Synthetic Application Of Endo-?-N-acetylglucosaminidases And Study Of The Catabolic Role Of ?-L-fucosidases

N-glycosylation is one of the most common decoration of eukaryotic protein and plays important roles in the folding,trafficking,stability,catalytic ability and interaction with other molecules of proteins.Eukaryotic N-glycans begin with a ?-linked GlcNAc to Asn residues of protein and share a common core pentaoligosaccharides,Manal,3(Man?1,6)Man?1,4GlcNAc?,4GlcNAc.This core pentaoligosaccharides could be extended with Man,GlcNAc and Gal to form different types of N-glycans.Further decoration,including ?1,6-fucosylation of inner GlcNAc(called "core fucosylation),terminal ?2,6-sialylation and variable antennary of glycan chains,form N-glycan with complex structures.Instead of assembled in a template process as DNA or protein,eukaryotic N-glycans are synthesized through corporation of an array of glycotransferases and glycosidases in the Endoplasmic reticulum and Golgi.The mature N-glycoproteins generated generally have significant glycan heterogeneity.Although the important roles of N-glycosylation have long been known,the heterogeneity of natural N-glycoproteins hinders studies of the function of N-glycans.Moreover,N-glycans of many glycoprotein drugs have been found having great importance for the function or efficiency of drugs.Immunoglobulin G(IgG)drugs are the most widely used glycoprotein drugs.The core fucosylation of Fc N-glycan of IgG can affect the affinity of IgG to its acceptor on cell face,while terminal?2,6-sialylation is responsible for the anti-inflammatory activity of IgG.Further study of the functions of N-glycans of glycoprotein drugs and the medical application of glycoprotein with specific N-glycoform require efficient ways to get homogeneous N-glycoprotein.For this purpose,researchers have tried strategies of organic synthesis and glycoengineering of cells,from which many imporvements are achieved.Howerver,as the reaction conditon of organic synthesis are generally harsh for glycosidic bond or peptide backbone,and the complexity of N-glycosylation process of eukaryotic cells is difficult to control,the synthesis of homogeneous N-glycoprotein is still a challeging area.Endo-?-N-Acetylglucosaminidases(ENGases,EC 3.2.1.96)are endo-glycosidases that can specifically hydrolyze the(?1,4-glycosidic linkage in theN,N'-diacetylchitobiose core of N-glycans.The hydrolytic ability of ENGases is frequently used to deglycosylate N-glycoprotein for structural and functional studies.While some ENGases also show transglycosylation activity.Using N-glycopeptide/glycoprotein with one GlcNAc moiety as acceptor,ENGases are able to transfer oligosaccharide to GlcNAc,reconstituting the natural ?31,4-glycosidic linkage.This reaction provides a new way for the synthesis of homogeneous N-glycoproteinin vitro.The appearance of glycosynthase derived from ENGases,toghether with the application of synthetic sugar oxazolines as donor substrates,greatly improved the yeild of transglycosylation reaction catalyzed by ENGases,making this methond an useful strategy for the synthesis homogeneous N-glycoprotein.Currently,ENGase from Arthrobactorprotophormiae(EndoA),Mucor hiemalis(EndoM)and their glycosynthases have been successfully applied for the synthesis of many homogeneous N-glycopeptide/glycoprotein.Nevertheless,there are still several restriction of this strategy.Concering the acceptor substrates,neither of EndoA,EndoM or their glycosynthases is able to use ?l,6-fucosylated GlcNAc as acceptor.As core fucosylation is one of the main decoration of N-glycans and Fc N-glycans of IgG drugs are mostly core fucoslyated,new ENGases with broad substrated specificity are required.Concering the donor substrates,currently the knowledge of the donor substrate specificity of ENGases are limited to serveral types of N-glycan that are commonly found,which restricts the applications of ENGases in the synthesis of N-glycopeptied/glycoprotein.There is no high-throughput method to study the donor