- Email: [email protected]
Study on thick-film PTC thermistor fabricated by micro-pen direct writing
International Journal of Biological Macromolecules 72 (2015) 699–705
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Depolymerized glycosaminoglycan and its anticoagulant activities from sea cucumber Apostichopus japonicus Jie Yang a,b , Yuanhong Wang a,b , Tingfu Jiang a,b , Lv Lv a,b , Boyuan Zhang a,b , Zhihua Lv a,b,∗ a b
Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China Shandong Provincial Key Laboratory of Glycoscience & Glycotechnology (Ocean University of China), Qingdao 266003, China
a r t i c l e
i n f o
Article history: Received 1 August 2014 Received in revised form 26 August 2014 Accepted 9 September 2014 Available online 23 September 2014 Keywords: Apostichopus japonicus Fucosylated chondroitin sulfate Free-radical depolymerization
a b s t r a c t A controlled Cu2+ catalytic free-radical depolymerization process of fucosylated chondroitin sulfate from sea cucumber Apostichopus japonicus was established. The results showed a good linear relationship between 1/Mw and time during the depolymerization. A series of fractions with different molecular weight were obtained, and the physicochemical properties of them were investigated and compared utilizing the chemical method, IR spectra and NMR spectra. The results showed no significant variations of the backbone and branches structures during the depolymerization. Furthermore, the anticoagulant activities of the depolymerized fractions were evaluated by the activated partial thromboplastin time (APTT). The APTT decreases in proportion to the molecular weight following a linear relationship and the prolongation of APTT activity requires at least oligosaccharide of 4 trisaccharide units (about 4000 Da). Their anticoagulant activity of low molecular weight fraction (Mw = 24,755 Da) is similar to LMWH with significantly less bleeding risk. The results suggest that the low molecular weight fraction could be used as a novel anticoagulant with less undesired side effects. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Sea cucumbers have been used as a traditional tonic food in Asian countries for centuries. Holothurian glycosaminoglycan was isolated from the body wall of sea cucumber, which had a backbone of chondroitin sulfate and large amount of sulfated fucose branches attaching to the O-3 positon of -d-glucuronic and O-4 and/or O-6 positions of N-acetyl--d-galactosamine [1–4]. It also could be named as fucosylated chondroitin sulfate (fCS). The fCS is a highly sulfated polysaccharide, which has attracted considerable interest in recent years due to their potential therapeutic application, such as antitumor, antiviral and antithrombotic properties [5–7]. Furthermore, the fCS had been demonstrated to possess a heparin-like anticoagulant activity [1] with an undesirable effect of platelet aggregation [8]. However, low molecular weight fractions of fCS obtained by using oxidative depolymerization with hydrogen peroxide were efficient to minimize the side effect [9]. Besides, it will avoid the risk of contamination with pathogenic agents as a therapeutics
∗ Corresponding author at: Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China. Tel.: +86 532 82032064; fax: +86 532 82032064. E-mail address: [email protected] (Z. Lv). http://dx.doi.org/10.1016/j.ijbiomac.2014.09.025 0141-8130/© 2014 Elsevier B.V. All rights reserved.
prepared from non-mammalian sources [7]. Depolymerized holothurian glycosaminoglycan (DHG) is in clinical trials in Japan as a new antithrombotic agent, which exerts its anticoagulant activities through two different inhibitory activities: one being the HCII-dependent inhibition of thrombin and the other the ATIII- and HCII-independent inhibition of the activation of factor X by the factor IXa-factor VIIIa complex [10]. However, there were no reports about the chemical characteristics and biological activity of different molecular weight fractions during the depolymerization of fCS from the sea cucumber A. japonicas available until now. There are generally two different processes to obtain the low molecular weight fCS: by the enrichment of low molecular weight fractions of intact polysaccharide and by the chemical depolymerization. The chemical depolymerization methods involved the free-radical depolymerization [11–14], acid hydrolysis [15] and enzymatic depolymerization [16]. The free-radical depolymerization has shown to be an advantageous method to produce large quantities of low molecular weight fractions of glycosaminoglycan with narrow molecular weight distribution, and barely induce the abscission of sulfate ester and fucose branches. The possible mechanism of the reaction was that the hydroxyl radicals generated by hydrogen peroxide with the presence of Cu2+ or Fe2+ preferentially attacks the non-sulfated D-glucuronic acid residues, which causes the depolymerization of glycosaminoglycan [11]. The depolymerized glycosaminoglycan with different molecular
700
J. Yang et al. / International Journal of Biological Macromolecules 72 (2015) 699–705
weights and structures could possess different pharmacological properties. Recently, we isolated a novel fCS from the sea cucumber Apostichopus japonicus originated from Qingdao sea area of China (the intact fCS named AHG), which consisted of N-acetylgalactosamine (GalNAc), glucuronic acid (GlcUA), fucose (Fuc) and ester sulfate approximately with the molecular ratio of 1:1:1:3.8, respectively. The chemical composition and structure of the glycosaminoglycan is different from that of glycosaminoglycan from Stichopus japonicus [9] and Ludwigothurea grisea [1]. Afterward, a novel controlled Cu2+ catalytic free-radical depolymerization of AHG has been established for the first time to obtain the low molecular weight fractions of AHG. And the productions have been studied by various tools to indicate their characteristics, compared with the intact AHG. Furthermore, the anticoagulant activities of the fucosylated chondroitin sulfate with different molecular weight are tested and compared with that of the low molecular weight heparin. And, the relationship between the molecular size of AHG and anticoagulant activities is discussed. 2. Materials and methods 2.1. Materials The sea cucumber A. japonicus was purchased in Nanshan market of Qingdao city, China. Diastase Vera (EC 3.3.21.4) was purchased from Xuemei Zymin Technology Ltd. (Wuxi, China). Chondroitin ABC lyase (EC 4.2.2.4) from Proteus vulgaris and low molecular weight heparin (Mw 4000–6000 Da, average molecular weight 3500 Da) were purchased from Sigma (USA). Chondroitin sulfates family disaccharide standards, including ␣-UA-1→3-GalNAc (Di-0S), ␣-UA-1→3-GalNAc(4-SO4 ) (Di-4S), ␣-UA-1→3-GalNAc(6-SO4 ) (Di-6S) and␣-UA1→3-GalNAc(4,6-diSO4 ) (Di-diSE ), were obtained from Iduron (Manchester, England). All other chemical reagents were of analytic grade from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). 2.2. Isolation and purification of intact AHG The AHG was extracted from the body wall of the sea cucumber A. japonicus, purchased from the market of Qingdao, China. The body wall of fresh sea cucumber A. japonicus (1 kg) was carefully separated from other tissues, grinded into homogenate and diluted to 1 L with water. The mixture were treated with KOH and kept at 60 ◦ C for 60 min. After neutralization with cold HCl, diastase vera (EC 3.3.21.4) was added to hydrolyze the protein. The crude polysaccharide was obtained by precipitation with 60% ethanol. The crude AHG was further fractionated by a Q Sepharose Fast Flow column (300 mm × 30 mm) coupled with peristaltic pump, eluted with a step-wise gradient of 0.75 and 1.5 M NaCl and detected by the phenol-sulfuric acid method [17]. The fractions eluted with 1.5 M NaCl were pooled, dialyzed and further purified on a Sephadex 25 column (100 cm × 2.6 cm) with deionized water at a flow rate of 0.3 mL/min. The major polysaccharides fractions were pooled and lyophilized. 2.3. Analysis of molecular weight The weight average molecular weights (Mw ) of glycosaminoglycans were determined by high performance gel permeation chromatography (HPGPC) and on a Waters UltrahydrogelTM Linear column (7.8 mm × 300 mm, Japan) with a Waters 2410 refractive index detector, eluted with 0.2 M Na2 SO4 at a flow rate of 0.5 mL/min. 20 L of 10 mg/mL sample dissolved in 0.2 M Na2 SO4 was injected. The molecular weight was calculated by a reference
