The Interactions Of Marine Polysaccharides And Their Derivatives With Proteins By Carbohydrate Microarray

Eleven sulfated galactans were successfully obtained from red alga Polysiphoniasenticulosa Harv. and Neosiphonia Japonica Harv., respectively. At the same time,fourteen polysaccharides were isolated and purified from two crude polysaccharides.Four pectins, one glucan and one xylan were prepared from Pyrus betulaefolia Bge.fruits, leaves and Ginkgo biloba Linn. sarcotesta. Nine derivatives of starch, agaroseand carrageenan were also prepared simultaneously by phosphorylation and oxidation.These forty polysaccharides we prepared were first discovered.The active research found that the polysaccharide PS3-2from P. senticulosa caneffectively extend the activeated partial thromboplastin time (APTT) and the effectwas stronger than the positive control low molecular weight heparin (LMWH) andpropylene glycol alginate sodium sulfate (PSS). PS3-2and polysaccharide NJ1-2from N. Japonica also could extend thrombin time (TT). Those results indicated thatthe two polysaccharides had good anticoagulant activity. The half inhibitoryconcentration (IC50) of the PBP from P. betulaefolia fruits was61.37μg·mL-1, whichwas less than the positive control Vitamin C (VC,197.60μg·mL-1). The PBP andPBPF from P. betulaefolia leaves had better cytopathic effect (CPE) inhibitory activity,and the further studies showed that the antiviral activity of PBPF might be exerted byinteracting with the Neuramidinase (NA). The GBC and GBH from G. bilobasarcotesta were able to inhibit the growth of tumor cells HCT-116, otherwise thetumor cells MDA-MB-231have been inhibited by the GBLA and GBHA. Agarose,ι-car phosphorylated and oxidized derivatives of the CPE’s inhibition rate issignificantly enhanced. Agarose oxidation product not only improved solubility butalso had inhibitory activity on tumor cells MDA-MB-231. The hydroxyl radicalscavenging activity of phosphorylated derivatives had been markedly improved, and κ-car derivative activity didn’t change significantly. Discovered by BaF3cellproliferation activity of FGF/FGFR mediated, red algae source of sulfated galactanPS3-0.5, PS3-1.5, NJ1-0.8, NJ1-1.4and NJ1-1.6, GBC and GBHA significantlyenhanced the cell proliferation. The oxidative and phosphorylative modificationcould increase the proliferation activity of ι-car and κ-car on BaF3cells, while thedesulfurization of kappa-car (dsκ-car) and after desulfurization phosphorylation ofκ-car (pκ-car) had no cell proliferation activity, showed that sulfate had a strongereffect than phosphate in the cell proliferation. Agarose and its modifications has thesimilar molecular frame with κ-car and ι-car, the difference between them is just inthe space configuration, which could enhance the activity of the saccharides. Theseresults demonstrated that except for the charge effect, molecular conformation is alsoan important fact in the structure-activity relationship.Fifty polysaccharides had been labeled by Fluoresceinisothiocyanate (FITC) or5-(((2-(Carbohydrazino)methyl)thio)acetyl) aminofluorescein (AF). The marinepolsaccharides chip with64polysaccharides probes,16×16square matrix,256sampledots was firstly built and the printing condition was also optimized. On thepolysaccharides chip, we could got obvious comining signal of fucoidans and itsspecific lectin, Aleuria aurantia lectin (AAL), which proved the feasibility of thecarbohydrate chip. Further studies were carried out based on that, and the interactionsbetween marine polysaccharides with Hemagglutinin (HA), NA and NS1protein ofinfluenza A/PR/8/34(H1N1) virus, hepatitis B virus (HBV), Amylin, Connectivetissue growth factor (CTGF) and alpha-Synuclein had been implemented especially.The results showed that structure of polysaccharides affects the binding activitydirectly, especially sulfuric acid, carboxyl and other acidic-based groups hadimportant influence on the activity. At the same time, the phosphorylation andoxidation modified of polysaccharides could significantly improve the bindingactivity of polysaccharides with proteins.According to the results of binding experiments from polysaccharides chips,PS3-2showed strong binding activity with proteins such as HA, NS1of H1N1, CTGFand α-synuclein. The structure of PS3-2was characterized by infrared spectroscopy, 1D and2D NMR techniques. The results proved that PS3-2is a linear polysaccharidewith repeating sequence of alternating (1â†'3)-linked6-O-sulfated-β-D-galactose(G6S) or (1â†'3)-linked-6-O-pyruvate-β-D-galactose (GPA) and (1â†'4)-linked-3,6-anhydro-β-L-galactose (A). The ratio between G6S and GPA is about4.2:1. Series ofoligosaccharides was prepared by mild acid degradation, and sulfated agarotriose,agaropentaose and agaroheptaose were determined using electrospray collisioninduced dissociation tandem mass spectrometry (ES-CID-MS/MS). Moreover,sulfated agarotriose, agaropentaose and agaroheptaose with xylose side chain werefound for the first time.In conclusion, we successfully created a marine polysaccharides chips for thefirst time by the polysaccharide sample preparation, carbohydrate microarray creating,interactions with proteins, results detection and analysis, structural characterization ofactive compound and so on. With this technique, we studied the interactions of marinepolysaccharides with several disease-related proteins. The fine structural feature ofpolysaccharides that binding with proteins were also determined. This study providesa technical reference for the carbohydrate microarray construction and application.