substrate specificity profile of ENGasse to date.To solve these problems,the first part of this thesis focused on three subjects,including mutagenesis of ENGases,synthesis of homogeneous IgG by ENGases and testing a high-throughput method for the study of donor substrate specificity profile of ENGases.ENGase from Streptococcuspneumonia(EndoD)is able to hydrolyze core fucosylated N-glycans,and the wild type of EndoD shows weak tranglycosylation activity.To get glycosynthase derived from EndoD,we performed site-directed mutagenesis to EndoD(amino acids 135-1047)based on the previous mutational study of EndoA and EndoM,and constructed five mutants(N322A,N322Q,E324Q,Y360Fand H371W).EndoD-N322A and EndoD-N322Q lost over 95%of the hydrolytic activity and EndoD-E324Q completely abolished the hydrolytic activity;while EndoD-Y360F and EndoD-H371 W had similar hydrolytic activity with wild type enzyme.Using Man3GlcNAc-oxazoline as donor,EndoD-N322A,EndoD-N322Q,EndoD-Y360F and EndoD-H371W showed transglycosylation activity to both core fucosylated GlcNAc acceptor and non-fucosylated GlcNAc acceptor,with overall higher transglycosylation efficiency for non-fucosylated GlcNAc acceptor.Among these mutants,EndoD-Y360F and EndoD-H371W showed obvious higher transglycosylation ability than that of wild type enzyme,but still hydrolyzed products,which suggested these two mutants are transglycosidases.EndoD-N322A and EndoD-N322Q were able to transfer glycan continually with only marginal hydrolytic ability on products,demonstrating they are glycosynthases.Especially,EndoD-N322Q was able to convert over 90%of non-fucosylated GlcNAc acceptor to product within one hour,or convert 87%of fucosylated GlcNAc acceptor to product within four hours,suggesting N322Q is an efficient glycosynthase for both acceptors.Study of the transglycosylation kinetic parameters of EndoD-N322A and EndoD-N322Q showed that both mutants had similar Km value for Man3GlcNAc,but the kcat value of N322Q was 47-fold higher than that of N322A.The kcat/Km value of N322Q for both acceptors were 25-fold higher than that of N322A,indicating that N322Q has much higher transglycosylation efficiency than N322A.Moreover,the Km value of N322Q for fucosylated GlcNAc acceptor was 24-folds higher than that for non-fucosylated GlcNAc acceptor,but N322A had similar Km values for these two acceptors.Therefore,replacement of Asn-322 by amino acids with different side chain leaded to significant change of affinity of EndoD to fucosylated GlcNAc acceptors.This result suggested that the ability of EndoD to accommodate the al,6fucose on GlcNAc is most likely due to a more flexible space at the catalytic cave of EndoD,rather than specific interactions between the fucose residue and protein.Then the capability of EndoD-N322Q to synthesize homogeneous N-glycoprotein was tested using an IgG drug(rituximab)as model protein.With(Fuc?1,6)GlcNAc-Fc fragment derived from deglycosylated rituximab as acceptor,EndoD-N322Q was able to transfer Man3GlcNAc-oxazoline to this acceptor with high transglycosylation efficiency(>95%yield),generating homogeneous Fc fragment of rituximab with fucosylated core pentaoligosaccharides.EndoD-N322Q represent the first glycosynthase derived from ENGases that could synthesis core fucosylated homogeneous N-glycoprotein.However,as the restriction of the substrate specificity of EndoD,EndoD-N322Q was unable to transfer complex-type N-glycan.ENGase from Streptococcus pyogenes(EndoS)is able to hydrolyze the bi-antennary complex-type N-glycan of IgG Fc domain specifically.We then performed site-directed mutagenesis to EndoS.Asp-233 of Endos was predicted to be responsible for promoting oxazolinium ion formation,and mutagenesis on generated two glycosynthases,EndoS-D233A and EndoS-D233Q.Using(Fuc?1,6)GlcNAc-rituximab as acceptor,EndoS-D233A and EndoS-D233Q were able to transfer sialylated complex-type N-glycan to rituximab efficiently(>95%yield),generating homogeneous IgG with full complex-type of Fc N-glycan for the first