From the Department of Cell and Molecular Biology, Lund University, BMC C13, SE-221 84, Lund, Sweden
Received for publication, October 28, 2002
, and in revised form, March 31, 2003.
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ABSTRACT |
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INTRODUCTION |
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Galactosaminoglycan synthesis is then initiated by the action of 1,4-N-acetylgalactosaminyltransferase I (GalNAcT I) (16). This enzyme catalyzes the addition of a GalNAc to the GlcUA in the linkage region, whereupon chondroitin is further polymerized by chondroitin synthase that catalyzes the addition of alternating -3GalNAc
1- and -4GlcUA
1-units (17). The subcellular location of GalNAcT I and chondroitin synthase have not yet been determined. Sulfotransferases catalyze the transfer of sulfate groups from the sulfate donor 3'-phosphoadenylyl 5'-phosphosulfate to various positions in the chain to complete the biosynthesis of CS. To form DS, GlcUA residues are epimerized to iduronic acid catalyzed by GlcUA C5 epimerase (18). The sulfotransferases are generally believed to reside in the trans-Golgi/trans-Golgi network (TGN), but the exact location appears to vary in different cell lines (1921).
The linkage region can be modified by sulfation of the Gal residues and by phosphorylation of the Xyl residue (22, 23). Sulfation of Gal has only been demonstrated in the CS linkage region, and it has been speculated that this might be a signal for CS biosynthesis (18, 23). Phosphorylation of Xyl has been shown to be a transient event during decorin biosynthesis in rat skin fibroblasts (24). In this case phosphorylation increased during Gal addition and was nearly stoichiometric at the trisaccharide level, followed by rapid dephosphorylation upon the addition of GlcUA. The enzymes responsible for the phosphorylation/dephosphorylation of Xyl are unknown.
The only phosphorylation of a structural sugar unit that has been studied in detail is the formation of mannose 6-phosphate in N-linked oligosaccharides on proteins destined for the lysosomes. This occurs in two steps during passage through the early secretory pathway. First GlcNAc-1-phosphate is transferred from UDP-GlcNAc to mannose. Subsequently, a hexosaminidase catalyzes removal of the GlcNAc portion leaving a 6-phosphate on mannose (25, 26). Lysosomal proteins with a C-terminal KDEL ER retention signal (27) accumulate the phosphodiester intermediate form (28). To study early events in the O-glycosylation of decorin, we have transiently expressed decorin with or without a C-terminal KDEL ER retention signal in COS-7 cells. We have determined the structure of the major O-linked saccharide including the phosphorylation of Xyl and chondroitin sulfation. This was performed in the absence or presence of the fungal metabolite brefeldin A (BFA) to manipulate the secretory pathway (29).
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EXPERIMENTAL PROCEDURES |
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Construction of VectorsEukaryotic expression vectors were constructed using PCR and rat decorin cDNA ligated in the eukaryotic expression vector pcDNA I neo (Invitrogen, San Diego, CA) (Dec) as template. To introduce KDEL we used the 3' primer (GGAATTCTTACAGTTCGTCCTTGTAGTTCCCAAGTTGAA) together with the 5' primer (CGGATCCATGAAGGCAACTCTCG). Dec was digested with BamHI/SacI (964). This fragment was ligated to PUC18 digested with BamHI/SacI. The PCR product was cleaved with SacI/EcoRI and ligated to the PUC18 construct digested with SacI/EcoRI. The whole decorin construct was then moved to pcDNA I neo (Dec-KDEL).
To construct the S34A mutation we used the 5' primer (TATCAGCTGCTGGCATAATCCCTTACGA) together with the 3' primer (CGTCTAGATGTAGTTCCCAAGTTGAATG). The PCR product was cleaved with PvuII/XbaI and ligated to Dec, from which the StuI(257)/XbaI fragment had been removed (Dec-S34A).
By exchanging the EcoRV(297)/XbaI fragment in Dec-S34A for the same fragment from Dec-KDEL, we obtained Dec-S34A-KDEL. The amplifications were performed for 30 cycles in a thermal cycler using Dynazyme Taq polymerase, 95 °C for 30 s, 56 °C for 45 s, and 72 °C for 60 s. All of the PCR products were sequenced to confirm their sequence identity.
