Proc. Natl. Acad. Sci. USAVol. 89, pp. 2267-2271, March 1992Biochemistry

A single mutation affects both N-acetylglucosaminyltransferase andglucuronosyltransferase activities in a Chinese hamster ovary cellmutant 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 IItDepartment of Biochemistry, Schools of Medicine and Dentistry, University of Alabama, Birmingham, AL 35294; *Depaltment of Veterinary MedicalChemistry, The Biomedical Center, Swedish University of Agricultural Sciences, S-751 23, Uppsala, Sweden; and §Department of Cell Biologyand 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 havebeen found that no longer produce heparan sulfate. Charac-terization of one of the mutants, pgsD-677, showed that it lacksboth N-acetylglucosaminyl- and glucuronosyltransferase, en-zymes required for the polymerization of heparan sulfatechains. pgsD-677 also accumulates 3- to 4-fold more chon-droitin sulfate than the wild type. Cell hybrids derived frompgsD-677 and wild type regained both transferase activities andthe capacity to synthesize heparan sulfate. Two segregantsfrom one of the hybrids reexpressed the dual enzyme defi-ciency, the lack of heparan sulfate synthesis, and the enhancedaccumulation of chondroitin sulfate, suggesting that all of thetraits were genetically linked. These fin gs indicate that thepgsD locus may represent a gene involved in the coordinatecontrol of glycosaminoglycan formation.

Proteoglycans consist of a core protein and one or morecovalently attached glycosaminoglycan chains. Typical ani-mal cells produce proteoglycans bearing chondroitin (derma-tan) sulfate or heparan sulfate chains, but the compositionvaries considerably among different cells (1, 2). To study theregulation of proteoglycan composition, we have isolatedChinese hamster ovary (CHO) cell mutants defective inglycosaminoglycan biosynthesis (3-6). Many of these mu-tants bear mutations in genes involved in the formation ofboth heparan sulfate and chondroitin sulfate chains (3, 5).Here we describe a CHO cell mutant, pgsD-677, that spe-cifically lacks heparan sulfate. The mutation in pgsD-677affects both N-acetylglucosaminyl (GlcNAc)- and glucurono-syl (GlcA)-transferase activities required for heparan sulfatepolymerization, suggesting that some form of coordinateregulation of these enzymes exists.

EXPERIMENTAL PROCEDURESCell Cultures. CHO cells (CHO-Ki) were obtained from

the American Type Culture Collection (CCL-61). All mutantswere identified by colony autoradiography (7), and the purityof each strain was ensured by its isolation from culturescontaining only mutant colonies. Cells were maintained inHam's F12 (8) medium (Mediatech, Washington) supple-mented with 10% (vol/vol) fetal bovine serum (HyClone) orin sulfate-deficient medium (4).

Cell fusion studies required the isolation of a CHO-K1subline resistant to thioguanine and ouabain (OT-1). Wild-type cells were treated with 10 ,uM 6-thioguanine in hypo-xanthine-free F12 medium supplemented with dialyzed fetal

bovine serum. A resistant mutant was isolated and thentreated with mutagen (7), and a ouabain-resistant clone wasselected in growth medium containing 1 mM ouabain. Theintroduction of these markers did not alter the proteoglycancomposition of the cells.

Cell hybrids were generated by co-plating 2 x 105 cells ofpgsD-677 and OT-1 in individual wells of a 24-well plate.After overnight incubation, the mixed monolayers weretreated for 1 min with 50% (wt/wt) poly(ethylene glycol)(PEG 3320) prepared in F12 medium without serum (9). After1 day the cells were harvested with trypsin, and multiple100-mm-diameter tissue culture plates were seeded withabout 103 cells in F12 medium containing 10 ALM aminopterinand 1 mM ouabain to counterselect parental cells. One daylater the medium was changed to remove dead cells, andthose remaining on the dish were overlaid with Whatman no.42 filter paper in order to obtain discrete colonies (7). Ninedays later, the disk was removed and resistant clones werepicked with glass cloning cylinders and trypsin. The inci-dence of drug-resistant colonies indicated that the hybridiza-tion efficiency was at least 1%. When each parental strainwas fused to itself, colonies of resistant cells were not found.To obtain segregants, about 20,000 colonies of hybrid 6.5

(pgsD-677 x OT-1) were screened by 35S autoradiography forthose exhibiting reduced incorporation of [35S]sulfate (7).Two strains (6.5.2 and 6.5.5) were identified in this mannerand repurified by replica plating.

