Biochimica et Biophysica Acta 1820 (2012) 989–1000
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Sequence determination and anticoagulant and antithrombotic activities of a novelsulfated fucan isolated from the sea cucumber Isostichopus badionotus
Shiguo Chen a, Yaqin Hu a, Xinqian Ye a, Guoyun Li b, Guangli Yu c, Changhu Xue b,⁎, Wengang Chai d,⁎⁎a College of Biosystem Engineering and Food Science, Zhejiang University, Hangzhou 310029, Chinab College of Food Science and Technology, Ocean University of China, Qingdao266003, Chinac School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, Chinad Glycosciences Laboratory, Department of Medicine, Imperial College London, Hammersmith Campus, London W12 0NN, United Kingdom
⁎ Correspondence to: C. Xue, College of Food Sciencversity of China, 5 Yushan Road, Qingdao, Shandong 26⁎⁎ Correspondence to: W. Chai, Glycosciences Laborat
Medicine, Northwick Park and St. Mark's Campus, HarroKingdom.
Background: The aim is to analyze the structure, anticoagulant and antithrombotic activities of a sulfatedfucan isolated from sea cucumber Isostichopus badionotus (fucan-Ib).Methods: Fucan-Ib was hydrolyzed under mild acid conditions. The oligosaccharide fragments were fraction-ated by gel-filtration chromatography and the structures were determined by negative-ion electrospray tan-dem mass spectrometry with collision-induced dissociation and two-dimensional NMR. Anticoagulantactivities were measured by activated partial thromboplastin, thrombin and prothrombin times, and by invitro inhibition experiments with factors IIa and Xa. Antithrombotic activities were determined in vitro bymeasuring the length and weight of the thrombus generated.Result: The linear polysaccharide sequence of fucan-Ib was deduced from the structures of its oligosaccharidefragments produced by acid hydrolysis. Undermild conditions, the glycosidic bonds between the non-sulfatedand 2,4-O-disulfated fucose residues were selectively cleaved and highly ordered oligosaccharide fragmentswith a tetrasaccharide repeating unit [→3Fuc(2S,4S)α1→3Fuc(2S)α1→3Fuc(2S)α1→3Fucα1→]n were
obtained. In in vitro assays fucan-Ib showed good anticoagulant and antithrombotic activities comparedwith heparin and the fucosylated chondroitin sulfate isolated from the same source (fCS-Ib). The two polysac-charides, fucan-Ib and fCS-Ib, differ in themechanism of action; the former exhibited activitymainly by poten-tiation of antithrombin acted on thrombin and factor Xawhereas the latter mainly through heparin cofactor II.Conclusion: Fucan-Ib has a well defined structure with tetrasaccharide tandem repeats and good anticoagu-lant and antithrombotic activities.General importance: Fucan-Ib has a well defined structure and can be readily quality-controlled, and thereforehas potential therapeutic value as an affective antithrombotic drug with low risk of bleeding.
Sea cucumber has been a traditional tonic food in China and otherAsian countries for thousands of years [1–3]. In traditional Chinesemedicine, sea cucumbers are used to treat a variety of symptoms,e.g. high blood pressure in humans and joint pain in pot-bellied pigs[4]. Acidic polysaccharides are the most important bioactive polymersin sea cucumbers. It has been reported recently that the acidic poly-saccharides isolated from sea cucumbers have anticoagulant andantithrombotic activities [5–7], and they can also modulate angiogen-esis [8] and inhibit metastasis of tumor [9]. Therefore, sulfated
e and Technology, Ocean Uni-6003, China.ory, Imperial College Faculty ofw, Middlesex HA1 3UJ, United
polysaccharides have attracted considerable interests in recentlyyears due to their potential therapeutic application [10,11]. Previousstudies indicated that the anticoagulant activity was not merely aconsequence of the charge density and the sulfate content [12–14].The structural requirement for these polysaccharides to interactwith coagulation cofactors and their target proteases are stereospecif-ic. The site of sulfation and the position of the glycosidic linkage areimportant for activities [15].
Two types of polysaccharides have been found in sea cucumbers:fucosylated chondroitin sulfate (fCS) [7,16] and fucan [17,18]. Al-though structures and bioactivities of the fCS have been describedin details [7,15,18,19], there has been limited reports on sea cucum-ber fucans [20,21]. The sequences of fucans from sea cucumbers arerelatively simple [21]; they are linear polysaccharides consisted ofregular tandem repeat, e.g. di-, tri- or tetrasaccharide repeating unit,with defined glycosidic linkages and distinctive sulfation patterns[21]. These features are in sharp contrast to the complex structuresof algae-derived fucans, which are composed of multiple fucose
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(Fuc) branches with different glycosidic linkages and sulfation pat-terns. As the structure and bioactivity studies of sulfated fucanshave been normally carried out with the polysaccharides, therehave been some ambiguities in the structural assignment due mainlyto the difficulties in detailed interpretation of NMR spectra and possi-ble desulfation during methylation linkage analysis of the sulfatedpolysaccharides [20].
Mass spectrometry has become increasingly important for structur-al analysis of carbohydrates, including glycan profiling [22] and oligo-saccharide sequencing [23], due to its high sensitivity, high accuracy,and fast processing compared with NMR and various chromatographicmethods [24]. Mass spectrometry has contributed considerably to thestructural analysis of sulfated oligosaccharides, e.g. using ionic liquidmatrices in matrix-assisted laser desorption/ionization mass spectrom-etry for analysis of sulfated heparin oligosaccharides [24], or usingsodium adducts and multiply charged ions as precursors in negative-ion electrospray ionization with collision-induced dissociation tandemmass spectrometry (ES-CID-MS/MS) [25] for analysis of carrageenanoligosaccharides. However, despite these advances, sequence assign-ment of highly-sulfated oligosaccharides by mass spectrometryremains a challenge area, especially for those derived from sulfatedfucans.
