Báo cáo khóa học: Acharan sulfate, the new glycosaminoglycan fromAchatina fulica Bowdich 1822 Structural heterogeneity, metabolic labeling and localization in the body, mucus and the organic shell matrix docx

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Báo cáo khóa học: Acharan sulfate, the new glycosaminoglycan fromAchatina fulica Bowdich 1822 Structural heterogeneity, metabolic labeling and localization in the body, mucus and the organic shell matrix docx

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Acharan sulfate, the new glycosaminoglycan from Achatina fulica Bowdich 1822 Structural heterogeneity, metabolic labeling and localization in the body, mucus and the organic shell matrix Tuane C. R. G. Vieira 1,2 , Adilson Costa-Filho 1,2 , Norma C. Salgado 3 , Silvana Allodi 4 , Ana-Paula Valente 2,5 , Luiz E. Nasciutti 4 and Luiz-Claudio F. Silva 1,2 1 Laborato ´ rio de Tecido Conjuntivo, Hospital Universita ´ rio Clementino Fraga Filho, 2 Departamento de Bioquı ´ mica Me ´ dica, 3 Laborato ´ rio de Malacologia, Museu Nacional, 4 Departamento de Histologia e Embriologia and 5 Centro Nacional de Ressona ˆ ncia Magne ´ tica Nuclear de Macromole ´ culas, Universidade Federal do Rio de Janeiro, Brazil Acharan sulfate, a recently discovered glycosaminoglycan isolated from Achatina fulica, has a major disaccharide repeating unit of fi4)-2-acetyl,2-deoxy-a- D -glucopyra- nose(1fi4)-2-sulfo-a- L -idopyranosyluronic acid (1fi,mak- ing it structurally related to both heparin and heparan sulfate. It has been suggested that this glycosaminoglycan is polydisperse, with an average molecular mass of 29 kDa and known minor disaccharide sequence variants containing unsulfated iduronic acid. Acharan sulfate was found to be located in the body of this species using alcian blue staining and it was suggested to be the main constituent of the mucus. In the present work, we provide further information on the structure and compartmental distribution of acharan sulfate in the snail body. Different populations of acharan sulfate presenting charge and/or molecular mass heterogeneities were isolated from the whole body, as well as from mucus and from the organic shell matrix. A minor glycosamino- glycan fraction susceptible to degradation by nitrous acid was also purified from the snail body, suggesting the presence of N-sulfated glycosaminoglycan molecules. In addition, we demonstrate the in vivo metabolic labeling of acharan sulfate in the snail body after a meal supplemented with [ 35 S]free sulfate. This simple approach might be applied to the study of acharan sulfate biosynthesis. Finally, we developed histo- chemical assays to localize acharan sulfate in the snail body by metachromatic staining and by histoautoradiography following metabolic radiolabeling with [ 35 S]sulfate. Our results show that acharan sulfate is widely distributed among several organs. Keywords: acharan sulfate; Achatina fulica; glycosamino- glycans; mucus; organic shell matrix; snail. Glycosaminoglycans (GAGs) consist of hexosamine and either hexuronic acid or galactose units that are arranged in alternating unbranched sequence, and carry sulfate substit- uents in various positions. The common GAGs include galactosaminoglycans (chondroitin sulfate and dermatan sulfate) and glucosaminoglycans (heparan sulfate, heparin, keratan sulfate and hyaluronic acid). Owing to the vari- ability in sulfate substitution, all GAGs display considerable sequence heterogeneity. Their strategic location and highly charged nature make them important biological players in the cell–cell and cell–matrix interactions that take place during normal and pathological events related to cell recognition, adhesion, migration and growth [1–4]. The new GAG, acharan sulfate, was first isolated and characterized by Kim et al. in 1996 [5], as a pure GAG from the giant African snail, Achatina fulica. Acharan sulfate occurs in large amounts and has a unique structure. This GAG is polydisperse with an average molecular mass of 29 kDa and has a major disaccharide repeating unit of fi4)-2-acetyl,2-deoxy-a- D -glucopyranose(1fi4)-2-sulfo-a- L - idopyranosyluronic acid (1fi, thereby relating it structurally both to heparin and heparan sulfate, although it is distinctly different from all known members of these classes of GAGs [5]. Kim et al. reported structural heterogeneity when they determined the structure of oligosaccharides prepared from acharan sulfate [6]. They identified two series of oligosac- charides, one derived from acharan sulfate’s major repeat- ing unit and a second minor group of undersulfated oligosaccharides. Oligosaccharides containing N-sulfated units were not detected [6]. A great number of biological roles were suggested for this molecule in the snail, but as this GAG was newly discovered it has only been evaluated on the basis of