Sulfate Transport-deficient Mutants of Chinese Hamster Ovary Cells

Val. 261, No. 33, Issue of November 25, pp. 15725-15733,1986 Printed in U.S.A.

T H EJOURNAL OF R1OLOGICAL CHEMISTRY D 1986 by The American Society of Biological Chemists, Inc

Sulfate Transport-deficient Mutants of Chinese Hamster Ovary Cells SULFATION OF GLYCOSAMINOGLYCANS DEPENDENT ON CYSTEINE* (Received for publication, April 23, 1986)

Jeffrey D. EskoS, AdaElgavishs, Tom Prasthofer, William

€3.Taylor, andJulie L. Weinke

From the Departments of Biochemistry and $Pharmacology, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama35294

We isolated 59 Chinese hamster ovary cell mutants incorporation into glycosaminoglydefective in 3BS04 cans. Thirty-five mutants incorporated[6-3H]glucosamine into glycosaminoglycans normally, suggesting that they were specifically impaired in sulfate incorporation. Cell hybridization studies revealed that the 35 mutants defined a unique complementation group. Pulse-labeling one of the mutants with 36S04showed that it possessed a defect in a saturable, 4-acetamido4-isothiocyanostilbene-2,2’-disulfonic acid-sensitive transport system required for sulfate uptake.Despite the dramaticreduction in 3sso4 incorporation, themutant synthesized sulfatedheparanandchondroitin chains. Incubation of the mutant with [36S]cysteine resulted in the formation of 35s04,which was subsequentlyincorporatedinto the glycosaminoglycans. Similar results were obtained when wild-type cells were incubated in sulfate-free growth medium containing 136S]cysteine,and isotope dilution analysis indicated that about 15 PM of sulfate was derived from cysteine catabolism. We also found that the sulfate transport deficiency rendered the mutant resistant to 5 I . ~ Msodium chromate, whereas wild-type cells did not divide under these conditions. However, the mutant also did not ~ when proliferate inmedium containing 5 f i chromate grown in the presence of wild-type cells, suggesting that chromate was transported through cell-cell contacts. Since co-cultivating sulfate transport-deficient mutants with mutantsdefective in xylosyltransferase or galactosyltransferase 1partially restored 35S04incorporation intoglycosaminoglycans, intercellular sulfate transport occurred as well. Therefore, the availability of sulfate for glycosaminoglycan synthesis depends on sulfate uptake,turnover of sulfur-containing amino acids, and sulfate transportbetween cells. ~~~~~~

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Animal cells takeup sulfatefromextracellular fluids through carrier-mediated transport (1).Much of the sulfate is subsequently incorporated into glycosaminoglycans, such as chondroitin sulfate and heparan sulfate. To a large extent, the charge density of these polysaccharides is determined by their content of sulfate, and theprecise arrangement of ester * This work was supported in part by Grant GM33063 from the National Institutes of Health, a n RDP-centered grant to theUniversity of Alabama from the Cystic Fibrosis Foundation, and a Basil O’Connor grant from the March of Dimes Birth Defects Foundation (to 3. D. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact. $ T o whom correspondence and reprint requests should be addressed.

sulfate and N-sulfate groups determines some of their biological properties (2,3). For example, the binding of heparin (4) and heparan sulfates( 5 ) to antithrombin III criticaily depends on thesynthesis of 3-0-sulfate estersof N-acetylglucosamine residues. Brachymorphic mice possess an inborn error of metabolism affecting the enzymatic formation of activated sulfate, PAPS (6). The mutation preferentially affects sulfation of cartilage chondroitin sulfate proteoglycans and resuits inabnormal bone development and disproportionate dwarfism (7). Nutritional studies of cultured cells established the importance of extracellular sulfate for the synthesis of glycosaminoglycans (8-11). Suzuki and co-workers (8, 9) showed that sulfate-deprivation of chick embryo cartilage led to the production of undersulfated chondroitin sulfate proteoglycans. Fukui et al. (10) reported that incubation of humanskin fibroblastsin medium containing 30 JLM inorganic sulfate resulted in the synthesis of chondroitin and dermatan sulfates that were undersulfated compared to theglycosaminoglycans produced by cells incubated with 0.52 mM sulfate. More recently, Humphries et al. (11) found that incubation of bovine aortic endothelial cells in medium containing reduced amounts of inorganic sulfate diminished the sulfation of chondroitin sulfate, whereas the sulfation of heparan sulfate was less dramatically affected. We found these observations surprising, since earlier in uiuo studies had demonstrated that sulfur-containing amino acids can provide all of the sulfate needed for growth and development (1, and references therein). Thefinding that sulfate starvation of chondrocytes, fibroblasts and endothelial cells caused the formation of undersulfated glycosaminoglycans (8-1 1) suggested that these tissues do not generate much sulfate from amino acid turnover. Previous studiesinourlaboratoryhavedealtwith the isolation and characterization of Chinese hamster ovary (CHO) cell mutants defective in sulfate incorporation into glycosaminoglycans (12). Some of the mutants failed to incorporate sulfateinto proteoglycans due to defects in xylosyltransferase (12) or galactosyltransferase I,’ enzymes required for the initiation of glycosaminoglycan chains (13).Our ability to isolate mutants also gave us the opportunity to study the relationship of sulfate metabolism and glycosaminoglycan biosynthesis. In this report we describe studies of mutants defective in sulfate transport. As shown below, the sulfation

’ The abbreviations used are: PAPS, 3’-phosphoadenyl-5’-phosphosulfate; CHO, Chinese hamster ovary; SITS, 4-acetsmido-4-bothiocyanostilbene-Z,2‘-disulfonicacid CPC, cetylpyridinium chloride; HPLC, high performance liquid chromatography; Hepes, 4-(2hydroxyethy1)-1-piperazineethanesulfonicacid. 3. D. Esko, J. L. Weinke, G. Ekborg, L. Roden, G. Anatharamaiah, and A. Gawish, manuscript submitted to J.Biol. Chern.

