Fucose-Rich Sulfated Polysaccharides from Two Vietnamese Sea Cucumbers Bohadschia argus and Holothuria (Theelothuria) spinifera: Structures and Anticoagulant Activity

Fucosylated chondroitin sulfates (FCSs) FCS-BA and FCS-HS, as well as fucan sulfates (FSs) FS-BA-AT and FS-HS-AT were isolated from the sea cucumbers Bohadschia argus and Holothuria (Theelothuria) spinifera, respectively. Purification of the polysaccharides was carried out by anion-exchange chromatography on DEAE-Sephacel column. Structural characterization of polysaccharides was performed in terms of monosaccharide and sulfate content, as well as using a series of non-destructive NMR spectroscopic methods. Both FCSs were shown to contain a chondroitin core [→3)-β-d-GalNAc-(1→4)-β-d-GlcA-(1→]n bearing sulfated fucosyl branches at O-3 of every GlcA residue in the chain. These fucosyl residues were different in pattern of sulfation: FCS-BA contained Fuc2S4S, Fuc3S4S and Fuc4S at a ratio of 1:8:2, while FCS-HS contained these residues at a ratio of 2:2:1. Polysaccharides differed also in content of GalNAc4S6S and GalNAc4S units, the ratios being 14:1 for FCS-BA and 4:1 for FCS-HS. Both FCSs demonstrated significant anticoagulant activity in clotting time assay and potentiated inhibition of thrombin, but not of factor Xa. FS-BA-AT was shown to be a regular linear polymer of 4-linked α-L-fucopyranose 3-sulfate, the structure being confirmed by NMR spectra of desulfated polysaccharide. In spite of considerable sulfate content, FS-BA-AT was practically devoid of anticoagulant activity. FS-HS-AT cannot be purified completely from contamination of some FCS. Its structure was tentatively represented as a mixture of chains identical with FS-BA-AT and other chains built up of randomly sulfated alternating 4- and 3-linked α-L-fucopyranose residues.


Results and Discussion
Crude extracts of sulfated polysaccharides were obtained from the body walls of sea cucumbers Bohadschia argus and Holothuria spinifera ( Figure S2) by conventional solubilization of biomass in the presence of papain [8] followed by treatment of the extract with hexadecyltrimethylammonium bromide to precipitate the sulfated components, which were then transformed into water-soluble sodium salts by dissolving in 2 M NaCl and precipitation with ethanol, giving rise to crude sulfated polysaccharides SP-BA and SP-HS, respectively. Both crude extracts were subjected to anion-exchange chromatography on DEAE-Sephacel column. The fractions obtained as the result of chromatographic resolution are listed in Table 1. Table 1. Characteristics of crude polysaccharide preparations SP-BA and SP-HS and the fractions obtained by their chromatography on DEAE-Sephacel and then used for structural analysis (composition in molar ratios relative to fucose). Isolation of FCS from Bohadschia argus has been described previously, and the polysaccharide was used to obtain oligosaccharides acting as anticoagulants by intrinsic factor Xase complex inhibition [40]. According primarily to NMR spectral data, this polysaccharide had a typical FCS structure that was wholly 3-O-fucosylated GlcA and 4,6-disulfated GalNAc in the core with Fuc3S4S (~95%) and Fuc2S4S (~5%) as branches. In our work three fractions appeared (FCS-BA1, FCS-BA2, and FCS-BA3) by elution with water, 0.75 M and 1.0 M NaCl, respectively. These preparations were obtained in comparable yields of about 10% and contained GlcA, GalNAc, Fuc and sulfate in ratios near to the majority of known holothurian FCSs. Detection of minor Gal and GlcN in hydrolysates may be explained by the possible presence of small amounts of other GAGs, which could not be eliminated by anion-exchange chromatography. Appearance of a part of FCS eluted with water, and hence not absorbed on anion-exchanger, may probably be explained by the high molecular weight of FCS-BA1 (cf. [38]), whereas FCS-BA2 and FCS-BA3, having similar NMR spectra, were eluted separately, probably due to slightly different position of sulfation. All three FCS fractions had similar behavior in agarose gel electrophoresis  (Table 1) was used further for structural analysis.