structural similarities to heparin and heparan sulfate, and the biological functions of GAGs. Therefore, intact or chemically modified acharan sulfate has been shown to present several in vitro biological activities, such as: inhibition of angiogenesis due to Correspondence to L C. F. Silva, Departamento de Bioquı ´ mica Me ´ dica, Centro de Cieˆ ncias da Sau´ de, Universidade Federal do Rio de Janeiro, Caixa Postal 68041, Rio de Janeiro, RJ, 21941-590, Brazil. Fax: + 55 21 2562 2090, E-mail: lclaudio@hucff.ufrj.br Abbreviations: GAG, glycosaminoglycan; HSQC, heteronuclear single quantum coherence. Enzymes: chondroitin AC lyase (EC 4.2.2.5) from Arthrobacter aurescens; chondroitin ABC lyase (EC 4.2.2.4) from Proteus vulgaris. (Received 17 November 2003, accepted 12 January 2004) Eur. J. Biochem. 271, 845–854 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.03989.x inhibition of vascular endothelial growth factor or the decrease of the mitogenic activity of fibroblast growth factor and other pharmacological activities [7–10]. In a recent study, Jeong et al. localized acharan sulfate in the snail body by alcian blue staining and showed that it is mainly secreted into the outer surface of the body as a mucous material from internal granules [11]. The mucus secreted on the body surfaces of mollusks is known to play crucial roles in locomotion, feeding, osmoregulation, repro- duction and protection of epithelial surfaces [12]. Achatina fulica is an intermediate host to Angiostrongylus cantonensis [13], the etiological agent of meningoencephalic angiostrongiliasis [14], and may act as a major source of human infection in places where it is commonly eaten by people, such as Taiwan [13]. The occurrence of Achatina fulica in Rio de Janeiro, Brazil, was recently reported [15]. In the localities visited, snails were found living freely, and larvae of Angiostrongylus cantonensis were not seen in any of them. The finding of Achatina fulica in the area may be related to its commercialization as a food item [15]. The giant African snail is considered a major pest in many parts of the world and as acharan sulfate is an important constituent of the snail body tissues, it might be involved in important biological roles for the survival of this animal. Therefore, a complete understanding of both biochemistry and compartmental distribution of this mole- cule might be important to develop methods of controlling this pest. Here, we provide new information on the biochemistry and properties of this interesting GAG molecule. Experimental procedures Materials Giant snails (Achatina (Lissachatina) fulica Bowdich 1822) were collected in Rio de Janeiro, Brazil. Animals were maintained in an air-conditioned laboratory at 25–28 °C under a 12-h light : 12-h dark cycle (natural light) in plastic tanks, and fed a vegetable diet. Chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, heparan sulfate and twice-crystallized papain (15 UÆmg )1 protein) were pur- chased from Sigma Chemical Co. Chondroitin AC lyase (EC 4.2.2.5) from Arthrobacter aurescens and chondroitin ABC lyase (EC 4.2.2.4) from Proteus vulgaris were pur- chased from Seikagaku American Inc. (Rockville, MD, USA). Radiolabeled carrier-free [ 35 S]Na 2 SO 4 was obtained from Instituto de Pesquisas Energe ´ ticas e Nucleares (Sa ˜ o Paulo, SP, Brazil). Preparation of GAGs from the soft body GAGs were isolated from the soft snail body following a previously described method, with modifications [5]. Briefly, the shell of the giant snail was removed, and the whole soft body was defatted using three 24 h extractions with acetone. The fat-free, dried snail was cut into very small pieces that were suspended in sodium acetate buffer (pH 5.5) containing 40 mg papain in the presence of 5 m M EDTA and 5 m M cysteine, and incubated at 60 °C for 24 h. The suspension was centrifuged at 3000 g for 20 min at room temperature and the supernatant, which contained the GAGs, was applied into a DEAE-cellulose column (34.0 cm · 2.5 cm; Sigma Chemical Co.), equilibrated with 0.05 M sodium acetate (pH 5.0). The column was washed with 200 mL of the same buffer and then eluted stepwise with 800 mL of 3.0 M NaCl in the same acetate buffer. The GAGs eluted from the column were exhaustively dialyzed against distilled water, lyophilized and dissolved in 10.0 mL of distilled water. The partially purified GAGs were applied to a Mono Q- FPLC column (HR 5/5; Pharmacia Biotech Inc., Uppsala, Sweden), equilibrated with 20 m M Tris/HCl (pH 8.0). The column was washed with 10 mL of the same buffer. Then, the column-bound GAGs were eluted in a stepwise gradient from 0.2 M NaCl to 3.0 M NaCl at intervals of 0.2 M NaCl in Tris buffer. Fractions of 0.5 mL were collected and the elution was monitored by the metachromatic property of the fractions using 1,9-dimethylmethylene blue [16]. The