The Development And Utilization Of Microbial Exopolysaccharides And Glycosyltransferases

Carbohydrates play important roles in a variety of essential biological processes.Glycans on bacterial proteins are involved in mediating host-pathogen interactions,and play a crucial role in bacterial adhesion,immune escape and host colonization.The changes of cell surface glycosylation are closely related to some immune diseases and tumorigenesis.More functional oligosaccharides,polysaccharides and glycoconjugates can be prepared by extraction,separation and chemo-enzymatic synthesis,which will contribute to the development of new carbohydrate drugs for the diagnosis and treatment of diseases.Because the surface of bacteria is covered with various polysaccharides with different structures,studying these polysaccharides and the enzymes involved in the bacterial glycosylation process will help to develope more functional polysaccharides and glycosyltransferases.Therefore,this study investigated the exopolysaccharides(EPS)and glycosyltransferases from bacteria,which will provides a theoretical basis for their development and utilization.Specifically,the study is divided into three parts:The first part is to isolate,identify and determine the antioxidant activity of EPS from Bacillus subtilis in Sichuan pickle;the second part is to explore the enzymatic properties of Aggregatibacter aphrophilus(AaNGT),a new N-glycosyltransferase and to synthesize glycopeptides and amino oligosaccharide derivatives;the third part concerns the preparation of an multifunctional transferrin nano drug delivery system galactosylated by AaNGTQ468G and the preliminary study on the antitumor activity effect on HepG2.Part 1:Extraction of functional polysaccharides from plants,animals and microorganisms has became an important way for people to develop natural medicines and health products.Due to its easy production and unique physical and chemical properties,EPS have attracted much attention in food and pharmaceutical industries.However,many EPS come from pathogenic microorganisms have the risk of endotoxin infection.Thus,it is urgent to develop bacterial EPS with high safety.The Sichuan pickle is a notable representative of fermented vegetables in China and is rich in microbial resources,including Bacillus and Lactobacillus.As a bioflocculant,bio-emulsifier,heavy metal chelator and antiviral immunomodulator,the EPS from Bacillus has-good physical,chemical and biological characteristics.However,few studies have focused on the protective effect of EPS from Bacillus against oxidative stress.Therefore,the antioxidant capacity of EPS extracted from Bacillus has been evaluated in the current study.As described in the first chapter,a high-yield EPS strain has been screened,isolated and cultured from Sichuan pickle.The strain was identified as Bacillus subtilis by 16S rDNA sequencing analysis and cluster analysis.Two kinds of EPS BS-1 and BS-2 were isolated and purified from the fermentation broth,in which BS-2 is the major component with average molecular weight(Mw)of 1.765×104 Da.As indicated by monosaccharides components analysis,methylation analysis,Fourier Transform infrared spectroscopy(FT-IR)and Nuclear Magnetic Resonance spectroscopy(NMR Spectroscopy),BS-2 mainly consisted of galactose,glucose,and mannose,and the main chain structure of the BS-2 was(1→4)-Glc、(1→4)-Gal and(1→4,6)-Glc.Galactose residue are terminal sugars existing in the sugar chain,and there may be a small amount of mannose might in the branched chain.In vitro antioxidant activity assay proved that the EPS possessed potent scavenging activity on free radicals and could reduce the apoptosis of RAW264.7 cells induced by H2O2 oxidative damage,and weaken the cell damage caused by oxidative damage.In conclusion,BS-2,a new type of EPS with high antioxidant capacity was isolated from Bacillus subtilis in Sichuan pickle,which provided basic theoretical guidance for the development of food grade EPS additives and the utilization of microbial polysaccharides.Part 2:Microbial glycosyltransferases not only regulate the synthesis of microbial polysaccharides,but also participate in the glycosylation of proteins and affect host-microbial interactions.Recently,a new N-glycosyltransferase(HMW 1C)from Haemophilus influenzae,has been found to directly transfer UDP-Glc and UDP-Gal to the asparagine residue of polypeptide.It is substantially different from the PglB-mediated a typical N-glycosylation,and it provides a new way for protein glycosylation.It was reported that Gln469 in the active pocket of N-glycosyltransferase(ApNGT)from Actinobacillus pleuropneumoniae is the key site affecting the catalytic activity of the substrate.AaNGT,an N-glycosyltransferase with high homology to ApNGT,was derived from Aggregatibacter aphrophilus was studied in this study.According to the SWISS-MODEL,the same catalytic site Gln468 was mutated to Glycine and Alanine with less steric hindrance.The results showed that the reaction of AaNGT does not need metal ion catalysis.The optimal reaction buffer was PBS with pH 8.0 and the optimum reaction temperature was 30℃.The reaction efficiency of AaNGT for UDP-Glc,UDP-Gal and UDP-GlcN were 100%,78%and 55%,respectively.Not only 