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 mutants, 35S-labeled material per ,ug of cell protein precipitated in the pgsD-677, was selected for further analysis. mutant, whereas 900 cpm/,ug precipitated in the wild type. pgsD-677 exhibited an 3-fold reduction in proteoglycan Thus, the enhanced solubility of pgsD-677 proteoglycans in synthesis by autoradiography. To obtain more quantitative TCA explained the reduced autoradiographic signal gener- data, cells were labeled to constant specific radioactivity ated by mutant colonies. ([35S]glycosaminoglycan per ,ug of cell protein) by growing pgsD-677 Is Defective in Heparan Sulfate Biosynthesis. Wild- them for 3 days in sulfate-deficient medium containing type CHO cells produce about 70% heparan sulfate and 30%6 [35S]sulfate (4). Isolation of [35S]glycosaminoglycans from chondroitin-4-sulfate (Fig. 2A and ref. 4). Analysis of [35S]- the cells and the medium showed that mutant and wild-type glycosaminoglycans from pgsD-677 by anion-exchange chro- cultures had accumulated 11,000 and 10,400 cpm of [35S]g- matography showed that they consisted almost entirely of lycosaminoglycans per ug of cell protein, respectively. Both material that was coeluted with chondroitin sulfate (Fig. 2B). strains synthesized similar amounts of [35S]glycosaminogly- All of the [35S]glycosaminoglycans in pgsD-677 were de- cans when cells were labeled with [35S]sulfate for only 4 hr polymerized by chondroitinase ABC, whereas in wild-type of cell in the mutant vs. 300 in (350 cpm/,ug protein cpm/,ug cells, only the material that was eluted at 0.6 M NaCl was the wild type). Separate analyses of the cell and medium depolymerized. Treatment of glycosaminoglycans from both compartments showed that the proteoglycans were distrib- strains with chondroitinase ABC generated disaccharides uted identically by mutant and wild-type cells as well (55- 59% in the medium and 41-45% in the cell layer). that did not bind to the resin and a small amount of material These findings were surprising since colonies ofpgsD-677 that was eluted at 0.35 M NaCl, which may represent core appeared about 3-fold defective in proteoglycan synthesis by protein-carbohydrate linkage regions. Over 90% of the di- autoradiography. Because the measurement of proteoglycan saccharides generated by chondroitinase treatment of sam- synthesis by colony autoradiography employed TCA to pre- ples labeled biosynthetically with [6_3H]glucosamine comi- cipitate [35S]proteoglycans (3), the proteoglycans produced grated on paper chromatograms with a 4,5-unsaturated chon- bypgsD-677 might have been more soluble in TCA than those droitin-4-sulfate disaccharide standard, and the remainder made by wild-type. To test this possibility, cells were incu- comigrated with nonsulfated chondroitin disaccharide (data bated with [35S]sulfate for 4 hr and treated with 10%o TCA to not shown). Thus, pgsD-677 does not make heparan sulfate Downloaded by guest on September 26, 2021 Biochemistry: Lidholt et al. Proc. Natl. Acad. Sci. USA 89 (1992) 2269 teoglycan form of f3-glycan by treatment with chondroitinase ABC. Thus, the mutant produced 83-glycan core protein normally but failed to assemble heparan sulfate chains. Mutant 677 Is Defective in Chain Polymerization. To test whether an early step in heparan sulfate synthesis was altered in pgsD-677, the mutant was fed estradiol /-D-xyloside and briefly labeled with [35S]sulfate (15). When added to pgsA- 745 cells, a CHO mutant defective in xylosyltransferase, estradiol 8-D-xyloside stimulated both heparan sulfate and CY 2 0 chondroitin sulfate (Table 1). In contrast, the addition of the primertopgsD-677 had no effecton heparan sulfate synthesis but did stimulate chondroitin sulfate synthesis. This finding suggested that mutant pgsD-677 was defective in a step downstream from core protein synthesis and xylosylation. 