15725

15726

Sulfation of Glycosaminoglycans

of glycosaminoglycansdoes notdependonsulfateuptake because cysteine is an abundantsource of sulfate.

aminoglycans-Enzymatic digestion of radioactive glycosaminoglycans was achieved with 50 milliunits of chondroitinase AC from Arthrobacter aurescens (Sigma), 50 milliunits of chondroitinase ABC from Proteus vulgaris (Sigma), 0.5 milliunit of heparitinase from EXPERIMENTALPROCEDURES Flavobacterium hparinurn (Miles Laboratories, Naperville, IL), 25 Cell Culture-Chinese hamster ovary cells (CHO-K1) were ob- milliunits of chondro-4-sulfatase, or 25 milliunits of chondro-6-sultained from the American Type Culture Collection (CCL-61), Rock- fatase from P. vulgaris (Sigma), according to established procedures ville,MD. The mutants described in this paper were isolated by (21-23). We tested various preparations of heparitinase prior to use replica plating and 35S04-colonyautoradiography, as described pre- to exclude batches contaminated with chondroitinases. Digestion viously (12). All of the mutants were recloned from cultures contain- products were analyzed by HPLC anion-exchange chromatography ing only deficient colonies in order to ensure their purity (14). In as described below. Nitrous acid degradation was performed at pH general, cells were maintained in Ham's F-12 (15) medium supple- 1.5 as described by Shively and Conrad (24). Prior to treatment, the mented with 10% (v/v) fetal bovine serum (Hyclone, Salt Lake City, samples were mixed with 2-5 mg of heparin from porcine intestinal UT), 100 pgof streptomycin sulfate/ml, and 100 units of penicillin mucosa (Sigma), converted to the free acid form by passage over G/ml. They were grownat 37 "Cf 0.2 "C under a 5% CO, atmosphere Dowex 50W-X2,200-400 mesh, hydrogen form (Bio-Rad),and lyopha t 100%relative humidity, and subcultured every 3-4 days with 0.25% ilized. In some experiments, protease-resistant peptides were removed trypsin (16). Fresh cells were revivedafter 15-20 passages from stocks from glycosaminoglycansby @-elimination(0.1 M NaOH, 1 M NaBH4 stored in liquid nitrogen in order to maintain their purity. a t 37 "C for 24 h) asdescribed by Oegema et al. (25). Some experiments required the use of specially formulated growth Analysis of chondroitin sulfate isomers was performed on samples medium. F-12 medium lacking sulfate was prepared from individual obtained from cells labeled with [6-3H]glucosamineand 35S04 or [:SI components (15), substituting chloride salts for sulfate salts and cysteine. Radioactive glycosaminoglycans,collected by CPC preclplomitting streptomycin sulfate. Defined medium was supplemented tation, were first separated by HPLC anion-exchange chromatograwith 10% (v/v) fetal bovine serum dialyzed 106-fold against phos- phy. Fractions containing chondroitin sulfate were pooled, concenphate-buffered saline(17). Proteoglycan-deficient serum was pre- trated by lyophilization, and desalted by passage over Sephadex Gpared by passing 100-ml portions of serum through a 3X 2.5-cm inner 25. A portion of the purified chondroitin sulfate was treated with diameter column of DEAE-Sephacel (Pharmacia, Uppsala, Sweden). chondroitinase ABC in the presence and absence of chondro-4-sulNonproteoglycan serum proteins were eluted from the column with fatase or chondro-6-sulfatase, and the digestion products were ana100 ml of 0.3 M NaCl containing 20 mM Tris-HC1 (pH 7.5) and pooled lyzed by ascending chromatography on Whatman No. l paper (26). with the material not bound to the resin. After concentrating the After autoradiography, the chromatogram was cut into 1-cm strips mixture against solid poly(ethy1ene glycol) (PEG 20,000; Fisher), it and counted by liquid scintillation spectrometry in order to quantitate was dialyzed against phosphate-buffered saline and filter-sterilized the distribution of radioactivity in individual disaccharides. (0.2 p pore diameter). This serum preparation was free of glycosamiHPLC Anion-exchange Chromatography-We analyzed glycosaminoglycans and supported CHO cell division. noglycans and their digestion products by HPLC using a 7.5-mm Radiochemical Labeling Studies-All radioisotopes were obtained inner diameter X 15 cm TSK-DEAE-3SW anion-exchange column from Amersham Corp. In order to label the cells with radioactive and a guard column containing DEAE-5PW resin (LKB, Sweden). precursors and obtain consistentpatterns of labeling, cell monolayers Analyses were conducted on an LKB microprocessor controlled were harvested with trypsin and centrifuged to remove spent medium. HPLC equipped with two pumps, an integrated fraction collector, The cells were washed once and resuspended in defined medium and a variable UV monitor. When intact glycosaminoglycans were lacking serum, sulfate, glucose, and cysteine. They were then added analyzed (containing up to 5 mgof carrier), the column was preto dishes containing growth medium and 10-20 pCi of Na3'S04 (25- equilibrated with 0.2 M NaCl containing 10 mM KH,P04 (pH 6.0). Ci/mmol)/ml, 40 Ci/mg)/ml, 10 pCi of ~-[6-~H]glucosamine-HCl(40 Five minutes after sample injection, the concentration of NaCl was 10 pCiof ~-[~'S]cysteine/ml,or 10 pCi of ~-[~'S]methionine/ml as increased 10 mM/min up to 0.7 M, and I-mlfractions were collected. indicated under "Results." Efficient incorporation of [6-3H]glucosa- When disaccharide analyses were performed, the column was premine was achieved by reducing the glucose concentrationin the equilibrated with 0.1 M NaCl containing 10 mM KHzPO, (pH 6.0), medium from 10 to 1 mM, as reported previously (12). When L-[3'Ss] and after 20 min the concentration of NaCl was increased 10 mM/ cysteine was added, the nonradioactive cysteine concentration in the min up to 0.2 M. After each analysis, the column was washed with 1 medium was reduced from 0.2 mM to 30 FM, the lowest concentration M NaCl, 10 mM KH,P04 and recycled to initial conditions. that supported cell proliferation. In general, incubation with radioSulfate Transport Assays-We measured radioactive sulfate uptake active precursors for 3 days permitted cells to achieve isotopic equias a function of time and sulfate concentration in late log-phase librium. After rinsing the labeled cell layers three times with phosphate- cultures. After incubation under the conditions indicated in the figure buffered saline, they were solubilized with 0.1 N NaOH a t 25 "C for legends, the cells were washed six times a t 4 "C with 3 ml of a stopping M sucrose, 0.1 M NaN03, 4 mM 15 min. An aliquot of the alkalineextract wasremoved for the solution (27) consisting of0.1 determination of protein using the method of Lowry et al. (18) with hemimagnesium gluconate, and 10 mM Tris-Hepes (pH 7.5). The bovine serum albumin as standard. The rest of the extract was Cells were then solubilized with 1 ml of 0.1 N NaOH for 20 min at adjusted to pH 5.5 with 10 N acetic acid and treated overnight a t 25 "C. Aliquots were taken for the determination of protein and for 40 "C with 2 mg of nonspecific protease (Boehringer Mannheim)/ml counting by liquid scintillation spectrometry. in the presence of 0.32 M NaCI, 0.14 M sodium acetate and 2 mg of RESULTS chondroitin sulfate (mixed isomers from shark and whale cartilage; Sigma)/ml. Growth medium was digested directly with protease, Identification of Sulfate Transport Mutants without prior treatment with base. When separate analyses of secreted and cellular glycosaminoglycans were not required, we added ComplementationAnalysis-Recently, we described a the medium back to theacidified cell extract and treated the mixture CHO cell mutants defective in with protease as described above. Collection of liberated glycosami- screening method for isolating noglycans was achieved by precipitation with 1% (w/v) cetylpyridin- proteoglycan synthesis (12). Briefly, this technique involved ium chloride (Aldrich). The precipitate was solubilized with heating the transferof animal cell coloniesfrom plastic tissue cultures in 2 M sodium acetate containing 10% ethanol and reprecipitated dishes t o overlying discs of polyester cloth. To measure prowith 3 volumes of cold ethanol, according to established procedures teoglycan synthesis in thecolonies on the replicas, they were (19). After washing the sample three times, the final preparations pulse-labeled with 35S04and treated with trichloroacetic acid. were dried briefly under vacuum and dissolved in a small volume of of the discs t o x-ray film revealed occasional coloExposure 20 mM Tris-HC1 (pH 7.4). Radioactivity was quantitated by liquid scintillation spectrometry using Aquasol (New England Nuclear) or nies that failed to incorporate 930, into proteoglycans. MuPatterson and Green scintillation fluids (20). The values obtained tant colonies detectedinthis way were picked from the were normalized to theamount of cell protein digested. When samples original master dishes and repurified through another round contained 3H and 35S, the values were corrected for approximately of replica plating (14). After screening about100,000colonies, 2% spillover of 3H and 30% spillover of 35Sinto theopposite counting we obtained 59 mutants tentatively defective in proteoglycan windows. Enzymatic and Chemical Characterization of Radioactive Glycos- synthesis.

