Biosynthesis and metabolism of sulfated glycosaminoglycans during ...

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band disappeared after incubation with chondroitin AC- or ABC-lyase but resisted degradation with nitrous acid. (Figure 1C). These results indicate that adult ...
Glycobiology vol. 14 no. 6 pp. 529±536, 2004 DOI: 10.1093/glycob/cwh070 Advance Access publication on March 24, 2004

Biosynthesis and metabolism of sulfated glycosaminoglycans during Drosophila melanogaster development

Daniela O. Pinto2,3, Paola L. Ferreira2, Leonardo R. Andrade4, Hilda Petrs-Silva3, Rafael Linden3, Eliana Abdelhay3, Helena M. M. ArauÂjo4, Carlos-Eloy V. Alonso3,5, and Mauro S.G. PavaÄo1,2 2

Laborat orio de Tecido Conjuntivo, Hospital Universit ario Clementino Fraga Filho and Departamento de BioquõÂmica Medica, Instituto de Ci^encias Biomedicas (ICB), Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brasil; 3Instituto de BiofõÂsica Carlos Chagas Filho, UFRJ, Rio de Janeiro, Brasil; 4Departamento de Histologia e Embriologia, ICB, UFRJ Rio de Janeiro, Brasil; and 5Stony Brook University, Department of Mathematics and Statistics and Center for Developmental Genetics, New York, NY Received on December 1, 2003; revised on February 3, 2004; accepted on February 26, 2004

We developed a simple methodology for labeling sulfated glycosaminoglycans (GAGs) in adult Drosophila melanogaster and studied some aspects of the biosynthesis and metabolism of these polymers during development. Adult D. melanogaster flies were fed with Na235SO4 for 72 h. During this period, 35S-sulfate was incorporated into males and females and used to synthesize 35S-sulfate±heparan sulfate (HS) and 35S-sulfate±chondroitin sulfate (CS). The incorporation of 35S-sulfate into HS was higher when compared to CS. In a pulse-chase experiment, we observed that 35S-sulfate incorporated into adult female was recovered in embryos and used for the synthesis of new 35S-sulfate-GAGs after 2 h of embryonic development. The synthesis of CS was higher than that of HS, indicating a change in the metabolism of these glycans from adult to embryonic and larval stages. Analysis of the CS in embryonic and larval tissues revealed the occurrence of nonsulfated and 4-sulfated disaccharide units in embryos, L1 and L2. In L3, in addition to these disaccharides, we also detected significant amount of 6sulfated units that are reported here for the first time. Immunohistochemical analysis indicated that HS and CS were present in nonequivalent structures in adult and larval stages of the fly. Overall, these results indicate that 35S-sulfate-precursors are transferred from adult to embryonic and larval tissues and used to assemble different morphological structures during development. Key words: biosynthesis/chondroitin sulfate/development/ Drosophila melanogaster/heparan sulfate

1

To whom correspondence should be addressed; e-mail: [email protected]