Detailed knowledge of the polysaccharide structure is importantfor better understanding of their biological roles. However, the highsulfate content poses a major challenge in sequence analysis andassignment of structure–function specificities of sulfated fucans.Traditionally, sequences of sulfated fucans were deduced from NMRspectroscopy of the polysaccharide and methylation linkage analysisof the monosaccharide components. For unambiguous sequence as-signment and for testing the activities in order to derive thestructure-function specificities it is important to prepare oligosaccha-ride fragments. Unfortunately, specific fucan-degrading enzymeshave not been generally available. Acid hydrolysis has been widelyused for partial depolymerization of fucan polysaccharides. However,acid hydrolysis is generally non-specific and it can also destroy someof the sulfates. Oligosaccharides thus obtained are complex and ex-tremely difficult to purify. Recently, in the structural characterizationof the linear sulfated fucan from the sea urchin Lytechinus variegates,Mourao and colleagues [26] were able to show, unexpectedly, thatunder mild conditions the apparently non-specific acid hydrolysis se-lectively removed a 2-O-sulfate from a mono-sulfated Fuc residue fol-lowed by preferential cleavage of the glycosidic bond between thisnewly created non-sulfated and the adjacent 2,4-O-disulfated Fuc res-idues. The hydrolysate containing the oligosaccharide mixture wasanalyzed directly by NMR to derive the sequence of the polysaccha-ride and the mechanism of the hydrolytic cleavage.
In the present study, prompted by the interesting work of acid hy-drolysis [26], we employed the mild conditions to cleave a sulfatedfucan isolated from the sea cucumber Isostichopus badionotus (fucan-Ib). The highly sulfated oligosaccharide fragments were isolated andtheir sequences were characterized by a unique ES-CID-MS/MS strat-egy together with 2D NMR. The structure of the polysaccharide wasunambiguously assigned and the mechanism of cleavage was pro-posed which was somewhat different from the previously described[26]. We further assessed the antithrombotic and anticoagulant activ-ities of fucan-Ib, including activated partial thromboplastin time(APTT) and thrombin time (TT), inhibition of thrombin (F.IIa) by hep-arin cofactor II (HC II) and antithrombin (AT), factor X (F.Xa) activa-tion by AT, were examined to investigate the mechanism of action.
2. Experimental Section
2.1. Materials
Dry sea cucumber I. badionotuswaspurchased from a localmarket inQingdao (China). Gel-filtration columns TSK G4000PWXL and G3000
PWXL were from TOSOH BIOSEP (Tokyo, Japan). DEAE ion-exchangeresin was fromWhatman (Brentford, England). Monosaccharides stan-dards and disaccharide lactose were purchased from Sigma (St. Louis,Missouri, USA). The derivatization reagent 1-phenyl-3-methyl-5-pyrazolone (PMP) was from Sinopharm Chemical Reagent (Shanghai,China). Low molecular weight heparin (LMWH) with an average MWof 4,000 Dawas prepared in our laboratory andheparinwas from JiangsuPharmacia (Jiangshu, China). The colorimetric substrates for F.IIa, F.Xa,AT, HC II and F.IIa were purchased from Calbiochem (Darmstadt,Germany) and that for F.Xa was from Sigma (St. Louis, MO, USA).
2.2. Isolation, purification and chemical composition analysis of fucan-Ib
Sulfated fucan was extracted from I. badionotus after papain diges-tion as previously described with some revision [12]. The crude poly-saccharide participated by cetylpyridinium chloride was dissolved ina solution of 2 M NaC1: ethanol (100:15, v/v). Additional ethanolwas added to a final concentration of 40% to precipitate fCS, whichwas purified using anion-exchange chromatography as describedpreviously [15]. After centrifugation (2000×g, 15 min) and removalof the precipitate, ethanol was added to the supernatant to a finalconcentration of 60%. The precipitate formed was collected by centri-fugation (2000×g, 15 min) and dissolved in water before dialysisagainst water for 24 h. The retained solution was lyophilized andcrude fucan was obtained.
The crude fucan was purified by anion-exchange chromatographyas previously described [15] (Supplementary Fig. 1). The fractionswhich showed single peaks on HPLC were pooled for further investi-gation. The polysaccharide collected, named as fucan-Ib, was withhigh viscosity, and the average molecular mass of fucan was deter-mined to be 450 kDa by gel filtration chromatography on a TSKG4000PWXL column.
Monosaccharide composition of fucan-Ibwas determined by HPLCafter derivatization with PMP [28], and sulfate content was deter-mined by ion-chromatography [29]. Fucose was the only monosac-charide detected. The sulfate content was determined to be about32.9%, and the molar ratio of Fuc to sulfates was 1.00 to 0.92, the pro-tein content was determined to be 7.2%.
2.3. Mild acid hydrolysis of fucan-Ib
For partial depolymerization of the polysaccharide fucan-Ib, mildacid hydrolysis was carried out with 0.05 M H2SO4 at 60 °C for4–9 h. The hydrolytic products were analyzed by polyacrylamide gelelectrophoresis (22%), and by HPLC with a TSK 3000 PWXL columnfor determinnation of the molecular weight distribution. The oligo-saccharide mixture was fractionated by gel filtration chromatographyon a Bio-Gel P-4 column (2.6×120 cm) eluted with 0.3 M NH4HCO3
at a flow rate of 0.3 mL/min, and monitored by refractive index. Thepooled oligosaccharide fractions were lyophilized.
