©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Accumulation of a Pentasaccharide Terminating in -N-Acetylglucosamine in an Animal Cell Mutant Defective in Heparan Sulfate Biosynthesis (*)

Lijuan Zhang , Jeffrey D. Esko (§)

From the (1) Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Heparan sulfate biosynthesis initiates by the transfer of -D-GlcNAc from UDP-GlcNAc to the D-GlcA moiety of the linkage tetrasaccharide, GlcA1-3Gal1-3Gal1-4Xyl1-core protein. The enzyme catalyzing this reaction differs from the -GlcNAc transferase involved in chain polymerization based on genetic and enzymatic studies of an animal cell mutant defective in chain polymerization (Fritz, T. A., Gabb, M. M., Wei, G., and Esko, J. D.(1994) J. Biol. Chem. 269, 28809-28814). In this report we show that this mutant also accumulates a pentasaccharide intermediate containing -GlcNAc. A fusion protein was made from the IgG-binding domain of protein A and a segment of the proteoglycan, betaglycan. This segment contained one glycosaminoglycan attachment site that primes only chondroitin sulfate and another that primes both heparan sulfate and chondroitin sulfate (Zhang, L., and Esko, J. D.(1994) J. Biol. Chem. 264, 19295-19299). Expression of the chimera in the mutant resulted in the accumulation of an oligosaccharide that labeled with [6-H]GlcN. The oligosaccharide comigrated with a pentasaccharide standard derived from chondroitin sulfate, but acid hydrolysis gave 98% [H]GlcN. Heparin lyase III digestion yielded [H]GlcNAc, suggesting that the GlcNAc residue was -linked to the nonreducing terminus. Enzymatic treatment of [6-H]Gal-labeled material yielded the tetrasaccharide, GlcA-[H]Gal-[H]Gal-xylitol. These findings suggest that pentasaccharide had the structure, GlcNAc1-4GlcA1-3Gal1-3Gal1-4Xyl. Its accumulation in a Chinese hamster ovary cell mutant defective in the polymerizing -GlcNAc transferase provides in vivo evidence that two -GlcNAc transferases catalyze the formation of heparan sulfate.


INTRODUCTION

Chondroitin sulfate and heparan sulfate are attached to specific serine residues in a proteoglycan through a common linkage tetrasaccharide (-GlcA1-3Gal1-3Gal1-4Xyl). Studies of Chinese hamster ovary mutants altered in glycosaminoglycan (GAG)() synthesis have shown that the pathways of heparan sulfate and chondroitin sulfate synthesis share at least the first two enzymes that initiate the chains, xylosyltransferase and galactosyltransferase I (Esko et al., 1985, 1987; Esko, 1991). The pathways diverge thereafter by the addition of the first N-acetylated hexosamine residue, -GlcNAc (heparan sulfate) or -GalNAc (chondroitin sulfate). Previous studies showed that the transferase that adds the first -GalNAc residue in chondroitin sulfate differs from the transferase involved in chain polymerization (Rohrmann et al., 1985). Recently, Fritz et al.(1994) reported that the enzyme transferring -GlcNAc to the linkage tetrasaccharide differs from the one involved in forming the repeating disaccharide units of heparan sulfate (GlcA1-4GlcNAc1-4). Based on this finding, we predicted that a Chinese hamster ovary mutant defective in heparan sulfate polymerization (Lidholt et al., 1992) might accumulate a pentasaccharide intermediate consisting of -GlcNAc attached to the linkage tetrasaccharide. Its accumulation in the mutant provides strong evidence that different enzymes catalyze the initiation and polymerization of heparan sulfate chains.


EXPERIMENTAL PROCEDURES

Cell Culture

Chinese hamster ovary cells (CHO-K1) were obtained from the American Type Culture Collection (CCL-61, Rockville, MD) and pgsD-677 was described previously (Lidholt et al., 1992). Cells were grown in Ham's F-12 medium supplemented with 7.5% fetal bovine serum (Hyclone, Salt Lake City, UT), penicillin G (100 units/ml), and streptomycin sulfate (100 µg/ml) under an atmosphere of 5% CO, 95% air and 100% relative humidity. Cells were passaged with trypsin every 3-4 days and revived periodically from frozen stocks.