Cell CultureCOS-7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, and 50 µg/ml gentamycin. One day prior to transfection, the cells were seeded in new bottles with fresh medium. The cells were transfected using electroporation (300 V, 500 microfarads in a Bio-Rad gene pulser) and then cultivated for 48 h prior to metabolic labeling.
Metabolic LabelingCOS-7 cells expressing decorin constructs were labeled with [35S]sulfate (100 µCi/ml) in low sulfate minimal Eagle's medium with Earle's salts, (Nord Vac, Sweden), [35S]methionine (20 µCi/ml) in methionine-free minimal Eagle's medium with Earle's salts, (Sigma), [32P]phosphate (200 µCi/ml) in phosphate-free Dulbecco's modified Eagle's medium (ICN), and D-[6-3H]galactose (50 µCi/ml) and D-[6-3H]glucosamine (50 µCi/ml) in culture medium. When deficient media were used, the cells were preincubated in labeling medium for 20 min prior to addition of labeling medium with radioactive precursors. Brefeldin A (Sigma) was used at 10 µg/ml.
Isolation of DecorinCulture medium was collected and cleared by centrifugation at 4000 x g for 10 min. The cell layer was washed three times with ice-cold phosphate-buffered saline and then extracted using 0.15 M NaCl2, 10 mM EDTA, 10 mM KH2PO4, pH 7.5, 2% (v/v) Triton X-100, 5 µg/ml ovalbumin followed by centrifugation. Complete Protease inhibitor (Roche Applied Science) was added to inactivate proteases. Both media and cell lysates were supplemented with 1/100 volume of a 50% slurry of protein A-Sepharose CL4B (Sigma) and were allowed to mix for several hours. After centrifugation, rabbit anti-rat decorin antiserum (30) (1/200 volume) was added to the supernatant and incubated overnight. After another addition of protein A-Sepharose and incubation for 1 h, bound material was collected by centrifugation at 1000 x g for 1 min. All of the steps were carried out at 4 °C. The immunoisolates were washed five times with 10 mM Tris, 150 mM NaCl, pH 7.4, 0.2% Tween 20, and decorin was eluted by boiling in SDS sample buffer for SDS-PAGE or boiling in 4 M guanidine HCl, 50 mM NaOAc, pH 5.8, 0.2% (v/v) Triton X-100 for subsequent size exclusion chromatography on Superose 6.
ChromatographySize exclusion chromatography was performed on Superose 6 (Amersham Biosciences) operated in 4 M guanidine HCl, 50 mM NaOAc, pH 5.8, 0.2% (v/v) Triton X-100 at a flow rate of 0.4 ml/min. Fractions of 1 min were collected. Biogel P10 was packed in Omnifit columns operated in 0.5 M NH4HCO3, 0.02% (w/v) NaN3 at a flow rate of 3 ml/h. Fractions of 30 drops were collected. The samples were supplemented with 100 µg of carrier consisting of CS, HS oligosaccharides, raffinose, sucrose, and glucose. Decorin [6-3H]galactose-labeled GAG chains, secreted by rat skin fibroblasts, were digested with chondroitin ABC lyase to generate linkage region hexasaccharides and further digested with chondroitin ACI lyase to generate linkage region tetrasaccharides. The Lichrosorb-NH2 column (Merck) was operated in 0.1 M acetate buffer, pH 5.0, at a flow rate of 0.5 ml/min. Three fractions/min were collected. The disaccharides were obtained by digestion of GAG chains with chondroitin ABC/AC-I lyases followed by purification on Biogel P10. The elution of non- and mono-sulfated unsaturated disaccharide standards were monitored by UV absorption at 232 nm. Radioactivity was measured using liquid scintillation counting (Beckman).
Degradation MethodsO-Linked glycan chains were released by alkaline scission using 0.4 M NaOH, 50 mM NaBH4 for 24 h at 4 °C. After neutralization with glacial HOAc, the samples were lyophilized and dissolved in the appropriate buffer. Enzymatic digestions were performed after repeated cycles of lyophilization and dissolving in water to evaporate all NH4HCO3. Digestion with chondroitin ABC/AC-I lyases (Seikagaku Fine Biochemicals, Tokyo, Japan) and 13-glycuronidase (gift from Dr. Yoshida, Seikagaku Corp.) was performed in 0.1 M Tris acetate, 10 mM EDTA, pH 7.3,
-galactosidase (Sigma) was used in 50 mM Tris acetate, 1 mM MgCl2, pH 7.3. Calf intestinal alkaline phosphatase (Roche Applied Science) was used according to the manufacturer's recommendation.