Sample
The structure of FCS-BA was characterized using 1D and 2D NMR spectroscopy (Figures 1, 2, and S4, Table S1). The presence of Fuc, GalNAc and GlcA units in both polysaccharides was confirmed by the characteristic values of chemical shifts of C-6 for Fuc (δ 17.2 ppm) and GlcA (δ 176.0 ppm), as well as of C-2 for GalNAc (δ 52.7 ppm) in 13 C NMR spectrum ( Figure 1). The anomeric region in 1 H NMR spectrum contained several low-field signals indicating the presence of different fucosyl branches ( Figure 2). These spectra gave no doubts that FCS-BA belongs to the well-known class of holothurian FCSs [1,2,41,42]. It should be emphasized that very small intensity of C-6 signal in 13 C NMR spectrum (δ 62.3 ppm, Figure 1) means that practically all the GalNAc residues are 4,6-disulfated. The ratio between residues B and C ( Figure 3) was found to be 14:1. According to intensities of anomeric signals of sulfated Fuc residues it is possible to conclude that Fuc3S,4S (E, δ 5.34 ppm) predominates considerably over the two other branches (D, δ 5.68 ppm, and F, δ 5.40 ppm) in the polysaccharide molecule ( Figure 3, the calculated ratio D:E:F = 1:8:2).
Chromatography of SP-HS gave rise to FCS-HS, which was eluted, as expected, with 1.0 M NaCl. Its electrophoretic mobility in agarose gel was identical to FCS-BA ( Figure S3). According to NMR spectra ( Figures 1 and 2), which are very similar to those of FCS-BA, the polysaccharide undoubtedly belongs to FCSs. There are some minor structural differences between these two samples. Thus, the more intense signal at δ 62.3 ppm ( Figure 1) corresponds to more substantial content of GalNAc residues non-sulfated at C-6 (the ratio between residues B and C, Figure 3, was found to be 4:1), whereas signals at δ 5.68 ppm and δ 5.34 ppm, having practically equal intensities (Figure 2), show the more substantial content of Fuc2S,4S in FCS-HS. The molar ratio between differently sulfated fucose residues D:E:F ( Figure 3) was calculated as 2:2:1.
Mar. Drugs 2022, 20, x FOR PEER REVIEW 4 of 13 ysates may be explained by the possible presence of small amounts of other GAGs, which could not be eliminated by anion-exchange chromatography. Appearance of a part of FCS eluted with water, and hence not absorbed on anion-exchanger, may probably be explained by the high molecular weight of FCS-BA1 (cf. [38]), whereas FCS-BA2 and FCS-BA3, having similar NMR spectra, were eluted separately, probably due to slightly different position of sulfation. All three FCS fractions had similar behavior in agarose gel electrophoresis ( Figure S3). Fraction eluted with 1.0 M NaCl and designated as FCS-BA (Table 1) was used further for structural analysis.
