Structural Elucidation and Biological Activity of a Highly Regular

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Structural elucidation and biological activity of a highly regular fucosylated glycosaminoglycan from the edible sea cucumber Stichopus herrmanni Xiaomei Li, Lan Luo, Ying Cai, Wenjiao Yang, Lisha Lin, Zi Li, Na Gao, Steven Purcell, Mingyi Wu, and Jinhua Zhao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03867 • Publication Date (Web): 04 Oct 2017 Downloaded from http://pubs.acs.org on October 5, 2017

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Journal of Agricultural and Food Chemistry

1

Structural elucidation and biological activity of a highly regular fucosylated

2

glycosaminoglycan from the edible sea cucumber Stichopus herrmanni

3 4

Xiaomei Li a, b, Lan Luo a, Ying Cai a, b, Wenjiao Yang a, b, Lisha Lin a,

5

Zi Li a, Na Gao a, Steven W. Purcell c, Mingyi Wu a,*, Jinhua Zhao a,*

6 7

a

8

Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China

9

b

University of Chinese Academy of Sciences, Beijing 100049, China

10

c

National Marine Science Centre, Southern Cross University, Coffs Harbour NSW

11

2450, Australia

State Key Laboratory of Phytochemistry and Plant Resources in West China,

12 13

∗

14

E-mail address: [email protected] (Mingyi Wu); [email protected]

15

(Jinhua Zhao).

Corresponding author. Tel.: +86 871 65226278; fax: +86 871 65226278.

16 17

Conflict of interest

18

The authors state no conflict of interest.

19 20 21 22 1

ACS Paragon Plus Environment


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Abstract

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Edible sea cucumbers are widely used as a health food and medicine. A

25

fucosylated glycosaminoglycan (FG) was purified from the high-value sea cucumber

26

Stichopus herrmanni. Its physicochemical properties and structure were analyzed and

27

characterized by chemical and instrumental methods. Chemical analysis indicated that

28

this FG with molecular weight of ~64 kDa is composed of N-acetyl-D-galactosamine,

29

D-glucuronic acid (GlcA) and L-fucose. Structural analysis clarified that the FG

30

contains the chondroitin-sulfate-E-like backbone and mostly 2, 4-di-O-sulfated (85%),

31

minor 3, 4-di-O-sulfated (10%) and 4-O-sulfated (5%) fucose side chains that link to

32

the C-3 position of GlcA. This FG is structurally highly regular and homogeneous

33

differing from other sea cucumber FGs, for its sulfation patterns are simpler.

34

Biological activity assays indicated that it is a strong anticoagulant by inhibiting

35

thrombin and intrinsic factor Xase. Our results expand knowledge on structural types

36

of FG and illustrate its biological activity as a functional food material.

37 38

Keywords: sea cucumber; fucosylated glycosaminoglycan; chemical structure;

39

anticoagulant; Stichopus herrmanni

40 41 42 43 44 2


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1. Introduction Sea cucumbers (Echinodermata, Holothuroidea) are a marine animal that are 1-6

47

important as human food source for their health and medicinal benefits.

The

48

processed body wall is mostly exported from producing countries to Asia, where it is

49

popular and of high value, especially in Chinese seafood markets.4

50

cucumber Stichopus herrmanni, called curryfish, occurs in most tropical countries of

51

the Indo-Pacific.4 This large species is one of many harvested in small-scale fisheries

52

throughout this broad distributional range. This species is sold commonly in Asian

53

markets and is used for the preparation of traditional medicinal products in Egypt. 7

The sea

54

Recent studies have indeed shown sea cucumbers to be high in protein, collagen

55

fibres, amino acids, and certain bioactive components such as glycosides and sulfated

56

polysaccharides.3,

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glycosaminoglycan found up to now exclusively in sea cucumbers, possesses

58

chondroitin sulfate-like backbone and is markedly different to typical mammalian

59

glycosaminoglycan (GAG) because of its unique sulfated fucose side chain.8,12,13 This

60

important polysaccharide is highly abundant in sea cucumbers comprising ~7% of dry

61

weight.14 FG is very likely to have beneficial activities for health, such as its

62

bioactivities against inflammation, angiogenesis, cancer metastasis, hyperglycemia,

63

atherosclerosis, and, above all, coagulation and thrombosis.8,15 Particularly, the special

64

GAG can be absorbed after oral administration and seems to retain its activity.16,17

8-11

Fucosylated

glycosaminoglycan

(FG),

a

distinct

65

Some teams including our research group have been searching for FGs from sea

66

cucumber, investigating their functions and developing them as functional food or/and 3



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medicines.3,13,18,19,20 Recently, we have obtained several FGs from various sea

68

cucumber species and analyzed their chemical structures.

69

macromolecule, besides its very complicated structure, the structure of FG is likely to

70

differ among sea cucumber species or regions from which they are source.20 Studies

71

including our work demonstrate that structural differences exist among FGs from

72

various sea cucumber species.9,12,20 Although structures of most reported FGs are very

73

complicated and heterogeneous, the structure of FGs from certain species, such as

74

Isostichopus badionotus

75

homogeneous.20,22 Similarly to the applications of polysaccharides, simpler chemical

76

structures could be favorable for pharmaceutical development because they are more

77

analyzable and their quality is more controllable. Therefore, further exploration of

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FGs in other sea cucumber species might provide a wider choice for studies on their

79

structures and functional activities.