2.4. Negative-ion ES-MS and ES-CID-MS/MS
Negative-ion ES-MS and CID-MS/MS were carried out on a WatersUltima mass spectrometer (Manchester, UK) with a Q-TOF configura-tion. Nitrogen was used as desolvation and nebulizer gas at a flowrate of 250 L/h and 150 L/h, respectively. Source temperature was80 °C, and the desolvation temperature 150 °C. A cone voltage of60–150 V was used for negative-ion detection, and the capillary volt-age was maintained at 3 kV. Product-ion spectra were obtained fromCID using argon as the collision gas at a pressure of 0.17 mPa. The col-lision energy was adjusted between 20 and 36 V for optimal fragmen-tation. A scan rate of 1.0 s/scan was used for both ES-MS and CID-MS/MS experiments, and the acquired spectra were summed for presen-tation. For analysis, oligosaccharides were dissolved in acetonitrile/water (1:1, v/v), typically at a concentration of 20 pmol/μL, of which
Fig. 1. Polyacrylamide gel electrophoretograms of the fucan-Ib hydrolytic products. Theproducts formed in the course of the mild acid hydrolysis were analyzed at different in-tervals with a 22% gel.
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5 μL was loop-injected. Solvent (acetonitrile/1 mM ammonium bicar-bonate, 1:1, v/v) was delivered by a Harvard syringe pump (HarvardApparatus, Holliston, MA, USA) at a flow rate of 10 μL/ min.
2.5. NMR spectroscopy
For NMR analysis, the polysaccharide (50 mg) and oligosaccharidefractions (10 mg) were co-evaporated with D2O (99.8%) twice by ly-ophilization before final dissolution in 500 μL high-quality D2O(99.96%), containing 0.1 μL acetone. 1H-NMR experiments were car-ried out at 600 MHz and 13 C-NMR at 150 MHz. Spectra were recordedat 25 °C for 13 C-NMR , and at 25 °C and 60 °C for 1H-NMR. The temper-atures were chosen in order to place the HDO signals with minimaldisturbance to saccharide protons. The observed 1H chemical shiftswere reported relative to internal acetone (2.23 ppm). COSY, HMBCand HMQC experiments were also carried out at 25 °C.
2.6. Anticoagulant assays
Healthy human blood was donated by a local young man aged24 years old and the investigation was performed according to the“Guidance for the Use of Human Blood” published by ShandongProvincial Government. Human blood was added into an aqueous solu-tion of 3.8% sodium citrate. The plasmawas separated by centrifugationat 3,000 rpm for 10 min. The anticoagulant assays, including APTT(assay kit from Organon-Tecknica, Fresnes, France), TT (5 NIH U/mlhuman thrombin, from Diagnostica Stago, AsnieAres, France) andprothrombin time (PT) with thromboplastin (from Diagnostica Stago,AsnieAres, France), were performed according to the manufacturer'srecommended procedures. The results were expressed as internationalunits/mg using a parallel standard curve based on the InternationalHeparin Standard (150 units/ mg).
2.7. Inhibition of F.IIa or F.Xa mediated by AT and HC II
The reactions were carried out in a 384-well micro-titer plate asdescribed [30]. The reactant solutions included AT (10 nmol/L) orHC II (30 nmol/L) and a polysaccharide (fucan-Ib, fCS-Ib or the stan-dard heparin) at different concentrations in 40 μL of TS/PEG buffer(0.02 M Tris/HCl, 0.15 M NaCl and 1.0 mg/mL PEG 8,000, pH 7.4).F.IIa or F.Xa (2 nmol/L) was added to initiate the reaction. After incu-bation at 37 °C for 60 s, 25 μL of TS/PEG buffer containing 0.4 mmol/Lcolorimetric substrate of F.IIa or F.Xa was added and the absorbanceat 405 nm was measured at an interval of 30 seconds within a periodof 300 s in a micro-plate reader. The absorbance change rate was pro-portional to the F.IIa or F.Xa activity remaining in the incubation mix-tures. Heparin was used as a control and the experimental resultswere expressed as the percent of control (n=3).
2.8. In vitro anti-thrombosis activity assays
Male Sprague–Dawley rats (Vital River Laboratories, Beijing, China)weighing 200–300 g were used for animal experiments. Animals wereinitially acclimated for 1 week, and food and water were provided adli-bitum. The protocols used for the experiments involving animals wereapproved by the Ethics Committee of the University Animal ServiceCenter. Rats were anesthetized by intraperitoneal injection of 20%ethyl p-aminobenzoate before intravenous administration of polysac-charides: fucan-Ib (at 0.50 mg/kg and 1.00 mg/kg), fCS-Ib (at 0.30 mg/kg and 0.50 mg/kg), standard heparin (at 0.30 mg/kg) or LWMH (at0.30 mg/kg and 0.50 mg/kg). One hour after injection of polysaccha-rides, blood was collected from abdominal aorta and anticoagulantedby 3.8% trisodium citrate solution (1:9 citrate/blood, v/v).
The in vitro thrombosis formation was measured in a thrombosismeasurement equipment [31]. Briefly, 1 mL of the blood sample wasinjected into the artificial blood vessel in a preheated bath at 37 °C
and the vessel was rotated for 10 min. The thrombus generated inthe artificial blood vessel was carefully pool out in a filter paper andthe length of thrombus was measured. The wet thrombus was driedat 60 °C for 1 h and weighed. Mean thrombus length and weightwas calculated by averaging 10 rats in each group.
3. Results and Discussion
3.1. Preparation of oligosaccharide fragments by mild acid hydrolysis
Mild acid hydrolysis (0.05 M H2SO4 at 60 °C) was employed forpartial depolymerization of fucan-Ib. Preliminary results indicatedthat mild acid gradually reduced the molecular size of fucan-Ib asshown by PAGE analysis which revealed a series of sharp bands(Fig. 1). As the time of hydrolysis proceeded from 4 to 9 h, the propor-tions of bands with higher electrophoretic mobility increased and thesame well-defined pattern of sharp bands was maintained (Fig. 1), in-dicative of preferential cleavage of a specific glycosidic linkage andpossible presence of an oligosaccharide repeating unit.
