Analysis,Identification And Catalytic Mechanism Of Glycosaminoglycans-Degrading Enzymes In The Intestinal Microorganisms Of Mice Chronically Fed Chondroitin Sulfate

Glycosaminoglycans（GAGs）are linear poly-anionic polysaccharides,including hyaluronic acid（HA）,chondroitin sulfate（CS）/dermatan sulfate（DS）,keratan sulfate（KS）and heparan sulfate（HS）/heparin（Hep）.Except for HA,all other GAGs are covalently bound to various core proteins to form proteoglycans（PGs）,which are widely distributed on the cell surface and in the extracellular matrix（ECM）and are involved in many physiological and pathological processes,such as Central Nervous System development,cell division,cell differentiation,growth factor signaling,morphogenesis,and inflammation.Numerous studies and clinical trials have shown that the rapid loss of CS from articular cartilage with aging is closely related to the development of Osteoarthritis（OA）,and that appropriate supplementation of CS can effectively alleviate and improve the symptoms of OA,so CS has been widely used as a drug and dietary supplement for OA treatment and symptom relief.However,because CS is a highly negatively charged poly-anionic polysaccharide,its oral bioavailability is very low.only about 0-13%of the total oralintake,and the majority of CS is degraded and metabolized by intestinal microorganisms,and its metabolites are believed to have positive effects on the treatment of OA and other body functions,although the exact mechanism of action remains to be elucidated.GAGs-degrading enzymes are critical in the degradation and metabolism of CS by intestinal microorganisms;however,there is a paucity of studies on the expression of GAGsdegrading enzymes and the enzymatic properties of key enzymes in host intestinal microorganisms as a result of long-term CS administration.We aimed at investigating the effect of long-term oral CS administration on the expression profile of GAGs-degrading enzymes in mice intestinal microorganisms,and investigating the enzymatic properties and catalytic mechanism of key GAGs-degrading enzymes with novel substrate degradation properties among them.On the one hand,it will help to reveal the metabolic regulation mechanism of CS and other related GAGs degradation by intestinal microorganisms,and on the other hand,it will provide the much needed novel tool enzymes for the structure-function study of complex GAGs and the preparation of functional oligosaccharides.The specific results of the study are as follows:（1）Effect of long-term feeding of CS on intestinal microorganisms and their GAGsdegrading enzymes expression in mice:In this study,metagenomic sequencing was performed on intestinal microorganisms of mice chronically drinking different concentrations of bovine cartilage-derived CS-A solution and water,respectively.At the microbial level,by comparing the composition and abundance of microorganisms in the intestinal flora of each group,it was found that the structure of the intestinal flora of mice consuming CS-A for a long period of time was significantly changed compared to the control group:at the phylum level,the abundance of Bacteroidetes was increased;at the genus level,the abundance of Bacteroides and Bifidobacterium increased;and at the species level,the proportion of Bifidobacterium pseudolongum and Bacteroides acidifaciens increased.At the probiotic level,long-term oral administration of CS-A resulted in Bifidobacterium pseudobifidum becoming the dominant probiotic in the intestinal microflora of mice.At the gene level,based on the correspondence between species and gene functions in the samples,we conducted an association analysis between the relative abundance of species and functional genes and found that the functions of polysaccharide lyases（PLs）were mainly performed through the genus Bacteroides.Further,this study analyzed the species and abundance of GAGs lyase and sulfatase genes in different groups of intestinal flora,and found that long-term feeding of CS-A did not cause significant changes in the species of GAGs lyases and sulfatases in the intestinal flora of mice;however,the analysis of the abundance of various GAGs degradation-related enzymes in the intestinal flora of CS-A-fed mice showed that the abundance of CSase ABC,CSase B,heparinase Ⅲ,△4,5 hexuronate-2-O-sulfatase,N-acetylgalactosamine-4-O-sulfatase and Hep/HS Nsulfamidase were significantly increased.Finally,sixty-one genes of suspected GAGsdegrading enzymes found in the gut microbial metagenomes of various groups were cloned for expression and activity verification,of which twenty-five en zymes were significantly active,including fifteen CS/DS lyases,seven heparinases,and three sulfatases.In the subsequent sections we will perform in-depth enzymatic characterization and catalytic mechanism studies on several of these enzymes with novel enzymatic properties.（2）Comparative study of two novel CS/DS lyases:Two novel CS/DS lyases（CSase ABC）,IM3796 and IM1634,were identified from the gut microbial metagenome of chronically fed CS mice by gene sequence alignment,recombinant expression and activity verification.Both enzymes belong to the PL8 family of polysaccharide lyases and have low sequence similarity（≤31%）to the identified CSase ABC,but have high sequence homology（query coverage:88.00%,percent identity:90.10%）between themselvesl The enzymatic characterization revealed that the two enzymes have very different substrate selectivity and product generation patterns:IM3796 prefers to degrade CS rich in 6-O-sulfated GalNAc residues into tetrasaccharides and disaccharides as the main products,while IM1634 can completely degrade CS/DS with various sulfation patterns to disaccharides with much higher enzymatic activity than IM3796.Structural modeling showed that IM1634 has an extra N-terminal structural domain consisting of two β-folds compared with IM3796,and the enzymatic properties of the mutant（IM1634-T109）of IM1634 deleted the N-terminal structural domain（Met1-His109）were similar to those of IM3796,and conversely,after grafting this structural domain to the Nterminal of IM3796,the mutant IM3796-A109 showed the enzymatic properties similar to IM1634.This study reveals,for the first time through biochemical evidence,the key role of the N-terminal structural domain of CSase ABC in regulating substrate binding and product generation,and provides two novel tool enzymes with clear enzymatic properties for CS/DS structure and function studies and oligosaccharide preparation.