The structure of FCS-BA was characterized using 1D and 2D NMR spectroscopy (Figures 1, 2, S4, Table S1). The presence of Fuc, GalNAc and GlcA units in both polysaccharides was confirmed by the characteristic values of chemical shifts of C-6 for Fuc (δ 17.2 ppm) and GlcA (δ 176.0 ppm), as well as of C-2 for GalNAc (δ 52.7 ppm) in 13 C NMR spectrum ( Figure 1). The anomeric region in 1 H NMR spectrum contained several low-field signals indicating the presence of different fucosyl branches ( Figure 2). These spectra gave no doubts that FCS-BA belongs to the well-known class of holothurian FCSs [1,2,41,42]. It should be emphasized that very small intensity of C-6 signal in 13 C NMR spectrum (δ 62.3 ppm, Figure 1) means that practically all the GalNAc residues are 4,6-disulfated. The ratio between residues B and C (Figure 3) was found to be 14:1. According to intensities of anomeric signals of sulfated Fuc residues it is possible to conclude that Fuc3S,4S (E, δ 5.34 ppm) predominates considerably over the two other branches (D, δ 5.68 ppm, and F, δ 5.40 ppm) in the polysaccharide molecule ( Figure 3   Chromatography of SP-HS gave rise to FCS-HS, which was eluted, as expected, with 1.0 M NaCl. Its electrophoretic mobility in agarose gel was identical to FCS-BA ( Figure S3). According to NMR spectra (Figures 1 and 2), which are very similar to those of FCS-BA, the polysaccharide undoubtedly belongs to FCSs. There are some minor structural differences between these two samples. Thus, the more intense signal at δ 62.3 ppm (Figure 1) corresponds to more substantial content of GalNAc residues non-sulfated at C-6 (the ratio between residues B and C, Figure 3, was found to be 4:1), whereas signals at δ 5.68 ppm and δ 5.34 ppm, having practically equal intensities (Figure 2), show the more substantial content of Fuc2S,4S in FCS-HS. The molar ratio between differently sulfated fucose residues D:E:F (Figure 3) was calculated as 2:2:1.
A rather unusual property of SP-BA is its incomplete solubility in water. The non-soluble gel-like fraction separated by centrifugation was treated with dilute acid in very mild conditions, giving rise to precipitate, which was shown to be mainly a protein. Neutralization of mother liquor followed by gel chromatography afforded FS-BA-AT ( Table 1). Application of 1D and 2D NMR experiments (COSY, HSQC, and ROESY) led to assigning all the signals in NMR spectra (Figure 4 and Figure 5, Figure S5, Table S2) and  Chromatography of SP-HS gave rise to FCS-HS, which was eluted with 1.0 M NaCl. Its electrophoretic mobility in agarose gel was identic ( Figure S3). According to NMR spectra (Figures 1 and 2), which are very si of FCS-BA, the polysaccharide undoubtedly belongs to FCSs. There are structural differences between these two samples. Thus, the more intense s ppm (Figure 1) corresponds to more substantial content of GalNAc residue at C-6 (the ratio between residues B and C, Figure 3, was found to be 4:1), w at δ 5.68 ppm and δ 5.34 ppm, having practically equal intensities (Figure more substantial content of Fuc2S,4S in FCS-HS. The molar ratio betwe sulfated fucose residues D:E:F (Figure 3) was calculated as 2:2:1.
A rather unusual property of SP-BA is its incomplete solubility i non-soluble gel-like fraction separated by centrifugation was treated with A rather unusual property of SP-BA is its incomplete solubility in water. The nonsoluble gel-like fraction separated by centrifugation was treated with dilute acid in very mild conditions, giving rise to precipitate, which was shown to be mainly a protein. Neutralization of mother liquor followed by gel chromatography afforded FS-BA-AT (Table 1). Application of 1D and 2D NMR experiments (COSY, HSQC, and ROESY) led to assigning all the signals in NMR spectra (Figures 4, 5, and S5, Table S2) and to establish the structure of FS-BA-AT as a regular linear polymer of 4-linked fucose 3-sulfate ( Figure 6). As mentioned above, similar polysaccharide was isolated previously from Holothuria fuscopunctata [22]. The signal assignments presented in [22] and our data are given in Table S2 for comparison. The systematic differences up to 2 ppm in carbon chemical shifts between spectra of two polymers probably arise due to alternative conditions of signal registration used in these two works. The structure of linear backbone built up of 4-linked α-L-fucopyranose in FS-BA-AT was confirmed by NMR spectra of its desulfated preparation FS-BA-AT-DS (Table S2).