Linkage Region Oligosaccharides

Decorin from bovine skin (a generous gift from M. Höök, Texas) was used to prepare oligosaccharide standards. The GAG chain was released by -elimination, and the terminal xylose residue was reduced to [H]xylitol with NaBH. Briefly, 0.5 µg of decorin was dissolved in 50 µl of water and mixed with 50 µl of a solution containing 0.5 M NaOH, 20 mM NaBH, and 5 mCi of NaBH (12.6 Ci/mmol, DuPont NEN). After 24 h at 4 °C, 10 µl of 10 N acetic acid was added in a fume hood to stop the reaction. Authentic chondroitin 4-sulfate (1 mg) was added as carrier. The sample was diluted with 9 volumes of water and applied to a 0.5-ml column of DEAE-Sephacel. The column was washed twice with 12.5 ml of a 20 mM sodium acetate buffer (pH 6.0) containing 0.25 M NaCl. The GAG chains were eluted with 1 M NaCl buffer (2.5 ml) and precipitated twice with 4 volumes of ethanol at 4 °C. The hexasaccha-ride, GlcA1-3GalNAc1-4GlcA1-3Gal1-3Gal1-4[H]xylitol, was generated by chondroitinase ABC digestion (Hascall et al. 1972; Oike et al., 1980). The tetrasaccharide, GlcA1-3Gal1-3Gal1-4[H]xylitol, was generated by chondroitinase ACII digestion (Hascall et al. 1972; Oike et al., 1980). The pentasaccharide, GalNAc1-4GlcA1-3Gal1-3Gal1-4[H]xylitol, and the trisaccharide, Gal1-3Gal1-4[H]xylitol, were generated by treating the hexasaccharide and the tetrasaccharide with mercuric acetate (Ludwigs et al., 1987), respectively. The completion of each reaction and the purity of each compound was confirmed by gel filtration and anion-exchange chromatography, as described below.

Transfection and Radiolabeling

A chimera containing a 45-amino acid segment of betaglycan fused to the IgG binding domain of staphylococcal protein A was expressed transiently in pgsD-677 mutant and wild-type cells as described previously (Zhang and Esko, 1994). In transfection experiments, a culture dish (150 mm diameter) was seeded with 5 10 cells in Ham's F-12 medium containing 10% (v/v) NuSerum (Collaborative Research, Inc., Bedford, MA). After 1 day, the medium was removed, and 10 ml of Ham's F-12 medium containing 0.25 mg/ml of DEAE-dextran (Sigma D-9885), 50 mM Tris-HCl (pH 7.4), 50 µg/ml of chloroquine, and 10 µg/ml of plasmid DNA was added. The cells were incubated at 37 °C for 2 h. The solution was aspirated, and 10 ml of 10% (v/v) dimethyl sulfoxide (Sigma) in phosphate-buffered saline was added. After 2 min at 23 °C, the solution was aspirated. The cells were washed with Ham's F-12 medium without serum and grown in 30 ml of complete Ham's F-12 medium for 12 h. The transfected cells were then labeled at 37 °C for 48 h with 50 µCi/ml [S]HSO (25-40 Ci/mg, Amersham Corp.) prepared in sulfate-free growth medium. In some experiments, the cells were labeled with 100 µCi/ml of D-[6-H]glucosamine (40 Ci/mmol) or 100 µCi/ml of D-[6-H]galactose (25.5 Ci/mmol) in medium containing 1 mM glucose (12 ml).

Fusion Protein Purification

Labeled culture medium was mixed with 120 µl of 1 M Tris-HCl (pH 7.5), 5% (v/v) Triton X-100, and 2% (w/v) sodium azide. The samples were centrifuged at 1,100 g for 10 min (IEC HN-SII centrifuge), and the supernatant was decanted into a fresh tube. IgG-Agarose beads (Sigma) were added (0.2 ml), and the sample was mixed end-over-end overnight at 4 °C. Samples were centrifuged 1,100 g for 10 min, and the supernatants were aspirated. The pellets were washed 3 times with buffer containing 50 mM Tris-HCl (pH 8), 0.15 M NaCl, and 0.02% (w/v) sodium azide (14 ml). S-Labeled samples were dissolved in 20 mM Tris buffer (pH 7.0), and an aliquot was treated at 37 °C for 4 h with 10 milliunits of chondroitinase ABC (Seikagaku), 2 milliunits of heparin lyase III (EC 4.2.2.8, Seikagaku), or both enzymes in a total volume of 50 µl. The reactions were stopped by adding SDS-PAGE sample buffer (Laemmli, 1970) and boiling the samples in a water bath for 7 min. The samples were loaded on a 5-16% linear gradient SDS-PAGE gel (200 160 1.5 mm) and electrophoresed at constant voltage (80 V) overnight (Laemmli, 1970). The gel was dried on a piece of filter paper, and the radioactive proteoglycans were visualized by autoradiography.