12, 21

As a bioactive

and Massinium magnum, could be regular and

80

In the process of searching for sea cucumber polysaccharides, we discovered a

81

new FG from S. herrmanni. In this paper, the structure of this GAG was analyzed by

82

chemical and instrumental methods such as Fourier transform infrared spectroscopy

83

(FT−IR), high performance liquid chromatography (HPLC), monosaccharide

84

composition analysis, and nuclear magnetic resonance (NMR) spectroscopy (1H, 13C,

85

COSY, TOCSY, ROESY, HSQC and HMBC). Our study shows that this FG is

86

structurally highly regular and a different than previously reported FGs in spite of

87

certain similarities. Moreover, we investigated its anticoagulant activity and the

88

activities in comparison with FG from the Japanese sea cucumber Apostichopus 4


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japonicus, a species farmed en masse in China. Our results provide novel information

90

to enrich knowledge on structural types of FG and to illustrate its functionality.

91

2. Materials and methods

92

2.1. Materials

93

Dried body wall of the sea cucumber S. herrmanni was collected from markets in

94

Zhanjiang, Guangdong Province, China. Amberlite FPA98 Cl ion exchange resin was

95

purchased from Rohm and Haas Company, USA. The monosaccharides including

96

D-glucuronic acid (GlcA), N-acetyl-2-deoxy-2-amino-galactose (GalNAc) and

97

L-fucose (Fuc) were purchased from Alfa Aesar. Dermatan sulfate (DS) (~41400 Da)

98

was purchased from Sigma (USA). LMWH (Enoxaparin, 0.4 mL × 4000 AXaIU) was

99

from Sanofi-Aventis (France). The activated partial thromboplastin time (APTT),

100

prothrombin time (PT), thrombin time (TT) reagents and standard human plasma were

101

from Teco Medical (Germany). Biophen FVIII: C kit, Biophen Heparin Anti-IIa kits,

102

Biophen Antithrombin Anti-Xa kits, Human HCII, AT, thrombin and thrombin

103

chromogenic substrate CS-01(38) were all from Hyphen Biomed (France). Human

104

factor VIII was from Bayer HealthCare LLC (Germany). All other chemicals were of

105

reagent grade and obtained commercially.

106

2.2. Purification of the Fucosylated Glycosaminoglycan

107

Polysaccharides were isolated using a previously described procedure with minor

108

modifications.21, 22 300 g tissue of the dried body wall was digested by 0.5 M sodium

109

hydroxide, and then core protein combined with polysaccharides was released by the

110

5% papain (EC 3.4.22.2). The crude polysaccharides were dissolved in deionized 5



111

water (~7.5% of yield), then potassium acetate and ethanol were added with the final

112

concentration of 0.5 M and 40% (v/v), respectively. The solutions were mixed and

113

centrifuged at 3000 × g for 15 min. The precipitate was dissolved with 2 L of distilled

114

water and decolorized with 3% H2O2 at pH 10 and 45 °C for 2 h. Then, ethanol was

115

added to a final concentration of 60% (v/v). After centrifugation, the interested

116

fraction was obtained and further purified by strong ion-exchange chromatography

117

using a FPA98 column, and the sample was eluted with distilled water, followed

118

sequentially by 0.5 M NaCl, 1.0 M NaCl and 2.0 M NaCl solution. Each fraction

119

eluted by water and solutions of gradient NaCl concentration was collected, dialyzed

120

by a dialysis bag with molecular weight cut-off of 3 kDa (Spectrum Laboratories Inc.,

121

USA) and lyophilized to obtain powders. The yield of fucosylated glycosaminoglycan

122

was 1.25% by dry weight.

123

The purity and molecular weight of the polysaccharide were examined by

124

high-performance gel permeation chromatography (HPGPC) using a Agilent

125

technologies 1200 series (Agilent Co., USA) apparatus equipped with RID, DAD

126

detectors and a Shodex OH-pak SB-804 HQ column (8 mm × 300 mm).

127

Chromatographic procedures and conditions were performed according to an

128

established method.12,21,23 For molecular weight determination, a standard curve was

129

calibrated by standard D-series Dextrans (D-0, 2, 3, 4, 5, 6, 7 and 8) and a FG from

130

another Indo-Pacific sea cucumber Thelenota ananas with known relative molecular

131

weight (Mw 65820 Da).19 Molecular weight calculations were performed by a GPC

132

software, version B01.01 (Agilent Co., USA). 6


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2.3. Deploymerization of the Fucosylated Glycosaminoglycan

134

To further elucidate the structure of the polysaccharide in detail, its depolymerized

135

product (~10 kDa) was prepared according to our previous methods with some

136

modifications.19 The FG (100 mg) and 1 mg of copper sulfate were dissolved in 3.65

137

mL H2O. A 140 µL 30% H2O2 solution was added and reacted at 35 °C for about 60

138

min. Depolymerized product was precipitated with ethanol (1:4 (v/v)). The precipitate

139

was collected by centrifugation (3000× g for 20 min) and washed with ethanol and

140

then dissolved in water, dialyzed by a dialysis bag with molecular weight cut-off of 3

141

kDa (Spectrum Laboratories Inc., USA) and lyophilized. The yield of the

142

depolymerized product was 77.8%.

143

2.4.Chemical Composition and Physicochemical Analysis

144

The monosaccharide composition of the polysaccharide was analyzed by

145

reverse-phase HPLC according to PMP derivatization procedures.21 The 300 µL 2

146

mg/mL polysaccharide solution and 300 µL trifluoroacetic acid (TFA) (4 M) were

147

mixed, then sealed and incubated at 110°C for 4 h. After evaporation, the dried

148

samples were reconstituted in deionized water. Then 50 µL of the sample solution,

149

100 µL of 0.5 M PMP in methanol and 50 µL of 0.6 M sodium hydroxide were mixed

150

and incubated at 70 °C for 30 min. After adjusting the pH to 7, 0.5 mL of chloroform

151

was added to extract PMP three times. The top aqueous layer was collected for HPLC

152

analysis. Analysis of the PMP-labeled saccharides was carried out using an Agilent

153

Technologies1260 series apparatus (Agilent Co., USA) equipped with DAD detectors

154

and an Agilent Eclipse XDB C18 (150 mm × 4.6 mm). The flow rate was 1 mL/min, 7



155

and UV absorbance of the effluent was monitored at 250 nm. Mobile phases A and B

156

(v/v, 85:15) consisted of 0.1 M ammonium acetate (pH 5.5) and acetonitrile,

157

respectively.

