Discovery Of Exolytic Heparinases And Their Catalytic Mechanism And Potential Application

Heparin(HP)/Heparan sulfate(HS),the linear polyanionic and acidic polysaccharides composed of repeating disaccharide units,is belonging to glycosaminoglycans(GAGs).HP/HS is widely distributed on the cell surface or in the intercellular matrix,even in some intracellular environments.Since 1935,HP has been widely used in clinical treatment due to its good anticoagulant activity.In the biosynthesis process of HP/HS,its polysaccharide backbone forms an unusual complex structure under the modification of several enzymes,which enables HP/HS to interact with a variety of bioactive proteins and participate in various physiological and pathological processes.Although the highly complex structure gives HP/HS a variety of biological activities,it also hinders researchers from studying the structure and function of HP/HS.Heparinase(Hepases)from bacteria is a type of polysaccharide lyase that specifically degrades HP/HS substrates.Hepases have important applications in the study on the structure-activity relationship of HP/HS,as well as preparation of HP/HS oligosaccharides and low-molecular-weight-heparin(LMWH).The identified heparinase is divided into three categories:Hepase I,Hepase? and Hepase ?,all of which are endo-type lyases.However,exolytic Hepases(exoHepase),which is very important for the study of HP/HS structure,has never been reported.In this study,we have screened five genes encoding the exoHepases from NCBI(The National Center for Biotechnology Information)database.These exoHepases were cloned and expressed,studied for their enzymatic properties,substrate specificity,structure and catalytic mechanism were studied in detial and preliminarily applied.The main results include the following aspects:(1)Screening and identification of Hepases from PL 15₂ subfamily:we screened 5 candidate genes designated as Hepase from the NCBI database and named BIexoHep,BCexoHep,BTexoHep,PAexoHep and BFexoHep,respectively.Through bioinformatics analysis,we found that BIexoHep,BCexoHep,BTexoHep,PAexoHep and BFexoHep all belonged to the PL15₂ subfamily.They are basically composed of N-terminal signal peptide,DUF4962 domain and C-terminal Hepase-II-III-family domain.Moreover,they have lower similarities with the identified Hepases(Hepase I,Hepase II and Hepase III),while more similar to a type of exotype alginate lyase Atu3025 from PL15₁ subfamily.After heterologous expression and activity characterization of BIexoHep,BCexoHep,BTexoHep,PAexoHep and BFexoHep,it was found that they only degrade HP/HS substrates,but not alginate substrates.Among them,the activities of the Hepases toward the HP substrate are much higher than that of the HS substrate,indicating that the Hepases are prefer to degrade high sulfated substrates.Therefore,we conclude that the PL15 family is divided into two subfamilies,namely the PL15₁ subfamily with exotype alginate lyase activity,and the PL15₂ subfamily showing HP/HS degradation activity.(2)Study on the enzymatic properties of the PL15₂ subfamily Hepases:Since BIexoHep,BC exoHep,BTexoHep,PAexoHep and BFexoHep have lower enzymatic activity on HP/HS substrates,we then prepared a substrate with higher sulfation degree,HP-F??