Synthesis And Biological Function Evaluation Of Complex Glycoconjugates

Carbohydrate is naturally widespread in a variety of organisms, playing various important functions; In organisms, the mainly form of carbohydrates is conjugation with proteins, lipid and other substances in covalent bonds forming glycoproteins/ proteoglycans, glycolipids and other conjugates. The position and configuration of the monosaccharide-linked glycosidic linkages in the sugar chains are different, and the sugar chains are modified in different ways, which confers a high degree of diversity and heterogeneity of the glycoconjugates; Various glycoconjugates presenting in different cells, tissues and organs provide different phenotypes and functions of cells, tissues and organs.There are glycocalyxes composed of various glycoconjugates on the surface of bacterial cells. These glycoconjugates play an important role in the cell adhesion and colonization process, and can protect aganist other organisms attack; It is noteworthy that of the sugar component on the bacterial surface has antigenicity, such as the lipopolysaccharide on gram-negative bacterias, can mediate the host cell immune response. Accordingly, the polysaccharide antigens on the bacterial surface become the focus of research and development of pathogenic vaccine.Lipopolysaccharide, presenting on the surface of Gram-negative bacteria, consists of lipid A, core oligosaccharide and O-polysaccharide; O-polysaccharide, also known as O-antigen, comprises the outermost domain of the lipopolysaccharide molecule, is an important target of the immune system recognition; O-antigen is the main basis for the research and development bacterial polysaccharide vaccine; However, The composition of O-antigen with high variability varies from strain to strain; therefore, the polysaccharide vaccines based on O-antigen have small broad-spectrum.The core oligosaccharide component attaches directly to lipid A and antigen. The structure of core oligosaccharide is relatively conserved among the strains compared with O-antigen, and can be researched as a broad-spectrum polysaccharide vaccine candidate due to its certain antigenicity. It is to be mentioned that the immune response induced by carbohydrate vaccine is independent on T cell with poor induction of O-PS-specific IgG and memory T cells, leading to low immune persistence. Therefore, it is needed to enhance the immunogenicity of carbohydrate vaccines.Bacterial surface polysaccharide have other activity except antigenicity, For example, previos researches have confirmed that the O-antigen of E.coli O86:B7 has human blood group B antigen activity because of the similar structure of the O-antigen and B antigen.The ABO blood group system is the most important blood type system in humans. Blood type incompatibility (ABO-incompatible,ABOi) means the exposure of A or B antigen to a person who has antibodies against these antigens. These antibodies act as haemagglutinins, which cause blood cells to clump and break apart, and can even cause death. Hyper acute rejection and graft necrosis induced by ABOi, can be decreased through removal of anti-A/B antibodies from plasma. At present, the commonly method to remove anti-A/B antibodies is immunoadsorption with A/B blood group antigens. Unfortunately, A and B blood group antigens are difficult to acquire and immobilize.Most A/B antigens are synthesized by chemical methods or enzymatic synthesis. One of the most difficult steps in the chemical synthesis of well-defined oligosaccharide antigens is the stereospecific formation of glycosidic linkages between monosaccharide units. Enzymatic synthesis utilizing the corresponding glycosyltransferase is limited by the availability of enzymes and the cost of activated sugar donors. Accordingly, it is necessary to find a low-cost and highly-effective method to produce A/B antigens to remove anti-A/B antibodies from plasma. Therefore, E. coli O86:B7 can be a potential cell factory of B antigens.The thesis is divided into two parts. The part one is the synthesis of E. coli core oliogsaccharides glycoconjugate and its immunological evaluation (Chapter Two); The part three is the synthesis of MBPmut-OPS and utilization of the adsorption blood group B antibody (Chapter Three).Part One:We designed rational chemical reaction scheme and synthsised E. coli R3 total outer core pentasaccharide after analysising the structure of E. coli R3, especially branched outer core. To improve the immunogenicity of outer core based vaccine, a propyl amino linker at the reducing end of pentasaccharide was added, to conjugate with carrier proteins after being actived. At last we got two glycogconjugates: pentasaccharide-CRM197 and pentasaccharide-BSA.Pentasaccharide-CRM197, as a immune antigen, was injected into mice to evaluate the immune activation. The ELISA assay was used to detected the titres of anti-pentasaccharide antibodies after inmmuned by Penta-CRM197. High titres specific anti-pentasaccharide antibodies were detected including