Chinese Hamster Ovary Cell Mutants Defective in Glycosaminoglycan Assembly and Glucuronosyltransferase I
1999; Elsevier BV; Volume: 274; Issue: 19 Linguagem: Inglês
10.1074/jbc.274.19.13017
ISSN1083-351X
AutoresXiaomei Bai, Ge Wei, Anjana Sinha, Jeffrey D. Esko,
Tópico(s)Cell Adhesion Molecules Research
ResumoThe proteoglycans of animal cells typically contain one or more heparan sulfate or chondroitin sulfate chains. These glycosaminoglycans assemble on a tetrasaccharide primer, -GlcAβ1,3Galβ1,3Galβ1,4Xylβ-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-β-d-xyloside (Xylβ-O-Np) resulted in accumulation of linkage region intermediates containing 1 or 2 mol of galactose (Galβ1, 4Xylβ-O-Np and Galβ1, 3Galβ1, 4Xylβ-O-Np) and sialic acid (Siaα2,3Galβ1, 3Galβ1, 4Xylβ-O-Np) but not any GlcA-containing oligosaccharides. Extracts of the mutants completely lacked UDP-glucuronic acid:Galβ1,3Gal-R glucuronosyltransferase (GlcAT-I) activity, as measured by the transfer of GlcA from UDP-GlcA to Galβ1,3Galβ-O-naphthalenemethanol (<0.2versus 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. The proteoglycans of animal cells typically contain one or more heparan sulfate or chondroitin sulfate chains. These glycosaminoglycans assemble on a tetrasaccharide primer, -GlcAβ1,3Galβ1,3Galβ1,4Xylβ-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-β-d-xyloside (Xylβ-O-Np) resulted in accumulation of linkage region intermediates containing 1 or 2 mol of galactose (Galβ1, 4Xylβ-O-Np and Galβ1, 3Galβ1, 4Xylβ-O-Np) and sialic acid (Siaα2,3Galβ1, 3Galβ1, 4Xylβ-O-Np) but not any GlcA-containing oligosaccharides. Extracts of the mutants completely lacked UDP-glucuronic acid:Galβ1,3Gal-R glucuronosyltransferase (GlcAT-I) activity, as measured by the transfer of GlcA from UDP-GlcA to Galβ1,3Galβ-O-naphthalenemethanol (<0.2versus 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. The assembly of the glycosaminoglycans (GAGs), 1The abbreviation used is: GAG, glycosaminoglycan; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; FGF2-SAP, basic fibroblast growth factor-saporin-6 chimera; GlcAT-I, UDP-glucuronic acid:Galβ1,3Gal-R glucuronosyltransferase; GlcAT-P, UDP-glucuronic acid:glycoprotein glucuronosyltransferase; MOPS, 3-[N-morpholino]propanesulfonic acid; XylT, xylosyltransferase; GalT-I, galactosyltransferase I.1The abbreviation used is: GAG, glycosaminoglycan; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; FGF2-SAP, basic fibroblast growth factor-saporin-6 chimera; GlcAT-I, UDP-glucuronic acid:Galβ1,3Gal-R glucuronosyltransferase; GlcAT-P, UDP-glucuronic acid:glycoprotein glucuronosyltransferase; MOPS, 3-[N-morpholino]propanesulfonic acid; XylT, xylosyltransferase; GalT-I, galactosyltransferase I. 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, GlcAβ1,3Galβ1,3Galβ1,4Xylβ-O-Ser (1Rodén L. 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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 (1Rodén L. Lennarz W.J. The Biochemistry of Glycoproteins and Proteoglycans. Plenum Publishing Corp., New York1980: 267-271Crossref Google Scholar). 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 (38Esko J.D. Stewart T.E. Taylor W.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3197-3201Crossref PubMed Scopus (484) Google Scholar, 39Esko J.D. Weinke J.L. Taylor W.H. Ekborg G. Rodén L. Anantharamaiah G. Gawish A. J. Biol. Chem. 1987; 262: 12189-12195Abstract Full Text PDF PubMed Google Scholar, 40Esko J.D. Curr. Opin. Cell Biol. 1991; 3: 805-816Crossref PubMed Scopus (183) Google Scholar). 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. 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 (38Esko J.D. Stewart T.E. Taylor W.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3197-3201Crossref PubMed Scopus (484) Google Scholar,39Esko J.D. Weinke J.L. Taylor W.H. Ekborg G. Rodén L. Anantharamaiah G. Gawish A. J. Biol. Chem. 1987; 262: 12189-12195Abstract Full Text PDF PubMed Google Scholar). 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 (41Ham R.G. Proc. Natl. Acad. Sci. U. S. A. 1965; 53: 288-293Crossref PubMed Scopus (645) Google Scholar), substituting chloride salts for sulfate, and supplemented with fetal bovine serum that had been dialyzed 106-fold against phosphate-buffered saline (PBS) (42Dulbecco R. Vogt M. J. Exp. Med. 1954; 99: 167-182Crossref PubMed Scopus (1993) Google Scholar). Low glucose medium containing 1 mm instead of 10 mmglucose 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. Wild-type CHO cells were mutagenized with ethylmethanesulfonate and frozen under liquid nitrogen (43Esko J.D. Methods Cell Biol. 1989; 32: 387-422Crossref PubMed Scopus (19) Google Scholar). 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, (44Lappi D.A. Ying W. Barthelemy I. Martineau D. Prieto I. Benatti L. Soria M. Baird A. J. Biol. Chem. 1994; 269: 12552-12558Abstract Full Text PDF PubMed Google Scholar). After 1 day, the cells were overlaid with a stack of polyester disks and glass beads in order to generate colony replicas (43Esko J.D. Methods Cell Biol. 1989; 32: 387-422Crossref PubMed Scopus (19) Google Scholar). 