Chinese Hamster Ovary Cell Mutants Defective in Glycosaminoglycan Assembly and Glucuronosyltransferase I*

Xiaomei Bai, Ge WeiDagger , Anjana Sinha, and Jeffrey D. Esko§

From the Division of Cellular and Molecular Medicine, Glycobiology Program, University of California, San Diego, La Jolla, California 92093-0687

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The proteoglycans of animal cells typically contain one or more heparan sulfate or chondroitin sulfate chains. These glycosaminoglycans assemble on a tetrasaccharide primer, -GlcAbeta 1,3Galbeta 1,3Galbeta 1,4Xylbeta -O-, attached to specific serine residues in the core protein. Studies of Chinese hamster ovary cell mutants defective in the first or second enzymes of the pathway (xylosyltransferase and galactosyltransferase I) show that the assembly of the primer occurs by sequential transfer of single monosaccharide residues from the corresponding high energy nucleotide sugar donor to the non-reducing end of the growing chain. In order to study the other reactions involved in linkage tetrasaccharide assembly, we have devised a powerful selection method based on induced resistance to a mitotoxin composed of basic fibroblast growth factor-saporin. One class of mutants does not incorporate 35SO4 and [6-3H]GlcN into glycosaminoglycan chains. Incubation of these cells with naphthol-beta -D-xyloside (Xylbeta -O-Np) resulted in accumulation of linkage region intermediates containing 1 or 2 mol of galactose (Galbeta 1, 4Xylbeta -O-Np and Galbeta 1, 3Galbeta 1, 4Xylbeta -O-Np) and sialic acid (Siaalpha 2,3Galbeta 1, 3Galbeta 1, 4Xylbeta -O-Np) but not any GlcA-containing oligosaccharides. Extracts of the mutants completely lacked UDP-glucuronic acid:Galbeta 1,3Gal-R glucuronosyltransferase (GlcAT-I) activity, as measured by the transfer of GlcA from UDP-GlcA to Galbeta 1,3Galbeta -O-naphthalenemethanol (<0.2 versus 3.6 pmol/min/mg). The mutation most likely lies in the structural gene encoding GlcAT-I since transfection of the mutant with a cDNA for GlcAT-I completely restored enzyme activity and glycosaminoglycan synthesis. These findings suggest that a single GlcAT effects the biosynthesis of common linkage region of both heparan sulfate and chondroitin sulfate in Chinese hamster ovary cells.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The assembly of the glycosaminoglycans (GAGs),1 heparan sulfate and chondroitin sulfate, initiates by the transfer of xylose to specific serine residues in core proteins, which then gives rise to the so-called linkage tetrasaccharide, GlcAbeta 1,3Galbeta 1,3Galbeta 1,4Xylbeta -O-Ser (1). In some proteoglycans, the linkage tetrasaccharide undergoes phosphorylation at C-2 of xylose (2-6) or sulfation at C-4 of the galactose residues (5, 7-13). The linkage tetrasaccharide serves as a primer for the formation of heparan sulfate and chondroitin sulfate chains, which initiate by the attachment of either GlcNAc or GalNAc, respectively (14, 15). The chains then assemble by the alternating addition of GlcA and GlcNAc (heparan sulfate) or GlcA and GalNAc (chondroitin sulfate). During polymerization, the chains undergo a series of modification reactions that include N-deacetylation and N-sulfation of GlcNAc residues, epimerization of some GlcA residues to iduronic acid, and addition of sulfate groups at various positions (1). The arrangement of sulfated and non-sulfated residues and the uronic acid epimers gives rise to specific binding sites for various protein ligands (reviewed in Refs. 16-19).

Several of the enzymes involved in glycosaminoglycan assembly have been purified and cloned, and some of them appear to be members of families of related genes. For example, at least three different GlcNAc N-deacetylase/N-sulfotransferases (20-24), three GlcNAc 6-O-sulfotransferases (25),2 and as many as five 3-O-sulfotransferases exist (26-28). Recent reports claim that multiple copolymerases forming the heparan sulfate backbone may exist as well (29, 30). Multiple isozymes make possible tissue-specific and developmentally regulated expression of the corresponding enzyme activities. Since the enzymatic properties of the isozymes may differ in subtle ways (27), their selective expression may be responsible for unique saccharide sequences found in different tissues (31-37).