For further detailed structural analysis, optimized conditions(0.05 M H2SO4, 60 °C and 9 h) were selected for large scale prepara-tion of oligosaccharides. The hydrolytic product was fractionated bygel chromatography on a Bio-Gel P-4 column (Fig. 2), and fivemajor fractions (OF-I to OF-V) were obtained. As OF-I was collectedat the void volume and contained multiple components, it was notsubjected to detailed structural analysis. The major fractions OF-II,-III and -IV were shown to contain sulfated fuco-dodeca-, octa- andtetrasaccharides, respectively, by negative-ion ES-MS (Table 1), withthe formula of (4Fuc.4SO3)n where n=1–3. This unambiguously indi-cated that a tetrasaccharide repeating unit was present in fucan-Ib,and mild acid acted on a specific glycosidic linkage of the polysaccha-ride chain and produced highly ordered oligosaccharide fragments.
Although all the main fractions of the hydrolysate contained mul-tiplies of the intact tetrasaccharide repeating unit, some minor com-ponents (totaling b25%) with losses of fucose residues or sulfateswere also detected (Table 1), e.g. fractions OF-IIIa, OF-IIIb and OF-IIIc were the hepta-, hexa-, and pentasaccharide, respectively, withdiffering numbers of sulfates (Table 1). These were presumably thedegraded products of the octasaccharide OF-III. OF-IVa and OF-IVbwere both tetrasaccharides but with three and two sulfates, respec-tively, which were likely derived from the tetrasaccharide OF-IV.The cleaved sulfated fucose was eluted in the fraction OF-V (Fig. 2and Table 1).
Fig. 2. Fractionation of fucan-Ib oligosaccharides by gel filtration chromatography. Oli-gosaccharide fragments were obtained by partial depolymerization of fucan-Ib with0.05 M H2SO4, fractionated on a Bio-Gel P4 column (1.6×100) with elution by 0.2 MNH4HCO3 at a flow rate of 18 mL/h and detection by RI.
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3.2. Sequence determination of the tetrasaccharide fragment OF-IV byES-CID-MS/MS
The sequence of the tetrasaccharide OF-IV was established initiallyby negative-ion ES-CID-MS/MS. Due to the lability of sulfate groups,ions generated from sulfate loss dominated the tandem mass spectraof multiply sulfated oligosaccharides and detailed structural informa-tion was rather limited [32]. It has been reported that sodiated [21]and multiply charged molecular ions [33,34] are generally more sta-ble than the singly charged free acid form. We here used a strategyof multi-step ES-CID-MS/MS using different precursors to derive thesequence of the tetra-sulfated tetrasaccharide OF-IV: 1) singlycharged ion of mono-sulfated molecule [M−3SO3−H]–, producedfrom partial desulfation during ionization, for sequence and fucoselinkage analyses; 2) [M−2SO3−H]– and [M−SO3−3H+2Na]– for lo-cation of the additional two sulfates; and 3) the doubly charged ion ofthe fully sulfated and sodiated molecule, [M−4H+2Na]2–, for assign-ment of the position of the remaining sulfate.
The partially desulfated fragment ion [M−3SO3−H]– (m/z 681) inthe ES-MS spectrum of OF-IV (Supplementary Fig. 2) was initially se-lected as the precursor for CID-MS/MS experiment. As shown inFig. 3a, a simple product-ion spectrumwas obtained. Full set of glyco-sidic fragment ions were present: B1/C1 at m/z 225/243, B2/C2 at m/z371/389, and B3/C3 at m/z 517/535 [35], which unambiguously iden-tified a linear tetrasaccharide sequence with a sulfate at the non-reducing terminus. Saccharide ring 1,4A cleavage also occurred andthis is characteristic of a 3-linkage for Fuc residue [36]. Three consec-utive 1,4A-ions (1,4A2 at m/z 315, 1,4A3 at m/z 461, and 1,4A4 at m/z607, Fig. 3a) indicated that all the glycosidic linkages are 3-linkedand a linear mono-sulfated fuco-tetrasaccharide was obtained (S: sul-fate group, SO3H):
FucðSÞ1→3Fuc1→3Fuc1→3Fuc
The di-sulfated fragment ion [M−2SO3−H]− was then selected asthe precursor (Fig. 3b) to assign the location of the second sulfate. The
Table 1Negative-ion ES-MS of oligosaccharide fractions obtained from sea cucumber fucan.
⁎: ‘-’, not detected (relative intensitiesb5%); ‘S’, sulfate group.