to a calibration curve made by a series of dextran T-series standards (Mw : 133.8, 84.4, 41.1, 21.4, 10.0, 7.1 kDa) [18]. 2.4. Preparation of different molecular weight fragments from AHG by Cu2+ catalytic free-radical depolymerization The depolymerized AHG fragments were prepared by controlled free-radical depolymerization of AHG induced by Cu2+ , while varying different parameters (concentration of H2 O2 and Cu2+ , temperature). The following protocol is representative of the different experiments: the intact AHG (10 mg) was dissolved in 1 mL 50 mM H2 O2 (diluted in 100 mM K2 HPO4 solution, pH 7.5) containing 1 mM CuAc2 . The mixture was maintained at different temperature. The reaction was stopped by addition of 2 M NaOH with the pH adjusted to 10–11. The excess Cu2+ was removed by centrifugation (8000 × g for 10 min at room temperature) and the supernatant was purified by a Q Sepharose Fast Flow column (300 mm × 30 mm) with a step-wise gradient of 0, 0.5 and 1.5 M NaCl and detected by the phenol-sulfuric acid method [17]. The fractions eluted by 1.5 M NaCl were concentrated and desalted on a Sephadex G-25 column (100 cm × 2.6 cm). The depolymerized fractions of AHG (named DAHG) were obtained by concentration and subsequently lyophilization. For analysis of depolymerization extent, 100 L of reaction mixture was withdrew and mixed with 100 L 20 mM disodium ethylenediamine tetra-acetate dihydrate (EDTA·2Na) to stop the reaction at different time. A threefold of the volume of 95% (v/v) ethanol was added and the crude products were collected by centrifugation at 8000 rpm for 10 min. The residues were dissolved in 0.2 M Na2 SO4 solution and ready for the analysis of molecular weight by HPGPC. 2.5. Composition analysis of AHG and DAHGs The aldohexuronic and acetamidoxyhexose contents of native AHG and DAHGs were estimated by methods as described [19,20]. The fucose content was determined by gas chromatography (GC) [21]. Sulfate ester content was estimated according to the high performance capillary electrophoresis method reported [22]. The purity of AHG and DAHGs were determined by cellulose acetate membrane electrophoresis [23]. 2.6. Analysis of unsaturated disaccharides composition 2.6.1. Partial acid hydrolysis Partial removal of sulfated fucose branches from the glycosaminoglycan was performed by the modified method as described [1]. Briefly, 20 mg of glycosaminoglycan was dissolved in 1.0 mL of 150 mM H2 SO4 , kept at 100 ◦ C for 30 min. The reaction solution was adjusted to pH 7.0 with saturated aqueous Ba(OH)2 . The mixture was centrifuged to remove the precipitate and the supernatant was dialyzed against distilled water for 24 h. Afterward, the defucosylated AHG and DAHGs were obtained after lyophilization. 2.6.2. Analysis of the products formed by digestion of partially defucosylated products of AHG and DAHGs with Chondroitin ABC lyase The partial defucosylated AHG and DAHG (5 mg) was dissolved in 100 L distilled water. The sample was incubated at 37 ◦ C for 6 h by addition of 800 L 50 mM Tris/HCl buffer (pH 8.0) and 0.1 unit of chondroitin ABC lyase (EC 4.2.2.4) from P. vulgaris (Seikagaku American Inc., Rockville, MD) dissolved in 100 L Tris/HCl buffer. The reaction was stopped by maintained at 100 ◦ C for 5 min. The mixture was centrifuged, and the supernatant was prepared for analysis.
J. Yang et al. / International Journal of Biological Macromolecules 72 (2015) 699–705
701
The identification and quantitation of each disaccharide were determined by high performance liquid chromatography as described [24]. The analysis was performed on a Waters Spherisorb S5 NH2 column (2.0 mm × 150 mm) at 30 ◦ C with detection at UV 232 nm. The mobile phase was 10 mM NaH2 PO4 solution (solvent A) and 400 mM NaH2 PO4 (solvent B). A gradient of B was performed as follow: 0% to 100% in 50 min. The identification and quantitation of each disaccharide was done by comparison with reference disaccharides (Di-0S, Di-4S, Di-6S, Di-diSE ). 2.7. Spectrometry analysis For IR spectroscopy, the samples were mixed with dried KBr, ground and made to a 1 mm pellets for Fourier-transform infrared (FT-IR) spectrum measurement in the frequency range of 4000–500 cm−1 . FT-IR spectrum was taken on a Nicolet Nexus 470 spectrometer. 1 H nuclear magnetic resonance (NMR) and 13 C NMR spectra of AHG and DAHGs were performed at 25 ◦ C using a JEOL-ECP 600 MHz spectrometer. The sample (30 mg) were co-evaporated with 500 L D2 O (99.9%, Sigma–Aldrich) twice followed by dissolution in 500 L D2 O containing TMS as internal standard. 2.8. Anticoagulant activity of AHG and DAHGs in vitro The anticoagulant activities of intact AHG and depolymerized AHG (DAHGs) were performed on a coagulometer. Activated partial thromboplastin time (APTT, assay kit from Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) was carried out according to the method as described [1,25]. Briefly, the lamb plasma (90 L) was mixed with 10 L of a solution of different amounts of polysaccharide in 0.9% NaCl, and incubated at 37 ◦ C for 5 min after addition of 100 L pre-warmed APTT assay regent. 100 L of pre-warmed calcium chloride (0.25 M) was added and the clotting time was recorded as the time for clot formation in a coagulometer. The results were compared with the low molecular weight heparin standard. 3. Results and discussion 3.1. Preparations of different molecular weight fragments of fucosylated chondroitin sulfate from A. japonicus A neutral pH is required for an optimized depolymerization to avoid the acidic or basic hydrolysis of the polysaccharides [11,14]. During depolymerization process, a sudden decrease of pH could be observed at the beginning due to the chelation process between the polysaccharide and the added Cu2+ . And, the pH was remained at 6.3–6.7 depending on the initial concentration of the added Cu2+ . With pH control by K2 HPO4 buffer, a good reproducibility was obtained by analyzing the molecular weight of depolymerized products at different time (the relative standard deviation (RSD %) ≤4.60, n = 3, data not shown). The influence of the concentration of Cu2+ on the depolymerization process was observed. It showed that high amount of Cu2+ did not improve the reaction, even suppress the depolymerization on the contrary (Fig. 1). As a catalyst of free radical reaction, trace amount of Cu2+ would catalyze the depolymerization of large quantity of samples. The concentration of H2 O2 and temperature were critical to the depolymerization process. The radical depolymerization was carried out through the generation of the free radicals from hydrogen peroxide catalyzed by Cu2+ . The amount of radicals would be efficiently affected by the concentration of H2 O2 and temperature. And, the rate of depolymerization would be increased with the increasing of H2 O2 concentration and temperature.
Fig. 1. Influence of Cu2+ concentration on depolymerization, for an AHG concentration of 10 mg/mL and H2 O2 concentration of 50 mM at 35 ◦ C.
The free radical cleavages rate of chain scission can be anticipated to be a linear function of time (first order reaction) as in the following equation [13,26]. 1 1 − = (k/m0 )t Mn (t) Mn (0) In the equation, Mn (0) is the initial weight average; Mn is obtained from regression; and Mn (t) is Mn during free radical depolymerization at t min, respectively. Meanwhile, k is the first order rate constant for change of weight average molecular weight, and the m0 is the molecular mass of a monomer unit. A good linear relationship of 1/Mn and reaction time was observed in present work (Fig. 2). The results indicated that the cleavage of the chain of AHG followed a first order reaction, and the rate constant k was supposed to be 1.276 × 10−5 min−1 by using m0 = 1019 g/mol. The previous reports [27] predicted that the change of polydispersity index (Q) resulting from random chain scission: whether if the polydispersity index of initial polymer is above/below 2.0, it will change to 2.0. The same results were obtained in present study (data not shown), which suggesting that the cleavage of the main chain of AHG is random. In present work, the different concentrations of H2 O2 with 25, 50 and 100 mM at different temperature (35, 45 and 55 ◦ C) were analyzed (data not shown). Ultimately, the reaction system of the intact AHG (10 mg/mL) dissolved in 50 mM H2 O2 (pH 7.5) containing 1 mM CuAc2 at 45 ◦ C was optimized to product a series of low
Fig. 2. Formation of change of the molecular weight (1/Mw ) against time upon depolymerization of glycosaminoglycan (10 mg/mL) in 50 mM H2 O2 (pH 7.5) with 1 mM CuAc2 at 45 ◦ C.