Introduction Proteoglycans are macromolecules composed of glycosaminoglycan (GAG) chains covalently bound to a protein core. GAGs are linear heteropolysaccharides consisting of repeating disaccharide units of hexuronic acid (L-iduronic acid or D-glucuronic acid) and hexosamine (D-glucosamine or D-galactosamine). The heterogeneity of these polymers results from variations in the degree of sulfation and occurrence of two types of hexuronic acid. The GAGs have a ubiquitous distribution in living organisms occurring in a great variety of organs and tissues (Conrad, 1989; Kjellen and Lindahl, 1991). GAG chain biosynthesis is a complex, multienzymatic process consisting of linker formation, chain elongation, and modification (For review see Esko and Selleck, 2002; Lindahl et al., 1998; Sugahara and Kitagawa, 2000). Linker formation in chondroitin (CS)/dermatan sulfate (DS) and heparan sulfate (HS)/heparin (Hep) involves common steps. First, a xylosyltransferase adds a single xylose residue to the core protein serine. Two galactose residues are then added by the actions of two separate galactosyltransferases, and finally, a single glucuronic acid (GlcA) residue is added to finish the linker region. Chain elongation in HS/Hep occurs through the sequential addition of N-acetyl-glucosamine (GlcNAc) and GlcA by the action of polymerases, encoded by the members of EXT gene family. In contrast, in CS/DS biosynthesis, N-acetyl-galactosamine (GalNAc) and GlcA are added sequentially by the alternate action of GalNAc transferase II and GlcA transferase II. In chain modification, N-deacetylation/N-sulfation of HS/hep is catalyzed by a family of enzymes known as N-deacetylase/N-sulfotransferases. Then C-5 epimerase converts some of the GlcA into iduronic acid (IdoA), which can then be modified by sulfation by a 2-O-sulfotransferase. This is followed by 6-O-sulfation of the GlcNAc catalyzed by a 6-O-sulfotransferase. 3-O-sulfation of the N-sulfoglucosamine can also occur by the action of a 3-O-sulfotransferase. Modification of CS/DS involves 4-O- and 6-O-sulfation of GalNAc, catalyzed by 4-O- or 6-O-sulfotransferase. C-5 epimerization of GlcA into IdoA with sequential 2-O-sulfation can also occur in DS. During CS/DS and HS/Hep chain modification, extracellular sulfate is taken up by the cells through a sulfate transporter and used to form 30 -phosphoadenosine50 -phosphosulfate by the action of ATP-sulfurylase and APS-kinase. This is the sulfate donor in the several sulfation reactions catalyzed by the different sulfotransferases. The fruit fly Drosophila melanogaster is a model organism that has been used to understand a wide range of molecular mechanisms involved in important biological phenomena, such as morphogenesis, differentiation, and cell growth.

Glycobiology vol. 14 no. 6 # Oxford University Press 2004; all rights reserved.

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Several studies in D. melanogaster reported the presence of proteoglycan or proteoglycan-like proteins, including glypican (Dally) (Nakato et al., 1995; Tsuda et al., 1999), syndecan (D-syndecan) (Spring et al., 1994), DROP-1 (Graner et al., 1994), Papilin (Campbell et al., 1987), and macrophage-derived proteoglycan-1 (Lin et al., 1999). Over the past decade, several studies revealed that mutations in genes encoding proteoglycan core proteins and GAG biosynthetic enzymes produce drastic morphological effects in the fly embryo (Haerry et al., 1997; Sen et al., 1998; The et al., 1998), indicating that these molecules are required for normal development of the invertebrate. Despite the extensive genetic analysis of proteoglycans and GAGs in D. melanogaster, biochemical analysis of these polymers in the fly has been less explored. A brief report described the incorporation of 35S-sulfate into extracellular HS (Cambiazo and Inestrosa, 1990). More recently, biochemical studies described the disaccharide composition of HS and CS in D. melanogaster (Toyoda et al., 2000a,b). In the present work, we extended the investigation of GAGs in D. melanogaster studying the biosynthesis and metabolism of these polymers during development. We developed a methodology to radiolabel sulfated GAGs in adult flies with 35S-sulfate and followed the 35S-sulfateGAGs in the embryo and in the three larval stages. Results Incorporation of 35S-sulfate into D. melanogaster glycans Drosophila adult flies were kept for 72 h in a medium containing Na235SO4 (35S-feeding mixture). During this period, the flies fed actively on the 35S-feeding mixture, allowing us to extract 35S-labeled glycans from adults. 35S-labeled molecules were extracted with proteolytic digestion and ethanol precipitation. The amount of radioactivity incorporated was estimated after washing out low-molecular-weight contaminants by paper chromatography and counting the radioactivity at the origin of the paper chromatogram, containing molecules free of low-molecular-weight contaminants. By this method, 35S-sulfate incorporation was about five times higher in females than in males (Figure 1A). The 35S-sulfate molecules extracted from male and female flies were analyzed by agarose gel electrophoresis, and autoradiography of the gel revealed two bands with well-distinguished electrophoretic mobilities. The low- and high-mobility bands migrated as standard HS and CS, respectively (Figure 1B). The nature of the 35 S-sulfate-glycans was verified by digestion with specific CS-lyases and deaminative cleavage with nitrous acid. The low-mobility band resisted incubation with chondroitin AC- and ABC-lyase but disappeared after deaminative cleavage with nitrous acid. In contrast, the high-mobility band disappeared after incubation with chondroitin ACor ABC-lyase but resisted degradation with nitrous acid (Figure 1C). These results indicate that adult flies incorporated 35S-sulfate into CS and HS chains. We analyzed the kinetics of 35S-sulfate incorporation in male and female for 24 h by our method. Incorporation began only after 5 h (Figure 2A). During the 24-h 530