158

The content of hexuronic acid was measured as described previously.24 0.2 mL of

159

the sample, 1.2 mL of sulfuric acid/tetraborate was added. The tubes were refrigerated

160

in crushed ice. The mixture was shaken and the tubes heated at 100 °C for 5 min.

161

After cooling in a water-ice bath, 20 µL of the hydroxydiphenyl reagent was added.

162

The tubes were shaken and, within 5 min, absorbance measurements made at 520 nm

163

in a Shimadzu UV-2450 spectrophotometer.

164

The ratio of GalNAc and Fuc was calculated according to integrals of their methyl

165

protons in the 1H NMR spectra. Acetylaminohexose was identified as described

166

previously.25 After strong acid hydrolysis of the polysaccharide (4.0 M HCl, 100 °C for

167

6 h), total hexosamine was determined by a modified Elson-Morgan reaction.26 The

168

ratio of sulfate and carboxyl groups of the polysaccharides were determined by a

169

conductimetric method.27

170

The specific rotation was determined by the optical rotation in Pharmacopoeia of

171

the People’s Republic of China.28 By this method, the concentration of the

172

polysaccharide was about 10 mg/mL and the detection temperature was 20 °C. The

173

intrinsic viscosity was measured according to determination of viscosity in

174

Pharmacopoeia of the People’s Republic of China.29

175 176 8


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177 178

2.5. UV, IR, and NMR Analysis The

UV−Vis

absorption

spectra

were

recorded

using

a

UV-2450

179

spectrophotometer (Shimadzu, Japan) in the wavelength range of 190−800 nm. The

180

FT-IR spectrum (KBr pellets) of the polysaccharide (2 mg) was recorded by a

181

Tensor-27 (Bruker, Germany) in a range of 400−4000 cm−1 at room temperature.

182

The structure elucidation was performed by NMR analysis at 298 K in D2O with

183

a Bruker Avance spectrometer of 600 or 800 MHz equipped with a 13C/1H dual probe in

184

FT mode, as previously described.13,

185

dissolved in D2O at a 20–30 g/L concentration. All spectra were recorded with HOD

186

suppression by presaturation. The 1H/1H correlated spectroscopy (COSY), total

187

correlation spectroscopy (TOCSY), rotating frame overhauser effect spectroscopy

188

(ROESY), 1H/13C heteronuclear single-quantum coherence (HSQC) and heteronuclear

189

multiple bond coherence (HMBC) spectra were recorded using state-time proportion

190

phase incrementation for quadrature detection in the indirect dimension. All chemical

191

shifts were relative to internal 3-trimethylsilyl-(2,2,3,3-2H4)-propionic acid sodium

192

(TSP, δH and δC = 0.00).

193

2.6. Anticoagulant Activity Assays

30, 31

The lyophilized samples were then

194

APTT, PT, and TT were determined with a coagulometer (TECO MC-4000,

195

Germany) using APTT, PT and TT reagents and standard human plasma as previously

196

described.12,23,32 The activity of intrinsic FXase inhibition was determined using the

197

previously described method with the reagents in the BIOPHEN FVIII: C kit.12,13 The

198

activity of inhibiting human factor IIa (FIIa) in the presence of HCII was detected by 9



199

the thrombin chromogenic substrate CS-01 using a Bio-Tek Microplate Reader.13, 23

200

The anti-FIIa activity in the presence of AT was tested using Biophen Heparin

201

Anti-FIIa kits.13, 23

202

3. Results and Discussion

203

3.1. Purification of the Fucosylated Glycosaminoglycan

204

The crude polysaccharides were extracted from the body wall of the sea cucumber

205

S. herrmanni by the method of alkaline hydrolysis and papain enzymolysis.21 The

206

yield of crude polysaccharides was 7.5% by dry weight. After being subjected to a

207

Shodex OH-pak SB-804 HQ column, the HPGPC of the extracted polysaccharides

208

showed two peaks (Supplementary Fig. S1), indicating a difference in molecular

209

weight.

210

Purification was achieved by strong anion exchange chromatography on a FPA98

211

column. The crude polysaccharides were separated into two major fractions (F-1 and

212

F-2 fraction), which corresponded to be eluted with 0-1 M and 2 M NaCl

213

concentrations, respectively. Based on a combination of their chemical compositions,

214

NMR analysis data and conductimetric titration curves (Table 1, Fig. S2 and see below

215

for discussion), the sea cucumber appears to contain fucan sulfate (peak F-1 in the Fig.

216

S1) and fucosylated glycosaminoglycan (peak F-2 in the Fig. S1), as reported for other

217

sea cucumber species.21,33 Additionally, the analysis of the sulfate and carboxyl by the

218

conductometric titration (Fig. S2) confirmed the high negative charge densities of the

219

two polysaccharides. The purity of the fucosylated glycosaminoglycan was above

10


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95% based on HPGPC analysis (Fig. S1). Structural analysis of fucan sulfate is

221

currently underway and the results will be published in due course.

222

3.2. Molecular Weight and Molecular Weight Distribution

223

The weight average (Mw) and number average (Mn) of molecular weight of the S.