Radiolabeling Studies. Na235SO4 (25-40 Ci/mg; 1 Ci = 37GBq) and D-[6-3H]glucosamine hydrochloride (40 Ci/mmol)were purchased from Amersham. Glycosaminoglycans werelabeled biosynthetically by incubating cells in sulfate-deficient medium containing [35S]sulfate (10-20 ,Ci/ml) orD-[6-3H]glucosamine (10 ,uCi/ml). The medium was removedand the cells were harvested in a small volume of 0.1 MNaOH. A portion of the alkaline cell extracts was used for thedetermination of protein by the method of Lowry et al. (10)with bovine serum albumin as standard. The cell extracts andmedia samples were digested with protease, and radioactiveglycosaminoglycans were purified by ion-exchange chroma-tography and ethanol precipitation (6). The disaccharidecomposition of chondroitin sulfate was determined by paper

Abbreviations: TCA, trichloroacetic acid; TGF-f3, transforminggrowth factor ,B.tPresent address: School of Basic Life Sciences, Division of Mo-lecular Biology and Biochemistry, University of Missouri, KansasCity, MO 64110.Present address: Department of Medical and Physiological Chem-istry, The Biomedical Center, University of Uppsala, S-751 23,Uppsala, Sweden.'1To whom reprint requests should be addressed.

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chromatography (4) using authentic standards (SeikagakuAmerica, St. Petersburg, FL).Enzyme Assays. N-Sulfotransferase was assayed using

N-desulfoheparin as substrate (6). GlcNAc- and GIcA-transferase were assayed using oligosaccharide acceptorsprepared from the capsular polysaccharide of Escherichiacoli K5 (11). The polysaccharide was partially N-deacety-lated with hydrazine and subjected to deaminative cleavagewith nitrous acid at pH 3.9 (12). The resulting mixture ofoligosaccharides, all having GlcA at their nonreducing ter-mini, was fractionated by gel filtration chromatography. Thedecasaccharide fraction was used as substrate for GlcNAc-transferase. Digestion of a tetradecasaccharide fraction withB3-D-glucuronidase yielded tridecasaccharides with nonre-ducing terminal GlcNAc residues, suitable as substrates forGlcA-transferase. Enzyme preparations were obtained bysolubilization of about 2 x 107 cells with 0.5 ml of 1%(vol/vol) Triton X-100/50 mM Tris-HCl, pH 7.2, containingphenylmethylsulfonyl fluoride (1 mM) and pepstatin (10pug/ml). After 30 min of gentle agitation at 40C, the sampleswere centrifuged. The supernatants were assayed for glyco-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

Identification of Heparan Sulfate-Deficient Mutants. A pre-vious study described a screening method for detectingmutants defective in proteoglycan biosynthesis (3). Thistechnique involves the transfer of CHO colonies from plastictissue culture dishes to disks of polyester cloth (7). Thetransferred colonies are incubated with [35S]sulfate, and theincorporation of radioactivity into trichloroacetic acid(TCA)-precipitable proteoglycans is measured by autora-diography. Mutant colonies defective in proteoglycan syn-thesis yield a reduced signal on the film and can be retrievedfrom the original plastic dish from which the replica wasgenerated. Several mutants exhibited a partial reductionin [35S]sulfate incorporation into proteoglycans, and in somecases this was due to incomplete inhibition of a specificenzyme in the biosynthetic pathway (e.g., ref. 6). Cellhybridization studies showed that four of these partial mu-tants (strains 623, 625, 677, and 803) comprised a newcomplementation group, pgsD (Fig. 1). One ofthese mutants,pgsD-677, was selected for further analysis.pgsD-677 exhibited an 3-fold reduction in proteoglycan