experiment 35S-sulfate was incorporated mainly into HS, as indicated by autoradiography of agarose gel electrophoresis of the glycans extracted from the flies in each time point (Figure 2B). Again, incorporation in females was higher than in males. Pulse and chase of 35S-GAGs during development To compare the 35S-sulfate-GAGs synthesized by adult flies to those synthesized by embryos and larvae, we performed a pulse-chase experiment. After a 72-h labeling period, already described, the embryos released by the females were collected at 24-h intervals for the analysis of embryonic 35S-sulfate-GAGs or transferred to the same feeding medium but without 35S-sulfate for the analysis of the 35Ssulfate-GAGs from the larvae. Larvae developed from embryos were collected from the 35S-sulfate-free medium after 24, 72, and 144 h, corresponding to first (L1), second (L2), and third (L3) instar stages, respectively. This procedure ensured that the incorporation of 35S-sulfate occurred only in adult stage. It is important to notice that D. melanogaster embryos do not feed; therefore during the labeling period they were not able to use 35S-sulfate from the medium and incorporate into GAGs. The relative proportions of 35S-sulfate-precursors that were obtained from adult female and used by embryos and larvae to synthesize GAGs was estimated by measuring the amount 35S-sulfate-GAGs in terms of CPM/mg dry tissue that was extracted from each stage of development (embryo, L1, L2, and L3). The amount of 35S-sulfate-GAGs in embryos and larvae represents about 50% and 30%, respectively, the amount of 35S-sulfate-GAGs in adult female (Figure 3A). The 35S-sulfate-GAGs extracted from embryos and larvae were analyzed by agarose gel electrophoresis before and after digestion with chondroitin lyases or deaminative cleavage with nitrous acid, as described for the 35S-sulfate-GAGs from adult flies. As shown in Figure 3B, the glycans from embryos, L1, L2, and L3 displayed bands with less-distinguished migration when compared to adults, but with a preponderant band migrating as CS in the three stages of larva. As indicated by incubation of the glycans by specific glycosidases or nitrous acid (Figure 3C), the embryos and larvae at different stages of development contained 35S-sulfate-HS and 35S-sulfate-CS. To obtain more information about the utilization of 35Ssulfate-precursors by the embryos, we labeled adult flies with 35S-sulfate for 24 h and collected embryos at 2, 4, 6, 8, 10, and 12 h of development. 35S-sulfate-GAGs were extracted from the embryos by proteolytic digestion and ethanol precipitation. The low-molecular-weight contaminants were washed out by paper chromatography, and the radioactivity of 35S-sulfate-GAGs was counted at the origin of the paper chromatogram. The amount of free 35S-sulfate in the embryos was estimated by the difference between the total radioactivity of the solution of the extracted glycans and that of 35S-sulfate-GAGs, present at the origin of the paper chromatogram. As shown in Figure 4A, 35S-sulfate-GAGs increased about fivefold in 4-h embryos when compared with 2-h embryos. Embryos of 6 to 12 h of development contained similar amounts of 35S-sulfate-GAGs. The increase in the 35S-sulfate-GAGs from 2- to 4-h embryos