702
J. Yang et al. / International Journal of Biological Macromolecules 72 (2015) 699–705
Table 1 Physicochemical characteristics of intact AHG and purified different molecular weight DAHGs. Polysaccharide
Intact AHG
DAHG-1
DAHG-2
DAHG-3
Mw (Da)
98070
41149
24755
8871
Chemical composition (molar ratio) 1.00 GalNAc GlcUA 1.06 Fuc 0.88 Sulfate 3.85
1.00 0.98 0.87 3.83
1.00 1.00 0.90 3.88
1.00 0.96 0.84 3.60
molecular weight fractions with Mw from 68.54 kDa to 8.87 kDa were obtained applying different reaction conditions. 3.2. Chemical composition of AHG and DAHGs Three different low molecular weight fractions of AHG were obtained for large quantities with Mw of 41149, 24755 and 8871 Da, respectively. The intact AHG and DAHGs have been analyzed by different analytical techniques to compare their physicochemical properties before and after free radical depolymerization as shown in Table 1. The monosaccharide and sulfation content analysis of AHG and DAHGs shows almost no variations. Only the content of GlcUA is very slightly lower in the DAHG-3. The fucose branches were more sensitive to acid than the core structure of chondroitin sulfate formed by aldohexuronic and acetamidoxyhexose, and removed from the polysaccharide by mild acid hydrolysis [1]. The analysis of unsaturated disaccharides formed by Chondroitin ABC digestion of acid-resistant fragments of intact AHG and DAHGs showed three sharp peaks, which agreed with those of Di-6S, Di-4S, Di-diSE in disaccharide standards, respectively (Fig. 3). The molar ratio of the disaccharides in the above order was 1.00: 0.20: 1.08 in AHG. However, the proportion of disaccharides generated from enzyme digestion of defucosylated DAHGs (DAHG-3 as representative) were similar to that of AHG, which confirmed the retention of the backbone structure of intact AHG during the depolymerization.
Fig. 3. HPLC chromatography for analysis of core disaccharide units of AHG and DAHGs. A: Disaccharide standards mixture; B: the disaccharides constitution of AHG. C: DAHG-3. The numbered peaks correspond to known disaccharide standards as follows: peak 1, Di-0S; peak 2, Di-6S; peak 3, Di-4S; peak 4, Di-diSE . x and y are the contaminants from the reaction system.
Fig. 4. The cellulose acetate membrane electrophoresis of chondroitin sulfate (1), AHG (2), DAHG-1 (3), DAHG-2 (4) and DAHG-3 (5).
The cellulose acetate membrane electrophoresis as in Fig. 4 showed that the basic physicochemical properties of AHG and DAHGs had no significant difference, which indicated that the primary structure was not destroyed during the depolymerization. The IR spectra of AHG and DAHGs (Fig. 5A) showed no variations between intact AHG and DAHGs. The absorptions at 1239 cm−1 (vS O ) and 820–860 cm−1 (vC–O–S ) confirmed the presence of sulfates, and the latter ones might indicate the pattern of the sulfates [28,29]: the absorption at 822 cm−1 for the presence of 2,4O-disulfated fucose or 6-O-sulfated GalNAc; and the signal of 852 cm−1 for the presence of 4-O-sulfated Fuc and/or GalNAc. The signal at 3461 cm−1 and 1031 cm−1 were attributed to the stretching vibration of O–H and C–O, respectively. Furthermore, signals at 1635 cm−1 was due to the asymmetric stretch vibration of C O of N-acetylgalactosamine and glucuronic acid; 1409 cm−1 , the symmetric stretch vibration of COO− of glucuronic acid and the stretch vibration of C–O within COOH; 2926 cm−1 , the stretch vibration of C–H. These data indicated that the structures of intact AHG and DAHGs were accordance and no major functional group transformations were found during the depolymerization. The structural consistency of intact AHG and DAHGs was further confirmed by 1 H NMR (Fig. 5B) and 13 C NMR (Fig. 5C) spectroscopy. The comparison of 1 H NMR between the intact AHG and DAHGs showed a coincidence among almost all signals. The chemical shift between 5.0 and 6.0 ppm were the signals of anomeric H of sulfated ␣-fucose: the signal of ı 5.56 ppm represented the H1 of 2,4-diSO3 -fucose, and that of ı 5.22 ppm was H1 of 3,4diSO3 -fucose. The chemical shift and proportion of them were almost equal. Signals of ı = 4.32 ppm (U-H1) and 4.45 ppm (AH1) were attribute to the anomeric H of GlcUA and GalNAc, which showed no differences between the intact AHG and DAHGs. And the same results could be found in the signals of methyl residue of GalNAc and Fuc with ı = 1.93 and 1.24 ppm. Furthermore, the comparison in 13 C NMR spectra showed a coincidence at the region of carbon ring (ı = 50–85 ppm) and anomeric C of GalNAc (A-C1, ı = 101.2 ppm), GlcUA (U-C1, ı = 105.3 ppm) and Fuc (F-C1, ı = 97.7 ppm). The spectra of AHG and DAHGs also showed signals assigned to carbonyl (C O), carbon-2 (A-C2), sulfated carbon-6 (A-C6) and acetamide-methyl (CO–CH3 ) of GalNAc moiety, which resonated at approximately ı = 176.9, 52.8, 67.8 and 23.9 ppm, respectively (ı = 176.9 and 23.9 ppm not shown).
J. Yang et al. / International Journal of Biological Macromolecules 72 (2015) 699–705
703
Fig. 5. IR (A), 1 H NMR (B) and 13 C NMR (C) spectra in D2 O of intact glycosaminoglycan AHG and its depolymerized products DAHGs. Signals labeled with “A” refer to those generated by acteamidodeoxyhexose residues, whereas those of uronosyl residues are labeled with “U”, and those of fucose residues are labeled with “F”.
704
J. Yang et al. / International Journal of Biological Macromolecules 72 (2015) 699–705
Fig. 6. (A) APTT of different molecular weight AHGs obtained by the free radical depolymerization as a function of Mw . The equation of experimental curve and its correlation coefficient is shown. The concentrations of the samples were 250 g/mL. (B) Comparison of the anticoagulant activities of DAHG-2 and LMWH as function of their concentrations. The equations of the linear curves and their coefficients are shown.
dermatan sulfate from beef mucosa expressed as APTT units [31] and the logarithmic-like function of their APTT activities and different molecular weight (Mw ) of chemical depolymerized fucosylated chondroitin sulfate from sea cucumber T. ananas [13]. The anticoagulant activities of the oligosaccharides with different molecular weight decreased with the reduction in molecular weight, while their primary structures were similar to those of the native AHG. Previous studies have indicated that the anticoagulant activity of glycosaminoglycan from sea cucumber mostly depends on the molecular size of the glycosaminoglycan and the sulfated fucose branches [1,2,4,5,13]. The potentiation of thrombin inhibition and prolongation of APTT activity by heparin require at least an oligosaccharide chain of about 16–18 units [32]. Fragments of dermatan sulfate containing a minimum of 12–14 monosaccharide residues are necessary to increase the inhibition of thrombin by heparin cofactor II (HCII) [31]. Meanwhile, the low molecular weight glycosaminoglycan needs at least an oligosaccharide chain of about 6–8 units (Mw = 6000–7000 Da), which obtained from chemical depolymerization of polysaccharide from sea cucumber T. ananas [13]. In present work, we prepared a broad series of oligosaccharides from chemical depolymerization of glycosaminoglycan obtained from sea cucumber A. japonicus. It is indicated that the potentiation of prolongation of APTT activity by AHG requires a minimum of 4–6 units (Mw = 4000–5000 Da). In contrast to heparin and low molecular weight heparin, depolymerized holothurian glycosaminoglycan lacks the antithrombin-dependent activities that contribute to increased bleeding risk [8]. The relationship of the anticoagulant activity in vitro of DTHG-2 (Mw = 24755 Da) and the concentration was investigated in APTT assay comparing to the low molecular weight heparin (LMWH, Mw = 3500 Da) with some concentration. The APTT activities of both DTHG-2 and LMWH have a linear relationship with their concentration (Fig. 6B). Their anticoagulant activity are almost the same, but the DAHG-2 could have significantly less bleeding risk compared with LMWH at equivalent therapeutic does. The results suggest that the DAHG could be used as a novel anticoagulant with less undesired side effects.