of

Sulfation

Glycosaminoglycans

Because sulfate addition to glycosaminoglycans is a late step inproteoglycan synthesis, screeningfor mutants by 35S04 incorporation allowed the isolation of mutants defective in different stagesof the pathway (12). To determine how many different types of mutants were present in the collection, we examined them by complementation analysis(Fig. 1).In these experiments, pairs of mutants were fused by treating mixed

745

604

15727

cell monolayers with poly(ethy1ene glycol) (28). The resulting population of cells, containing some hybrids,were replated at low density in order to obtain300-1000 colonies per dish. If two mutants bore mutations in different genes, then they should complement each other and the resultanthybrid colonies should incorporate 35S04 to a much greater extent than colonies composed of a single mutant. As shown in Fig. 1,

605

745

604

FIG. 1. Complementationanalysis of mutants 604, 605, and 745 by autoradiography.Individual 20mm diameter wells of a 24-well dish were filled with 0.5 ml of complete growth medium and seeded with 2 X lo5 cells of each indicated strain. After one day a t 33 "C the cells were treated for 1 min with 0.5 ml of 50% (w/w) poly(ethyleneglyco1) (PEG 3350; Fisher) prepared in F-12 medium without serum (28). The fusion medium was then aspirated, and the cells were rinsed three times with 0.5 ml of regular growth medium. Following overnight incubation, the cells in each wellwere harvested with trypsin and replated a t different dilutions in 100-mm diameter tissue culture plates incomplete growth medium. After 10-12 days a t 33 "C, colonies had formed on the dishes. At this time, the spent medium was replaced with sulfate-deficient medium a t 40 "C containing 10 pCi/ml 3sS04.Four hours later, the medium was removed and thecells were washed three times with phosphate-buffered saline a t 4 "C.Treatment with 10%trichloroacetic acid-precipitated radioactive proteoglycans associated with the colonies on the dish. After washing the colonies three times with 2% trichloroacetic acid, the plates were air-dried, and thebottom surfaces were excised and exposed to x-ray film for 2 days. The exposures lying along the diagonal in the figure represent results obtained from fusions of each mutant to itself. The other exposures resulted from fusions between the pairs of mutants, as indicated above and to theright of the panels.


15728

fusion of mutants 604 or 605with strain 745 resultedin hybrid colonies that incorporated more 35S04than colonies derived from either parental strain. Since strain745 is defective in xylosyltransferase(12),the appearanceof colonies that indicated that mutants604 and 605 incorporated more 35s04 were defective in a different step involved in proteoglycan synthesis. Fusion of mutant 604 to mutant 605 did not give rise to colonies that incorporated more %04,and therefore did not complement. Since mutant 604 and 605 varied quanbitatively in 35S04incorporation,theypresumably defined different mutant alleles of the same gene. Fusion of each mutantto itselfalso didnotresult in highly radioactive colonies, except in rare instances whena revertant was present in the original population of cells (notice the one dark colonyin thefusion of 605 to itself). Of the 59 mutants available, 35 belonged to the complementation groupdefined by mutants 604 and 605. Glycosaminoglycan Content of the Mutants-To characterize the new mutants further, we measured the amount of glycosaminoglycans produced by mutant and wild-type cells. Quantitation of 35s04 incorporation, under conditions that labeled the glycosaminoglycans to constant specific radioactivity, showed that mutant 605 incorporated about 40-fold less sulfate than the wild-type, CHO-K1 (Table I). Mutant 604 incorporated about %fold less 35s04 than the wild-type, as originally indicated by autoradiography (Fig. 1).When we incubated cells with [6-3H]glucosamine, we found that mutants 604, 605, and wild-typecells incorporated label into glycosaminoglycans to the same extent. Both mutant and wild-type cells secreted about two-thirds of the 3H-glycosaminoglycan into the growth medium and retained one-third in the cell monolayer. These findings suggested that mutants 604 and 605 synthesized the carbohydrate backbones of glyto the newly cosaminoglycans, but were unable to add 35s04 made chains. These mutants behaved very differently than and [6mutant 745, which failed to incorporate both 35s04 :'H]glucosamine duetotheabsence of xylosyltransferase. Thus mutants604 and 605 behaved biochemically and genetically distinct from mutant 745. Sulfate Transport Is Deficient in Mutant 605-The initial step involved in "so4incorporation intoglycosaminoglycans is transport of sulfate across the plasma membrane. To test whether transport mightbe defective in the mutants,we first examined sulfate uptake by wild-type cells. In this experi-