Oligosaccharide Purification

All purification steps were performed at room temperature unless otherwise indicated.

Step 1: Trypsin Treatment

[6-H]Gal and [6-H]GlcN-labeled betaglycan chimeras bound to IgG-agarose beads were washed with 0.1 M ammonium bicarbonate and suspended in bicarbonate buffer (0.5 ml) containing 0.1 mM CaCl and 50 µg/ml L-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Sigma). After the samples were incubated at 37 °C, they were centrifuged. The proportion of total counts in the supernatant increased from less than 1% at the beginning of the reaction to 65-100% by 40 min. The reaction was continued for another 40 min and then stopped by boiling the samples in a water bath for 10 min. The beads were removed by centrifugation, and the supernatants were collected. Chondroitin 4-sulfate (1 mg, Calbiochem, La Jolla, CA) was added to each sample.

Step 2: DEAE-Sephacel Chromatography

DEAE-Sephacel columns (0.5 ml) were prepared in a disposable pipette tip and equilibrated with 0.25 M NaCl in 50 mM sodium acetate buffer (pH 6). The samples were loaded, and the columns were washed with 12.5 ml of buffer containing 0.25 M NaCl to remove protein A fragments and any betaglycan segments without GAG chains. Peptides containing chains were eluted with 2.5 ml of buffer containing 1 M NaCl and precipitated by adding 4 volumes of ethanol.

Step 3: -Elimination

The precipitated glycopeptides were resuspended in 0.5 M NaOH containing 1 M sodium borohydride (0.1 ml) and incubated at 4 °C for 24 h to liberate any O-linked GAG or oligosaccharide chains. After -elimination, the samples were neutralized with acetic acid and diluted 500-fold with water.

Step 4: DEAE-Sephacel Chromatography

The -eliminated material was subjected to another round of chromatography on DEAE-Sephacel to separate small oligosaccharides from large GAG chains. The flow-through (50 ml) and 0.25 M NaCl wash fraction (10 ml) were collected. The GAG chains were eluted with 2.5 ml of 1 M NaCl buffer and precipitated with ethanol.

Step 5: AG1-X2 Anion-exchange Chromatography

The flow-through sample was loaded on a 0.5-ml column of AG1-X2 resin (200-400 mesh, Bio-Rad, Hercules, CA) equilibrated with water. The column was eluted stepwise with 5 ml each of water, 0.015, 0.05, 0.1, 0.15, 0.2, and 1 M ammonium acetate (pH 7.0). Each fraction was collected and lyophilized.

Step 6: TSK-G2500 Gel Filtration Chromatography

Samples collected from AG1-X2 were analyzed by gel filtration high performance liquid chromatography (Progel TSK-G2500, 300 7.8 mm, inner diameter, Toso Haas, Montgomeryville, PA). The column was equilibrated in 0.5 M pyridinium acetate buffer (pH 5.0), and radioactivity in the effluent was detected by in-line liquid scintillation counting (Packard Instrument Company, Downers Grove, IL). The column was calibrated with tri-, tetra-, penta- and hexasaccharide standards prepared from chondroitin sulfate as described above. Material that migrated in the position of the pentasaccharide standard was collected for further analysis.

Compositional Analysis

[6-H]GlcN-labeled material from the mutant was acid hydrolyzed for 4 h at 100 °C as described by Hardy et al.(1988). A sample was mixed with standard GlcN and GalN (10 nmol) and applied to a CarboPac PA-1 column (Dionex Corp) equilibrated with 16 mM NaOH. The column was run at 1 ml/min, and 0.5-ml fractions were collected. The elution position of sugar standards was monitored by pulsed amperometric detection. The radiolabeled sugars were quantitated by liquid scintillation spectrometry, and their elution positions were compared with that of the sugar standards.