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 (43Esko J.D. Methods Cell Biol. 1989; 32: 387-422Crossref PubMed Scopus (19) Google Scholar), the disks were dried and exposed to x-ray film (Fuji). Tentative mutants were identified as blue-stained colonies that failed to incorporate35SO4. Candidates were picked from the original master plates using cloning cylinders and trypsin (43Esko J.D. Methods Cell Biol. 1989; 32: 387-422Crossref PubMed Scopus (19) Google Scholar). 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 tests were carried out by cell hybridization (43Esko J.D. Methods Cell Biol. 1989; 32: 387-422Crossref PubMed Scopus (19) Google Scholar). Approximately 2 × 105cells 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 (M r = 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 with35SO4 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. 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 (45Bame K.J. Esko J.D. J. Biol. Chem. 1989; 264: 8059-8065Abstract Full Text PDF PubMed Google Scholar) 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 mmKH2P04 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. CHO cells were grown to near confluence and then incubated with low glucose F-12 medium supplemented with different concentrations of naphthol-β-d-xyloside (46Fritz T.A. Lugemwa F.N. Sarkar A.K. Esko J.D. J. Biol. Chem. 1994; 269: 300-307Abstract Full Text PDF PubMed Google Scholar) 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 0.5m 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. The dried samples were resuspended in 2 mmTris 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 (47Roux L. Holojda S. Sundblad G. Freeze H.H. Varki A. J. Biol. Chem. 1988; 263: 8879-8889Abstract Full Text PDF PubMed Google Scholar), 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 C18cartridges. 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 β-galactosidase (bovine testes, Oxford Glycosystems) in 0.1m 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 β-glucuronidase (bovine liver, Sigma) overnight in 100 mmsodium acetate (pH 5.0) buffer containing 0.3 m NaCl and 8 mm d-galactonic acid γ-lactone to inhibit contaminating β-galactosidase activity; and (iii) 1 milliunit of α-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 β-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 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 mNaCl. 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 of125I-FGF2 (∼4 × 105 cpm, Ref. 48Bai X. Esko J.D. J. Biol. Chem. 1996; 271: 17711-17717Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) showed that the overall recovery of material was 30–40%. A cDNA for glucuronosyltransferase I (GlcAT-I) was cloned from CHO cells and inserted into pCDNA3 (49Wei G. Bai X.M. Sarkar A.K. Esko J.D. J. Biol. Chem. 1999; 274: 7857-7864Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). 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. 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 (49Wei G. Bai X.M. Sarkar A.K. Esko J.D. J. Biol. Chem. 1999; 274: 7857-7864Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), 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. 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 msucrose, 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, Galβ1,3Galβ-O-naphthalenemethanol, were synthesized as described (49Wei G. Bai X.M. Sarkar A.K. Esko J.D. J. Biol. Chem. 1999; 274: 7857-7864Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). We have identified previously a number of CHO cell mutants altered in GAG biosynthesis using a "brute force" replica plating technique (43Esko J.D. Methods Cell Biol. 1989; 32: 387-422Crossref PubMed Scopus (19) Google Scholar). 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 or125I-antithrombin to colonies could be measured as well, providing a way to obtain mutants in steps that affect sulfation of the chains (48Bai X. Esko J.D. J. Biol. Chem. 1996; 271: 17711-17717Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 50Colliec-Jouault S. Shworak N.W. 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Chem. 1996; 271: 17711-17717Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar,50Colliec-Jouault S. Shworak N.W. Liu J. de Agostini A.I. Rosenberg R.D. J. Biol. Chem. 1994; 269: 24953-24958Abstract Full Text PDF PubMed Google Scholar, 51Esko J.D. Elgavish A. Prasthofer T. Taylor W.H. Weinke J.L. J. Biol. Chem. 1986; 261: 15725-15733Abstract Full Text PDF PubMed Google Scholar, 52Elgavish A. Esko J.D. Knurr A. J. Biol. Chem. 1988; 263: 18607-18613Abstract Full Text PDF PubMed Google Scholar, 53Lidholt K. Weinke J.L. Kiser C.S. Lugemwa F.N. Bame K.J. Cheifetz S. Massagué J. Lindahl U. Esko J.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2267-2271Crossref PubMed Scopus (234) Google Scholar). 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
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