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RESULTS |
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Phosphorylation of Core ProteinsNo [32P]phosphate-labeled core protein could be detected by using SDS-PAGE when Dec was transiently expressed in COS-7 cells, presumably because of the low level of intracellular 43-kDa core protein (Fig. 2, compare lanes 1 and 3). Expression of decorin-KDEL yielded a 43-kDa [32P]phosphate-labeled core protein, which migrated slightly more retarded on SDS-PAGE than [35S]methionine-labeled decorin-KDEL (Fig. 2, compare lanes 2 and 4). This shows that the vast majority of intracellular core proteins are not substituted with [32P]phosphate. The [32P]phosphate-labeled decorin-KDEL was also sensitive to endoglycosidase H degradation, indicating similar locations of phosphorylated and nonphosphorylated decorin-KDEL in ERGIC (data not shown). Retention of lysosomal enzymes by tagging the C terminus with the KDEL ER retention signal leads to accumulation of a GlcNAc-phosphate-mannose phosphodiester intermediate that is resistant to digestion with alkaline phosphatase (28). However, all phosphate groups in decorin-KDEL were sensitive to alkaline phosphatase, demonstrating the absence of a phosphodiester intermediate (Fig. 2, lane 5). Ser-34 in decorin is the amino acid residue that accepts Xyl, initiating GAG synthesis. To examine whether incorporation of phosphate was linked exclusively to O-glycosylation and did not represent amino acid phosphorylations, we used a mutated decorin, where Ser-34 was substituted with Ala (Dec-S34A). The Dec-S34A construct yielded no detectable sulfate-labeled decorin PG in the medium (Fig. 3A). A cDNA construct Dec-S34A-KDEL encoding mutated decorin with a KDEL ER retention signal in the C terminus was transiently expressed in COS-7 cells. After labeling of cells with [32P]phosphate, no phosphorylated 43-kDa decorin core protein could be detected (Fig. 3B, lane 1). These results show that Ser-34 is indirectly essential for the phosphorylation by providing for the preceding O-glycosylation.
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Phosphorylated O-Linked OligosaccharidesTo analyze the O-linked GAG precursor of decorin-KDEL, immunoisolated core protein labeled with [32P]phosphate and [6-3H]galactose was subjected to alkaline -elimination and chromatographed on Biogel P10 (Fig. 4). The major product eluted at a position corresponding to a pentasaccharide, but longer oligosaccharides were also obtained. The putative phosphorylated pentasaccharide was degraded by chondroitin AC I lyase, which is known to erode chondroitin chains to the linkage tetrasaccharide. The phosphorylated linkage oligosaccharide, resulting from the chondroitin AC I lyase digestion of the putative pentasaccharide, eluted at fractions 4852 in Fig. 4. (data not shown). This oligosaccharide could be further reduced in size by the sequential degradation with linkage region degrading enzymes (see below). This indicates that the major linkage region GAG precursor is a phosphorylated pentasaccharide. The vast majority of the oligosaccharides were phosphorylated, because the 3H- and 32P-labeled saccharides coeluted on the Biogel P10 column (compare elution positions for phosphorylated and nonphosphorylated monosaccharide in Fig. 4). The 3H-labeled material in the peak at fraction 59 was not sensitive to chondroitinase AC I,
13-glucuronidase nor to
-galactosidase. Therefore, this material does not appear to represent the O-linked GAG precursor and was not further characterized.
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GAG Chain PolymerizationTo examine whether O-linked saccharides on decorin-KDEL could serve as primers for GAG elongation, we treated cells with BFA. BFA causes mixing of the ER and Golgi compartments proximal to TGN and could expose the decorin-KDEL intermediate to the polymerizing and sulfating enzymes present in the medial-and the trans-Golgi compartments (29). When BFA was added to cells labeled with [32P]phosphate, a larger and more heterogeneous decorin-KDEL proteoglycan was obtained (Fig. 5, lane 2). This PG was smaller than decorin secreted by cells transfected with Dec (compare Figs. 1A and 3A, lane 2) and indicate a partial elongation of the GAG chain.