time.Moreover,EndoS-D233A could transfer azide-Man3GlcNAc to(Fuc?1,6)GlcNAc-rituximab efficiently.The introduction of azide group will allow further site-specific modifications of IgG,which may be explored for the labeling,targeting,or further glycosylation of IgG.Researches on EndoD-N322Q,EndoS-D233A and EndoS-D233Q provide efficient enzymes for the synthesis of homogeneous N-glycoprotein with core fucosylation and for the glycosylation remodelling of IgG.Then we test the ability of ENGases to transfer a group of N-glycans with known composition in single reaction(named "block-transfer" reaction)to study the donor substrate specificity of ENGases.Chicken albumin was chosen as model protein because the composition and structure of its N-glycan have been well studied.The hydrolytic ability of EndoA,EndoM,EndoD and ENGase from Enterococcus faecalis V583(EndoE2)to N-glycans of chicken albumin were studied.Then a group of N-glycans were released from chicken albumin by the combination of these four ENGases,and converted to sugar oxazoline.Using the sugar oxazoline mixture as donor,the transglycosylation activities of EndoA-N171A,EndoM-N175A and EndoD-N322Q were tested.Each of these three glycosynthase was able to transfer different groups of glycans to GlcNAc acceptor,and quantification of products revealed new catalytic properties of these three glycosynthases.EndoA-N171 A was able to transfer tetra-antennary complex-type N-glycans,suggesting this enzyme has relative broad donor substrate specificity,but it preferred to transfer small glycans such as Man3GIcNAc and GlcNAcMan3GlcNAc.EndoM-N175A showed obvious transglycosylation efficiency on GlcNAcMan5GlcNAc and GlcNAc2Man5GlcNAc,suggesting EndoM-N175A prefers to transfer hybrid-type N-glycans.EndoD-N322Q was able to transfer several small glycans such as Man3-5GlcNAc,but it showed significant transglycosylation efficiency on GlcNAcMan3GlcNAc and GlcNAc2Man3GlcNAc,which suggested EndoD-N322Q has transglycosylation preference for small glycans with terminal GlcNAc.These results could guide the further applications of EndoA-N171A,EndoM-N175A and EndoD-N322Q for the synthesis of N-glycoprotein.Meanwhile,these finding also demonstrated that "block transfer" reaction is an applicable way to study the donor substrate specificity profile of ENGases.Besides,EndoD-N322Q was able to transfer a group of N-glycans to glycopeptide acceptor,GlcNAc-CD52,suggesting that "block transfer" reaction is a potential way for the synthesis of N-glycopeptide.The second part of this thesis focused on the hydrolytic ability of glycosidases and their roles in the utilization of carbon sources by gut microbes.Fucosylated glycans are abundant on terminal of O-glycans of various intestinal glycoconjugates or oligosaccharides,including mucins,surface glycoproteins on epithelial cells and human milk oligosaccharide(HMOs).On intestinal mucin O-glycan of colon,fucose residues are mostly ?1,3/4-linked;while in HMOs fucose residues are mainly ?1,2-linked.The removal of terminal fucose residue is one key step for the utilization of these carbon sources by gut microbes.?-L-fucosidases are presented in various organisms and grouped into glycoside hydrolase(GH)family 29 and 95.Many researches focused on the roles of ?-L-fucosidase in the consumption of HMOs by gut microbes,but relation of ?-L-fucosidase and the utilization of mucin O-glycans by gut microbes are still largely unknown.In this work,three ?-L-fucosidases from Clostridium perfringens ATCC 13124(CpAfc1,CpAfc2 and CpAfc3)were cloned,expressed and characterized.CpAfc1 had ?-L-fucosidase domain but showed no hydrolytic activity to common fucosylated substrates.CpAfc2 was determined to be a 1,3-1,4-?-L-fucosidase of GH29-B subfamily,and CpAfc3 was determined to be a 1,2-?-L-fucosidase of GH95 family.Both CpAfc2 and CpAfc3 showed hydrolytic ability on porcine gastric mucin(PGM).To study the roles of ?-L-fucosidases in the utilization of mucin O-glycans by C.perfringens ATCC 13124,this strain was cultured in basal medium(BM),BM with 0.5%PGM(BM-PGM)and BM with 0.5%glucose(BM-Glc)separately.The growth and the production of ?