Isolation, purification and structural characteristics of glycosaminoglycans from sea cucumber stichopus horrens

Crude glycosaminoglycans were fractionated and purified by using anion-exchange chromatography on the DEAE-Macro Prep column to give two fractions of fucosylated chondroitin sulfate FCS

ISOLATION, PURIFICATION AND STRUCTURAL

CHARACTERISTICS OF GLYCOSAMINOGLYCANS FROM SEA

Pham Duc Thinh1, 2, *, Duong Khanh Minh2, 3, Dinh Thanh Trung1

1

Nhatrang Institute of Research and Application, VAST, 02 Hung Vuong, Nha Trang, Viet Nam

2

Graduate University of Science and Technology, VAST, 18 Hoang Quoc Viet, Ha Noi, Viet Nam

3

Institue of Vaccine and Medical Biologicals, 9 Pasteur, Nha Trang, Viet Nam

*

Email: ducthinh.nitra@gmail.com,; ducthinh@nitra.vast.vn

Received: 20 September 2020; Accepted for publication: 7 January 2020

Abstract Sea cucumber glycosaminoglycans have been well known as potential anticoagulant

and antithrombin agents In this investigation, glycosaminoglycans were isolated from sea

cucumber Stichopus horrens (S horrens) by papain enzymatic digestion Crude

glycosaminoglycans were fractionated and purified by using anion-exchange chromatography on

the DEAE-Macro Prep column to give two fractions of fucosylated chondroitin sulfate (FCS)

and fucan sulfate (FS) Structural characteristics of F1 and F2 fractions were elucidated using

chemical and IR, NMR spectroscopic methods The results showed that the monosaccharide

compositions of F1 consists of N-Acetyl-Galactosamine (glcnac), D-Glucuronic acid (glca) and

Fucose (Fuc) residues with different molar ratios, while F2 content only fucose residues Sulfate

contents of F1 and F2 were 47.4 % and 48.1 %, respectively F1 and F2 fractions were different

in the pattern of sulfation of N-Acetyl-Galactosamine and fucose residues IR and NMR spectra

of two frations showed that sulfate groups were primarily occupied at C4 of pyranose residues in

F2 and C6, C2 and/or C3 of pyranose residues in F1 fraction Our results contributed to the

knowledge on structural types of glycosaminoglycan from sea cucumbers in Viet Nam The

establishment of structural features plays an important role in further studies of the

structure-bioactivity relationship of sea cucumber glycosaminoglycan

Keywords: glycosaminoglycans, fucosylated chondroitin sulfate, fucan sulfate, sea cucumber, Stichopus

horrens

Classification numbers: 1.1.1, 1.5.1

1 INTRODUCTION

Glycosaminoglycans present in sea cucumbers are especially interesting since they possess

anticoagulant, antithrombotic, antiangiogenic, anti-inflammatory, anti-oxidant, antivirus,

antitumor and many other activities [1] Holothurian glycosaminoglycans classified into two

main types including fucosylated chondroitin sulfate (FCS) and fucan sulfate (FS) [2] The

sulfated fucose branches of FCS play an important role for biological activities

Glycosaminoglycan extracted from various sea cucumbers have different sulfation patterns of

Glycosaminoglycans were obtained from the body wall of some sea cucumbers, such as

Holothuria nobillis, Acaudina molpadioidea [2]; Pearsonothuria graeffei, Holothuria vagabunda, Stichopus tremulus and Isostichopus badionotus [3]; Stichopus horrens [4]; Holothuria edulis, Ludwigothurea grisea [5]; Stichopus japonicus [6]; Apostichopus japonicus