In contrast to the amount of information available for these late reactions, relatively little is known about the structure and expression of the transferases that act early in the pathway (1). Mutational studies of GAG assembly in Chinese hamster ovary cells have shown that mutants in the first two steps (catalyzed by xylosyltransferase (XylT) and galactosyltransferase I (GalT-I)) fail to make both heparan sulfate and chondroitin sulfate chains (38-40). This finding suggests that a single set of enzymes catalyze the biosynthesis of the linkage region, without regard to the eventual composition of the chain. To obtain information about other enzymes involved in linkage region formation, we have developed a powerful selection and screening method to find mutants in these reactions. One new class of mutants does not synthesize glycosaminoglycan chains due to a deficiency of glucuronosyltransferase I (GlcAT-I). Like mutants defective in XylT or GalT-I, the new mutants fail to make both heparan sulfate and chondroitin sulfate, suggesting that each of the early steps in linkage region formation is catalyzed by a single isozyme.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Cultures-- Chinese hamster ovary cells (CHO-K1) were obtained from the American Type Culture Collection (CCL-61, Rockville, MD). Mutants pgsA-745 (xylosyltransferase-deficient) and pgsB-761 (galactosyltransferase I-deficient) were characterized previously (38, 39). Cells were grown under an atmosphere of 5% CO2 in air and 100% relative humidity in Ham's F-12 growth medium (Life Technologies, Inc.) supplemented with 7.5% (v/v) fetal bovine serum (HyClone Laboratories), 100 µg/ml streptomycin sulfate, and 100 units/ml penicillin G. Defined F-12 medium was prepared from individual components (41), substituting chloride salts for sulfate, and supplemented with fetal bovine serum that had been dialyzed 106-fold against phosphate-buffered saline (PBS) (42). Low glucose medium containing 1 mM instead of 10 mM glucose was used for labeling cells with radioactive sugars. Deprivation of the cells for sulfate and glucose in this way had no effect on GAG composition but raised the level of radiolabeling by 10-fold or more.

Mutant Screening-- Wild-type CHO cells were mutagenized with ethylmethanesulfonate and frozen under liquid nitrogen (43). A portion of cells was thawed, propagated for 3 days, and used to screen for mutants. Approximately 1 × 105 mutagen-treated cells were added to multiple 100-mm diameter tissue culture dishes filled with complete growth medium containing 2 µg/ml FGF2-SAP, a recombinant chimera consisting of FGF2 fused to the plant cytotoxin saporin-6 (Selective Genetics, Inc., La Jolla, (44). After 1 day, the cells were overlaid with a stack of polyester disks and glass beads in order to generate colony replicas (43). The plates were incubated at 37 °C, and the FGF2-SAP-containing medium was changed every 4 days. Under these conditions, 50-100 colonies arose on each plate after ~10 days. The disks were then removed from the plates and transferred to a fresh plate containing sulfate-deficient medium supplemented with 10 µCi/ml 35SO4. After 4 h incubation at 37 °C, radiolabeled proteoglycans in each colony were precipitated by incubating the disks in a few milliliters of 10% (w/v) trichloroacetic acid. The disks were washed by replacing the solution three times with 2% trichloroacetic acid and then water. After staining the colonies with Coomassie Blue (43), the disks were dried and exposed to x-ray film (Fuji). Tentative mutants were identified as blue-stained colonies that failed to incorporate 35SO4. Candidates were picked from the original master plates using cloning cylinders and trypsin (43). While the disks were being screening, the master plates were stored at 33 °C filled with complete F-12 growth medium supplemented with 2.5 µg/ml Fungizone and 10 units/ml Nystatin (Life Technologies, Inc.).

Complementation Assay-- Complementation tests were carried out by cell hybridization (43). Approximately 2 × 105 cells of each strain were added to individual wells of a 24-well plate along with an equal number of pgsA-745 or pgsB-761 cells. After overnight incubation, the mixed cell monolayers were treated for 1 min with 50% (w/w) polyethylene glycol (Mr = 3350) in F-12 medium. The cells were incubated for 1 day, replated in 100 mm-diameter tissue culture dishes to obtain ~300 colonies per dish, and then overlaid with polyester cloth. The replica-plated colonies were incubated with 35SO4 as described above. Complementation was assessed by the appearance of colonies that regained the capacity to incorporate 35SO4 into acid-precipitable material, as judged by autoradiography.

Purification of Glycosaminoglycan Chains-- Cells were labeled for 24 h with 10 µCi/ml Na35SO4 (25-40 Ci/mg, NEN Life Science Products) in growth medium containing serum or with 10 µCi/ml D-[6-3H]glucosamine HCl (40 Ci/nmol, NEN Life Science Products) in low glucose medium. Radiolabeled GAG chains were isolated as described (45) and analyzed by anion-exchange high pressure liquid chromatography using a 7.5-mm diameter × 7.5-cm column of DEAE-3SW (Tosohaas, Montgomeryville, PA). The column was equilibrated in 10 mM KH2P04 buffer (pH 6.0) containing 0.2% (w/v) Zwittergent 3-12 and 0.2 M NaCl. GAGs were eluted with a linear gradient of NaCl (0.2-1 M) in the same buffer using a flow rate of 1 ml/min and by increasing the NaCl concentration by 10 mM/min. The effluent from the column was monitored for radioactivity with an in-line radioactivity detector (Radiomatic Flo one/beta, Packard Instruments) with sampling rates every 6 s and data averaged over 1 min.

Isolation of Metabolically Labeled Xyloside-primed Oligosaccharides-- CHO cells were grown to near confluence and then incubated with low glucose F-12 medium supplemented with different concentrations of naphthol-beta -D-xyloside (46) and 20 µCi/ml D-[6-3H]galactose (34.6 Ci/mmol) (NEN Life Science Products). After 4 h incubation, the medium was collected, centrifuged to remove floating cells, adjusted to 0.5 M NaCl, and applied to Sep-Pak Vac C18-cartridges (Waters, 100 mg). The cartridges were washed with 10-25 ml of deionized water, and bound material was eluted with 50% methanol and dried. The cell layers were rinsed three times with PBS and lysed in 0.1 N NaOH. An aliquot was taken for protein assay using a kit (Bio-Rad) with bovine serum albumin as standard.