Glycosaminoglycans during Drosophila development

Fig. 1. Incorporation of 35S-sulfate into GAGs in adult male and female D. melanogaster and characterization of the 35S-sulfate-glycans. Adult flies (male and female) were fed with Na235SO4 for 72 h, as described in Materials and methods. (A) After the feeding period, 35S-sulfate-glycans were extracted and the amount of 35S-sulfate incorporated was estimated by cpm/mg dry tissue (mean  SD, n ˆ 3), after washing out low-molecular-weigh contaminants by paper chromatography, as described in Materials and methods. (B) 35S-sulfate-glycans (~20,000 cpm) from adult flies and a mixture of standard GAGs containing CS, DS, and HS (20 mg each) were applied to a 0.5% agarose gel in 0.05 M 1,3 diaminopropane/acetate (pH 9.0), and run for 1 h at 110 mV. After electrophoresis, the glycans were fixed and stained, as described in Materials and methods. The radioactive bands corresponding to the 35S-sulfate-glycans were detected by autoradiography of the stained gel. (C) 35S-sulfate-glycans were identified by agarose gel electrophoresis before (ÿ) or after (‡) incubation with chondroitinase (Chase) AC or ABC or deaminative cleavage with nitrous acid. M, adult male; F, adult female.

Fig. 2. Time course of 35S-sulfate incorporation into GAGs in male and female. (A) Adult male and female flies were fed separately with Na235SO4 for different times. 35S-sulfate-glycans were extracted from the flies obtained from each feeding time, and the amount of radioactivity incorporated was estimated by cpm/mg dry tissue, as described in the legend of Figure 1. (B) 35S-sulfate-glycans extracted from the flies from different feeding times were analyzed by agarose gel electrophoresis, as described in Materials and methods. The electrophoretic mobility of standard CS and HS are indicated.

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Fig. 3. Pulse-chase of 35S-sulfate-glycans during embryonic and larval development. (A) Adult flies (male and female) were fed for 72 h with Na235SO4. During this period 35S-sulfate-labeled embryos were collected in acetone at 24-h intervals and 35S-sulfate-labeled adult flies collected in acetone at the end of the 72-h period. To obtain 35S-sulfate-labeled larvae, adult flies were fed with the feeding mixture for 72 h as described. The 35S-sulfate-labeled embryos from the first 48 h were removed and the embryos from the last 24 h transferred to a medium containing the feeding mixture without Na235SO4. L1, L2, and L3 35S-sulfate-labeled larvae were collected in acetone after 24 h, 72 h, or 144 h, respectively. 35S-sulfate-glycans from adult females, embryos, and larvae were extracted and the amount of incorporation in the different stages of development estimated by cpm/mg dry tissue (mean  SD, n ˆ 3), as described. The content of 35S-sulfate-glycans obtained from embryo are significantly different from those of adult female (p 5 0.01). Similarly, the content of 35S-sulfate-glycans from larvae (L1, L2, and L3) are significantly different from those of embryo (p 5 0.05). (B) 35S-sulfate-glycans extracted from adult female, embryos, L1, L2, and L3 were analyzed by gel electrophoresis. (C) The identity of the glycans was confirmed by agarose gel electrophoresis before (ÿ) or after (‡) incubation with chondroitinase-AC and -ABC and deaminative cleavage with nitrous acid.

was followed by a decrease in the amount of free 35S-sulfate, as observed by the ratio 35S-sulfate-GAGs/free 35S-sulfate in 4-h (35S-sulfate-GAGs/free 35S-sulfate ˆ 0.6) and 2-h embryos (35S-sulfate-GAGs/free 35S-sulfate ˆ 0.2) (data not shown). The synthesis of CS is preponderant during the first 10 h of development. HS begins to be synthesized only after 10 h (Figure 4B). Analysis of chondroitin disaccharides during D. melanogaster development We noticed that there was an increase in the relative synthesis of CS in the three stages of larva, when compared to adult tissues. To verify if the composition of CS changes during development, we analyzed the disaccharide composition of this GAG obtained from embryo, L1, L2, and L3. After proteolytic digestion and ethanol precipitation, total GAGs were incubated exhaustively with chondroitinase ABC. The disaccharides were then purified by gel-filtration 532

chromatography on a peptide column and analyzed by high-performance liquid chromatography (HPLC) on an Spherisorb Sax column. As shown in Figure 5, we detected very low amounts of nonsulfated disaccharides and the prevalence of DDi-4S in the embryo, L1, and L2. In L3, we detected significant amounts of DDi-6S, in addition to small quantities of DDi-OS and the preponderance of DDi-4S. These results indicate that the composition of CS changes during development and suggest the presence of different sulfotransferase activities during larval development. Immunolocalization of sulfated GAGs To localize CS and HS in the tissues of the fruit fly at various developmental stages, we used monoclonal antibodies that recognize specific epitopes generated by the action of chondroitin ABC-lyase on cryo-sections from adult female and third larval stage (L3). HS was localized by

