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J. Biol. Chem., Vol. 279, Issue 7, 5693-5698, February 13, 2004

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Temperature-sensitive Glycosaminoglycan Biosynthesis in a Chinese Hamster Ovary Cell Mutant Containing a Point Mutation in Glucuronyltransferase I^*

Ge Wei, Xiaomei Bai, and Jeffrey D. Esko

From the Department of Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California, San Diego, La Jolla, California 92093-0687

Received for publication, October 23, 2003

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
In previous studies, we reported the isolation and characterization of a Chinese hamster ovary cell mutant (pgsG) defective in glucuronyltransferase I (GlcATI). This enzyme adds the terminal GlcA residue in the core protein-linkage tetrasaccharide (GlcA1,3Gal1,3Gal1, 4Xyl-O-) on which glycosaminoglycan assembly occurs (Bai, X. M., Wei, G., Sinha, A., and Esko, J. D. (1999) J. Biol. Chem. 274, 13017–13024; Wei, G., Bai, X. M., Sarkar, A. K., and Esko, J. D. (1999) J. Biol. Chem. 274, 7857–7864). Here we show that incorporation of ³⁵SO₄ into glycosaminoglycans in the mutant is temperature-sensitive, with greater synthesis occurring at 33 °C compared with 37 °C. Wild-type cells show the opposite thermal dependence. Rabbit antiserum to hamster GlcATI failed to detect cross-reactive material in pgsG cells by immunofluorescence and Western blotting. Furthermore, expression of chimeric proteins composed of mutant GlcATI fused to IgG binding domain of protein A or to green fluorescent protein did not yield the proteins at the expected mass. The green fluorescent protein-tagged version appeared as a truncated protein, and immunofluorescence showed large perinuclear bodies at 30 °C. At 37 °C, the fusion protein was not readily detectable. Sequencing cDNAs from mutant and wild-type cells revealed a single base transition (G331A) in the open reading frame in pgsG cells, which resulted in a Val-111 Met substitution. These data suggest that pgsG cells contain a labile form of GlcATI that causes conditional expression of glycosaminoglycans dependent on temperature.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Proteoglycans play important roles as regulators in many biologic processes, such as cell adhesion, proliferation, differentiation, and cytokine and growth factor action (1, 2). Most of these activities depend on interactions of proteins with sulfated oligosaccharide sequences present in the glycosaminoglycan (GAG)¹ chains that distinguish proteoglycans from other glycoconjugates. GAG assembly initiates by the formation of a tetrasaccharide, GlcA1,3Gal1,3Gal1,4Xyl-O-attached to the side chain hydroxyl group of specific serine residues (3). This oligosaccharide serves as a primer for chain elongation, forming either heparan sulfate or chondroitin sulfate depending upon the addition of a GlcNAc or GalNAc residue, respectively (3, 4). The chains then polymerize by the alternating addition of GlcA and GlcNAc or GalNAc residues and undergo modifications by multiple sulfotransferases and epimerases, thus generating binding sites for various ligands (5, 6). Nearly all of the enzymes involved in GAG assembly have now been identified, molecularly cloned, and in many cases mutated in cells or model organisms (6).

Glucuronyltransferase I (GlcATI) catalyzes the final biosynthetic step in the formation of the linkage region tetrasaccharide. The enzyme has been purified and cloned from several sources (7–12). It belongs to a family of three glucuronyltransferases that catalyze the formation of GlcA1,3Gal-linkages in different glycoconjugates (13–16). Negishi and co-workers (17, 18) have crystallized GlcATI with bound trisaccharide acceptor (Gal1,3Gal1,4Xyl) and UDP-GlcA donor. The enzyme appears as a dimer in the crystal structure (17, 18). In vivo, it is located in the Golgi along with other enzymes involved in GAG assembly (19).