synthesis by autoradiography. To obtain more quantitativedata, cells were labeled to constant specific radioactivity([35S]glycosaminoglycan per ,ug of cell protein) by growingthem for 3 days in sulfate-deficient medium containing[35S]sulfate (4). Isolation of [35S]glycosaminoglycans fromthe cells and the medium showed that mutant and wild-typecultures had accumulated 11,000 and 10,400 cpm of [35S]g-lycosaminoglycans per ug of cell protein, respectively. Bothstrains synthesized similar amounts of [35S]glycosaminogly-cans when cells were labeled with [35S]sulfate for only 4 hr(350 cpm/,ug of cell protein in the mutant vs. 300 cpm/,ug inthe wild type). Separate analyses of the cell and mediumcompartments showed that the proteoglycans were distrib-uted identically by mutant and wild-type cells as well (55-59% in the medium and 41-45% in the cell layer).These findings were surprising since colonies ofpgsD-677

appeared about 3-fold defective in proteoglycan synthesis byautoradiography. Because the measurement of proteoglycansynthesis by colony autoradiography employed TCA to pre-cipitate [35S]proteoglycans (3), the proteoglycans producedbypgsD-677 might have been more soluble inTCA than thosemade by wild-type. To test this possibility, cells were incu-bated with [35S]sulfate for 4 hr and treated with 10%o TCA to

a** .*~~~

803 x 745 745 x 745

FIG. 1. Autoradiographic analysis of cell hybrids. Mixed mono-layers of the indicated strains were treated with poly(ethylene glycol)to induce cell fusion. The treated cells were replated into 100-mmtissue culture dishes to obtain 300-1000 colonies per dish. After 9days, the colonies were labeled for 4 hr with 35SO4 and radioactiveproteoglycans were precipitated in situ with TCA. The bottom of thedish was excised and exposed to x-ray film. Complementation hadoccurred if occasional colonies yielded a strong signal comparable tothat given by wild-type colonies (not shown).

precipitate 35S-labeled macromolecules. About 200 cpm of35S-labeled material per ,ug of cell protein precipitated in themutant, whereas 900 cpm/,ug precipitated in the wild type.Thus, the enhanced solubility of pgsD-677 proteoglycans inTCA explained the reduced autoradiographic signal gener-ated by mutant colonies.pgsD-677 Is Defective in Heparan Sulfate Biosynthesis. Wild-

type CHO cells produce about 70% heparan sulfate and 30%6chondroitin-4-sulfate (Fig. 2A and ref. 4). Analysis of [35S]-glycosaminoglycans from pgsD-677 by anion-exchange chro-matography showed that they consisted almost entirely ofmaterial that was coeluted with chondroitin sulfate (Fig. 2B).All of the [35S]glycosaminoglycans in pgsD-677 were de-polymerized by chondroitinase ABC, whereas in wild-typecells, only the material that was eluted at 0.6 M NaCl was

depolymerized. Treatment of glycosaminoglycans from bothstrains with chondroitinase ABC generated disaccharidesthat did not bind to the resin and a small amount of materialthat was eluted at 0.35 M NaCl, which may represent coreprotein-carbohydrate linkage regions. Over 90% of the di-saccharides generated by chondroitinase treatment of sam-

ples labeled biosynthetically with [6_3H]glucosamine comi-grated on paper chromatograms with a 4,5-unsaturated chon-droitin-4-sulfate disaccharide standard, and the remaindercomigrated with nonsulfated chondroitin disaccharide (datanot shown). Thus, pgsD-677 does not make heparan sulfate

677 x 8O 677 x 677

9

677 x 745 803 x 803

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CY2 0

01 00 10 20 30 40

%I-160

B

CL12

8

4

0 10 20 30 40

Fraction Number

FIG. 2. Anion-exchange HPLC of glycosaminoglycans derivedfrom wild-type (A) and pgsD-677 (B) cells. Cells were labeled with3"SO4 (10 uCi/ml) for 3 days. [35S]Glycosaminoglycans were re-leased from cell and media proteoglycans and collected by anion-exchange chromatography and ethanol precipitation (6). A portion of[35S]glycosaminoglycans was digested with chondroitinase ABC,and each sample was analyzed by anion-exchange HPLC (6). Theamount of radioactivity in each fraction was normalized to theequivalent amount of protein that had been analyzed. The broken linerepresents the programmed gradient of NaCl. Filled symbols, un-treated samples; open symbols, after treatment with chondroitinaseABC.