to establish the structure of FS-BA-AT as a regular linear polymer of 4-linked fucose 3-sulfate ( Figure 6). As mentioned above, similar polysaccharide was isolated previously from Holothuria fuscopunctata [22]. The signal assignments presented in [22] and our data are given in Table S2 for comparison. The systematic differences up to 2 ppm in carbon chemical shifts between spectra of two polymers probably arise due to alternative conditions of signal registration used in these two works. The structure of linear backbone built up of 4-linked α-L-fucopyranose in FS-BA-AT was confirmed by NMR spectra of its desulfated preparation FS-BA-AT-DS (Table S2).   to establish the structure of FS-BA-AT as a regular linear polymer of 4-linked fucose 3-sulfate ( Figure 6). As mentioned above, similar polysaccharide was isolated previously from Holothuria fuscopunctata [22]. The signal assignments presented in [22] and our data are given in Table S2 for comparison. The systematic differences up to 2 ppm in carbon chemical shifts between spectra of two polymers probably arise due to alternative conditions of signal registration used in these two works. The structure of linear backbone built up of 4-linked α-L-fucopyranose in FS-BA-AT was confirmed by NMR spectra of its desulfated preparation FS-BA-AT-DS (Table S2).    Preparation SP-HS was also partially soluble in water, but in this case a rather sm insoluble fraction (12%) was not investigated further. Anion-exchange chromatograp afforded the main fraction FS-HS, not absorbed on the column and eluted with water. T improve the resolution of spectral signals, this fraction was treated with dilute acid, above, to give FS-HS-AT. The NMR spectra of FS-HS-AT were shown to be rather com plicated for detailed analysis due to overlapping of many important signals (Figures 4,  S6). This complexity may be explained by the random distribution of sulfates along t polymeric chains, since sulfation at every position causes a change in chemical shifts n only in its own residue, but also in both neighboring glycosylated and glycosylati residues. Nevertheless, the assignment of many signals and the corresponding corre tions in the anomeric region of the spectra has been suggested (Table S3) based on t assumption about the simultaneous presence of two types of polymers containing ra domly sulfated repeating units shown in Figure 6. Further attempts to resolve the com plex preparation FS-HS-AT are now in progress.
Since FCSs and FSs are known to demonstrate anticoagulant and antithrombotic a tivities [16,43], we have studied three isolated samples FCS-BA, FCS-HS and FS-BA-A as anticoagulant agents in vitro. Low molecular weight heparin (enoxaparin) was used standard (Figure 7). In the clotting time assay (APTT-test) the effects of branched FCS-B and FCS-HS were higher than that of enoxaparin, while linear FS-BA-AT was almo inactive at the same concentrations ( Figure 7A). The values of 2APTT (the concentratio that led to two-time increasing of clot formation) were 2.6 ± 0.1 μg/mL for FCS-HS, 3.1 0.1 μg/mL for FCS-BA, and 3.8 ± 0.2 μg/mL for enoxaparin. The active samples FCS-B and FCS-HS were studied further in the experiments with purified proteins thromb and factor Xa ( Figure 7B,C). It was shown that in the presence of anti-thrombin III (ATI FCS-BA and FCS-HS potentiate the inhibition of thrombin, although their effect w slightly lower than that of enoxaparin ( Figure 7B). This activity may be explained specific formation of the ternary complex between thrombin, antithrombin III and FC The possibility of such complexation was demonstrated by computer docking studies one of our previous works [44]. At the same time both samples had very low activi against factor Xa ( Figure 7C). Preparation SP-HS was also partially soluble in water, but in this case a rather small insoluble fraction (12%) was not investigated further. Anion-exchange chromatography afforded the main fraction FS-HS, not absorbed on the column and eluted with water. To improve the resolution of spectral signals, this fraction was treated with dilute acid, as above, to give FS-HS-AT. The NMR spectra of FS-HS-AT were shown to be rather complicated for detailed analysis due to overlapping of many important signals (Figures 4, 5, and S6). This complexity may be explained by the random distribution of sulfates along the polymeric chains, since sulfation at every position causes a change in chemical shifts not only in its own residue, but also in both neighboring glycosylated and glycosylating residues. Nevertheless, the assignment of many signals and the corresponding correlations in the anomeric region of the spectra has been suggested (Table S3) based on the assumption about the simultaneous presence of two types of polymers containing randomly sulfated repeating units shown in Figure 6. Further attempts to resolve the complex preparation FS-HS-AT are now in progress.