0 0 110 20 30 400 To test whether chain polymerization was altered, assays %I-16 for GlcA-transferase and GlcNAc-transferase were estab- 0 B lished for CHO cells using oligosaccharides derived from E. coli K5 capsular polysaccharide as sugar acceptors (Table 2). In wild-type cell extracts, the extent of GlcNAc and GlcA CL12 transfer was proportional to protein concentration and de- pendent on the addition of acceptor polysaccharide (data not shown). However, in pgsD-677 cell extracts, neither enzyme

8 activity was detectable (Table 2). Mixtures composed of equal amounts of mutant and wild-type extracts contained 58% and 47% ofthe wild-type GlcNAc- and GlcA-transferase activities, respectively, demonstrating that the mutant did 4 not produce a soluble inhibitor. Although pgsD-677 cells lacked both glycosyltransferases, they contained normal levels of N-sulfotransferase (46 pmol of sulfate transferred per min per mg of cell protein in the 0 10 20 30 40 mutant vs. 37 pmol per min per mg in the wild type), as Fraction Number measured by the transfer of [35S]sulfate from 3'-phospho- adenosine 5'-phosphosulfate to N-desulfated heparin prepa- FIG. 2. Anion-exchange HPLC of glycosaminoglycans derived rations (6). Thus, the mutation in pgsD-677 affects chain from wild-type (A) and pgsD-677 (B) cells. Cells were labeled with formation but not a chain modification reaction involved in 3"SO4 (10 uCi/ml) for 3 days. [35S]Glycosaminoglycans were re- heparan sulfate biosynthesis. When the lectin sensitivity of leased from cell and media proteoglycans and collected by anion- the cells was measured (Table 3), only small differences were exchange chromatography and ethanol precipitation (6). A portion of noted, suggesting that the synthesis of Asn-linked oligosac- [35S]glycosaminoglycans was digested with chondroitinase ABC, charides, glycolipids, and other O-linked glycoconjugates are and each sample was analyzed by anion-exchange HPLC (6). The amount of radioactivity in each fraction was normalized to the normal in the mutant as well. equivalent amount ofprotein that had been analyzed. The broken line The Various Phenotypes Are Genetically Linked. To test represents the programmed gradient of NaCl. Filled symbols, un- whether the decline in heparan sulfate synthesis was a treated samples; open symbols, after treatment with chondroitinase recessive trait, pgsD-677 was fused to OT-1, a thioguanine- ABC. resistant, ouabain-resistant subline of wild-type cells. Anal- ysis of the glycosaminoglycan composition of four hybrids and accumulates 3-4 times more chondroitin-4-sulfate than showed that they produced heparan sulfate normally and the wild type. accumulated somewhat more chondroitin sulfate than wild To determine whether the pgsD mutation affected the type or hybrids of wild-type and OT-1 cells (Table 4). One of synthesis of core proteins destined to contain heparan sulfate the hybrids, 6.5, was tested and shown to contain both GlcA- chains, we examined the composition of p-glycan, a proteo- and GlcNAc-transferase (Table 2). The moderate depression glycan that binds transforming growth factor 83 (TGF-3). of enzyme activities in the hybrid compared with wild-type Receptors for TGF-,f were affinity labeled and crosslinked and OT-1 cells may reflect differences in protein content of with 125I-TGF-13 (Fig. 3). Wild-type CHO cells contain three hybrid cells, since the hybrids were noticeably larger than types of TGF-,3 receptors (14). Type I and type II receptors cells of the parental strains. The complete recovery of do not contain glycosaminoglycan chains, whereas type III heparan sulfate synthesis in the hybrids suggests that the lack proteoglycan receptors (J3-glycan) contain mostly heparan of heparan sulfate in the mutant is recessive. sulfate chains. Treatment of the cells with heparitinase prior To obtain evidence that the dual enzyme deficiency and the to affinity labeling and crosslinking shifted >90%o of(3-glycan lack of heparan sulfate were due to the same mutation, from a characteristically heterogeneous species at about 250 segregation of the mutation was studied in hybrid 6.5. About kDa to labeled species migrating at 120-140 kDa. When 20,000 colonies ofhybrid 6.5 were screened by replica plating TGF-P was crosslinked to pgsA-745 cells, a mutant that does and [35S]sulfate colony autoradiography (7). Two clones were not make any glycosaminoglycan chains (3), f8-glycan mi- identified that took up about one-third as much [35S]sulfate as grated as 120- to 140-kDa species. This is the nonproteogly- other colonies (strains 6.5.2 and 6.5.5). Labeling of the cells can form of 8-glycan (14) and is unaffected by heparitinase with [35S]sulfate showed that they did not synthesize any treatment. In pgsD-677 cells >90% of /8-glycan migrated like detectable heparan sulfate and accumulated 2- to 5-fold more the nonproteoglycan form of ,B-glycan found in pgsA-745 or chondroitin sulfate than parental OT-1 cells (Table 4). These in wild-type cells after heparitinase treatment. About 10%o isolates also lacked both GlcNAc- and GlcA-transferase migrated as polydisperse proteoglycan. These proteoglycan activities (Table 2). Although only two isolates were identi- receptors did not change mobility when cells were treated fied, the incidence ofstrains like 6.5.2 and 6.5.5 was very high with heparitinase, but they were converted to the nonpro- (-0.01%) in the hybrid 6.5 cell population. Since strains like Downloaded by guest on September 26, 2021 2270 Biochemistry: Lidholt et al. Proc. Natl. Acad. Sci. USA 89 (1992)

Treatment Wild-type Chondroitinase + Heparitinase

Mr H Type 111 H,- H 100-

- 200 2010

'. ,w ii 11 iA I 1 6 - 116 9. TyJpe 11 - 9? 618 - Offer"" Type I -E: - 68

FIG. 3. Autoradiographic visualization of TGF-f3 crosslinking to receptors. Confluent monolayers of CHO cells in 24-well dishes were incubated at 37°C for 3 hr with no additions (-), with 10 milliunits of chondroitinase ABC, with 100 milliunits of heparitinase, or with a combination of both enzymes as indicated (+). Cells were treated with enzymes in 0.2 ml of Krebs-Ringer solution containing 5 mM MgCl2 and buffered with 20 mM Hepes (pH 7.5). After the cells were rinsed, TGF-P receptors were affinity labeled with 100 pM 125I-TGF-P and processed for SDS/PAGE and autoradiography (14).

these have not been found among wild-type CHO cells never In pgsD-677, a mutation has occurred in a gene involved in treated with mutagen, we infer that they arose from a polymerization of heparan sulfate chains. This mutant lacks segregation-like event (17) that separated the pgsD mutant heparan sulfate because both the GlcNAc- and the GlcA- allele from the wild-type allele. Thus, the findings suggest transferase involved in chain polymerization are altered. that the failure to produce heparan sulfate and the lack ofboth Since chemical mutagenesis was used to increase the likeli- transferases are linked. hood of finding interesting mutants, pgsD-677 could have multiple mutations. However, the probability of obtaining a DISCUSSION strain with mutations in two genes involved in the same CHO cells, like