Separation and Characterization of Neutral and Charged Products-- The dried samples were resuspended in 2 mM Tris base and separated on a 0.5-ml column of QAE-Sephadex (Amersham Pharmacia Biotech) equilibrated with 2 mM Tris base and packed into a disposable 1-ml pipette tip. The flow-through and wash fraction (10 ml of 2 mM Tris base) were collected as neutral products (47), and the bound material (charged products) was eluted with 0.4 M NaCl in 2 mM Tris base. The fractions were desalted and concentrated on Sep-Pak C18 cartridges.

The oligosaccharides were characterized by treating samples with various enzymes at 37 °C in a final volume of 25-100 µl. Neutral species were incubated for 4 and 24 h with 5 milliunits of beta -galactosidase (bovine testes, Oxford Glycosystems) in 0.1 M citrate/phosphate buffer (pH 4.0). Charged materials were treated with (i) 10 milliunits of sialidase (Arthrobacter ureafaciens, Oxford Glycosystems) for 16 h in 100 mM sodium acetate (pH 5.0); some samples were desialylated by boiling in 10 mM HCl for 30 min; (ii) 100 units of beta -glucuronidase (bovine liver, Sigma) overnight in 100 mM sodium acetate (pH 5.0) buffer containing 0.3 M NaCl and 8 mM D-galactonic acid gamma -lactone to inhibit contaminating beta -galactosidase activity; and (iii) 1 milliunit of alpha -N-acetylgalactosaminidase (chicken liver, Oxford Glycosystems) overnight in 0.1 M citrate/phosphate buffer (pH 4.0). In some samples, the incubation was adjusted to pH 5.0 and incubated with beta -glucuronidase as described above. After enzymatic treatment, samples were boiled for 10 min, dissolved in 200 µl of water, and injected into a reverse phase C18 column (Tosohaas RP18, 4.6 mm × 25 cm) connected to Rainin HPXL solvent delivery system. The column was first washed with water (flow rate 0.5 ml/min) and then with increasing concentrations of acetonitrile in water. Radioactivity in the eluate was monitored by Radiomatic Flo-one Beta detector connected in line to the column. Some samples were analyzed by QAE-Sephadex anion-exchange chromatography

Biotinylation of FGF2-- FGF2 (0.5 mg, Escherichia coli recombinant material, Selective Genetics, Inc.) was protected with heparin (0.4 mg) in 0.2 M HEPES buffer (pH 8.4) and mixed with biotin hydrazide (Pierce Chemical, Long arm, water-soluble, 40 µg) in a final volume of 0.2 ml. After 2 h at room temperature, 40 µl of 10 mg/ml glycine solution was added to stop the reaction. The sample was diluted with 30 ml of 20 mM HEPES buffer (pH 7.4) containing 0.5 M NaCl and 0.2% bovine serum albumin and loaded onto a 1-ml column of heparin-Sepharose CL-6B (Amersham Pharmacia Biotech). The column was washed with 30 ml of buffer and eluted with 2.5 ml of solution adjusted to 3 M NaCl. The sample was desalted on a PD-10 column (Amersham Pharmacia Biotech) equilibrated with 20 mM HEPES buffer (pH 7.4) containing 0.2% bovine serum albumin. Inclusion of 125I-FGF2 (~4 × 105 cpm, Ref. 48) showed that the overall recovery of material was 30-40%.

Stable Transfection of Glucuronosyltransferase I-- A cDNA for glucuronosyltransferase I (GlcAT-I) was cloned from CHO cells and inserted into pCDNA3 (49). Mutant cells were transfected using Lipofectin (Life Technologies, Inc.) according to the manufacturer's instructions, and stable transfectants were selected with geneticin (G418). Drug-resistant colonies were isolated and further characterized by measuring the binding of biotinylated FGF2 in the following way. Cells were detached with 5 mM EDTA in PBS, washed twice with 10 ml of PBS, and resuspended in 0.2 ml of F-12 medium containing ~0.5 µg/ml biotin-FGF2 and 1% bovine serum albumin. The cells were incubated for 1 h at 4 °C with constant shaking, washed twice with 0.5 ml of cold 20 mM NaH2PO4 (pH 7.4) buffer containing 150 mM NaCl, and resuspended in 0.2 ml of buffer containing 5 µg/ml fluorescein/avidin/DCS (Vector Laboratories). After shaking the cells in the dark at 4 °C for 20 min, they were washed and resuspended in cold phosphate buffer. Cell sorting was done on a FACS Star Sorter (Becton Dickinson) and strongly positive clones were picked for further analysis.