ment, we pulse-labeled cells with 35S04and quantitated the incorporation of label into CPC-soluble material as a measure of free and activated sulfate(Fig. 2). As shown, the uptakeof 35s04was rapid and reached steady-state conditions after about 30 min. However, incorporation of label into CPCprecipitable glycosaminoglycan lagged for about 5 min, and thereafter accumulated in proportion totime. We also examinedtheincorporation of 35S04in the xylosyltransferasedeficient mutant, 745. Sulfate uptake into CPC-soluble material occurred with kinetics virtually identical to that observed in the wild-type, but little radioactive glycosaminoglycan accumulated due to the missing enzyme activity (Fig. 2). This findingshowed that uptake and intracellular accumulation of sulfate did notdependontheability of cells to synthesize glycosaminoglycan. Examination of sulfate uptake in mutant605 revealed that the initial rate was very low compared to the wild-type (Fig. 3). Moreover, uptake was insensitive to the anion transport inhibitor, SITS. Since SITS reduced sulfate uptake in the wild-type to the level observed in mutant 605, the mutant apparently lacked the SITS-sensitivecomponent. To confirm that sulfate uptake was carrier-mediated in CHO cells, we examineditssaturability (Fig. 4). Inthisexperiment, we for only 2 min to eliminate pulse-labeled the cells with 35s04 incorporation of label into glycosaminoglycan. Under these conditions, the rateof sulfate uptake in mutant 605 increased in proportion to the sulfate concentration in themedium up to 10 mM, the highest concentration examined in this experiment. In contrast, the wild-type took up sulfate 10-20-fold more rapidly than the mutant at all sulfate concentrations and reached a maximal rate at about 5 mM. Replotting the data obtained from the wild-type according to the methodof Woolf (S/V versus S, Ref. 29) yielded an apparent K,,, of 3.6 mM and an apparentV,,, o f 1.5 nmol/min/mg of cell protein. Inclusion of SITS intheincubation mediumreduced the level capacity of wild-typecells to take up sulfate to the observed in mutant 605, whereas addition of SITS to the mutant had no effect (Fig. 4). These findings suggested that -nl

201

\

TABLEI Distribution of radioactive glycosaminoglycanin wild-type cellsand mutants 604, 605, and 745 Multiple 60-mm diameter tissue culture dishes were each seeded with approximately 10' cells in 5 ml of sulfate-free growth medium containing 1.0 mM glucose and 10 pCi each of 35S04and [6-3H] glucosamine/ml. After 3 days a t 37 "C, aliquots of the medium and the cells were treated with protease, and the liberated glycosaminoglycans were precipitated with cetylpyridinium chloride (see "Experimental Procedures"). The values obtained were normalized to the amount of cellular protein analyzed. Shown are the average of duplicate determinations which varied by less than 20%. Radioactive glycosaminoglycan content Strain

[6-3H1Glucosarnine Cells Medium

Total

3 6 ~ 0 ,

Cells

Medium Total

cprn/@gcell protein

Wild-type CHO-K1 580 230 Mutants 604 605 745

290 280 110

810 2100 3100 5200 600 550 110

890 830 220

520 80 20

1040 50 70

1560 130 90

Minutes

FIG. 2. Sulfate transport in CHO cells. Multiple 30-mm diameter tissue culture dishes were each seeded with approximately 10' cells in complete growth medium. After 2 days the mediumwas removed from the dishes and replaced with growth medium supplemented with 10% (v/v) dialyzed fetal bovine serum. After 20 min, the medium wasremoved and the cells were rinsed three times with sulfate-deficient medium. Uptake assays were initiated by adding 1 ml of growth medium supplemented with dialyzed fetal bovine serum M 3'S04(1.67 mCi/pmol). After incubation for the indicated and 6 ~ L of times, the cells were washed rapidly with stopping solution (see "Experimental Procedures") and solubilized in 1 ml of 0.1 N NaOH for 20 min at 25 "C. One portion of the extract was used for protein determination. Another portion was neutralized with acetic acid, digested with protease, and treated with 1% CPC in 0.3 M NaCl. After centrifugation, a portion of the supernatants (0,A) and the pellets (0,A) were counted by liquid scintillation spectrometry. 0, 0, wildtype cells; A, A,xylosyltransferase-deficientmutant (745). The values shown are the average of duplicate determinations which varied by less than 10%.