We described previously a set of Chinese hamster ovary cell mutants designated pgsG that lack GlcATI enzyme activity and fail to make both heparan sulfate and chondroitin sulfate (20). In the course of studying the Caenorhabditis elegans ortholog of GlcATI (21), we discovered that GAG assembly in the pgsG mutants depended on the temperature at which the cells were grown. Here we show that the pgsG mutants contain a point mutation in the GlcATI gene, which renders GAG synthesis temperature-sensitive.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Cell Culture—Chinese hamster ovary cells (CHO-K1, ATCC CCL-61) were obtained from the American Type Culture Collection (Manassas, VA). The isolation of mutants pgsG-110, -114, and -224 was described previously (20). The cells were grown under an atmosphere of 5% CO₂ in air and 100% relative humidity and maintained in Ham's F-12 growth medium (Hyclone Laboratories) supplemented with 7.5% (v/v) fetal bovine serum (Hyclone Laboratories), 100 µg/ml streptomycin sulfate, and 100 units/ml penicillin G. Sulfate-free medium was prepared from individual components (22), substituting chloride salts for sulfate, and fetal bovine serum that had been dialyzed against phosphate-buffered saline (23).

Purification of Glycosaminoglycan Chains—Cells were labeled for 6–24 h with 10 µCi/ml H₂³⁵SO₄ (1600 Ci/mmol, PerkinElmer Life Sciences) in sulfate-free medium as indicated in the figure legends. Radiolabeled GAGs were isolated from the growth medium and the cells by anion-exchange chromatography as described (24) and quantified by liquid scintillation chromatography, and the counts were expressed relative to the amount of cell protein that was analyzed. In some experiments, the radiolabeled GAGs were analyzed by anion-exchange high pressure liquid chromatography (20). The elution position of chondroitin sulfate and heparan sulfate was determined by enzymatic treatment of the chains (24).

In Vitro Assay of GlcATI Activity—GlcATI activity was assayed under optimized conditions as described (10, 20). The standard reaction (25 µl) contained 10–30 µg of total cell homogenates, 0.05% Triton X-100, 50 mM MES, pH 6.5, 10 mM MnCl₂, 0.2–0.4 µCi of UDP-[1-³H]GlcA, 100 µM UDP-GlcA, and 10 mM synthetic Gal1,3Gal-O-naphthalenemethanol as acceptor (25). The reaction was diluted with 1 ml of 0.5 M NaCl and applied to a Sep-Pak C18 cartridge (100 mg, Waters). After washing the cartridge with 2 ml of water, the product was eluted with 50% methanol, dried, and counted by liquid scintillation. The reaction was proportional to protein and time at 37 °C for at least 4 h.

DNA Sequencing—mRNA from mutant and wild-type CHO cells was purified, and first-strand cDNA was produced using random primers (Superscript^TM Preamplification System for First Strand cDNA synthesis, Invitrogen). The 5'- and 3'-primers are 5'-TCTAAAGCGGTCCCCGCGGACCCC-3' and 5'-TGGAGGGGCTGCCTCTGGTTCTGA-3'. PCR was carried out with Pfu polymerase (Clontech; 25 cycles at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min, followed by a final incubation at 72 °C for 7 min) in a 2400 Thermocycler (PerkinElmer Life Sciences). The PCR products were cloned into pCR-Script Amp SK(+) (Stratagene). At least two clones from each PCR were sequenced on both strands by the dideoxy chain termination method using Taq polymerase (dye terminator cycle sequencing, PerkinElmer Life Sciences) with a DNA automatic sequencer (ABI PRISM genetic analyzer).