4. Conclusions The fucose branches were essential to the anticoagulant activity of the fucosylated chondroitin sulfate [1,2,30]. The signals of the anomeric carbons, ring carbons and CH3 of ␣-fucose among AHG and DAHGs have no variation, which resonate at ı = 97.7, 85.0–65.0 and 17.1 ppm, respectively. All data above indicate that the basic structure of DAHGs is similar to the native AHG. There were no significant variations detected for the monosaccharide composition, the sulfate content, the core disaccharide composition, IR spectra, and 1 H/13 C NMR spectra between intact AHG and DAHGs. The results showed that the primary structure of intact AHG was well retained during the depolymerization, as previously reported for heparin [12] and depolymerized glycosaminoglycan from Thelenata ananas [13]. The Cu2+ induced free radical depolymerization reaction indicated to be an efficient way to obtain the depolymerized different molecular weight fragments without destroying the primary structure of fucosylated chondroitin sulfate from A. japonicus. 3.3. Anticoagulant activity The APTT of intact AHG and DAHGs with different molecular weight were evaluated on their anticoagulant activities. The APTT activities decreased in proportion to the weight-average molecular weight (Mw ) of AHG following a linear relationship (Fig. 6A). The results are different to that of different relative molecular mass (Mr )
In present work, we firstly prepared a broad series of glycosaminoglycans with different molecular weight from the controlled free-radical depolymerization of polysaccharide obtained from the sea cucumber A. japonicas originated from Qingdao, China. The physicochemical properties of the depolymerized fragments are similar to the intact glycosaminoglycan isolated from A. Japonicus. The Cu2+ catalyzed free-radical depolymerization process showed to be an efficient way to prepare the low molecular weight glycosaminoglycan without destroying the primary structure of polysaccharide from sea cucumber A. Japonicus. Moreover, the results of anticoagulant activities indicate that the APTT activities of different molecular weight fragments are followed a linear relationship in proportion to the molecular weight (Mw ), and the activity requires molecular weight above 4000 Da. The low molecular weight glycosaminoglycan could inhibit the multiple pathways of coagulant and thrombin activity or conversion of fibrinogen to fibrin with slight bleeding risks, which makes it a potential therapeutic anticoagulant.
Acknowledgment This research was funded by Natural Science Foundation of China (31171665).
J. Yang et al. / International Journal of Biological Macromolecules 72 (2015) 699–705
References [1] P.A.S. Mourão, M.S.G. Pereira, B. Mulloy, D.M. Tollefsen, M.C. Mowinckel, U. Abildgaard, J. Biol. Chem. 271 (1996) 23973–23984. [2] S.G. Chen, C.H. Xue, L.A. Yin, Q.J. Tang, G.L. Yu, W.G. Chai, Carbohydr. Polym. 83 (2011) 688–696. [3] R.P. Vieira, B. Mulloy, P.A.S. Mourão, J. Biol. Chem. 266 (1991) 13530–13536. [4] Y. Kariya, S. Watabe, M. Kyogashima, M. Ishihara, T. Ishii, Carbohydr. Res. 297 (1997) 273–279. [5] L. Borsig, L. Wang, M.C. Cavalcante, L. Cardilo-Reis, P.L. Ferreira, P.A.S. Mourão, J.D. Esko, M.S.G. Pavão, J. Biol. Chem. 282 (2007) 14984–14991. [6] N. Huang, M.Y. Wu, C.B. Zheng, L. Zhu, J.H. Zhao, Y.T. Zheng, Carbohydr. Res. 380 (2013) 64–69. [7] P.A.S. Mourão, B. Giumaraes, B. Mulloy, S. Thomas, E. Gray, Br. J. Haematol. 101 (1998) 647–652. [8] J.Z. Li, E.C.Y. Lian, J. Thromb. Haemost. 59 (1988) 435–439. [9] K. Yoshida, Y. Minami, H. Nemoto, K. Numata, E. Yamanaka, Tetrahedron Lett. 33 (1992) 4959–4962. [10] H. Nagase, K. Enjyoji, K. Minamiguchi, K.T. Kitazato, K. Kitazato, H. Saito, H. Kato, Blood 85 (1995) 1527–1534. [11] A.C. Petit, N. Noiret, C. Sinquin, J. Ratiskol, J. Guézennec, S.C. Jouault, Carbohydr. Polym. 64 (2006) 597–602. [12] N. Volpi, G. Mascellani, P. Bianchini, Anal. Biochem. 200 (1992) 100–107. [13] M.Y. Wu, S.M. Xu, J.H. Zhao, H. Kang, H. Ding, Food Chem. 122 (2010) 716–723. [14] M.Y. Wu, S.M. Xu, J.H. Zhao, H. Kang, H. Ding, Carbohydr. Res. 345 (2010) 649–655.
705
[15] A. Karlsson, S.K. Singh, Carbohydr. Polym. 38 (1999) 7–15. [16] Y. Cheng, K.M. Brown, R.K. Prud’homme, Int. J. Biol. Macromol. 31 (2002) 29–35. [17] T. Masuko, A. Minami, N. Iwasaki, T. Majima, S. Nishimura, Y.C. Lee, Anal. Biochem. 339 (2005) 69–72. [18] W.J. Mao, F. Fang, H.Y. Li, X.H. Qi, H.H. Sun, Y. Chen, S.D. Guo, Carbohydr. Polym. 74 (2008) 834–839. [19] J.L. Reissig, T.L. Strominger, I.F. Leloir, J. Biol. Chem. 217 (1955) 959–966. [20] N. Blumenkrantz, G. Asboe-Hansen, Anal. Biochem. 54 (1973) 484–489. [21] W. Zhang, H.B. He, X.D. Zhang, Soil Biol. Biochem. 39 (2007) 2665–2669. [22] X.Q. Zhan, Z.J. Zhu, J.W. Kang, Chin. J. Chromatogr. 29 (2011) 362–364. [23] M. Yokoyama, H. Tanaka, S. Yoshihara, M. Endo, J. Biochem. Biophys. Methods 12 (1986) 239–246. [24] K. Yoshida, S. Miyauchi, H. Kikuchi, A. Tawada, K. Tokuyasu, Anal. Biochem. 177 (1989) 327–332. [25] H.J. Zhang, W.J. Mao, F. Fang, H.Y. Li, H.H. Sun, Y. Chen, X.H. Qi, Carbohydr. Polym. 71 (2008) 428–434. [26] V.M.F. Lai, C. Lii, W.L. Hung, T.J. Lu, Food Chem. 68 (2000) 319–325. [27] J.S. Yoon, I.J. Chin, M.N. Kim, C. Kim, Macromolecules 29 (1996) 3303–3307. [28] G. Bernardi, G.F. Springer, J. Biol. Chem. 237 (1962) 75–80. [29] M.E.R. Duarte, M.A. Cardoso, M.D. Noseda, A.S. Cerezo, Carbohydr. Res. 333 (2001) 281–293. [30] M.Y. Wu, R.R. Huang, D.D. Wen, N. Gao, J.B. He, Z. Li, J.H. Zhao, Carbohydr. Polym. 87 (2012) 862–868. [31] N. Volpi, Carbohydr. Res. 255 (1994) 133–144. [32] J. Hirsh, T.E. Warkentin, S.G. Shaughnessy, S.S. Anand, J.L. Halperin, R. Raschke, et al., Chest 119 (2001) 64S–94S.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Depolymerized glycosaminoglycan and its anticoagulant activities from sea cucumber Apostichopus japonicus Jie Yang a,b , Yuanhong Wang a,b , Tingfu Jiang a,b , Lv Lv a,b , Boyuan Zhang a,b , Zhihua Lv a,b,∗ a b
Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China Shandong Provincial Key Laboratory of Glycoscience & Glycotechnology (Ocean University of China), Qingdao 266003, China
a r t i c l e
i n f o
Article history: Received 1 August 2014 Received in revised form 26 August 2014 Accepted 9 September 2014 Available online 23 September 2014 Keywords: Apostichopus japonicus Fucosylated chondroitin sulfate Free-radical depolymerization
a b s t r a c t A controlled Cu2+ catalytic free-radical depolymerization process of fucosylated chondroitin sulfate from sea cucumber Apostichopus japonicus was established. The results showed a good linear relationship between 1/Mw and time during the depolymerization. A series of fractions with different molecular weight were obtained, and the physicochemical properties of them were investigated and compared utilizing the chemical method, IR spectra and NMR spectra. The results showed no significant variations of the backbone and branches structures during the depolymerization. Furthermore, the anticoagulant activities of the depolymerized fractions were evaluated by the activated partial thromboplastin time (APTT). The APTT decreases in proportion to the molecular weight following a linear relationship and the prolongation of APTT activity requires at least oligosaccharide of 4 trisaccharide units (about 4000 Da). Their anticoagulant activity of low molecular weight fraction (Mw = 24,755 Da) is similar to LMWH with significantly less bleeding risk. The results suggest that the low molecular weight fraction could be used as a novel anticoagulant with less undesired side effects. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Sea cucumbers have been used as a traditional tonic food in Asian countries for centuries. Holothurian glycosaminoglycan was isolated from the body wall of sea cucumber, which had a backbone of chondroitin sulfate and large amount of sulfated fucose branches attaching to the O-3 positon of -d-glucuronic and O-4 and/or O-6 positions of N-acetyl--d-galactosamine [1–4]. It also could be named as fucosylated chondroitin sulfate (fCS). The fCS is a highly sulfated polysaccharide, which has attracted considerable interest in recent years due to their potential therapeutic application, such as antitumor, antiviral and antithrombotic properties [5–7]. Furthermore, the fCS had been demonstrated to possess a heparin-like anticoagulant activity [1] with an undesirable effect of platelet aggregation [8]. However, low molecular weight fractions of fCS obtained by using oxidative depolymerization with hydrogen peroxide were efficient to minimize the side effect [9]. Besides, it will avoid the risk of contamination with pathogenic agents as a therapeutics
∗ Corresponding author at: Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China. Tel.: +86 532 82032064; fax: +86 532 82032064. E-mail address: [email protected] (Z. Lv). http://dx.doi.org/10.1016/j.ijbiomac.2014.09.025 0141-8130/© 2014 Elsevier B.V. All rights reserved.
prepared from non-mammalian sources [7]. Depolymerized holothurian glycosaminoglycan (DHG) is in clinical trials in Japan as a new antithrombotic agent, which exerts its anticoagulant activities through two different inhibitory activities: one being the HCII-dependent inhibition of thrombin and the other the ATIII- and HCII-independent inhibition of the activation of factor X by the factor IXa-factor VIIIa complex [10]. However, there were no reports about the chemical characteristics and biological activity of different molecular weight fractions during the depolymerization of fCS from the sea cucumber A. japonicas available until now. There are generally two different processes to obtain the low molecular weight fCS: by the enrichment of low molecular weight fractions of intact polysaccharide and by the chemical depolymerization. The chemical depolymerization methods involved the free-radical depolymerization [11–14], acid hydrolysis [15] and enzymatic depolymerization [16]. The free-radical depolymerization has shown to be an advantageous method to produce large quantities of low molecular weight fractions of glycosaminoglycan with narrow molecular weight distribution, and barely induce the abscission of sulfate ester and fucose branches. The possible mechanism of the reaction was that the hydroxyl radicals generated by hydrogen peroxide with the presence of Cu2+ or Fe2+ preferentially attacks the non-sulfated D-glucuronic acid residues, which causes the depolymerization of glycosaminoglycan [11]. The depolymerized glycosaminoglycan with different molecular
700
J. Yang et al. / International Journal of Biological Macromolecules 72 (2015) 699–705
weights and structures could possess different pharmacological properties. Recently, we isolated a novel fCS from the sea cucumber Apostichopus japonicus originated from Qingdao sea area of China (the intact fCS named AHG), which consisted of N-acetylgalactosamine (GalNAc), glucuronic acid (GlcUA), fucose (Fuc) and ester sulfate approximately with the molecular ratio of 1:1:1:3.8, respectively. The chemical composition and structure of the glycosaminoglycan is different from that of glycosaminoglycan from Stichopus japonicus [9] and Ludwigothurea grisea [1]. Afterward, a novel controlled Cu2+ catalytic free-radical depolymerization of AHG has been established for the first time to obtain the low molecular weight fractions of AHG. And the productions have been studied by various tools to indicate their characteristics, compared with the intact AHG. Furthermore, the anticoagulant activities of the fucosylated chondroitin sulfate with different molecular weight are tested and compared with that of the low molecular weight heparin. And, the relationship between the molecular size of AHG and anticoagulant activities is discussed. 2. Materials and methods 2.1. Materials The sea cucumber A. japonicus was purchased in Nanshan market of Qingdao city, China. Diastase Vera (EC 3.3.21.4) was purchased from Xuemei Zymin Technology Ltd. (Wuxi, China). Chondroitin ABC lyase (EC 4.2.2.4) from Proteus vulgaris and low molecular weight heparin (Mw 4000–6000 Da, average molecular weight 3500 Da) were purchased from Sigma (USA). Chondroitin sulfates family disaccharide standards, including ␣-UA-1→3-GalNAc (Di-0S), ␣-UA-1→3-GalNAc(4-SO4 ) (Di-4S), ␣-UA-1→3-GalNAc(6-SO4 ) (Di-6S) and␣-UA1→3-GalNAc(4,6-diSO4 ) (Di-diSE ), were obtained from Iduron (Manchester, England). All other chemical reagents were of analytic grade from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). 2.2. Isolation and purification of intact AHG The AHG was extracted from the body wall of the sea cucumber A. japonicus, purchased from the market of Qingdao, China. The body wall of fresh sea cucumber A. japonicus (1 kg) was carefully separated from other tissues, grinded into homogenate and diluted to 1 L with water. The mixture were treated with KOH and kept at 60 ◦ C for 60 min. After neutralization with cold HCl, diastase vera (EC 3.3.21.4) was added to hydrolyze the protein. The crude polysaccharide was obtained by precipitation with 60% ethanol. The crude AHG was further fractionated by a Q Sepharose Fast Flow column (300 mm × 30 mm) coupled with peristaltic pump, eluted with a step-wise gradient of 0.75 and 1.5 M NaCl and detected by the phenol-sulfuric acid method [17]. The fractions eluted with 1.5 M NaCl were pooled, dialyzed and further purified on a Sephadex 25 column (100 cm × 2.6 cm) with deionized water at a flow rate of 0.3 mL/min. The major polysaccharides fractions were pooled and lyophilized. 2.3. Analysis of molecular weight The weight average molecular weights (Mw ) of glycosaminoglycans were determined by high performance gel permeation chromatography (HPGPC) and on a Waters UltrahydrogelTM Linear column (7.8 mm × 300 mm, Japan) with a Waters 2410 refractive index detector, eluted with 0.2 M Na2 SO4 at a flow rate of 0.5 mL/min. 20 L of 10 mg/mL sample dissolved in 0.2 M Na2 SO4 was injected. The molecular weight was calculated by a reference
