Identification And Signature Sequence Of Chondroitin Sulfate/Dermatan Sulfate Sulfatases From Bacteria

Glycosaminoglycans(GAGs)are a complex family of linear polysaccharides,composed of repeating disaccharide units of hexuronic acid and N-acetyl-hexosamine.They are synthesized as free chains or as sidechains of proteoglycans(PGs)and exist at the cell surface or extra cellular matrix participating in and regulate a number of cellular events and physiological/pathological processes.Chondroitin Sulfate/Dermaton Sulfate(CS/DS)and Heparin/Heparan Sulfate(Hep/HS)are important sulfated GAGs.They are not only tissue organizers but also key functional molecules regulating a wide variety of biological functions,such as cell division,central nervous system development,viral and bacterial infections,and tumor progression.Many studies have shown that CS/DS chains exert these functions through interactions with various proteins,such as adhesion molecules,growth factors and cytokines,where the sulfation pattern of CS/DS chains is crucial for these interactions.CS/DS sulfatases specifically catalyze the hydrolysis of sulfate groups from CS/DS chains and are potential tools for structural and functional studies of CS/DS.Furthermore,they participate in the metabolic pathways of bacteria and animal.The desulfation of saccharides by sulfatases facilitates CS/DS degradation.In human,deficiencies in these enzymes results in a series of genetic disorders,such as multiple sulfatase deficiency(MSD).CS/DS sulfatases are also essential for the bioremediation of organosulfur compounds derived from plants and animals.However,despite being an important class of enzymes,only a few CS/DS sulfatases from prokaryotes and eukaryotes have been studied in detail.Some previously reported CS/DS sulfatases,such as N-acety;ga;actoamine-4-O-sulfatase and N-acetylgalactoamine-6-O-sulfatase from vulgaris,have not been annotated in the GenBank database,and their gene sequences are not yet clear.The rare protein sequences characterized in databases do not provide enough information for the reliable assignment of GAG sulfatases via bioinformatic analysis.Therefore,although some candidate genes are predicted to be GAG sulfatases,their degradation specificity may be falsely annotated.Furthermore,we found that the specific subfamily of potential CS/DS sulfatase genes could not be correctly identified and predicted while using it as the query sequence to blast the database such as GenBank.Moreover,the sulfatase substrate is extremely complex,including not only a large family of structurally diverse GAGs,but also other biomolecules such as glucosinolates,choline sulfate,steroids,and glycolipids.The CS/DS sulfatase could only be determined through the recombinant expression and enzymatic activity analysis of all the potential sulfatase,which will require a lot of time and is a heavy workload.As GAG sulfatase is an essential enzyme in the degradative metabolism and structural analysis of GAGs,it is important to identify additional types of sulfatases to enrich the sulfatase database.In this study,we report a ?4,5hexuronate-2-O-sulfatase PB2SF and an exolytic N-acetylgalactosamine-4-O-sulfatase exoPB4SF from Photobacterium sp.FC615,and an exolytic N-acetylgalactosamine-6-O-sulfatase from Photobacterium damselae.Furthermore,a comparative study was performed on the basic biochemical features,substrate-degrading patterns and catalytic mechanisms between the exoPB4SF and another N-acetylgalactosamine-4-O-sulfatase endoVB4SF who shares an 83%identity with exoPB4SF but shows endolytic activity:(1)?4.5Hexuronate-2-O-sulfatase,designated PB2SF,specific catalyzes the hydrolysis of the sulfate ester from the C-2 position of unsaturated uronic acid residues of CS/DS and Hep/HS disaccharides produced by GAG lyases.It exhibited the highest reaction rate at 40? in NaAc-HAc(pH 6.0)buffer.A structural model of PB2SF was constructed through a homology-modeling method.Six conserved amino acids around the active site were chosen for further analysis using site-directed mutagenesis.N113A,K141A,K141H,H143A,H143K,H205A and H205K mutants exhibited only feeble activity,while the H310A,H310K and D52A mutants were totally inactive,indicating that these conserved residues,particularly Asp52 and His310,were essential in the catalytic mechanism.The three-dimensional structure model shows that the active center amino acid of PB2SF are highly conserved to the bacteria and human sulfatases whose structure have been reported,suggesting that CS/DS sulfatases should have similar sulfate hydrolysis mechanism to other sulfatases.