224

herrmanni fucosylated glycosaminoglycan were 63700 Da and 50670 Da, respectively

225

(Table 1). Furthermore, the result obtained by HPGPC (Fig. S1B) shows differential

226

and cumulative molecular weight distributions, representing the homogeneity and

227

dispersibility of the polymer respectively, which are narrow, suggesting that the natural

228

biomacromolecule may be homogeneous. Compared with FGs from other species of

229

sea cucumbers (Table 1),19,34 the S. herrmanni FG exhibited higher molecular weight

230

and intrinsic viscosity [η], which is related to molecular weight of the polymer

231

according to the Mark–Houwink–Sakurada relationship.35

232

3.3. Chemical Composition Analysis

233

The monosaccharide compositions of the S. herrmanni FG were qualitatively

234

identified by reverse-phase HPLC according to PMP derivatization procedures.21 The

235

HPLC profiles (Fig. S3) showed that the three monosaccharides, namely D-glucuronic

236

acid (GlcA), N-acetyl-D-galactosamine (GalNAc) and L-fucose (Fuc), were observed

237

as expected, and a molar ratio of three monosaccharide was 1:1.13:1.09. Additionally,

238

the classical spectrophotometric analysis indicated a molar ratio of chemical

239

composition of 1:0.96:1.02 (Table 1), consistent with the data based on the

240

reverse-phase HPLC analysis after PMP derivatization. The FG could be assigned to

241

chondroitin sulfate since the ratio of GlcA and GalNAc is around 1:0.96, which is 11



242

similar to the chondroitin sulfate backbone structure.36 Cleavage of the sea cucumber

243

FG by digestion with specific chondroitin lyases was followed by HPGPC analysis. The

244

polysaccharide could not be depolymerized by digestion with chondroitin lyases, due to

245

the steric hindrance caused by their sulfated fucose branches that link to the chondroitin

246

sulfate backbone.25,33

247

Among other diagnostic information, the content of possible charged groups, such

248

as sulfate and uronic groups, is essential to evaluate the charge distribution along the

249

polyelectrolyte chain. Thus, the charge of the polysaccharide was measured by

250

conductometric titration.21,27 The result showed that the FG had two inflection points

251

(V1 and V2) and the molar ratio of sulfate to uronic groups (V1/(V2-V1)) was calculated to

252

be 3.82:1 (Fig. S2). By comparing the chemical composition of FGs among five sea

253

cucumbers, we found that the fucose and sulfate contents of these FGs varied from each

254

other (Table 1), which may account for their structural complexity.

255

3.4. UV and IR Spectra Analysis

256

The fucosylated glycosaminoglycan showed negative response to the Lowry test

257

and no absorption at 280 or 260 nm in the UV spectrum (Fig. S4A), indicating the

258

absence of protein and nucleic acids. When a dye (1, 9-dimethylmethylene blue) was

259

added to FG solution, a high increase in absorbance at 531 nm was observed. In

260

contrast, the absorption spectrum of the dye solution was not observed. The formation

261

of soluble blue dye-GAG complexes could alter the absorption spectra.37 Thus, this

262

indicated that FG, as a sulfated GAG, can form a complex with this blue dye.

263 12


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264

IR spectra of four fucosylated glycosaminoglycans from different sea cucumbers

265

all showed several bands corresponding to sulfate ester: the peaks at 1253 and 853

266

cm-1 are derived from the stretching vibration of S=O of sulfate and the bending

267

vibration of C-O-S of sulfate in axial position, respectively (Fig. S4B). The signals at

268

3448 and 1031 cm-1 are from the stretching vibration of O-H and C-O, respectively.38

269

In addition, signals at 2923 cm-1were due to the stretch vibration of C-H; 1419 cm-1,

270

the symmetric stretch vibration of COO- of glucuronic acid and the stretch vibration of

271

C-O within COOH. The characteristic absorptions of the native FG are similar to

272

those of the FGs from other sea cucumber species that had been reported in literature.

273

19, 39

274

of sea cucumber are the same.

275

3.5. NMR Analysis

These data showed that the major functional groups of FGs from various species

276

The structural features of the FG were further elucidated by NMR spectral

277

analysis. In the 1H NMR spectra (Fig. S5), the signals observed in the region

278

approximating to 5.2–5.7 ppm could be assigned to anomeric protons of α-L-fucose

279

residues.12 Notably, the proton signals of Fuc in S. herrmanni FG are simpler,

280

compared with the FG from Apostichopus japonicus, which is the most famous

281

high-value sea cucumber in China and is a predominant species in markets in China

282

and Japan. The data based on 1H NMR spectra indicated that the S. herrmanni FG

283

might be more regular than the A. japonicus FG (see below for detailed discussion),

284

though their chemical composition and physicochemical properties are similar to each

285

other (Table 1). 13



Page 14 of 33

286

The NMR analysis of the FG produced overlapping spectra with broad signals, with

287

line widths of several Hz (Fig. S5), which hampered the resolution. To further

288

elucidate the structures in detail, chemical shifts of individual residues of the

289

depolymerized FG were assigned (Table 2, Table S1) according to 1D (1H, 13C) (Fig. 1)

290

and 2D COSY, TOCSY, ROESY, HSQC, and HMBC experiments (Fig. 2), based on

291

some previous studies.25, 30,31,40 In the 1H NMR spectrum, the signals at about 1.20–1.30

292

ppm and 2.00–2.10 ppm could be readily assigned to the methyl protons of fucose (CH3)

293

and GalNAc (CH3CO) residues, respectively (Fig. 1A). The three signals observed in

294

the region of approximately 5.20−5.70 ppm could be assigned to anomeric protons of