other mammalian cells, produce a mixture of Table 2. GlcNAc- and GIcA-transferase activities sulfate and sulfate and heparan chondroitin proteoglycans, Transfer the overall composition of the mixture remains unchanged (pmol/mg of through many cell generations. By treating cells with a cell protein) chemical mutagen, we obtained stable mutants altered in proteoglycan biosynthesis (3-7). In some cases, mutations Strain GlcNAc GlcA occurred in genes affecting the activity of xylosyltransferase Wild-type (CHO-Ki) 1.8 100 or galactosyltransferase I, enzymes required for the assembly Mutant pgsD-677 <0.03 <0.7 of the carbohydrate-protein linkage tetrasaccharide, Parental OT-1 1.7 74 D-GlcA(,8 ,3)D-Gal( 31 ,3)D-Gal(f31 ,4)D-Xyl-f3-O-[t-Ser], Hybrid 6.5 1.1 30 that links heparan sulfate and chondroitin sulfate chains to (pgsD-677 x OT-1) proteoglycan core proteins. These mutants fail to produce Segregant 6.5.2 <0.03 <0.7 any glycosaminoglycan chains, indicating that different core Segregant 6.5.5 <0.03 <0.7 proteins utilize the same enzymes for the initiation of both GIcNAc-transferase was assayed by incubating 0.1 mM of deca- heparan sulfate and chondroitin sulfate chains (1, 2). saccharide substrate with 0.5 AGCi of UDP-[6-3H]GIcNAc, 10 mM MnCI2, 10 mM MgCl2, 5 mM CaCl2, 1% Triton X-100, 50mM Hepes Table 1. Glycosaminoglycan synthesis in mutant cells (pH 7.2), and 0.25 mg of solubilized cell protein in a total volume of 25 Al. GlcA-transferase was assayed under similar conditions except [35S]Glycosaminoglycan, that 0.5 mM tridecasaccharide and 0.68 AGCi of UDP-[14CJGlcA were (cpm/,ug X 10-3) used. After incubation at 37C for 40 min, 25 tl of 10%6 TCA was added to precipitate protein-bound endogenous acceptors. The mix- Heparan Chondroitin tures were centrifuged, and the supernatants were neutralized with Strain EDX Total sulfate sulfate 14 Al of 1 M NaOH. Carrier heparin (0.5 mg) was added and, after pgsD-677 - 0.58 0.02 0.56 a second centrifugation, the samples were passed through a Sepha- + 2.2 0.02 2.2 dex G-25 column [1 cm (inner diameter) x 40 cm] equilibrated with 1 M Triton were pgsA-745 - 0.02 ND ND NaCl/0.1% X-100/50 mm Tris HCI, pH 8. Fractions collected at a rate of 15 ml/hr and the amount of radioactivity + 1.8 0.50 1.3 recovered in oligosaccharides was divided by the specific radioac- Multiple 60-mm culture dishes were seeded with 2 x 10' cells in 2 tivity of the nucleotide sugar donors. Control incubations in the ml of growth medium. After 1 day, the medium was replaced with 1 absence of added oligosaccharide acceptors yielded <5% of the ml of sulfate-free medium with or without 30 j.M estradiol P-D- counts incorporated into exogenous acceptors. These values were xyloside (EDX, ref. 15). One hour later, 20 ,Ci of 35S04 was added, subtracted from the data obtained from incubations with exogenous and after 3 hr [35(iglycosaminoglycans in the medium and the cells acceptors. The large difference in activity ofthe GlcA- and GlcNAc- were isolated. Amounts of chondroitin sulfate and heparan sulfate transferase is somewhat misleading because the concentration of were determined by chondroitinase ABC digestion and nitrous acid UDP-[3H]GIcNAc was well below its apparent Km (to increase the treatment (15). Average values for duplicate determinations varied yield of 3H counts), whereas the concentration of UDP-[14C]GlcA by <10o. ND, not determined. was within a factor of 2 of its apparent Km. Downloaded by guest on September 26, 2021 Biochemistry: Lidholt et al. Proc. Natl. Acad. Sci. USA 89 (1992) 2271 Table 3. Lectin sensitivity of pgsD-677 and wild-type cells between mutant and wild-type cells synthesize wild-type