and accumulates 3-4 times more chondroitin-4-sulfate thanthe wild type.To determine whether the pgsD mutation affected the

synthesis of core proteins destined to contain heparan sulfatechains, we examined the composition of p-glycan, a proteo-glycan that binds transforming growth factor 83 (TGF-3).Receptors for TGF-,f were affinity labeled and crosslinkedwith 125I-TGF-13 (Fig. 3). Wild-type CHO cells contain threetypes of TGF-,3 receptors (14). Type I and type II receptorsdo not contain glycosaminoglycan chains, whereas type IIIproteoglycan receptors (J3-glycan) contain mostly heparansulfate chains. Treatment of the cells with heparitinase priorto affinity labeling and crosslinking shifted >90%o of(3-glycanfrom a characteristically heterogeneous species at about 250kDa to labeled species migrating at 120-140 kDa. WhenTGF-P was crosslinked to pgsA-745 cells, a mutant that doesnot make any glycosaminoglycan chains (3), f8-glycan mi-grated as 120- to 140-kDa species. This is the nonproteogly-can form of 8-glycan (14) and is unaffected by heparitinasetreatment. In pgsD-677 cells >90% of /8-glycan migrated likethe nonproteoglycan form of ,B-glycan found in pgsA-745 orin wild-type cells after heparitinase treatment. About 10%omigrated as polydisperse proteoglycan. These proteoglycanreceptors did not change mobility when cells were treatedwith heparitinase, but they were converted to the nonpro-

teoglycan form of f3-glycan by treatment with chondroitinaseABC. Thus, the mutant produced 83-glycan core proteinnormally 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 alteredin pgsD-677, the mutant was fed estradiol /-D-xyloside andbriefly 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 andchondroitin sulfate (Table 1). In contrast, the addition of theprimertopgsD-677 had no effecton heparan sulfate synthesisbut did stimulate chondroitin sulfate synthesis. This findingsuggested that mutant pgsD-677 was defective in a stepdownstream from core protein synthesis and xylosylation.To test whether chain polymerization was altered, assays

for GlcA-transferase and GlcNAc-transferase were estab-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 GlcAtransfer was proportional to protein concentration and de-pendent on the addition of acceptor polysaccharide (data notshown). However, in pgsD-677 cell extracts, neither enzymeactivity was detectable (Table 2). Mixtures composed ofequal amounts of mutant and wild-type extracts contained58% and 47% of the wild-type GlcNAc- and GlcA-transferaseactivities, respectively, demonstrating that the mutant didnot produce a soluble inhibitor.Although pgsD-677 cells lacked both glycosyltransferases,

they contained normal levels of N-sulfotransferase (46 pmolof sulfate transferred per min per mg of cell protein in themutant vs. 37 pmol per min per mg in the wild type), asmeasured by the transfer of [35S]sulfate from 3'-phospho-adenosine 5'-phosphosulfate to N-desulfated heparin prepa-rations (6). Thus, the mutation in pgsD-677 affects chainformation but not a chain modification reaction involved inheparan sulfate biosynthesis. When the lectin sensitivity ofthe cells was measured (Table 3), only small differences werenoted, suggesting that the synthesis of Asn-linked oligosac-charides, glycolipids, and other O-linked glycoconjugates arenormal in the mutant as well.The Various Phenotypes Are Genetically Linked. To test

whether the decline in heparan sulfate synthesis was arecessive trait, pgsD-677 was fused to OT-1, a thioguanine-resistant, ouabain-resistant subline of wild-type cells. Anal-ysis of the glycosaminoglycan composition of four hybridsshowed that they produced heparan sulfate normally andaccumulated somewhat more chondroitin sulfate than wildtype or hybrids of wild-type and OT-1 cells (Table 4). One ofthe hybrids, 6.5, was tested and shown to contain both GlcA-and GlcNAc-transferase (Table 2). The moderate depressionof enzyme activities in the hybrid compared with wild-typeand OT-1 cells may reflect differences in protein content ofhybrid cells, since the hybrids were noticeably larger thancells of the parental strains. The complete recovery ofheparan sulfate synthesis in the hybrids suggests that the lackof heparan sulfate in the mutant is recessive.To obtain evidence that the dual enzyme deficiency and the