15729

with 355S0 and 4 [6-3H]glucosaminefor several days and analyzed the radioactive glycosaminoglycans by anion-exchange chromatography. In preliminary experiments, we found that chromatography on conventional ion-exchange resins, such as DEAE-Sephacel (Pharmacia), did not effectively resolve CHO cell heparan and chondroitin sulfate chains (data not shown). However, HPLC anion-exchange chromatography resolved the radioactive glycosaminoglycans into two peaks, one eluting at 0.45 M NaCl and the other eluting at 0.6 M NaCl (Fig. 6A). Prior treatment of the glycosaminoglycans with chondroitinase ABC or AC resulted in thedisappearance of the more retarded peak (Fig. 6 B ) and in the appearance of Minutes radioactive disaccharides in the fraction not bound to the FIG. 3. Sulfate transport in mutant 605. Cells were grown as resin. Ascending paper chromatography of the disaccharides described in the legend to Fig. 2, except 0.5 mM SITS was added to some of the cultures 20 min before initiating the uptake assay. SITS (see "Experimental Procedures") indicated that the material was not present during the incubation with %O,. Sulfate uptake was eluted at 0.6 M NaCl was composed of over 95% chondroitin quantitated by counting an aliquot of the alkaline extracts prior to 4-sulfate. Treatment of crude glycosaminoglycans with heCPC precipitation. 0,0,wild-type cells; A, A,mutant 605 cells. Filled paritinase caused the peak at 0.45 M NaCl to elute at lower symbols are results obtained after the cells were treated with SITS, saltand in the appearance of radioactive material in the whereas open symbols are results obtained when the cells were not unbound fraction (Fig. 6C). Furthermore,treatment with treated with SITS. nitrous acid at pH 1.5 degraded over 80% of the radioactive mutant 605 was defective in a saturable, SITS-sensitive car- material in this region (data not shown), demonstrating that rier required for the uptake of inorganic sulfate from the it was composed mostly of heparan sulfate. Quantitation of the areas under the profiles shown in Fig. 6A indicated that medium. It was knownfrom studies of sulfate transportin other cells CHO cells contain about 70% heparan sulfate and 30% chon(30-34) that chromate is also taken up through the sulfate droitin 4-sulfate. Analysis of the xylosyltransferase-deficient transporter. Since chromate is toxic to cells, we predicted that mutant, 745, showed that nearly all of the radioactive material sulfate transport-deficientmutants isolated by replica plating produced by the wild-type consisted of glycosaminoglycans would be resistant to the cytotoxic effects of chromate. As (Fig. 6D). Mutant 745 synthesized a small residue of labeled shown in Fig. 5, the LD5, for chromate in mutant 605 was material (about 1%)which eluted within the chondroitin 4about 15 PM. Both the wild-type and the xylosyltransferase- sulfate region. Its composition has not yet been determined. Having established the identity of CHO cell glycosaminodeficient mutant had an LDSoof about 0.7 WM. glycans, we next analyzed by HPLC the radioactive glycosL-Cysteine Is a Precursor of Glycosaminoglycan Sulfate aminoglycans derived from the sulfate transport-deficient Glycosaminoglycan Synthesis in Mutant 605-Having es- mutant, 605 (Fig. 6E). As shown, two peaks of 3H-labeled tablished that mutant 605 was deficient in sulfate transport, material eluted in the same positions as glycosaminoglycans we studied the effect of inhibitingsulfateuptake on the derived from the wild-type. We initially found this result assembly of glycosaminoglycans. We labeled wild-type cells puzzling, since the lack of sulfate incorporation in the mutant

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0

2.5

5.0

7.5

10.0

Sulfate(mM) FIG. 4. Saturability of sulfate uptake. Multiple 30-mm diameter tissue culture plates were seeded with approximately lo6 cells of wild-type (0,O) and mutant 605 (A, A) cells under the conditions given in the legend of Fig. 2. Uptake assays were initiated using Na3%04 (1-100 rCi/ml) at theindicated concentrations and stopped after 2 min. 0, A, results obtained in the absence of SITS; 0, A, results obtained in its presence. Shown are the averages of duplicate determinations which varied by less than 10%.In the inset,the uptake values obtained from wild-type cells in the absence of SITS were replotted using S/V as the ordinate and S as the abscissa. A best fit, linear regression program was used to obtain the indicated line. The calculated values of K,,, and V,, are given in the text. Mutant605 did not takeup enou-ghradioactive sulfate to permit further kinetic analysis.

15730


ChromatebM)

FIG. 5. Chromate sensitivity of wild-type and mutant cells. Approximately 500 cells were added to 100-mm diameter tissue cul-

ture plates containing regular growth medium. Sodium dichromate

was added to achieve the indicated concentration of chromate ion. Each dish was then overlayed with Whatman No. 42 filter paper and glass beads in order to obtain discrete colonies (14). After 12 days’ incubation at 37 “C, the colonies were fixed with trichloroacetic acid and stained with Coomassie Brilliant Blue (14). The relative plating efficiency of each strain was determined by dividing the number of coloniesobservedin the presence of chromate by the number of colonies observed in its absence. 0, wild-type CHO-Kl; A, xylosyltransferase-deficient mutant 745; 0, sulfate transport-deficientmutant 605.