Northern Analysis-- mRNA was isolated from mutant and wild-type cells using QuickPrep Micro mRNA Purification Kit (Amersham Pharmacia Biotech). The mRNA was denatured at 65 °C in a solution of 50% (v/v) formamide, 6% (v/v) formaldehyde, and 20 mM MOPS (pH 7.0) and separated on a 1.2% (w/v) agarose gel containing 6% (v/v) formaldehyde. The gels were blotted for 18 h onto a Nytran plus nylon membrane (Schleicher & Schuell). The blotted RNA was fixed by UV-catalyzed cross-linking and prehybridized for 2 h at 42 °C in a solution containing 50% formamide, 20 mM sodium phosphate (pH 6.8), 5× SSC, 1× Denhardt's solution, 1% SDS, 5% dextran sulfate, and 100 µg/ml denatured salmon sperm DNA. A double-stranded DNA probe was labeled with [32P]dCTP with random oligonucleotide primers (Prime-IT II labeling kit, Stratagene) using the GlcAT-I cDNA (1.5 kilobase pairs) as template (49), and the probe was purified on an Elute-tip (Schleicher & Schuell). Hybridization was carried out overnight at 42 °C in the same buffer as the prehybridization but containing ~1 × 106 cpm/ml 32P-labeled probe. The membrane was washed twice for 30 min at 42 °C with 2× SSC containing 0.1% SDS and then twice for 30 min at 65 °C with 0.2× SSC and 0.1% SDS. Bound probe was visualized on a PhosphorImager (Storm 860, Molecular Dynamics) after overnight exposure.

Glucuronosyltransferase I Assay-- The activities of GlcAT-I was measured in crude cell-free homogenates. Cells were grown to confluence, rinsed three times with cold PBS, and detached with a rubber policeman in 50 µl of solution containing 0.25 M sucrose, 50 mM Tris-HCl (pH 7.4), 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 1 mM phenylmethylsulfonyl fluoride. Aliquots of the cell extracts were stored at -20 °C. The donor, UDP[1-3H]glucuronic acid, and the substrate, Galbeta 1,3Galbeta -O-naphthalenemethanol, were synthesized as described (49).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Selection of Glycosaminoglycan-deficient Mutants-- We have identified previously a number of CHO cell mutants altered in GAG biosynthesis using a "brute force" replica plating technique (43). In this procedure, cell colonies were grown on plastic tissue culture dishes in the presence of an overlaying disk of polyester cloth. Colonies forming on the plate also developed on the cloth, thus providing two copies of each colony. The cloth "replica" was then incubated in growth medium containing 35SO4, and incorporation of 35SO4 into proteoglycans was measured by autoradiography after acid precipitating radioactive proteoglycans on the disk. Binding of 125I-FGF2 or 125I-antithrombin to colonies could be measured as well, providing a way to obtain mutants in steps that affect sulfation of the chains (48, 50). Using these techniques, mutants altered in linkage region assembly (designated pgsA and pgsB), sulfate transport (pgsC), heparan sulfate polymerization (pgsD), and GlcN N-sulfation (pgsE), uronic acid 2-O-sulfation (pgsF), and GlcNAc 3-O-sulfation of heparan sulfate chains (38-40, 45, 48, 50-53). pgsA and pgsB mutants, defective in xylosyltransferase (XylT) and galactosyltransferase I (GalT-I), respectively, fail to make both chondroitin sulfate and heparan sulfate. Mutants in the second galactosylation step (GalT-II) and glucuronosyltransferase I (GlcAT-I), however, have not yet been identified, possibly due to their low incidence in mutagen-treated populations of cells.

In order to identify these rarer mutants, a higher capacity screening method was needed. Previous studies have shown that cells lacking fully sulfated heparan sulfate did not bind FGF2 (54, 55). Thus, heparan sulfate-deficient mutants should also be resistant to a recombinant mitotoxin composed of FGF2 and saporin-6, a type 1 ribosome-inactivating protein from Saponaria officinalis seeds (44, 56). To test if this idea was correct, wild-type CHO cells and GAG-deficient pgsA-745 cells were treated with different concentrations of the mitotoxin (FGF2-SAP). As shown in Fig. 1, wild-type CHO cells succumbed with an ED50 of ~1 µg/ml for the mitotoxin, whereas the GAG-deficient mutant pgsA-745 survived under these conditions.


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Fig. 1.   FGF2-saporin inhibition of cell growth. Mutant pgsA-745 and wild-type CHO cells were plated in a 96-well dish at 1 × 103 cells/well. Different concentrations of FGF2-saporin were added. After 10 days incubation, the cells were fixed with 10% trichloroacetic acid and stained with Coomassie Blue.

After chemical mutagenesis ("Experimental Procedures"), the incidence of FGF2-SAP-resistant mutants was ~10-4, which therefore makes possible direct selection of large numbers of mutants. Approximately 105 mutagen-treated cells were added to individual tissue culture plates, treated with 2 µg/ml FGF2-SAP, and overlaid with polyester cloth. The resulting colonies that arose on the replica disks were screened by 35SO4 incorporation into proteoglycans using the colony autoradiography procedure described above (see "Experimental Procedures") (43). Thirteen colonies showed dramatic loss of 35SO4 incorporation (data not shown) and had stable phenotypes after repurification by limiting dilution. Many more colonies exhibited partial reduction in 35SO4 incorporation presumably from defects in specific sulfotransferases or mutations that did not completely inactivate a particular enzyme. Only the mutants that showed dramatic reduction in 35SO4 incorporation were characterized further.