295

α-L-fucose residues (Fig. 1A). The coupling system in the 1H−1H COSY and 1H−13C

296

HSQC spectra (Fig. 2) showed that H-2 and H-4 chemical shifts (δH 4.496 and 4.848

297

ppm) of the Fuc residue were shifted downfield by approximately 0.6–0.7 ppm and that

298

the C-2 and C-4 chemical shifts (δC 78.3 and 84.0 ppm) of the Fuc residue were shifted

299

downfield by approximately 5−7 ppm compared with those from standard Fuc,41

300

indicating that this Fuc residue was sulfated at both the C-2 and C-4 positions,

301

respectively. Similarly, the very weak signals at 5.395 and 5.340 ppm were from H-1 of

302

Fuc residues that were sulfated at C-4 and at both the C-3 and C-4 positions,

303

respectively. Thus, the three major signals at 5.688, 5.395 and 5.340 ppm in the

304

spectrum could be assigned to H-1 of 2, 4-O-disulfated fucose (Fuc2S4S),

305

4-O-monosulfated

306

respectively (Table S1). According to the integrals of the anomeric protons, a molar

307

ratio of Fuc2S4S, Fuc4S and Fuc3S4S was 0.85:0.05:0.10, indicating that Fuc2S4S is

fucose

(Fuc4S)

and

3,4-O-disulfated

14


fucose

(Fuc3S4S),

Page 15 of 33


308

the main type of fucose branches of the S. herrmanni FG. Although these sulfation

309

types of fucoses in S. herrmanni FG are the same as A. japonicus FG (Fig. S5), the

310

sulfation distribution differs markedly from that of A. japonicus FG, which in contrast

311

exhibited higher Fuc4S and Fuc3S4S.

312

The proton signals from GlcA and GalNAc of the S. herrmanni FG could also be

313

assigned according to the 1H-1H 2D NMR spectra (Fig. 2). For instance, the detailed

314

proton signals (Table 2) of these systems from H-1 to H-6 were assigned using the

315

1

H−1H COSY (Fig. 2A) and TOCSY detections (Fig. 2B) as shown in Table 2.

316

The carbon signals were assigned based on the assignment of the protons (Table

317

2), using 1H−13C HSQC spectrum (Fig. 2D). The 13C NMR spectra in Fig. 1B showed

318

two signals at 175−180 ppm ascribed to two carbonyl groups of GlcA and GalNAc.

319

The signals in the anomeric region (δC 95−110 ppm) were assigned to GlcA (δC

320

106.59 ppm), GalNAc (δC 102.58 ppm) and Fuc (δC 99.32 ppm) from the low field to

321

the high field, respectively, which were confirmed by cross peaks in the 1H−13C

322

HSQC spectrum. The up-field C-2 resonance of GalNAc (δC 54.19 ppm) was assigned

323

to the presence of the amino group.12 The chemical shifts at δ 70−80 ppm, showing

324

overlapping peaks, were assigned to other carbons of glycosidic rings. Notably, the

325

signal at approximately 70.1 ppm in distortionless enhancement by polarization

326

transfer (DEPT)-135º

327

whereas unsubstituded C-6 signals were almost absent,19 indicating that all the C-6 of

328

the GalNAc was sulfated. The data based on 1H-1H and 1H-13C 2D NMR spectra

329

suggested that the major GalNAc in the backbone was 4, 6-O-disulfated

13

C NMR spectrum was from the sulfated C-6 of GalNAc,

15



330

N-acetyl-D-galactosamine (GalNAc4S6S).

331

According to the correlated signals shown in the ROESY (Fig. 2C) and HMBC (Fig.

332

2E) spectra, GlcA and GalNAc residues in the S. herrmanni FG were linked as a

333

chondroitin sulfate-like core with alternating β(1→3) and β(1→4) bonds, whereas Fuc

334

branches were linked to GlcA through α(1→3) glycosidic linkages. Obviously, the

335

ROESY and HMBC spectra showed several long-range correlations corresponding to

336

the sugar connectivities (see red annotation in Fig. 2C and E). Configurations of the

337

glycosidic linkages were further confirmed by the direct coupling constants (1JC–H) of

338

C-1 of each sugar. The large values of ~175 Hz and ~165 Hz for these sugar residues

339

indicated that the protons at C-1 are equatorial and axial, respectively.34,42

340

Based on the above analysis, the proposed structure of the sea cucumber FG can

341

be trustworthily depicted (Fig. 3A). Its backbone is →4)-D-GlcA-β(1→3)-

342

D-GalNAc4S6S-β(1→, and monosaccharide L-Fuc side chains are linked to GlcA

343

through α(1→3) glycosidic bonds whose sulfation types are mostly Fuc2S4S. This

344

sequence units of the S. herrmanni FG seem to be more regular and homogeneous

345

compared with those of FGs from other species of sea cucumber.12,19,22,30,31

346

3.6. Biological Activity Analysis

347

In order to confirm whether the new FG affect the coagulation system,

348

anticoagulant activities were investigated by measuring the APTT, PT and TT in the

349

presence of the sea cucumber GAG, and compared with that of low-molecular-weight

350

heparin (LMWH). These assays indicated that the polysaccharide exhibited strong

351

APTT and TT prolonging activities in a dose dependent manner (the concentrations 16


Page 16 of 33

Page 17 of 33


352

that are required to double the APTT and TT are 2.72 µg/mL and 9.4 µg/mL,

353

respectively), and did not affect PT of human plasma at the concentrations tested

354

(1280 µg/mL) (Table 3). As expected, LMWH has strong anticoagulant activities.