Since FCSs and FSs are known to demonstrate anticoagulant and antithrombotic activities [16,43], we have studied three isolated samples FCS-BA, FCS-HS and FS-BA-AT as anticoagulant agents in vitro. Low molecular weight heparin (enoxaparin) was used as standard ( Figure 7). In the clotting time assay (APTT-test) the effects of branched FCS-BA and FCS-HS were higher than that of enoxaparin, while linear FS-BA-AT was almost inactive at the same concentrations ( Figure 7A). The values of 2APTT (the concentrations that led to two-time increasing of clot formation) were 2.6 ± 0.1 µg/mL for FCS-HS, 3.1 ± 0.1 µg/mL for FCS-BA, and 3.8 ± 0.2 µg/mL for enoxaparin. The active samples FCS-BA and FCS-HS were studied further in the experiments with purified proteins thrombin and factor Xa ( Figure 7B,C). It was shown that in the presence of anti-thrombin III (ATIII) FCS-BA and FCS-HS potentiate the inhibition of thrombin, although their effect was slightly lower than that of enoxaparin ( Figure 7B). This activity may be explained by specific formation of the ternary complex between thrombin, antithrombin III and FCS. The possibility of such complexation was demonstrated by computer docking studies in one of our previous works [44]. At the same time both samples had very low activity against factor Xa ( Figure 7C

General Methods
Procedures for determination of neutral monosaccharides, amino sugars, uronic acids and sulfate content of polysaccharides have been described previously [45][46][47]. Solvolytic desulfation [45] was used to prepare FS-HS-AT-DS from FS-HS-AT. Molecular weights of polysaccharides were evaluated by chromatographic comparison with standard pullulans [48].

Isolation of Polysaccharides
Wild sea cucumbers Holothuria (Theelothuria) spinifera (Theel, 1886) and Bohadschia argus (Jaeger, 1833) with size range of 20-30 cm in length and 300-500 g in weight were collected in October 2020 at the coast-line of Nhatrang Bay, Vietnam. The samples were stored in sea water and transported to the laboratory in a day. After removing the viscera and washing, the fresh sea cucumber body walls were treated with 96% ethanol for 3 to 5 days, followed by soaking in acetone overnight at room temperature. Finally, the defatted samples were chopped and air-dried. Sulfated polysaccharides were isolated from the body wall of the sea cucumber using a method of Pham Duc Thinh et al., 2018 [39] (Figure 2). The defatted residue (50 g) was incubated with papain (10 g) in 1 L of 0.1 M sodium acetate buffer (pH 6) containing 5 mM EDTA and 5 mM cysteine at 60 • C for 24 h. The obtained mixture was heated at 100 • C for 20 min to inactivate the enzyme and to remove a small precipitate. The supernatant was treated with aqueous 10% hexadecyltrimethylammonium bromide solution (Cetavlon) up to complete precipitation of sulfated polysaccharides and left overnight at 4 • C. The resulting precipitate was centrifuged, washed with distilled water, dissolved in a mixture of 2 M NaCl and EtOH (4:1), precipitated with 3 volumes of 96% ethanol, and left at 4 • C for 24 h. The precipitate was centrifuged, washed with ethanol, dissolved in water, dialyzed, concentrated under a vacuum, and lyophilized to obtain crude sulfated polysaccharide SP-HS from Holothuria spinifera (yield 3,63%) and SP-BA from Bohadschia argus (yield 3,57%). Composition of crude polysaccharides is given in Table 1.