Heparin Lyase III Digestion

An aliquot of the intermediate labeled either with [6-H]GlcN or [6-H]Gal was dissolved in 50 µl of 20 mM Tris-HCl buffer (pH 7.0) containing 1 mM CaCl and 10 milliunits of heparin lyase III (Seikagaku, heparitinase I) and incubated at 37 °C overnight. The enzyme was inactivated by boiling the sample for 10 min. Samples were filtered through a microcon 10 microconcentrator (Amicon, Inc., Beverly, MA), and the filtrates were applied to the CarboPac PA-1 column.

The CarboPac PA-1 column was equilibrated in 0.1 M NaOH and calibrated with 10 cpm of each linkage region standards. The samples and standards were eluted with a gradient of sodium acetate as described by Shibata et al.(1992). Fractions (1 ml) were collected at a flow rate of 1 ml/min, and the radiolabeled sugar was quantitated by liquid scintillation spectrometry.


RESULTS

Intermediate Purification

We have described a mutant designated pgsD-677 that lacks the -GlcNAc transferase involved in the polymerization of heparan sulfate chains (Lidholt et al., 1992). Enzymatic studies showed that the mutant transferred -GlcNAc residues to a disaccharide resembling the terminal sugars of the linkage region (GlcA1-3Gal-). This finding suggested that cells use two -GlcNAc transferases to assemble heparan sulfate, one to initiate the chain (-GlcNAc transferase I) and another to polymerize the chain (-GlcNAc transferase II) (Fritz et al., 1994). If this idea is correct, then pgsD-677 cells should accumulate an intermediate consisting of -GlcNAc attached to the linkage tetrasaccharide (GlcNAc1-4GlcA1-3Gal1-3Gal1-4Xyl-).

Attempts to identify oligosaccharide intermediates on native core proteins in the mutant did not meet with success because their concentration was too low, and O-linked and N-linked oligosaccharides interfered with the analyses (data not shown). To circumvent this problem, a fusion protein was made consisting of a 45-amino acid segment of betaglycan fused by its N terminus to the 286-amino acid IgG-binding domain of Staphylococcal protein A and the signal peptide from the secreted metalloprotease, transin. This segment of betaglycan (residues 518-562) has two GAG attachment sites at Ser and Ser (Zhang and Esko, 1994; López-Casillas et al., 1994). The site at Ser primes both heparan sulfate and chondroitin sulfate, whereas the site at Ser primes only chondroitin sulfate (Zhang and Esko, 1994; López-Casillas et al., 1994). Mutant and wild-type cells were transiently transfected with the construct and labeled with [6-H]GlcN. Chimeras secreted into the growth medium were purified by affinity chromatography using IgG-agarose (see ``Experimental Procedures''). Both cell lines glycosylated the chimeras, but the amount of [6-H]GlcN-labeled material was higher in wild-type cells than in the mutant (). This result was not unexpected since the mutant makes chondroitin sulfate, whereas the wild-type makes both chondroitin sulfate and heparan sulfate and most of the label was found in the repeating disaccharide units of the GAG chains. In contrast, comparable amounts of material was made in mutant and wild-type cells labeled with [6-H]Gal. This precursor labels the internal Gal residues found in the linkage region, suggesting that the chimeras expressed by mutant cells might contain linkage fragments.

Analysis of SO-labeled chimeras by SDS-PAGE (Fig. 1) revealed that wild-type cells produced sulfated material that separated into two broad bands, typical of proteins containing variably sized GAG chains (lane3). The upper, more slowly migrating material consisted of chimeras containing two GAG chains since none was made in chimeras containing only one attachment site.() Digesting the samples with chondroitinase ABC (lane4) or heparin lyase III (lane5) collapsed the upper band from wild-type cells, indicating that most of these chimeras contained one chondroitin sulfate and one heparan sulfate chain. Samples treated with both enzymes yielded only a residual core protein (45 kDa) that labeled with SO due to sulfate-containing linkage fragments resistant to enzyme digestion (lane6). The chimeras expressed in pgsD-677 cells mostly contained one chondroitin sulfate chain (lane1), and all of the material collapsed into the 45-kDa range after chondroitinase ABC treatment (lane2). The reduced amount of chimeras containing two GAG chains in the mutant suggested that some molecules containing a chondroitin sulfate chain might bear an oligosaccharide intermediate at the site where heparan sulfate assembled in wild-type cells.