to a calibration curve made by a series of dextran T-series standards (Mw : 133.8, 84.4, 41.1, 21.4, 10.0, 7.1 kDa) [18]. 2.4. Preparation of different molecular weight fragments from AHG by Cu2+ catalytic free-radical depolymerization The depolymerized AHG fragments were prepared by controlled free-radical depolymerization of AHG induced by Cu2+ , while varying different parameters (concentration of H2 O2 and Cu2+ , temperature). The following protocol is representative of the different experiments: the intact AHG (10 mg) was dissolved in 1 mL 50 mM H2 O2 (diluted in 100 mM K2 HPO4 solution, pH 7.5) containing 1 mM CuAc2 . The mixture was maintained at different temperature. The reaction was stopped by addition of 2 M NaOH with the pH adjusted to 10–11. The excess Cu2+ was removed by centrifugation (8000 × g for 10 min at room temperature) and the supernatant was purified by a Q Sepharose Fast Flow column (300 mm × 30 mm) with a step-wise gradient of 0, 0.5 and 1.5 M NaCl and detected by the phenol-sulfuric acid method [17]. The fractions eluted by 1.5 M NaCl were concentrated and desalted on a Sephadex G-25 column (100 cm × 2.6 cm). The depolymerized fractions of AHG (named DAHG) were obtained by concentration and subsequently lyophilization. For analysis of depolymerization extent, 100 L of reaction mixture was withdrew and mixed with 100 L 20 mM disodium ethylenediamine tetra-acetate dihydrate (EDTA·2Na) to stop the reaction at different time. A threefold of the volume of 95% (v/v) ethanol was added and the crude products were collected by centrifugation at 8000 rpm for 10 min. The residues were dissolved in 0.2 M Na2 SO4 solution and ready for the analysis of molecular weight by HPGPC. 2.5. Composition analysis of AHG and DAHGs The aldohexuronic and acetamidoxyhexose contents of native AHG and DAHGs were estimated by methods as described [19,20]. The fucose content was determined by gas chromatography (GC) [21]. Sulfate ester content was estimated according to the high performance capillary electrophoresis method reported [22]. The purity of AHG and DAHGs were determined by cellulose acetate membrane electrophoresis [23]. 2.6. Analysis of unsaturated disaccharides composition 2.6.1. Partial acid hydrolysis Partial removal of sulfated fucose branches from the glycosaminoglycan was performed by the modified method as described [1]. Briefly, 20 mg of glycosaminoglycan was dissolved in 1.0 mL of 150 mM H2 SO4 , kept at 100 ◦ C for 30 min. The reaction solution was adjusted to pH 7.0 with saturated aqueous Ba(OH)2 . The mixture was centrifuged to remove the precipitate and the supernatant was dialyzed against distilled water for 24 h. Afterward, the defucosylated AHG and DAHGs were obtained after lyophilization. 2.6.2. Analysis of the products formed by digestion of partially defucosylated products of AHG and DAHGs with Chondroitin ABC lyase The partial defucosylated AHG and DAHG (5 mg) was dissolved in 100 L distilled water. The sample was incubated at 37 ◦ C for 6 h by addition of 800 L 50 mM Tris/HCl buffer (pH 8.0) and 0.1 unit of chondroitin ABC lyase (EC 4.2.2.4) from P. vulgaris (Seikagaku American Inc., Rockville, MD) dissolved in 100 L Tris/HCl buffer. The reaction was stopped by maintained at 100 ◦ C for 5 min. The mixture was centrifuged, and the supernatant was prepared for analysis.
J. Yang et al. / International Journal of Biological Macromolecules 72 (2015) 699–705
701
The identification and quantitation of each disaccharide were determined by high performance liquid chromatography as described [24]. The analysis was performed on a Waters Spherisorb S5 NH2 column (2.0 mm × 150 mm) at 30 ◦ C with detection at UV 232 nm. The mobile phase was 10 mM NaH2 PO4 solution (solvent A) and 400 mM NaH2 PO4 (solvent B). A gradient of B was performed as follow: 0% to 100% in 50 min. The identification and quantitation of each disaccharide was done by comparison with reference disaccharides (Di-0S, Di-4S, Di-6S, Di-diSE ). 2.7. Spectrometry analysis For IR spectroscopy, the samples were mixed with dried KBr, ground and made to a 1 mm pellets for Fourier-transform infrared (FT-IR) spectrum measurement in the frequency range of 4000–500 cm−1 . FT-IR spectrum was taken on a Nicolet Nexus 470 spectrometer. 1 H nuclear magnetic resonance (NMR) and 13 C NMR spectra of AHG and DAHGs were performed at 25 ◦ C using a JEOL-ECP 600 MHz spectrometer. The sample (30 mg) were co-evaporated with 500 L D2 O (99.9%, Sigma–Aldrich) twice followed by dissolution in 500 L D2 O containing TMS as internal standard. 2.8. Anticoagulant activity of AHG and DAHGs in vitro The anticoagulant activities of intact AHG and depolymerized AHG (DAHGs) were performed on a coagulometer. Activated partial thromboplastin time (APTT, assay kit from Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) was carried out according to the method as described [1,25]. Briefly, the lamb plasma (90 L) was mixed with 10 L of a solution of different amounts of polysaccharide in 0.9% NaCl, and incubated at 37 ◦ C for 5 min after addition of 100 L pre-warmed APTT assay regent. 100 L of pre-warmed calcium chloride (0.25 M) was added and the clotting time was recorded as the time for clot formation in a coagulometer. The results were compared with the low molecular weight heparin standard. 3. Results and discussion 3.1. Preparations of different molecular weight fragments of fucosylated chondroitin sulfate from A. japonicus A neutral pH is required for an optimized depolymerization to avoid the acidic or basic hydrolysis of the polysaccharides [11,14]. During depolymerization process, a sudden decrease of pH could be observed at the beginning due to the chelation process between the polysaccharide and the added Cu2+ . And, the pH was remained at 6.3–6.7 depending on the initial concentration of the added Cu2+ . With pH control by K2 HPO4 buffer, a good reproducibility was obtained by analyzing the molecular weight of depolymerized products at different time (the relative standard deviation (RSD %) ≤4.60, n = 3, data not shown). The influence of the concentration of Cu2+ on the depolymerization process was observed. It showed that high amount of Cu2+ did not improve the reaction, even suppress the depolymerization on the contrary (Fig. 1). As a catalyst of free radical reaction, trace amount of Cu2+ would catalyze the depolymerization of large quantity of samples. The concentration of H2 O2 and temperature were critical to the depolymerization process. The radical depolymerization was carried out through the generation of the free radicals from hydrogen peroxide catalyzed by Cu2+ . The amount of radicals would be efficiently affected by the concentration of H2 O2 and temperature. And, the rate of depolymerization would be increased with the increasing of H2 O2 concentration and temperature.
Fig. 1. Influence of Cu2+ concentration on depolymerization, for an AHG concentration of 10 mg/mL and H2 O2 concentration of 50 mM at 35 ◦ C.
The free radical cleavages rate of chain scission can be anticipated to be a linear function of time (first order reaction) as in the following equation [13,26]. 1 1 − = (k/m0 )t Mn (t) Mn (0) In the equation, Mn (0) is the initial weight average; Mn is obtained from regression; and Mn (t) is Mn during free radical depolymerization at t min, respectively. Meanwhile, k is the first order rate constant for change of weight average molecular weight, and the m0 is the molecular mass of a monomer unit. A good linear relationship of 1/Mn and reaction time was observed in present work (Fig. 2). The results indicated that the cleavage of the chain of AHG followed a first order reaction, and the rate constant k was supposed to be 1.276 × 10−5 min−1 by using m0 = 1019 g/mol. The previous reports [27] predicted that the change of polydispersity index (Q) resulting from random chain scission: whether if the polydispersity index of initial polymer is above/below 2.0, it will change to 2.0. The same results were obtained in present study (data not shown), which suggesting that the cleavage of the main chain of AHG is random. In present work, the different concentrations of H2 O2 with 25, 50 and 100 mM at different temperature (35, 45 and 55 ◦ C) were analyzed (data not shown). Ultimately, the reaction system of the intact AHG (10 mg/mL) dissolved in 50 mM H2 O2 (pH 7.5) containing 1 mM CuAc2 at 45 ◦ C was optimized to product a series of low
Fig. 2. Formation of change of the molecular weight (1/Mw ) against time upon depolymerization of glycosaminoglycan (10 mg/mL) in 50 mM H2 O2 (pH 7.5) with 1 mM CuAc2 at 45 ◦ C.
