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dermatan sulfate hybrid chain in shark and ray tissues
Carbohydrate Research 424 (2016) 54–58
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Carbohydrate Research j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a r r e s
Facile analysis of contents and compositions of the chondroitin sulfate/dermatan sulfate hybrid chain in shark and ray tissues Naoko Takeda a, Sawako Horai b, Jun-ichi Tamura b,* a b
Organization for Regional Industrial Academic Cooperation, Tottori University, 4-101 Koyamacho-Minami, Tottori 680-8550, Japan Department of Regional Environment, Faculty of Regional Sciences, Tottori University, 4-101 Koyamacho-Minami, Tottori 680-8551, Japan
A R T I C L E
I N F O
Article history: Received 2 December 2015 Received in revised form 15 February 2016 Accepted 16 February 2016 Available online 24 February 2016 Keywords: Glycosaminoglycan Chondroitin sulfate Dermatan sulfate Shark Ray
A B S T R A C T
The chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chain was extracted from specific tissues of several kinds of sharks and rays. The contents and sulfation patterns of the CS/DS hybrid chain were precisely analyzed by digestion with chondroitinases ABC and AC. All samples predominantly contained the A- and C-units. Furthermore, all samples characteristically contained the D-unit. Species-specific differences were observed in the contents of the CS/DS hybrid chain, which were the highest in Mako and Blue sharks and Sharpspine skates, but were lower in Hammerhead sharks. Marked differences were observed in the ratio of the C-unit/A-unit between sharks and rays. The contents of the CS/DS hybrid chain and the ratio of the C-unit/A-unit may be related to an oxidative stress-decreasing ability. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction The demand for chondroitin sulfate (CS) in health food supplements and for medical use is increasing. CS is obtained from the tissues of sharks and porcine animals. However, since the shark population is decreasing due to overfishing and porcine animals are susceptible to infectious diseases, we have been seeking alternative resources to these animals. Marine products such as fishes and squids are promising because they are abundant and not markedly affected by infectious diseases. The contents and sulfation patterns of the CS/dermatan sulfate (DS) hybrid chain as well as hyaluronic acid derived from fishes and squids have already been clarified.1,2
Abbreviations: CS, chondroitin sulfate; DS, dermatan sulfate; GAG, glycosaminoglycan; GlcA, glucuronic acid; GalNAc, N-acetyl-galactosamine; ΔO, ΔHexA(1– 3)β-d-GalNAc; ΔA, ΔHexA(1–3)β-d-GalNAc(4S); ΔC, ΔHexA(1–3)β-d-GalNAc(6S); ΔD, ΔHexA(2S)(1–3)β-d-GalNAc(6S); ΔE, ΔHexA(1–3)β-d-GalNAc(4,6-diS); ΔK, ΔHexA(3S)(1–3)β-d-GalNAc(4S); ΔT, ΔHexA(2S)(1–3)β-d-GalNAc(4,6-diS); O, -4)βd-GlcA(1–3)β-d-GalNAc(1-; iO, -4)α-l-IdoA(1–3)β-d-GalNAc(1-; A, -4)β-d-GlcA(1– 3)β-d-GalNAc(4S)(1-; iA, -4)α-l-IdoA(1–3)β-d-GalNAc(4S)(1-; B, -4)β-d-GlcA(2S)(1– 3)β-d-GalNAc(4S)(1-; iB, -4)α-l-IdoA(2S)(1–3)β-d-GalNAc(4S)(1-; C, -4)β-d-GlcA(1– 3)β-d-GalNAc(6S)(1-; iC, -4)α-l-IdoA(1–3)β-d-GalNAc(6S)(1-; D, -4)β-d-GlcA(2S)(1– 3)β-d-GalNAc(6S)(1-; iD, -4)α-l-IdoA(2S)(1–3)β-d-GalNAc(6S)(1-; E, -4)β-d-GlcA(1– 3)β-d-GalNAc(4,6-diS)(1-; iE, -4)α-l-IdoA(1–3)β-d-GalNAc(4,6-diS)(1-; T, -4)β-dGlcA(2S)(1–3)β-d-GalNAc(4,6-diS)(1-; iT, -4)α-l-IdoA(2S)(1–3)β-d-GalNAc(4,6diS)(1-. * Corresponding author. Department of Regional Environment, Tottori University, Tottori 680-8551, Japan. Tel.: +81 857 31 5108; fax: +81 857 31 5108. E-mail addresses: [email protected] (N. Takeda), [email protected] (S. Horai), [email protected] (J. Tamura). http://dx.doi.org/10.1016/j.carres.2016.02.006 0008-6215/© 2016 Elsevier Ltd. All rights reserved.