Figure 1: SDS-PAGE of [S]betaglycan chimeras. Mutant and wild-type cells were transfected with the betaglycan chimera (see ``Experimental Procedures'') and labeled with SO. Chimeras were purified by IgG-affinity chromatography and dissolved in SDS-PAGE protein sample buffer. The samples were analyzed on SDS-polyacryamide (5-16%) gradient gels. The positions of chimeras containing one and two GAG chains are indicated to the right of the gel, and the positions of protein molecular weight standards are indicated to the left. Each lane contained the chimera with the insert VVYYNSIVVQAPSPGDSSGWPDGYEDLESGDNGFPGDGDEGETAP derived from betaglycan (López-Casillas et al., 1991). Lane1, chimera expressed in pgsD-677 cells; lane2, chimera from pgsD-677 cells after treatment with chondroitinase ABC; lane3, chimera expressed in wild-type cells; lane4, chimera from wild-type after treatment with chondroitinase ABC; lane5, chimera from wild-type cells after treatment with heparin lyase III; lane6, chimera from wild-type cells after treatment with both chondroitinase ABC and heparin lyase III.



A purification scheme for finding intermediates was devised (Fig. 2). Chimeras were first affinity purified from cells labeled with [6-H]Gal or [6-H]GlcN. They were then treated with trypsin to remove the protein A domain. This region contains potential N-linked and O-linked glycosylation sites that might interfere with the identification of linkage region intermediates. The peptide derived from betaglycan does not contain Arg or Lys residues, and the closest cleavage site was 5 amino acids into the protein A domain (Nilsson et al., 1985). Thus, trypsin should have generated a fragment containing 45 amino acids from betaglycan and 5 amino acids from protein A.


Figure 2: Purification scheme for oligosaccharides.



Next, the mixture was separated by DEAE-Sephacel chromatography to select fragments containing a GAG chain. This step ensured that any oligosaccharides found subsequently would have been made on fragments that also supported the assembly of a complete GAG chain. Treating this material with alkali liberated the O-linked chains. A second round of chromatography on DEAE-Sephacel separated the GAG chains from neutral or weakly charged oligosaccharides. The latter material was then separated by strong anion-exchange chromatography on a column of AG1-X2 resin (see ``Experimental Procedures''). Prior calibration of the column with linkage region fragments showed that oligosaccharides containing a charge of -1 or -2 would bind to the column and elute with 50 mM ammonium acetate. Material eluting under these conditions was used for the analytical studies described below.

Analysis of the Intermediates

Gel filtration chromatography showed that some of the oligosaccharides labeled with [6-H]GlcN from mutant cells migrated in the same position as a pentasaccharide standard derived from authentic chondroitin sulfate (Fig. 3A). Lesser amounts of tetrasaccharide and trisaccharide were also present. In contrast, wild-type cells contained mostly short oligosaccharides and little if any material migrating like pentasaccharides (Fig. 3B). Another aliquot was analyzed by chromatography on a CarboPac PA-1 column under alkaline conditions (Shibata et al., 1992). Most of the labeled oligosaccharides from the mutant eluted like the pentasaccharide standard, except that its position was shifted by one fraction (Fig. 4A). Wild-type cells did not produce any material migrating in this position (Fig. 4B).


Figure 3: An unique oligosaccharide exists in pgsD-677. Chimeras containing two GAG sites were labeled with [6-H]GlcN and purified from mutant and wild-type cells. An aliquot of material that eluted with 50 mM ammonium acetate from the AG1-X2 column was analyzed by TSK-G2500 gel filtration chromatography (see ``Experimental Procedures''). The fractions indicated by shading were pooled for further analysis. A, mutant; B, wild-type; 3, Gal1-3Gal1-4xylitol; 4, GlcA1-3Gal1-3Gal1-4xylitol; and 5, GalNAc1-4GlcA1-3Gal1-3Gal1-4xylitol.