702
J. Yang et al. / International Journal of Biological Macromolecules 72 (2015) 699–705
Table 1 Physicochemical characteristics of intact AHG and purified different molecular weight DAHGs. Polysaccharide
Intact AHG
DAHG-1
DAHG-2
DAHG-3
Mw (Da)
98070
41149
24755
8871
Chemical composition (molar ratio) 1.00 GalNAc GlcUA 1.06 Fuc 0.88 Sulfate 3.85
1.00 0.98 0.87 3.83
1.00 1.00 0.90 3.88
1.00 0.96 0.84 3.60
molecular weight fractions with Mw from 68.54 kDa to 8.87 kDa were obtained applying different reaction conditions. 3.2. Chemical composition of AHG and DAHGs Three different low molecular weight fractions of AHG were obtained for large quantities with Mw of 41149, 24755 and 8871 Da, respectively. The intact AHG and DAHGs have been analyzed by different analytical techniques to compare their physicochemical properties before and after free radical depolymerization as shown in Table 1. The monosaccharide and sulfation content analysis of AHG and DAHGs shows almost no variations. Only the content of GlcUA is very slightly lower in the DAHG-3. The fucose branches were more sensitive to acid than the core structure of chondroitin sulfate formed by aldohexuronic and acetamidoxyhexose, and removed from the polysaccharide by mild acid hydrolysis [1]. The analysis of unsaturated disaccharides formed by Chondroitin ABC digestion of acid-resistant fragments of intact AHG and DAHGs showed three sharp peaks, which agreed with those of Di-6S, Di-4S, Di-diSE in disaccharide standards, respectively (Fig. 3). The molar ratio of the disaccharides in the above order was 1.00: 0.20: 1.08 in AHG. However, the proportion of disaccharides generated from enzyme digestion of defucosylated DAHGs (DAHG-3 as representative) were similar to that of AHG, which confirmed the retention of the backbone structure of intact AHG during the depolymerization.
Fig. 3. HPLC chromatography for analysis of core disaccharide units of AHG and DAHGs. A: Disaccharide standards mixture; B: the disaccharides constitution of AHG. C: DAHG-3. The numbered peaks correspond to known disaccharide standards as follows: peak 1, Di-0S; peak 2, Di-6S; peak 3, Di-4S; peak 4, Di-diSE . x and y are the contaminants from the reaction system.
Fig. 4. The cellulose acetate membrane electrophoresis of chondroitin sulfate (1), AHG (2), DAHG-1 (3), DAHG-2 (4) and DAHG-3 (5).
The cellulose acetate membrane electrophoresis as in Fig. 4 showed that the basic physicochemical properties of AHG and DAHGs had no significant difference, which indicated that the primary structure was not destroyed during the depolymerization. The IR spectra of AHG and DAHGs (Fig. 5A) showed no variations between intact AHG and DAHGs. The absorptions at 1239 cm−1 (vS O ) and 820–860 cm−1 (vC–O–S ) confirmed the presence of sulfates, and the latter ones might indicate the pattern of the sulfates [28,29]: the absorption at 822 cm−1 for the presence of 2,4O-disulfated fucose or 6-O-sulfated GalNAc; and the signal of 852 cm−1 for the presence of 4-O-sulfated Fuc and/or GalNAc. The signal at 3461 cm−1 and 1031 cm−1 were attributed to the stretching vibration of O–H and C–O, respectively. Furthermore, signals at 1635 cm−1 was due to the asymmetric stretch vibration of C O of N-acetylgalactosamine and glucuronic acid; 1409 cm−1 , the symmetric stretch vibration of COO− of glucuronic acid and the stretch vibration of C–O within COOH; 2926 cm−1 , the stretch vibration of C–H. These data indicated that the structures of intact AHG and DAHGs were accordance and no major functional group transformations were found during the depolymerization. The structural consistency of intact AHG and DAHGs was further confirmed by 1 H NMR (Fig. 5B) and 13 C NMR (Fig. 5C) spectroscopy. The comparison of 1 H NMR between the intact AHG and DAHGs showed a coincidence among almost all signals. The chemical shift between 5.0 and 6.0 ppm were the signals of anomeric H of sulfated ␣-fucose: the signal of ı 5.56 ppm represented the H1 of 2,4-diSO3 -fucose, and that of ı 5.22 ppm was H1 of 3,4diSO3 -fucose. The chemical shift and proportion of them were almost equal. Signals of ı = 4.32 ppm (U-H1) and 4.45 ppm (AH1) were attribute to the anomeric H of GlcUA and GalNAc, which showed no differences between the intact AHG and DAHGs. And the same results could be found in the signals of methyl residue of GalNAc and Fuc with ı = 1.93 and 1.24 ppm. Furthermore, the comparison in 13 C NMR spectra showed a coincidence at the region of carbon ring (ı = 50–85 ppm) and anomeric C of GalNAc (A-C1, ı = 101.2 ppm), GlcUA (U-C1, ı = 105.3 ppm) and Fuc (F-C1, ı = 97.7 ppm). The spectra of AHG and DAHGs also showed signals assigned to carbonyl (C O), carbon-2 (A-C2), sulfated carbon-6 (A-C6) and acetamide-methyl (CO–CH3 ) of GalNAc moiety, which resonated at approximately ı = 176.9, 52.8, 67.8 and 23.9 ppm, respectively (ı = 176.9 and 23.9 ppm not shown).
J. Yang et al. / International Journal of Biological Macromolecules 72 (2015) 699–705
703
Fig. 5. IR (A), 1 H NMR (B) and 13 C NMR (C) spectra in D2 O of intact glycosaminoglycan AHG and its depolymerized products DAHGs. Signals labeled with “A” refer to those generated by acteamidodeoxyhexose residues, whereas those of uronosyl residues are labeled with “U”, and those of fucose residues are labeled with “F”.
704
J. Yang et al. / International Journal of Biological Macromolecules 72 (2015) 699–705
Fig. 6. (A) APTT of different molecular weight AHGs obtained by the free radical depolymerization as a function of Mw . The equation of experimental curve and its correlation coefficient is shown. The concentrations of the samples were 250 g/mL. (B) Comparison of the anticoagulant activities of DAHG-2 and LMWH as function of their concentrations. The equations of the linear curves and their coefficients are shown.
dermatan sulfate from beef mucosa expressed as APTT units [31] and the logarithmic-like function of their APTT activities and different molecular weight (Mw ) of chemical depolymerized fucosylated chondroitin sulfate from sea cucumber T. ananas [13]. The anticoagulant activities of the oligosaccharides with different molecular weight decreased with the reduction in molecular weight, while their primary structures were similar to those of the native AHG. Previous studies have indicated that the anticoagulant activity of glycosaminoglycan from sea cucumber mostly depends on the molecular size of the glycosaminoglycan and the sulfated fucose branches [1,2,4,5,13]. The potentiation of thrombin inhibition and prolongation of APTT activity by heparin require at least an oligosaccharide chain of about 16–18 units [32]. Fragments of dermatan sulfate containing a minimum of 12–14 monosaccharide residues are necessary to increase the inhibition of thrombin by heparin cofactor II (HCII) [31]. Meanwhile, the low molecular weight glycosaminoglycan needs at least an oligosaccharide chain of about 6–8 units (Mw = 6000–7000 Da), which obtained from chemical depolymerization of polysaccharide from sea cucumber T. ananas [13]. In present work, we prepared a broad series of oligosaccharides from chemical depolymerization of glycosaminoglycan obtained from sea cucumber A. japonicus. It is indicated that the potentiation of prolongation of APTT activity by AHG requires a minimum of 4–6 units (Mw = 4000–5000 Da). In contrast to heparin and low molecular weight heparin, depolymerized holothurian glycosaminoglycan lacks the antithrombin-dependent activities that contribute to increased bleeding risk [8]. The relationship of the anticoagulant activity in vitro of DTHG-2 (Mw = 24755 Da) and the concentration was investigated in APTT assay comparing to the low molecular weight heparin (LMWH, Mw = 3500 Da) with some concentration. The APTT activities of both DTHG-2 and LMWH have a linear relationship with their concentration (Fig. 6B). Their anticoagulant activity are almost the same, but the DAHG-2 could have significantly less bleeding risk compared with LMWH at equivalent therapeutic does. The results suggest that the DAHG could be used as a novel anticoagulant with less undesired side effects.
