Glycosaminoglycan Composition of the Large ... - ACS Publications

Biomacromolecules 2005, 6, 3174-3180

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Glycosaminoglycan Composition of the Large Freshwater Mollusc Bivalve Anodonta anodonta Nicola Volpi* and Francesca Maccari Dipartimento di Biologia Animale, University of Modena and Reggio Emilia, Modena, Italy Received July 18, 2005; Revised Manuscript Received September 8, 2005

In this paper, glycosaminoglycans from the body of the large freshwater mollusc bivalve Anodonta anodonta were recovered at about 0.6 mg/g of dry tissue, composed of chondroitin sulfate (approximately 38%), nonsulfated chondroitin (about 21%), and heparin (41%). This last polysaccharide was found to consist of a large percentage (approximately 88%) of a fast-moving species possessing a lower molecular mass and sulfate group amount and about 12% of a more sulfated, slow-moving component having a greater molecular mass. The chondroitin sulfate was composed of approximately 28% of the 6-sulfated disaccharide, 46% of the 4-sulfated disaccharide, and about 26% of the nonsulfated disaccharide, with a charge density value of 0.74. Heparin was subjected to the oligosaccharide mapping after treatment with heparinase and then separation of the resulting unsaturated oligosaccharides by SAX-HPLC. A heparin sample from Anodonta anodonta showed a degree of sulfation similar to that of bovine mucosal heparin because of the presence of approximately the same mol % of the trisulfated disaccharide (∆UA2S(1f4)-R-D-GlcN2S6S), a slight modification of the other oligosaccharides, and a significant increase of the disaccharide bearing the sulfate group in position 3 of the N-sulfoglucosamine 6-sulfate (f4)-β-D-GlcA(1f4)-R-D-GlcN2S3S6S(1f) part of the ATIII-binding region. However, the anticoagulant activity of mollusc heparin was quite similar to that of pharmaceutical grade heparin. The data obtained again emphasize the heterogeneity of GAGs from molluscs. Introduction Glycosaminoglycans (GAGs) are acidic, highly sulfated, complex, linear polysaccharides. There are many types of GAGs generally grouped into four classes: (1) hyaluronan (HA), (2) keratan sulfate, (3) chondroitin sulfate (CS) and dermatan sulfate (DS), and (4) heparan sulfate (HS) and heparin. They are synthesized as polymers of repeating disaccharides with an N-acetylhexosamine (N-acetylgalactosamine or N-acetylglucosamine) as one of the sugars. The alternating sugar is glucuronic acid, with the exception of keratan sulfate which contains galactose instead. HA, containing N-acetylglucosamine, is not further modified, whereas the other classes are modified by (1) the addition of O-sulfate groups on various hydroxyls (the three classes), (2) 5-epimerization of some glucuronic acid residues to form iduronic acid residues (DS, HS, heparin), and (3) removal of acetyl residues from some hexosamines replaced with N-sulfates (HS and heparin). These modifications introduce microheterogeneity within the chains and often have major roles in a wide variety of biological and pharmacological processes.1,2 With the exception of HA, GAGs are covalently bound to core proteins, forming macromolecules called proteoglycans. Proteoglycans, found inside cells, on their surfaces, or in extracellular matrices, play an important role in a variety * Prof. Nicola Volpi. Department of Biologia Animale, University of Modena and Reggio Emilia, Via Campi 213/D, 41100 Modena, Italy. E-mail: [email protected]. Fax number: 0039 59 2055548. Tel. number: 0039 59 2055543.

of diseases.3-5 GAGs, produced by extraction and purification from different animal tissues, have several fundamental biological activities, as well as pharmacological properties, making them important drugs for use in clinical and pharmaceutical fields.6-8 The presence of sulfated GAGs in some taxa of invertebrates is now well-documented.9-18 A comprehensive survey of different classes of invertebrates has shown that CS/DS, HS-like, and/or heparin-like compounds are present in many species.16 Previous studies have also shown that heparin is present in several species of molluscs. A compound from the clam Mercenaria mercenaria14 exhibits several structural similarities to heparin. Heparins with high anticoagulant activity have been isolated from the molluscs Anomalocardia brasiliana,11,13 TiVela mactroides,11 and Tapes phylippinarum.17 CS,12,19 DS,9 and acharan sulfate20 have also been isolated and characterized from different families of molluscs. Thanks to our knowledge of the sulfated polysaccharides in vertebrates and invertebrates, it is now possible to draw a phylogenetic tree of the distribution of sulfated GAGs in the animal kingdom.21 Commercial manufacture of CS/DS and heparin relies, in particular, on mammalian tissues as raw material. However, the appearance of bovine spongiform encephalopathy and its apparent link to the similar prion-based Creutzfeldt-Jakob disease in humans22 has limited the use of bovine CS and heparin, and porcine GAGs also have problems associated with religious restrictions on their use. Furthermore, nonanimal sources of GAGs, such as chemically synthesized,

10.1021/bm0505033 CCC: $30.25 © 2005 American Chemical Society Published on Web 10/13/2005