-L-fucosidase of this strain were then determined.The cell density of C.perfringens ATCC 13124 in stationary phase increased 25%in BM-PGM,but decreased 18%in BM-Glc.Transcriptional analysis of afc1,afc2 and afc3 revealed that all these three genes were transcribed in BM.Transcription of afc2 and afc3 in BM-PGM were one-fold and 0.3-fold higher than that in BM,while the transcription of afc1 was not affect by PGM obviously.In BM-Glc,the transcription of all these three genes decreased over four-fold.Therefore,the transcription of afc1 afc2 and afc3 could be induced by PGM moderately,and subjected to carbon catabolite repression by glucose.Noteworthily,the transcription levels of CpAfc2 were 2-to 5.8-fold higher than CpAfc3 through all the tests.We then determined ?-L-fucosidase activity produced by C.perfringens ATCC 13124.When cultured in BM-PGM,the 1,3-1,4-?-L-fucosidase activity was 1.4-fold higher than that in BM,the 1,2-?-L-fucosidase activity was 1.7-fold higher than that in BM.Conversely,the 1,3-1,4-?-L-fucosidase activity and the 1,2-?-L-fucosidase activity in BM-Glc were over 12-fold lower than those in BM.In addition,the 1,3-1,4-?-L-fucosidase activity were over five-fold higher than the 1,2-?-L-fucosidase activity in all the tests.These results were consistent with that of transcriptional analysis,suggesting that CpAfc2 and CpAfc3 were important glycosidases in the utilization of mucin O-glycans by C.perfringens ATCC13124,and CpAfc2 plays a primary role.Correspondingly,C.perfringens ATCC 13124 showed stronger degradation ability on ?1,3/4-fucosyloligosaccharides than on ?1,2-fucosyloligosaccharides during growth,suggesting that the high 1,3-1,4-?-L-fucosidase activity provides this pathogen catalytic preference for ?1,3/4-fucosylated substrates.As intestinal mucin O-glycans of human have increasing ?1,3/4-fucosylation level from ileum to rectum,the high production of 1,3-1,4-?-L-fucosidase by C.perfringens ATCC13124 will make this strain an efficient pathogen to utilize mucin O-glycans,which could be favorable for its colonization in colonic mucins.Moreover,as 50%-92%oligosaccharides in HMOs are ?1,2-fucosylated,the low 1,2-?-L-fucosidase activity could be unfavorable for the utilization of HMOs by C.perfringens ATCC13124.This deduction was consistent with the weak growth of this strain in HMOs reported previously,indicating that the catalytic preference for?1,3/4-fucosylated substrates is one important factor for its weak growth in HMOs.This work represented the first report that revealed the important role of ?-L-fucosidases in the utilization of mucin O-glycans and the colonization of enteric pathogens in human intestine,expanding our knowledge about the relation between the catabolism and inhabitation of gut microbes.Overall,we performed mutagenesis study for two ENGases from Streptococcus(EndoD and EndoS)and generate three efficient glycosynthases,EndoD-N322Q,EndoS-D233A and EndoS-D233Q,providing efficient tools for the synthesis of homogeneous N-glycoprotein with core fucosylation and for the glycosylation remodelling of IgG.We performed "block-transfer" reaction and found new transglycosylation properties of EndoA-N171A,EndoM-N175A and EndoD-N322Q,which suggest that "block-transfer" reaction could be used as a high-throughput method for the study of donor substrate specificity profile of ENGases.We also performed molecular cloning,heterogenous expression and enzymatic characterization of three ?-L-fucosidases from C.perfringens ATCC13124,together with the studies the growth and the production of ?-L-fucosidases of this strain in different carbon sources.The important role of 1,3-1,4-?-L-fucosidase in the utilization of mucin O-glycans and colonization in gut of this strain were revealed,which gives us a deeper understanding for the relation between catabolism and inhabitation of gut microbes,as well as provides guidance for the prevention of this pathogen.