Chemical Structure And Heparanase Inhibitory Activity Of FG From Holothuria Floridana

Fucosylated glycosaminoglycan（FG）,a glycosaminoglycan（GAG）derivative,found up to now exclusively in sea cucumber,generally possesses a chondroitin sulfate（CS）-like backbone,fucose（Fuc）side chains and some sulfate groups.The high molecular weight and complex structure of native FG make it difficult to determine its exact structure.Studies on the exact structure of some native FGs have made positive progress,especially in the structural types and connection position of Fuc side chains.However,the precise structures of FGs vary in a species-specific manner.Therefore,the structural diversity of native FG worth further studying.In our previous work,three oligosaccharide fragments（Fr1–Fr3）were obtained from FG derived from Holothuria floridana（HfFG）by a deacetylation–deaminative cleavage method.The structural characteristics and physicochemical properties of HfFG were studied.However,the ¹H NMR spectra of the depolymerized products of HfFG showed that the signals located in the 5.35 ppm and 5.62 ppm did not appear in Fr1–Fr3,suggesting that complex and novel side chains may exist in HfFG.In our previous studies,aβ-elimination depolymerization method has been successfully used in the structural analysis of FG with complex side chains.Therefore,in this paper,the exact structure of HfFG was systematically studied by the peroxidative depolymerization,partial acid hydrolysis andβ-elimination depolymerization methods,so as to provide a reference for studying the exact structure of complex FGs.Furthermore,the heparanase inhibitory activities of native HfFG and oligosaccharide fragments were evaluated to provide basis for the study of structure-activity relationship of heparanase inhibitors.The main research methods and results are as follows:（1）Structural characteristics of HfFGHfFG was extracted and purified by enzymatic-alkaline hydrolysis,alcohol precipitation,hydrogen peroxide decolorization,anion exchange chromatography and gel permeation chromatography.According to the content calculated by area normalization method,the purity of HfFG was greater than 99%.HPGPC analysis showed that its molecular weight was 47.3k Da and its basic compositions were analyzed by the ¹H NMR,which were consistent with our previous report.The product dHfFG-1 was prepared by the peroxidative depolymerization of HfFG,and its molecular weight（Mw）was 6994 Da.1D/2D NMR showed that dHfFG-1 had the basic structural characteristics of FG,and theα-anomeric protons signal in ¹H NMR showed the presence of four sulfated types of fucoses on the side chains.The two signals at about 5.0-5.2ppm indicated the possible existence of novel types of Fuc side-chains.In view of the sensitivity of glycosidic bonds of methyl pentose to acids,partial acid hydrolysis was used to analyze the structure of HfFG.The backbone（Bb）and side chain（Sc）products,released from the hydrolysis product of HfFG treated by 0.1 M H₂SO₄,were separated and obtained.The structure of Sc was analyzed by 1D/2D NMR spectra and four types of side chains could be observed clearly(Fuc S=Fuc_2S4S,Fuc_3S4S,Fuc_4S or D-Gal NAc_4S-α（1,2）-Fuc_3S4S).（2）Analysis of the precise chemical structure of oligosaccharidesdHfFG-2 was obtained by theβ-eliminative depolymerization,a glycosidic bond-selective depolymerization method.Three size-homogeneous fragments（Fr1–Fr3）were isolated by gel permeation chromatography from dHfFG-2.Their ¹H NMR spectra showed that the chemical structures of these components were complex.The SAX-HPLC analysis showed that Fr2 contains a series of oligosaccharide fragments.Fr2 was further purified by a semi-preparative SAX-HPLC to yield purified compound 1 and 2.The chemical structures of those size-homogeneous oligosaccharide fragments（Fr1–Fr3）and purified oligosaccharides（compound 1 and 2）were analyzed by 1D/2D NMR.The results