355

Interestingly, the anticoagulant activities of the S. herrmanni FG were observed to be

356

similar to those of the A. japonicus FG,

357

structures and molecular sizes (Table 1). Notably, the depolymerized FG (~10 kDa)

358

still showed strong APTT prolonging activity, but it could not exhibit PT and TT

359

prolonging activity up to concentrations as high as 1280 µg/ml, which means that they

360

have no or little effect on the extrinsic and common coagulation pathways (Table 3).19

361

Anti-FIIa activity in the presence of heparin cofactor II (HCII) or antithrombin

362

(AT), and anti-factor Xase (FXase) activity were further examined with

363

corresponding chromogenic substrates (Fig. 3B-D). The sea cucumber polysaccharide

364

displayed strong anti-FXase activity and anti-FIIa by HCII or AT activity in a dose

365

dependent manner (their IC50 values were 11.1, 624, and 593 ng/mL, respectively),

366

comparable to those of two positive anticoagulant compounds LMWH and DS (Table

367

3). The anti-FXase and anti-FIIa activities of the S. herrmanni FG were markedly

368

different from those of the A. japonicus FG, 12 possibly due to their different sulfation

369

patterns (Table 1).21 Interestingly, the inhibition of intrinsic FXase displayed by the

370

depolymerized S. herrmanni FG was still as strong as that of the native

371

polysaccharide, while the ability to activate factor IIa by AT decreased (Table 3),

372

suggesting that the low-molecular-weight FG might exhibit a higher selectivity for

373

intrinsic FXase.

12

possibly due to their similar chemical

17



Page 18 of 33

374

These above results lead to the conclusion that the native FG and its

375

low-molecular-weight product have strong anticoagulation ability. These in vitro data

376

encourage a much more detailed screening of the in vivo anticoagulant effects of the

377

sulfated polysaccharides. More interestingly, the FG from the sea cucumber

378

Holothuria grisea is absorbed after oral administration in vivo and seems to retain its

379

activity.16,17 This gives promise for further intragastric experiments in animals to test

380

the health function of FG, and to investigate the feasibility of FG as a functional food

381

material.

382

4. Conclusions

383

The sea cucumber S. herrmanni is valued for its health benefits and is rich in a

384

variety of biological active ingredients with high nutritional value and medicinal

385

value. This study isolated and characterized a unique glycosaminoglycan FG from the

386

edible sea cucumber. This FG is mainly composed of D-glucuronic acid,

387

N-acetyl-D-galactosamine, L-fucose and sulfate with a molar ratio of 1:0.96:1.02:3.82.

388

The

389

→4)-D-GlcA-β(1→3)-D-GalNAc4S6S-β(1→, and monosaccharide L-Fuc side chains

390

are linked to GlcA through α(1→3) glycosidic bonds and their sulfation types are

391

mostly 2, 4-di-O-sulfated (85%), minor 3, 4-di-O-sulfated (10%) and 4-O-sulfated

392

(5%). This GAG is structurally highly regular and homogeneous which distinguishes

393

it from FGs from other sea cucumber species. The FG in S. herrmanni exhibits strong

394

anticoagulant activities by inhibiting thrombin and intrinsic factor Xase. The findings

395

warrant further investigation of the feasibility of FG as a functional food material.

structural

analysis

showed

that

its

backbone

18


sequence

is

Page 19 of 33


are:

Glycosaminoglycan;

FG,

fucosylated

Abbreviations

397

glycosaminoglycan; LMWH, low-molecular-weight heparin; DS, dermatan sulfate;

398

CS, chondroitin sulfate; TSP, trimethylsilyl-propionic acid; GlcA, glucuronic acid;

399

GalNAc,

400

GalNAc; Fuc, fucose; Fuc4S, 4-O-sulfated fucose; Fuc3S4S, 3, 4-di-O-sulfated

401

fucose; Fuc2S4S, 2, 4-di-O-sulfated fucose; APTT, activated partial thromboplastin

402

time; PT, prothrombin time; TT, thrombin time; HCII, heparin cofactor II; AT,

403

antithrombin; FXase, intrinsic tenase complex; FIIa, thrombin.

404

Acknowledgements

405

used

GAG,

396

N-acetyl-2-deoxy-2-amino-galactose;

GalNAc4S6S,

4,6-di-O-sulfated

Bo Li and Jianchao Chen are acknowledged for performing the NMR experiments.

406

Funding sources

407

This work was funded in part by the National Natural Science Foundation of China

408

(81373292, 81673330 and 81773737), Yunnan Provincial Science and Technology

409

Department in China (2010CI116 and 2016FA050), Institutes for Drug Discovery and

410

Development (CASIMM0220151008), Youth Innovation Promotion Association

411

(2017435), and Discovery, Evaluation and Transformation of Active Natural

412

Compounds, Strategic Biological Resources Service Network Programme (ZSTH-020)

413

of Chinese Academy of Sciences.

414

Supporting Information: Information on HPGPC profiles, conductimetric titration

415

curve, UV absorption spectra, FT-IR spectra and 1H NMR spectra of the fucosylated

416

glycosaminoglycan are available.

417 19



418

References

419

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420 421 422 423 424 425 426

1999, 11, 3–9. (2) To, A.W. L.; Shea, S. K. H. Patterns and dynamics of Bȇche-de-mer trade in Hong Kong and mainland China. Traffic Bull. 2012, 24, 65–76. (3) Bordbar, S.; Anwar, F.; Saari, N. High-value components and bioactives from sea cucumbers for functional foods–A review. Mar. Drugs 2011, 9, 1761–1805. (4) Purcell, S.W. Value, market preferences and trade of Bȇche-de-mer from Pacific Island sea cucumbers. PLoS ONE 2014, 9, e95075.

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chondroitin sulfates and their derivatives as selective inhibitors of intrinsic factor

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Xase. Eur. J. Med. Chem. 2015, 92, 257–269.