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The structure of O-linked GAG chain precursors synthesized in the absence or presence of BFA was determined. The cells transfected with Dec-KDEL were labeled with [32P]phosphate and [3H]glucosamine. Decorin-KDEL was immunoisolated from the cell layer, and alkali-released O-linked chains were separated on Biogel P10 (Fig. 6). In the absence of BFA, phosphorylated oligosaccharides with a major product corresponding to a pentasaccharide were again obtained (Fig. 6A). These saccharides incorporated no [3H]glucosamine, because no extensive GAG polymerization is taking place. Pooled saccharides, representing mostly pentasaccharides, were cleaved with chondroitin ABC/AC-I lyases, which should erode the saccharides down to HexUA
13Gal
13Gal
14XylOH-2-phosphate. Accordingly, chromatography on Biogel P10 yielded a single tetrasaccharide peak (Fig. 6B). This tetrasaccharide was first cleaved with
13-glycuronidase generating Gal
13Gal
14XylOH-2-phosphate (Fig. 6C) and then with
-galactosidase generating XylOH-2-phosphate (Fig. 6D). Alkaline phosphatase treatment of this product generated inorganic phosphate (data not shown).
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In the presence of BFA, all O-linked phosphorylated chains migrated in the void volume. This product contained large amounts of radiolabeled sugars derived from [3H]glucosamine, indicating extensive polymerization (Fig. 6E). When these chains were digested with chondroitin ABC/AC-I lyases, the resulting phosphorylated peak chromatographed in the expected position of a linkage tetrasaccharide (Fig. 6, compare F and B). Most of the 3H label appeared in disaccharides that could be cleaved into monosaccharides using 13-glycuronidase (data not shown). A minor peak containing 3H label and overlapping with the [32P]phosphate-labeled peak probably represented some residual tetrasaccharide from the repetitive part of the polymer not cleaved by the chondroitin lyases. The phosphorylated tetrasaccharides obtained from the linkage region of GAG primed in the presence of BFA were also sensitive to
13-glycuronidase and
-galactosidase (Fig. 6, G and H). All [32P]saccharides were sensitive to alkaline phosphatase digestion (data not shown).
GAG Chain SulfationTo analyze whether the GAG polymer generated on decorin-KDEL in BFA-treated cells contained sulfate, we labeled cells expressing decorin-KDEL with [3H]glucosamine in the presence of BFA. GAG chains derived from decorin-KDEL were digested with chondroitin ABC/AC-I lyases and analyzed on a Lichrosorb column (data not shown). All of the disaccharides were unsulfated. This indicates that the sulfotransferases and the polymerase are not located in the same Golgi compartment in these cells. No linkage-region Gal sulfation was detected (data not shown).
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DISCUSSION |
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The phosphorylated decorin-KDEL core protein was slightly larger than core protein labeled with [35S]methionine. No phosphorylation could be detected when cells expressed a core protein lacking GAG attachment site (decorin-S34A-KDEL), showing that the phosphate is exclusively a substituent of the Xyl residues linked to Ser-34 in decorin. GAG synthesis appears to be initiated only in a minor fraction of the decorin-KDEL core proteins, and the majority of the decorin-KDEL core proteins substituted with O-linked oligosaccharides are phosphorylated.
Lysosomal enzymes tagged with KDEL contain phosphodiester intermediates resistant to phosphatase digestion (28). However, the phosphate on decorin-KDEL could be removed by phosphatase, suggesting a different mechanism, possibly indicating the existence of a kinase.
