Identification And Application Of A Chondroitin Sulfate E-resisted Glycosaminoglycan Lyase

The backbone of the chondroitin sulfate(CS)chain is composed of repeating disaccharide units of glucuronic acid(G1cUA)and N-acetyl-D-galactosamine(GalNAc).CS is a negatively charged polysaccharide synthesized as the glycosaminoglycan(GAG)side chain of proteoglycans and widely distributed on cell surfaces and in the extracellular matrix.CS participates in various important biological processes,such as neuronal growth,morphogenesis,inflammation,tumor progression,virus infection,and cytokinesis.These various functions of CS are attributed to its structural diversity.The disaccharide is further modified by specific sulfotransferases at C-2 of GlcUA/L-iduronic acid and/or C-4 and/or C-6 of GaIN Ac to yield substantial structural diversity via differential sulfation patterns.Moreover,some GlcUA residues are epimerized into L-iduronic acid residues(IdoUA)by glucuronyl C-5 epimerase,and correspondingly the chain containing repeating disaccharide units of-IdoUA-GaIN Ac-is designated dermatan sulfate(DS).Thus,CS and DS often exist in a single chain in a hybrid form(CS-DS)and are usually periodically distributed in a cell/tissue-specific manner.Several different sulfation modifications by a number of sulfotransferases result in the creation of a variety of CS disaccharide units,such as the O unit(GlcUA?1-3GalNAc),the A unit(GlcUA?1-3GalNAc(4S)),the C unit(GlcUA?3-3GalNAc(6S)),the D unit(GlcUA(2S)?1-3GalNAc(6S)),the E unit(GlcUA?1-3GalNAc(4S,6S))and the T unit(GlcUA(2S)?1-3GalNAc(4S,6S)),where 2S,4S,and 6S represent 2-O-,4-0-,and 6-O-sulfate groups,respectively.Glycosaminoglycan(GAG)is a very important bioactive molecule,because of its critical biological function,and widely used in medicine and functional foods.Same GAG,especially sulfated GAGs from different sources or growth period have different activity because of their prominent structural heterogeneity.More and more studies have shown that GAG polysaccharides exhibit biological functions by binding with specific proteins through special structures and sequences of GAGs.Furthermore,interactions of different structural sequences in single GAG chain with different proteins result in different functions.Therefore,uniform or relative homogenous structure with specific sequence is activity specificity and easy-to-absorption,which is very critical for the development of CS-related medicine.Chondroitin sulfate chains containing E units have been found in mammalian tissues,including brain,liver,spleen,kidney,and malignant tumors,and have been shown to be involved in various physiological and pathological processes through interactions with various growth factors or chemokines.Interestingly,these interactions were specifically inhibited by CS or DS chains containing the E unit and iE unit(IdoUAal-3GalNAc(4S,6S)),respectively.Furthermore,E unit-rich CS polysaccharides,such as CS-E from squid cartilage,showed remarkable biological activities,such as promotion of neurite outgrowth,inhibition of viral infection,and anti-tumor metastasis.Studies on the structure-function relationship of E unit-containing polysaccharides have shown that E unit-rich domains in these CS/DS chains play key roles in various biological functions,and the minimal essential structures for various bioactivities of CS-E have been identified.Compared to the complexity of polysaccharides,CS-E oligosaccharides with specific sequence are much more homogeneous in size and structure,which will greatly exclude the ambiguity of polysaccharides in a specific bioactivity,and they are more easily absorbed and utilized due to their low molecular mass.Thus,the ability to selectively prepare E unit-rich oligosaccharides from CS-E polysaccharides will be critical for the development of CS therapeutic agents.E unit-rich domains have been shown to play key roles in various biological functions of chondroitin sulfate(CS).However,an enzyme that can specifically isolate such domains through the selective digestion of other domains in polysaccharides has not yet been reported.Here,we prepared novel chondroitin sulfate E(CS-nE)from Dosidicus Gigas cartilage.And we identified a glycosaminoglycan lyase from a marine bacterium Vibrio sp.FC509 with CS-nE as substrate.This enzyme efficiently degraded hyaluronic acid(HA)and CS variants(CS-A,CS-C and CS-D),but not E unit-rich CS,into unsaturated disaccharides;therefore,we designated this enzyme a CS-E-resisted HA/CS lyase(HCLase Er).The main results are summarized as follows:1.Preparation,purification,disaccharide compositon and bioactivity analysis of CS-nE:We used enzyme hydrolysis,alcohol precipitation and ion-exchange chromatography technology to prepare CS-nE with Dosidicus Gigas cartilage as material A standard curve of molecular mass and elution time was created by using a series of standard dextrans