Production of Rabbit Polyclonal Antiserum against GlcAT-1—A polyclonal antiserum was prepared against recombinant GlcATI fused to glutathione S-transferase. A cDNA fragment consisting of GlcATI without its transmembrane and cytosolic domains (residues 29–335) was amplified by PCR using Pfu polymerase (Clontech) and cloned into the EcoRI and XhoI sites of pGEX-KG expression vector (Amersham Biosciences). The resulting cDNA encoded a fusion protein composed of GST, a thrombin cleavage site, and the catalytic domain of hamster GlcATI. The GST fusion protein was produced after isopropyl-1-thio--D-galactopyranoside induction of a 1-liter culture of Escherichia coli DH5 containing the plasmid. The cells were recovered by centrifugation and resuspended in 20 ml of cold PBS containing 1 mM EDTA, 0.1% -mercaptoethanol, 0.2 mM phenylmethylsulfonyl fluoride, and 5 mM benzamidine. The sample was sonicated twice for 30 s on ice, adjusted to 1% (v/v) Triton X-100, and centrifuged at 4 °C at 10,000 x g for 10 min. Glutathione-agarose beads (Sigma) were equilibrated with PBS, and 1 ml of a 50% slurry was added to the supernatant. The suspension was mixed gently at 4 °C for 30 min using a rotary mixer and then briefly centrifuged to recover the beads. After washing the beads 4 times with cold PBS, the fusion protein was eluted by incubating the beads twice with 1 ml of fresh, reduced glutathione (10 mM) followed by centrifugation. The eluant was diluted 10-fold with cold PBS and repurified on fresh beads. GST-GlcATI was injected into New Zealand White rabbits (0.5 mg/injection, Robert Sargeant, Ramona, CA). The antiserum was obtained after 5–6 injections, spaced apart by 6–12 weeks.

To make affinity columns, purified GST and GST-GlcATI were cross-linked to CNBr-activated Sepharose 4B (Amersham Biosciences). Rabbit antiserum was diluted 1:3 with PBS and mixed with GST-GlcATISepharose (1 ml) at 4 °C overnight. The resin was collected by low speed centrifugation and washed with PBS until the absorbance at 280 nm of the supernatant was 0.01. The antibodies were eluted twice with 4 ml of a buffer containing 0.05 M glycine and 0.15 M NaCl, pH 2.3, neutralized immediately with 1 M Tris base, pH 9.5, and dialyzed against PBS at 4 °C. Antibodies to GST were removed by passing the antiserum through a column containing GST, and the material was then concentrated by membrane centrifugation (Centricon 10) to 0.5 mg/ml. The antiserum appeared to be specific based on its strong reaction with purified GST-GlcATI fusion protein by Western blotting and the reaction of a single band in extracts from wild-type CHO cells.

Expression of Wild-type and Mutant GlcATI Fusion Proteins—cDNA encoding amino acids 30–335 of GlcATI (stem region and catalytic domain) was prepared and fused in-frame to the C terminus of the IgG-binding domain of protein A in pRK5-F10-PROTA (26). The fusion proteins were transiently expressed in COS-7 cells, isolated by IgG affinity chromatography from the medium, and analyzed by SDS-PAGE.

The full-length coding sequences of mutant and wild-type GlcATI were prepared by PCR and fused in-frame to the N terminus of green fluorescent protein (GFP) in vector pEGFP-N1 (Clontech). These constructs were transiently transfected into CHO-K1 cells using LipofectAMINE 2000 (Invitrogen). For Western blots, the cells were harvested 48 h after transfection. For labeling studies, [³⁵S]methionine was added 24 h after transfection, and 1 day later the cells were harvested and analyzed by SDS-PAGE, autoradiography, and Western blotting

SDS-PAGE Analysis—Samples were boiled in reducing SDS sample buffer (Invitrogen) and analyzed by SDS-PAGE on a 4–20% Tris-glycine gel. Gels containing [³⁵S]methionine-labeled samples were dried and exposed to x-ray film. Other samples were blotted on polyvinylidene difluoride membrane. The blots were incubated with anti-GlcATI antiserum (1:500 dilution) and goat anti-rabbit IgG conjugated to horseradish peroxidase or with anti-GFP monoclonal antibody (1:1000 dilution of 0.4 mg/ml stock) and horseradish peroxidase-conjugated goat antimouse IgG.