glycosidic ions B1/C1 at m/z 327/345 clearly identified disulfation atthe non-reducing terminal fucose and the further glycosidc ions (B2/C2 at m/z 473/491, and B3/C3 at m/z 619/637) indicated that theother three fucose residues were non-sulfated. The 1,4A-type ions(m/z 417, m/z 563 and m/z 709) also confirmed the linear and 3-linked fucose sequence:
FucðS2Þ1→3Fuc1→3Fuc1→3Fuc
We further examined the product-ion spectrum of the singlycharged mono-desulfated molecule of the tetrasaccharide OF-IV assodiated ion, [M−SO3−3H+2Na]− at m/z 885 (Fig. 3c). Similarly,the B1/C1 ions at m/z 327/345 indicated a disulfated fucose at thenon-reducing terminus. The mass difference of 248 Da (146+80+22: Fuc+Sulfate+Na) between B2/C2 (m/z 473/491) and B3/C3
(m/z 721/739) indicated that the third sulfate was at the residuenext to the reducing terminal fucose. The unique 1,4A ions (m/z 417,m/z 563 and m/z 811, Fig. 3c) again confirmed the 3-glycosidic link-age of fucose. The sequence of the trisulfated tetrasaccharide frag-ment can therefore be assigned as:
FucðS2Þ1→3Fuc1→3FucðSÞ1→3Fuc
Finally, we used the doubly charged ion of the fully sulfated andsodiated molecule, [M−4H+2Na]2– at m/z 482, as the precursor forCID-MS/MS to assign the remaining sulfate, as doubly chargedsodiated ions are generally more stable and extensive desulfationcan be largely prevented. In the product-ion spectrum (Fig. 3d), theglycosidic cleavage ions B1/C1 at m/z 327/345 indicated two sulfatesat the non-reducing end, and B2/C2 at m/z 575/593 indicated thatthe non-reducing sub-terminal residue was mono-sulfated. Loss of asulfate from B3/C3 (B3/C3−S: m/z 721/739) prevented further assign-ment of the location of the fourth sulfate and a complete assignmentof the sulfation pattern from a single product-ion spectrum was notpossible. However, taking the information obtained from the threeMS/MS spectra described above, the sequence of the OF-IV can beassigned as:
FucðS2Þ1→3FucðSÞ1→3FucðSÞ1→3Fuc
3.3. Complete structural assignment of the fucan-Ib oligosaccharidesby NMR
After the major structural features were established, NMR wasused to corroborate the sequence assignment by mass spectrometryand to further complete the structural characterization by determina-tion of the anomeric configurations of the Fuc residues and the posi-tions of sulfate substitutions.
NMR experiments of OF-IV were carried out first. In the COSYspectrum of OF-IV (Fig. 4a) three narrow doublet peaks were
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observed at 5.22 ppm (J=3.92, A-α), 5.26 ppm (J=3.60, B-α) and5.20 ppm (J=3.81, C-α), which were attributed to the anomeric pro-tons of threeα-configurations of the fucose residues A, B, and C (Fig. 4and Table 2). The anomeric peaks at 5.09 ppm (J=3.22, D-α) and4.43 ppm (J=6.84, D-β), with a ratio of 1:2, were from the reducingterminal residue D. The full assignments of other signals of OF-IVwere shown in Table 2, assisted by HMQC (Supplementary Fig. 3a),TOCSY (Supplementary Fig. 3b). Sulfate substitutions were assignedby careful comparison of each proton chemical shift of the Fuc resi-dues in OF-IV with that of a standard monosaccharide fucose. The po-sition of sulfate was deduced from the apparent down-field shiftbetween 0.3-1.0 ppm (Table 2). H-2 (4.30 ppm) and H-4 (4.71 ppm)of residue A were all had down-field shift compared with H-2(4.00 ppm) and H-4 (3.96 ppm) of the standard non-sulfated fucose,indicating a 2,4-disulfation of residue A. Similarly, the H-2 of residuesB and C (4.42 ppm and 4.33 ppm, respectively) also had down-fieldshifts and the two residues when were all 2-O-sulfated. HMBC(Fig. 4b) was used to assign the glycosdic linkages. We could find cor-relation signals indicating unit C linked to the C-3 position of unit B,unit B linked to the C-3 of unit A as well as unit A linked to the
(a)
OCH3
OCH3
O
O
HO3S
225 243
389
B1C1
1,4A2
1,4A3
371
315
B2 C2
461
0
100
%225
243389
B1
C1
1,4A2
1,4A3
371
315
B2
C2461
(b)
OCH3
SO3H
OCH3
O
HO3S
327345B1 C1
1,4A2
417
0
100
%
225
4
B1-S
371B2-S
C327
B1
345C1
247
B1-S+Na1,4A2
417
473
B2
B2-S+Na
393
200 250 300 350 400 450
150 200 250 300 350 400 450
Fig. 3. Negative-ion ES-CID-MS/MS product-ion spectra of tetrasaccharide OF-IV using (a)cursors. Structures are shown to indicate the proposed fragmentation. The nomenclaturesents sulfate.
C-3unit D (D (α) and D’(β). The complete structure of the OF-IVwas thus identified as:
Fucð2S; 4SÞα1→3Fucð2SÞα1→3Fucð2SÞα1→3Fucα;β
Further 2D-NMR analyses of the octa- and dodecasaccharide frac-tions, OF-III and OF-II, respectively, confirmed the tetrasaccharide se-quence as a repeating unit of the polysaccharide. Both fractions gavesimilar COSY spectra (Fig. 5) as that of OF-IV (Fig. 4a). For example,the COSY spectrum of OF-III showed cross-peaks protons H-1 to H-4for the fucose residues (Fig. 5a). Together with TOCSY (Supplementa-ry Fig. 4a) and HMQC (Supplementary Fig. 4b), the chemical shifts ofOF-III were assigned as shown in Table 3. Similarly, the chemicalshifts of OF-II (Supplementary Table 1) were assigned using COSY(Fig. 5b), TOCSY and HMQC (data not shown). The positions of sulfateof the two fraction were also deduced from the down-field shifts oftheir proton signals compared with the standard fucose (Table 3and Supplementary Table 1). The structures of OF-III and OF-II weretherefore identified as octa- and dodecasaccharides, containing twoand three tetrasaccharide repeating units, respectively.