4. Conclusions The fucose branches were essential to the anticoagulant activity of the fucosylated chondroitin sulfate [1,2,30]. The signals of the anomeric carbons, ring carbons and CH3 of ␣-fucose among AHG and DAHGs have no variation, which resonate at ı = 97.7, 85.0–65.0 and 17.1 ppm, respectively. All data above indicate that the basic structure of DAHGs is similar to the native AHG. There were no significant variations detected for the monosaccharide composition, the sulfate content, the core disaccharide composition, IR spectra, and 1 H/13 C NMR spectra between intact AHG and DAHGs. The results showed that the primary structure of intact AHG was well retained during the depolymerization, as previously reported for heparin [12] and depolymerized glycosaminoglycan from Thelenata ananas [13]. The Cu2+ induced free radical depolymerization reaction indicated to be an efficient way to obtain the depolymerized different molecular weight fragments without destroying the primary structure of fucosylated chondroitin sulfate from A. japonicus. 3.3. Anticoagulant activity The APTT of intact AHG and DAHGs with different molecular weight were evaluated on their anticoagulant activities. The APTT activities decreased in proportion to the weight-average molecular weight (Mw ) of AHG following a linear relationship (Fig. 6A). The results are different to that of different relative molecular mass (Mr )
In present work, we firstly prepared a broad series of glycosaminoglycans with different molecular weight from the controlled free-radical depolymerization of polysaccharide obtained from the sea cucumber A. japonicas originated from Qingdao, China. The physicochemical properties of the depolymerized fragments are similar to the intact glycosaminoglycan isolated from A. Japonicus. The Cu2+ catalyzed free-radical depolymerization process showed to be an efficient way to prepare the low molecular weight glycosaminoglycan without destroying the primary structure of polysaccharide from sea cucumber A. Japonicus. Moreover, the results of anticoagulant activities indicate that the APTT activities of different molecular weight fragments are followed a linear relationship in proportion to the molecular weight (Mw ), and the activity requires molecular weight above 4000 Da. The low molecular weight glycosaminoglycan could inhibit the multiple pathways of coagulant and thrombin activity or conversion of fibrinogen to fibrin with slight bleeding risks, which makes it a potential therapeutic anticoagulant.
Acknowledgment This research was funded by Natural Science Foundation of China (31171665).
J. Yang et al. / International Journal of Biological Macromolecules 72 (2015) 699–705
References [1] P.A.S. Mourão, M.S.G. Pereira, B. Mulloy, D.M. Tollefsen, M.C. Mowinckel, U. Abildgaard, J. Biol. Chem. 271 (1996) 23973–23984. [2] S.G. Chen, C.H. Xue, L.A. Yin, Q.J. Tang, G.L. Yu, W.G. Chai, Carbohydr. Polym. 83 (2011) 688–696. [3] R.P. Vieira, B. Mulloy, P.A.S. Mourão, J. Biol. Chem. 266 (1991) 13530–13536. [4] Y. Kariya, S. Watabe, M. Kyogashima, M. Ishihara, T. Ishii, Carbohydr. Res. 297 (1997) 273–279. [5] L. Borsig, L. Wang, M.C. Cavalcante, L. Cardilo-Reis, P.L. Ferreira, P.A.S. Mourão, J.D. Esko, M.S.G. Pavão, J. Biol. Chem. 282 (2007) 14984–14991. [6] N. Huang, M.Y. Wu, C.B. Zheng, L. Zhu, J.H. Zhao, Y.T. Zheng, Carbohydr. Res. 380 (2013) 64–69. [7] P.A.S. Mourão, B. Giumaraes, B. Mulloy, S. Thomas, E. Gray, Br. J. Haematol. 101 (1998) 647–652. [8] J.Z. Li, E.C.Y. Lian, J. Thromb. Haemost. 59 (1988) 435–439. [9] K. Yoshida, Y. Minami, H. Nemoto, K. Numata, E. Yamanaka, Tetrahedron Lett. 33 (1992) 4959–4962. [10] H. Nagase, K. Enjyoji, K. Minamiguchi, K.T. Kitazato, K. Kitazato, H. Saito, H. Kato, Blood 85 (1995) 1527–1534. [11] A.C. Petit, N. Noiret, C. Sinquin, J. Ratiskol, J. Guézennec, S.C. Jouault, Carbohydr. Polym. 64 (2006) 597–602. [12] N. Volpi, G. Mascellani, P. Bianchini, Anal. Biochem. 200 (1992) 100–107. [13] M.Y. Wu, S.M. Xu, J.H. Zhao, H. Kang, H. Ding, Food Chem. 122 (2010) 716–723. [14] M.Y. Wu, S.M. Xu, J.H. Zhao, H. Kang, H. Ding, Carbohydr. Res. 345 (2010) 649–655.
705
[15] A. Karlsson, S.K. Singh, Carbohydr. Polym. 38 (1999) 7–15. [16] Y. Cheng, K.M. Brown, R.K. Prud’homme, Int. J. Biol. Macromol. 31 (2002) 29–35. [17] T. Masuko, A. Minami, N. Iwasaki, T. Majima, S. Nishimura, Y.C. Lee, Anal. Biochem. 339 (2005) 69–72. [18] W.J. Mao, F. Fang, H.Y. Li, X.H. Qi, H.H. Sun, Y. Chen, S.D. Guo, Carbohydr. Polym. 74 (2008) 834–839. [19] J.L. Reissig, T.L. Strominger, I.F. Leloir, J. Biol. Chem. 217 (1955) 959–966. [20] N. Blumenkrantz, G. Asboe-Hansen, Anal. Biochem. 54 (1973) 484–489. [21] W. Zhang, H.B. He, X.D. Zhang, Soil Biol. Biochem. 39 (2007) 2665–2669. [22] X.Q. Zhan, Z.J. Zhu, J.W. Kang, Chin. J. Chromatogr. 29 (2011) 362–364. [23] M. Yokoyama, H. Tanaka, S. Yoshihara, M. Endo, J. Biochem. Biophys. Methods 12 (1986) 239–246. [24] K. Yoshida, S. Miyauchi, H. Kikuchi, A. Tawada, K. Tokuyasu, Anal. Biochem. 177 (1989) 327–332. [25] H.J. Zhang, W.J. Mao, F. Fang, H.Y. Li, H.H. Sun, Y. Chen, X.H. Qi, Carbohydr. Polym. 71 (2008) 428–434. [26] V.M.F. Lai, C. Lii, W.L. Hung, T.J. Lu, Food Chem. 68 (2000) 319–325. [27] J.S. Yoon, I.J. Chin, M.N. Kim, C. Kim, Macromolecules 29 (1996) 3303–3307. [28] G. Bernardi, G.F. Springer, J. Biol. Chem. 237 (1962) 75–80. [29] M.E.R. Duarte, M.A. Cardoso, M.D. Noseda, A.S. Cerezo, Carbohydr. Res. 333 (2001) 281–293. [30] M.Y. Wu, R.R. Huang, D.D. Wen, N. Gao, J.B. He, Z. Li, J.H. Zhao, Carbohydr. Polym. 87 (2012) 862–868. [31] N. Volpi, Carbohydr. Res. 255 (1994) 133–144. [32] J. Hirsh, T.E. Warkentin, S.G. Shaughnessy, S.S. Anand, J.L. Halperin, R. Raschke, et al., Chest 119 (2001) 64S–94S.