Study On Structure And Activity Of Fucosylated Glycosaminoglycan Based On Its Purified Resulting Fragments

Objectives:Fucosylated glycosaminoglycan(FG)is a novel GAG derivative extracted from sea cucumber,which is composed of three types of monosaccharides,D-GlcA,D-GalNAc and L-Fuc.Native FG has a strong FXase inhibitory activity,receiving extensive attention from researchers.However,it also activates FXII and induces platelet aggregation,which limits its application.In recent years,the selective depolymerization technology has been used to the structural research of FG and made great progress in the structural study of FG with only one type of sulfated Fuc branch.However,the structural study of FG with complex sulfated Fuc branches has not made significant progress.Moreover,the activities are associated with the structural features such as sulfation patterns of Fuc branches and molecular mass.Apparently,the exact structural analysis of AjFG is the basis for investigating its pharmacological mechanism and structure-activity relationship,and for further exploring its application value.The selective-glycosidic degradation method,combined with the analysis of obtained oligosaccharides with well-defined structures,is a reliable way to study the exact structure of native FG.In this study,partial deacetylation-deaminative depolymerization,a highly selective degradation method for FG,was used to study the structures of two complex FGs from sea cucumbers ApostichopuLs japonica,s(AjFG)and Holothuria floridana(HfFG).The definite structures of two FGs were elucidated through the high-regular oligosaccharides obtained from their depolymerized FGs,which might provide some references for the exact structure of complex sulfate-substituted FG.The anticoagulant activities of these depolymerized fragments,including inhibition of FXase,and side effects(effect on FXII and platelets),were also analyzed to provide a reference for development of new anticoagulant drugs.Methods:The polysaccharides from A.japonicas and H.floridana were extracted by enzymatic digestion combined with alkali treatment.Proteins were removed at their isoelectric point,compounds with low molecular weights and fat-soluble impurities were removed by grading-ethanol precipitation,and pigments were removed by hydrogen peroxide treatment.Moreover,other methods,such as ion exchange chromatography and gel permeation chromatography,were used to obtain pure AjFG and HfFG.The purity and molecular weight were analyzed by high performance liquid chromatography(HPLC),the molar ratio of sulfate to uronic acid groups was determined by conductimetric titration method,and the monosaccharide composition was analyzed by PMP derivative-HPLC.The basic chemical constitution was analyzed by IRand 1H NMR spectra.Deaminative depolymerization was used to depolymerize AjFG and HfFG,which selectively cleaves hexosamine glycosidic bond,and the regular oligosaccharides with various degree of polymerization(dp)were then separated by gel chromatography.The structures of Fr-1-Fr-4(the fragments of depolymerized AjFG)and dp-3-dp-9(the fragments of depolymerized HfFG)were elucidated by the 1D/2D NMR and ESI-Q-TOF-MS analysis,then the exact structures of AjFG and HfFG could be inferred.The APTT prolonging activity and the effects on FXase,FXII and platelets of AjFG,dAjFG and Fr-1-Fr-4 were studied by the methods in vitro.Results:The yield of purified AjFG was 0.3%in relation to dry weight of sea cucumber,and the molecule weight was 76.4 kDa.The molar ratio of sulfate to uronic acid groups was determined to be 3.59.AjFG was composed of three types of monosaccharides—GlcA,GalNAc and Fuc—with the molar ratio of 1:0.94:1.07.The specific rotation was determined to be-68 °,which is levorotatory.Based on the analysis of 1D/2D NMR and ESI-MS spectra,the structure of Fr-1 was established as L-FucS-?l,3-D-GlcA-?1,3-D-anTal-diol4S6S(FucS=Fuc2S4S or Fuc3S4s),and the molar ratio of two types of trisaccharides is approximately 2:1.The structure of Fr-2 was established as L-FucS-?l,3-D-GlcA-?1,3-D-GalNAc4S6S-?1,4-[L-FucS-?1,3-]D-G1cA-?1,3-D-anTal-dio14S6S(FucS=Fuc2S4S~51%,Fuc3S4S~30%and