Identification And Application Of Two Novel Dermatan Sulfate Lyases

Chondroitin Sulfate/Dermatoderin Sulfate(CS/DS)is a linear polyanionic polysaccharide consisting of repeated disaccharide units.The structure of the CS disaccharide unit is made up of glucuronic acid(G1cUA)and N-acetyl-D-galactosamine(GalNAc).The DS disaccharide unit is formed when the GlcUA in the CS disaccharide unit is changed to L-iduronic acid(IdoUA)under the action of glucuronyl C-5 epimerase.Among them,the C-2 and C-3 positions of GlcUA or IdoUA and C-4 and C-6 positions of GalNAc will undergo different degrees of sulfation under the action of various sulfotransferases and form different disaccharide units thus resulting in the highly complex structure of CS/DS polysaccharide chain.CS/DS is an important class of Glycosaminoglycans(GAGs),which often exists as the side chain of proteoglycans(PGs).The complex and variable structure of CS/DS chain also endows it with different biological activities,such as participating in cell growth,cell adhesion,virus infection,nervous system development etc.The heterogeneity of the sequences,the variability of disaccharide arrangement,the isomerization of uronic acid and especially the difference of sulfation patterns make the structural and functional studies of CS/DS extremely difficult.Specific CD/DS-degrading enzymes are an indispensable tools for the structure-function studies of CS/DS.Currently,the available types and numbers of CS/DS-degrading enzymes are extremely limited,especially the enzyme specifically degrading DS,only one called Chondroitinase B(CSase B)being reportedIn this study,we found and identified two enzymes that specifically degrade DS and named them DLase 1 and DLase 2,respectively.We found that both DLase 1 and DLase 2 have low homology with the known CSase B and they are endo-type lyases In addition,their enzyme activity under optimum conditions are much higher than that of CSase B.In this study,CS/DS polysaccharides were first extracted from the skin of Paralichthys olivaceus,Thamnaconus modestus and Cynoglossus semilaevis.Their composition and structure were analyzed by using the newly identified DLase.The detailed research results are as follows1.Two potential DS-specific lyase genes,dlasel and dlase 2,were found from GenBank database by bioinformatics method,and their similarity with CSase B was 57%and 43%respectively.Analysis of the two suspected genes and their coding protein sequences showed that the full length of DLase 1 gene was 1473bp,the GC content was 49.08%,the predicted protein contained 491 amino acids,the theoretical molecular weight was 55.35 kDa,the Pl was 8.60,and the N-terminal signal peptide contained 21 amino acids.The full length of DLase 2 gene is 1401 bp,the GC content is 48.61%,the predicted protein contains 467 amino acids,the theoretical molecular weight is 52.00 kDa,the PI was 6.1 8,and the N-terminal signal peptide contains 19 amino acids.It is noteworthy that although the two new enzymes have similar molecular weights to CSase B(55.20 kDa),their pl values are lower,especially DLase 2.After whole-gene synthesis,we performed heterologous recombinant expression of two suspected genes Both proteins could be well water-soluble expression and purified by Ni column affinity SDS-PAGE electrophoresis analysis showed that the purity of the purified recombinant proteins DLase 1 and DLase 2 was greater than 95%.2.The substrate degradation specificity of DLase 1 and DLase 2 was studied.the biochemical properties were analyzed and the enzyme activity was measured.It was found that DLase 1 and DLase 2 were the same as CSase B,and could only degrade DS specifically.The optimal reaction buffer of DLase 1 was 50 mM Tris-HCI(pH 10.0),and the optimal reaction temperature was 40?.Ca2+could significantly promote its activity.When Ca2+of different concentrations was added to the reaction system,it was found that when the concentration of Ca2+was 20 mM,the promotion of the degradation activity of DLase 1 was the strongest,which was up to 155%of the Ca2+unadded activity.The optimal reaction buffer of DLase 2 was 50 mM Tris-HCl(pH 9.0),and the optimal reaction temperature was 40?.Similar to DLase 1,Ca2+ could significantly promote its degradation activity.When the concentration of Ca2+was 20 mM,the degradation activity of DLase 2 reached 191%of the activity without adding metal ions The optimal buffer for CSase B reaction was 50mM Tris-HCl(pH 8.0),similar to those of DLase 1 and DLase 2,and the optimal reaction temperature was 30?,lower than that(40?)of DLase 1 and DLase 2.Under the optimal conditions the enzymatic activity of DLase 1 and DLase 2 were 911 U/mg and 1630 U/mg respectively,which was much higher than that(84.6 U/mg)of CSase B.In addition,we also measured the effects of other GAGs on the substrate-degrading activities of DLase 1 and DLase 2,and found that HA,Hep,HS,CS-A and CS-C could competitively inhibit the degradation of DS by DLase 1 and DLase 2,and Hep had the most obviously inhibitory effect,which may be due to the high sulfaion and rich IdoUA residues in Hep chains.Through the analysis of the resistant structures to DLase 1 and DLase 2,it can be known that DLase 1 and DLase 2 could not degrade a small portion of CS structures in DS chains.3.In order to find and identify more novel CS/DS,three novel CS/DS polysaccharides were isolated and purified from the skin of Paralichthys olivaceus,Thamnaconus modestus and Cynoglossus semilaevis by means of proteolysis,ethanol precipitation and column chromatography.Gel filtration analysis showed that the average molecular mass of CS/DS from the skin of Cynoglossus semilaevis was 56.24 kDa,the average molecular mass of CS/DS from the skin of thamnaconus modestus was 60.52 kDa,and the average molecular mass of CS/DS from the skin of Paralichthys olivaceus was 65.19 kDa.Furthermore,the composition and proportion of disaccharides of CS and DS in three novel CS/DS polysaccharides were analyzed by using CS-specific degrading enzyme CSase AC I and DS-specific degrading enzyme DLase 1 discovered in this study.The results showed that although the new CS/DS from the three fish skin sources all contained high levels of A-unit(GIcUA?-3GalNAc(4S))and iA-unit(IdoAal-3GalNAc(4S)),the content and disaccharide composition of CS and DS were significantly different?which provide a possibility for the bioactivity research and utilization of the new CD/DS preparations.In conclusion,this study found and identified two novel DS-specific lyases,whose enzymatic activities are much higher than that of CSase B,the only identified DS-specific lyase so far.Undoubtedly,they should have very important application value in the structural and functional studies of CS/DS and the preparation of active CS/DS ligosaccharides.At the same time,the separation and purification of the new CS/DS derived from the skin of three kinds of fish and the analysis of their composition and structre have provided the possibility for the further study of biological activity and the development and utilization of relevant resources.