(2)N-acetylgalactosamine-4-O-sulfatase,designated exoPB4SF,specific catalyzes the hydrolysis of the sulfate ester from the C-4 position of CS/DS disaccharides.Similar to endoVB4SF,exoPB4SF exhibited the maximal rate in NaH2PO4-Na2HPO4(pH 8.0)buffer at 30?.The degradation pattern analysis of exoPB4SF and endoVB4SF toward hexasaccharide showed that exoVB4SF could only remove the C-4 sulfate at the reducing end of the hexasaccharide,and therefore is a typical exolytic sulfatase.This property makes exoPB4SF a perfect tool for preparing a specific structural oligosaccharide with no sulfate at the C-4 position of the reducing end.Interestingly,the endolytic sulfatase endoVB4SF was observed to preferentially hydrolyze the sulfate esters from the reducing to nonreducing ends of the hexasaccharide ?A-A-A,suggesting that endosulfatases act upon CS/DS chains in a more orderly manner than endolytic lyases,which typically cleave CS/DS chains at random.To further analyze the substrate degradation mechanism of exoPB4SF and endoVB4SF,the crystal structure of endoVB4SF was obtained and refined at 1.95 A resolution.This is the first endolytic sulfatase whose structure has been reported.The three-dimensional structure shows that the key amino acid at the catalytic center is a C?-formylglycine(FGly)residue which is modified from the oxidation of a conserved Cys residue.This structure reveals that CS/DS sulfatase shares the same hydrolysis mechanism with other sulfatase during the cleavage of sulfate group.Moreover,there is a long strip of trench on the surface of the catalytic center,suggesting that this trench may provide spatial conditions for the binding of polysaccharide chains.In order to better understand the substrate binding modes and catalytic mechanisms of endo-and exo-4-O-sulfatases,we constructed the structural model of exoPB4SF using homology modeling method and performed docking simulations.Docking results revealed that the substrate-binding capacity of exoPB4SF and endoVB4SF were different.The endoVB4SF can well interface with its substrate disaccharide ?A and hexasaccharide ?A-A-A.Although exoPB4SF can degrade one sulfate at the reducing end of the hexasaccharide ?A-A-A,the whole structure of hexasaccharide ?A-A-A cannot be properly docked into its active site.We ascribe this result to the steric hindrance effect.Maybe one part of hexasaccharide could interact with the catalytic center of exoPB4SF.The docking model revealed that amino acids around the catalytic center are highly conserved in both exoPB4SF and endoVB4SF.Therefore,we propose that the primary factor influencing the substrate selectivity of endoVB4SF and exoPB4SF is the conformation of the catalytic center in the tertiary structure.The residues at the active site of endoVB4SF adopt a more favorable conformation for the interaction of the enzyme with longer CS/DS chains than that of exoPB4SF,which may explain why endoVB4SF exhibits significant endolytic activity but exoPB4SF does not.(3)N-acetylgalactosamine-6-O-sulfatase,designated exoPB4SF,specific catalyzes the hydrolysis of the sulfate ester from the C-6 position of CS/DS disaccharides.It exhibited the highest reaction rate at 30? in NaAc-HAc(pH 6.0)buffer.ExoPM6SF is the second reported bacterial N-acetylgalactosamine-6-O-sulfatase which could act on CS/DS disaccharides.The first reported N-acetylgalactosamine-6-O-sulfatase was from Proteus vulgaris,but its gene sequence has not been annotated in the database and therefore cannot provide reference in the sequence analysis of CS/DS sulfatase.The sequence of exoPM4SF will provide a basis for analysis of N-acetylgalactosamine-6-O-sulfatase.After enzymatic property and structural assay,we analyzed the primary sequence of CS/DS sulfatase.As the sequences of N-acetylgalactosamine-4-O-sulfatase and N-acetylgalactosamine-6-O-sulfatase from Proteus vulgaris were not clear,we recombinantly expressed the sulfatases in this strain and identified the sequences of both N-acetylgalactosamine-4-O-sulfatase and N-acetylgalactosamine-6-O-sulfatase.After sequence alignment of all identified bacterial CS/DS sulfatases,we found that CS/DS sulfatases with specific substrate specificity have highly conserved regions,respectively.In this study,we report the signature sequences of ?4,5 hexuronate-2-O-sulfatases,N-acetylgalactosamine-4-O-sulfatases and N-acetylgalactosamine-6-O-sulfatases,which will be very helpful for the proper prediction and identification of GAG sulfatases.Notably,although these signature motifs are decentralized in the primary structure,they surround the catalytic key residue and are tightly concentrated at the catalytic center in the tertiary structure,which indicates that these conserved regions may influence the substrate selectivity of GAG sulfatases.Therefore,we propose that these conserved regions play essential roles during catalysis and have therefore been highly conserved during biological evolution.Overall,this study systematically analyzed CS/DS sulfatases from primary structure to tertiary structure.The enzymatic properties and substrate degradation patterns of three novel CS/DS sulfatases ?4,5 hexuronate-2-O-sulfatase PB2SF,N-acetylgalactosamine-4-O-sulfatase exoPB4SF and N-acetylgalactosamine-6-O-sulfatase exoPM6SF were investigated,respectively.The structure of endoVB4SF and molecular modeling of exoPB4SF provide structural and biochemical insight into the interaction of CS/DS chains with endo-/exo-type sulfatases.The signature sequences of different type of CS/DS sulfatase were reported for the first time,which solved the problem that CS/DS sulfatases cannot be correctly predicted and classified in the database.In conclusion,the results of this study should aid in the identification and further application of novel GAG sulfatases.