lack of heparan sulfate were due to the same mutation,segregation of the mutation was studied in hybrid 6.5. About20,000 colonies of hybrid 6.5 were screened by replica platingand [35S]sulfate colony autoradiography (7). Two clones wereidentified that took up about one-third as much [35S]sulfate asother colonies (strains 6.5.2 and 6.5.5). Labeling of the cellswith [35S]sulfate showed that they did not synthesize anydetectable heparan sulfate and accumulated 2- to 5-fold morechondroitin sulfate than parental OT-1 cells (Table 4). Theseisolates also lacked both GlcNAc- and GlcA-transferaseactivities (Table 2). Although only two isolates were identi-fied, the incidence of strains like 6.5.2 and 6.5.5 was very high(-0.01%) in the hybrid 6.5 cell population. Since strains like

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I 1

9.

61

Treatment Wild-typeChondroitinase +

Heparitinase

Type 111

0

6

TyJpe 11

H,- H 100-

- 200

'.

,w ii 11 iA- 116

- 9?

- 68Type I

FIG. 3. Autoradiographic visualization of TGF-f3 crosslinking to receptors. Confluent monolayers of CHO cells in 24-well dishes wereincubated at 37°C for 3 hr with no additions (-), with 10 milliunits of chondroitinase ABC, with 100 milliunits of heparitinase, or with acombination of both enzymes as indicated (+). Cells were treated with enzymes in 0.2 ml of Krebs-Ringer solution containing 5 mM MgCl2and 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 andprocessed for SDS/PAGE and autoradiography (14).

these have not been found among wild-type CHO cells nevertreated with mutagen, we infer that they arose from a

segregation-like event (17) that separated the pgsD mutantallele from the wild-type allele. Thus, the findings suggestthat the failure to produce heparan sulfate and the lack ofbothtransferases are linked.

DISCUSSIONCHO cells, like other mammalian cells, produce a mixture ofheparan sulfate and chondroitin sulfate proteoglycans, andthe overall composition of the mixture remains unchangedthrough many cell generations. By treating cells with achemical mutagen, we obtained stable mutants altered inproteoglycan biosynthesis (3-7). In some cases, mutationsoccurred in genes affecting the activity of xylosyltransferaseor galactosyltransferase I, enzymes required for the assemblyof the carbohydrate-protein linkage tetrasaccharide,D-GlcA(,8 ,3)D-Gal( 31 ,3)D-Gal(f31 ,4)D-Xyl-f3-O-[t-Ser],that links heparan sulfate and chondroitin sulfate chains toproteoglycan core proteins. These mutants fail to produceany glycosaminoglycan chains, indicating that different coreproteins utilize the same enzymes for the initiation of bothheparan sulfate and chondroitin sulfate chains (1, 2).

Table 1. Glycosaminoglycan synthesis in mutant cells

[35S]Glycosaminoglycan,(cpm/,ug X 10-3)Heparan Chondroitin

Strain EDX Total sulfate sulfate

pgsD-677 - 0.58 0.02 0.56+ 2.2 0.02 2.2

pgsA-745 - 0.02 ND ND+ 1.8 0.50 1.3

Multiple 60-mm culture dishes were seeded with 2 x 10' cells in 2ml of growth medium. After 1 day, the medium was replaced with 1ml of sulfate-free medium with or without 30 j.M estradiol P-D-xyloside (EDX, ref. 15). One hour later, 20 ,Ci of 35S04 was added,and after 3 hr [35(iglycosaminoglycans in the medium and the cellswere isolated. Amounts of chondroitin sulfate and heparan sulfatewere determined by chondroitinase ABC digestion and nitrous acidtreatment (15). Average values for duplicate determinations variedby <10o. ND, not determined.