[7] The structure of these holothurian glycosaminoglycans were found to be species-specific [3, 4] Sea cucumbers are used as healthy food and traditional medicine in Viet Nam Sea

cucumbers are widely distributed in most of the coastal provinces [8] The sea cucumber S

horrens is a species widely distributed in tropical waters Fucosylated chondroitin sulfate (FCS)

represents the major glycosaminoglycans found in sea cucumbers; other glycosaminoglycans

isolated are sulfated fucans or fucoidans from the body wall of the Vietnamese sea cucumber S

horrens Glycosaminoglycans from different species of holothuria vary in pattern of sulfation,

presence of branching, and molecular weight [3, 4]

In this paper, structural characteristics of two glycosaminoglycan fractions (F1 and F2)

isolated from sea cucumber S horrens were studied using chemical methods, infrared (IR), and

nuclear magnetic resonance (NMR) spectroscopy Fucan sulfate (FS) was built of the repeating

α-L-fucopyranosyl residues

2 MATERIALS AND METHODS

2.1 Isolation and purification of glycosaminoglycans from sea cucumber S horrens

* Isolation of glycosaminoglycans: glycosaminoglycans were isolated from the body wall

of sea cucumber S horrens using a modification of a previously described method [9] The fresh

body wall (1500 g) was washed with tape water, minced, homogenized, and pretreated by 4.5 L ethanol 96 % for 6 hours at 50 oC to remove lipids and pigments, and then dried at room temperature The defatted dried sea cucumber (50 g) was incubated with papain 2000 IU/gram (5 g) in 1 L of 0.1M sodium acetate buffer (pH 6.0) containing 5 mM EDTA and 5 mM cysteine at

60 oC for 24 hours The obtained mixture was centrifuged at 6000 rpm for 30 mins, discarded the pellet Glycosaminoglycans from the supernatant were precipitated with 10 % hexadecyltrimethylammonium bromide solution (cetavlon) and left overnight at 4 oC The precipitate was collected by centrifugation at 6000 rpm for 30 min, washed with distilled water then redissolved with a solution of 3N NaCl in 20 % ethanol (5 × 100 mL) for 3 days After that adding 3 volumes of chilled ethanol to reprecipitate This precipitate was collected by centrifugation, washed with ethanol 75 % (3 × 250 mL) and dissolved in distilled water, dialyzed, concentrated under a vacuum and lyophilized to obtain crude glycosaminoglycans

* Purification of glycosaminoglycans: the crude glycosaminoglycans were fractionated and

purified by using anion-exchange chromatography on the DEAE-Macro Prep column (1.6 × 40 cm) equilibrated with 0.1M sodium acetate buffer (pH 5) The column was washed with 200 ml

of sodium acetate buffer Then, the glycosaminoglycanfractions were successively eluted with a linear gradient of NaCl (from 0.1 to 2 M) in 0.5 M sodium acetate (pH 5) The fractions were collected at 10 mL, each eluted fraction was tested for carbohydrate content using the phenol-sulphuric acid method [10] Two glycosaminoglycans fractions (F1 and F2) were obtained from the fractionation of crude glycosaminoglycans (Figure 1)

2.2 Structural characteristics

2.2.1 Chemical composition

 Sulfate content: Sulfate content was determined by turbidity method with BaCl2/Gelatin [11], using dipotassium sulfate (K2SO4) as a standard The standard curve was plotted for different concentrations of K2SO4 (Merck)

 Protein content: The protein content of each glycosaminoglycan fraction was determined

by Lowry assay [12]

 Uronic acid content: Uronic acid content was determined by the colorimetric method with

carbazole reagent and D-glucuronic acid (GlcA) as a standard [13]

 Monosaccharide composition: The monosaccharide composition of the glycosaminoglycan

fractions was determined by ion chromatography (HPAEC-PAD), with 10 mg of each fraction was hydrolyzed with 2M trifluoroacetic acid (TFA) at 100 oC for 6 hours The Dionex ICS-6000 system with a guard MA1 (50 mm × 4) and analytical column MA1 (250

mm × 4), AS-DV module, Pulsed Amperometric Detector The operating conditions were mobile phase NaOH 1M with flow rate 0.4 ml/min and the time analysis was 30 min The standard curve was plotted for different concentrations of N-Acetyl-Galactosamine and Fucose