Glycosaminoglycan Composition in A. anodonta

enzymatically synthesized, or recombinant polyanions are currently not available for pharmaceutical purposes. These concerns have motivated us to look for alternative, nonmammalian sources of complex polysaccharides from the large freshwater mollusc bivalve Anodonta anodonta. Materials and Methods Heparin from bovine intestinal mucosa, CS from bovine trachea, DS from porcine intestinal mucosa, and HA from rooster comb were from Sigma. The SM-heparin and FMheparin components of heparin were purified as their barium salts at different temperatures, as previously reported.23-25 The first species had an Mr of about 14 900 and a sulfateto-carboxyl ratio of 2.66, while the second heparin component had an Mr of 7920 and a charge density of 2.11. Heparin lyase I, heparinase, from FlaVobacterium heparinum (EC 4.2.2.7), specific activity of 1.5 units/mg protein, and heparan sulfate lyase, heparitinase, from FlaVobacterium heparinum (EC 4.2.2.8), specific activity of 1.5 units/mg protein, were from Seikagaku. Heparin lyase II, heparinase II, from FlaVobacterium heparinum (no EC number), 100-300 units/ mg solid, was from Sigma. Papain from papaya latex (EC 3.4.22.2), specific activity of 16-40 units/mg protein, was from Sigma. Deoxyribonuclease I, DNase I (EC 3.1.21.1) from bovine pancreas, specific activity of 10 000 units/mL, chondroitinase ABC, chondroitin ABC lyase, from Proteus Vulgaris (EC 4.2.2.4), specific activity of 0.5-2 units/mg, and hyaluronate lyase from Streptomyces hyalurolyticus (hyaluronidase, EC 4.2.2.1) were from Sigma. Hyaluronidase SD from Streptococcus dysgalactiae and chondroitinase B, chondroitin B lyase, from FlaVobacterium heparinum (EC 4.2.2.) were from Seikagaku. Unsaturated heparin/HS and CS/DS disaccharides were from Seikagaku. Stains-All (1ethyl-2-[3-(1-ethylnaphtho[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl] naphtho[1,2-d]thiazolium bromide, 3,3′-diethyl9-methyl-4,5,4′,5′-dibenzothiacarbocyanine bromide) was from Sigma. QAE Sephadex A-25 anion-exchange resin was from Pharmacia Biotech, Uppsala, Sweden. Microcon YM-3 filters having a molecular mass cutoff of 3000 Da were from Amicon. Spectrapore dialysis tubing (Mr 1000 Da cutoff) was from Spectrum. All other reagents were of analytical grade. The carbazole assay for uronic acids was performed according to Cesaretti et al.23 Purification of the Glycosaminoglycan Component. Adult specimens of Anodonta anodonta were collected from a local river near Reggio Emilia, Italy. After each collection, the molluscs were killed and the shell removed. The bodies of individual specimens were separately defatted by acetone. After centrifugation at 10 000 g for 10 min and drying at 60 °C for 24 h, the pellet (580 mg) was solubilized (1 g/20 mL) in 100 mM NaOAc buffer (pH 5.5) containing 5 mM EDTA and 5 mM cysteine. Papain (20 mg) was added, and the solution incubated for 24 h at 60 °C in a stirrer. After boiling for 10 min, the mixture was centrifuged at 5 000 g for 15 min, and the pellet treated as reported above. After boiling for 10 min, the mixture was centrifuged at 5 000 g for 15 min, and supernatants collected together. Three

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volumes of ethanol saturated with sodium acetate were added to the pooled supernatants and stored at +4 °C for 24 h. The precipitate was recovered by centrifugation at 5 000 g for 15 min and dried at 60 °C for 6 h. The dried precipitate (about 205 mg) was dissolved in 10 mL of distilled water by prolonged mixing. After centrifugation at 5 000 g for 15 min, 1 mL of 20% trichloroacetic acid was added to 5 mL of the supernatant. After 2 h at 4 °C, the mixture was centrifuged at 5 000 g for 15 min, and supernatant recovered and lyophilized. After solubilization in 1 mL of 0.05 M NaCl and centrifugation at 10 000 g for 10 min, the supernatant was applied to a column (1 cm × 20 cm) packed with QAE Sephadex A-25 anion-exchange resin equilibrated with the same NaCl solution. GAGs were eluted with a linear gradient of NaCl from 0.05 to 1.2 M from 0 to 150 min using a lowpressure liquid chromatography (Biologic LP chromatography system from BioRad) at a flow of 1 mL/min. Fractions of 2 mL were collected and further analyzed. Agarose-Gel Electrophoresis. Agarose-gel electrophoresis in barium acetate/1,2-diaminopropane was performed as reported elsewhere24,25 with minor modifications. A Pharmacia Multiphor II (from Pharmacia LKB Biotechnology, Uppsala, Sweden) electrophoretic cell instrument was used. Agarose gel was prepared at a concentration of 0.5% in 0.04 M barium acetate buffer (pH 5.8). The run was in 0.05 M 1,2-diaminopropane (buffered at pH 9.0 with acetic acid) for 150 min at 50 mA. After migration, the plate was soaked in cetyltrimethylammonium bromide 0.1% solution for at least 6 h, dried, and stained with toluidine blue for sulfated GAGs or toluidine blue and Stains-All for nonsulfated polymers.25 Sulfated GAGs, heparin, DS, and CS show strong metachromatic properties when stained with toluidine blue, showing a purple color. On the contrary, because of the absence of sulfate groups, HA or chondroitin are not detected with toluidine blue, while they are stained with Stains-All after toluidine blue treatment, showing a strong blue color.25 Strong Anion-Exchange High-Performance Liquid Chromatography. High-performance liquid chromatography (HPLC) equipment was from Jasco (pump model PU-1580, UV detector model UV-1570, Rheodyne injector equipped with a 100 µL loop, software Jasco-Borwin release 1.5). The unsaturated oligosaccharides generated from HA or GAG extract samples treated with hyaluronate lyase or hyaluronidase SD were analyzed by strong anion-exchange (SAX)HPLC separation using a 150 × 4.6 mm stainless steel column spherisorb 5-SAX (5 µm, trimethylammoniopropyl groups Si-CH2-CH2-CH2-N+(CH3)3 in Cl- form, from Phase Separations Limited, Deeside Industrial Park, Deeside Clwyd, U.K.) and detection at 232 nm. Isocratic separation was performed using 50 mM NaCl (pH 4.00) for 5 min followed by a linear gradient from 5 to 60 min of 50 mM NaCl to 1.2 M NaCl (pH 4.00), at a flow rate of 1.2 mL/ min. Oligosaccharide Mapping of Heparin. Anodonta anodonta heparin was treated for 12 h at 37 °C with 15 milliunits of heparinase in 100 µL of 50 mM pH 7.3 acetate buffer containing 25 mmol calcium acetate, after which it was frozen and stored at -70 °C. Oligosaccharides labeled