To sort the various strains into different groups, we tested the ability of the cells to prime oligosaccharides on naphthol-beta -D-xyloside added to the culture medium. Xylosides are known to stimulate GAG synthesis by substituting for the natural xylosylated core protein intermediates (57). Thus, providing xyloside to XylT-deficient pgsA-745 CHO cells stimulates GAG synthesis by bypassing the GAG deficiency in this strain (38). In contrast, providing xyloside to pgsB-761 cells does not stimulate GAG synthesis since these cells lack GalT-I that adds the next sugar in the linkage region (39). Thus, xylosides can be used to map if a defect in a mutant lies upstream or downstream of XylT. Of the 13 mutants identified above, 10 were responsive to xyloside treatment and three were not (Table I). The three unresponsive strains were then fused pair-wise to mutants pgsA-745 and pgsB-761 to test for genetic complementation. All three strains complemented both pgsA-745 and pgsB-761, indicating that the defects were downstream from GalT-I. When fused to each other, the mutants failed to complement, suggesting that they belonged to the same complementation group, which was formally designated pgsG. When metabolically labeled with both 35SO4 and [6-3H]glucosamine, all three strains failed to incorporate the precursors into GAG chains to the same extent as the pgsA mutant (Table I). Thus, the new mutants did not make either chondroitin sulfate or heparan sulfate, consistent with the idea that they have defects in a shared step early in the pathway.

                              
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Table I
GAG content of mutant and wild-type cells
Cells were metabolically labeled with either [6-3H]glucosamine or 35SO4 in the presence or absence of naphthol-beta -D-xyloside (Xylbeta -O-Np), and the GAG was isolated from the cells and the growth medium (see "Experimental Procedures"). The amount of radioactive material was normalized to the amount of cell protein analyzed.

The Oligosaccharides Assembled on beta -D-Xylosides Suggest a Defect in GlcA Addition-- In addition to priming mature GAG chains, many cell types accumulate biosynthetic intermediates on xylosides that resemble those made during chain assembly (58-64). Thus, knowing the composition of these intermediates could help pinpoint the enzymatic defect in the mutants. When incubated with 3 µM naphthol-beta -D-xyloside and [6-3H]galactose, mutant and wild-type cells secreted labeled oligosaccharides covalently linked to the primer. The amount of secreted material in all three pgsG mutants was approximately the same as in wild-type cells (Table II). As expected, the formation of the oligosaccharides was not dependent on XylT, since comparable amounts of material were recovered in pgsA-745 cells. However, it was entirely dependent on GalT-I, since pgsB cells, which lack the enzyme, did not accumulate any primed oligosaccharides.

                              
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Table II
Xyloside-stimulated oligosaccharide formation in mutant and wild-type cells
Cells were fed 3 µM naphthol-beta -D-xyloside and labeled with [6-3H]galactose for 4 h. The conditioned medium was passed through Sep-Pak C18 cartridges, and the oligosaccharides generated on the primer were eluted and separated into neutral and charged species by anion-exchange chromatography (see "Experimental Procedures").

Analysis of the material by anion-exchange chromatography revealed neutral and anionic oligosaccharides bearing one or two charges (see "Experimental Procedures"). The neutral species from both wild-type and mutant cells resolved into two peaks when analyzed by reverse phase-high pressure liquid chromatography (Fig. 2, top two panels). Both peaks were sensitive to overnight digestion with beta -galactosidase, with the counts shifting to the position of unbound [3H]galactose (Fig. 2, lower panel). However, the individual peaks differed significantly in their sensitivity to beta -galactosidase; peak II was completely digested after only 4 h, whereas peak I was only partially hydrolyzed in this time period. Some of the counts shifted to the same position as peak II, suggesting that peak I consisted of a galactosylated form of material in peak II. Since peak II comigrated exactly in the same position as authentic Galbeta 1,4Xylbeta -O-naphthol and was sensitive to beta -galactosidase, the findings suggested that peak I was most likely Galbeta 1,3Galbeta 1,4Xylbeta -O-naphthol. The actual linkages were not directly determined but inferred from the established structure of the linkage region tetrasaccharide (1). The proposed structure is also consistent with the relative resistance of Gal-linked beta 1,3 versus beta 1,4 (65). The presence of galactosylated oligosaccharides in the mutant indicated that the second galactosyltransferase (GalT II) was functioning normally in pgsG cells (Table III).


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Fig. 2.   Analysis of neutral oligosaccharides primed on naphthol-beta -D-xyloside. Cells were fed 3 µM naphthol-beta -D-xyloside and [6-3H]galactose (see "Experimental Procedures"). Radioactive oligosaccharides generated on the primer were concentrated from the growth medium and analyzed by reverse phase-high pressure liquid chromatography. Peak II comigrated with authentic Galbeta 1,4Xylbeta -O-naphthol. The individual peaks were collected and digested with beta -galactosidase for 4 h and rechromatographed. In the lower panel, the material from both peaks was combined and treated with beta -galactosidase overnight.