L-PHA, WGA, Con A, Ricin, LCA, levels of heparan sulfate and still accumulate chondroitin Strain ug/ml iLg/ml Ag/ml tg/ml Ag/ml sulfate, although to a lesser extent than in the original mutant. This suggests that pgsD may define a gene that modulates the Wild type 10 1-3 10-20 0.01-0.025 15-20 expression of enzymes involved in both chondroitin sulfate pgsD-677 10-15 3 15-20 0.025-0.05 20-30 and heparan sulfate synthesis. Additional studies are needed pgsD-677 and wild-type (CHO-Ki) cells were challenged with to establish whether the mutant expresses altered levels of various concentrations of the lectins (16). The indicated values are GlcA- and GalNAc-transferases involved in chondroitin sul- the concentrations of lectin at which cell growth was about 10o of fate that observed in the absence of lectin. The lectins (and their known synthesis. specificities) were as follows: L-PHA, leukoagglutinin from Phase- Strains like pgsD-677 should prove extremely useful for olus vulgaris (galactose in P31,4 branches of complex Asn-linked studying the biological function of heparan sulfate proteo- oligosaccharides); WGA, wheat germ agglutinins from Triticum glycans. It has already been shown that mutants that are vulgaris (terminal sialic acid or GlcNAc residues); Con A, con- grossly deficient in glycosaminoglycan synthesis have altered canavalin A from Canavalia ensiformis (mannose residues); ricin adhesion characteristics (22-24); lack binding sites for throm- toxin from Ricinus communis (terminal galactose or GalNAc resi- bospondin (25), herpes simplex virus (26), and basic fibro- dues); and LCA, Lens culinaris agglutinin (mannose residues in blast growth factor (27); and fail to form tumors in nude mice fucosylated Asn-linked oligosaccharides). (28). Use of strains like pgsD-677 will make it possible to pathway seems unlikely. pgsD-803, another member of the study whether these and other biological properties depend pgsD complementation group (Fig. 1), also displays the dual specifically on heparan sulfate proteoglycans. enzyme deficiency expressed by pgsD-677 (data not shown). We thank Dr. Pamela Stanley (Albert Einstein) for analysis of the Since pgsD-803 was obtained from a different population of iectin sensitivity ofthe strains and Drs. K. Rostand, R. Montgomery, mutagenized cells, it seems unlikely that the identical pair of and R. LeBaron for many helpful discussions regarding this work. This research was supported by Grants GM33063 and CA46462 from mutations would have occurred in both strains. However, the National Institutes of Health (J.D.E.), by Grant 2309 from the revertants of the mutants are needed to exclude this possi- Swedish Medical Research Council (U.L.), and by Grant CA34610 bility with certainty. from the National Institutes of Health (J.M.). Other more intriguing explanations should be considered 1. Lindahl, U. & Kjellen, L. (1991) Annu. Rev. Biochem. 60, 443-475. for the dual enzyme deficiency. The pgsD locus could encode 2. Esko, J. D. (1991) Curr. Opin. Cell Biol. 3, 805-816. a factor that regulates the expression of both glycosyltrans- 3. Esko, J. D., Stewart, T. E. & Taylor, W: H. (1985) Proc. Natl. ferase genes. Alternatively, if the two enzymes formed Acad. Sci. USA 82, 3197-3201. complexes required for catalytic activity, then a mutation in 4. Esko, J. D., Elgavish, A., Prasthofer, T., Taylor, W. H. & Weinke, J. L. (1986) J. Biol. Chem. 261, 15725-15733. a shared subunit or in one of the enzymes could alter the 5. Esko, J. D., Weinke, J. L., Taylor, W. H., Ekborg, G., Roddn, L., activity of the entire complex. A third possibility is that both Anantharamaiah, G. & Gawish, A. (1987) J. Biol. Chem. 262, enzyme activities are associated in a single protein. Although 12189-12195. GlcNAc- and GicA-transferase have been solubilized, they 6. Bame, K. J. & Esko, J. D. (1989) J. Biol. Chem. 264, 8059-8065. 