In order to find an alternative animal to sharks as a resource of CS, it is important to know the contents and sulfation patterns of CS from sharks. CS belongs to the proteoglycan family, each member of which has a linear glycan chain composed of the repeating disaccharide unit, -4)β-d-GlcA(1–3)β-d-GalNAc(1-. Some of the C-5 of GlcA in the CS chain is epimerized to IdoA in order to form a DS unit, and, thus, the repeating disaccharide unit is expressed as -4)α-l-IdoA(1– 3)β-d-GalNAc(1-. Naturally occurring CS often forms a CS/DS hybrid chain. CS/DS hybrid chains in fish are mostly composed of A- and C-units at concentrations of several hundred milligrams per 100 g of dry-defatted tissue. CS from squids characteristically contains ~ 30% of the E-unit. The amount of CS in squids is similar to that in fish. CS has been investigated in shark and ray species by other groups. Fürth and Bruno initially isolated CS from the cartilage of sharks and rays in 1937.3 In 1963, Seno and Meyer isolated CS-B and hyaluronic acid from the skin of sharks (Blue sharks and Sandbar sharks).4 However, a precise compositional analysis was not performed until much later. Sugahara’s group reported the isolation of specific oligosaccharides from shark cartilage.5a–i Oligosaccharides containing the D structure have been shown to exhibit neurite outgrowth-promoting activities 5e and binding activities to CS antibodies.5h,i Sugahara’s group subsequently determined the molar ratio of CS disaccharides in the cartilage and liver of Blue sharks (Prionace glauca). The chondroitinase ABC digestion of CS afforded ΔO, ΔC, ΔA, ΔD, ΔB, and ΔT.5f,g The cartilage and liver of Blue sharks were found to contain 30~50% of the A-unit, but smaller amounts of the D-unit. These parts also contained 6~18% of the B-unit and
N. Takeda et al./Carbohydrate Research 424 (2016) 54–58
10~23% of the E-unit. In contrast, fin cartilage has been reported to contain small amounts of the B- and E-units, but 15% of the D-unit (species not shown).5i Toida’s group recently isolated the CS/DS hybrid chain from the fins of deep-sea sharks, and demonstrated that the fins contained hyaluronic acid and the O-, C-, A-, B-, and D-units.6 It has not yet been determined whether these differences are due to the species of shark or tissue examined. Tsegenidis and co-workers investigated CS in ray species. The chondroitinase digestion of the skin from Raja clavata afforded ΔO, ΔC, ΔA, ΔD, and ΔK at 3, 4, 61, 13, and 20%, respectively.7 Dellias et al examined the ratio of CS disaccharides in the skin of 4 ray species. Enzymatic digestion of the skins from D. americana, D. guttata, and A. narinari mainly afforded ΔA (50, 51, and 47%, respectively) and ΔB (30, 33, and 24%, respectively), and, to a lesser extent, ΔD (3, 2, and 8%, respectively), while the skin from P. motoro afforded ΔA (75%), ΔB (6%), but not ΔD.8 To the best of our knowledge, the CS/DS hybrid chain has not yet been examined in other tissues of sharks and rays. Although analytical studies have been performed using a limited number of shark species, the total contents of the CS/DS hybrid chain in these tissues currently remain unknown. Therefore, we herein isolated the CS/ DS hybrid chain from a number of tissues in five shark species and three ray species, some of which have been utilized for the man-
ufacture of CS, and then analyzed the contents and compositions of disaccharide units. 2. Materials and methods Blue sharks (Prionace glauca) were caught off the coast of Fukuejima, Nagasaki, Mako sharks (Isurus oxyinchus) off the coast of Ishinomaki, Miyagi, Smooth hammerhead (Sphyrna zygaena) and Scalloped hammerhead (Sphyrna lewini) sharks off the coast of Yushima, Kumamoto, Dusky sharks (Carcharhinus obscurus) off the coast of Narushima, Nagasaki, Stingrays (Dasyatis akajei) at Nakaumi, Tottori, and Sharpspine skates (Okamejei acutispina) and Kwangtung skates (Dipturus kwangtungensis) in the Sea of Japan, off the coast of Tottori, Japan. The tissues of sharks and rays examined in this study were summarized in Table 1. Each material was minced before use. DEAE-cellulose was purchased from Wako Pure Chemical (Osaka, Japan). Dialysis cellulose tubing (MWCO 12,000–16,000) was from Viskase Companies, Inc. (Darien, IL). Chondroitinase ABC (EC 4.2.2.4) and chondroitinase AC (EC 4.2.2.5) were from Sigma Aldrich (Tokyo, Japan) and IBEX Pharmaceuticals Inc. (Montréal, Canada), respectively. The LH-20 gel and amino-bound PA-03 (PA-G) column were from GE Healthcare, Bio-Science AB (Uppsala, Sweden), and YMC
Table 1 Contents and sulfation patterns of the CS/DS hybrid chain in shark and ray tissues
Name
Tissue
Content of Sufation patterns of the disaccharide unit GlcACS/DS hybrid GlcA-type IdoA-type IdoA-type GlcA- + IdoA-type (%) type (mg) /100 g (%) (%) (%) (%) defatted dry * * A C D E A C D E A C D E iA iC iD iE A C iA iC iD iE tissue
Tail fin
955
27 57 13 3
11 49 0 0 16 8 13 3
Dorsal fin
1061
27 56 14 3
8 45 0 0 19 11 14 3
Pectoral fin
1863
32 48 20 0
7 33 0 0 25 15 20 0
Spine
1823
20 67 13 0
5 52 0 0 15 15 13 0
Tail fin
1582
37 44 17 2
13 42 0 0 24 2 17 2
Dorsal fin
1670
37 40 20 3
11 35 0 0 26 5 20 3
Blue shark
Mako Shark Pectoral fin
1376
38 38 21 3
13 30 0 0 25 8 21 3
Spine
1330
33 48 18 1
11 46 0 0 22 2 18 1
Tail fin
767
39 36 20 5
11 25 0 0 28 11 20 5
Dusky shark Dorsal fin Spine Tail fin Smooth Dorsal fin Hammerhead Spine Dorsal fin Scalloped Pectoral fin Hammerhead Spine
Stingray
41 33 21 5
6 16 0 0 35 17 21 5
38 41 19 2
16 36 0 0 22 5 19 2
502
42 32 22 4
10 22 0 0 32 10 22 4
189
42 31 23 4
11 23 0 0 31 8 23 4
1064
43 36 19 2
14 28 0 0 29 8 19 2
395
53 23 19 5
20 21 0 0 33 2 19 5
997
57 17 21 5
15 17 0 0 42 0 21 5
368
52 24 21 3
10 12 0 0 42 12 21 3
1608
24 66 10 0
9 55 0 0 15 11 10 0
Spine
573
26 62 11 1
9 49 0 0 17 13 11 1
Fin
628
27 59 12 2
7 41 0 0 20 18 12 2
1296
28 61 10 1
10 47 0 0 18 14 10 1
1172
31 55 12 2
12 48 0 0 19 7 12 2
882
32 57 9 2
11 44 0 0 21 13 9 2
597
35 52 12 1
12 41 0 0 23 11 12 1
Kwangtung Head-Tail skate Fin a
439 458
Head
Sharpspine Head-Tail skate Fin
55
The value of the iD-unit needs to be considered as a mixture of the D- and iD-units (see details in the text).
56
N. Takeda et al./Carbohydrate Research 424 (2016) 54–58
Fig. 1. a, b. Representative HPLC patterns of (a) chondroitinase ABC and (b) chondroitinase AC digestion (1 M of the LiCl fraction, tail fin of a Blue shark).
(Kyoto, Japan), respectively. Extra pure grade organic solvents and reagents were obtained commercially.