Some samples were immunoprecipitated to enrich for fusion proteins. The spent medium was decanted from cells transiently transfected with GlcATI-GFP fusion proteins, and the cell layers were washed three times with 50 mM Tris-HCl, pH 7.5, containing 0.15 M NaCl. Lysis buffer (1 ml) was added (50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride, and 5 mM EDTA). After incubation for 20 min at room temperature, the cell lysates were incubated with 10 µg of anti-GFP monoclonal antibody (Roche Applied Science, catalog number 1814460) on ice for 1 h. Protein G-agarose (50 µl, Roche Applied Science) was added, and the mixture was incubated for 3 h at 4 °C with gentle rocking. The pellet of absorbed immunoprecipitate was washed with lysis buffer and collected for SDS-PAGE.

Immunostaining GlcATI in Wild-type and Mutant CHO Cells—Both wild-type and pgsG mutant cells were grown on glass coverslips at low density. The cells were first made permeable by treatment with PBS containing 0.1% Triton and 0.1% bovine serum albumin (10 min) and then blocked with 1% bovine serum albumin in PBS for 1 h. The cells were then stained with antibodies to GlcATI or -mannosidase II (kindly provided by Dr. Marilyn Farquhar, University of California, San Diego) and FITC-conjugated goat anti-rabbit IgG secondary antibodies. Cells were viewed by epifluorescence under a Zeiss microscope fitted with a green barrier filter for FITC emission. The digitized images were captured using a DKC-5000 digital camera (Sony) and rendered in Adobe Photoshop.

Wild-type and mutant GlcATI-GFP were transiently transfected into wild-type and mutant CHO cells on coverslips using LipofectAMINE^TM (Invitrogen). Three days later, cells were made permeable with 0.1% Triton X-100 in PBS and then fixed with 1% paraformaldehyde in PBS. The cells were stained with Hoechst dye, and photomicrographs were taken and processed as described (19).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Glycosaminoglycan Synthesis Is Conditionally Defective in pgsG Cells—In the course of studying the GlcATI ortholog of C. elegans (SQV-8), a cDNA clone for the C. elegans gene was introduced into pgsG mutant cells to test if it corrected the GAG deficiency (21). In these studies, the cells were grown at 30 °C because some enzymes from nematodes are more active at lower temperatures. pgsG cells made GAGs after transfection with SQV-8 GlcATI, but the same results were obtained when the cells were transfected using a control vector lacking any insert. Further analysis showed that the mutant produced GAGs to the same extent as wild-type cells at all temperatures below 30 °C, whereas it produced significantly less GAG at higher temperatures (35–37 °C) (Fig. 1A). Some residual synthesis occurred at elevated temperatures presumably due to a small amount of residual enzyme activity (as shown below). Analysis of the composition of the [³⁵S]GAGs generated at 30 °C showed that pgsG cells made the normal amount of heparan sulfate and chondroitin sulfate as compared with wild-type cells grown under identical conditions (Fig. 2). At 37 °C, the mutant made a small amount of ³⁵S-labeled material that migrated like GAG chains. Furthermore, the heparan sulfate chains from both mutant and wild-type cells grown at 30 °C bound FGF-2 to the same extent (data not shown and see Ref. 27).

View larger version (14K): [in this window] [in a new window]   FIG. 1. Glycosaminoglycan biosynthesis in pgsG cells at different temperatures. A, cells were cultured at 37 °C to near confluence and then shifted to the indicated temperatures and labeled with 35SO4 for 6 h. [35S]GAGs were isolated from the cells and the medium. , wild-type; , pgsG. B, wild-type and pgsG cells were cultured at 30 °C with 35SO4. After 3 days, one set of cultures was shifted to 37 °C. At the indicated times [35S]GAGs were isolated from the cells and conditioned medium ("Experimental Procedures"). •, wild-type at 30 °C; , wild-type at 37 °C; , pgsG at 30 °C; , pgsG at 37 °C.