A suspension of 400 mg of SP-BA in 90 mL of water was stirred at room temperature for several hours and centrifuged at 6.000 rpm for 30 min. The gel-like precipitate was washed several times with water and lyophilized to give FS-BA-ins. The supernatant was placed on a column (3 × 10 cm) with DEAE-Sephacel (Saint Louis, MO, USA) in Cl -form and eluted with water followed by NaCl solution of increasing concentration (0.5, 0.75, 1.0 and 1.5 M), each time up to the absence of a positive reaction of eluate for carbohydrates [49]. Fractions were desalted on Sephadex G-15 (Saint Louis, MO, USA) column and lyophilized. Fractions eluted with water, 0.75 M NaCl and 1.0 M NaCl with the yields of 7.3, 11.8 and 11.8%, respectively, had similar composition and NMR spectra. The latter fraction was designated as FCS-BA and used further for structural analysis. Similar treatment of SP-HS gave rise to a rather small water-insoluble fraction (yield 12%), which was not investigated further. Chromatography of the water-soluble part of a sample on DEAE-Sephacel afforded the main fraction FS-HS eluted with water (yield 34.7%) and the second considerable fraction FCS-HS eluted with 1.0 M NaCl (yield 24.5%, Table 1).

Dilute Acid Treatment of Samples
A suspension of 170 mg of FS-BA-ins in 20 mL of 0.1 M HCl was stirred at 50 • C for 3 h, the insoluble material was centrifuged, washed and lyophilized to give a preparation (35.3%) containing 77.6% of protein and no carbohydrates. A viscous supernatant was neutralized by NaHCO 3 , desalted on Sephadex G-15 and chromatographed on DEAE-Sephacel, as above. The main polysaccharide fraction eluted with 1 M NaCl was desalted and lyophilized to afford FS-BA-AT (Table 1). Preparation FS-HS-AT was obtained after similar mild acid treatment of FS-HS (Table 1).

NMR Spectroscopy
The NMR spectra were recorded using the facilities of Zelinsky Institute Shared Center. Sample preparation and the conditions of experiments were described previously [50].

Anticoagulant Activity Measured in Clotting Time Test
The activated partial thromboplastin time assay for samples FCS-BA, FCS-HS, FS-BA and enoxaparin (Clexane ® , Sanofi, Paris, France) was performed using Coatron ® M2 coagulation analyzer (TECO, Munich, Germany) as described previously [4,51]. Briefly, a solution (10 µL) with concentration of 75 µg/mL, 37.5 µg/mL or 19.0 µg/mL of a polysaccharide sample (FCS-BA, FCS-HS, FS-BA, or enoxaparin) in purified water was added to 50 µL of normal plasma (Cormay, Poland). The mixture was incubated for 2 min at 37 • C, then 50 µL of APTT reagent (Cormay, Poland) was added, and the mixture was incubated again for 3 min at 37 • C. Then 50 µL of CaCl 2 solution (0.025M) was added, and the time of clot formation was recorded. Purified water instead of a saccharide solution was used as a control.

Statistical Analysis
All biological experiments were performed in quadruplicate (n = 4). The results are presented as Mean ± SD. Statistical significance was determined with Student's t test. The p values less than 0.05 were considered as significant. Moreover, the polysaccharides differ also in GalNAc4S6S and GalNAc4S units content, the ratios being 14:1 for FCS-BA and 4:1 for FCS-HS. Both polysaccharides demonstrated significant anticoagulant activity in clotting time assay. This activity is probably connected with the ability of these FCSs to potentiate inhibition of thrombin by formation of ternary complex with thrombin and antithrombin III. Such complexation was confirmed previously by computer docking experiments [44]. At the same time activity of these FCSs as inhibitors of Xa was rather low. Fucan sulfate FS-BA was shown to be a linear polymer of 4-linked α-L-fucopyranose 3-sulfate, structure being confirmed by NMR spectra of the native polysaccharide and its desulfated derivative. It is interesting to mention that FS-BA, despite its rather substantial sulfate content, was practically devoid of anticoagulant activity. FS-HS had much more complex NMR spectra, and its structure was tentatively represented as a polysaccharide containing fragments which coincide with FS-BA, together with other fragments built up of randomly sulfated alternating 4-and 3-linked α-L-fucopyranose residues.