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depolymerization of a fucosylated chondroitin sulphate from sea cucumber

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and anticoagulant activities of fucosylated chondroitin sulfates from different sea

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polysaccharide account for its high anticoagulant action. J. Biol. Chem. 1996, 271,

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Two fucosylated chondroitin sulfates from the sea cucumber Eupentacta

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fraudatrix. Carbohydr. Polym. 2017, 164, 8–12.

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coagulation proteases or inhibitors. Thromb. Res. 2016, 146, 59–68.

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chondroitin sulfate from sea cucumber: Evidence for the presence of

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3-O-sulfo-β-D-glucuronosyl residues. J. Biol. Chem. 1991, 266, 13530–13536.

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DHG, a depolymerized glycosaminoglycan from sea cucumber Stichopus

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japonicus. Tetrahedron Lett. 1992, 33, 4959–4962.

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(35) Hiemenz C. P.; Timothy L. P. Polymer Chemistry (Second ed.); Boca Raton: CRC 2007; 336–339.

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(36) Kariya, Y.; Watabe, S.; Hashimoto, K.; Yoshida, K. Occurrence of chondroitin

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sulfate E in glycosaminoglycan isolated from the body wall of sea cucumber

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Stichopus japonicus. J. Biol. Chem. 1990, 265, 5081–5085.

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(37) Gold, E. W. The quantitative spectrophotometric estimation of total sulfated

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glycosaminoglycan levels. Formation of soluble alcian blue complexes. Biochim. 24


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Biophys. Acta. 1981, 673, 408–415. (38) Romdhane, M. B.; Haddar, A.; Ghazala, I.; Jeddou, K. B.; Helbert, C. B.;

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Ellouz-Chaabouni,

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watermelon rinds: Structure, functional and biological activities. Food Chem.

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(39) Hoshino, H.; Heiwamachi, M. Anti-HIV drug. 1990, EP 0410002A1.

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(40) Santos, G. R. C.; Glauser, B. F.; Parreiras, L. A.; Vilanova, E.; Mourão, P. A. S.

535

Distinct structures of the α-fucose branches in fucosylated chondroitin sulfates do

536

not affect their anticoagulant activity. Glycobiology 2015, 10, 1043–1052.

537

(41) Wu, M.; Huang, R.; Wen, D.; Gao, N.; He, J.; Li, Z.; Zhao. J. Structure and effect

538

of sulfated fucose branches on anticoagulant activity of the fucosylated

539

chondroitin sulfate from sea cucumber Thelenota ananas. Carbohydr. Polym.

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2012, 87, 862–868.

541 542

(42) Bubb, W. A. NMR spectroscopy in the study of carbohydrates: Characterizing the structural complexity, Concepts in Magnetic Resonance Part A, 2003, 19A, 1–19.

543

544

545

546

547

548 25



549

Figure captions

550

Figure 1. 1H (A) and

551

glycosaminoglycan with molecular weight of ~10 kDa. Chemical shifts are relative to

552

external trimethylsilylpropionic acid at 0 ppm for 1H, and 13C. The peaks are labeled as

553

U, A and F for GlcA, GalNAc and Fuc, respectively. The number after these letters

554

indicate the positions of 1H and 13C.

555

Figure 2. Expansions of the COSY (A), TOCSY (B), ROESY (C), HSQC (D) and

556

HMBC (E) spectra of the depolymerized fucosylated glycosaminoglycan from the sea

557

cucumber S. herrmanni. The peaks are labeled as U, A and F for GlcA, GalNAc and

558

Fuc, respectively.

559

Figure 3. Proposed structure of the fucosylated glycosaminoglycan from the sea

560

cucumber S. herrmanni (A) and its anti-FIIa and anti-FXase activities (B~D). Effects

561

of FG on FIIa activity in the presence of HCII (B) or AT (C), and human intrinsic

562

FXase inhibition (D).

13

C (B) NMR spectra of the depolymerized fucosylated

26


Page 26 of 33

Page 27 of 33


Table 1. Chemical composition and physicochemical properties of polysaccharides from the body wall of five sea cucumbers. Chemical composition [η]

Specific

(mL/g)

rotation

Molecular weight

(molar ratios) a

Species

References

GlcA : GalNAc : Fuc : -SO3-

Mn

Mw

Mw/Mn

Stichopus herrmanni

1 : 0.96 : 1.02 b: 3.82

42.7

-55.6

50670

63700

1.26

Holothuria edulis

1 : 1.28 : 0.82 b: 3.50

24.6

-37.9

41340

51090

1.24

This work Luo et al., 2013 (Ref. 21) Luo et al., 2013

Holothuria nobilis

1 : 0.96 : 0.82 b : 2.99

34.0

-45.9

42460

55320

1.30 (Ref. 21) Luo et al., 2013

Apostichopus japonicus

1 : 1.05 : 1.03 b: 2.96

32.5

-56.2

46760

56820

1.22 (Ref. 21)

Thelenota ananas d

1 : 1.02 : 0.98 : 3.81

48.1

-59.1

60944

65820

1.08

Wu et al., 2010 (Ref. 19) Yoshida et al. ,

Apostichopus japonicus d

1 : 1.01 : 0.95 : 3.77

NDc

NDc

NDc

66500

NDc

1992 (Ref. 34)

a

GlcA: D-glucuronic acid; GalNAc: N-acetyl-D-galactosamine; Fuc: α-L-Fucose.

b

The ratios of GlcA and GalNAc were determined by the chemical methods [Materials and methods], and the ratios

of GalNAc and Fuc were determined by their –CH3 integrals analysis of 1H-NMR spectra . c

ND, Not determined.

d

Molecular weights were determined by low-angle laser light scattering (LALLS).