In pgsD-677, a mutation has occurred in a gene involved inpolymerization of heparan sulfate chains. This mutant lacksheparan sulfate because both the GlcNAc- and the GlcA-transferase involved in chain polymerization are altered.Since chemical mutagenesis was used to increase the likeli-hood of finding interesting mutants, pgsD-677 could havemultiple mutations. However, the probability of obtaining astrain with mutations in two genes involved in the same

Table 2. GlcNAc- and GIcA-transferase activitiesTransfer

(pmol/mg ofcell protein)

Strain GlcNAc GlcA

Wild-type (CHO-Ki) 1.8 100Mutant pgsD-677 <0.03 <0.7Parental OT-1 1.7 74Hybrid 6.5 1.1 30(pgsD-677 x OT-1)

Segregant 6.5.2 <0.03 <0.7Segregant 6.5.5 <0.03 <0.7

GIcNAc-transferase was assayed by incubating 0.1 mM of deca-saccharide substrate with 0.5 AGCi of UDP-[6-3H]GIcNAc, 10 mMMnCI2, 10mM MgCl2, 5 mM CaCl2, 1% Triton X-100, 50mM Hepes(pH 7.2), and 0.25 mg of solubilized cell protein in a total volume of25 Al. GlcA-transferase was assayed under similar conditions exceptthat 0.5 mM tridecasaccharide and 0.68 AGCi of UDP-[14CJGlcA wereused. After incubation at 37C for 40 min, 25 tl of 10%6 TCA wasadded to precipitate protein-bound endogenous acceptors. The mix-tures were centrifuged, and the supernatants were neutralized with14 Al of 1 M NaOH. Carrier heparin (0.5 mg) was added and, aftera second centrifugation, the samples were passed through a Sepha-dex G-25 column [1 cm (inner diameter) x 40 cm] equilibrated with1 M NaCl/0.1% Triton X-100/50mm Tris HCI, pH 8. Fractions werecollected at a rate of 15 ml/hr and the amount of radioactivityrecovered in oligosaccharides was divided by the specific radioac-tivity of the nucleotide sugar donors. Control incubations in theabsence of added oligosaccharide acceptors yielded <5% of thecounts incorporated into exogenous acceptors. These values weresubtracted from the data obtained from incubations with exogenousacceptors. The large difference in activity of the GlcA- and GlcNAc-transferase is somewhat misleading because the concentration ofUDP-[3H]GIcNAc was well below its apparent Km (to increase theyield of 3H counts), whereas the concentration of UDP-[14C]GlcAwas within a factor of 2 of its apparent Km.

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WGA, Con A,iLg/ml Ag/ml1-3 10-203 15-20

Ricin,tg/ml

0.01-0.0250.025-0.05

LCA,Ag/ml15-2020-30

pgsD-677 and wild-type (CHO-Ki) cells were challenged withvarious concentrations of the lectins (16). The indicated values arethe concentrations of lectin at which cell growth was about 10o ofthat observed in the absence of lectin. The lectins (and their knownspecificities) were as follows: L-PHA, leukoagglutinin from Phase-olus vulgaris (galactose in P31,4 branches of complex Asn-linkedoligosaccharides); WGA, wheat germ agglutinins from Triticumvulgaris (terminal sialic acid or GlcNAc residues); Con A, con-canavalin A from Canavalia ensiformis (mannose residues); ricintoxin from Ricinus communis (terminal galactose or GalNAc resi-dues); and LCA, Lens culinaris agglutinin (mannose residues infucosylated Asn-linked oligosaccharides).

pathway seems unlikely. pgsD-803, another member of thepgsD complementation group (Fig. 1), also displays the dualenzyme deficiency expressed by pgsD-677 (data not shown).Since pgsD-803 was obtained from a different population ofmutagenized cells, it seems unlikely that the identical pair ofmutations would have occurred in both strains. However,revertants of the mutants are needed to exclude this possi-bility with certainty.

Other more intriguing explanations should be consideredfor the dual enzyme deficiency. ThepgsD locus could encodea factor that regulates the expression of both glycosyltrans-ferase genes. Alternatively, if the two enzymes formedcomplexes required for catalytic activity, then a mutation ina shared subunit or in one of the enzymes could alter theactivity of the entire complex. A third possibility is that bothenzyme activities are associated in a single protein. AlthoughGlcNAc- and GicA-transferase have been solubilized, theyhave not been purified to homogeneity (18, 19). Thus, infor-mation about their subunit structure is not yet available.Recent genetic and biochemical studies ofthe heparan sulfatemodification enzymes N-sulfotransferase and N-deacetylasesuggest that they may exist in complexes whose integrity isessential for N-deacetylation to occur (20, 21).The accumulation of chondroitin sulfate in pgsD mutants

also is intriguing. It may simply reflect the occurrence ofexcess UDP-sugars in the mutant caused by the cessation ofheparan sulfate synthesis. However, cell hybrids prepared