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1-8 produced by the action of heparinase were separated and quantified by SAX-HPLC separation at 232 nm as reported above. The structures of standards 1-8 have been fully determined26-28 and are as follows: 1, ∆UA(1f4)-R-D-GlcN2S6S. 2, ∆UA2S(1f4)-R-D-GlcN2S. 3, ∆UA2S(1f4)-R-D-GlcN2S6S. 3a, ∆UA2S(1f4)-R-D-GlcN2S(1f4)-R-L-IdoA2S(1f4)R-D-GlcN2S. 3b, ∆UA2S(1f4)-R-D-GlcN2S(1f4)-β-D-GlcA(1f4)-RD-GlcN2S6S. 4, ∆UA2S(1f4)-R-D-GlcN2S6S(1f4)-R-L-IdoA2S(1f4)R-D-GlcN2S. 4a, ∆UA2S(1f4)-R-D-GlcN2S6S(1f4)-R-L-IdoA(1f4)R-D-GlcN2S6S. 5, ∆UA2S(lf4)-R-D-GlcN2S6S(lf4)-β-D-GlcA(1f4)-RD-GlcN2S6S. 6, ∆UA2S(1f4)-R-D-GlcN2S6S(1f4)-R-L-IdoA2S(1f4)R-D-GlcN2S6S. 6a, ∆UA2S(1f4)-R-D-GlcN2S6S(1f4)-R-L-IdoA(1f4)R-D-GlcNAc6S(lf4)-β-D-GlcA(1f4)-R-D-GlcN2S6S. 7, ∆UA2S(1f4)-R-D-GlcN2S6S(1f4)-β-D-GlcA(1f4)R-D-GlcN2S3S6S. 8, ∆UA2S(1f4)-R-D-GlcN2S6S(1f4)-R-L-IdoA(1f4)R-D-GlcNAc6S(1f4)-β-D-GlcA(1f4)-R-D-GlcN2S3S6S. Major peaks were identified and quantified on the basis of their comigration with oligosaccharide standards prepared according to literature26-28 and the structure established by comparison with disaccharide standards after treatment with heparin lyase I (EC 4.2.2.7), heparin lyase II (no EC number), and heparan sulfate lyase (EC 4.2.2.8) and SAX-HPLC separation. The major peaks were first assigned by the coinjection of oligosaccharide standards, and the peaks were integrated to obtain the oligosaccharide composition.17,18 The disaccharide composition was calculated from the oligosaccharide map with ∆UA2S being assigned to R-L-IdoAp2S consistent with the known specificity of heparinase.17,18,29 Anticoagulant Properties. The activated partial thromboplastin time (APTT) and the amidolytic antifactor Xa assay of Anodonta anodonta heparin was determined as previously described17,18 in comparison with pharmaceutical-grade heparin. Specific activities were calculated as international units (IU) per milligram. Results After defatting with organic solvents and extraction by proteolytic treatment, GAGs from the body of Anodonta anodonta were fractionated on a anion-exchange resin and eluted with a linear NaCl gradient of increasing molarity. A strong absorbance at 210 nm was detected for fractions from 33 to 55 (not shown), and the carbazole test for uronic acids23 and agarose-gel electrophoresis stained with toluidine blue and Stains-All confirmed the presence of different polysaccharides in the extract (Figure 1). The fractions from 33 to 55 were collected, and GAGs were precipitated at 4 °C by adding 2 volumes of ethanol saturated with sodium acetate. After centrifugation at 10 000

Volpi and Maccari

Figure 1. Agarose-gel electrophoresis stained with toluidine blue and Stains-All25 of the collected fractions from 30 to 55 separated by means of a column (1 cm × 20 cm) packed with QAE Sephadex A-25 anion-exchange resin equilibrated with NaCl 0.05 M solution. GAGs were eluted with a linear gradient of NaCl from 0.05 to 1.2 M from 0 to 150 min using low-pressure liquid chromatography (Biologic LP chromatography system from BioRad) at a flow of 1 mL/min. Fractions of 2 mL were collected.

