Proc. Natl. Acad. Sci. USA Vol. 89, pp. 2267-2271, March 1992 Biochemistry A single mutation affects both N-acetylglucosaminyltransferase and glucuronosyltransferase activities in a Chinese hamster ovary cell mutant defective in heparan sulfate biosynthesis (glycosaminoglycans/proteoglycans/glycosyltransferases/replica plating) KERSTIN LIDHOLT*, JULIE L. WEINKEt, CHERYL S. KISERt, FULGENTIUS N. LUGEMWAt, KAREN J. BAMEtt, SELA CHEIFETZ§, JOAN MASSAGUO§, ULF LINDAHL*¶1, AND JEFFREY D. ESKOt II tDepartment of Biochemistry, Schools of Medicine and Dentistry, University of Alabama, Birmingham, AL 35294; *Depaltment of Veterinary Medical Chemistry, The Biomedical Center, Swedish University of Agricultural Sciences, S-751 23, Uppsala, Sweden; and §Department of Cell Biology and Genetics, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021 Communicated by Marilyn G. Farquhar, December 10, 1991 ABSTRACT Mutants of Chinese hamster ovary cells have bovine serum. A resistant mutant was isolated and then been found that no longer produce heparan sulfate. Charac- treated with mutagen (7), and a ouabain-resistant clone was terization of one of the mutants, pgsD-677, showed that it lacks selected in growth medium containing 1 mM ouabain. The both N-acetylglucosaminyl- and glucuronosyltransferase, en- introduction of these markers did not alter the proteoglycan zymes required for the polymerization of heparan sulfate composition of the cells. chains. pgsD-677 also accumulates 3- to 4-fold more chon- Cell hybrids were generated by co-plating 2 x 105 cells of droitin sulfate than the wild type. Cell hybrids derived from pgsD-677 and OT-1 in individual wells of a 24-well plate. pgsD-677 and wild type regained both transferase activities and After overnight incubation, the mixed monolayers were the capacity to synthesize heparan sulfate. Two segregants treated for 1 min with 50% (wt/wt) poly(ethylene glycol) from one of the hybrids reexpressed the dual enzyme defi- (PEG 3320) prepared in F12 medium without serum (9). After ciency, the lack of heparan sulfate synthesis, and the enhanced 1 day the cells were harvested with trypsin, and multiple accumulation of chondroitin sulfate, suggesting that all of the 100-mm-diameter tissue culture plates were seeded with traits were genetically linked. These fin gs indicate that the about 103 cells in F12 medium containing 10 ALM aminopterin pgsD locus may represent a gene involved in the coordinate and 1 mM ouabain to counterselect parental cells. One day control of glycosaminoglycan formation. later the medium was changed to remove dead cells, and those remaining on the dish were overlaid with Whatman no. Proteoglycans consist of a core protein and one or more 42 filter paper in order to obtain discrete colonies (7). Nine covalently attached glycosaminoglycan chains. Typical ani- days later, the disk was removed and resistant clones were mal cells produce proteoglycans bearing chondroitin (derma- picked with glass cloning cylinders and trypsin. The inci- tan) sulfate or heparan sulfate chains, but the composition dence of drug-resistant colonies indicated that the hybridiza- varies considerably among different cells (1, 2). To study the tion efficiency was at least 1%. When each parental strain regulation of proteoglycan composition, we have isolated was fused to itself, colonies of resistant cells were not found. Chinese hamster ovary (CHO) cell mutants defective in To obtain segregants, about 20,000 colonies of hybrid 6.5 glycosaminoglycan biosynthesis (3-6). Many of these mu- (pgsD-677 x OT-1) were screened by 35S autoradiography for tants bear mutations in genes involved in the formation of both heparan sulfate and chondroitin sulfate chains (3, 5). those exhibiting reduced incorporation of [35S]sulfate (7). Here we describe a CHO cell mutant, pgsD-677, that spe- Two strains (6.5.2 and 6.5.5) were identified in this manner cifically lacks heparan sulfate. The mutation in pgsD-677 and repurified by replica plating. affects both N-acetylglucosaminyl (GlcNAc)- and glucurono- Radiolabeling Studies. Na235SO4 (25-40 Ci/mg; 1 Ci = 37 syl (GlcA)-transferase activities required for heparan sulfate GBq) and D-[6-3H]glucosamine hydrochloride (40 Ci/mmol) polymerization, suggesting that some form of coordinate were purchased from Amersham. Glycosaminoglycans were regulation of these enzymes exists. labeled biosynthetically by incubating cells in sulfate- deficient medium containing [35S]sulfate (10-20 ,Ci/ml) or D-[6-3H]glucosamine (10 ,uCi/ml). The medium was removed EXPERIMENTAL PROCEDURES and the cells were harvested in a small volume of 0.1 M Cell Cultures. CHO cells (CHO-Ki) were obtained from NaOH. A portion ofthe alkaline cell extracts was used for the the American Type Culture