OCH3
O
OCH3
OH
517 535B3 C3
1,4A4
607
m/z
517
535
B3
C3
1,4A4
607[M-3S-H]-
681
O
OCH3
O
OCH3
OH
619 637
491B3 C3
1,4A3
1,4A4
473B2 C2
563709
m/z
[M-2S-H]-
619
63791
B3
C31,4A42
709
1,4A3
563 761
500 550 600 650 700 750
500 550 600 650 700 750 800
[M-H-3SO3]–, (b) [M-H-2SO3]–, (c) [M-H-SO3]–, and (d) [M−4H+2Na]2– as the pre-used to define the cleavage is based on that introduced previously36, and “S” repre-
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Taken together, the polysaccharide can be deduced to contain anovel sequence with the tetrasaccharide repeating unit:
½→3Fucð2S; 4SÞα1→3Fucð2SÞα1→3Fucð2SÞα1→3Fucα1→�n:
3.4. Comparison of the structural features of the intact polysaccharideand the oligosaccharide fragments by NMR
To ensure that the acid hydrolysis conditions employed for partialdepolymerization did not alter the structural features and the oligo-saccharide fragments obtained had the same repeating sequence asthe parent fucan-Ib, 1H-NMR spectra of fucan-Ib and the oligosaccha-ride fractions were compared. The spectrum of fucan-Ib showed fouranomeric signals (Fig. 6a) which were assigned to residues A–Dshown in Fig. 6. The spectra of oligosaccharides OF-II, -III and -IV(Fig. 6b, c and d) showed an overall similarities as that of the parentfucan polysaccharide (Fig. 6a). The main difference was the anomericsignal of unit D. In the polysaccharide this was a broad peak at4.90 ppm. From OF-II to OF-IV the intensity of this peak was de-creased and new anomeric signals at 5.09 ppm (D'α) and 4.43 ppm
(D'β), in a ration of 1:2, appeared. The signal of these two newanomeric signals increased as the DP of oligosaccharides decreased,indicating that residue D became a reducing terminius as previouslyindentified in OF-IV, -III and -II. The results demonstrated that duringhydrolysis the acid preferentially cleaved the glycosidic bond be-tween the nonsulfated (residue D, Fig. 6) and 2,4-O-disulfated resi-dues (residue A, Fig. 6) without apparent alteration of the structuralfeature and that the tetrasaccharide repeating unit was indeed thestructural feature of fucan-Ib.
3.5. Anticoagulant activities
Anticoagulant activities of fucan-Ib were assessed by APTT and TTassays, and compared with fCS-Ib, the other major polysaccharidepresent in sea cucumber I. badionotus, and with standard heparin.The result showed that ATPP (9 IU/mg) and TT (6 IU/mg) of fucan-Ibwere much lower than fCS-Ib (183 IU/mg in APTT and 157 IU/mg inTT, Table 4) and standard heparin (150 IU/mg in APTT and 150 IU/mg in TT, Table 4), but a little higher than the fucan from Ludwi-gothurea grisea [27] in APTT (2 IU/mg).
O
HO3SOHO3SO
HO
CH3
O
HO3SOHO
O
CH3
O
HO3SOHO
O
CH3
O
HOHOOH
O
CH3
A
B
C
D'
(a)
(b)
Fig. 4. 2D-NMR spectra of tetrasaccharide OF-IV. Oligosaccharide OF-IV (10 mg) was used to obtain the COSY (a) and HMBC (b) spectra. Experiment was carried out at 25 °C and the1H chemical shifts were reported relative to internal acetone (2.03 ppm).
995S. Chen et al. / Biochimica et Biophysica Acta 1820 (2012) 989–1000
On the basis of the in vitro assays, we further investigated theinfluence of the fucan-Ib on F.IIa/ F.Xa inactivation by AT/HC II,in comparison with unfractionated heparin. In the anti-F-IIa/ F.Xaexperiments mediated by AT (Fig. 7a and c), increased concentra-tions of fucan-Ib lead to nearly complete inhibition of F.IIa andF.Xa activation by AT, at a concentration of 100–250 μg/ml nearly100% F.IIa used in assays was inhibited. The concentrations ofhalf inhibition of F.IIa and F.Xa (EC50) were 0.32 μg/ml and0.53 μg/ml for fucan-Ib, respectively (Table 4). Higher EC50 were
required for fCS-Ib , which were 0.56 μg/ml and 2.58 μg/ml forF.IIa and F.Xa mediated by AT (Table 4), respectively. These resultindicated fucan-Ib may have better effect than fCS-Ib on F.IIa andF.Xa inactivation when mediated by AT. These results were verysimilar to those obtained with the fucan from sea cucumber L.grisea [27]. No inhibition occurred in control experiments inwhich F.IIa or F.Xa was incubated with AT or HC II in the absenceof both polysaccharides, no matter whether the target proteasewas F.IIa or F.Xa.
Table 21H and 13 C chemical shifts and the coupling constants of H1 and H2 in the 1D and 2DNMR spectra of tetrasaccharide OF-IV.
⁎Residues A, B, C and D were defined in Fig. 6; ⁎⁎ The chemical shifts highlighted in boldwere down-field shifted, compared with the 1H chemical shifts of the standard non-sulfated fucose, and therefore were the positions of sulfation; ⁎⁎⁎ The chemical shifts ofFuc0Swere froma standard non-sulfatedmonosaccharide fucose, cited from literature (16).
Fig. 5. COSY spectra of octasaccharide OF-III (a) and dodecasaccharides OF-II (b
996 S. Chen et al. / Biochimica et Biophysica Acta 1820 (2012) 989–1000
In the anti-F.IIa experiment mediated by HC II (Fig. 7b) was alsoconcentration dependent. Increase concentrations of the fucan-Ibcan also result in essentially higher percentage inhibition of F.IIa acti-vation, but the inhibitory effect wasmuch lower thanmediated by AT.At a concentration of 10 μg/ml, it can only inhibit about 60% of F.IIaactivation. The EC50 for F.IIa activation by HC II was 2.55 μg/ml forfucan-Ib (Table 4), which was much higher than that of fCS-Ib(0.05 μg/ml). This indicated fCS-Ib achieved inactivation of F.IIa main-ly by HC II, whereas fucan-Ib was mainly by AT.
