| Chondroitin sulfate(CS),as a representative glycosaminoglycans(GAGs),is widely distributed on the surface of vertebrate cells or surrounding cells in the form of proteoglycan(CSPGs)side sugar chains.The repeating disaccharide unit structure is:[-4-GlcA-β1-3-GalNAc-β1-]n.CS participates in many physiological processes in organisms and is also closely related to many pathological processes.Clinically,CS based preparations have been widely used in the treatment of osteoarthritis(OA),cartilage damage and dry eye,and can also be used as antiviral and anti-infective drugs;studies have shown that CS can effectively shorten the wound healing process or for central nervous system repair;in addition,CS alone or in combination with glucosamine and other ingredients as a dietary supplement has been widely used in nutritional supplements in Europe and the United States.In another hand,CS can also be used to diagnose or treat malignant tumors-Many literatures have reported changes in chain length and sulfation patterns of CS in tumor cells/tissue,so CS has potential application value as a biomarker for early cancer detection;Injection of imide-modified CS into the tumor tissue of nude mice model of breast cancer can effectively slow or eliminate the growth of cancer cells without causing significant toxicity to adjacent normal tissues;CS-F also inhibits lung colonization of adenocarcinoma MC-38 cells in an experimental transfer mouse model.It is a hot issue in the development of CS applications to synthesize homogenous polysaccharides or structurally uniform deterministic oligosaccharide molecules.At present,the large-scale sales of CS raw materials are still using the traditional animal tissue extraction method.The obtained polysaccharides have poor homogeneity,are easily contaminated by impurities in the production process,and may even be contaminated by interspecies viruses and/or prions.In addition,CS polysaccharides extracted from chicken,cattle or fish cartilage have different chemical structures:the repeat disaccharide unit composition is different,and the sulfate group modification sites and distribution are also inconsistent.The shortcomings such as the inconsistent degree of uniformity of the sugar chain length of the final product not only limit the improvement of the quality of CS clinical drugs,but also the safety of such drugs is questioned,and it also restricts its effectiveness as an effective clinical drug.With the rapid development of glycobiology,the chemical enzymatic method of glycosaminoglycan oligosaccharides has been developed recently.Compared with traditional chemical synthesis methods,the process is simple,the product yield is high,and the reaction conditions are mild and environmentally friendly.With the deep research on the mechanism of CS polysaccharide synthesis,the production of CS polysaccharide by genetic engineering strains has also made great progress by means of synthetic biology.Chondroitin synthase(ChnS,EC 2.4.1.175)belongs to the CAZy(Carbohydrate Active enZymes database)GT2 family,which has both GlcA and GalNAc glycosyltransferase(GlcA-T and GalNAc-T)activities.The GalNAc and GlcA residues are transferred from their sugar nucleotides to the non-reducing ends of the CS sugar chain to catalyze the extension of the chondroitin chain.As a key catalytic tool molecule for artificially synthesizing CS polysaccharide,the discovery,catalytic mode and substrate specificity of this kind of enzyme molecule have important theoretical and practical value.Among them,the microbial-derived chondroitin synthase for the synthesis of capsular polysaccharide has the characteristics of being easy to be recombinantly expressed and having a wide selective substrate,and is more advantageous for artificial use than the isoenzyme for synthesizing chondroitin sulfate in vertebrates.There are three types of bacterial-derived chondroitin synthase that have been successfully expressed and identified to date:PmCS from Pasteurella type F,KfoC from E.coli K4,and CpCS from Chlorobium phaeobacteroides.In this paper,three novel chondroitin synthase enzymes(ApCS,McCS and AuCS)with GalNAc and GlcA diglycosyltransferase functions were identified from the genomes of Avibacterium paragallinarum,Actinobacillus Ureae and Moraxella canis,and their recombinant expression and catalytic activity were confirmed;In addition,based on the above-mentioned novel chondroitin synthase substrate specificity study,ApCS was selected to carry out a preliminary study on the selection mechanism of monosaccharide donors.The content includes:(1)By amino acid sequence alignment,three coding genes with similar molecular sequence homology to known chondroitin synthase were found,which were ApCS of Avibacterium paragallinarum and.AuCS of Actinobacillus Ureae,McCS of Moraxella canis.(2)Soluble expression and purification of recombinant protein of suspected microbial-derived chondroitin synthase:The soluble recombinant expression of ApCS,AuCS and McCS genes in E.coli expression system and the recombinant protein purification method was established.(3)It was determined that ApCS,AuCS and McCS have the catalytic activity of chondroitin synthase:using GlcA(GlcA-pNP)with pNP group as a receptor,it was confirmed that three recombinant proteins can transfer GalNAc group to GlcA in the non-reducing end with pNP,which produces a chondroitin-structured disaccharide GalNAc-GlcA-pNP;and GalNAc-GlcA-pNP as a receptor,the GlcA transferase activities of the three recombinant proteins were determined.(4)The determination of the optimal reaction conditions and enzymatic dynamic constants of recombinant ApCS,AuCS and McCS was completed.(5)Analysis and mechanism study of substrate specificity of recombinant ApCS,AuCS and McCS was completed.In this experiment,the shortest substrate sugar chain that can be utilized by chondroitin synthase was first explored.The results showed that other cartilage complexes except PmCS enzyme can react with the monosaccharide GlcA-pNP as a receptor,and the the shortest acceptor sugar chain required by PmCS is CS-trisaccharide;the mechanism of the difference in the length of the chondroitin synthase receptor sugar chain is preliminarily analyzed by Biacore 3000;In addition,it was confirmed that ApCS can still exert GalNAc-T catalytic activity when HPtrisaccharide is a substrate.(6)Preliminary study on the substrate specificity and mechanism of recombinant ApCS,AuCS and McCS:Three kinds of novel chondroitin synthase enzymes were analyzed for the donor substrate specificity,it was found that McCS showed the broadest monosaccharide donor selectivity;molecular mechanism of ApCS exhibiting stringent monosaccharide donor selectivity was analyzed by protein-molecular docking simulation.And the result shows that when the substrate is UDP-GalNAz,the azide group in the molecule forms a hydrogen bond with the 402th asparagine of the protein molecule,the molecule deviates from the active center of Mn2+,and the coordination with Mn2+ changes,which may be the reason why the substrate cannot be catalyzed.In summary,this paper found three new microbial-derived chondroitin synthases,which have increased the identified enzyme molecules into six species.As a typical polysaccharide sugar chain synthase,the study of the enzymatic properties and receptor/monosaccharide donor selectivity of chondroitin synthase has important theoretical significance for the study of the Leloir family of glycosyltransferases,and the relevant conclusions are directly conducive to the development of CS synthetic systems,application value is obvious. |
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