                              
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Table III
Oligosaccharides primed on naphthol-beta -D-xyloside
Cells were labeled with [6-3H]Gal, and the oligosaccharides were quantified (see Figs. 2-4 and "Experimental Procedures").

Approximately 50% of the xyloside-primed oligosaccharides bore a charge in pgsG mutants, somewhat less than found in wild-type cells (~70%).3 The charged oligosaccharides obtained from the mutant separated into two peaks of material after reverse phase chromatography (Fig. 3B). This material was completely sensitive to mild acid or sialidase (Fig. 3C), but it was resistant to beta -glucuronidase, alpha -N-acetylgalactosaminidase, or a combination of both enzymes (Fig. 3, D, E and F). Sialidase shifted the counts to material eluting in the position of the neutral trisaccharide (compare Fig. 3, A and C), suggesting the structure, Siaalpha 2,3Galbeta 1,3Galbeta 1,4Xylbeta -O-naphthol. The faster eluting, relatively minor peak in Fig. 3B also was sensitive to sialidase treatment, suggesting that this material may represent desialylated oligosaccharide.


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Fig. 3.   Analysis of charged oligosaccharides primed on naphthol-beta -D-xyloside by mutant cells. A, the distribution of neutral oligosaccharides for comparison; B, the elution position of charged oligosaccharides; C, after treatment with A. ureafasciens sialidase; D, after treatment with beta -glucuronidase; E, after treatment with alpha -N-acetylgalactosaminidase; and F, after treatment with both alpha -N-acetylgalactosaminidase and beta -glucuronidase. All of the charged material was sensitive to sialidase and resistant to the other enzymes.

Wild-type cells also generated charged oligosaccharides (Fig. 4B), but these were >95% resistant to sialidase treatment (and mild acid hydrolysis) (Fig. 4C). The material was also resistant to beta -glucuronidase (Fig. 4D) and alpha -N-acetylgalactosaminidase (Fig. 4E). However, it was sensitive to the combination of these two enzymes (Fig. 4F), resulting in the counts shifting to the position of the neutral trisaccharide (compare with Fig. 4A). These findings suggested that the majority of charged intermediates in the wild-type most likely had the structure, GalNAcalpha 1,3GlcAbeta 1,3Galbeta 1,3Galbeta 1,4Xylbeta -O-naphthol, which was described previously (62, 66-69). In contrast the mutant lacked this oligosaccharide. The proportion of neutral and charged oligosaccharides in mutant and wild-type cells is summarized in Table III. Taken together, the data suggested that the mutant probably lacked GlcAT-I.


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Fig. 4.   Analysis of charged oligosaccharides primed on naphthol-beta -D-xyloside by wild-type cells. A, the distribution of neutral oligosaccharides for comparison; B, the elution position of charged oligosaccharides; C, after treatment with A. ureafasciens sialidase; D, after treatment with beta -glucuronidase; E, after treatment with alpha -N-acetylgalactosaminidase; and F, after treatment with both alpha -N-acetylgalactosaminidase and beta -glucuronidase. Only a small amount of material was sensitive to sialidase. The majority of material was cleaved by a combination of alpha -N-acetylgalactosaminidase and beta -glucuronidase.

Deficient GlcAT-I in pgsG Mutants-- An optimized assay for GlcAT-I in CHO cell-free extracts was developed using synthetic Galbeta 1,3Galbeta -O-naphthalenemethanol as an acceptor (see "Experimental Procedures"). Enzyme activity was undetectable in the mutants compared with the wild-type (<0.2 pmol/min/mg in the mutant versus 3.7 pmol/min/mg in wild-type cells) (Table IV). When equal amounts of wild-type and mutant cell extract were assayed together, the enzyme-specific activity was 1.7 pmol/min/mg, exactly the amount predicted by simple mixing. This finding indicated that the mutant did not produce a soluble inhibitor that acted on wild-type GlcAT-I in the extracts. Likewise, the wild type did not seem to produce a soluble activator of the enzyme that might have been missing in the mutant.

                              
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Table IV
GlcAT-I activity in mutant and wild-type CHO cells
GlcAT-I activity was assayed in crude cell-free extracts using Galbeta 1, 3Gal-naphthalenemethanol as acceptor (see "Experimental Procedures"). Each value represents the average at least two independent determinations plus and minus the standard error from the mean.

Recently, GlcAT-I was cloned from human placenta and CHO cells (49, 70). One of the mutants was stably transfected with the CHO cDNA clone, and individual colonies were screened (see "Experimental Procedures"). Assay for enzyme activity in the transfectants showed that they contained normal levels of GlcAT-I (Table IV). As shown in Fig. 5, the synthesis of heparan sulfate and chondroitin sulfate was also restored and in this example to levels that were somewhat higher than wild type. Northern blot analysis of poly(A+) mRNA from mutant and wild-type cells showed that the level of transcript was comparable, suggesting that both transcription and message stability were comparable in mutant and wild-type cells (Fig. 6). These findings support the idea that the defect in GAG synthesis in pgsG cells was due to the loss of GlcAT-I and that the mutant most likely has a defect in the structural gene encoding the enzyme.