3.6. Antithrombotic activities
The anti-thrombotic activity of fucan-Ib was measured in an invitro experimental and was compared with that of fCS-Ib, heparinand LMWH. As showed in Table 5, although sulfated fucan-Ib hadlower anticoagulant activity than that of fCS-Ib, it reduced the forma-tion of thrombus by 63% at a dose of 0.5 mg/kg, slightly less effective
). 1H chemical shifts were reported relative to internal acetone (2.03 ppm).
Table 31H and 13 C chemical shifts and coupling constants of H1 and H2 in the 1D and 2D NMRNMR spectra of octasaccharide OF-III.
⁎Residues A, B, C and D were defined in Fig. 6; ⁎⁎ The chemical shifts highlighted inbold were down-field shifted, compared with the 1H chemical shifts of the standardnon-sulfated fucose (Table 2), and therefore were the positions of sulfation.
O
HO3SOHO3SO
O
CH3
O
HO3SOHO
O
CH3
HOHO
O
CH3
A
B
Fig. 6. Comparison of 1H NMR spectra of the fucan-Ib polysaccharide (a)
Table 4Anticoagulant properties of sulfated polysaccharides isolated from sea cucumbers I.badionotus.
⁎The activity is expressed as international units/mg using a parallel standard curvebased on the International Heparin Standard (150 IU/mg); ⁎ ⁎ “Anti-F.IIa/HCII” meanseffect of fCSs on generation of thrombin by HCII; “Anti-F.IIa/AT” means generation ofthrombin by AT. “Anti-F.Xa/AT” means generation of F.Xa by AT.
997S. Chen et al. / Biochimica et Biophysica Acta 1820 (2012) 989–1000
than fCS-Ib (70% inhibition at the same dose). When compared withLMWH, the dose at 0.5 mg/kg of fucan gave a similar effect asLMWH at a dose of 0.5 mg/kg. There was no thrombus formed afterinjection of heparin (0.3 mg/kg). There was no apparent differenceon the length of the generated thrombus in fucan-Ib, fCS-Ib andLWMH treated rats.
O
3SO
O
HOHOO
O
CH3
C
D
with its oligosaccharide fractions OF-II (b), OF-III (c) and OF-IV (d).
Fig. 7. Effect of fucan-Ib and fCS-Ib on inhibition of F.IIa and F.Xa activities. (a) F.IIa/AT;(b) F.IIa/HC II; (c) F.Xa/AT. AT (10 nmol/L) or HC II (30 nmol/L) were incubated withF.IIa or F.Xa (2 nmol/L each) in the presence of sulfated polysaccharides (fucan-Ib:×;fCS-Ib: ■; heparin:◆) at various concentrations. After 60 s of incubation at 37 °C, theremaining F.IIa or F.Xa was determined with a chromogenic substrate (A405 nm/min). Results were expressed as mean±SD (n=3/group).
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4. Discussion
In the present study, a novel sulfated fucan with a repeating tet-rasaccharide unit→3Fuc(2S,4S)α1→3Fuc(2S)α1→3Fuc(2S)α1→3Fucα1→was isolated from the sea cucumber I. badionotus. The se-quence is different from the previously reported fucans isolated fromthe sea cucumbers S. japonicus and L. grisea [16,17], either in the sulfatecontent or the sulfation pattern of the repeating unit. The structure wasestablished using a unique strategy based on negative-ion ES-CID-MS/MS and NMR of the oligosaccharide fragments obtained after mild acidhydrolysis of the polysaccharide. Conventionally, the sequences ofthese sulfated polysaccharides are assigned by a combination of NMRof the polysaccharides and GC-MS methylation analysis of the mono-saccharide components. Due to the heterogeneity and complexity ofeach polysaccharide structure, some ambiguities in spectral interpreta-tion may occur and can lead to incorrect structural assignment. The labil-ity of the sulfate groups can also result in sulfate loss during methylationanalysis procedure. The advantage to use properly prepared oligosaccha-ride fragments are two folds: firstly, in NMR analysis better spectral reso-lution can be obtained and assignment of individual signals can be morereadily achievable; secondly, the advanced mass spectrometric method-ologies developed recently for oligosaccharide sequencing can beemployed. In the present study, the negative-ion ES-CID-MS/MS wasused and by selecting different ions as precursors the sequence of thetetra-sulfated tetrasaccharide was derived. The unique 1,4A-type frag-mentation was important to establish the 3-linked fucose in the polysac-charide backbone and the position of the sulfation.
Interestingly, in the attempt to obtain the fucan oligosaccharides,the apparently non-specific acid hydrolysis, prompted by the workreported byMourao and colleagues(26), produced oligosaccharide frac-tions with ordered sequences. The mild acid conditions preferentiallycleaved the 3-linked glycosidic bond between the nonsulfated residue(D in Fig. 5) and the 2,4-O-disulfated residue (A in Fig. 5), producing ol-igosaccharides with varying numbers of the tetrasaccharide repeatingunit. The reactionwas carried out with 0.05 MH2SO4 at 60 °C. Althoughthe conditionwas stronger than that employed in the literature [26,37],no apparent sulfate loss or sequence alteration was observed. Thesewere different from the results obtained with mild acid hydrolysis offucans from sea urchin L. variegates [25] and Strongylocentrotus pallidus(37). In the previous work, different repeating tetrasaccharides,→3Fuc(2S)α1→3Fuc(2S)α1→3Fuc(4S)α1→3Fuc(2S,4S)α1→(25)and→3Fuc(2S)α1→3Fuc(2S)α1→3Fuc(4S)α1→3Fuc(4S)α1→ [37],were obtained (See Scheme 1). The cleavage by the acid conditions used(0.01 MHCl at 60 °C) were different. In the publishedwork, a 2-O-sulfatewas initially removed from a monosulfated fucose before selective cleav-age of the glycosidic bond between the newly created nonsulfated Fucand the adjacent reducing side 4-O-sulfated residue [26,37]. However,in the present work, based on the detailed structural analysis of the oligo-saccharide fragments, we did not observe removal of 2-O-sulfate groupand the preferential cleavage was at the glycosidic bond between thenonsulfated and the 2,4-O-disulfated Fuc residues (Scheme 1).