LiCl. Carbazole-positive fractions were subjected to dialysis and further desalted through a column for gel permeation (LH-20, H2O, ϕ1.1 × 80 cm).
2.1. Preparation of defatted dry powder of shark and ray tissues The prepared materials described above were defatted with double their weight of acetone. The solutions were filtered, and the filtrates were defatted again in the same manner. The filtrates were dried in vacuo. 2.2. Preparation of crude glycans from defatted dry tissue Crude glycans were prepared as previously described.1 Briefly, a suspension of the defatted dry material (10 g) was treated in 80 mL of boiling water for 10 min, followed by the addition of 0.5 M borate buffer (100 mL), pH 7.0. The suspension was then incubated with protease N Amano G (0.67 g) at 55 °C for 7 d. The incubated suspension was quenched with trichloroacetic acid at a concentration of 5%. The soluble fraction obtained by centrifugation at 0 °C was collected and subjected to dialysis for 7 d. Insoluble materials were filtered off, and subsequent precipitation with 80% ethanol with 1.25% NaOAc afforded crude glycans, which were collected by centrifugation at 0 °C and dried in vacuo. 2.3. DEAE-cellulose chromatography One portion of crude glycans (100 mg) was diluted with a small amount of 0.15 M LiCl/0.05 M acetic acid, pH 4.0, and applied to a column (ϕ2.2 × 15 cm) of DEAE-cellulose. The column was washed stepwise with 160 mL of buffers containing 0.15, 0.5, 1.0, and 2.0 M
2.4. Digestion with Chondroitinases ABC and AC followed by a HPLC analysis The pure glycans (50 μg) obtained above were diluted with H2O (50 μL), BSA solution (0.05 mg/5 μL), and 250 mM Tris-HCl(AcOH) buffer (10 μL), pH 8.0 (solution A). Solution A was digested with chondroitinase ABC (25 mU/25 μL) or chondroitinase AC (25 mU/ 25 μL) at 37 and 30 °C, respectively, for 8 h. The unsaturated disaccharides derived from the CS/DS hybrid chain produced by this digestion were analyzed on a PA-03 (PA-G) column by HPLC according to a previously described method.9 The contents of the CS/ DS hybrid chain and the ratios of the GlcA-GalNAc and IdoAGalNAc units as well as the sulfation patterns in pure glycans were calculated using calibration curves based on the HPLC peak area of the standard unsaturated disaccharide. Representative HPLC patterns are shown in Fig. 1a and b, and the results obtained are summarized in Table 1. 2.5. Statistical analysis Relationships between the contents of the CS/DS hybrid chain and ΔC/ΔA were determined using Spearman’s rank correlation test (Fig. 2). Similarly, relationships between the values of ΔC/ΔA and (GlcA-GalNAc)/(IdoA-GalNAc) were determined (Fig. 3). A p value less than 0.05 was considered to be significant. All statistical analyses were executed using the Statcel 3 program.
N. Takeda et al./Carbohydrate Research 424 (2016) 54–58
57
2000 Blue shark
1800
Blue shark
Contents ( mg ) / 100 g of defatted dry tissue
Mako Shark
1600
Stingray
Mako Shark
Mako Shark
1400
Sharpspine skate
Mako Shark
r = 0.467
Sharpspine skate
1200 Smooth Hammerhead
1000
A
Blue shark
Scalloped Hammerhead Kwangtung skate
B
800
600
Blue shark
Dusky shark Kwangtung skate
Stingray Stingray
Smooth Hammerhead Dusky shark
Scalloped Hammerhead
400
Dusky shark Scalloped Hammerhead
200
Smooth Hammerhead
0 0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
C/ A Fig. 2. The relationship between contents of the CS/DS hybrid chain and ΔC/ΔA in each tissue.
3.00
2.50
Blue shark
Stingray
Mako Shark
(GlcA-GalNAc) / (IdoA-GalNAc)
Sharpspine skate
r = 0.740
Mako Shark
2.00 Dusky shark
Stingray
Blue shark
Kwangtung skate
Blue shark
Sharpspine skate
Mako Shark
1.50
Kwangtung skate
Mako Shark Scalloped Hammerhead
Stingray Smooth Hammerhead
1.00
Blue shark Dusky shark Smooth Hammerhead Smooth Hammerhead Scalloped Hammerhead
0.50 Dusky shark Scalloped Hammerhead
0.00 0.00
0.50
1.00
1.50
2.00
2.50
C/ A Fig. 3. The relationship between ΔC/ΔA and (GlcA-GalNAc unit)/(IdoA-GalNAc unit) in each tissue.