Fuc4S~19%).The structures of Fr-3 and Fr-4 were deduced to be D-FucS-?1,3-D)-GlcA-?1,3-{D-GalNAc4S6s-?1,4-[L-FucS-?l,3]-D-GlcA-?1,3}n-D-anTal-diol4S6S(n=2,3).The structures of these oligosaccharides had a common structural formula of L-FucS-?1,3-D-GlcA-pi,3-{D-GalNAc4S6s-?1,4-[D-FucS-?l,3-]D)-GlcA}n-D-anTal-d iol(n 0-3,FucS=Fuc2S4s,Fucs4s or Fuc4s).Therefore,the structure of native AjFG could be rationally deduced as a repeating trisaccharide unit-{4-[L-FucS-al,3]-D-GlcA-?1,3-D-GalNAc4S6s-?1}n-,which possessed a CS-E-like backbone,and the proportion of three kinds of sulfated Fuc branches was 54%,29%,and 17%.The yield of purified HFIfFG was 0.9%in relation to dry weight of sea cucumber.The molecule weight was 82.7 kDa,and the molar ratio of sulfate to uronic acidgroups was determined to be 3.756.FIfFG was composed of three types of monosaccharides—GlcA,GalNAc and Fuc—with the molar ratio of 1:0.91:1.04.The specific rotation was determined to be-50 °,which is levorotatory.Based on the analysis of 1D/2D NMR,the structure of dp-3 was established as L-FucS-al,3-D-GlcA-pi,3-D-anTal-diol4S6s(FucS=Fuc2S4s or Fuc3s4s),and the molar ratio of two types of trisaccharides was approximately 2:1.The structure of dp-6 was established as L-FucS-al,3-D-GlcA-?1,3-D-GalNAc4S6s-?1,4-[L-FucS-al,3-]D-GlcA-?1,3-D-anTal-diol4S6S(FucS=Fuc2S4S~53%,Fuc3S4S~28%and Fuc4s~19%).The structure of dp-9 was deduced to be L-FucS-al,3-D-GlcA-pl,3-{D-GalNAc4S6S-pl,4-[L-FucS-al,3]-D-GlcA-?1,3}2-D-anTal-diol4S6S(FucS=Fuc2S4S~47%,Fuc3S4S~29%and Fuc4S~24%).The structures of these oligosaccharides had a common structural formula of L-FucS-al,3-D-GlcA-pi,3-{D-GalNAc4S6s-?1,4-l[L-FucS-al,3-]D-GlcA}n-D-anTal-diol(n=0-2,FucS F'uc.'sts,FUC3S4S or Fucts).The backbone of FIfFG was similar tothat of AjFG,which possessed a CS-E-like backbone,but the proportions of three kinds of sulfated Fuc branches were 40%,31%,and 29%,respectively.Anticoagulant activities and side effects of FG with complex sulfated Fuc branches,depolymerized FG,and the oligosaccharide fragments were investigated.The native AjFG showed more potent APTT prolonging activity than LMWH,and its concentration required for double APTT was about 1/3 of that of LMWH.The concentrations of dAjFG Fr-3,and Fr-4 exhibited comparable or less potent APTT prolonging activity than LMWH.However,Fr-1 and Fr-2 showed no significant APTT prolonging activity at the concentration up to 128 ?g/mL.The anti-FXase activity of AjFG was more than 10-fold higher than that of LMWH,while the activities of dAjFGQ Fr-3 and Fr-4 were slightly weaker than that of LMWH.However,Fr-1 and Fr-2 had no significant anti-FXase activity at concentrations as high as 1000 ?g/mL.Compared with OSCS or ADP,dAjFG,Fr-1-Fr-4 exhibited no FXII activation and platelet aggregation(P>0.05)within the experimental concentration range.Conclusions:The results of this study clearly showed that all the FucS side chains in AjFG existed as a monosaccharide,and the fucobiose or fucotriose side chains were not found.The FucS side chain only attached to GlcA in an a-1,3 manner,and no FucS side chain linked to Ga1NAc of the backbone.Furthermore,O-4 and O-6 positions of the GalNAc were all replaced by sulfate esters,and monosulfated GalNAc was not obseIrved.The backbone of HfFG was similar to that of AjFG,but their proportion of three kinds of sulfated Fuc branches was different.The proportion of Fuc4S side chain contained in HfFG was higher than that of AjFGThe native AjFG,dAjFG,Fr-3,and Fr-4 showed strong inhibitory effects on endogenous coagulation,which displayed strong APTT prolonging activities and inhibition of intrinsic FXase,and the activity study showed that the anticoagulant activity of FG decreased with the decrease of molecular weight of the fragments.The necessary structure of AjFG for effectively anti-FXase and anticoagulant activities might contain at least three or more trisaccharide structural units.The side effects decreased with the decrease of molecular weight of the fragment.These results indicated that dAjFQ nonasaccharides and dodecasaccharides might be safe and effective anticoagulants.