2.2.2 Structural analysis

 FTIR method: The glycosaminoglycans fractions were pressed with KBr in a mass ratio of

1:2 The FTIR infrared spectrum of glycosaminoglycans was recorded on Tensor 27 spectrometer (Bruker, Germany) of Institute of Chemistry, Vietnam Academy of Science and Technology

 NMR method: Nuclear magnetic resonance (NMR) spectra 1

recorded on Bruker 500 MHz NMR spectrometer (Germany) at the Institute of Chemistry, Vietnam Academy of Science and Technology The glycosaminoglycan fractions were mixed in D2O with a concentration of 20 μg/ml, measured at a frequency of 500 MHz at a temperature of 70 oC

3 RESULTS AND DISCUSSION 3.1 Isolation and purification

Figure 1 Fractionation of glycosaminoglycans from body wall of sea cucumber S horrens by anion

exchange chromatography on the DEAE-Macro Prep column

Glycosaminoglycans were extracted from the body wall of sea cucumber S horrens by

using papain to destroy bonds between glycosaminoglycans and proteins [14] The crude glycosaminoglycans obtained from the extract by precipitate with four volumes of ethanol 98 %, the yield of glycosaminoglycan was 4.2 % by dry weight equivalent polysaccharide sulfate

content (4.41 %) from S horrens collected in Russia [4] This result was significantly higher

than the content of glycosaminoglycan (polysaccharide sulfate) from other sea cucumbers such

as: Apostichopus japonicas 1.7 % [15]; Stichopus japonicas 0.62 % [6]; but lower than the GAG content obtained from sea cucumbers of the same genus Stichopus tremulus 7.0 %, Stichopus

variegatus 6.8 % [3, 9]

Glycosaminoglycans were fractionated and purified by anion-exchange chromatography on the DEAE-Macro Prep column to give two fractions F1 and F2 (Figure 1) with yield ratios were 16.7 % (w/w) and 22.6 % (w/w), respectively These fractions were further analyzed of

compositions and structural characteristics by different analytical methods

3.2 Structural characteristics

3.2.1 Chemical composition

Monosaccharide composition determined by HPAEC-PAD (High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detector) after glycosaminoglycan fractions hydrolyzed with TFA (Figure 2) Glucuronic acid (GlcA) content was determined by the colorimetric method with carbazole reagent The results were given in Table 1 Three monosaccharides including fucose (Fuc), N-Acetyl-Galactosamin (GalNAc) and Glucuronic acid (GlcA) with different molar ratio (1: 0.63: 0.41) constituted in fraction F1 On the contrary, fraction F2 content only one fucose residue

Figure 2 HPAEC-PAD chromatogram of monosaccharide composition: standards (A), fraction F1 (B)

and fraction F2 (C)

A

B

C

Table 1 Chemical composition of glycosaminoglycan fractions

Glycosaminoglycans

fractions

Protein (% w/w)

Sulfate (% w/w)

Uronic acid (% w/w)

Monosaccharide composition

(molar ratio) Fuc GalNAc GlcA

nd: not detected;

Beside of monosaccharide composition, F1 contained different amounts of sulfate, uronic acid and protein were 47.4 %, 4.7 %, and5.01 %, respectively Uronic acid and protein were not detected in F2, sulfate content of this fraction was 48.1 % F2 fraction consisted of fucose and sulfate groups assigned fucan sulfate; while, F1 contained three monosaccharide residues (Fuc, GalNAc, GlcA) and sulfate groups classified as fucosylated chondroitin sulfate [3, 4, 15] Previous studies reported that sea cucumber glycosaminoglycans usually divided into two types (Fucosylated Chondroitin Sulfate-FCS and Fucan Sulfate-FS) such as glycosaminoglycans