Figure 4: A pentasaccharide exists in pgsD-677 mutant but not in wild-type cells. An aliquot of material purified by AG1-X2 chromatography was analyzed on a CarboPac PA-1 column (Shibata et al., 1992). The amount of radioactivity in each fraction was monitored. A, pgsD-677; B, wild-type; 3, Gal1-3Gal1-4xylitol; 4, GlcA1-3Gal1-3Gal1-4xylitol; and 5, GalNAc1-4GlcA1-3Gal1-3Gal1-4xylitol.



The putative pentasaccharide intermediate represented 0.1% of the starting material from chimeras labeled with [6-H]GlcN and 2% of material labeled with [6-H]Gal. The labeling of the pentasaccharide with [6-H]GlcN suggested that it contained GlcNAc, but some GalNAc may have been present since GalNAc can be made from GlcNAc through epimerization of the corresponding nucleotide sugars (Kingsley et al., 1986). Acid hydrolysis, however, released 98% [H]GlcN and less than 2% [H]GalN (Fig. 5).


Figure 5: Acid hydrolysis liberates [H]GlcN. [6-H]GlcN-labeled oligosaccharide from the mutant was hydrolyzed in acid and analyzed by CarboPac PA-1 chromatography. The arrows indicate the position of authentic GlcN and GalN standards mixed with the sample and detected by pulsed amperometric detection.



Fractions containing pentasaccharides were pooled from gel filtration (Fig. 3, shadedarea). This material migrated on the Carbopac PA-1 column like the major oligosaccharide peak found in the material prior to gel filtration (Fig. 4). The pentasaccharide was resistant to -hexosaminidase (data not shown), suggesting that the GlcNAc residue was not -linked or that it was inaccessible. When [6-H]GlcN-labeled material was treated with 10 milliunits of heparin lyase III, [6-H]GlcNAc was released (Fig. 6A). Heparin lyase III recognizes N-acetyl or N-sulfated GlcN residues linked -(1-4) to GlcA in heparan sulfate chains (Nader et al., 1990). The reaction normally liberates disaccharides with a nonreducing terminal -D--unsaturated glucopyranosyluronic acid. Apparently, the enzyme also acts on the terminal GlcNAc residues. Treating [6-H]Gal-labeled intermediate with heparin lyase III should liberate radiolabeled tetrasaccharide with a unsaturated glucopyranosyluronic acid at the nonreducing end (-GlcA1-3[H]Gal1-3[H]Gal1-4xylitol). Analysis of the products of the reaction showed a single radiolabeled product that migrated exactly at the position of standard tetrasaccharide on the CarboPac PA-1 column (Fig. 6B). Together, these data indicated that the pentasaccharide accumulating in pgsD-677 cells had the structure GlcNAc1-4GlcA1-3Gal1-3Gal1-4Xyl.


Figure 6: Heparin lyase III digestion demonstrates that the pentasaccharide in the mutant is GlcNAc1-4GlcA1-3Gal1-3Gal1-4xylitol. The pooled pentasaccharides labeled with [6-H]GlcN or [6-H]Gal were applied to a CarboPac PA-1 column before () or after () heparin lyase III digestion. Fractions were monitored for radioactivity. A, samples labeled with [6-H]GlcN; B, samples labeled with [6-H]Gal; 4, GlcA1-3Gal1-3Gal1-4xylitol; 5, GalNAc1-4GlcA1-3Gal1-3Gal1-4xylitol.




DISCUSSION

The GAG chains of proteoglycans assemble by the stepwise addition of monosaccharides to the nonreducing terminus of nascent chains (Rodén, 1980). The sequential addition of sugars suggests that discrete oligosaccharide intermediates should exist in vivo. Vertel et al.(1994) found xylose on a truncated aggrecan precursor that fails to be secreted. However, more complex intermediates have not been detected on core proteins, possibly because of their rapid and efficient conversion to full-length chains.