27



Table 2. 1D (1H and

Page 28 of 33

13

C) NMR chemical shifts and 2D NMR assignments for the

fucosylated glycosaminoglycan from the sea cucumber S. herrmanni. 1 1

Fuc2S4S

GlcA

GalNAc4S6S

H

δ (ppm)

H1

H-1H

1

H-1H

1

H-1H ROESY

13

C

δ (ppm)

13

C-1H HMBC

COSY

TOCSY

5.688

H2

H2,H3,H4

U3,c U1,H2,H3

C1

99.32

U3, H5

H2

4.496a

H1,H3

H1,H3,H4

H1

C2

78.30

H3,H4

H3

4.133

H2,H4

H1,H2,H4

H4,H5

C3

69.52

H2,H4,H5

H4

4.848

H3

H1,H2,H3

H3

C4

84.01

H5,H6

H5

4.898

H6

H6

H3,H6

C5

69.10

H1,H4,H6

H6

1.365

H5

H5

H4,H5

C6

18.70

H4,H5

H1

4.475

H2

H2,H3,H4,H5

A3,H3,H5

C1

106.59

H2

H2

3.665

H1,H3 H1,H3,H4,H5

H4

C2

76.56

H3

H3

3.746b

H2,H4 H1,H2,H4,H5

F1,H1,H4

C3

80.23

F1, H2

H4

3.963

H3,5

H1,H2,H3,H5

H4

C4

77.97

H3

H5

3.692

H4

H1,H2,H3,H4

H1,H4

C5

79.81

C6

177.84

H1

4.628

H2

H2

U4,H2,H3,H5

C1

102.58

U4

H2

4.028

H1,H3

H1

H1

C2

54.19

H4

H3

3.946

H2

H1

C3

77.97

H4,H5

H4

4.826

H5

H3,H5

C4

79.23

H3

H5

3.982

H6,H6’

H6,H6

H1,H6

C5

74.60

H6,H6

H6

4.276

H5,H6’

H5,H6

H5

C6

70.13

H6’

4.178

H5,H6

H5,H6

C7

177.84

H8

H8

2.056

C8

25.49

H8

a

Values in bold type indicate positions of sulfation.

b

Values in italic type indicate glycosylated positions.

c

The GlcA GalNAc and Fuc are labeled as U, A and F, respectively. The number after these letters

indicate the positions of 1H and 13C. 28


Page 29 of 33


Table 3. Sulfation patterns of fucose residues, anticoagulant activity and IC50 of the fucosylated glycosaminoglycans for FXase or thrombin inhibition in the presence of HCII or AT. Sulfation patterns of fucose residues (percentage of molar ratio )

APTT

TT

Anti-FXase

Anti-FIIa/HCII

Anti-FIIa/AT

(µg/mL) b

(µg/mL) b

(ng/mL)c

(ng/mL)c

(ng/mL)c

Glycosaminoglycans a

a

a

Fuc4S

Fuc2S4S

Fuc3S4S

5

85

10

2.72

9.4

11.1

624

593

5

85

10

4.14

>1280

15.1

354

3145

7.80

4.0

67.6

494

33.7

S. herrmanni (native FG)

S. herrmanni (depolymerized FG) d

LMWH a

Fuc4S, 4-O-sulfated fucose; Fuc3S4S, 3, 4-di-O-sulfated fucose; Fuc2S4S, 2, 4-di-O-sulfated fucose;

b

The concentration required to double the APTT or TT of human plasma;

c

IC50 value, the concentration required to inhibit 50% of protease activity;

d

Molecular weight of the depolymerized S. herrmanni FG is about 10 kDa.

29



Figure 1.

1

H (A) and

13

C (B) NMR spectra of the depolymerized fucosylated

glycosaminoglycan with molecular weight of ~10 kDa. Chemical shifts are relative to external trimethylsilylpropionic acid at 0 ppm for 1H, and 13C. The peaks are labeled as U, A and F for GlcA, GalNAc and Fuc, respectively. The number after these letters indicate the positions of 1H and 13C.

30


Page 30 of 33

Page 31 of 33


Figure 2. Expansions of the COSY (A), TOCSY (B), ROESY (C), HSQC (D) and HMBC (E) spectra of the depolymerized fucosylated glycosaminoglycan from the sea cucumber S. herrmanni. The peaks are labeled as U, A and F for GlcA, GalNAc and Fuc, respectively.

31



A

OSO3 - OSO 3

-

OOC O

O

O O

O

NHAc n

OH H3 C

Page 32 of 33

O R1

R 1 = -OSO 3- (85%) or -OH R 2 = -OSO 3- (10%) or -OH n = ~ 60

R2 OSO3 -

FIIa activity by HCII (% )

B 100 80

60

40 FG DS LMWH

20

0 -2 10

-1

10

0

10

1

10

2

10

3

10

3

10

3

10

10

4

10

5

4

10

4

10

Concentration (ng/mL)

FIIa activity by AT (%)

C

100 80 60 40 20

FG LMWH

0 -2

10

-1

10

0

10

1

10

2

10

10

5


D 100 Xa generation (%)

80 60 40 20

FG LMWH

0 -2

10

-1

10

0

10

1

10

2

10

10

5


Figure 3. Proposed structure of the fucosylated glycosaminoglycan from the sea cucumber S. herrmanni (A) and its anti-FIIa and anti-FXase activities (B~D). Effects of FG on FIIa activity in the presence of HCII (B) or AT (C), and human intrinsic FXase inhibition (D). 32


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Table of Contents Graphic

33


Structural Elucidation and Biological Activity of a Highly Regular

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