Glycosaminoglycans during Drosophila development

Fig. 4. Biosynthetic activity in embryos of different ages. Adult flies were labeled with 35S-sulfate for 24 h, as described in Materials and methods. (A) Embryos at 2, 4, 6, 8, 10, and 12 h of age were collected; the 35 S-sulfate-glycans were extracted by proteolytic digestion and ethanol precipitation. Low-molecular-weight contaminants were washed out by paper chromatography and the amount of 35S-sulfate-glycans recovered in the embryos counted and estimated as cpm/mg dry tissue (mean  SD, n ˆ 3). (B) Agarose gel electrophoresis autoradiography of the 35 S-sulfate-glycans extracted from embryos at different ages. Similar amount of radioactivity in terms of cpm/mg dry tissue of each glycan was applied to the agarose gel.

monoclonal antibody against intact HS chains. Chondroitin 4-sulfate (C4S) epitopes were detected in the ovary and intestine of adult flies and in several structures of the larva, occurring in different locations (Figure 6). In adult flies, C4S epitopes abounded in oocytes and were also present in high amounts at the intestinal epithelium. In larval tissues, C4S epitopes were intensively detected in salivary gland (mostly at the membranes facing the lumen), in fat bodies, and in stomach (Figure 6). It was also detected in the proventriculus, being the inner cell layer of the foreintestine more heavily stained than the outer cell layer of the midintestine. In adult females, HS was intensively detected in oocytes, nurse cells, and intestinal epithelium. In larval tissues, HS occurred in brain hemispheres, in the inner cell layer of the foreintestine, in the stomach and fat bodies (Figure 6). Discussion After ingestion of Na235SO4 by the flies, 35S-sulfate was taken up by adult tissues, both in males and in females, and used to synthesize 35S-sulfate-HS and 35S-sulfate-CS but not DS. Incorporation in females was five times higher than in males, which may reflect a higher biosynthetic activity in females due to an intense GAG synthesis in the ovary. Previous work reported the presence of high

Fig. 5. Strong anion-exchange HPLC analysis of the disaccharides formed by a specific lyase on the glycans obtained from different stages of development. A mixture of standard chondroitin disaccharides and the disaccharides formed by exhaustive action of chondroitinase ABC on the glycans extracted from embryos, L1, L2, and L3, were applied to a 250 mm  4.6 mm Spherisorb-SAX column linked to an HPLC system. The column was eluted with a linear gradient of NaCl, as described in Materials and methods. The chondroitin disaccharide standards used were: DDi-0S, DUA-1!3-GalNAc; DDi-4S, DUA-1!3GalNAc(4SO4); DDi-6S, DUA-1!3-GalNAc(6SO4). Arrows indicate the position of elution of the standards disaccharides. The results were reproduced in duplicate experiments.

amounts of HS and CS in the ovary of adult female (Toyoda et al., 2000a). However, the possibility that the higher incorporation in female was due to a difference in the feeding habits between males and females cannot be ruled out. The kinetic of 35S-sulfate incorporation into GAGs in males and females (see Figure 2) showed that HS is the predominant glycan to appear during the first 24 h of incorporation. CS can only be seen after 72 h of 35S-sulfate incorporation in both males and females (see Figure 1). This can be due to a higher metabolism of HS in adult tissues when compared to CS. After feeding female Drosophila on medium with 35Ssulfate, high amounts of 35S-sulfate-GAGs were detected in embryos and larvae. Probably, 35S-sulfate taken up by adult female equilibrated among the tissues, including the oocytes in the ovary. After fertilization of the oocytes, 35Ssulfate was used during the de novo synthesis of GAGs 2 h after embryonic development. During the first 2 h of development, most of the molecules are synthesized and provided by the female parent. Only at the onset of zygotic expressions (>2 h) new biosynthetic activities increase (Demereck, 1993). In fact, we observed a sharp increase in 533