3.00
3.50
58
N. Takeda et al./Carbohydrate Research 424 (2016) 54–58
3. Results and discussion Chondroitinase ABC digests every kind of GalNAc-GlcA/IdoA linkage, while chondroitinase AC specifically digests GalNAc-GlcA, but does not cleave the GalNAc-IdoA linkage or GalNAc-GlcA linkage at the non-reducing side of the D-unit, GalNAc-(GlcA2S-GalNAc6S). Since the ratio of the GlcA-GalNAc and IdoA-GalNAc units was calculated for the above reason, the value of the D-unit was 0%. The value of the iD-unit needs to be considered as a mixture of D- and iD-units. The results obtained revealed that all samples from the tissues of sharks and rays were composed of the A-, iA-, C-, iC-, and D/iDunits (Table 1). Some tissues contained the iE-unit, but at less than 6%. Other sulfation patterns including O-units, iO-units, and the hyaluronic acid disaccharide unit (GlcA-GlcNAc) were not detected. The contents of the CS/DS hybrid chain in bony fish were approximately several hundred milligrams.1 Our results indicate that the contents of the CS/DS hybrid chain were higher in cartilaginous fish than in bony fish. The relationship between the contents of the CS/DS hybrid chain and the ratios of ΔC and ΔA were plotted in Fig. 2. A positive correlation was observed between the contents of the CS/DS hybrid chain and the value of ΔC/ΔA (r = 0.467, n = 24, p < 0.05; *). Blue sharks, Mako sharks, Stingrays, Kwangtung skates, and Sharpspine skates were categorized into the A group, namely, those with “a high content of the CS/DS hybrid chain and a highΔC/ΔA value”. Other sharks close to the zero point were grouped into B. Blue and Mako sharks belonging to the A group are pelagic and, thus, require a large amount of oxygen for aerobic exercise. As a result, their cells are exposed to radical oxygen more than those of slow moving coastal sharks. Previous studies reported that CS suitably coordinates metal ions such as Fe and inhibits radical oxygen, with the A-unit of CS being more effective than the C-unit for this purpose.10,11 In Fig. 2, sharks in category A had higher contents of the CD/DS hybrid chain. Although the ΔC/ΔA values were relatively high, the larger amount of CS may inhibit radical oxygen. In contrast, pelagic bony fish such as tuna, mackerel, and jack mackerel contain smaller amounts of the CS/DS hybrid chain (12~370 mg per 100 g of defatted dry tissue), but always possess abundant amounts of the A-unit (ΔA:ΔC = 46:35~100:0).1 Thus, these bony fish may not need as much CS/DS as sharks. On the other hand, coastal sharks in category B contain a relatively low amount of CS/DS, but are predominantly composed of the A-unit (ΔC:ΔA < 1), which may effectively protect cells from oxidative stress. Most rays belong to category A. They inhabit the bottom of the coast. Rays are often caught by flatfish fishermen as a by-catch. The results obtained for group A are in good agreement with those for bottom-dwelling bony fish such as flatfish.1 Flatfish and rays are completely different species, but inhabit a similar environment with quasi motilities. We hypothesized that a relationship exists between the contents of the CS/DS hybrid chain and ratio of the isomeric sulfate from the point of view of the inhibition of radical oxygen. We also evaluated the relationship between the ratios of (GlcAGalNAc unit)/(IdoA-GalNAc unit) and ΔC/ΔA in each tissue in Fig. 3. A positive correlation was observed between these two elements (r = 0.740, n = 24, p < 0.001; ***). In the biosynthesis of the CS/DS hybrid chain, the CS chain is epimerized at C-5 of the GlcA residue with the assistance of epimerase in order to form the CS/DS hybrid chain.12 OH-4 and OH-6 on the GalNAc residue are sulfated by the
corresponding sulfotransferases. Fig. 3 indicates that the level of expression and/or activities of 6-O- and 4-O-sulfotransferases are greater for GlcA-GalNAc and IdoA-GalNAc units, respectively. 4. Conclusions We herein demonstrated that the contents of the CS/DS hybrid chain in five shark species and three ray species differed in speciesand tissue-dependent manners. Sharks and rays contain 189– 1863 mg of the CS/DS hybrid chain per 100 g of defatted dry tissue. The sulfation patterns of sharks and rays mainly comprised the Aand C-units, and also characteristically contained the D-unit at 9% to 23%. Blue sharks and Mako sharks, which are categorized as pelagic sharks, contained large contents of the CS/DS hybrid chain and high ratios of ΔC/ΔA. Oxidative stress caused by more intense aerobic exercise in pelagic sharks may affect the contents of CS/DS and sulfation patterns. Commercially available CS is often derived from sharks, which have been overfished. We herein clarified the contents of the CS/ DS hybrid chain and composition of the disaccharide unit including sulfation patterns in sharks and rays. These results will contribute to the identification of animals as alternative sources of CS in the market. Acknowledgments This study was supported by a Grant-in-Aid for scientific research in innovative areas (23110003) from MEXT, Japan. N.T. is grateful for the JSPS Research Fellowship for Young Scientists (25•8319). We thank Mr. Eiichi Hiroiwa (Tottori Fishery port) for the kind gift of fish. The authors also thank Ryosuke Toita, Kento Ueda, Yumi Irie, Nozomi Kidoguchi, Daisuke Kimino, Naoya Goto, and Hiroya Takada (Tottori University) for their helpful assistance. References 1. Arima K, Fujita H, Toita R, Imazu-Okada A, Tsutsumishita-Nakai N, Takeda N, et al. Carbohydr Res 2013;366:25–32. 2. Tamura J, Arima K, Imazu A, Tsutsumishita N, Fujita H, Yamane M, et al. Biosci Biotechnol Biochem 2009;73:1387–91. 3. Fürth O, Bruno T. Biochem Z 1937;294:153–73. 4. Seno N, Meyer K. Biochim Biophys Acta 1963;78:258–64. 5. (a) Sugahara K, Kojima T. Eur J Biochem 1996;239:865–70; (b) Sugahara K, Tanaka Y, Yamada S. Glycoconj J 1996;13:609–19; (c) Sugahara K, Nadanaka S, Takeda K, Kojima T. Eur J Biochem 1996;239:871–80; (d) Nadanaka S, Sugahara K. Glycobiology 1997;7:253–63; (e) Nadanaka S, Clement A, Masayama K, Faissner A, Sugahara K. J Biol Chem 1998;273:3296–307; (f) Nandini CD, Itoh N, Sugahara K. J Biol Chem 2005;280:4058–69; (g) Li F, Shetty AK, Sugahara K. J Biol Chem 2007;282:2956–66; (h) Deepa SS, Yamada S, Fukui S, Sugahara K. Glycobiology 2007;17:631–45; (i) Mizumoto S, Murakoshi S, Kalayanamitra K, Deepa SS, Fukui S, Kongtawelert P, et al. Glycobiology 2013;23:155–68. 6. Higashi K, Takeuchi Y, Mukuno A, Tomitori H, Miy M, Linhardt RJ, et al. PLoS ONE 2015;10:e0120860. 7. Chatziioannidis CC, Karamanos NK, Tsegenidis T. Comp Biochem Physiol Part B 1999;124:15–24. 8. Dellias JMM, Onofre GR, Werneck CC, Landeira-Fernandez AM, Melo FR, Farias WRL, et al. Biochemie 2004;86:677–83. 9. Yoshida K, Miyauchi S, Kikuchi H, Tawada A, Tokuyasu K. Anal Biochem 1989;177:327–32. 10. Campo GM, D’Ascola A, Avenoso A, Campo S, Ferlazzo AM, Micali C, et al. Glycoconj J 2004;20:133–41. 11. Campo GM, Avenoso A, Campo S, Ferlazzo AM, Calatroni A. Mini Rev Med Chem 2006;6:1311–20. 12. Malmström A, Bartolini B, Thelin MA, Pacheco B, Maccarana M. J Histochem Cytochem 2012;60:916–25.