View larger version (26K): [in this window] [in a new window]   FIG. 2. pgsG cells produce a normal complement of glycosaminoglycans at 30 °C. Wild-type (A) and pgsG (B) cells were cultured at 37 °C for 2 days. One set of cultures was left at 37 °C and another was shifted to 30 °C. Both sets were labeled with 35SO4 overnight. [35S]GAGs were isolated from the conditioned medium and the cells and analyzed by anion-exchange high pressure liquid chromatography ("Experimental Procedures"). •, wild-type at 30 °C; , wild-type at 37 °C; , pgsG at 30 °C; , pgsG at 37 °C.

GlcATI Activity in pgsG Cells—The labeling studies suggested that GlcATI activity should be present in pgsG cells cultured at 30 °C and that it might be thermolabile. To test this possibility, cell-free extracts were prepared from mutant and wild-type cells cultured at 30 °C and assayed at 30 and 37 °C using conditions optimized for the wild-type enzyme (10, 20). Wild-type cells expressed robust activity at both temperatures (Fig. 3, solid bars). In contrast, the mutant had low activity at both temperatures compared with the wild type, but the values were significantly above background (i.e. the signal obtained in the absence of added acceptor; open bars). Somewhat higher activity was observed at 30 °C compared with 37 °C in several experiments, but this difference was not statistically significant due to variation in the assay. Attempts to stabilize the residual activity in the mutant by altering the concentration of substrates and cofactors were not successful, making further analysis of the enzyme difficult. Although enzyme activity was low in the mutant at permissive conditions, it could account for GAG biosynthesis. CHO cells produce about 1 µg of GAG/mg of cell protein, which is 50 pmol of GAG chains/mg of cell protein assuming on average 100 disaccharides/chain. Residual enzyme activity in the mutant was reported to be 0.2 pmol/min/mg cell protein (10, 20). Given a generation time of 14 h, the mutant could generate about 0.2 pmol/min/mg x (60 min/h x 14 h) = 170 pmol of chains/mg cell protein, more than enough to account for the level of residual synthesis. In the wild type, GlcATI is expressed in large catalytic excess.

View larger version (15K): [in this window] [in a new window]   FIG. 3. Defective enzyme activity in pgsG cells. Wild-type and mutant cells were cultured at 30 °C, and cell homogenates were assayed for GlcATI activity at 30 and 37 °C. The open bars refer to samples assayed in the absence of added acceptor Gal1,3Gal-O--naphthalenemethanol ("Experimental Procedures"). The filled bars refer to samples of wild-type (black) and mutant cells (shaded) assayed in the presence of acceptor.

View larger version (56K): [in this window] [in a new window]   FIG. 4. pgsG cells lack GlcATI. Wild-type (A and C) and pgsG (B and D) cells were incubated with anti--mannosidase II antibodies (A and B) or affinity-purified rabbit polyclonal anti-GlcATI antibodies (C and D) followed by fluorescein isothiocyanate-conjugated goat ant-rabbit IgG antibody.

View larger version (25K): [in this window] [in a new window]   FIG. 5. A protein A-pgsG GlcATI fusion protein is not stable. Mutant and wild-type forms of GlcATI lacking their transmembrane and cytoplasmic domains were fused to the IgG binding domain of protein A and expressed in COS-7 cells ("Experimental Procedures"). The fusion proteins were affinity-purified from the conditioned medium using IgG-agarose beads and analyzed by SDS-gel electrophoresis. The gels were silver-stained or analyzed by Western blotting using rabbit polyclonal anti-GlcA T1 antibody. + and - refer to the orientation of GlcATI (sense or antisense) in the expression vector pRK5. MW, molecular weight markers.