(the final product of HP degraded by Hepase III),to study the enzymatic properties of these Hpases.The experimental results show that the Hepases of the PL15₂ subfamily are mostly adapted to neutral pH and low temperature environments,such as 20?.When the temperature is higher than 30?,their enzyme activities drop sharply,indicating that they are more sensitive to temperature.Studies have shown that heavy metal ions such as Co2+,Hg2+,Ni2+,Pb2+,Cu2+and 2n2+can strongly inhibit the enzymatic activities of the Hepases in the PL15₂ subfamily.Divalent metal ions such as Ca2+,Ba2+and Mg2+can significantly promote the activities of these Hepases.In contrast,both EGTA and EDTA can show more or less inhibitory effects on the activities of these Hepases.The results indicate that the divalent metal ions play very important roles in the enzymatic activities of the Hepases in the PL15₂ subfamily.In addition,the appropriate concentration of salt(NaCl or KC1)can also significantly promote the above-mentioned Hepases'enzymatic activities.After testing,the enzyme activities of BIexoHep,BCexoHep,BTexoHep,PAexoHep and BFexoHep toward HP-Fa? were 49.89,61.86,26.75,24.25 and 12.15 U/mg,respectively.The enzymatic activities of BlexoHep and BCexoHep against HP were 0.75 and 1.14 U/mg,respectively.The enzymatic activity of BTexoHep,PAexoHep and BFexoHep against HP is lower than 0.5 U/mg.In addition,the Hepases of the PL 15₂ subfamily in this study has extremely lower activities on HS,and the enzymatic activity is lower than 0.5 U/mg.The above results indicate that the Hepases of the PL15₂ subfamily tends to degrade substrates with a higher degree of sulfation.(3)Study on the substrate degradation mode of the PL15₂ subfamily Hepases:we used BIexoHep,BCexoHep,BTexoHep,PAexoHep,and BFexoHep to degrade HP polysaccharides in a time gradient,and found that only unsaturated HP disaccharide products were generated during the degradation process;later we used them to degrade saturated HP DP 13 in a time gradient,similarly,only unsaturated HP disaccharide products are produced during the degradation process.The results show that they are all exolytic heparinases,that is,exoHepases.Further studies have shown that the exoHepases in this paper cannot degrade 2-AB-labeled HP DP 13,which means that the fluorescent group labeled at the reducing end of HP DP 13 prevents the degradation of the substrate by exoHepases,which proves that the degradation of the substrate by exoHepase is started from the reducing end.This study identified Hepases with exolyase activity for the first time,which not only fills the gap in this field,but also provides a powerful tool enzyme for the structure-activity relationship study of HP/HS.(4)Study on the substrate specificity of the exoHepases:in order to study the substrate specificity of exoHepases,we prepared some structure-defined HP tetrasaccharides:P4-4(?U Al-4GlcN Ac6S1-4GlcA 1-4GlcNS6S and ?UA1-4GlcNAc6S1-4IdoAl-4GlcNS6S),P4-5(AUA1-4G1cNSl-4G1cAl-4G1cNS6S and?UA1-4GlcNS 1-4IdoA 1-4GlcNS6S),P4-6(?UA1-4GlcNS6S1-4GlcA1-4GlcNS6S),P4-7(?UA2S1-4GlcNS6S1-4GlcA1-4GlcNS6S)and P4-8(?UA2S1-4GlcNS6S 1-4IdoA2S 1-4GlcNS6S).The study found that the tetrasaccharides P4-4,P4-5,P4-6,P4-7 and P4-8 can be completely degraded by the exoHepases,indicating that the exoHepases has no selectivity for the uronic acid type in the substrate.While,we also found that exoHepase has a different tendency to the uronic acid type.The exoHepases tends to degrade substrates containing IdoA2S,and then for the GlcA-containing substrates.The tendency of these enzymes toward IdoA is the lowest.In addition,exoHepases tend to degrade substrates with higher sulfation degree.ExoHepases exhibit higher enzymatic activity toward the substrates with higher sulfation degree,such as P4-8,and lower enzymatic activity toward substrates with lower sulfation degree,such as P4-4.This result explains the reason why exoHepases have good degradation efficiency for HP substrates,but poor degradation efficiency for HS substrates.Although exoHepases have higher enzyme activity toward the substrates with high sulfation degree,when the substrate has 3-O sulfation modification,such as fondaparinux sodium,it becomes resistant to the activity of exoHepase and cannot be completely degraded.