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

Vietnam Journal of Science and Technology 58 (6A) (2020) 199-208 doi:10.15625/2525-2518/58/6A/15527 ISOLATION, PURIFICATION AND STRUCTURAL CHARACTERISTICS OF GLYCOSAMINOGLYCANS FROM SEA CUCUMBER STICHOPUS HORRENS Pham Duc Thinh1, 2, *, Duong Khanh Minh2, 3, Dinh Thanh Trung1 Nhatrang Institute of Research and Application, VAST, 02 Hung Vuong, Nha Trang, Viet Nam Graduate University of Science and Technology, VAST, 18 Hoang Quoc Viet, Ha Noi, Viet Nam Institue of Vaccine and Medical Biologicals, Pasteur, Nha Trang, Viet Nam * Email: ducthinh.nitra@gmail.com,; ducthinh@nitra.vast.vn Received: 20 September 2020; Accepted for publication: 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 structurebioactivity relationship of sea cucumber glycosaminoglycan Keywords: glycosaminoglycans, fucosylated chondroitin sulfate, fucan sulfate, sea cucumber, Stichopus horrens Classification numbers: 1.1.1, 1.5.1 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 Pham Duc Thinh, Duong Khanh Minh, Dinh Thanh Trung Glycosaminoglycan extracted from various sea cucumbers have different sulfation patterns of fucose and N-Acetyl-galactosamine leading to different bioactivities [2 - 4] 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 (1→3)-linked 4-O-sulfated α-L-fucopyranosyl and (1→3)-linked 2-O-sulfated α-Lfucopyranosyl residues 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 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 L of 0.1M sodium acetate buffer (pH 6.0) containing mM EDTA and 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 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 days After that adding 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 glycosaminoglycan fractions were successively eluted with a linear gradient of NaCl (from 0.1 to 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 phenolsulphuric acid method [10] Two glycosaminoglycans fractions (F1 and F2) were obtained from the fractionation of crude glycosaminoglycans (Figure 1) 2.2 Structural characteristics 200 Isolation, purification and structural characteristics of glycosaminoglycans from sea 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 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 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 1H-NMR and 13C-NMR were 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 RESULTS AND DISCUSSION 3.1 Isolation and purification Figure 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 201 Pham Duc Thinh, Duong Khanh Minh, Dinh Thanh Trung 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 AnionExchange 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 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 A B C Figure HPAEC-PAD chromatogram of monosaccharide composition: standards (A), fraction F1 (B) and fraction F2 (C) 202 Isolation, purification and structural characteristics of glycosaminoglycans from sea Table Chemical composition of glycosaminoglycan fractions Glycosaminoglycans fractions Protein (% w/w) Sulfate (% w/w) Uronic acid (% w/w) F1 F2 5.01 nd 47.4 48.1 4.7 nd Monosaccharide composition (molar ratio) Fuc 1 GalNAc 0.63 nd GlcA 0.41 nd nd: not detected; Beside of monosaccharide composition, F1 contained different amounts of sulfate, uronic acid and protein were 47.4 %, 4.7 %, and 5.01 %, 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 A C-O-S C=O S=O OH B C4-O-S S=O OH Figure The IR spectra of F1 (A) and F2 (B) fractions from sea cucumber S horrens 203 Pham Duc Thinh, Duong Khanh Minh, Dinh Thanh Trung * 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 (4-O-sulfated-Fuc or 4-O-sulfatedgalnac), 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 1HNMR 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 5.05.8 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] B A Fuc-CH3 Fuc-CH3 H2 - H5 Fuc3,4S-H1 Fuc2S4S-H1 GalNAc-CO-CH3 Fuc-H1 Figure 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-Odisulfated 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 204 Isolation, purification and structural characteristics of glycosaminoglycans from sea 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 A COO- CH2 OR B O R3 O H O H O H H O NH OH CH3 R, R1 = H or SO3R2 = H, SO3- or Fuc -O3 SO R1 R2 OSO3- CH3 O Me O Me O O R O H HO R = H or SO3- n O Me Figure Structures of fucosylated chondroitin sulfate F1 (A) and Fucan sulfate F2 (B) OH O Me HO OSO 3Na Similarly, the 1H-NMR 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] H HO OSO 3Na OSO 3- R O H O O Me O Me HO O O Me OSO 3Na HO HO C2 – C5 Fuc-C6 Fuc-C1 Figure 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 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 205 HO O Pham Duc Thinh, Duong Khanh Minh, Dinh Thanh Trung 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] 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-LFucose and sulfation groups F2 fraction classified to fucan sulfate composed of 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 REFERENCES Chen S G., Xue C H., Yin L A., Tang Q J., Yu G L., Chai W G - Comparison of structures and anticoagulant activities of fucosylated chondroitin sulfates from different sea cucumbers, Carbohydrate Polymers 83 (2) (2011) 688-896 http://doi.org/10.1016/j.carbpol.2010.08.040 Dong X, Pan R, Deng X, Chen Y, Zhao G, Wang C - Separation purification, anticoagulant activity and preliminary structural characterization of two sufated polysaccharides from sea cucumber Acaudina molpadioidea and Holothuria nobilis, Process Biochemistry 49 (8) (2014) 1352-1361 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composition: standards (A), fraction F1 (B) and fraction F2 (C) 202 Isolation, purification and structural characteristics of glycosaminoglycans from sea Table Chemical... information due to the 204 Isolation, purification and structural characteristics of glycosaminoglycans from sea overlapping of signals [1, 2] So, the structural backbone of F1 (Figure 5A) provide