2 Laboratório de Tecido Conjuntivo, Hospital Universitário Clementino Fraga Filho and Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas (ICB), Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brasil; 3 Instituto de Biofísica Carlos Chagas Filho, UFRJ, Rio de Janeiro, Brasil; 4 Departamento de Histologia e Embriologia, ICB, UFRJ Rio de Janeiro, Brasil; and 5 Stony 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
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Abstract |
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Key words: biosynthesis / chondroitin sulfate / development / Drosophila melanogaster / heparan sulfate
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Introduction |
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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 3'-phosphoadenosine-5'-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. 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-sulfate-GAGs in the embryo and in the three larval stages.
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Results |
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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.
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Discussion |
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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 35S-sulfate, 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, 35S-sulfate 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 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 6-sulfated 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.
Overall, the findings of the present study indicate that 35S-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.
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Materials and methods |
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Materials
HS from human aorta was extracted and purified as described previously (Cardoso and Mourão, 1994). C4S from whale cartilage, DS from bovine intestinal mucosa, twice-crystallized papain (15 U/mg protein), and the standard disaccharides
4,5 unsaturated hexuronic acid
UA)-1
3-GalNAc,
UA-1
3-GalNAc(4SO4),
UA-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 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 µg yeast, 200 µl distilled water, and 50 µCi 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 35S-sulfate labeled embryos were collected in acetone and the feeding mixture exchanged at 24-h intervals. 35S-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 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. 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 tetra-acetic acid (EDTA), and 5 mM cysteine and incubated at 60°C for 12 h. The incubation mixture was then centrifuged (2000 x 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 x 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 x g for 10 min at room temperature), freeze-dried, and suspended in 1 ml distilled water.
Agarose gel electrophoresis
35S-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 µg 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 35S-sulfate-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 acidtreated 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 anion-exchange chromatography on a Supelco 4.5x250 mm Spherisorb-SAX column, using a linear gradient of 01.0 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.
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 µm) were obtained in cryostat operated at 40°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 TrisHCl 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 µg/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 < 0.05.
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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