Table 4. Glycosaminoglycan content of hybrids and segregants

[35S]Glycosaminoglycan,(cpm/,ug x 10-3)

Heparan ChondroitinStrain Total sulfate sulfate

Cell hybrid experimentWT (CHO-Ki) 5.8 ± 0.2 4.2 ± 0.1 1.6 ± 0.1W x OT-1 (n = 5) 6.9 ± 1.2 4.9 ± 0.9 2.0 ± 0.4pgsD-677 x OT-1 (n = 4) 7.0 ± 0.7 3.8 ± 0.4 3.3 ± 0.4

Segregation experimentParental OT-1 3.3 2.5 0.8Hybrid 6.5 3.9 1.7 2.2Segregant 6.5.2 4.2 0 4.2Segregant 6.5.5 1.6 0 1.6

Cell hybrids and segregants were generated as described in Ex-perimental Procedures. Strains were labeled with [35S]sulfate (10,uCi/ml) for 3 days, and radioactive glycosaminoglycans were ana-lyzed. n denotes the number of individual hybrid strains that wereexamined. Each strain was analyzed in duplicate and the mean values(± 1 SD) obtained for all strains within each group are given. WT,wild type.

between mutant and wild-type cells synthesize wild-typelevels of heparan sulfate and still accumulate chondroitinsulfate, although to a lesser extent than in the original mutant.This suggests that pgsD may define a gene that modulates theexpression of enzymes involved in both chondroitin sulfateand heparan sulfate synthesis. Additional studies are neededto establish whether the mutant expresses altered levels ofGlcA- and GalNAc-transferases involved in chondroitin sul-fate synthesis.

Strains like pgsD-677 should prove extremely useful forstudying the biological function of heparan sulfate proteo-glycans. It has already been shown that mutants that aregrossly deficient in glycosaminoglycan synthesis have alteredadhesion characteristics (22-24); lack binding sites for throm-bospondin (25), herpes simplex virus (26), and basic fibro-blast growth factor (27); and fail to form tumors in nude mice(28). Use of strains like pgsD-677 will make it possible tostudy whether these and other biological properties dependspecifically on heparan sulfate proteoglycans.We thank Dr. Pamela Stanley (Albert Einstein) for analysis of the

iectin sensitivity of the strains and Drs. K. Rostand, R. Montgomery,and R. LeBaron for many helpful discussions regarding this work.This research was supported by Grants GM33063 and CA46462 fromthe National Institutes of Health (J.D.E.), by Grant 2309 from theSwedish Medical Research Council (U.L.), and by Grant CA34610from the National Institutes of Health (J.M.).1. Lindahl, U. & Kjellen, L. (1991) Annu. Rev. Biochem. 60, 443-475.2. Esko, J. D. (1991) Curr. Opin. Cell Biol. 3, 805-816.3. Esko, J. D., Stewart, T. E. & Taylor, W: H. (1985) Proc. Natl.

Acad. Sci. USA 82, 3197-3201.4. Esko, J. D., Elgavish, A., Prasthofer, T., Taylor, W. H. & Weinke,

J. L. (1986) J. Biol. Chem. 261, 15725-15733.5. Esko, J. D., Weinke, J. L., Taylor, W. H., Ekborg, G., Roddn, L.,

Anantharamaiah, G. & Gawish, A. (1987) J. Biol. Chem. 262,12189-12195.

6. Bame, K. J. & Esko, J. D. (1989) J. Biol. Chem. 264, 8059-8065.7. Esko, J. D. (1989) Methods Cell Biol. 32, 387-422.8. Ham, R. G. (1965) Proc. Natl. Acad. Sci. USA 53, 288-293.9. Davidson, R. L. & Gerald, P. S. (1976) Somatic Cell Genet. 2,

165-176.10. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.

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Table 3. Lectin sensitivity of pgsD-677 and wild-type cells

L-PHA,Strain ug/ml

Wild type 10pgsD-677 10-15

Biochemistry: Lidholt et al.

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