GAGs eluted from the column were exhaustively dialyzed against distilled water, lyophilized and dissolved in 1.0 mL of distilled water. Collection and analysis of mucus GAGs Mucus was collected from the surface of live snails. The collected mucus was mixed with three volumes of sodium acetate buffer (pH 5.5) containing 40 mg papain in the presence of 5 m M EDTA and 5 m M cysteine, and incubated at 60 °C for 24 h. GAGs were isolated and purified from the papain-digested mucus by sequential anion-exchange chro- matograpy firstly in DEAE-cellulose column followed by fractionation on a Mono Q-FPLC column, as described above. Extraction of GAGs from the organic shell matrix The preparation of an EDTA-free extraction of organic matrix was performed as described previously, with modi- fications [17]. Briefly, clean, dry, powdered shells were decalcified during 24 h at 4 °C in 200 mL of 1 M HCl. The solution was stirred constantly. After centrifugation at 3000 g for 20 min at room temperature, the supernatant was dialyzed against water for four days and then lyo- philized. This material was named soluble organic shell matrix. GAGs were isolated from the soluble matrix and purified as described above for the mucous material. A purified preparation of acharan sulfate isolated from the whole body was submitted to decalcification by the same protocol described above, and analyzed by the same methodology used to characterize acharan sulfate from the other snail tissues. In vivo metabolic 35 S-labeling of snail body GAGs We performed a simple method for the metabolic labeling of snail GAGs by wetting small pieces of leaves of lettuce with 1.48 mBq [ 35 S]Na 2 SO 4 , leaving it to dry, mixing it with unlabeled lettuce and offering it as food to snails that were 1 day without food. Isolation of the radiolabeled GAGs Twenty four hours after a meal containing the radioactive precursor, the snails were killed, the shell was removed and 846 T. C. R. G. Vieira et al. (Eur. J. Biochem. 271) Ó FEBS 2004 GAGs were isolated and purified as described above for the unlabeled snails. The 35 S-labeled GAGs were detected in the Mono Q-FPLC fractions by scintillation counting, exhaust- ively dialyzed against distilled water and re-suspended in 1.0 mL of distilled water. Identification of the snail GAGs GAGs were characterized by a set of current biochemical methods that included: agarose gel electrophoresis, diges- tion with chondroitin lyases, heparin lyase I and deamin- ative cleavage with nitrous acid, PAGE and NMR spectroscopy [18,19], as described below. Agarose gel electrophoresis Agarose gel electrophoresis was carried out as previously described [20]. Approximately 10 lg of GAGs, before and after chondroitin lyase or heparin lyase I digestions or deaminative cleavage with nitrous acid (see below), as well as a mixture of standard chondroitin4-sulfate, dermatan sulfate and heparan sulfate (10 lg of each) were applied to 0.5% agarose gels in 0.05 M 1,3-diaminopropane/acetate buffer (pH 9.0). Following electrophoresis, GAGs were fixed in the gel with 0.1% N-cetyl-N,N,N-trimethylammonium bromide in water, and stained with 0.1% toluidine blue in acetic acid/ ethanol/water (0.1 : 5 : 5, v/v/v). The 35 S-labeled GAGs were visualized by autoradiography of the stained gels. Digestion with chondroitin lyases Digestions with chondroitin ABC lyase were performed according to Saito et al. [21]. Approximately 100 lgofsnail GAGs were incubated with 0.3 units of chondroitin ABC lyase for 8 h at 37 °Cin100lLof50m M Tris/HCl (pH 8.0), containing 5 m M EDTA and 15 m M sodium acetate. Digestion with heparin lyase I Enzymatic digestion with heparin lyase I was performed with addition of the enzyme (1 mU) in 100 lL of 100 m M sodium acetate and 10 m M calcium acetate (pH 7.0), over a 36hperiodat37°C[22]. Deamination with nitrous acid Deamination by nitrous acid at pH 1.5, was performed as described by Shively & Conrad [23]. Briefly,  100 lgof snail GAGs were incubated with 200 lL of fresh generated HNO 2 at room temperature for 10 min. The reaction mixtures were then neutralized with 1.0 M Na 2 CO 3 . N-acetylation of purified GAG fractions N-acetylation of purified snail GAG fractions was per- formed by the addition of 0.1 mL of acetic anhydride [5]. Polyacrylamide gel electrophoresis Fractionated GAGs from both mucus and the whole snail body (see above for methodology) were applied to a 1 mm thick, 6% polyacrylamide slab gel. Following electrophor- esis at 100 V for  1 h in 0.06 M sodium barbital (pH 8.6), the gel was stained with 0.1% toluidine blue in 1% acetic acid. After staining, the gel was washed for  6hin1% acetic acid [24]. Superose-12 HPLC gel filtration chromatography Intact, b-eliminated or papain-digested total GAGs obtained from mucus were applied separately into a Superose-12 (HR 10/30) column linked to a