Collection (CCL-61). All mutants determination of protein by the method of Lowry et al. (10) were identified by colony autoradiography (7), and the purity with bovine serum albumin as standard. The cell extracts and of each strain was ensured by its isolation from cultures media samples were digested with protease, and radioactive containing only mutant colonies. Cells were maintained in glycosaminoglycans were purified by ion-exchange chroma- Ham's F12 (8) medium (Mediatech, Washington) supple- tography and ethanol precipitation (6). The disaccharide mented with 10% (vol/vol) fetal bovine serum (HyClone) or composition of chondroitin sulfate was determined by paper in sulfate-deficient medium (4). Cell fusion studies required the isolation of a CHO-K1 Abbreviations: TCA, trichloroacetic acid; TGF-f3, transforming subline resistant to thioguanine and ouabain (OT-1). Wild- growth factor ,B. type cells were treated with 10 ,uM 6-thioguanine in hypo- tPresent address: School of Basic Life Sciences, Division of Mo- xanthine-free F12 medium supplemented with dialyzed fetal lecular Biology and Biochemistry, University of Missouri, Kansas City, MO 64110. Present address: Department of Medical and Physiological Chem- The publication costs of this article were defrayed in part by page charge istry, The Biomedical Center, University of Uppsala, S-751 23, payment. This article must therefore be hereby marked "advertisement" Uppsala, Sweden. in accordance with 18 U.S.C. §1734 solely to indicate this fact. '1To whom reprint requests should be addressed. 2267 Downloaded by guest on September 26, 2021 2268 Biochemistry: Lidholt et al. Proc. Natl. Acad. Sci. USA 89 (1992) chromatography (4) using authentic standards (Seikagaku America, St. Petersburg, FL). Enzyme Assays. N-Sulfotransferase was assayed using N-desulfoheparin as substrate (6). GlcNAc- and GIcA- transferase were assayed using oligosaccharide acceptors prepared from the capsular polysaccharide of Escherichia coli K5 (11). The polysaccharide was partially N-deacety- lated with hydrazine and subjected to deaminative cleavage with nitrous acid at pH 3.9 (12). The resulting mixture of 677 x 8O oligosaccharides, all having GlcA at their nonreducing ter- 677 x 677 mini, was fractionated by gel filtration chromatography. The decasaccharide fraction was used as substrate for GlcNAc- transferase. Digestion of a tetradecasaccharide fraction with B3-D-glucuronidase yielded tridecasaccharides with nonre- 9 ducing terminal GlcNAc residues, suitable as substrates for GlcA-transferase. Enzyme preparations were obtained by solubilization of about 2 x 107 cells with 0.5 ml of 1% (vol/vol) Triton X-100/50 mM Tris-HCl, pH 7.2, containing phenylmethylsulfonyl fluoride (1 mM) and pepstatin (10 pug/ml). After 30 min of gentle agitation at 40C, the samples were centrifuged. The supernatants were assayed for glyco- 677 x 745 803 x 803 syltransferase activities. UDP-[6-3H]GlcNAc (27 Ci/mmol) was from New England Nuclear. UDP-["4C]GlcA (321 mCi/ mmol) was prepared from D-[14C]glucose (13). RESULTS ** .*~~~a Identification of Heparan Sulfate-Deficient Mutants. A pre- vious study described a screening method for detecting mutants defective in proteoglycan biosynthesis (3). This technique involves the transfer of CHO colonies from plastic tissue culture dishes to disks of polyester cloth (7). The 803 x 745 745 x 745 transferred colonies are incubated with [35S]sulfate, and the incorporation of radioactivity into trichloroacetic acid (TCA)-precipitable proteoglycans is measured by autora- FIG. 1. Autoradiographic analysis of cell hybrids. Mixed mono- layers ofthe indicated strains were treated with poly(ethylene glycol) in syn- diography. Mutant colonies defective proteoglycan to induce cell fusion. The treated cells were replated into 100-mm thesis yield a reduced signal on the film and can be retrieved tissue culture dishes to obtain 300-1000 colonies per dish. After 9 from the original plastic dish from which the replica was days, the colonies were labeled for 4 hr with 35SO4 and radioactive generated. Several mutants exhibited a partial reduction proteoglycans were precipitated in situ with TCA. The bottom ofthe in [35S]sulfate incorporation into proteoglycans, and in some dish was excised and exposed to x-ray film. Complementation had cases this was due to incomplete inhibition of a specific occurred if occasional colonies yielded a strong signal comparable to enzyme in the biosynthetic pathway (e.g., ref. 6). Cell that given by wild-type colonies (not shown). hybridization studies showed that four of these partial mu- tants (strains 623, 625, 677, and 803) comprised a new precipitate 35S-labeled macromolecules. About 200 cpm of complementation group, pgsD (Fig. 1). One ofthese