(polysaccharides) from sea cucumbers H nobilis, A japonica [16]; H Polii [17]; A

molpadioidea, H Nobilis [2]; P graeffei, H vagabunda, S tremulus, I badionotus [4], and H atra, H arenicola, S horrens [14], except polysaccharides from H edulis obtained three

fractions including two FCS fractions and one FS fraction [14]

3.2.2 Structural characteristics

* FT-IR

Figure 3 The IR spectra of F1 (A) and F2 (B) fractions from sea cucumber S horrens

C = O

C-O-S

OH

A

C 4 -O-S

S = O

OH

B

* IR spectroscopy: The IR spectrum of F1 and F2 fraction were shown in Figure 3, the

signals at 3448.95 and 3446.46 cm-1 with a broad intense peak that attributed to the stretching of O-H [18] The band at 2927.69 and 2937.36 cm-1 reflect the stretching vibration of C-H representing methyl group of fucose, that suggesting the presence of fucose in both fraction F1 and F2 [19] Signals at 1643 cm-1 and 1033 cm-1 (Figure 3A) were assigned to the asymmetric stretching vibration of C=O (carboxyl) and C-O-C, respectively, that belong to uronic acid group [18] The signal at 1641 cm-1 (Fig 3B) might be assigned to vibrations of crystallized water [18] Three signal groups in IR spectrum of F1 (Figure 3A) were assigned to the sulfate groups, particularly, those appeared at 1250.32 cm-1 correspond to S=O asymmetric stretching vibration, those at 825.37, 848.38 cm-1 were assigned to the symmetric C–O–S stretching vibration [18], which was the C4 axial position of the fucopyranose ring (Fuc or 4-O-sulfated-galnac), and those at 586.03 cm-1 was caused by S–O stretching vibration, which showed that the sulfate group also had a small amount at the C2 and/or C3 positions [18] IR spectrum of F2 (Figure 3B) showed strong absorptions at 1233 cm-1 (S=O) and 842 cm-1 to indicate the presence

of 4-O-sulfated-Fuc, as according to previous reports of fucan sulfate [2, 18]

* NMR spectroscopy: Similarly, the NMR spectrum of fucosylated chondroitin sulfate

(FCS) of other sea cucumber species in the world has been published [1, 14, 15, 17] The 1 H-NMR spectra of F1 and F2 fractions were also very complicated with many different signal regions since the structural composition contained many different sugars, besides, different degrees of sulfate and O-glycoside bonds The 1H-NMR spectrum (Figure 4A) showed strong signals at 2.05 ppm and 1.26 ppm, which were identified as the characteristic signals of methyl protons of GalNAc and Fuc, respectively The chemical shifts of proton anomeric signals at 5.0-5.8 ppm considered the presence of sulfated fucose residues [1] Two signals at 5.67 ppm and 5.4 ppm assigned to 2,4-O-disulfated fucose (Fuc2,4S) and 3,4-O-disulfated fucose (Fuc3,4S) [2] Thus, F1 contained two types of fucose residue with different sulfation patterns content,

which were similar to fucosylated chondroitin sulfate from sea cucumnbers H vaganbuda, H

polii [1, 17]

Figure 4 The 1H-NMR spectra of F1 (A) and F2 (B) fractions from sea cucumber S horrens

However, FCS from S horrens collected in Russia only consisted of one type of

2,4-O-disulfated fucose residue [20], it may be explained as due to the difference of extraction method and localization to chemical composition and structural characteristic of glycosaminoglycan [1,

2, 20] The proton signals at the region of 3.3 - 4.4 ppm (Figure 4A) were attributed to protons

of C2-C5 of glycosidic ring So that, it is difficult to give accurate information due to the

