and Genetics, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Communicated by Marilyn G. Farquhar, December 10, 1991.
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 and Genetics, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021
§Department of Cell Biology
Communicated by Marilyn G. Farquhar, December 10, 1991
bovine serum. A resistant mutant was isolated and then treated with mutagen (7), and a ouabain-resistant clone was selected in growth medium containing 1 mM ouabain. The introduction of these markers did not alter the proteoglycan composition of the cells. Cell hybrids were generated by co-plating 2 x 105 cells of pgsD-677 and OT-1 in individual wells of a 24-well plate. After overnight incubation, the mixed monolayers were treated for 1 min with 50% (wt/wt) poly(ethylene glycol) (PEG 3320) prepared in F12 medium without serum (9). After 1 day the cells were harvested with trypsin, and multiple 100-mm-diameter tissue culture plates were seeded with about 103 cells in F12 medium containing 10 ALM aminopterin and 1 mM ouabain to counterselect parental cells. One day later the medium was changed to remove dead cells, and those remaining on the dish were overlaid with Whatman no. 42 filter paper in order to obtain discrete colonies (7). Nine days later, the disk was removed and resistant clones were picked with glass cloning cylinders and trypsin. The incidence of drug-resistant colonies indicated that the hybridization efficiency was at least 1%. When each parental strain was 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 for those exhibiting reduced incorporation of [35S]sulfate (7). Two strains (6.5.2 and 6.5.5) were identified in this manner and repurified by replica plating. Radiolabeling Studies. Na235SO4 (25-40 Ci/mg; 1 Ci = 37 GBq) and D-[6-3H]glucosamine hydrochloride (40 Ci/mmol) were purchased from Amersham. Glycosaminoglycans were labeled biosynthetically by incubating cells in sulfatedeficient medium containing [35S]sulfate (10-20 ,Ci/ml) or D-[6-3H]glucosamine (10 ,uCi/ml). The medium was removed and the cells were harvested in a small volume of 0.1 M NaOH. A portion of the alkaline cell extracts was used for the determination of protein by the method of Lowry et al. (10) with bovine serum albumin as standard. The cell extracts and media samples were digested with protease, and radioactive glycosaminoglycans were purified by ion-exchange chromatography and ethanol precipitation (6). The disaccharide composition of chondroitin sulfate was determined by paper
ABSTRACT Mutants of Chinese hamster ovary cells have been found that no longer produce heparan sulfate. Characterization of one of the mutants, pgsD-677, showed that it lacks both N-acetylglucosaminyl- and glucuronosyltransferase, enzymes required for the polymerization of heparan sulfate chains. pgsD-677 also accumulates 3- to 4-fold more chondroitin sulfate than the wild type. Cell hybrids derived from pgsD-677 and wild type regained both transferase activities and the capacity to synthesize heparan sulfate. Two segregants from one of the hybrids reexpressed the dual enzyme deficiency, the lack of heparan sulfate synthesis, and the enhanced accumulation of chondroitin sulfate, suggesting that all of the traits were genetically linked. These fin gs indicate that the pgsD locus may represent a gene involved in the coordinate control of glycosaminoglycan formation.
Proteoglycans consist of a core protein and one or more covalently attached glycosaminoglycan chains. Typical animal cells produce proteoglycans bearing chondroitin (dermatan) sulfate or heparan sulfate chains, but the composition varies considerably among different cells (1, 2). To study the regulation of proteoglycan composition, we have isolated Chinese hamster ovary (CHO) cell mutants defective in glycosaminoglycan biosynthesis (3-6). Many of these mutants bear mutations in genes involved in the formation of both heparan sulfate and chondroitin sulfate chains (3, 5). Here we describe a CHO cell mutant, pgsD-677, that specifically lacks heparan sulfate. The mutation in pgsD-677 affects both N-acetylglucosaminyl (GlcNAc)- and glucuronosyl (GlcA)-transferase activities required for heparan sulfate polymerization, suggesting that some form of coordinate regulation of these enzymes exists.