Figure 2. Agarose-gel electrophoresis stained with toluidine blue and Stains-All of Anodonta anodonta GAGs, untreated and treated with various enzymes. GAGs, purified polysaccharides untreated; +ABC, extract treated with chondroitinase ABC; +HA lyase, extract treated with hyaluronidase SD from Streptococcus dysgalactiae; +ABC +Hep I, extract treated with chondroitinase ABC and heparin lyase I; HA, HA from rooster comb; HA + HA lyase, HA from rooster comb treated with hyaluronidase SD from Streptococcus dysgalactiae; HA: hyaluronic acid; CS: chondroitin sulfate; C: nonsulfated chondroitin; HS, heparan sulfate; FM, fast-moving heparin; SM, slow-moving heparin; o, origin.

g for 10 min, the pellet was dried at 60 °C and solubilized in distilled water for further characterization. Analyses performed by means of the carbazole test for uronic acids23 and agarose-gel electrophoresis25 yielded approximately 0.6 mg GAGs/g of dry tissue. Identification of the GAG Species. To identify the species of polysaccharides extracted from Anodonta anodonta, the GAG extract was subjected to treatment with specific enzymes, i.e., chondroitin ABC lyase, chondroitinase B, heparin lyase I, hyaluronate lyase from Streptomyces hyalurolyticus or hyaluronidase SD from Streptococcus dysgalactiae, and agarose-gel electrophoresis. Furthermore, the products of the enzymatic analyses were also qualitatively and quantitatively evaluated by HPLC. Figure 2 shows the agarose-gel electrophoresis stained with toluidine blue and Stains-All of Anodonta anodonta GAGs, both untreated and treated with various enzymes. The lanes marked as GAGs in the figure consist of the extract which was not subjected to the enzymatic procedures. The lane indicated as +ABC represents the extract treated with chondroitin ABC lyase, showing the disappearance of the bands having the same mobility as CS and nonsulfated chondroitin (see below). The use of chondroitinase B resulted in no effect on the pattern of Anodonta anodonta GAGs (not shown), supporting the absence of DS in the extract. After treatment with chondroitinase ABC, two main bands were visible, one having no capacity to migrate into the gel


(indicated as SM and localized on the position of the loading of the gel, the origin) and the second one possessing the same migrational properties as either HS or FM-heparin.24,25 As a consequence, we supposed the presence of heparin with its two components, the SM-heparin having high molecular mass and degree of sulfation and the FM-heparin possessing a lower molecular mass and sulfate group amount. This was confirmed by simultaneous treatment with chondroitinase ABC and heparinase I, showing the resultant disappearance of the two bands (marked in Figure 2 as +ABC+Hep I), and also by the analysis of the heparinase I oligosaccharides by means of HPLC (see below). Because of the possible presence in the extract of nonsulfated polysaccharides, such as HA or nonsulfated chondroitin (C), which are stained by the adopted procedure25 and which possess a mobility intermediate between FM-heparin and CS (see the lane marked HA showing the migration of HA in Figure 2), we treated the GAGs extract with hyaluronidase SD from Streptococcus dysgalactiae, which is able to degrade HA and nonsulfated chondroitin (C) so as to yield unsaturated nonsulfated disaccharides.30 As is evident from the agarosegel separation in Figure 2 (see the lane + HA lyase), a band intermediate between CS and FM-heparin disappears. The nature of this nonsulfated species was determined as nonsulfated chondroitin and not as HA by using other analytical approaches (see below). In addition to the qualitative evaluation of the GAG species in the body of Anodonta anodonta, by using specific calibration curves for CS, SMheparin, FM-heparin, and HA and densitometric analysis25 and the treatment with enzymes illustrated above, we were able to give a quantitative profile of the mollusc polysaccharides. CS was present in a percentage of approximately 38%, nonsulfated chondroitin was about 21%, while heparin was 41% (88% of the FM-heparin species and 12% of the SM-heparin component). As is evident, no keratan sulfate was detected in the Anodonta anodonta extract. Characterization of the Mollusc Nonsulfated Polysaccharide. The GAG extract from mollusc was treated with hyaluronate lyase from Streptomyces hyalurolyticus or hyaluronidase SD from Streptococcus dysgalactiae. After treatment, the unsaturated products of the enzymatic treatments were recovered by filtering on filters with a molecular mass cutoff of 3 000 Da, while the undigested polysaccharides, i.e., CS and heparin, were recovered in the unfiltered materials and further analyzed (see below). Hyaluronidase SD from Streptococcus dysgalactiae, able to degrade HA and nonsulfated chondroitin (C),30 yielded unsaturated nonsulfated disaccharides as shown by SAXHPLC analysis (Figure 3). On the contrary, hyaluronate lyase from Streptomyces hyalurolyticus is highly specific for HA producing unsaturated tetra- and hexasaccharides, but it is inert with nonsulfated chondroitin. This enzyme was unable to degrade the nonsulfated polysaccharide species from Anodonta anodonta, evaluated by SAX-HPLC31 (Figure 4B), thus confirming the nature of this GAG as nonsulfated chondroitin. Determination of the Nature of Anodonta anodonta CS. The GAG extract composed of CS and heparin remaining