Contents lists available at ScienceDirect
Carbohydrate Research j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a r r e s
Facile analysis of contents and compositions of the chondroitin sulfate/dermatan sulfate hybrid chain in shark and ray tissues Naoko Takeda a, Sawako Horai b, Jun-ichi Tamura b,* a b
Organization for Regional Industrial Academic Cooperation, Tottori University, 4-101 Koyamacho-Minami, Tottori 680-8550, Japan Department of Regional Environment, Faculty of Regional Sciences, Tottori University, 4-101 Koyamacho-Minami, Tottori 680-8551, Japan
A R T I C L E
I N F O
Article history: Received 2 December 2015 Received in revised form 15 February 2016 Accepted 16 February 2016 Available online 24 February 2016 Keywords: Glycosaminoglycan Chondroitin sulfate Dermatan sulfate Shark Ray
A B S T R A C T
The chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chain was extracted from specific tissues of several kinds of sharks and rays. The contents and sulfation patterns of the CS/DS hybrid chain were precisely analyzed by digestion with chondroitinases ABC and AC. All samples predominantly contained the A- and C-units. Furthermore, all samples characteristically contained the D-unit. Species-specific differences were observed in the contents of the CS/DS hybrid chain, which were the highest in Mako and Blue sharks and Sharpspine skates, but were lower in Hammerhead sharks. Marked differences were observed in the ratio of the C-unit/A-unit between sharks and rays. The contents of the CS/DS hybrid chain and the ratio of the C-unit/A-unit may be related to an oxidative stress-decreasing ability. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction The demand for chondroitin sulfate (CS) in health food supplements and for medical use is increasing. CS is obtained from the tissues of sharks and porcine animals. However, since the shark population is decreasing due to overfishing and porcine animals are susceptible to infectious diseases, we have been seeking alternative resources to these animals. Marine products such as fishes and squids are promising because they are abundant and not markedly affected by infectious diseases. The contents and sulfation patterns of the CS/dermatan sulfate (DS) hybrid chain as well as hyaluronic acid derived from fishes and squids have already been clarified.1,2
Abbreviations: CS, chondroitin sulfate; DS, dermatan sulfate; GAG, glycosaminoglycan; GlcA, glucuronic acid; GalNAc, N-acetyl-galactosamine; ΔO, ΔHexA(1– 3)β-d-GalNAc; ΔA, ΔHexA(1–3)β-d-GalNAc(4S); ΔC, ΔHexA(1–3)β-d-GalNAc(6S); ΔD, ΔHexA(2S)(1–3)β-d-GalNAc(6S); ΔE, ΔHexA(1–3)β-d-GalNAc(4,6-diS); ΔK, ΔHexA(3S)(1–3)β-d-GalNAc(4S); ΔT, ΔHexA(2S)(1–3)β-d-GalNAc(4,6-diS); O, -4)βd-GlcA(1–3)β-d-GalNAc(1-; iO, -4)α-l-IdoA(1–3)β-d-GalNAc(1-; A, -4)β-d-GlcA(1– 3)β-d-GalNAc(4S)(1-; iA, -4)α-l-IdoA(1–3)β-d-GalNAc(4S)(1-; B, -4)β-d-GlcA(2S)(1– 3)β-d-GalNAc(4S)(1-; iB, -4)α-l-IdoA(2S)(1–3)β-d-GalNAc(4S)(1-; C, -4)β-d-GlcA(1– 3)β-d-GalNAc(6S)(1-; iC, -4)α-l-IdoA(1–3)β-d-GalNAc(6S)(1-; D, -4)β-d-GlcA(2S)(1– 3)β-d-GalNAc(6S)(1-; iD, -4)α-l-IdoA(2S)(1–3)β-d-GalNAc(6S)(1-; E, -4)β-d-GlcA(1– 3)β-d-GalNAc(4,6-diS)(1-; iE, -4)α-l-IdoA(1–3)β-d-GalNAc(4,6-diS)(1-; T, -4)β-dGlcA(2S)(1–3)β-d-GalNAc(4,6-diS)(1-; iT, -4)α-l-IdoA(2S)(1–3)β-d-GalNAc(4,6diS)(1-. * Corresponding author. Department of Regional Environment, Tottori University, Tottori 680-8551, Japan. Tel.: +81 857 31 5108; fax: +81 857 31 5108. E-mail addresses: [email protected] (N. Takeda), [email protected] (S. Horai), [email protected] (J. Tamura). http://dx.doi.org/10.1016/j.carres.2016.02.006 0008-6215/© 2016 Elsevier Ltd. All rights reserved.