EXPERIMENTAL PROCEDURES Cell Cultures. CHO cells (CHO-Ki) were obtained from the American Type Culture Collection (CCL-61). All mutants were identified by colony autoradiography (7), and the purity of each strain was ensured by its isolation from cultures containing only mutant colonies. Cells were maintained in Ham's F12 (8) medium (Mediatech, Washington) supplemented with 10% (vol/vol) fetal bovine serum (HyClone) or in sulfate-deficient medium (4). Cell fusion studies required the isolation of a CHO-K1 subline resistant to thioguanine and ouabain (OT-1). Wildtype cells were treated with 10 ,uM 6-thioguanine in hypoxanthine-free F12 medium supplemented with dialyzed fetal
Abbreviations: TCA, trichloroacetic acid; TGF-f3, transforming growth factor ,B. tPresent address: School of Basic Life Sciences, Division of Molecular Biology and Biochemistry, University of Missouri, Kansas City, MO 64110. Present address: Department of Medical and Physiological Chemistry, The Biomedical Center, University of Uppsala, S-751 23, Uppsala, Sweden. '1To whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
2267
2268
Proc. Natl. Acad. Sci. USA 89 (1992)
Biochemistry: Lidholt et al.
chromatography (4) using authentic standards (Seikagaku America, St. Petersburg, FL). Enzyme Assays. N-Sulfotransferase was assayed using N-desulfoheparin as substrate (6). GlcNAc- and GIcAtransferase were assayed using oligosaccharide acceptors prepared from the capsular polysaccharide of Escherichia coli K5 (11). The polysaccharide was partially N-deacetylated with hydrazine and subjected to deaminative cleavage with nitrous acid at pH 3.9 (12). The resulting mixture of oligosaccharides, all having GlcA at their nonreducing termini, was fractionated by gel filtration chromatography. The decasaccharide fraction was used as substrate for GlcNActransferase. Digestion of a tetradecasaccharide fraction with B3-D-glucuronidase yielded tridecasaccharides with nonreducing 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 glycosyltransferase 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 previous 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 transferred colonies are incubated with [35S]sulfate, and the incorporation of radioactivity into trichloroacetic acid (TCA)-precipitable proteoglycans is measured by autoradiography. Mutant colonies defective in proteoglycan synthesis yield a reduced signal on the film and can be retrieved from the original plastic dish from which the replica was generated. Several mutants exhibited a partial reduction in [35S]sulfate incorporation into proteoglycans, and in some cases this was due to incomplete inhibition of a specific enzyme in the biosynthetic pathway (e.g., ref. 6). Cell hybridization studies showed that four of these partial mutants (strains 623, 625, 677, and 803) comprised a new complementation 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 quantitative data, cells were labeled to constant specific radioactivity ([35S]glycosaminoglycan per ,ug of cell protein) by growing them for 3 days in sulfate-deficient medium containing [35S]sulfate (4). Isolation of [35S]glycosaminoglycans from the cells and the medium showed that mutant and wild-type cultures had accumulated 11,000 and 10,400 cpm of [35S]glycosaminoglycans per ug of cell protein, respectively. Both strains synthesized similar amounts of [35S]glycosaminoglycans when cells were labeled with [35S]sulfate for only 4 hr (350 cpm/,ug of cell protein in the mutant vs. 300 cpm/,ug in the wild type). Separate analyses of the cell and medium compartments showed that the proteoglycans were distributed identically by mutant and wild-type cells as well (5559% in the medium and 41-45% in the cell layer). These findings were surprising since colonies of pgsD-677 appeared about 3-fold defective in proteoglycan synthesis by autoradiography. Because the measurement of proteoglycan synthesis by colony autoradiography employed TCA to precipitate [35S]proteoglycans (3), the proteoglycans produced bypgsD-677 might have been more soluble in TCA than those made by wild-type. To test this possibility, cells were incubated with [35S]sulfate for 4 hr and treated with 10%o TCA to
677 x 8O
677 x 677
9
803 x 803
677 x 745
* *
.*~~~ a
803 x 745
745
x
745
FIG. 1. Autoradiographic analysis of cell hybrids. Mixed monolayers of the indicated strains were treated with poly(ethylene glycol) to induce cell fusion. The treated cells were replated into 100-mm tissue culture dishes to obtain 300-1000 colonies per dish. After 9 days, the colonies were labeled for 4 hr with 35SO4 and radioactive proteoglycans were precipitated in situ with TCA. The bottom of the dish was excised and exposed to x-ray film. Complementation had occurred if occasional colonies yielded a strong signal comparable to that given by wild-type colonies (not shown).