HPLC system (Shimadzu, Tokyo, Japan). Ammonium bicarbonate 0.2 M (pH 5.0) was the buffer used. The column was eluted with the same solution, at a flow rate of 0.5 mLÆmin )1 ,and fractions of 0.5 mL were collected and assayed for meta- chromasia or for the protein content, measured by auto- matically recording the absorption at 280 nm. b-elimination of GAGs from mucus b-elimination was performed by the incubation of GAGs in 0.1 M NaOH at 37 °C for 12 h. NMR spectroscopic analysis 1 Hand 13 C spectra were recorded using a Bruker DRX 600 with a triple resonance probe (Bruker, Germany). About 2 mg each of purified mucus and soft body GAGs were dissolved in 0.5 mL of 99.9% D 2 O (CIL). All spectra were recorded at 60 °C with HOD suppression by presaturation. 1 H/ 13 C heteronuclear single quantum coherence (HSQC) spectra were run with 1024 · 300 points and globally optimized alternating phase rectangular pulses for decou- pling. All chemical shifts were relative to external trimethyl- silylpropionic and [ 13 C]methanol. Radioautographic and metachromatic detection of sulfated GAGs After the in vivo metabolic labeling of snail GAGs, the snails were killed, the shell was removed and several organs were dissected and fixed in 4% formaldehyde (freshly prepared from paraformaldehyde) in Sorensen phosphate buffer (0.1 M ,pH7.4)at4°C overnight. After fixation and washing, the tissues were dehydrated in ethanol and embedded in paraffin. Tissue sections (7 lm) were obtained and collected on gelatin-coated slides. These were then immersed in NTB 2 liquid emulsion (Kodak) and left in a dark box for 2 weeks at 4 °C. They were developed, washed several times, and stained with cationic dye 1,9-dimethyl- methylene blue [16] in 0.1 M HCl, containing 0.04 m M glycine and 0.04 m M NaCl [25]. The sections were then examined and photographed using a light microscope (Zeiss, Axioskop 2). Results Heterogenic forms of acharan sulfate are present in the soft body, mucus and the organic shell matrix The GAG component of the soft body, mucus and the organic shell matrix of the giant African snail was isolated Ó FEBS 2004 Acharan sulfate structure and distribution (Eur. J. Biochem. 271) 847 and purified by anion-exchange chromatography on a Mono Q-FPLC column (Fig. 1A–C, respectively). Five metachro- matic peaks were eluted from 0.2 to 1.0 M NaCl at intervals of 0.2 M and designated as peaks R1 to R5 (Fig. 1A–C). Differences in the proportion and occurrence of R-peaks were observed among the three sources of snail GAGs. The most drastic differences were observed in the profile obtained for GAGs from the organic shell matrix when compared to the other profiles, where peaks R3 and R4 predominate over the other R-peaks, which are either present in a very small amount or are lacking (Fig. 1C). In order to verify whether the conditions used to dissolve the shell would alter the structure of acharan sulfate, a control experiment using acharan sulfate isolated from the whole body was carried out to show that it was stable under these conditions. Similar patterns of elution of snail body acharan sulfate subpopu- lations, regardless of incubation with or without the presence of the decalcifying agents, were obtained in a Mono Q-FPLC column using the same conditions described in the legend of Fig. 1 (data not shown). GAGs present in the R-peaks were first analyzed by agarose gel electrophoresis (Fig. 1D–F). Peaks R1 to R5 presented a major metachromatic band with an electro- phoretic migration similar to that of the dermatan sulfate standard. This band was not detected in peak R1 from the organic shell matrix (Fig. 1F). As acharan sulfate was shown to be the only GAG species expressed by the giant African snail [5], we assigned this band as acharan sulfate in Fig. 1D–F. However, a second minor band, with an electrophoretic migration similar to that of the heparan sulfate standard, was present only in peak R1 from the snail body (Fig. 1D). Fig. 1. Fractionation of snail GAGs. Purification of GAGs from whole body (A), mucus (B) and the organic shell matrix (C) of Achatina fulica on a Mono Q-FPLC column. The DEAE-cellulose-purified GAGs were applied to a Mono Q-FPLC and purified as described. Fractions were monitored by the metachromatic property (k). The NaCl concentration in the fractions was determined by measuring the conductivity. The fractions corresponding to GAGs (peaks R1 to R5) as indicated by horizontal bars, were pooled, dialyzed against distilled water and lyophilized. Peaks R1 to R5 from whole body (D), mucus (E) and the organic shell matrix (F) were analyzed by agarose gel electrophoresis. HS, heparan sulfate; DS, dermatan sulfate; CS, chondroitin 4/6 sulfate. The major band in (D) to (F) was assigned as acharan sulfate (see text). 848 T. C. R. G. Vieira et al. (Eur. J. Biochem. 