Table 5In vitro antithrombotic activities of sulfated polysaccharides isolated from sea cucum-bers I. badionotus.
I. badionotus--2S---2S---0S---24S-- --2S---2S---0S 24S--
de-2S cleavage
X
X
X
--2S---0S 4S---4S--
--[4S---24S---2S---0S] --n
--[4S---4S---2S---0S] --n
--[24S---2S---2S---0S] --n
X
?
X
X
X
Scheme 1. Mild acid hydrolysis of 3-linked linear sulfated fucan polysaccharides.
999S. Chen et al. / Biochimica et Biophysica Acta 1820 (2012) 989–1000
The difference in the results may indicate that the removal of thelabile sulfate can be prevented by using H2SO4 instead of HCl or re-quire specific sulfation partterns. The latter may also be importantfor the cleavage of the 3-linked glycosidic bond of the non-sulfatedFuc residues. Mourao and coworkers [37] considered that a 2-O-sul-fate is labile compared with 4-O-sulfate and can be removed bymild acid hydrolysis if located next to a 4-O-sulfated Fuc. In the pre-sent case there is no 4-O-sulfated Fuc adjacent to the two 2-O-sulfat-ed residues and this may explain the reason for the result obtainedhere, in which none of the two 2-O-sulfates was removed. In themechanism proposed by Mourao et al. [37], a 3-linked glycosidicbond between a nonsulfated and a 4-O-sulfated Fuc residues can bereadily cleaved. In fucan-Ib the cleavage takes place at a glycosidicbond between a nonsulfated and a 2,4-O-disulfated Fuc residues(Scheme 1). It is also interesting to note that a tetrasaccharide tan-dem repeating unit of a sulfated fucan isolated from sea cucumberLeptopentacta grisea is very similar to the tetrasaccharide unitobtained here but the order of the nonsulfated and the 2,4-O-disul-fated Fuc residues was reversed and no glycosidic cleavage has beenobserved [37]. Therefore, our observation extended the conclusionon the sulfation pattern required for cleavage. The cleavage can takeplace at a linkage between a nonsulfated and a 4-O-mono- or 2,4-O-disulfated Fuc residues and the 2,4-O-disulfated Fuc has to be at thereducing side (Scheme 1). It has been recently observed that someapparently non-specific chemical hydrolysis methods can lead to spe-cific cleavage of polysaccharides under carefully controlled condi-tions. For example, under mild acid hydrolysis conditions sulfatedgalactan polysaccharides, which contain alternating α1-3 and β1-4glycosidic bonds, can be cleaved at preferentially the α1-3 bond togive even-numbered oligosaccharide fragments followed by immedi-ate removal of the newly created unstable 3,6-anhydro-galactose atthe reducing end to give odd-numbered galactan oligosaccharides[38]. Therefore, specificities of various mild acid hydrolysis conditionsfor marine-derived polysaccharides and their wide applicability de-serve further re-investigation.
The in vitro anticoagulant and antithrombotic activities of thefucan-Ib were assessed and compared with fSC-Ib. Although fromthe same source, the two sulfated polysaccharides had some differ-ences in activities. Our result showed that the fucan-Ib had muchlower anticoagulant activity whereas only slight lower antithrombo-tic activity compared with fSC-Ib. Their anticoagulant mechanismswere also different. The anticoagulant activity of fucan-Ib wasthrough potentiation of AT activity on F.IIa and F.Xa whereas the ac-tivity of the fCS-Ib was primarily due to potentiation of HC II on F.IIaand F.Xa. These results were very similar to those of fucan and fCSobtained from the sea cucumber L. grisea [27], although some struc-ture features of the sulfated polysaccharides were different. As hasbeen well documented previously [39], some sulfated polysaccha-rides, particularly those isolated from marine invertebrates, exhibitanticoagulant and antithrombotic activities similarly to heparin. Clas-sically, potentiation of antithrombin activity by sulfated polysaccharide
requires a specific heparin pentasaccharide sequence. However, thispentasaccharide may not be the only sulfated polysaccharide motif tointeract with high affinity with antithrombin. The negatively chargedgroups present in other sulfated polysaccharide may have the requiredthree dimensional structure able to interact with the specific positivelycharged amino groups of Lys and Arg residues non-contiguously dis-tributed along the antithrombin polypeptide chain.
5. Conclusion
Our research indicated that fucan-Ib had high antithrombotic ac-tivity but low anticoagulant activity, indicating that fucan-Ib is agood candidate as a highly potent antithrombotic drug with verylow risk of bleeding. Furthermore, the widely used antithromboticdrug heparin and LMWH or the well documented fucoidan fromalgae all contain very heterogeneous components with complexstructures, which pose considerable difficulty in quality control fortheir clinical use in humans as highlighted in the recent incident ofheparin contamination [40]. Fucan-Ib has a well defined structurewith tetrasaccharide repeating unit and can be readily quality-controlled. Therefore, the therapeutic potential of fucan-Ib deservesfurther investigation for its clinical application as an antithromboticdrug candidate.
Acknowledgements
This work was supported by the International Science & Technolo-gy Cooperation Program of China (2010DFA31330), the Marine Scien-tific Research and Specific Public Service Project (201105029 and201005024), the National Natural Science Foundation of China(30871944&30972284). We thank Xiuli Zhang for NMR analysis andShumei Ren for ES-MS analysis. We also thank Dr. Xue Zhao for helpfuldiscussion and revision of the manuscript on the thrombosis andanticoagulation aspects.
Appendix A. Supplementary data
Supplementary data to this article can be found online at doi:10.1016/j.bbagen.2012.03.002.
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