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Fig. 6. Histochemistry for chondroitin 4 epitopes and HS in adult female and L3 larvae. Sections from adult female and L3 larvae were treated with anti-C4S or anti-HS monoclonal antibodies, as described in Materials and methods. e, oocytes; i, intestine; n, nurse cells; sgl, salivary gland; pv, proventriculum; b, brain hemisphere; f, fat body; s, stomach. Control was obtained omitting the primary antibody.

the amount of 35S-sulfate-GAGs in 4-h embryos, followed by a decrease in the amount of free 35S-sulfate. Overall, these results indicate that 35S-sulfate in the embryos is the source of sulfated GAGs during embryonic and larval stages. The presence of a significant amount of 35S-sulfate-GAG in 2-h embryos is intriguing (see Figure 4). At this stage of development there is little transcriptional activity (Demereck, 1993). Currently we do not have an explanation for that, but it raises the possibility that small amounts of GAGs are transferred from female to oocytes, probably through the nurse cells. Further experiments are required to properly address this hypothesis. Different from what we observed in adult flies, CS was the preponderant GAG synthesized in larval tissues, suggesting that the metabolism of these polymers is differently regulated in adult and developing stages. The disaccharide analysis of CS in embryos, L1, and L2 revealed the presence of unsaturated 4-sulfated and nonsulfated units, previously reported (Toyoda et al., 2000a,b). In L3, we detected significant amounts of unsaturated 6sulfated units, indicating the occurrence, in the fly, of a specific sulfotransferase not reported yet. Our data on the disaccharide composition of CS differ from previous report that detected mainly nonsulfated disaccharides followed by smaller amounts of the 4-sulfated forms (Toyoda et al., 2000a,b). This difference may be related to the different methodologies used in the studies. It remains to be determined whether the 4- and 6-sulfated disaccharides represent individual forms of chondroitin or are components of a heterogeneous polymer. 534

Overall, the findings of the present study indicate that S-sulfate is transferred from adult to embryonic and larval tissues and used in the de novo synthesis of GAGs to assemble different morphological structures during development. The data also imply the occurrence in D. melanogaster of a previously unidentified 6-O-sulfotransferase involved in the biosynthesis of CS. 35

Materials and methods Flies D. melanogaster, Berlin WT strain (Faculdade de Medicina de RibeiraÄo Preto, Universidade de SaÄo Paulo, SP, Brazil) has been maintained in the laboratory for several years. The flies were maintained in glass bottles with 70 ml standard yeast agar medium at room temperature, as described (Demereck, 1993). Materials HS from human aorta was extracted and purified as described previously (Cardoso and MouraÄo, 1994). C4S from whale cartilage, DS from bovine intestinal mucosa, twicecrystallized papain (15 U/mg protein), and the standard disaccharides D4,5 unsaturated hexuronic acid DUA)-1! 3-GalNAc, DUA-1!3-GalNAc(4SO4), DUA-1!3-GalNAc(6SO4) were purchased from Sigma (St. Louis, MO); chondroitin AC lyase (E.C. 4.2.2.5) from Arthrobacter aurenses, chondroitin ABC lyase (E.C. 4.2.2.4) from