IgG and IgM, which is one of important indicator to evaluate immune effects.In order to illuminate the nature of immune response produced in mice immunized with pentasaccharide-CRM197 and its potential relevance to overall T cell phenotype, IgG subclass profiles were evaluated by ELISA assay. The IgG subtypes of the organism have IgG1, IgG2, IgG3 and so on. IgG subtypes with different functions and characteristics can reflecte the direction of Th cell immune response (Th1 or Th2). The results of our experiments indicated that not only Thl-type response was evoked in mice immunized with pentasaccharide-CRMi97 but also Th2-type response was observed.To confirm whether the immune system activated by Penta-CRM197 has the ability to kill pathogenic bacteria containing R3 outer core structure, E. coli O157:H7 was incubated with the immuned serum by pentasaccharide-CRM197. The results confirmed the killing ability, ans The highest serum dilution fold yielding 50% killing of E. coli O157:H7 in pentasaccharide-CRM197 group was almost 160.In summary, a new glycoconjugate was obtained after rational designation, and its immune activation was confirmed in mice, which provide new ideas on the design of bacterial polysaccharide conjugate vaccine. Moreover, this report represented a new perspective in design and synthesis of carbohydrate antigens to be explored as broad-spectrumvaccine candidates.Part Two:This part focued on other activation of lipopolysaccharides on bacteria surface, such as the human blood group B antigen activation of E. coli O86:B7 lipopolysaccharides. In consideration of the difficultied to obtain group B antigen and the the urgency of preparing universal blood group, we planed to used E. coli O86:B7 as a cell factory to product O-antigen which has B antigen activation, and to conjugate it to a certain carrier protein easily to separat and purify.Howerver, natural E. coli O86:B7 can not conjugate O-antigen with a certain carrier protein, because of no associated glycosyltransferase. Therefore, we applied an in vivo enzymatic approach based on N-glycosylation system in Campylobacter jejuni (C. jejuni) to produce glycoconjugate. PglB, a kind of oligosaccharyltransferase, as the key enzyme of N-glycosylation system in C. jejuni, transfers glycan chain from a polyisoprenoid carrier to the asparagine (N) residue in the consensus sequence D/E-X-N-Y-S/T (X, Y≠P) of the carrier protein.Maltose-binding protein (MBP) was choosed as the carrier protein of O-antigen, which is coded by gene malE, and is responsible for maltose/maltodextrin transport, have high affinity for maltose or maltodextrin; MBP signal peptide at the N-terminus can direct MBP to E. coli periplasm space. MBP is usally used as a label proteins in the E. coli system, which prevents the aimed protein into inclusion bodies.To establish the E. coli N-glycosylation system, two genes were transforme into E. coli O86:B7 including C. jejuni glycosyltransferase PglB and malE coding MBP, respectively carried by plasma pACT3 and pBAD24. In order to be glycosylated under PglB with O-antigen, a glycosylation site D/E-X-N-Y-S/T (X, Y≠P) was added into MBP C terminal, named MBPmut. Thus, PglB transformed into E. coli O86:B7 catalyzes the syhthsis of MBPmut-OPS using O-antigen as sugar donor and MBPmut as protein donor.To be more effectively conjugate O-antigen to MBPmut, gene waal in E. coli 086 was successfully knocked out using Red recombination System (including three plasmas:pKD4, pSIM19 and pCP20), which made no conjugation between O-antigen and Lipid A core sugar residue, leading the accumulation of O-antigen in E. coli periplasmic spaceper.Overall, E. coli N-glycosylation system has be successfully established, which expressed a glycolconjugate MBPmut-OPS. We got the glycoconjugatie after expression, purification and detection, including Coomassie blue staining, Western blot and MALDI-TOF, with a purity of 95%, and a yield of about 1.5 mg/L (quantified by the protein concentration).The group B activation of MBPmut-OPS was confirmed by Westernblot, and the ability of specific binging to human anti-B antibodies was detected by ELISA assay, constantly no binding with human anti-A antibodies. After MBPmut-OPS interacted with human A blood group and O blood group plasma, the results indicated that MBPmut-OPS could effectively absorb anti-B antibodies from plasma, through the contrast of anti-B antibody titers before and after MBPmut-OPS treatment. Furtherly, the absortion by MBPmut-OPS made no effection on coagulation function.To summariz, this study successfully glycosylated MBP with B antigen using E. coli N-glycosylation system. MBPmut-OPS was applied to remove the anti-B antibodies from human plasma as we expected. A new method to get human group B antigen was provided to use E. coli O86:B7 as the factory. In the system, large quantities of glycoproteins are produced and have great potential for further clinical development in many fields including ABOi organ transplantation and universal blood transfusion.