271) Ó FEBS 2004 GAGs present in peaks R1 to R5 were further analyzed by agarose gel electrophoresis, before and after incubation with condroitin ABC lyase (which degrades both chondroitin and dermatan sulfate), or deaminative cleavage with nitrous acid (which degrades N-sulfated GAGs such as heparan sulfate and heparin). The major electrophoretic band in the R-peaks from the whole body was resistant to both treatments (Fig. 2A). This is as expected for acharan sulfate, as this GAG was previously shown to be substan- tially degraded only by heparin lyase II, thus it is resistant to all other enzymatic treatments that degrade GAGs [5]. The same pattern was obtained for snail GAGs in the R-peaks from both mucus and the organic shell matrix (data not show). Interestingly, the minor band detected only in peak R1 from whole body (Fig. 1D) was resistant to treatment with chondroitin ABC lyase, but was surprisingly degraded by nitrous acid (Fig. 2A). This result suggests the existence of either N-sulfated or free amino groups in the minor GAG fraction of peak R1. In order to address this issue, the R1 fraction was chemically N-acetylated and subsequently analyzed by agarose gel electrophoresis before and after nitrous acid treatment. The N-acetylation procedure did not affect the electrophoretic migration of GAGs in R1, but the minor GAG band remained susceptible to the nitrous acid treatment (Fig. 2B), indicating that it presents N-sulfated groups. Additionally, this GAG fraction was also suscept- ible to degradation with heparin lyase I, as expected for N-sulfated GAGs (Fig. 2B). The spread of elution observed to snail GAGs with increasing salt concentration in the Mono Q-FPLC chro- matographies (Fig. 1A–C) may be explained by increasing molecular mass. In order to address this issue, we evaluated their molecular mass distribution by polyacrylamide gel electrophoresis. The obtained results revealed marked differences between the mobility of GAGs from the R-peaks of the polysaccharides extracted from the whole body compared to mucus. In the snail body fraction, we found four different metachromatic bands with increasing mole- cular mass, from R2 to R5 (Fig. 3A), showing that acharan sulfate is composed of heterogeneous and polydisperse GAG chains. GAGs from R1 of the snail body were not analyzed due to the presence of a contaminating brown pigment that caused distortions in the metachromatic bands. An additional incubation of GAGs present in peaks R2 to R5 with papain did not modify the patterns of electrophoretic mobility on polyacrylamide gel (not shown). Less pronounced differences were observed among the GAGs present in peaks R1 to R5 from mucus (Fig. 3B). This analysis was not performed for GAGs present in the R-peaks from the organic shell matrix because sufficient material was lacking. Another interesting detail revealed by these experiments is that the molecular masses of GAGs from both body and mucus (Fig. 3A,B) correspond to those of chondroitin 4- and 6-sulfate standards (S2 and S3 in Fig. 3C, respectively) in this system (molecular mass range of 40–60 kDa) (compare Fig. 3A and C). NMR analysis confirms the structural identity of snail acharan sulfate The 1 H one-dimensional NMR spectra of the major GAG fractions from the snail whole body and from mucus (Fig. 4A and B, respectively) and interpretations of 1 H/ 13 C HSQC spectra of whole body GAG (Fig. 5), confirm the identification of these GAG fractions as the previously Fig. 2. Characterization of GAGs from the snail body. (A) Agarose gel electrophoresis of GAGs present in peaks R1 to R5 from whole body, before (–) and after (+) chondroitin ABC lyase digestion or deamin- ative cleavage by nitrous acid. After enzymatic or chemical incubation, the GAGs were applied to 0.5% agarose gel and electrophoresis was carried out as described. (B) Samples of peak R1 were subjected to an N-acetylation protocol and subsequently analyzed by agarose gel electrophoresis, before (–) and after (+) deaminative cleavage by nitrous acid. An intact sample of peak R1 was also analyzed by gel electrophoresis after incubation with heparin lyase 1. CS, chondroitin 4/6-sulfate; DS, dermatan sulfate; HS, heparan sulfate. Fig. 3. Polyacrylamide gel electrophoresis of GAGs present in peaks R1 to R5 from whole body (A), mucus (B) and standards (C). Standard sulfated polysaccharides are: S1, high molecular mass dextran sulfate (500 kDa); S2, chondroitin 6-sulfate (60 kDa); S3, chondroitin 4-sul- fate (40 kDa); S4, low molecular mass dextran sulfate (8 kDa). Peaks R1 to R5 were obtained as described for Fig. 1. Ó FEBS 2004 Acharan sulfate structure and distribution (Eur. J. Biochem. 