Figure 3. SAX-HPLC of the unsaturated disaccharide produced by HA (from rooster comb) (A) and the nonsulfated polysaccharide species extracted from Anodonta anodonta (B) treated with hyaluronidase SD from Streptococcus dysgalactiae.

after treatment with hyaluronate lyase was subjected to treatment with chondroitinase ABC, and the unsaturated disaccharides produced recovered by passage through 3000 Da filters. The undigested heparin was recovered in the retentate and further analyzed (see below), while the CS disaccharides were qualitatively and quantitatively analyzed by SAX-HPLC (Figure 5). The disaccharides nonsulfated ∆UA1f3GalNAc, 6-sulfated ∆UA1f3GalNAc6(SO4), and 4-sulfated ∆UA1f3GalNAc4(SO4) accounted for 26%, 28%, and 46% of the total products derived from Anodonta anodonta CS. This polysaccharide showed a charge density value of 0.74 and a 4-sulfated/6-sulfated ratio of approximately 1.6. Characterization of the Anodonta anodonta Heparin. After elimination of the CS and nonsulfated CS from the purified mollusc GAGs, heparin was subjected to qualitative and quantitative oligosaccharide characterization, as previously reported.17,18 Oligosaccharide mapping26-29 was performed by depolymerizing Anodonta anodonta heparin (Figure 6A) with heparinase (EC 4.2.2.7) and then separating the resulting unsaturated oligosaccharides by SAX-HPLC in comparison with the bovine mucosal heparinase-depolymerized heparin (Figure 6B). A mass balance close to 95% (Table 1) was calculated for Anodonta anodonta heparin due to the presence of approximately 5% of unidentified oligosaccharides. Accord-

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Figure 4. SAX-HPLC of the unsaturated tetrasaccharide (T) and hexasaccharide (H) produced by HA (from rooster comb) (A) and the polysaccharides extracted from Anodonta anodonta showing no products (B), treated with hyaluronate lyase from Streptomyces hyalurolyticus. No peak possessing an unsaturated bond able to absorb at 232 nm was detected.

Figure 5. SAX-HPLC of unsaturated disaccharides from Anodonta anodonta CS treated with chondroitinase ABC. 1. Nonsulfated disaccharide ∆UA1 f 3GalNAc. 2. 6-Sulfated disaccharide ∆UA 1 f 3 GalNAc6 (SO4). 3. 4-Sulfated disaccharide ∆UA 1 f 3 GalNAc4 (SO4).

ing to previous data,17,18,26 bovine heparin showed a mass balance of approximately 92%. Differences between bovine and Anodonta anodonta heparins lay in the absence of the oligosaccharides 3a, 3b, and 4a, in an increase of the oligosaccharides 5, 7, and 8,

Volpi and Maccari

Figure 6. SAX-HPLC chromatograms of heparin lyase-treated (A) Anodonta anodonta and (B) bovine mucosal heparin samples. The major peaks corresponding to oligosaccharides 1-8 are indicated, and they were assigned by coinjection with standards (see their structures in Materials and Methods section). Table 1. Oligosaccharide Analysis of Heparins after Heparinase Treatmenta oligosaccharide

Anodonta anodonta heparin

bovine mucosal heparin

1 2 3 3a 3b 4 4a 5 6 6a 7 8 total (1-8)

n.d. 7.0 64.1 n.d. n.d. 3.2 n.d. 9.4 4.2 n.d. 3.3 3.8 95.0

n.d. 5.3 62.2 2.6 2.0 3.7 0.8 6.5 4.0 0.3 1.9 2.2 91.5

a Total mol % of the oligosaccharides is calculated by summing the mol % for the oligosaccharides in each column. n.d. ) not detected.

and in the lack of modification of the content of the trisulfated disaccharide (∆UA2S(1f4)-R-D-GlcN2S6S) (Table 1). The results of these analyses were used to calculate the disaccharide composition (Table 2). As expected from the oligosaccharide compositional analysis, heparin from Anodonta anodonta has a degree of sulfation similar to that of bovine mucosal heparin due to the presence of approximately



Table 2. Disaccharide Composition of Anodonta anodonta and Bovine Mucosal Heparins Performed by Considering the Oligosaccharide Quantitative Analysis Reported in Table 1a disaccharide sequence f4)-R-L-IdoA2S(1 f 4)-R-D-GlcN2S6S(1f f4)-β-D-GlcA(1 f 4)-R-D-GlcN2S6S(1f f4)-R-L-IdoA2S(1 f 4)-R-D-GlcN2S(1f f4)-R-L-IdoA(1 f 4)-R-D-GlcNAc6S(1f f4)-β-D-GlcA(1 f 4)-R-D-GlcN2S3S6S(1f f4)-R-L-IdoA(1 f 4)-R-D-GlcN2S6S(1f total mol % (measured in oligosaccharides 1-8)