should have reduced the charge of the glycosaminoglycans, causing them to elute from the resinat lower ionic strength. T o clarify this anomaly, we mixed 35S-glycosaminoglycans isolated from the wild-type with 3H-glycosaminoglycans derived from mutant 605 and treated the mixture with chon1 droitinase ABC. Analysis of the mixed radioactive disaccha0 rides by HPLC anion-exchange chromatographyshowed that they eluted together in the same position as standard chondroitin sulfate disaccharides(Fig. 7). Less than 5%of the 3H6 labeled disaccharides (derived from the mutant) co-migrated with standard chondroitin disaccharide lacking sulfate. Thus, 4 the mutant synthesized chondroitin sulfate chains thatwere sulfated to the same extent as those synthesized by the wild2 type. Treatment with heparitinase digested the 3H-glycosaminoglycan that eluted in the position of heparan sulfate, indicating that the mutant also synthesized N-sulfate groups 0 (data not shown). Fraction Cysteine Utilization-The ability of sulfate transport-defiFIG. 6. HPLC anion-exchange chromatography of glycoscient mutants to generate sulfated glycosaminoglycan indi- aminoglycans. Radioactive glycosaminoglycan chains obtained cated that thecells obtained sulfate from a source other than from wild-type cellswere isolated by CPC precipitation and analyzed inorganic sulfate in the growth medium. Because CHO cells by HPLC anion-exchange chromatography (see “Experimental Proare normally bathed medium in containing fetalbovine serum, cedures”). A , no treatment; B, after treatment with chondroitinase we reasoned that uptake and catabolismof serum proteogly- ABC; C, after treatment with heparitinase; D,results obtained from the analysis of mutant 745 glycosaminoglycans; E, results obtained cans might provide sulfate to the mutant. However, incuba- from the analysis of mutant 605. The broken line in panel A indicates tion of cells in growth medium supplemented with proteogly- the concentration gradientof sodium chloride.0,35Scpm; 0,3H cpm. can-deficient serum (see “Experimental Procedures”) did not alter the ionic properties of the glycosaminoglycans derived from the mutant and did not increase the extent of 35s04of the profile in Fig. 8A indicated that the ratioof 35Sto 3H was not constant across the individual peaks, suggesting that incorporationin wild-type cells. Thus,little if anyserum [35S]cysteine presentin proteoglycan contributed sulfate for de nouo glycosaminogly- some 35S might beduetointact peptides resistant to proteolysis. Therefore, we treated the can synthesis. Because many tissues can liberate sulfate through theoxi- sample with NaOH/NaBH, and reprecipitated the resistant dation of sulfur-containingaminoacids (l), we examined material with CPC. We recovered about 40% of the 35Sand whether CHOcells generated 36S-glycosaminoglycans fromL- about 60% of the 3H after treating the glycosaminoglycans in the original [35S]cysteine.After incubating mutant605 and wild-type cells with base, suggesting that about 70% of the groups. in labeled medium for 3 days, we collected radioactive glycos- preparation was present as ester sulfate and N-sulfate aminoglycan by CPC precipitation. Quantitationof the radio- As shown in Fig. 8B, the HPLC elution pattern of the baseactivity showed that mutant andwild-type cells accumulated stable %-labeled material was virtually identical to the discomparable amountsof 35Scounts in these crude preparationstribution of 3H-labeled material. Digestion of the 35S-glycosof glycosaminoglycans (Table 11). Further fractionationof the aminoglycans with chondroitinaseABC and heparitinase conglycosaminoglycans by HPLC anion-exchange chromatogra- firmed that the majority of the 35Swas present as ester sulfate phy showed that the pattern of labeled glycosaminoglycans and N-sulfategroups (Fig. 8, C and D, respectively). (comThese findings showed that cysteine provided some of the was similar to thatobserved in cells labeled with 35s04 pare Figs. 8A and 6A, respectively). However, close inspection sulfate needed for glycosaminoglycansynthesis. Labeling cells

3000

15731


3 1 .o

2

Fraction

0.5

FIG. 7. HPLC anion-exchange chromatography of chondroitin sulfate disaccharides obtained from mutant 605 and wild-typecells. Portions of 35S-labeled glycosaminoglycans ob-

1

0

tained from wild-type cells were mixed with an equal amount of 3Hlabeled glycosaminoglycans isolated from mutant 605. The mixture was then treated with chondroitinase ABC and the resulting disaccharides were chromatographed by HPLC anion-exchange chromatography (see “Experimental Procedures”) using the indicated gradient of NaCl (broken line).0, 3H radioactivity recovered; 0, recovery of :’AS radioactivity. The hatchedareas mark the positions where authentic chondroitin and chondroitin sulfatedisaccharides migrated.

0

4

1.5

3 1.o

2

0.5

1

0 1.5

0

TABLE I1 Incorporation of 35Scpm from (JSSlcysteine and35S04into mutant 605 and wild-type glycosaminoglycans Multiple 60-mm diameter tissue culture dishes were each seeded with approximately lo5 mutant 605 or wild-type cells in 5 mlof sulfate-free growth medium containing 10 pCi/ml either 35S04or [35Ss] cysteine (see “Experimental Procedures”). After 3 days’ incubation at 37 “C, the radioactive glycosaminoglycans were isolated by precipitation with cetylpyridinium chloride, and theamount of radioactivity recovered was normalized to the amount of cell protein that was analyzed. Shown are the averages of two different experiments, each performed in duplicate. The average error in these measurements was approximately 10% of the indicated values. Strain

FIG. 8. HPLC anion-exchange chromatography of labeled glycosaminoglycans obtained from wild-type cells after incubation with [36S]cysteine.A portion of labeled glycosaminoglycans obtained from wild-type cells grown in the presence of 10 pCi/ ml each [6-3H]glucosamineand [35S]cysteinewas analyzed by HPLC anion-exchange chromatography (see “Experimental Procedures”). A, no treatment; B, after treatment with base and sodium borohydride; C, after diges$ion with chondroitinase ABC; and D, after treatment with heparitinase. 0, recovery of 35Scpm; 0, recovery of 3H cpm.In panel B,the recovery of heparan [35S]sulfatewas lessthan the recovery of 35S-chondroitinpresumably due to the greater baselability of 6-0-sulfate esters (35).

Radioactive glycosaminoglycan content

=so,

[35S]Cysteine

cprnfflg cell protein

450

Wild-type CHO-K1 Mutant 605

5300

3200

TABLE I11 Restoration of 35S04sulfate incorporation by co-cultivation of mutants Multiple 60-mm diameter tissue culture dishes were seeded with IO5 cells in sulfate-free growth medium containing 10 pCi/ml 35S04. Some of the plates containedonly individual mutants, whereas others contained an equal mixture of two mutants. After 4 days’ incubation at 33 “C, the cells and the medium were harvested together and the amount of radioactive glycosaminoglycans was quantitated (see “Experimental Procedures”). Shown are the average of duplicate deterI nminations which varied by less than 20%. Mutant 745 is deficient in xylosyltransferase, strain 761 is defective in galactosyltransferase I, and 693 belongs to the same complementation group as 605, the sulfate transport-deficient mutant.

with 30 p~ ~-[~‘S]methionine also resulted in 35S04incorporation into glycosaminoglycans, but after correcting for differences in radiospecific activities, we concluded that 6-fold less sulfate originated from this aminoacid. We were able to estimate indirectlyhow much sulfatewas available for glycosaminoglycan synthesis through isotope dilution a n a l y ~ i s . ~ this experiment, cells were incubated in growth medium containing 20 pCi/ml “so4and varying concentrations of inorganic sulfate. After quantitating “5S04incorporation into glycosaminoglycans, we calculatedthatthe effective pool of sulfateavailabletothe cells was 15 3 p ~ Thus, . ifwe assume that cysteine was the primary source of sulfate, then about 8% of the cysteine in the medium (0.2380 mM) had been210 catabolized. Isotope dilution analysiswas conducted by measuring the amount of radioactive sulfate incorporated into proteoglycans in medium containing 20 pCi of 35S04/ml and 0, 0.01, 0.1, and 1.0 mM added sodium sulfate. The results were interpreted using the equation: C, = C.S/(S, - S ) , where C, = the concentration of sulfate available to the cells in the absence of added sulfate, So= cpm/pg of cell protein obtained in the absence of added sulfate, C = the concentration of sulfate added to themedium, and S = cpm/gg of cell protein obtained in the presence of added sulfate.