Glycosaminoglycans during Drosophila development

Proteus vulgaris, HS lyase (E.C. 4.2.2.8), and heparin lyase (EC4.2.2.7) from Flavobacterium heparinum were from Seikagaku America (Rockville, MD); agarose (standard low Mr) was from BioRad (Richmond, CA); toluidine blue was from Fisher Scientific (NJ); 1,9-dimethylmethylene blue from Serva Feinbiochimica (Heidelberg, Germany); and cetyltrimethylammonium bromide from Merck (Darmstadt, Germany). Metabolic labeling of GAGs D. melanogaster flies (50 couples) were kept in a plastic recipient, containing 5 ml medium for ovoposition (Demereck, 1993) with a feeding mixture containing 200 mg yeast, 200 ml distilled water, and 50 mCi Na235SO4 placed at the center of the recipient. Adult flies were fed with the feeding mixture for a period of 24 or 72 h, during which 35 S-sulfate labeled embryos were collected in acetone and the feeding mixture exchanged at 24-h intervals. 35 S-sulfate-labeled adult flies were collected in acetone at the end of the period. To obtain 35S-sulfate-labeled larvae, adult flies were fed with the feeding mixture for 72 h as described. The 35Ssulfate-labeled embryos from the first 48 h were removed, and the embryos from the last 24 h transferred to a medium containing the feeding mixture without Na235SO4. L1, L2, and L3 35S-sulfate-labeled larvae were collected in acetone after 24 h, 72 h, or 144 h, respectively. During these periods, the feeding mixture with cold Na2SO4 was exchanged at 24-h intervals. Extraction of GAGs After metabolic labeling, approximately 100 adult flies (male or female), 500 embryos, and 500 larvae (L1, L2, and L3) were immediately immersed in acetone, kept for 12 h at 4 C, and dried at 60 C. The dried material was suspended in 10 ml 0.1 M sodium acetate buffer (pH 5.5), containing 25 mg papain, 5 mM ethylenediamine tetraacetic acid (EDTA), and 5 mM cysteine and incubated at 60 C for 12 h. The incubation mixture was then centrifuged (2000  g for 10 min at room temperature) and another 25 mg papain in 10 ml of the same buffer containing 5 mM EDTA and 5 mM cysteine was added to the pellet and incubated at 60 C for another 12 h. The mixture was then centrifuged (2000  g for 10 min at room temperature) and the pellet incubated one more time with papain, as described. The clear supernatants from the three extractions were combined and the GAGs precipitated with three volumes of 95% ethanol at 4 C for 12 h. The precipitate formed was collected by centrifugation (2000  g for 10 min at room temperature), freeze-dried, and suspended in 1 ml distilled water. Agarose gel electrophoresis 35 S-sulfate-GAGs (~20,000 cpm), obtained by proteolytic extraction from adult (male or female), embryos, and larvae (L1, L2, and L3) either before or after degradation with specific enzymes or deaminative cleavage with nitrous acid, and a mixture of standard GAGs containing CS, DS, and HS (20 mg each) were applied to a 0.5% agarose gel in 0.05 M 1,3 diaminopropane/acetate (pH 9.0) and run for 1 h at

110 mV. After electrophoresis, the glycans were fixed with aqueous 0.1% cetyltrimethylammonium bromide solution and stained with 0.1% toluidine blue in acetic acid/ethanol/ water (0.1:5:5, v/v). The radioactive bands corresponding to the 35S-sulfate-labeled molecules were detected by autoradiography of the stained gel. Quantification of the 35S-sulfate incorporation into the D. melanogaster tissues A solution of the 35S-sulfate-GAGs, extracted from adult (male or female), embryos, and larvae (L1, L2, and L3) was applied to a Whatman No. 3 chromatographic paper and developed for 48 h in 1-butanol/pyridine/water (3:2:1, v/v). At the end of the developing period, the origin of the paper chromatogram containing the glycans free of low-molecular-weight contaminants was cut out, added to 10 ml 0.2% PPO/toluene solution, and counted in a liquid scintillation counter. The 35S-sulfate incorporation was estimated by cpm/dry weight of the tissue. The identification of the 35Ssulfate-labeled molecules was carried out by agarose gel electrophoresis before or after incubation with specific glycosidases or deaminative cleavage with nitrous acid, as described earlier. Enzymatic treatment The 35S-sulfate-GAGs (~30,000 cpm) were incubated with 0.1 U of chondroitin ABC- or AC-lyase (Seikagaku) in 0.1 ml 50 mM ethylenediamine:acetate buffer, pH 8.0. After incubation at 37 C for 12 h, another 0.01 U of enzyme was added for an additional 12-h period. Agarose gel electrophoresis of the control or enzyme-incubated glycans was used to assess enzymatic activity. Deaminative cleavage Deaminative cleavage with nitrous acid at pH 2.0 was performed as described by Shively and Conrad (1976). The extent of deaminative cleavage was assessed by agarose gel electrophoresis of the control or nitrous acid±treated glycans, as described earlier. Analysis of the products formed by digestion of the glycans with chondroitin ABC-lyase Embryos (~100 mg), L1, L2, and L3 (~200 mg each) were subjected to papain extraction, as described earlier. Total GAGs from embryos, L1, L2, and L3 were incubated exhaustively with 0.1 U chondroitin ABC lyase, as described earlier. Thereafter, the products formed were separated on a Superdex-Peptide column (Amersham Biosciences, Piscataway, NJ), equilibrated with 20% acetonitrile (pH 3.5). The column was developed in the same buffer at a flow rate of 0.5 ml/min. Fractions of 0.5 ml were collected and checked for absorbance at 232 nm. The fractions corresponding to disaccharides (>90% of the degraded material) were pooled and analyzed by strong anionexchange chromatography on a Supelco 4.5  250 mm Spherisorb-SAX column, using a linear gradient of 0±1.0 M aqueous NaCl (pH 3.5) at a flow rate of 0.5 ml/min. The elution of the disaccharides was followed by absorbance at 232 nm, and they were identified by comparison with elution positions of known disaccharide standards. 535