7. Esko, J. D. (1989) Methods Cell Biol. 32, 387-422. have not been purified to homogeneity (18, 19). Thus, infor- 8. Ham, R. G. (1965) Proc. Natl. Acad. Sci. USA 53, 288-293. mation about their subunit structure is not yet available. 9. Davidson, R. L. & Gerald, P. S. (1976) Somatic Cell Genet. 2, Recent genetic and biochemical studies ofthe heparan sulfate 165-176. modification enzymes N-sulfotransferase and N-deacetylase 10. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. suggest that they may exist in complexes whose integrity is (1951) J. Biol. Chem. 193, 265-275. 11. Vann, W. F., Schmidt, M. A., Jann, B. & Jann, K. (1981) Eur. J. essential for N-deacetylation to occur (20, 21). Biochem. 116, 359-364. The accumulation of chondroitin sulfate in pgsD mutants 12. Shively, J. E. & Conrad, H. E. (1976) Biochemistry 15, 3932-3942. also is intriguing. It may simply reflect the occurrence of 13. Jacobsson, I., Backstr6m, G., Hook, M., Lindahl, U., Feingold, excess UDP-sugars in the mutant caused by the cessation of D. S., Malmstr6m, A. & Roden, L. (1979) J. Biol. Chem. 254, heparan sulfate synthesis. However, cell hybrids prepared 2975-2982. 14. Cheifetz, S. & Massague, J. (1989) J. Biol. Chem. 264, 12025-12028. 15. Lugemwa, F. N. & Esko, J. D. (1991) J. Biol. Chem. 266, 6674- Table 4. Glycosaminoglycan content of hybrids and segregants 6677. [35S]Glycosaminoglycan, 16. Stanley, P. (1985) in Molecular Cell Genetics, ed. Gottesman, (cpm/,ug x 10-3) M. M. (Wiley Interscience, New York), pp. 745-772. 17. Worton, R. G. & Grant, S. G. (1985) in Molecular Cell Genetics, ed. Heparan Chondroitin Gottesman, M. M. (Wiley Interscience, New York), pp. 831-867. Strain Total sulfate sulfate 18. Helting, T. (1972) J. Biol. Chem. 247, 4327-4332. 19. Forsee, W. T. & Roden, L. (1981) J. Biol. Chem. 256, 7240-7247. Cell hybrid experiment 20. Pettersson, I., Kusche, M., Unger, E., Wlad, H., Nylund, L., WT (CHO-Ki) 5.8 ± 0.2 4.2 ± 0.1 1.6 ± 0.1 Lindahl, U. & Kjellen, L. (1991) J. Biol. Chem. 266, 8044-8049. W x OT-1 (n = 5) 6.9 ± 1.2 4.9 ± 0.9 2.0 ± 0.4 21. Bame, K. J., Reddy, R. V. & Esko, J. D. (1991) J. Biol. Chem. 266, pgsD-677 x OT-1 (n = 4) 7.0 ± 0.7 3.8 ± 0.4 3.3 ± 0.4 12461-12468. Segregation experiment 22. LeBaron, R. G., Esko, J. D., Woods, A., Johansson, S. & Hook, M. (1988) J. Cell Biol. 106, 945-952. Parental OT-1 3.3 2.5 0.8 23. Kaesberg, P. R., Ershler, W. B., Esko, J. D. & Mosher, D. F. Hybrid 6.5 3.9 1.7 2.2 (1989) J. Clin. Invest. 83, 994-1001. Segregant 6.5.2 4.2 0 4.2 24. LeBaron, R. G., Hook, A., Esko, J. D., Gay, S. & Hook, M. (1989) Segregant 6.5.5 1.6 0 1.6 J. Biol. Chem. 264, 7950-7956. 25. Murphy-Ullrich, J. E., Westrick, L. G., Esko, J. D. & Mosher, Cell hybrids and segregants were generated as described in Ex- D. F. (1988) J. Biol. Chem. 263, 6400-6406. perimental Procedures. Strains were labeled with [35S]sulfate (10 26. Shieh, M.-T., WuDunn, D., Montgomery, R. I., Esko, J. D. & ,uCi/ml) for 3 days, and radioactive glycosaminoglycans were ana- Spear, P. G. (1992) J. Cell Biol. 283, 208-209. lyzed. n denotes the number of individual hybrid strains that were 27. Yayon, A., Klagsbrun, M., Esko, J. D., Leder, P. & Ornitz, D. M. examined. Each strain was analyzed in duplicate and the mean values (1991) Cell 64, 841-848. (± 1 SD) obtained for all strains within each group are given. WT, 28. Esko, J. D., Rostand, K. S. & Weinke, J. L. (1988) Science 241, wild type. 1092-1096. Downloaded by guest on September 26, 2021