271) 849 described acharan sulfate [5]. The 1 H chemical shifts of the residue found in the major snail body GAG fraction, characterized as acharan sulfate, are presented in Table 1, and are based on interpretations of 1 H/ 13 CHSQC(Fig.5). The assignment was performed based on comparison with the previously described acharan sulfate (Table 1). The values obtained here are in agreement with a sulfated H2 of the a- L -iduronic acid residue as the major disaccharide unit. This conclusion was reinforced by data from literature, which show that a- L -iduronic acid residues from the two types of disaccharide units composed of glucosamine linked to either 2-O-sulfated or nonsulfated a- L -iduronic acid residues, have approximately the same chemical shifts for H4 and H5, but that the H2 is 0.69 p.p.m. downfield in the 2-O-sulfated unit (Table 1). NMR analyses of GAGs derived from the organic shell matrix were not performed due to the scarce amount of purified material. In view of the very small amount of Fig. 4. 1 H NMR spectra at 600 MHz of snail GAG from whole body (A) and mucus (B). The samples were dissolved in 500 lLofD 2 O 100% and the spectra recorded at 60 °C with suppression of residual HOD signal by presaturation. Fig. 5. 1 H/ 13 C HSQC spectrum of snail GAG from whole body. The assignment was based on comparison with the previously described acharan sulfate. The spectrum was acquired with 1024 · 300 points and 128 scans. Table 1. Proton chemical shifts for acharan sulfate (from whole body), heparin and modified heparins. Chemical shifts are referenced to internal trimethylsilylpropionic acid at 0 p.p.m. Protons designated ÔIÕ refer to those of a- L -iduronic acid residues, whereas those of a- D -glucosamine are designed as ÔAÕ. Proton Chemical shift (p.p.m.) Acharan sulfate This work Acharan sulfate Kim et al. [5] IdoAp2SGlcNpS(6S) Mulloy et al. [29] IdoApGlcNpS(6S) Jaseja et al. [30] IdoApGlcNpAc Mulloy et al. [29] I-1 5.20 5.19 5.22 5.00 4.89 I-2 4.34 a 4.34 a 4.35 a 3.80 3.65 I-3 4.19 4.28 4.20 4.10 3.83 I-4 4.05 4.03 4.10 4.10 4.04 I-5 5.08 4.93 4.81 4.80 4.68 A-1 5.07 5.11 5.39 5.30 5.11 A-2 3.97 4.02 3.29 3.30 4.00 A-3 3.70 3.74 3.67 3.70 3.73 A-4 3.70 3.74 3.77 3.80 3.73 A-5 3.71 3.87 4.03 4.00 4.00 A-6 3.82 3.87 4.27 a 4.20 a 3.85 Acetyl 2.04 2.08 – – 2.04 a Values indicate positions bearing sulfate ester. 850 T. C. R. G. Vieira et al. (Eur. J. Biochem. 271) Ó FEBS 2004 purified nitrous acid-sensitive snail GAG and the difficulty in separating it from acharan sulfate (Figs 1A,D and 2), we did not further characterize this GAG by NMR analysis. Acharan sulfate appears to exist as a protein-free polysaccharide in mucus Unfractionated mucus samples were analyzed by poly- acrylamide gel electrophoresis before or after incubation with papain, a protease of broad specificity (Fig. 6A), in order to investigate whether mucus acharan sulfate is covalently linked to proteins as a proteoglycan. No changes on the electrophoretic migration of both intact and papain-digested mucus were observed (Fig. 6A). Additionally, intact, b-eliminated or papain-digested GAGs from mucus were analyzed by gel filtration chromatography (Fig. 6B). No significant differences were observed in the metachromatic pattern of eluted GAG among the three samples (Fig. 6Ba–c). The pattern of protein elution was only modified in the papain-digested sample (Fig. 6Bd–f). Together, these experiments suggest that acharan sulfate in mucus is present as a protein-free polysaccharide, because no modifications occurred on either the metachromatic or the protein patterns of elution after b-elimination (a procedure that separates the GAG chain from the core protein) or papain digestion (except by the change in the profile of protein elution after treatment with this enzyme, as expected). However, we cannot exclude the possibility that these molecules may be synthesized as proteoglycans in the snail body tissue and that protein-free GAG chains would be produced from proteoglycans by strong protease activities present in the mucus. In vivo metabolic labeling of acharan sulfate Snails were fed with leaves of lettuce coated with 1.48 mBq [ 35 S]Na 2 SO 4 . After the labeling period, snails were killed and GAGs were extracted and purified from the soft body, as described in Experimental procedures. The labeled GAG component of the soft body of Achatina fulica was purified by anion-exchange chromatography on a Mono Q-FPLC column (Fig. 7A). In a similar situation to that found for the unlabeled snail GAGs (Fig. 1A), five radiolabeled peaks were eluted from 0.2 M to 1.0 M NaCl at intervals of 0.2 M andreferredtoaspeaksR1toR5(Fig.7A).However, unlike that observed for the unlabeled GAGs, R3 and R4 were the predominant peaks (compare Figs 1A and 7A). Agarose gel electrophoresis followed by autoradiography of the stained gels revealed a similar electrophoretic pattern to both metachromatic and radiolabeled bands (Fig. 7B,C, respectively). These results show that all GAG species in the snail body were metabolically labeled through assimilation