The majority of the decorin-KDEL O-linked oligosaccharides were GalNAc14GlcUA
13Gal
13Gal
14Xyl-2-phospate, but longer chains were also present. This indicates that XylT, GalT I and II, GlcUAT I, and GalNAcT I are present and active in the ERGIC compartment. Recent immunolocalization results indicate that the bulk of GlcUAT I enzymes reside in the medial-Golgi. Our results demonstrate GlcUAT I activity also proximal to the medial-Golgi stacks. The nature of the first N-acetyl-hexosamine residue added determines whether further CS or HS polymerization will ensue. Our results indicate that this choice is made in the ERGIC. Despite the formation of a GalNAc-containing pentasaccharide, radiolabeling of the terminal GalNAc in the linkage region was low. This is because a long time is needed for [3H]glucosamine to equilibrate with UDP-GlcNAc/UDP-GalNAc present in the Golgi, and then with UDP-GalNAc transported back to the ER. Different intracellular pools of UDP sugar precursors have been reported (35). GalNAcT I also possesses GalNAcT II activity, i.e. catalyzes transfer of GalNAc onto nonreducing terminal GlcUA of chondroitin in
14-linkage (16). The longer oligosaccarides obtained, which appear to represent odd-numbered oligosaccharides, could thus be explained by the presence of minute amounts of GlcUAT II activity of CS synthase. When a terminal GalNAc acquires a GlcUA residue, it will immediately be substituted with another GalNAc residue resulting in odd-numbered oligosaccharides.
All oligosaccharides were phosphorylated, indicating that dephosphorylation is not obligatory for addition of the first GlcUA. It has been reported2 that Gal13Gal
14Xyl-2-phosphate is a better acceptor for GlcUA than Gal
13Gal
14Xyl, indicating that phosphorylation promotes elongation (36). Xyloside-primed GAG chains can retain Xyl-2-phosphate when produced in large amounts (37), and extracellular PGs often contain residual amounts of Xyl-2-phosphate (38, 39). Hence, dephosphorylation of Xyl-2-phosphate can be postponed until decorin has reached compartments distal to the ERGIC. However, Xyl phosphorylation was not detected in secreted decorin (data not shown), supporting the notion that the phosphorylation is transient (24).
BFA is known to block anterograde but not retrograde vesicular transport in the secretory pathway. Using BFA it was demonstrated that CS synthase is located in the TGN of rat ovarian granulosa cells (20) and that both CS polymerase and CS sulfotransferases are located proximal to TGN in mouse mastocytoma cells (21) and in M21 human melanoma cells (19). BFA treatment of COS-7 cells resulted in partial polymerization of GAG chains in decorin. Sulfation could not be detected in the presence of BFA. Our results indicate that CS synthase resides in the medial- and/or trans-Golgi stacks and that CS sulfotransferases reside in the TGN in COS-7 cells. The latter compartment does not merge with the ER when cells are exposed to BFA. In the presence of BFA, Xyl remained phosphorylated during elongation. In the artificial environment created in the fused compartment, dephosphorylation may have been inactivated. Also, the simultaneous presence of both a phosphatase and a phosphorylating enzyme system could give the same result. Dephosphorylation could also be a TGN event, and Dec-KDEL would not be exposed to a putative phosphatase.
Sulfation of the linkage region Gal residues has only been reported for CS, suggesting a potential role in regulating the substitution of the linkage region with CS or HS (18, 23). However, we could not detect any sulfated Gal residues in decorin-KDEL even in the presence of BFA because sulfotransferases were apparently not relocated to the ER.
Sulfation has been shown to stimulate further elongation by the GalNAcT II activity of chondroitin synthase that adds GalNAc to GlcUA during CS polymerization (40). The GAG synthesized on decorin-KDEL in the presence of BFA contained no sulfate groups, which could be the reason why it is not polymerized up to the full length of CS in secreted decorin.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 46-46-222-8577; Fax: 46-46-222-3128; E-mail: Ake.Oldberg{at}medkem.lu.se.
1 The abbreviations used are: PG, proteoglycan; ERGIC, endoplasmic reticulum-Golgi intermediate compartment; GalNAc, N-acetyl-D-galactosamine; GlcUA, D-glucuronic acid; Gal, D-galactose; Xyl, xylose; GAG, glycosaminoglycan; CS, chondroitin sulfate; DS, dermatan sulfate; HS, heparan sulfate; XylT, -D-xylosyltransferase; ER, endoplasmic reticulum; UDP, uridine diphosphate; GalT I,
1,4-galactosyltransferase I; GalT II,
1,3-galactosyltransferase II; GlcUAT I,
1,3-glucuronyltransferase I; GalNAcT I,
1,4-N-acetylgalactosaminyltransferase I; TGN, trans-Golgi network; BFA, brefeldin A; Dec, decorin.
2 Y. Tone, J. Nishihara, J. Tamura, H. Kitagawa, and K. Sugahara, unpublished data.
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REFERENCES |
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