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Fig. 5.   Anion-exchange chromatography of GAG from cells. Mutant pgsG-110, wild-type, and mutant cells stably transfected with GlcAT-I cDNA were labeled with 35SO4. The 35S-GAG chains were isolated from proteoglycans in the medium and the cells and analyzed by anion-exchange high pressure liquid chromatography (see "Experimental Procedures"). open circle , mutant pgsG-110; triangle , GlcAT-I transfected pgsG-110; , wild-type cells.


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Fig. 6.   Northern blot analysis of the GlcAT-I in pgsG mutants and wild-type CHO cells. ~5 µg of poly(A+) RNA from each strain was electrophoresed and blotted to nitrocellulose ("Experimental Procedures"). A, hybridization results using 32P-labeled full-length GlcAT-I cDNA as probe; B, hybridization with the gamma -glyceraldehyde-3-phosphate dehydrogenase (gamma -GADPH) probe. Kb, kilobase pair.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

FGF2-SAP Selection Yields New GAG-deficient Mutants-- In previous studies, we identified GAG-deficient mutants of CHO cells using a replica plating technique in which individual colonies were screened for failure to incorporate sulfate into GAG chains or by altered binding of 125I-FGF2 (38-40, 45, 48, 51-53). Although this technique has yielded interesting mutants, replica plating has a relatively low capacity (~105 colonies) due to the physical requirements of colony transfer, assay, and autoradiography. In this report, we have described a direct selection protocol using a chimeric mitotoxin prepared from FGF2 and saporin-6, a toxic plant lectin. Direct selection has the advantage that very large populations of cells can be manipulated (~108), which should make it possible to detect even rare mutations in GAG biosynthesis. Other selection techniques have been described that depend on resistance to viruses that bind to heparan sulfate during infection (71-74). The advantage of the present method is that it targets GAG synthesis selectively.

In theory, conjugates composed of any GAG-binding protein and a suitable toxin can be used to identify interesting mutants in GAG synthesis. By careful selection of the binding component, it should be possible to obtain mutants in the fine structure of the chains in addition to the more dramatic mutants described here. For example, chimeras prepared with antithrombin might yield mutants altered in 3-O-sulfation of GlcNAc, uronic acid epimerization, and other reactions required for the generation of the high affinity binding site (50, 75). Although we have not yet characterized all of the mutants resistant to FGF2-SAP, preliminary studies indicate the most prevalent class of mutations affects the processing of the heparan sulfate chains (the so-called "partial" mutants). Some of the these strains may bear defects in other previously unidentified genes required for binding site assembly.

The dramatic deficiency in GAG synthesis in the 13 mutants reported here indicates that they have defects in early steps common to heparan sulfate and chondroitin sulfate assembly. Three of 13 mutants identified lacked GlcAT-I. Presumably, the other strains have defects in other early steps, such as XylT (76, 77), the decarboxylase that converts UDP-GlcA to UDP-xylose (78, 79), or the UDP-xylose transporter (80-83). Other studies have suggested that the linkage region of both heparan sulfate, chondroitin sulfate, and dermatan sulfate may undergo sulfation of the Gal residues (5, 7-13) or phosphorylation at C-2 of the xylose (2-6). If these reactions are essential for GAG synthesis, then in theory some mutants altered in GAG assembly could have defects in one of these steps as well.

GAG Synthesis Depends on the Linkage Region Tetrasaccharide-- The analysis of pgsG strains supports our earlier conclusion that only single isozymes of each transferase are involved in linkage region formation. Defects in XylT, GalT-I, and GlcAT-I fail to produce the two major GAG chain types in CHO cells, chondroitin sulfate and heparan sulfate. These findings indicate that the initial steps in the synthesis of these GAGs are catalyzed by a shared set of enzymes, at least in CHO cells. Since heparin and dermatan sulfate are more highly modified forms of heparan sulfate and chondroitin sulfate, respectively, we conclude that all of the major GAGs linked by way of xylose (and containing uronic acids) probably use a common set of enzymes to form the linkage region. Mutants in the second galactosyltransferase (GalT-II) are needed to confirm this conclusion, and efforts are underway to determine if such strains are present in the new collection of mutants. Divergence of the pathways presumably occurs at the addition of the first unique sugar, alpha -GlcNAc in the case of heparan sulfate and heparin and beta -GalNAc in the case of chondroitin and dermatan sulfate (14, 15, 84).

Recent cloning experiments have yielded cDNA clones for human and Chinese hamster GlcAT-I (49, 70). This gene has high homology with GlcAT-P, an enzyme selectively expressed in brain and neurons that is responsible for the formation of HNK-1 determinants (3OSO3GlcAbeta 1,3Gal-) (85). Substrate specificity studies showed that GlcAT-I is highly selective for substrates that resemble the linkage region tetrasaccharide, namely Galbeta 1,3Gal-terminated oligosaccharides (49). In contrast, GlcAT-P tends to be more promiscuous in vitro, using these substrates just as well as (or better than) glycoproteins bearing N-linked or O-linked (mucin-type) chains with terminal beta -linked Gal residues (49). Transfection experiments showed that GlcAT-P will in fact substitute for GlcAT-I by enabling pgsG-110 cells to form GAG chains (49). Thus, in brain and other tissues that express GlcAT-P, two isozymes may participate in forming the linkage region of proteoglycans. Determining the relative contribution of each of these enzymes in neural tissues will require additional experiments, including mutation of the genes in neuronal cell lines or in mice by tissue-specific gene ablation.