of labeled free sulfate from the food by the digestion process. Acharan sulfate is widely distributed in the snail body Radioautography and metachromatic staining were per- formed to evaluate the distribution of GAGs in internal Fig. 6. Evaluation of the molecular mass of acharan sulfate from mucus. Polyacrylamide gel electrophoresis (A) of GAGs present in unfractionated snail mucus, before (–) and after (+) papain digestion. After enzymatic incubation, the GAGs were applied to polyacrylamide gel and electro- phoresis was carried out as described. Standard sulfated polysaccharides (Standards) are: S1, high molecular mass dextran sulfate (500 kDa); S2, chondroitin 6-sulfate (60 kDa); S3, chondroitin 4-sulfate (40 kDa); S4, low molecular mass dextran sulfate (8 kDa). (B) The chromatographic profiles of intact (a and d), b-eliminated (b and e) or papain-digested (c and f) GAGs from mucus samples. GAGs were detected by the metachromatic property of the fractions (a to c), while proteins were detected by the absorption at 280 nm of the fractions (d–f). Ó FEBS 2004 Acharan sulfate structure and distribution (Eur. J. Biochem. 271) 851 and external sites of the snail body (Fig. 8). The metachromatic staining (purple) showed the presence of sulfated compounds in several organs: kidney, salivary glands, esophagus, stomach, albumen gland, feet and spermatheca. Light micrographs for stained feet and kidney are shown (Fig. 8A; feet and Fig. 8B,C; kidney). Radioautographic silver grains were mostly observed on the purple-stained structures indicating an efficient incor- poration of [ 35 S]sulfate. Discussion Acharan sulfate was first isolated from the body of the giant African snail, Achatina fulica,byKimet al.[5]bymeansof protease digestion of the dried fat-free snail tissues. The GAG was precipitated initially by ethanol, re-dissolved and subsequently precipitated by a quaternary ammonium salt, cetylpyrydinium chloride, and characterized as a new GAG species. Based on the pattern of susceptibility of this GAG to degradation by specific mucopolysaccharidases, disac- charide analysis by capillary electrophoresis and NMR spectrometry [5], the authors showed that acharan sulfate has an average molecular mass of 29 kDa and a major disaccharide repeating unit of fi4)-2-acetyl,2-deoxy-a- D - glucopyranose(1fi4)-2-sulfo-a- L -idopyranosyluronic acid (1fi, making it structurally related to both heparin and heparan sulfate but distinctly different from all known members of these classes of GAGs. Acharan sulfate also presents minor disaccharide sequence variants containing unsulfated iduronic acid [6]. Here, using a different protocol to isolate and purify GAGs from the soft body of Achatina,wewereabletoshow that acharan sulfate from the snail body presents a great Fig. 8. Light micrographs of the Achatina feet and kidney stained with 1,9-dimethylmethylene blue dye and labeled with silver grains obtained by autoradiography. (A) Feet displaying metachromatic purple color on the epithelium surface (arrowhead) and on glands with secretory portions deeply located (asterisks). The radioautography technique strongly labeled the connective tissue associated to the glandular compartment (arrows). (B) Low magnification of the kidney showing metachromatic material distributed by the organ. (C) High magnifi- cation of the kidney. In this micrograph silver grains are clearly seen concentrated on the metachromatic structures. Scale bar in C is 90 lm (A); 25 lm(B);5.5lm(C). Fig. 7. Incorporation of [ 35 S]sulfate into GAGs from the snail body. (A) Purification of 35 S-labeled GAGs from snail whole body on a Mono Q-FPLC column. Fractions were monitored by scintillation counting (k). The NaCl concentration in the fractions (–) was determined by measuring the conductivity. The fractions corresponding to the 35 S-labeled GAGs, as indicated by horizontal bars, were pooled, dialyzed against distilled water and lyophilized. Toluidin blue stained (B) and autoradiogram (C) of agarose gel electrophoresis of GAGs present in peaks R1 to R5. HS, heparan sulfate; DS, dermatan sulfate; CS, chondroitin 4/6 sulfate. The majorbandin(B)and(C)wasassignedasacharansulfate(seetext). 852 T. C. R. G. Vieira et al. (Eur. J. Biochem. 