（3）Expression and identification of two novel HA-specific lyases:We identified and characterized two HA-specific lyases IM1805 and IM7373 from the gut microbial metagenome of mice chronically fed CS-A solution,both of which belong to the PL8 family of polysaccharide lyases,and their,highest similarity to the identified GAGs lyases is less than 24%;in terms of evolutionary status,the two enzymes are not clustered with other PL8 family enzymes and are independent branches,which are novel enzymes with important research value.Further studies on the biochemical characteristics and substrate specificity of the two enzymes showed that both IM1805 and IM7373 are HA-specific degradation enzymes with disaccharide as the final products,unlike the previously identified HA-specific lyases,which degrade HA into tetrasaccharides and hexasaccharides as the minimal products.The specific enzyme activity of IM7373 reached 109.80 U/mg,which was about twenty times that of IM 1805.In addition,the two enzymes have good thermostability at 0-30 ℃,which makes the two enzymes have important application potential.In the present study,the treatment of various commercial GAGs with these two enzymes as tool enzymes revealed that all commercial GAGs contained varying degrees of HA contamination,and the two enzymes could degrade HA impurity to disaccharides in the presence of CS,which could be used to remove the mixed HA from CS to improve the purity of current commercial CS.Therefore,the discovery and identification of this novel HAspecific lyases not only further enriches the library of GAGs lyases,but also provides an important enzymatic tool for the specific detection and removal of HA in GAG products,the enzymatic preparation of bioactive HA oligosaccharides and the treatment of related diseases.（4）Structural resolution and catalytic mechanism of N-acetylgalactosamine-6-O-sulfatase（GalNAc-6-O-sulfatase）:In this study,to address the problems that the number of identified GalNAc-6-O-sulfatases is extremely limited and the structure of GalNAc-6-O-sulfatase which acts on CS/DS oligosaccharides has not been resolved,based on the recombinant expression and activity validation of two GalNAc-6-O-sulfatases from the mice intestinal flora and their six homologs,a preliminary screening of the crystallization of the eight enzymes was performed,and PB₃682 was found to be easy to grow regular shaped and high-quality crystals under various conditions.Therefore,in this study,the basic enzymatic properties of PB₃682 was thoroughly investigated,and its structure and preliminary catalytic mechanism were further analyzed.PB₃682 selectively removes the 6-O-sulfate group from the GalNAc residue of the reducing end of many CS disaccharides as well as tetrasaccharides,which is typical of CS/DS exo-type GalNAc-6-O-sulfatase;by optimizing the crystallization conditions,we successfully prepared the crystals of PB₃682 with high quality and resolved its structure to obtain the first crystal structure of GalNAc-6-O-sulfatases acting on CS/DS oligosaccharides with a resolution of 2.0 （?）,The structure-based catalytic mechanism studies:showed that PB₃682 shares the same hydrolase catalytic mechanism as the identified CS/DS sulfatases,and the presence of positively charged amino acids near the catalytic center makes it easier for negatively charged substrates to approach the catalytic site,thus allowing the hydrolysis reaction to proceed smoothly.The docking results showed that the catalytic cavity of PB₃682 only allowed the entry of single sugar residues,and thus could only remove the 6-O-sulfate group of GalNAc residue located at the reducing end of disaccharide and oligosaccharide,which well supported its exo-type characteristics.This study greatly enriched the library of GalNAc-6-O-sulfatases and resolved its structure for the first time,which is an important reference for the discovery and identification of this class of enzymes and their engineering modification.In this study,the effect of long-term feeding of CS on the structure of mice intestinal flora and the expression of related GAGs-degrading enzymes was firstly investigated by means of metagenome,and a large number of GAGs-degrading enzymes found in the metagenome of intestinal flora were further cloned for expression and activity verification,and the basic enzymatic properties,substrate degradation patterns and catalytic mechanisms of several enzymes with novel substrate degradation characteristics were investigated in depth.We found that long-term feeding of CS to mice resulted in a significant increase in the abundance of the probiotic Bifidobacterium and a significant increase in the abundance of the associated GAGsdegrading enzymes in their intestinal microorganisms;the key role of the N-terminal β-folding region of CSase ABC in regulating substrate binding and product production was demonstrated for the first time by biochemical analysis;two novel HA-specific lyases with important applications were identified;the discovery and identification of eight CS/DS-specific GalNAc6-O-sulfatases not only greatly enriched the enzyme library of this class,but also resolved the crystal structure of GalNAc-6-O-sulfatases acting on CS/DS oligosaccharidesfor the first time.In summary,this study not only investigated the effects of long-term oral CS on host gut microbes and related GAGs-degrading enzymes from a.new perspective,but also further enriched the library of GAGs-degrading enzymes through the in-depth study of a series of novel GAGs-degrading enzymes,and provided novel enzymatic tools for the structural and functional studies of GAGs and the development and production of related products.