View larger version (49K): [in this window] [in a new window]   FIG. 6. Immunoprecipitation blot of GFP-GlcATI fusion proteins. GFP-GlcATI fusion proteins were transiently expressed in CHO cells and immunoprecipitated with anti-GFP monoclonal antibody. The fusion proteins were analyzed by SDS-PAGE, blotted, and visualized either by autoradiography or by Western blotting with anti-GFP or anti-GlcATI antiserum ("Experimental Procedures"). The arrows mark the migration of GFP and GlcATI-GFP fusion proteins, and the arrowheads indicate the position of an unusual cleavage product generated from the mutant GlcATI-GFP fusion.

View larger version (98K): [in this window] [in a new window]   FIG. 7. Thermolabile expression of GFP-tagged GlcATI in vivo. Wild-type CHO cells were transfected with the wild-type and mutant GlcATI-GFP fusion proteins and cultured at 30 (A, C, and E) or 37 °C (B, D, and F). The cells were fixed and counterstained with Hoechst dye (blue). A and B, expression of EGFP; C and D, expression of wild-type GlcATI-GFP construct; E and F, expression of mutant GlcATI-GFP construct.

To test this idea, cDNAs for the full-length transcripts were prepared by reverse transcriptase-PCR and sequenced by dideoxy chain termination. Three mutants identified in the original screening had a single base transition in the cDNA at the same position (G331A) (Fig. 8). Three clones from each PCR were sequenced for each mutant and the wild-type, suggesting that this was the only mutation present in the coding sequence. All three strains were obtained from a single pot of cells that had been exposed to mutagen and subsequently treated with a selective agent to find cells lacking heparan sulfate (20). Because it is unlikely that the mutagen (ethylmethanesulfonate) induced identical mutations in three independent lines, the three strains were most likely siblings arising from the same mutagen-treated parent.

View larger version (40K): [in this window] [in a new window]   FIG. 8. pgsG cells contain a point mutation in GlcATI. Dideoxy chain termination sequencing electrophoretograms of wild-type and pgsG cDNAs for GlcATI are shown. Only the region around nucleotide 331 is shown (non-sense strand).

Mutant cells exhibiting temperature-sensitive GAG synthesis should prove useful for several types of studies. The mutant can be used for biosynthetic studies to synchronize the expression of GAGs and proteoglycans on the cell surface. Turnover studies can be performed in the mutants in the absence of biosynthesis, thus simplifying the analysis of proteoglycan turnover kinetics and distribution. The dependence of binding or activation of processes in which GAGs play a role (e.g. receptor activation) can be titrated by using cells at different times after shift to permissive conditions or by growing cells at intermediate temperatures. Temperature-sensitive mutants also provide a tool for studying enzyme folding and oligomerization. Although conditional alleles in other genes involved in GAG assembly are not yet available, it should be possible to phenocopy the mutant described here by expressing transgenes that can be activated pharmacologically. This latter approach could be used in other CHO mutants non-conditionally defective in GAG biosynthesis (24, 27, 30–33), thus expanding the utility of existing mutants.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grant R37GM33063 (to J. D. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work. Present address: Neose West, 6330 Nancy Ridge Rd., Ste. 102-130, San Diego, CA 92121.

To whom correspondence should be addressed: Dept. of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Dr., CMM-East 1055, La Jolla, CA 92093-0687. Tel.: 858-822-1100; Fax: 858-534-5611; E-mail: jesko{at}ucsd.edu.

¹ The abbreviations used are: GAG, glycosaminoglycan; CHO, Chinese hamster ovary; GlcA, D-glucuronic acid; GlcATI, UDP-GlcA: Gal1,3Gal-R 1,3glucuronyltransferase; PBS, phosphate-buffered saline; GFP, green fluorescent protein; MES, 2-(N-morpholino) ethanesulfonic acid; GST, glutathione S-transferase; FITC, fluorescein isothiocyanate.

	ACKNOWLEDGMENTS

 
UDP-[1-³H]GlcA was prepared by the Glycotechnology Core in the Glycobiology Research and Training Center at the University of California, San Diego, through grant R24 GM61894 from the National Institutes of Health.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 

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