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Immunohistochemistry Adult females and L3 larvae were anesthetized with CO2 and fixed in 4% paraformaldehyde in sodium phosphate buffer 0.1 M (pH 7.3) under microwave irradiation (Laboratory Microwaves Processor, Pelco Model RFS59MP, 2.45 GHz), for 15 s at 45 C, followed by a 30-min period at room temperature. The samples were then cryoprotected in 20% sacarose in phosphate buffer 0.1 M (pH 7.3) for 24 h at room temperature, embedded in OCT (Miles, Elkhart, IN), and frozen in liquid nitrogen. Semi-thin sections (10 mm) were obtained in cryostat operated at ÿ40 C. For immunostaining using chain-specific chondroitin antibodies against C4S (Seikagaku, Tokyo), the reaction with primary antibodies was preceded by digestion with chondroitinase ABC (Seikagaku) to unmask the specific antigen epitope. Sections were incubated with 0.5 U chondroitinase ABC in 1 ml 0.25 M Tris±HCl buffer (pH 8.0) for 1 h at 37 C in a moist chamber. Slides with (C4S) or without predigestion (HSPG, from Seikagaku) were carefully washed in 0.1 M phosphate buffered saline (PBS), followed by 50 mM NH4Cl and PBS, and incubated with PBS, 1% bovine serum albumin in PBS (pH 7.4) for 1 h. This procedure avoids nonspecific binding of antibody. The sections were then incubated with the primary antibodies (dilution 1:100) overnight at 4 C in a moist chamber, washed with PBS, and incubated with a secondary biotinylated antibody (mouse IgG). A mouse-PAP complex was then applied to the sections after a further wash and incubated with an ABC complex. The peroxidase reaction was visualized by incubation with 0.05% diaminobenzidine and 4 mg/ml glucose oxidase. The sections were then mounted in glycerol and examined under an optical microscope (Zeiss, Axioplan). High-resolution images were obtained on a digital camera (Zeiss) coupled to an image acquisition program (Axiovision, Zeiss). Statistical analysis Statistical differences among the experimental groups were evaluated with the one-tailed paired t-test. The level of significance was set at p 5 0.05. Acknowledgments We express appreciation to Silvania and Nilson for technical assistance. This work was supported by PRONEX, FAPERJ, and the Fogarty International Center (R03 TW05775). M.S.G.P., E.A., and R.L. are research fellows from CNPq. Abbreviations DUA, D4,5 unsaturated hexuronic acid; CS, chondroitin sulfate; DS, dermatan sulfate; EDTA, ethylenediamine tetra-acetic acid; GAG, glycosaminoglycan; HPLC, high-performance liquid chromatography; HS, heparan sulfate; PBS, phosphate buffered saline.

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