are as follows:Fr1:L-Fuc S-α（1,3）-L-Δ^4,5Glc UA-α（1,3）-D-Gal NAc_4S6S-ol,Fuc S=Fuc_2S4S（90%）,Fuc_3S4S（10%）Fr2:L-Fuc S-α（1,3）-L-Δ^4,5Glc A-α（1,3）-D-Gal NAc_4S6S-β（1,4）-[L-Fuc S-α（1,3）]-D-Glc A-ol(Fuc S=Fuc_2S4S,D-Gal NAc_4S-α（1,2）-L-Fuc_3S4S,Fuc_3S4S or Fuc_4S)Fr3:L-Fuc S-α（1,3）-L-Δ^4,5Glc A-α（1,3）-D-Gal NAc_4S6S-β（1,4）-[L-Fuc S-α（1,3）]-D-Glc A-D-Gal NAc_4S6S-β（1,4）-[L-Fuc S-α（1,3）]-D-Glc A-ol(Fuc S=Fuc_2S4S,D-Gal NAc_4S-L-α（1,2）-Fuc_3S4S,Fuc_3S4S or Fuc_4S)Compound 1:D-Gal NAc_4S-α（1,2）-L-Fuc3S4S-α（1,3）-L-Δ^4,5Glc A-α（1,3）-D-Gal NAc4S6S-β（1,4）-[L-Fuc2S4S-α（1,3）]-D-Glc A-olCompound 2:L-Fuc2S4S-α（1,3）-L-Δ^4,5Glc A-α（1,3）-D-Gal NAc4S6S-β（1,4）-D-Gal NAc_4S-α（1,2）-L-Fuc3S4S-α（1,3）]-D-Glc A-ol（3）The exact structure analysis of the native HfFG（1）The structure characteristics of two oligosaccharides were analyzed.Compound 1and 2 contained the main chain structure similar to CS-E,i.e.,{D-Glc UA-β（1,3）-D-Gal NAc_4S6S-β1-}_n.Both Fuc_2S4Sand D-Gal NAc_4S-α（1,2）-L-Fuc_3S4S side chains were connected to D-Glc UA or L-Δ^4,5Glc UA of the main chain by anα（1,3）glycosidic bond.（2）The 1D/2D NMR spectra of Sc clearly showed four types of side chains,including Fuc_2S4S,Fuc_3S4S,Fuc_4S and D-Gal NAc_4S-α（1,2）-Fuc_3S4S,indicating the existence of disaccharide side chains in the native FG.（3）Based on the basic structural features of FG oligosaccharides,especially the presense of the special disaccharide side chain D-Gal NAc_4S-α（1,2）-L-Fuc_3S4S,the 1D/2D NMR spectra of dHfFG-1 could be fully assigned.The side chain Fuc S is Fuc_2S4S,Fuc_3S4S,Fuc_4S and D-Gal NAc_4S-α（1,2）-Fuc_3S4S,with a ratio of 8:2:3:7.（4）The ¹H NMR spectra of oligosaccharides and size homogeneous oligosaccharides components Fr1-Fr3 were analyzed and comparised.Although the ¹H NMR signals of Fr1–Fr3 are complex,almost all signals can be retrospect to the corresponding oligosaccharide signals.The information of oligosaccharides structure fully showed the chemical structural characteristics of the homogeneous components Fr1–Fr3,which also reflected the basic structural features of native HfFG.Based on the structure analyses of size-homogeneous oligosaccharides fragments Fr1–Fr3,purified FG oligosaccharide,side chains after partial acid hydrolysis and peroxidation depolymerization product dHfFG-1,it could be decuced that the chemical structure of native HfFG is:{-4-[L-Fuc S-α（1,3）-]-D-Glc UA-β（1,3）-D-Gal NAc_4S6S-β1-}_n(L-Fuc S=L-Fuc_2S4S,L-Fuc_3S4S,L-Fuc_4S or D-Gal NAc_4S-α（1,2）-L-Fuc_3S4S).（4）Assay of heparanase inhibitory activityThe heparanase inhibitory activities of HfFG and its oligosaccharide fragments were determined by a HTRF method.The native HfFG and its oligosaccharides exhibited heparanase inhibitory activity,which increased with the increase of molecular weight.Conclusion:（1）The results of this study clearly showed that native HfFG possessed a CS-E-like backbone and each Glc UA in the backbone was branched with the Fuc S at C-3,and there was no other position substituted by Fuc S in the main chain.Furthermore,O-4 and O-6 positions of the Gal NAc were all substituted by sulfate esters.The side chains were monosaccharide(Fuc_2S4S,Fuc_3S4S or Fuc_4S)or disaccharide(D-Gal NAc_4S-α（1,2）-L-Fuc_3S4S).Among them,the disaccharide side chain D-Gal NAc_4S-α（1,2）-L-Fuc_3S4S was found in the native FG for the first time.In addition,the content of disaccharide side chains in HfFG was higher than that of FG from other sea cucumber species currently known.（2）The heparanase inhibitory activity assay showed that the native FG possessed potent inhibitory activity.The anti-heparanase activities of the three homogeneous oligosaccharides increased gradually with the increase of molecular weight.Additionally,structural characteristics such as sulfation patterns,the terminal structure of oligosaccharides and the presence of fucosyl branches may be important factors affecting heparanase inhibitory activity.