precipitate 35S-labeled macromolecules. About 200 cpm of 35S-labeled material per ,ug of cell protein precipitated in the mutant, whereas 900 cpm/,ug precipitated in the wild type. Thus, the enhanced solubility of pgsD-677 proteoglycans in TCA explained the reduced autoradiographic signal generated by mutant colonies. pgsD-677 Is Defective in Heparan Sulfate Biosynthesis. Wildtype CHO cells produce about 70% heparan sulfate and 30%6 chondroitin-4-sulfate (Fig. 2A and ref. 4). Analysis of [35S]glycosaminoglycans from pgsD-677 by anion-exchange chromatography showed that they consisted almost entirely of material that was coeluted with chondroitin sulfate (Fig. 2B). All of the [35S]glycosaminoglycans in pgsD-677 were depolymerized by chondroitinase ABC, whereas in wild-type cells, only the material that was eluted at 0.6 M NaCl was depolymerized. Treatment of glycosaminoglycans from both strains with chondroitinase ABC generated disaccharides that did not bind to the resin and a small amount of material that was eluted at 0.35 M NaCl, which may represent core protein-carbohydrate linkage regions. Over 90% of the disaccharides generated by chondroitinase treatment of samples labeled biosynthetically with [6_3H]glucosamine comigrated on paper chromatograms with a 4,5-unsaturated chondroitin-4-sulfate disaccharide standard, and the remainder comigrated with nonsulfated chondroitin disaccharide (data not shown). Thus, pgsD-677 does not make heparan sulfate
Biochemistry: Lidholt et al.
CY
Proc. Natl. Acad. Sci. USA 89 (1992)
0
2
0
10
20
30
400
10
20
30
40
1
0
%I-16 0 B
CL12
8
4
0
Fraction Number FIG. 2. Anion-exchange HPLC of glycosaminoglycans derived from wild-type (A) and pgsD-677 (B) cells. Cells were labeled with 3"SO4 (10 uCi/ml) for 3 days. [35S]Glycosaminoglycans were released from cell and media proteoglycans and collected by anionexchange 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). The amount of radioactivity in each fraction was normalized to the equivalent amount of protein that had been analyzed. The broken line represents the programmed gradient of NaCl. Filled symbols, untreated samples; open symbols, after treatment with chondroitinase ABC.
and accumulates 3-4 times more chondroitin-4-sulfate than the wild type. To determine whether the pgsD mutation affected the synthesis of core proteins destined to contain heparan sulfate chains, we examined the composition of p-glycan, a proteoglycan that binds transforming growth factor 83 (TGF-3). Receptors for TGF-,f were affinity labeled and crosslinked with 125I-TGF-13 (Fig. 3). Wild-type CHO cells contain three types of TGF-,3 receptors (14). Type I and type II receptors do not contain glycosaminoglycan chains, whereas type III proteoglycan receptors (J3-glycan) contain mostly heparan sulfate chains. Treatment of the cells with heparitinase prior to affinity labeling and crosslinking shifted >90%o of(3-glycan from a characteristically heterogeneous species at about 250 kDa to labeled species migrating at 120-140 kDa. When TGF-P was crosslinked to pgsA-745 cells, a mutant that does not make any glycosaminoglycan chains (3), f8-glycan migrated as 120- to 140-kDa species. This is the nonproteoglycan form of 8-glycan (14) and is unaffected by heparitinase treatment. In pgsD-677 cells >90% of /8-glycan migrated like the nonproteoglycan form of ,B-glycan found in pgsA-745 or in wild-type cells after heparitinase treatment. About 10%o migrated as polydisperse proteoglycan. These proteoglycan receptors did not change mobility when cells were treated with heparitinase, but they were converted to the nonpro-
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 pgsA745 cells, a CHO mutant defective in xylosyltransferase, estradiol 8-D-xyloside stimulated both heparan sulfate and 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. To test whether chain polymerization was altered, assays for GlcA-transferase and GlcNAc-transferase were established 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 transfer was proportional to protein concentration and dependent on the addition of acceptor polysaccharide (data not shown). However, in pgsD-677 cell extracts, neither enzyme activity was detectable (Table 2). Mixtures composed of equal amounts of mutant and wild-type extracts contained 58% and 