4-azide and 4-methylated sugar derivates as well as 2-Deoxy and 2-fluoro sugar derivates could not be recognized,but also sugar derivates with group larger than NH2-,such as UDP-GlcNAc and UDP-GlcNPr could not be recognized.Compared with wild-type AaNGT,the catalytic efficiency of AaNGTQ468G and AaNGTQ468A for UDP-GlcN was not significantly improved.After 6 h reaction,the catalytic efficiency of AaNGTQ468G for UDP-Gal was 93%,which is significantly higher than AaNGT and AaNGTQ468A.Based on the fact that AaNGT can catalyze the covalent coupling of UDP-GlcN with Asn on peptide sequence,we propose a simple three-step method for the synthesis of N-glycopeptides.Initially,the activated GlcN(UDP-GlcN)was transferred to the Asn residue of the polypeptide by AaNGT to form GlcN-peptide.Then GlcN was acetylated to GlcNAc by a glucosamine N-acetyltransferase(GlmA)from Clostridium acetobutylicum to form GlcNAc linked peptide.Finally,endo-β-N-acetylglucosaminidase M mutant(EndoMN175Q)was used to transfer N-glycan oxazoline to GlcNAc linked peptide to form N-glycan glycopeptide.This will provide a novel idea to produce homogeneous glycopeptides and glycoproteins.In addition,based on the similar electronic radius and polarity of amino NH2-and HO-,the recognition of UDP-GalN by different galactosyltransferase(LgtB,LgtC andα1,3 GalT)was explored,and a series of amino oligosaccharide derivatives were synthesized,providing a new way for the chem-enzymatic synthesis of oligosaccharide analogues.Part 3:Based on the fact that the mutant enzyme AaNGTQ468G can galactosylated DANYTK peptide with high efficiency,human heavy chain transferrin(HFn)with good biocompatibility and self-assembly function was modified with DANYTK and RGDS(Arg-Gly-Asp-Ser)targrting peptide at C-terminal domain,and was galactosylated by AaNGTQ468G to form RGDS/Gal-HFn nanoparticle carrier protein.Asialoglycoprotein receptor(ASGPR)is highly expressed on the surface of liver cancer cells.Galactosylated HFn can target to ASGPR to achieve liver targeting,and RGDS mainly target to integrin receptor αVβ3 on hepatoma carcinoma cells,so it can achieve a dual-targeting effect of HFn.A liver targeted drug delivery system can effectively deliver drugs to the lesion locations,improve therapeutic effect and reduce side effects.It has been reported that 2-Deoxy-2-fluoro-L-fucose(2FF)as a specific fluorinated analog of fucose,has a dramatically antitumor activity.However,the effect of 2FF on hepatoma carcinoma cells remains unclear.Therefore,based on the self-assembly characteristics of RGDS/gal HFn under different pH conditions,a liver targeting drug delivery system(RGDS/Gal-HFn/2FF)with 2-Deoxy-2-fluoro-L-fucosc was constructed,and then the mechanism of antitumor effect was preliminarily explored.The results showed that cell proliferation and migration of HepG2 could be effectively suppressed by RGDS/Gal-HFn/2FF.The fucosylation level on the surface of HepG2 was also decreased by using RGDS/Gal-HFn/2FF.The results from RT-PCR indicated that expression level of immune-suppressive cytokines including TGF-β,IL-2,IL-10 and VEGF were also significantly decreased by using RGDS/Gal-HFn/2FF RGDS/Gal-HFn/2FF.Western blot results showed that the levels of phospho-ERK and phospho-Akt,which play important role in tumor growth,were patently decreased after using RGDS/Gal-HFn/2FF.After co-incubation with HepG2 and immature dendritic cells(DC),the drug system could significantly promote the maturation of DC.The In vitro experiment indicated that RGDS/Gal-HFn/2FF was effective to inhibit growth of tumor.There were abnormal changes in HE staining of tumor tissues,but no obvious changes was observed in the heart,liver,spleen,lung and kidney.After the tail intravenous injection of cy5.5 labled nanoparticles,targeted imaging of tumor site in mice can be realized.According to the flow cytometric analysis,RGDS/Gal-HFn/2FF could increase the ratio of CTLs/Treg cells,and the levels of interleukin 12(IL-12p40),interferon y(IFN-γ),and TNF-α in serum from mice The results of this part suggested that RGDS/Gal-HFN/2FF could improve the immunity of tumor model mice and played a better anti-tumor activity.The targeted imaging of tumor can also be carried out by modifying fluorescent molecules.Our study has provided a new way for the development of glycoprotein conjugates and nanoparticle drug delivery systems,and it has a certain practical value in the diagnosis and treatment of tumors.In conclusion,we have described the extraction,separation and activity determination of a new EPS from Bacillus extracted from Sichuan pickle in the first part Moreover,the substrate specificity of glycosyltransferase for aminesaccharide bas been explored,and the application of site-specific modification of glycopeptide and oligosaccharide fragments extends the chemical enzymatic synthesis of oligosaccharide analog conjugates.Furthermore,the preparation and application of liver targeted drug delivery system(RGDS/Gal-HFn/2FF)has been described,which provides a new way to the development of nanoparticle drug delivery systems in cancer diagnosis and treatment.These findings provide new ideas for the development and utilization of exopolysaccharides and glycosyltransferase.