35

S-Glycosaminoglycan content

Strain

745

761

693

cpmfdish

745 761 693

6800 3600

640 710

Intercellular Transport of Sulfate During the course of these studies we found that co-cultivation of certain pairsof mutants restored“so4incorporation into glycosaminoglycans. As shown in Table 111, co-cultivation of mutant 605 with mutant 745 (xylosyltransferasedeficient) or mutant 761 (galactosyltransferase I-deficient*) for 5 days resulted in 6-10-fold more label incorporated than

15732


expected from summing the residual 35S0, incorporation in the individual mutants, The extent of %304incorporation in the co-cultures was typically 1540% of that observed in cultures containing only wild-type cells. We did not observe this "cross-feeding" phenomenon during eo-cultivation of mutants 745 and 761, suggesting that the resumption of sulfate inco~orationdid not result from transfer of proteoglycan intermediates between the strains.Cross-feeding studies using conditioned medium or sonicated cell preparations also did notrestoresulfate incorporation, However, restoration of sulfate incorporation required the mixture of cells to reach confluency, suggesting that cell-cell contact was necessary. These findings were consistent with the formation of interceltular gap junctions which permitted the transfer of small molecules such as sulfate or PAPS. To confirm that some form of communication was taking place between the cells, we took advantage of the chromate resistance of the sulfate transport-deficient mutant (Fig. 5). If junctions formed, then co-incubation of sulfate transportdeficient mutant andwild-type cells would render the mutant more sensitive to chromate. indeed, the addition of 5 PM chromate to cultures containing 5 X lo5 wild-type cells and 500 mutant cells inhibited the formation of colonies by the mutant (7 coIonies in the presence of wild-type cells versus 360 colonies in their absence). DISCUSSION

Sulfute Transport-Our study of mutants deficient in sulfate transport indicates that CHO cells take up sulfate by a SITS-sensitive carrier. Since all 35 mutants belong to a single complementation group, the sulfate transporter most likely consists of a single gene product, as originally suggested by Campbell et al. (34). However, because the carrier has not yet been purified, little information about its structure and mechanism of action is available. Studies of sulfate transport in other cells indicates that the carrier catalyzes "S04/CI exchange (36, 37), Na+/S04 cotransport (38, 39), or H+/SOr cotransport (40), depending on theorigin of the cell. Although we do not know whether sulfate uptake is coupled to the transport of other ions in CKO cells, preliminary studies indicate that the CHO cell transporter is very sensitive to pH.4 Interestingly, residual sulfate uptakein mutant 605 does not respond to pH changes, whereas uptake in mutant 604 remains sensitive. Additional studies of themutantsare needed to establish whether this finding means that sulfate transport is coupled to proton translocation or a pHgradient. Kineticstudies of the sulfatetransporter in CHO cells yielded an apparent K, of about 3.6 mb%under physiological conditions (Fig. 4). Most tissues have low affinity, high capacity carriers possessing an apparent K, in the millimolar range (36-40). Since the serum concentration of sulfate varies between 0.3 and 2.5 mM, depending on the species (l),the carriers potentiallylimit the uptake of sulfate. Thus, sulfation of glycosaminoglycans might vary according to the extracellular concentrationof sulfate. This would only occur in tissues that utilize sulfate more rapidly than the rate of sulfate transport across the plasma membrane. It would not occur in cells, like CHO, which generate enough sulfate from amino acid turnover to make sulfated glycosaminoglycans. Cysteine as u Major Source of Sulfate-Our finding that cysteine provides sulfate required for glycosaminoglycan synthesis is consistent with nutritional studiesof animals showing that sulfur-containing amino acids provide adequate SUIfate for normal growth and development (1).In contrast to .___

A. Elgavish and J. D. Esko, unpublishedresults.

CHO cells, chick chondrocytes (8,9), human fibroblasts (lo), and bovine endothelial cells (11) synthesize undersu~fat~d chondroitin sulfate chains when incubated in sulfate-defic~ent growth medium. The different response of chondrocytes to sulfate deprivation may reflect the enormous amount of sulfate required for cartilage proteoglycan synthesis. Measurement of "SO4 incorporation by cultured cells derived from chondrosarcoma indicates that cartilage-producing cells make at least 1000-foldmore sulfated glycosaminoglycan than CHO cells.5 Unfortunately, the amount of ~ l y ~ o s a m i ~ o g ~proycan duced by endothelial cells and fibroblasts relative to CHO cells is not yet available. We suspect that the limited extent to which sulfate starvationaffects glycosaminoglycan synthesis in endothelia1cells and fibroblasts indicates that they also generate sulfate from cysteine. However, the amount of sulfate generated in this way is apparently insufficient to satisfy the sulfate requirement for glycosaminoglycan synthesis. In order to estimate how much sulfate is derived from cysteine, we performed an isotope dilution analysis of 35S0, incorporation into glycosaminoglycans, This experiment yielded an endogenous pool of about 15 I . ~ Msulfate. If we use this value to correct the specific radioactivity of added 35S04, we calculate that CHO cells contain about 4.5 nmol of glycosaminoglycan sulfate/mg of cell protein. If, instead, we calculate the content of glycosaminog~ycansulfate from the radiospecific activity of [35S]cysteine,we find that CHO cells containabout 5 nmol of sulfatelmg of cell protein. The similarity of these values confirms that, in the absence of inorganic sulfate, most glycosamino~~~can sulfate derives from cysteine turnover, and little comes from methionine or other s u l f ~ - c o n ~ i n i compounds ng in the growth medium. It is interesting to speculate whether sulfate transport and sulfate production from cysteine are coordinately regulated. In the isotope dilution experiment described above, we incubated cells in medium containing 0.2 mniI cysteine and variable concentrations of inorganic ~ u l f a t e We . ~ found that the endogenous pool of sulfate availabie for glycosaminoglycan sulfation was independent of the concentration of added inorganic sulfate. If amino acid turnover decreased when inorganic sulfate in the medium wasplentiful, then theeffective pool of sulfate calculated by dilution analysis should have declined as the sulfate concentrationwas raised in the medium. Thus, the generation of sulfate from cysteine appears to be constitutive in CHO cells. In a separate experiment, we found that the addition of 0.1 mM inorganic sulfate to medium containing ~-$S]cysteine inhibited the incorporation of 35Sinto glycosaminoglycans by a factor of 8 (2900 cpm/pg of cell protein in the absence of sulfate versus 360 cpm/,ug of cell protein in its presence). The observed dilution is very close to thatpredict,ed if the two sources of sulfate were to mix ((100 pM f 15 pM)/ 15 p~ = 7.7).This finding also suggests that CHO cells do not coordinately control sulfate uptake and cysteine turnover. We do not know whether other tissues regulate amino acid metabolism and sulfate uptake. In IMR-90 lung fibroblasts, the addition of sulfur-containing amino acids to the growth medium partiallyinhibits the incorporation of 35S04into macromolecules.6 However,supplementation of smooth muscle cells with methionine and cysteine has little effect on 35s04incorporation? Thus, some tissues may differentially coordinate the uptake and metabolism of inorganic sulfate and sulfur-containing amino acids, perhaps in response $0 other metabolic requirements, such as protein synthesis, or the sulfation of glycoproteins and glycolipids. Intercelk&zr Sulfate Transport-Our finding that co-culti-

