| With the development of glycochemistry and glycobiology, the glycan-based medicine research has captured researcher’s interests worldwide. However, the number of glycan-based drug for treatment and/or diagnosis was much smaller than small molecular-based drug. The complexity of polysaccharide structure and difficulty of targeting bioactive study were main reason which causes such situation. The interaction study between the structural elucidated polysaccharide and the key protein could promote the research and development of glycan-based drug. Glycosaminoglycan, such heparin and heparan sulfate, played extensive and key roles in organisms. Accordingly, this project was expected to characterize the negative charged polysaccharides structure and understand the bioactivities. Through the interaction between glycan and protein, we could reveal the polysaccharides bioactive mechanism and develop the glycanbased new drug candidates.Nemacystus decipiens is a brown seaweed which is served as food and traditional medicine herb. According to record in Chinese alternative medicine book, Ben-CaoShi-Yi, N. decipiens showed a lot of bioactivities. However, up to date, the structure of polysaccharides and bioactivity of natural product from N. decipiens are still largely unknown. Based on the 1 H and 13 C NMR, researcher had deduced that the polysaccharide’s linkage type. Nevertheless, the fine structure and bioactivity of fucoidan from N. decipiens have not been reported yet.In order to reveal the bioactive mechanism of N. decipiens, the polysaccahrides in this seaweed was extracted and analyzed. Firstly, the sundried N. decipiens was extracted with 80℃ water to give the crude polysaccharide NDH. The monosaccharide composition analysis and infrared spectroscopy(IR) spectrum indicated that crude polysaccharide DNH was sulfated fucoidan. The brownish crude polysaccharide was applied to Q sepharose Fast Flow column and Sephacryl S-300 HR column, to give the 6 homogeneous polysaccharides, NDH01, NDH221, NDH231, NDH241, NDH31 andNDH41.Using infrared spectroscopy(IR), nuclear magnetic resonance(NMR), sugar composition, methylation analysis, partial hydrolysis, etc techniques, we showed that NDH01 was composed of mannose(Man), glucuronic acid(Gla A), fucose(Fuc), sulfate group and acetyl group in the molar ratio of 3.0: 14.4: 82.6: 34.3: 13.9. The backbone of NDH01 was fucose-free core, composed of α-D-1,2-Manp and β-D-1,4-Glcp A disaccharide repeat unit. The branches were attached at the C3, C4 and C6 of α-D-1,2-Manp. The branch was composed of α-L-1,3,4-Fucp, α-L-1,4-Fucp, α-L-1,3-Fucp and α-L-1,4-Glcp A. The sulfate group was linked to C4 of α-L-1,3,4-Fucp, whereas, acetyl group was branched on C2 of α-L-1,2,3-Fucp.The bioactivity study indicated that NDH01 could disrupt tube formation and inhibit the migration as well as cell growth of human microvascular endothelial cells(HMEC-1). Besides, phosphorylation of Smad/1/5/8, Erk and FAK was significantly inhibited by NDH01. Further studies uncovered that NDH01 blocked Smad1/5/8 signaling via interacting with bone morphogenetic protein 4 and downregulating bone morphogenetic protein 4(BMP4) expression. In addition, NDH01 could bind to bone morphogenetic protein(BMP2) and BMP4 strongly. The KD values ware evaluated to respectively 8.93 × 10-6, 4.06 × 10-8 by surface plasmon resonance(SPR).The tube formation assay indicated that the other homogeneous polysaccharides, NDH221, NDH231, NDH241, NDH32 and NDH41 in this seaweed also could inhibit the HMEC-1 cells tube formation. The interaction between NDH221, NDH231, NDH32, NDH41 and BMP2 was investigated and their binding affinity KD values were calculated to 7.67 × 10-6, 1.16 × 10-5, 1.5 × 10-5, 2.55 × 10-6, respectively by SPR analysis. Besides, the binding between NDH221, NDH231, NDH241, NDH32, NDH41 and BMPR- I A, BMPR-II was also detected. The KD values between fucoidans and BMPR- I A were 3.34 × 10-5,2.64 × 10-5,2.95 × 10-5,2.07 × 10-5,1.83 × 10-5, respectively. Moreover, the KD values of the binding between fucoidans and BMPR-II were 3.13 × 10-5,2.70 × 10-5,2.15 × 10-5,9.33 × 10-6,7.67 × 10-6, respectively. Thefurther study revealed that the polysaccharides could not only bind to BMP but also bind to BMPR- I A,BMPR- II. The cellular level bioactivity was positive correlated with the binding affinity between fucoidans and BMPR- I A,BMPR- II. The result suggested that the interaction between fucoidans and BMPR seemly played more significant role on anti-angiogenesis than the interplay between polysaccharides and BMP2.To understand the structure basis of targeting polysaccharide, the fucoidan oligosaccharides were prepared through fucoidan polysaccharides degradation by H2O2/Vc(vitamin C) and diluted TFA. Furthermore, the rh BMP2 was expressed in BL21 E. coli. system. The inclusion body BMP2 was harvested and denatured. The purified BMP2 monomer was refolded to form the functional dimer. The glycan-BMP2 co-crystal culture condition was screened by Hampton crystal kits. Fortunately, the acicular crystal was cultured in PEG-isopropanol-MES buffer system.Cynomorium songaricum is a traditional Chinese medicine herb which has showed a lot of medicinal function, such as, reproductive function, HIV-1-protease inhibitory, and immunity modulation. A lot of natural products and its bioactivities have been reported. However, most of bioactive compounds elucidated from this plant are small molecular, such as, triterpenoid, steroid, lignan, alkaloid and flavonoid. Only two neutral polysaccharides were analyzed.To better understand the structure and function of complex negative charged polysaccharide in this plant, a novel rhamnogalacturonan I pectin, named CSW2S2, was purified from traditional Chinese medicine C. songaricum by DEAE-cellulose anion-exchange chromatography and Sephacryl S-300 gel permeation chromatography. CSW2S2 was composed of rhamnose(Rha), galacturonic acid(Gal A), galactose and arabinose(Ara) in a molar ratio of 7.7: 8.2: 26.7: 57.4. The IR, NMR, methylation analysis and partial hydrolysis data revealed the detail structure of CSW2S2. The backbone of the polysaccharide consisted of alternate 1,2-linked α-L-Rhap and 1,4-linked α-D-Glcp A which branched at C4 of 1,2-Rhap. The arabinan sidechain wasmainly composed of 1,5-Ara which had the T-Ara attached at C3 and C2. Another sidechain was galactan which had the Ara non-reducing terminal. The galactan was composed of 1,4-Gal and 1,6-Gal in which the T-Gal attached at C3. The bioactivity study showed that the polysaccharide could protect INS-1E cells from high glucoseinduced apoptosis.Taken all together, this is the first thesis in which describes structures and their anti-angiogenesis bioactivities, targeting molecules and their underlying mechanism of polysaccharides in Nemacystus decipiens. The results suggested that the polysaccharides with different structure motifs of fucoidan from this seaweed may target distinguish functional proteins. These provides the fundmental for fucoidanbased new drug development. |
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