaccharides chip method. It was shown that all36kinds of marine-derivedoligosaccharides showed a higher binding ability to NA proteins than theendogenous GLS, and the binding strength was related to sorts of residue, linkages,sulfation degree and position, and the degree of polymerization ofoligosaccharides.(5). The interaction of endogenous GAGs and marine acidic polysaccharide withtype2diabetic nephropathy related factor-CTGF was first analyzed bypolysaccharides chip method. The results showed that the sulfation degree and theuronic acid content of the polysaccharides significantly affected the bondingstrength to CTGF.In conclusion, the method of the system analysis of trace amount of GLS,N-glycans and GAGs in animal tissues was established, and this could be a reliablemethod for the glycomics study. It was also useful for new markers development fromtissues and new treatment for the early diagnosis of diseases. Meanwhile, it was beneficial to the validity of drug evaluation by analysis the fine structural differenceof GAGss. Furthermore, it could provide methodological foundation for further revealthe biological function of glycans in the occurrence and development of relateddiseases and new drugs development.