___

K. Rostand and J. D. Esko, unpublished results. e A. Elgavish, unpublishedresults.

Sulfation of Glycosaminoglycans 9. vation of mutants defective in glycosaminoglycan synthesis (mutants 745 and 761) with sulfate transport-deficient mu10. tants stimulated35S04 incorporation intoglycosaminoglycans is intriguing. Because we found that chromate ionsalso pass 11. between cells, as measured by cytotoxicity, we suggested that the formation of cell-cell contacts, possibly gap junctions, 12. underliestherestoration of sulfateuptake.Kriegerand co-workers (41, 42) also concluded thatCHO cells form junc- 13. tions from their studies of mutants deficient in low density lipoprotein uptake. They found that co-cultivation of ldlD 14. mutantswith cells bearingmutationsinthe low density 15. 16. lipoprotein receptor structural gene ( M U )restored the synthesis of fully functional receptors (41). Recently, Kingsley et al. (42) showed that ldlD mutants lack the 4-epimerase re17. sponsible for the interconversion of UDP-esters of glucose 18. (and N-acetylglucosamine) and galactose (and N-acetylgalactosamine). These findings suggested that nucleotide sugars 19. present in ldk4 cells passed through organized cell contacts and permitted ldlD cells to synthesize fully functional recep- 20. tors. 21. The importance of intercellular transport of sulfate and nucleotide sugars in glycosaminoglycan synthesis has not yet 22. been explored. Intercellular transfer of precursors may help 23. regulate glycosaminoglycan synthesis in tissuesby equilibrat- 24. ing intermediates among cells (43). Spatial control over glycosaminoglycan synthesis would be important in organized 25. celllayers, such as epithelia, which deposit proteoglycans 26. into basement membranes. Since extracellular matrices are thought to modulate cell differentiation and tissue morpho- 27. genesis (44), maintaining an even distribution of proteoglycan biosynthesis may prove essential for the spatial and temporal 28. needed 29. control of tissue differentiation. Additional studies are to determine whether the transferof glycosaminoglycan precursors between cells plays a role in theseprocesses. 30.

15733

[to, K., Kimata, K.,Sobue, M., and Suzuki, S. (1982) J. Biol. Chem. 257,917-923 Fukui, S., Yoshida, H., Tanaka, T., Sakano, T., Usui, T., and Yamashina, I. (1981) J. Biol. Chem. 256, 10313-10318 HumDhries, D. E., Silbert, C. K., and Silbert, J. E. (1986) J. Biol. Chem. 261,9122-9127. Esko. J. D.. Stewart. T. E.. and Tavlor.W . H. (1985) Proc. Natl. A c k . Sci. U. S. A,' 82, 3197-3201 Rod& L. (1980) in The Biochemistry of Glycoproteins and Proteoglycans (Lennarz, w. J., ed) pp. 267-371, PlenumPress, New York Esko, J. D. (1986) Methods Enzymol. 129, 237-253 Ham, R. G. (1965) Proc. Natl. Acud. Sci. U. S. A. 53, 288-293 Litwin, J. (1973) in Tissue Culture-Methods and Applications (Kruse. P. F.. and Patterson, M. K., eds) pp. 671-673, Academic Press, New York Dulbecco. R.. and Voet. M. (1954) J. E m . Med. 99. 167-182 Lowry, 0 . H:, Rosebrough, N. J.,'Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 Rodh, L., Baker, J. R., Cifonelli, J. A., and Matthews, M. B. (1972) Methods Enzymol. 28, 73-140 Patterson, M. S., and Green, R. C. (1965) Anal. Chem. 37, 854'

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Linker, A., and Hovingh, P. (1972) Methods EnzymoL 28, 902911

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Oegema, T. R., Jr., Kraft, E. L., Jourdian, G. W., and Van Valen, T. R. (1984) J. Biol. Chem. 259, 1720-1726 Yamagata, T., Saito, H., Habuchi, O., and Suzuki, S. (1968) J. Biol. Chem. 243,1523-1535 Levinson, C., and Villereal, M. C. (1974) J. Cell. Physiol. 85, 114

Davidson, R. L., and Gerald, P. S. (1976) Somatic Cell Genet. 2, 165-176

Haldane, J. B. S., and Stern, K. B. (1932) in Allgemeine Chemie der Enzyme p. 119, Verlag von Theodor Steinkopff, Dresden Arst, H. N., Jr. (1968) Nature 219,268-270 31. Roberts. K. R., and Marzluf,G . A. (1971) Arch. Biochem. Biophvs. _ .

Acknowledgments-We express our appreciation to S. Barnes for his help in analyzingthe kinetics of sulfate uptake andto F. Rahemtulla and L. R0dL.n for their careful readingof this manuscript.

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