Anodonta bovine mol % mol % 77.5 4.7 8.6 1.3 2.9 0.0 95.0

73.5 4.4 10.7 0.8 1.7 0.4 91.5

a The mol % of each oligosaccharide was the total absorbance % corresponding to each peak. The oligosaccharides each contain a ∆UA2S at their reducing termini that is used for their detection. The ∆UA2S residue is assumed to arise from an R-L-IdoA2S residue on the basis of the specificity of heparinase.

the same mole percent of the trisulfated disaccharide (∆UA2S(1f4)-R-D-GlcN2S6S) (Table 2). On the contrary, there is a significant increase (approximately 70%) of the disaccharide bearing the sulfate group in position 3 of the N-sulfoglucosamine 6-sulfate (f4)-β-D-GlcA(1f4)-R-DGlcN2S3S6S(1f) part of the ATIII-binding region (Table 2). The anticoagulant activity of the Anodonta anodonta heparin was also evaluated after its further purification. The ATIII-mediated antifactor Xa of pharmaceutical-grade heparin obtained from bovine intestine shows an activity of 125 IU/mg and an APTT activity of 145 IU/mg.17,18 Heparin isolated from the Anodonta anodonta body in our laboratory showed an antifactor Xa activity of 120 IU/mg and an APTT activity of 137 IU/mg. Discussion Anodonta anodonta is an elongated, oval, and fairly swollen freshwater mussel having a total length of approximately 12-16 cm. GAGs from the body of this clam were recovered at about 0.6 mg/g of dry tissue, composed of CS (approximately 38%), nonsulfated chondroitin (about 21%), and heparin (41%). This last polysaccharide was found to consist of a large percentage (approximately 88%) of the fast-moving species possessing a lower molecular mass and sulfate group amount and about 12% of the more sulfated, slow-moving component having a greater molecular mass.24,25 No keratan sulfate was detected in the Anodonta anodonta extract, but to our knowledge, the presence of keratan sulfate in molluscs has never been demonstrated. The CS macromolecule was determined to be composed of a low-sulfated polysaccharide made up of approximately 28% of the 6-sulfated disaccharide, 46% of the 4-sulfated disaccharide, and about 26% of the nonsulfated disaccharide, with a charge density value of 0.74 lower than mammalian polymers.32 Furthermore, we found the presence of a large amount, approximately 21%, of nonsulfated chondroitin. The extraction and purification methods adopted are commonly used to purify GAGs from various tissues and sources.17,18,24 This excludes the possibility that the CS from Anodonta anodonta might be desulfated during the purification process. This polysaccharide shows, in particular, a low charge density

due to the presence of 21% nonsulfated chondroitin and approximately 26% nonsulfated disaccharides inside the CS chains probably caused by an enzymatic desulfation in the mollusc. Low-sulfated CS molecules have been purified and characterized from other species of Mollusca gastropoda 12,33 and in Drosophila melanogaster and Caenorhabditis elegans.34 Pharmaceutical heparin is typically prepared from bovine or porcine intestinal mucosa or beef lung.35 However, heparin samples with peculiar structures and properties have been purified from several species of molluscs.13-15,17,18,21 Heparin from Anodonta anodonta is quite similar to the heparin from bovine mucosa, in particular, for the amount of SM-heparin and FM-heparin species and for the percentage of the oligosaccharides derived from the treatment with heparinase and the related disaccharides (see Table 2). When bovine heparin is treated with heparin lyase I, six major oligosaccharides (2, 3-6, 8) are observed on SAX-HPLC, accounting for 85-90 mol % of the oligosaccharide products, and the structures of additional minor oligosaccharides (1, 3a, 3b, 4a, 6a and 7) have also been reported.26-29 Furthermore, specific 3-O-sulfated oligosaccharides corresponding to part of the ATIII-binding site have been identified as the tetrasaccharide 7 (∆UA2S(1f4)-R-D-GlcN2S6S(1f4)-β-DGlcA(1f4)-R-D-GlcN2S3S6S) and the hexasaccharide 8 (∆UA2S(1f4)-R-D-GlcN2S6S(1f4)-R-L-IdoA(1f4)-R-DGlcNAc6S(1f4)-β-D-GlcA(1f4)-R-D-GlcN2S3S6S).26 By using the same methodological approach, we analyzed the structure of the heparin sample purified from the clam Anodonta anodonta in comparison with bovine mucosal heparin. This heparin was found to be composed of approximately equal mole percent of the disaccharide sequence in comparison with bovine mucosal heparin (see Table 2) and a similar degree of sulfation. However, a larger amount of the oligosaccharide sequences 7 and 8 bearing a portion of the ATIII binding site was detected in the clam heparin than in the bovine sample (see Table 1), as shown by the higher amount of the disaccharide bearing the sulfate group in position 3 of the N-sulfoglucosamine 6-sulfate (f4)-βD-GlcA(1f4)-R-D-GlcN2S3S6S(1f). Despite the higher percentage of the oligosaccharides bearing part of the ATIIIbinding site, the anticoagulant activity of mollusc heparin was quite similar to that of bovine mucosal heparin. Because of the similar structure and anticoagulant activity with pharmaceutical grade heparin, Anodonta anodonta heparin may be a suitable alternative source of this complex polysaccharide. Whereas HSs and CSs are ubiquitous components of all tissue-organized metazoans, heparin has shown a very peculiar distribution in mammalian and other vertebrate tissues, as well as invertebrates.16 Furthermore, a large variation of the concentration of heparin among species is evident, with the non-mammalian vertebrate tissues showing considerably lower amounts.21 In invertebrates, heparin is found in few taxa, namely molluscs, crustaceans, annelida, echinoderma, and cnidaria,21 with the anticoagulant activity varying according to the species analyzed. By using several analytical approaches, we were able to demonstrate the presence of heparin in the Anodonta anodonta mollusc with