Intermediates accumulate when GAG synthesis is altered by drugs. Spiro et al.(1991) characterized accumulated Gal1-3Gal1-4Xyl stubs on a chondroitin sulfate proteoglycan core protein in M21 human melanoma cells after blocking chondroitin sulfate elongation with brefeldin A. Takagaki et al.(1991) and Freeze and co-workers (Freeze et al., 1993; Manzi et al., 1995; Salimath et al., 1995) have characterized several oligosaccharides primed on -D-xylosides, including Gal1-4Xyl, Gal1-3Gal1-4Xyl, GlcA1-4Xyl, NeuAc2-3Gal1-4Xyl, and GalNAc1-4GlcA1-3Gal1-3Gal1-4Xyl, although only the first two lie along the pathway of GAG synthesis. The accumulation of these compounds may reflect detoxification reactions (glucuronidation) or utilization of alternate substrates by enzymes belonging to other biosynthetic pathways.

Mutants provide another way to find intermediates, since they should accumulate metabolites upstream from the block imposed by the mutation. Quentin et al.(1990) showed that xylose residues occur on a natural proteoglycan core protein in human fibroblasts deficient in galactosyltransferase I. The accumulation of the pentasaccharide, GlcNAc1-4GlcA1-3Gal1-3Gal1-4Xyl1-, in a mutant altered in heparan sulfate polymerization suggests that the assembly of chains normally goes through this intermediate. Its absence in wild-type cells suggests that the conversion of pentasaccharide to heparan sulfate occurs rapidly and quantitatively under normal conditions. Because the mutant is defective in chain polymerization (Lidholt et al., 1992), the presence of -GlcNAc-terminated pentasaccharide provides another piece of evidence that a unique -GlcNAc transferase catalyzes the addition of the first -GlcNAc residue (Fritz et al., 1994).

The pentasaccharide that accumulates in the mutant does not contain phosphate at C-2 of xylose (Oegema et al., 1984; Fransson et al., 1985; Shibata et al., 1992), sulfate at C-4 or C-6 of the Gal residues (Sugahara et al., 1988; de Waard et al., 1992), or sulfate on the -GlcNAc residue (Sugahara et al., 1992a). Hexasaccharides from chondroitin sulfate containing these modifications would have eluted from the AG1-X2 column with 1 M ammonium acetate. Thus, if phosphorylation or sulfation had occurred, we would have detected the modified oligosaccharides. The lack of phosphorylation and sulfation is not surprising since it does not occur universally or stoichiometrically in chondroitin sulfate and heparan sulfate (Sugahara et al., 1988; de Waard et al., 1992; Fransson et al., 1990; Shibata et al., 1992; Sugahara et al., 1992a, 1992b).

The chimera used in this study makes 40% heparan sulfate and 60% chondroitin sulfate in wild-type cells, and about one-half of the material contains two chains (Fig. 1). Therefore, the amount of pentasaccharide (plus tetrasaccharide and trisaccharide) should have represented 40% of material labeled with [6-H]Gal in pgsD-677 cells instead of only 2% (). Several possibilities may explain this discrepancy. Core proteins with immature chains may be degraded in the endoplasmic reticulum/Golgi by proteolysis (Su et al., 1993), by endolytic cleavage of the carbohydrate chains (Villers et al., 1994), or by removal of the terminal -GlcNAc residue by an unknown mechanism. Addition and removal of sugars occurs normally during the processing of N-linked oligosaccharides (Kornfeld and Kornfeld, 1985), in the modification of cytosolic proteins (Kearse and Hart, 1991), and in the sorting of lysosomal glycoproteins (Reitman and Kornfeld, 1981; Varki and Kornfeld, 1980). Additional experiments are needed to determine if linkage fragments from GAG chains also undergo dynamic processing reactions.

  
Table: Yield of [6-H]GlcN or [6-H]Gal-labeled oligosaccharides in wild-type and mutant pgsD-677 cells

Transfected cells were labeled with [6-H]GlcN or [6-H]Gal, and the chimeras were purified by IgG-agarose affinity chromatography (see ``Experimental Procedures''). The recovery of radioactive material through the various preparation steps was measured. The data represent a typical experiment (n = 3).



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 205-934-6034; Fax: 205-975-2547; E-mail: jesko@bmg.bhs.uab.edu.

The abbreviations used are: GAG, glycosaminoglycan; PAGE, polyacrylamide gel electrophoresis; GlcA, D--unsaturated glucopyranosyluronic acid.

L. Zhang, G. David, and J. D. Esko, submitted for publication.


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