47% of the wild-type GlcNAc- and GlcA-transferase activities, respectively, demonstrating that the mutant did 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 mutant vs. 37 pmol per min per mg in the wild type), as measured by the transfer of [35S]sulfate from 3'-phosphoadenosine 5'-phosphosulfate to N-desulfated heparin preparations (6). Thus, the mutation in pgsD-677 affects chain formation but not a chain modification reaction involved in heparan sulfate biosynthesis. When the lectin sensitivity of the cells was measured (Table 3), only small differences were noted, suggesting that the synthesis of Asn-linked oligosaccharides, glycolipids, and other O-linked glycoconjugates are normal in the mutant as well. The Various Phenotypes Are Genetically Linked. To test whether the decline in heparan sulfate synthesis was a recessive trait, pgsD-677 was fused to OT-1, a thioguanineresistant, ouabain-resistant subline of wild-type cells. Analysis of the glycosaminoglycan composition of four hybrids showed that they produced heparan sulfate normally and accumulated somewhat more chondroitin sulfate than wild type or hybrids of wild-type and OT-1 cells (Table 4). One of the hybrids, 6.5, was tested and shown to contain both GlcAand GlcNAc-transferase (Table 2). The moderate depression of enzyme activities in the hybrid compared with wild-type and OT-1 cells may reflect differences in protein content of hybrid cells, since the hybrids were noticeably larger than cells of the parental strains. The complete recovery of heparan sulfate synthesis in the hybrids suggests that the lack of 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. About 20,000 colonies of hybrid 6.5 were screened by replica plating and [35S]sulfate colony autoradiography (7). Two clones were identified that took up about one-third as much [35S]sulfate as other colonies (strains 6.5.2 and 6.5.5). Labeling of the cells with [35S]sulfate showed that they did not synthesize any detectable heparan sulfate and accumulated 2- to 5-fold more chondroitin sulfate than parental OT-1 cells (Table 4). These isolates also lacked both GlcNAc- and GlcA-transferase activities (Table 2). Although only two isolates were identified, 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
2270
Biochemistry: Lidholt et al. Treatment
Proc. Natl. Acad. Sci. USA 89 (1992) Wild-type
Chondroitinase
+
Heparitinase
Mr
Type 111
H
H,-
201
H
100-
-
200
-
116
-
9?
-
68
0
'.
I
,w
16 9. TyJpe
618
11
i
1
iA
Offer""
Type
-E:
I
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 treated with mutagen, we infer that they arose from a segregation-like event (17) that separated the pgsD mutant allele from the wild-type allele. Thus, the findings suggest that the failure to produce heparan sulfate and the lack ofboth transferases are linked.
DISCUSSION CHO cells, like other mammalian cells, produce a mixture of heparan sulfate and chondroitin sulfate proteoglycans, and the overall composition of the mixture remains unchanged through many cell generations. By treating cells with a chemical mutagen, we obtained stable mutants altered in proteoglycan biosynthesis (3-7). In some cases, mutations occurred in genes affecting the activity of xylosyltransferase or galactosyltransferase I, enzymes required for the assembly of 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 to proteoglycan core proteins. These mutants fail to produce any glycosaminoglycan chains, indicating that different core proteins utilize the same enzymes for the initiation of both heparan sulfate and chondroitin sulfate chains (1, 2). Table 1. Glycosaminoglycan synthesis in mutant cells
[35S]Glycosaminoglycan,
(cpm/,ug Heparan
X
10-3)
Chondroitin Total sulfate sulfate 0.58 0.02 0.56 + 2.2 0.02 2.2 0.02 ND pgsA-745 ND + 1.8 0.50 1.3 Multiple 60-mm culture dishes were seeded with 2 x 10' cells in 2 ml of growth medium. After 1 day, the medium was replaced with 1 ml of sulfate-free medium with or without 30 j.M estradiol P-Dxyloside (EDX, ref. 15). One hour later, 20 ,Ci of 35S04 was added, and after 3 hr [35(iglycosaminoglycans in the medium and the cells were isolated. Amounts of chondroitin sulfate and heparan sulfate were determined by chondroitinase ABC digestion and nitrous acid treatment (15). Average values for duplicate determinations varied by