(5)Structure study of the exoHepases:through the structural studies of a typical exoHepase:BIexoHep,we found that the structure of BIexoHep is mainly composed of three domains:the small N-terminal ?-sheet domain,the center(a/a)5 incomplete barrel domain and C-terminal ?-sandwich domain.The crystal structure of BIexoHp appears as a monomer rather than dimer,and each BlexoHep protein molecule contains two Ca2+ions.The structural similarity between BlexoHep with the identified Hepase ?,Hepase ? and Hepase ? is extremely low,but they can overlap at a certain degree,especially the active centers of BlexoHep and Hepase II are particularly similar.Through experimental research,we speculate that the catalytic mechanism of BlexoHep should be:(1)when the substrate containing IdoA,the residue His337 acts as Bronsted base to attract the proton of C5 in IdoA,and Tyr390 acts as the Bronsted acid to provide protons to the oxygen in the glycosidic bond,and thus to complete ? elimination reaction.(2)When degrading the substrate containing GlcA,Tyr390 will simultaneously act as Br(?)nstGd base and acid to complete the ? elimination reaction.Further,via preparing and analyzing the co-crystal structure of BIexoHep with HP tetrasaccharide substrate,we found that there is a semi-open,narrow,elongated and L-shaped catalytic cavityin the structure of BlexoHep.BIexoHep's unique semi-open L-shaped tunnel consists of two almost perpendicular parts:the positively charged entrance and the negatively charged exit.The existence of this special reaction tunnel endows BIexoHep the exolytic characters.We speculate that the exocytosis mechanism of BIexoHep is:1)first,the reducing end of the free HP chain in the environment binds to the positively charged entrance of the L-shaped tunnel via charge attraction;2)through electrostatic interactions,the bound HP chain moves into the tunnel,and once the reducing terminal disaccharide reaches the+1 and+2 subsites,the disaccharides will be cleaved via a ?-elimination mechanism;3)with the aid of the negatively charged residue Asp335,the resulting disaccharide product is pushed into the negatively charged exit and rapidly released with assistance from Pro67 and Asp281.(6)Preliminary application of the exoHepases:based on the exolytic feature of BlexoHep that sequentially cleave HP chains from the reducing ends,we have developed a novel enzymatic sequencing method.The sequencing method uses BlexoHep to incompletely degrade HP unsaturated oligosaccharides,and then the corresponding non-reducing end UDP4,UDP6,UDP8...UDP2n were isolated.By analyzing and comparing the disaccharide composition of these unsaturated oligosaccharides,the complete sequence of HP oligosaccharide can be deduced.In this paper,we have used this method to successfully sequence five HP octasaccharides,namely:P8-1?UA1-4GlcNS6S 1-4HexA2S 1-4GlcNS6S-4HexA2S 1-4GlcNS6S 1-4HexA2S 1-4Glc NS,P8-2?UAl-4GlcNS6S 1-4HexA2S 1-4GlcNS-4HexA2S 1-4GlcNS6S 1-4HexA2S 1-4GlcNS 6S,P8-3?UA1-4GlcNAc(S)1-4HexA2S 1-4GlcNS6S-4HexA2S 1-4GlcNS6S 1-4HexA 1-4GlcN S6S,P8-4?UA1-4GlcNS6S 1-4HexUA2S1-4GlcNS6S1-4HexUA2S1-4GlcNS6S1-4HexUA2S1-4GlcNS6S and P8-5?UA1-4GlcNAc(S)1-4HexA2S1-4GlcNS6S-4HexA2S1-4GlcNS6S1-4HexA2S1-4G1 cNS6S.The method is easy to operate,and detect the disaccharides as units,which greatly reduces the operating procedures.Moreover,because it is detected under fluorescent conditions,the amount of sample required is greatly reduced.We believe that this method can also used to sequence the larger oligosaccharides.In this thesis,five exoHepases from the PL15 2 subfamily have been studied fortheir enzymatic properties,substrate specificity,three-dimensional structure and catalytic mechanism in detial as well as preliminary application.The study of exoHepases in this paper not only fills the gap in the field of Hepases,but also provides important tool enzymes for the structural study of HP/HS.