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its two components, FM-heparin and SM-heparin detected by agarose-gel electrophoresis, and not HS. Finally, this research and previous studies conducted in our laboratory17,18 and the data available in the literature16,21 again imply that heparins have considerable structural variation depending on their origin. Acknowledgment. The authors are grateful to Dr. Mirko Iotti for supplying the molluscs. Abbreviations Ac, acetate; CS, chondroitin sulfate; DS, dermatan sulfate; FM-heparin, fast-moving heparin; GlcA, β-D-glucopyranosyluronic acid; GlcN, 2-deoxy-2-amino-R-D-glucopyranose; GAG, glycosaminoglycan; HA, hyaluronan, hyaluronic acid; HPLC, high-pressure liquid chromatography; HS, heparan sulfate; IdoA, R-L-idopyranosyluronic acid; Mr, molecular mass; S, sulfate; SAX, strong anion exchange; SM-heparin, slow-moving heparin; ∆UA, 4-deoxy-R-L-threo-hex-4enopyranosyluronic acid. References and Notes (1) Lindahl, U.; Kusche-Gullberg, M.; Kjellen, L. Regulated diversity of heparan sulfate. J. Biol. Chem. 1998, 273, 24979-24982. (2) Jackson, R. J.; Busch, S. J.; Cardin, A. D. Glycosaminoglycans: molecular properties, protein interactions, and role in physiological processes. Physiol. ReV. 1991, 71, 481-539. (3) Hardingham, T. E.; Fosang, A. J. Proteoglycans: many forms and many functions. FASEB J. 1992, 6, 861-870. (4) Ruoslahti, E. Structure and biology of proteoglycans. Annu. ReV. Cell Biol. 1988, 4, 229-255. (5) Poole, A. R. Proteoglycans in health and disease: structures and functions. Biochem. J. 1986, 236, 1-14. (6) Mammen, E. F., Walenga, J. M., Fareed, J., Eds. Seminars in Thrombosis and Hemostasis; Theme Medical Publishers: New York/ Stuttgart, 1991; Vol. 17. (7) Heparin and related polysaccharides. Structure and activities. In Annals of the New York Academy of Sciences; Ofosu, F. A., Danishefsky, I., Hirsh, J., Eds.; New York Academy of Sciences: New York, 1989; Vol. 556. (8) Lane, D. A., Lindahl, U., Eds. Heparin. Chemical and Biological properties. Clinical applications; Edward Arnold: London/Melbourne/Auckland, 1989. (9) Cassaro, C. M.; Dietrich, C. P. Distribution of sulfated mucopolysaccharides in invertebrates. J. Biol. Chem. 1977, 252, 2254-2261. (10) Hovingh, P.; Linker, A. An unusual heparan sulfate isolated from lobsters (Homarus americanus). J. Biol. Chem. 1982, 257, 98409844. (11) Pejler, G.; Danielsson, A.; Bjork, I.; Lindahl, U.; Nader, H. B.; Dietrich, C. P. Structure and antithrombin-binding properties of heparin isolated from the clams Anomalocardia brasiliana and TiVela mactroides. J. Biol. Chem. 1987, 262, 11413-11421. (12) Nader, H. B.; Ferreira, T. M. P. C.; Paiva, J. F.; Medeiros, M. G. L.; Jeronimo, S. M. B.; Paiva, V. M. P.; Dietrich, C. P. Isolation and structural studies of heparan sulfates and chondroitin sulfates from three species of molluscs. J. Biol. Chem. 1984, 259, 1431-1435. (13) Dietrich, C. P.; de-Paiva, J. F.; Moraes, C. T.; Takahashi, H. K.; Porcionatto, M. A.; Nader, H. B. Isolation and characterization of a heparin with high anticoagulant activity from Anomalocardia brasiliana. Biochim. Biophys. Acta 1985, 843, 1-7. (14) Jordan, R. E.; Marcum, J. A. Anticoagulantly active heparin from clam (Mercenaria mercenaria). Arch. Biochem. Biophys. 1986, 248, 690-695. (15) Chavante, S. F.; Santos, E. A.; Oliveira, F. W.; Guerrini, M.; Torri, G.; Casu, B.; Dietrich, C. P.; Nader, H. B. A novel heparan sulphate with high degree of N-sulphation and high heparin cofactor-II activity from the brine shrimp Artemia franciscana. Int. J. Biol. Macromol. 2000, 27, 49-57.