with different molecular mass eluted on a gel filtration column,and based on this method the molecular mass of CS-nE was determined to be about 623.3 kDa.CS-nE was digested with commercial CSase ABC to determine the disaccharide composition by HPLC and MS.The disaccharides of CS-nE are mainly O unit(6.9%),C unit(12.1%),A unit(35.4%),and E unit(45.6%),and the m/z of each unsaturated disaccharide(378.1009(1),458.0558(1),or 538.0120(1)/268.50332(2))was consistant with theoretical molecular masses of ?O(?4,5HexUA?1-3GalNAc),AA/C(?4,5HexUA?1-3GalNAc(4S/6S)),and ?E(?4.5HexUA?1-3GalNAc(4S,6S)),respectively.To determine the interaction activity of CS-nE polysaccharide with growth factors and selectins,CS-nE was biotinylated and immobilized on a streptavidin chip.For HGF,bFGF,PTN,and HB-EGF,the ka values were(5.67±1.84)× 106,(3.26±1.19)x 106,(8.14±0.31)× 103,and(2.36±1.56)× 108 m-1S-1,respectively;the kd values were(2.01 ±0.46)x 10-3,(6.81 ±0.7)× 10-5,(2.43±0.73)×10-4,and(9.6±1.48)×10-2 S-1,respectively;and the Kd values were 0.26±0.004,0.03±0.01,27.38±7.95,and 0.27±0.16 nM,respectively.The kinetic curves and constants indicate that HGF,bFGF,PTN,and HB-EGF have high affinity to CS-nE,and in contrast selectines bind CS-nE with a relatively low affinity.These results suggest that CS-nE can interact with a variety of growth factors,play a regulatory role in cell signal transduction,and has important biological activities in a variety of physiological and pathological processes.2.Study of HCLase Er:The full length gene and protein sequence of HCLase Er were analyzed by online database and bioinformatic softwares.The putative GAG-degrading gene named hclase er(GenBankTM accession number MF458894),was 2973 bp in length and had a GC content of 50.6%.The predicted full length protein was composed of 990 amino acid residues with a theoretical molecular mass of 108.4 kDa and an isoelectric point of 5.34.The full length sequence of the HCLase Er was cloned into pET-30a(+)to form the recombined plasmid,then the recombined protein was purified and its biological activities was analyzed detailedly.The optimal reaction condition is Tris-HCl,pH 8.0 at 30?.Interestingly,this enzyme did not show halophilic characteristic as previously found for HCLase,and even its activity was significantly inhibited when the concentration of NaCl was higher than 500 mM.This enzyme could efficiently degraded hyaluronic acid(HA)and CS variants(CS-A,CS-C and CS-D),but not E unit-rich CS into unsaturated disaccharides;therefore,we designated this enzyme a CS-E-resisted HA/CS lyase(HCLase Er).We isolated a series of resistant oligosaccharides from the final products of the low-sulfated CS-nE exhaustively digested by HCLase Er,and found that the E units were dramatically accumulated in these resistant oligosaccharides.By determining the structures of several resistant tetrasaccharides,we observed that all of them possessed a?4,5 HexUA?1-3GalNAc(4S,6S)at their nonreducing ends.Furthermore,?E-E was easily cleaved by HCLase Er after 4-O-edousulfatase pretreatment.The results indicating that the disulfation of GalNAc abrogates HCLase Er activity on the ?1-4 linkage between the E unit and the following disaccharide.?4,5 HexUA?1-3GalNAc(4S,6S)?1-4GlcUA?1-3GalNAc(4S,6S)was most strongly resistant to HCLase Er,and it could partially inhibit the action of HCLase Er on HA/CS-A.To our knowledge,this study is the first reporting a glycosaminoglycan lyase specifically inhibited by both 4-0-and 6-O-sulfation of GaINAc.Site-directed mutation(rHCLase Er-H243A,rHCLase Er-Y252A,rHCLase Er-R306A and rHCLase Er-R310A)and truncation mutagenesis experiments indicate that HCLase Er may use a general acid-base catalysis mechanism and that an extra domain(Gly739-Gln796)is critical for its activity.This enzyme will be a useful tool for the structural analysis and oligosaccharides preparation of HA and CS variants in particular E unit-rich CS chains.3.The application of HCLase Er:The CS-nE polysaccharide was digested by rHCLase Er,and the products were collected by ethanol precipitation and centrifugation.And then,the size-defined CS-nE oligosaccharides were fractionated and collected by gel filtration chromatography.The anti-tumor metastasis activity of these CS-nE oligosaccharides was investigated by using a experimental metastatic model prepared by injecting rat breast cancer cell line 4T1 to BALB/c female mice through tail vein.The results showed that the tumor inhibition rate of CS-nE hexasaccharide,octasaccharide,decasaccharide,dodecasaccharide and polysaccharide was 45.84%,31.95%,77.78%,72.12%,and 64.85%,respectively,indicating that decasaccharide and dodecasaccharide exhibited observably anti-tumor metastasis activity while CS-nE hexasaccharide and octasaccharide has weak actibity.Among the bioactive oligosaccharides,CS-nE decasaccharide has the stongest anti-tumor metastasis activity.