In order to find an alternative animal to sharks as a resource of CS, it is important to know the contents and sulfation patterns of CS from sharks. CS belongs to the proteoglycan family, each member of which has a linear glycan chain composed of the repeating disaccharide unit, -4)β-d-GlcA(1–3)β-d-GalNAc(1-. Some of the C-5 of GlcA in the CS chain is epimerized to IdoA in order to form a DS unit, and, thus, the repeating disaccharide unit is expressed as -4)α-l-IdoA(1– 3)β-d-GalNAc(1-. Naturally occurring CS often forms a CS/DS hybrid chain. CS/DS hybrid chains in fish are mostly composed of A- and C-units at concentrations of several hundred milligrams per 100 g of dry-defatted tissue. CS from squids characteristically contains ~ 30% of the E-unit. The amount of CS in squids is similar to that in fish. CS has been investigated in shark and ray species by other groups. Fürth and Bruno initially isolated CS from the cartilage of sharks and rays in 1937.3 In 1963, Seno and Meyer isolated CS-B and hyaluronic acid from the skin of sharks (Blue sharks and Sandbar sharks).4 However, a precise compositional analysis was not performed until much later. Sugahara’s group reported the isolation of specific oligosaccharides from shark cartilage.5a–i Oligosaccharides containing the D structure have been shown to exhibit neurite outgrowth-promoting activities 5e and binding activities to CS antibodies.5h,i Sugahara’s group subsequently determined the molar ratio of CS disaccharides in the cartilage and liver of Blue sharks (Prionace glauca). The chondroitinase ABC digestion of CS afforded ΔO, ΔC, ΔA, ΔD, ΔB, and ΔT.5f,g The cartilage and liver of Blue sharks were found to contain 30~50% of the A-unit, but smaller amounts of the D-unit. These parts also contained 6~18% of the B-unit and
N. Takeda et al./Carbohydrate Research 424 (2016) 54–58
10~23% of the E-unit. In contrast, fin cartilage has been reported to contain small amounts of the B- and E-units, but 15% of the D-unit (species not shown).5i Toida’s group recently isolated the CS/DS hybrid chain from the fins of deep-sea sharks, and demonstrated that the fins contained hyaluronic acid and the O-, C-, A-, B-, and D-units.6 It has not yet been determined whether these differences are due to the species of shark or tissue examined. Tsegenidis and co-workers investigated CS in ray species. The chondroitinase digestion of the skin from Raja clavata afforded ΔO, ΔC, ΔA, ΔD, and ΔK at 3, 4, 61, 13, and 20%, respectively.7 Dellias et al examined the ratio of CS disaccharides in the skin of 4 ray species. Enzymatic digestion of the skins from D. americana, D. guttata, and A. narinari mainly afforded ΔA (50, 51, and 47%, respectively) and ΔB (30, 33, and 24%, respectively), and, to a lesser extent, ΔD (3, 2, and 8%, respectively), while the skin from P. motoro afforded ΔA (75%), ΔB (6%), but not ΔD.8 To the best of our knowledge, the CS/DS hybrid chain has not yet been examined in other tissues of sharks and rays. Although analytical studies have been performed using a limited number of shark species, the total contents of the CS/DS hybrid chain in these tissues currently remain unknown. Therefore, we herein isolated the CS/ DS hybrid chain from a number of tissues in five shark species and three ray species, some of which have been utilized for the man-
ufacture of CS, and then analyzed the contents and compositions of disaccharide units. 2. Materials and methods Blue sharks (Prionace glauca) were caught off the coast of Fukuejima, Nagasaki, Mako sharks (Isurus oxyinchus) off the coast of Ishinomaki, Miyagi, Smooth hammerhead (Sphyrna zygaena) and Scalloped hammerhead (Sphyrna lewini) sharks off the coast of Yushima, Kumamoto, Dusky sharks (Carcharhinus obscurus) off the coast of Narushima, Nagasaki, Stingrays (Dasyatis akajei) at Nakaumi, Tottori, and Sharpspine skates (Okamejei acutispina) and Kwangtung skates (Dipturus kwangtungensis) in the Sea of Japan, off the coast of Tottori, Japan. The tissues of sharks and rays examined in this study were summarized in Table 1. Each material was minced before use. DEAE-cellulose was purchased from Wako Pure Chemical (Osaka, Japan). Dialysis cellulose tubing (MWCO 12,000–16,000) was from Viskase Companies, Inc. (Darien, IL). Chondroitinase ABC (EC 4.2.2.4) and chondroitinase AC (EC 4.2.2.5) were from Sigma Aldrich (Tokyo, Japan) and IBEX Pharmaceuticals Inc. (Montréal, Canada), respectively. The LH-20 gel and amino-bound PA-03 (PA-G) column were from GE Healthcare, Bio-Science AB (Uppsala, Sweden), and YMC
Table 1 Contents and sulfation patterns of the CS/DS hybrid chain in shark and ray tissues
Name
Tissue
Content of Sufation patterns of the disaccharide unit GlcACS/DS hybrid GlcA-type IdoA-type IdoA-type GlcA- + IdoA-type (%) type (mg) /100 g (%) (%) (%) (%) defatted dry * * A C D E A C D E A C D E iA iC iD iE A C iA iC iD iE tissue
Tail fin
955
27 57 13 3
11 49 0 0 16 8 13 3
Dorsal fin
1061
27 56 14 3
8 45 0 0 19 11 14 3
Pectoral fin
1863
32 48 20 0
7 33 0 0 25 15 20 0
Spine
1823
20 67 13 0
5 52 0 0 15 15 13 0
Tail fin
1582
37 44 17 2
13 42 0 0 24 2 17 2
Dorsal fin
1670
37 40 20 3
11 35 0 0 26 5 20 3
Blue shark
Mako Shark Pectoral fin
1376
38 38 21 3
13 30 0 0 25 8 21 3
Spine
1330
33 48 18 1
11 46 0 0 22 2 18 1
Tail fin
767
39 36 20 5
11 25 0 0 28 11 20 5
Dusky shark Dorsal fin Spine Tail fin Smooth Dorsal fin Hammerhead Spine Dorsal fin Scalloped Pectoral fin Hammerhead Spine
Stingray
41 33 21 5
6 16 0 0 35 17 21 5
38 41 19 2
16 36 0 0 22 5 19 2
502
42 32 22 4
10 22 0 0 32 10 22 4
189
42 31 23 4
11 23 0 0 31 8 23 4
1064
43 36 19 2
14 28 0 0 29 8 19 2
395
53 23 19 5
20 21 0 0 33 2 19 5
997
57 17 21 5
15 17 0 0 42 0 21 5
368
52 24 21 3
10 12 0 0 42 12 21 3
1608
24 66 10 0
9 55 0 0 15 11 10 0
Spine
573
26 62 11 1
9 49 0 0 17 13 11 1
Fin
628
27 59 12 2
7 41 0 0 20 18 12 2
1296
28 61 10 1
10 47 0 0 18 14 10 1
1172
31 55 12 2
12 48 0 0 19 7 12 2
882
32 57 9 2
11 44 0 0 21 13 9 2
597
35 52 12 1
12 41 0 0 23 11 12 1
Kwangtung Head-Tail skate Fin a
439 458
Head
Sharpspine Head-Tail skate Fin
55
The value of the iD-unit needs to be considered as a mixture of the D- and iD-units (see details in the text).
56
N. Takeda et al./Carbohydrate Research 424 (2016) 54–58
Fig. 1. a, b. Representative HPLC patterns of (a) chondroitinase ABC and (b) chondroitinase AC digestion (1 M of the LiCl fraction, tail fin of a Blue shark).
(Kyoto, Japan), respectively. Extra pure grade organic solvents and reagents were obtained commercially.
LiCl. Carbazole-positive fractions were subjected to dialysis and further desalted through a column for gel permeation (LH-20, H2O, ϕ1.1 × 80 cm).