Volpi and Maccari (16) Medeiros, G. F.; Mendes, A.; Castro, R. A.; Bau, E. C.; Nader, H. B.; Dietrich, C. P. Distribution of sulfated glycosaminoglycans in the animal kingdom: widespread occurrence of heparin-like compounds in invertebrates. Biochim. Biophys. Acta 2000, 1475, 28794. (17) Cesaretti, M.; Luppi, E.; Maccari, M.; Volpi, N. Isolation and characterization of a heparin with high anticoagulant activity from the clam Tapes phylippinarum. Evidence for the presence of a high content of antithrombin III-binding site. Glycobiology 2004, 14, 1275-1284. (18) Luppi, E.; Cesaretti, M.; Volpi, N. Purification and characterization of heparin from the italian clam Callista chione. Biomacromolecules 2005, 6, 1672-1678. (19) Oliveira, F. W.; Chavante, S. F.; Santos, E. A.; Dietrich, C. P.; Nader, H. B. Appearance and fate of a beta-galactanase, alpha, betagalactosidases, heparan sulfate and chondroitin sulfate degrading enzymes during embryonic development of the mollusc Pomacea sp. Biochim. Biophys. Acta 1994, 1200, 241-246. (20) Kim, Y. S.; Jo, Y. Y.; Chang, I. M.; Toida, T.; Park, Y.; Linhardt, R. J. A new glycosaminoglycan from the giant African snail Achatina fulica. J. Biol. Chem. 1996, 271, 11750-11755. (21) Nader, H. B.; Lopes, C. C.; Rocha, H. A. O.; Santos, E. A.; Dietrich, C. P. Heparins and heparinoids: occurrence, structure and mechanism of antithrombotic and hemorrhagic activities. Curr. Pharm. Des. 2004, 10, 951-66. (22) Schonberger, L. B. New variant Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Infect. Dis. Clin. North Am. 1998, 12, 111-121. (23) Cesaretti, M.; Luppi, E.; Maccari, F.; Volpi, N. A 96-well assay for uronic acid carbazole reaction. Carbohydr. Polymers 2003, 54, 5961. (24) Volpi, N. “Fast moving” and “slow moving” heparins, dermatan sulfate, and chondroitin sulfate: qualitative and quantitative analysis by agarose-gel electrophoresis. Carbohydr. Res. 1993, 247, 263278. (25) Volpi, N.; Maccari F. Detection of submicrogram quantities of glycosaminoglycans on agarose-gels by sequential staining with toluidine blue and Stains-All. Electrophoresis 2002, 23, 4060-4066. (26) Linhardt, R. J.; Wang, H. M.; Loganathan, D.; Bae, J. H. Search for the heparin antithrombin III-binding site precursor. J. Biol. Chem. 1992, 267, 2380-2387. (27) Rice, K. G.; Linhardt, R. J. Study of structurally defined oligosaccharide substrates of heparin and heparan monosulfate lyases. Carbohydr. Res. 1989, 190, 219-233. (28) Linhardt, R. J.; Rice, K. G.; Kim, Y. S.; Lohse, D. L.; Wang, H. M.; Loganathan, D. Mapping and quantification of the major oligosaccharide components of heparin. Biochem. J. 1988, 254, 781-787. (29) Linhardt, R. J.; Turnbull, J. E.; Wang, H. M.; Loganathan, D.; Gallagher, J. T. Examination of the substrate specificity of heparin and heparan sulfate lyases. Biochemistry 1990, 29, 2611-2617. (30) Ototani, N.; Yosizawa, Z. Purification of chondroitinase B and chondroitinase C using glycosaminoglycan-bound AH-Sepharose 4B. Carbohydr. Res. 1979, 70, 295-306. (31) Maccari, F.; Tripodi, F.; Volpi, N. High-performance capillary electrophoresis separation of hyaluronan oligosaccharides produced by Streptomyces hyalurolyticus hyaluronate lyase. Carbohydr. Polymers 2004, 56, 55-63. (32) Mucci, A.; Schenetti, L.; Volpi, N. 1H- and 13C-nuclear magnetic resonance identification and characterization of components of chondroitin sulfates of various origin. Carbohydr. Polymers 2000, 41, 37-45. (33) Volpi, N.; Mucci A. Characterization of a low-sulfated chondroitin sulfate from the body of ViViparus ater (Mollusca gastropoda). Modification of its structure by lead pollution. Glycoconjugate J. 1998, 15, 1071-1078. (34) Toyoda, H.; Kinoshita-Toyoda, A.; Selleck, S. B. Structural analysis of glycosaminoglycans in Drosophila and Caenorhabditis elegans and demonstration that tout-velu, a Drosophila gene related to EXT tumor suppressors, affects heparan sulfate in vivo. J. Biol. Chem. 2000, 275, 2269-2275. (35) Casu, B. Structure and biological activity of heparin. AdV. Carbohydr. Chem. Biochem. 1985, 43, 51-134.

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