Fuc-CH 3

GalNAc-CO-CH 3

Fuc2S4S-H1

Fuc3,4S-H1

Fuc-CH 3

Fuc-H1

H2 - H5

overlapping of signals [1, 2] So, the structural backbone of F1 (Figure 5A) provide similar to

the backbone of fucosylated chondroitin sulfate from sea cucumber species [1, 20], the main

difference is in the sulfate group density and the position of the internal sulfate group in each

pyranose residues

H H

O O

R 3

O NH

CH 2 OR O

H O

O OH

COO-O -O 3 SO

R 2 R 1

CH 3

O

CH 3

OSO3

-H

O

H

R

Me

O

OSO3Na O OH

O H Me O

OSO3Na O

O H Me O

OSO3Na O

O H Me O H

OSO3

-H

O

H

R

Me

O

OSO3Na O

OH

O

Me

O

OSO3Na O

O

Me

O

OSO3Na O

O

Me

O

n

Figure 5 Structures of fucosylated chondroitin sulfate F1 (A) and Fucan sulfate F2 (B)

Similarly, the 1HNMR spectrum (Figure 4B) of F2 had the strong signals at about 1.20

-1.27 ppm assigned to the methyl protons of fucose residues (-CH3) [3] The chemical shifts of

the envelope of anomeric signals at 5.1 - 5.4 ppm were consistent with the existence of different

types of α-L-fucose units [7] The intense signals at 5.19 ppm assigned to 4-O-sulfated-Fucose

(Fuc4S), other weak signals at 5.3 - 5.4 ppm identified as 2-O-sulfated-Fucose (Fuc2S) [3, 7]

This result was also confirmed by the FT-IR spectrum Previous studies indicated that sea

cucumber fucans sulfate were built of different types of fucose residues [2, 3, 7, 9], except for

fucan sulfate from S horrens having only one type of Fuc2S residue [4]

Figure 6 The 13C-NMR spectrum of fraction F2 from sea cucumber S horrens

The 13C-NMR spectrum of F2 was shown in Figure 6, revealing that signals at 90 - 100 ppm

and 16.00 ppm chemical shift regions were characterized for anomeric carbon (C1) and C6

carbon methyl group (Fuc-CH3) of the fucose rings [3, 21] Simultaneously, the NMR spectra

(Figure 6 and Figure 4B) also appeared some signals at the chemical shifts 92.81 ppm (C1); 5.4

ppm (H1) and 96.84 ppm (C1); 5.19 (H1) confirmed the presence of (1→3)-linked α-Fuc2S and

(1→3)-linked α-Fuc4S units, respectively [5] The signals in the region 65 - 75 ppm showed that

Fuc-C1

Fuc-C6 C2 – C5

A

B

-the residual of carbon C2-C5 of -the fucose ring, -the similar result indicated in -the previous report

of fucan sulfate of S horrens [4]

4 CONCLUSION

Two fractions of glycosaminoglycans contained sulfate were isolated and purified from the

sea cucumber S.horrens These glycosaminoglycans were characterized by chemical and

spectroscopy methods F1 fraction consist of D-glucuronic acid, N-Acetyl-galactosamine, α-L-Fucose and sulfation groups F2 fraction classified to fucan sulfate composed of α-L-α-L-Fucose and sulfation groups, it’s structure was built of (1→3)-linked α-Fuc2S and (1→3)-linked α-Fuc4S residues (Figure 5B) Our results contributed to the knowledge on structural types of

glycosaminoglycans from sea cucumber S horrens in Viet Nam The establishment of structural

features plays an important role in further studies of the structure-bioactivity relationship of sea cucumber glycosaminoglycans

Acknowledgements: The research funding of this work supported by the National Foundation of Science

and Technology Development (NAFOSTED) under Grant number: 106.02-2019.34

Author contributions: Pham Duc Thinh and Duong Khanh Minh: designed the experiment, analyzed the

data and wrote the manuscript; Dinh Thanh Trung: obtained and purified glycosaminoglycans; Pham Duc

Thinh: revised the manuscript

Conflict statements: The authors declare no conflict of interest

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