2.1. Preparation of defatted dry powder of shark and ray tissues The prepared materials described above were defatted with double their weight of acetone. The solutions were filtered, and the filtrates were defatted again in the same manner. The filtrates were dried in vacuo. 2.2. Preparation of crude glycans from defatted dry tissue Crude glycans were prepared as previously described.1 Briefly, a suspension of the defatted dry material (10 g) was treated in 80 mL of boiling water for 10 min, followed by the addition of 0.5 M borate buffer (100 mL), pH 7.0. The suspension was then incubated with protease N Amano G (0.67 g) at 55 °C for 7 d. The incubated suspension was quenched with trichloroacetic acid at a concentration of 5%. The soluble fraction obtained by centrifugation at 0 °C was collected and subjected to dialysis for 7 d. Insoluble materials were filtered off, and subsequent precipitation with 80% ethanol with 1.25% NaOAc afforded crude glycans, which were collected by centrifugation at 0 °C and dried in vacuo. 2.3. DEAE-cellulose chromatography One portion of crude glycans (100 mg) was diluted with a small amount of 0.15 M LiCl/0.05 M acetic acid, pH 4.0, and applied to a column (ϕ2.2 × 15 cm) of DEAE-cellulose. The column was washed stepwise with 160 mL of buffers containing 0.15, 0.5, 1.0, and 2.0 M
2.4. Digestion with Chondroitinases ABC and AC followed by a HPLC analysis The pure glycans (50 μg) obtained above were diluted with H2O (50 μL), BSA solution (0.05 mg/5 μL), and 250 mM Tris-HCl(AcOH) buffer (10 μL), pH 8.0 (solution A). Solution A was digested with chondroitinase ABC (25 mU/25 μL) or chondroitinase AC (25 mU/ 25 μL) at 37 and 30 °C, respectively, for 8 h. The unsaturated disaccharides derived from the CS/DS hybrid chain produced by this digestion were analyzed on a PA-03 (PA-G) column by HPLC according to a previously described method.9 The contents of the CS/ DS hybrid chain and the ratios of the GlcA-GalNAc and IdoAGalNAc units as well as the sulfation patterns in pure glycans were calculated using calibration curves based on the HPLC peak area of the standard unsaturated disaccharide. Representative HPLC patterns are shown in Fig. 1a and b, and the results obtained are summarized in Table 1. 2.5. Statistical analysis Relationships between the contents of the CS/DS hybrid chain and ΔC/ΔA were determined using Spearman’s rank correlation test (Fig. 2). Similarly, relationships between the values of ΔC/ΔA and (GlcA-GalNAc)/(IdoA-GalNAc) were determined (Fig. 3). A p value less than 0.05 was considered to be significant. All statistical analyses were executed using the Statcel 3 program.
N. Takeda et al./Carbohydrate Research 424 (2016) 54–58
57
2000 Blue shark
1800
Blue shark
Contents ( mg ) / 100 g of defatted dry tissue
Mako Shark
1600
Stingray
Mako Shark
Mako Shark
1400
Sharpspine skate
Mako Shark
r = 0.467
Sharpspine skate
1200 Smooth Hammerhead
1000
A
Blue shark
Scalloped Hammerhead Kwangtung skate
B
800
600
Blue shark
Dusky shark Kwangtung skate
Stingray Stingray
Smooth Hammerhead Dusky shark
Scalloped Hammerhead
400
Dusky shark Scalloped Hammerhead
200
Smooth Hammerhead
0 0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
C/ A Fig. 2. The relationship between contents of the CS/DS hybrid chain and ΔC/ΔA in each tissue.
3.00
2.50
Blue shark
Stingray
Mako Shark
(GlcA-GalNAc) / (IdoA-GalNAc)
Sharpspine skate
r = 0.740
Mako Shark
2.00 Dusky shark
Stingray
Blue shark
Kwangtung skate
Blue shark
Sharpspine skate
Mako Shark
1.50
Kwangtung skate
Mako Shark Scalloped Hammerhead
Stingray Smooth Hammerhead
1.00
Blue shark Dusky shark Smooth Hammerhead Smooth Hammerhead Scalloped Hammerhead
0.50 Dusky shark Scalloped Hammerhead
0.00 0.00
0.50
1.00
1.50
2.00
2.50
C/ A Fig. 3. The relationship between ΔC/ΔA and (GlcA-GalNAc unit)/(IdoA-GalNAc unit) in each tissue.