Unusual Xyloside-primed Oligosaccharides-- The accumulation of unusual oligosaccharides on beta -D-xylosides deserves comment. Previous studies from a number of laboratories have shown that xylosides will induce the formation of GAG chains and various intermediates that lie along the pathway (58-64). PgsG mutants accumulate normal intermediates, such as Galbeta 1,4Xylbeta -O-R and Galbeta 1,3Galbeta 1,4Xylbeta -O-R (Fig. 3). Interesting, linkage tetrasaccharides (GlcAbeta 1,3Galbeta 1,3Galbeta 1, 4Xylbeta -O-R) were not observed in this study (in wild-type cells) but have been found at low concentrations in other cell types fed other types of xylosides (58, 62, 86). These findings suggest that once GlcA is added to the nascent chain, the intermediate is efficiently consumed by downstream enzymes in the pathway and that in CHO cells GlcAT-I may be rate-limiting. As shown in Fig. 5, transfection of the mutant with GlcAT-I cDNA augmented GAG synthesis to levels about 2-fold higher than seen in wild-type cells, consistent with this idea.

In addition to these intermediates, xylosides will stimulate the formation of unusual oligosaccharides, such as Siaalpha 2,3Galbeta 1,4Xylbeta -O-R, Xylbeta 1,3Xylbeta -O-R, GlcAbeta 1,3Xylbeta -O-R, 3OSO3GlcAbeta 1,3Xylbeta -O-R, and GalNAcalpha 1,3GlcAbeta 1,3Galbeta 1,3Galbeta 1,4Xylbeta -O-R (60-64, 66). In this report, we found that CHO cells also make Siaalpha 2,3Galbeta 1,3Galbeta 1,4Xylbeta -O-R and possibly a disialylated species of unknown structure (86). To date, none of these oligosaccharides have been found on endogenous proteoglycan, glycoprotein, or glycolipids. Presumably, their accumulation reflects the alternate use of substrates by resident transferases under conditions where the substrate concentration is driven to very high levels with exogenously added xyloside. The formation of these compounds depends to a certain extent on the concentration of xyloside provided to the cells, aglycone, and the type of cell (62, 64, 86). However, at the lowest concentration tested (3 µM), the unusual alpha -GalNAc-capped linkage tetrasaccharide actually predominates over GAG chains and other precursors. This finding suggests that the alpha -GalNAc transferase (68, 69) must be quite abundant in CHO cells, but somehow it is normally sequestered from endogenous proteoglycan intermediates since this unusual modification has not yet been found on a native proteoglycan.

PgsG Mutations May Help Define the Catalytic Domain-- Northern blot analysis of pgsG cell mRNA indicates that all of the mutants make normal amounts of message for GlcAT-I. Although transcriptional rates were not measured, the data suggest that the mutants most likely have a missense or nonsense mutation that does not alter transcription or message stability. Sequencing cDNA clones prepared from pgsG mRNA will help confirm this idea. Point mutations altering GlcAT-I are quite desirable since they may help define the catalytic domain of the protein. The three mutants reported here represent a good starting point for structure/function studies of this interesting and essential enzyme for GAG biosynthesis.

    ACKNOWLEDGEMENTS

We thank Mark D'Andrea and Barbara Sosnowski of Selective Genetics, Inc., for their preparation and generous provision of FGF2-saporin and Hud Freeze for many helpful discussions.

    FOOTNOTES

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

Dagger On leave from the Dept. of Biochemistry and Molecular Genetics, University of Alabama, Birmingham, AL 35294.

§ To whom correspondence should be addressed: Division of Cellular and Molecular Medicine, Dept. of Medicine, The Glycobiology Program, University of California, San Diego, 9500 Gilman Dr., CMM East 1055, La Jolla, CA 92093-0687. Tel.: 619-822-1100; Fax: 619-534-5611; E-mail: jesko{at}ucsd.edu.

2 K. Kimata, personal communication.

3 The relative amount of charged material was dependent on the concentration of xyloside in the growth medium; ~50% of the oligosaccharides in wild-type cells and ~20% in the mutant were charged when the xyloside concentration was increased to >= 10 µM.

    ABBREVIATIONS

The abbreviations used are: GAG, glycosaminoglycan; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; FGF2-SAP, basic fibroblast growth factor-saporin-6 chimera; GlcAT-I, UDP-glucuronic acid:Galbeta 1,3Gal-R glucuronosyltransferase; GlcAT-P, UDP-glucuronic acid:glycoprotein glucuronosyltransferase; MOPS, 3-[N-morpholino]propanesulfonic acid; XylT, xylosyltransferase; GalT-I, galactosyltransferase I.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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