271) Ó FEBS 2004 degree of heterogeneity regarding negative net charge density and/or molecular mass (Figs 1A and 3A, respect- ively). In addition, we identified the presence of a GAG species, not described before, that occurs in minor amounts and that was susceptible to deamination by nitrous acid, even after it had been subjected to an N-acetylation protocol. This GAG species was also sensitive to degrada- tion by the action of heparin lyase I, indicating that this GAG presents N-sulfate groups at the glucosamine residue. Kim et al. also commented in their previous paper, which described the snail body GAG composition [5], that at least 95% of the GAG that was purified corresponded structur- ally to acharan sulfate, whereas the remaining 5% either corresponded, with minor structural heterogeneity, to this GAG, or was due to a small amount of a contaminant GAG of a different structure [5]. These findings raise the interesting question of whether the balance between acharan sulfate and this other GAG species remains unchanged from birth to the adult life of the snail. The presence of this unique GAG, acharan sulfate, with its simple but unusual sequence poses interesting questions about its biosynthesis. Our results, which establish that it is possible to label GAGs in the snail body by supplementing the food with [ 35 S]sulfate (Experimental procedures and Fig. 7), may, in the near future, turn out to be an important tool in developing experiments to follow the biosynthesis of acharan sulfate. Jeong et al. conducted a histochemical analysis of the distribution of GAGs in tissue sections of the snail body [11]. They analyzed the alcian blue staining of GAGs from tissue samples of exterior sites and interior spaces of the snail body. Positive staining was visualized only on the exterior sites, and GAGs were primarily located inside granules and were secreted onto the surface as a mucous material [11]. Here, we extend their basic findings by analyzing the distribution of GAGs, by means of both metachromatic staining and autoradiography, of several internal organs and also over the exterior surface of the snail body. We found that sulfated compounds are widely distributed among the tissues examined (Fig. 8). This finding adds new possible biological roles for snail GAGs, especially acharan sulfate, according to the source of the tissue, which included the digestive tract, kidney, albumen gland, feet and spermatheca. GAGs are reported to be present in the mollusk shell organic matrix [17,26,27] and these molecules may be responsible for fixation of calcium in the shell. Marxen & Becker [16] recently suggested that a material that was isolated from the organic shell matrix of the freshwater snail, Biomphalaria glabrata, which was stainable with alcian blue, would probably be sulfated proteoglycans or GAGs. Alcian blue stainable compounds were also identified in aqueous [26] or acidic [27] extracts isolated from the nacreous layer of the shell from the pearl oyster, Pinctada maxima. Although these and other reports suggest the presence of GAGs or proteoglycans in the mollusk shell organic matrix, the composition and structure of these compounds have never been reported. Here, we were able to extract significant amounts of acharan sulfate from the organic shell matrix of Achatina fulica (Fig. 1). Therefore, our results are the first biochemical description of the identity of GAGs from a mollusk shell organic matrix. The location of acharan sulfate in the organic shell matrix of Achatina fulica suggests that it may be involved in the promotion of shell mineralization. As we can see, acharan sulfate is widely distributed in the body of Achatina fulica suggesting that this GAG may be extremely important to the physiology of this gastropod. Altogether, our findings open interesting possibilities to investigate the biological roles of acharan sulfate by analyzing the effects of GAG-modifying agents, such as chlorate or selenate (sulfation inhibitors), on the physiology, shell formation, mucus production and growth rates of individuals at different ages. In this case, the metabolic labeling protocol would be of interest to assess the alteration in the degree of sulfation of GAGs synthesized under the influence of the inhibitors. Finally, Achatina fulica is an intermediate host to Angiostrongylus cantonensis [13], the etiological agent of meningoencephalic angiostrongiliasis [14], and may act as a major source of human infection in places where people commonly eat it. Interestingly, heparin-binding adhesion proteins were reported to be expressed in adult forms of Strongyloides venezuelensis [28]. As Achatina fulica may act as an intermediate host for some species of Strongyloides and also express acharan sulfate (a heparin-related GAG) throughout the body, it is tempting to speculate that this GAG might be involved in the processes of interaction between the parasite and the snail. Acknowledgements We would like to thank Selma Pacheco Ramos and Jorge Luis da Silva for technical assistance. 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Acharan sulfate, the new glycosaminoglycan from Achatina fulica Bowdich 1822 Structural heterogeneity, metabolic labeling and localization in the body,. [5]. The same pattern was obtained for snail GAGs in the R-peaks from both mucus and the organic shell matrix (data not show). Interestingly, the minor band

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