3.00
3.50
58
N. Takeda et al./Carbohydrate Research 424 (2016) 54–58
3. Results and discussion Chondroitinase ABC digests every kind of GalNAc-GlcA/IdoA linkage, while chondroitinase AC specifically digests GalNAc-GlcA, but does not cleave the GalNAc-IdoA linkage or GalNAc-GlcA linkage at the non-reducing side of the D-unit, GalNAc-(GlcA2S-GalNAc6S). Since the ratio of the GlcA-GalNAc and IdoA-GalNAc units was calculated for the above reason, the value of the D-unit was 0%. The value of the iD-unit needs to be considered as a mixture of D- and iD-units. The results obtained revealed that all samples from the tissues of sharks and rays were composed of the A-, iA-, C-, iC-, and D/iDunits (Table 1). Some tissues contained the iE-unit, but at less than 6%. Other sulfation patterns including O-units, iO-units, and the hyaluronic acid disaccharide unit (GlcA-GlcNAc) were not detected. The contents of the CS/DS hybrid chain in bony fish were approximately several hundred milligrams.1 Our results indicate that the contents of the CS/DS hybrid chain were higher in cartilaginous fish than in bony fish. The relationship between the contents of the CS/DS hybrid chain and the ratios of ΔC and ΔA were plotted in Fig. 2. A positive correlation was observed between the contents of the CS/DS hybrid chain and the value of ΔC/ΔA (r = 0.467, n = 24, p < 0.05; *). Blue sharks, Mako sharks, Stingrays, Kwangtung skates, and Sharpspine skates were categorized into the A group, namely, those with “a high content of the CS/DS hybrid chain and a highΔC/ΔA value”. Other sharks close to the zero point were grouped into B. Blue and Mako sharks belonging to the A group are pelagic and, thus, require a large amount of oxygen for aerobic exercise. As a result, their cells are exposed to radical oxygen more than those of slow moving coastal sharks. Previous studies reported that CS suitably coordinates metal ions such as Fe and inhibits radical oxygen, with the A-unit of CS being more effective than the C-unit for this purpose.10,11 In Fig. 2, sharks in category A had higher contents of the CD/DS hybrid chain. Although the ΔC/ΔA values were relatively high, the larger amount of CS may inhibit radical oxygen. In contrast, pelagic bony fish such as tuna, mackerel, and jack mackerel contain smaller amounts of the CS/DS hybrid chain (12~370 mg per 100 g of defatted dry tissue), but always possess abundant amounts of the A-unit (ΔA:ΔC = 46:35~100:0).1 Thus, these bony fish may not need as much CS/DS as sharks. On the other hand, coastal sharks in category B contain a relatively low amount of CS/DS, but are predominantly composed of the A-unit (ΔC:ΔA < 1), which may effectively protect cells from oxidative stress. Most rays belong to category A. They inhabit the bottom of the coast. Rays are often caught by flatfish fishermen as a by-catch. The results obtained for group A are in good agreement with those for bottom-dwelling bony fish such as flatfish.1 Flatfish and rays are completely different species, but inhabit a similar environment with quasi motilities. We hypothesized that a relationship exists between the contents of the CS/DS hybrid chain and ratio of the isomeric sulfate from the point of view of the inhibition of radical oxygen. We also evaluated the relationship between the ratios of (GlcAGalNAc unit)/(IdoA-GalNAc unit) and ΔC/ΔA in each tissue in Fig. 3. A positive correlation was observed between these two elements (r = 0.740, n = 24, p < 0.001; ***). In the biosynthesis of the CS/DS hybrid chain, the CS chain is epimerized at C-5 of the GlcA residue with the assistance of epimerase in order to form the CS/DS hybrid chain.12 OH-4 and OH-6 on the GalNAc residue are sulfated by the
corresponding sulfotransferases. Fig. 3 indicates that the level of expression and/or activities of 6-O- and 4-O-sulfotransferases are greater for GlcA-GalNAc and IdoA-GalNAc units, respectively. 4. Conclusions We herein demonstrated that the contents of the CS/DS hybrid chain in five shark species and three ray species differed in speciesand tissue-dependent manners. Sharks and rays contain 189– 1863 mg of the CS/DS hybrid chain per 100 g of defatted dry tissue. The sulfation patterns of sharks and rays mainly comprised the Aand C-units, and also characteristically contained the D-unit at 9% to 23%. Blue sharks and Mako sharks, which are categorized as pelagic sharks, contained large contents of the CS/DS hybrid chain and high ratios of ΔC/ΔA. Oxidative stress caused by more intense aerobic exercise in pelagic sharks may affect the contents of CS/DS and sulfation patterns. Commercially available CS is often derived from sharks, which have been overfished. We herein clarified the contents of the CS/ DS hybrid chain and composition of the disaccharide unit including sulfation patterns in sharks and rays. These results will contribute to the identification of animals as alternative sources of CS in the market. Acknowledgments This study was supported by a Grant-in-Aid for scientific research in innovative areas (23110003) from MEXT, Japan. N.T. is grateful for the JSPS Research Fellowship for Young Scientists (25•8319). We thank Mr. Eiichi Hiroiwa (Tottori Fishery port) for the kind gift of fish. The authors also thank Ryosuke Toita, Kento Ueda, Yumi Irie, Nozomi Kidoguchi, Daisuke Kimino, Naoya Goto, and Hiroya Takada (Tottori University) for their helpful assistance. References 1. Arima K, Fujita H, Toita R, Imazu-Okada A, Tsutsumishita-Nakai N, Takeda N, et al. Carbohydr Res 2013;366:25–32. 2. Tamura J, Arima K, Imazu A, Tsutsumishita N, Fujita H, Yamane M, et al. Biosci Biotechnol Biochem 2009;73:1387–91. 3. Fürth O, Bruno T. Biochem Z 1937;294:153–73. 4. Seno N, Meyer K. Biochim Biophys Acta 1963;78:258–64. 5. (a) Sugahara K, Kojima T. Eur J Biochem 1996;239:865–70; (b) Sugahara K, Tanaka Y, Yamada S. Glycoconj J 1996;13:609–19; (c) Sugahara K, Nadanaka S, Takeda K, Kojima T. Eur J Biochem 1996;239:871–80; (d) Nadanaka S, Sugahara K. Glycobiology 1997;7:253–63; (e) Nadanaka S, Clement A, Masayama K, Faissner A, Sugahara K. J Biol Chem 1998;273:3296–307; (f) Nandini CD, Itoh N, Sugahara K. J Biol Chem 2005;280:4058–69; (g) Li F, Shetty AK, Sugahara K. J Biol Chem 2007;282:2956–66; (h) Deepa SS, Yamada S, Fukui S, Sugahara K. Glycobiology 2007;17:631–45; (i) Mizumoto S, Murakoshi S, Kalayanamitra K, Deepa SS, Fukui S, Kongtawelert P, et al. Glycobiology 2013;23:155–68. 6. Higashi K, Takeuchi Y, Mukuno A, Tomitori H, Miy M, Linhardt RJ, et al. PLoS ONE 2015;10:e0120860. 7. Chatziioannidis CC, Karamanos NK, Tsegenidis T. Comp Biochem Physiol Part B 1999;124:15–24. 8. Dellias JMM, Onofre GR, Werneck CC, Landeira-Fernandez AM, Melo FR, Farias WRL, et al. Biochemie 2004;86:677–83. 9. Yoshida K, Miyauchi S, Kikuchi H, Tawada A, Tokuyasu K. Anal Biochem 1989;177:327–32. 10. Campo GM, D’Ascola A, Avenoso A, Campo S, Ferlazzo AM, Micali C, et al. Glycoconj J 2004;20:133–41. 11. Campo GM, Avenoso A, Campo S, Ferlazzo AM, Calatroni A. Mini Rev Med Chem 2006;6:1311–20. 12. Malmström A, Bartolini B, Thelin MA, Pacheco B, Maccarana M. J Histochem Cytochem 2012;60:916–25.