Proc. Natl. Acad. Sci. USA Vol. 82, pp. 3197-3201, May 1985 Biochemistry Animal cell mutants defective in glycosaminoglycan biosynthesis (Chinese hamster ovary cells/replica plating/proteoglycans/sulfation/xylosyltransferase) JEFFREY D. ESKO, TOD E. STEWART, AND WILLIAM H. TAYLOR Department of Biochemistry, University of Alabama in Birmingham, Birmingham, AL 35294 Communicated by Phillips W. Robbins, January 18, 1985 ABSTRACT We have obtained Chinese hamster ovary glycosaminoglycan biosynthesis. One of these mutants has a cell mutants defective in the biosynthesis of glycosaminogly- defect in the xylosyltransferase (2), the first sugar transfer cans by screening replicate colonies immobilized on polyester reaction in glycosaminoglycan formation (Fig. 1). cloth. Depending upon the strain, the mutants accumulated less 35S-labeled glycosaminoglycans per ,ug of cell protein by a EXPERIMENTAL PROCEDURES factor of 6-60 compared to the wild type. Some of the mutants incorporated [6-3H]glucosamine into glycosaminoglycans to Materials. Na35SO4 (25-40 Ci/mg; 1 Ci = 37 GBq) and D- the same extent as the wild type, suggesting that sulfate addi- [6-3H]glucosamine HCl (25-30 Ci/mmol) were obtained tion was specifically altered. In contrast, five strains failed to from Amersham. UDP-[1-3H]xylose was purchased from generate 'H-labeled glycosaminoglycans normally. In four of New England Nuclear. All nonradioactive chemicals were these, the initiation of glycosaminoglycan assembly was specif- generally obtained from Sigma. Chondroitinase AC from ically altered, since the addition of p-nitrophenyl-f-xyloside Arthrobacter aurescens (Sigma), chondroitinase ABC from restored sulfation to normal. Enzymatic assay of the xylosyl- Proteus vulgaris (Sigma), and heparitinase from Flavobac- transferase in extracts prepared from these mutants revealed terium heparinum (Miles) were stored in 50 mM Tris HCl that one of the strains, S745, contained less enzyme activity by (pH 7.0) at -20'C. a factor of 15 than the wild type. This mutant provides genetic Cell Cultures. CHO cells (CHO-Ki) were obtained from evidence that the xylosyltransferase assayed in vitro is respon- the American Type Culture Collection (CCL-61) and grown sible for the initiation of chondroitin sulfate and heparan sul- in an atmosphere of 5% CO2 in air and 100% relative humid- fate biosynthesis in vivo. ity in Ham's F12 growth medium (GIBCO) supplemented with 100 ,ug of streptomycin sulfate per ml, 100 units of peni- Proteoglycans are ubiquitous components of mammalian cillin G per ml, and 10% (vol/vol) fetal bovine serum (Hy- cells and tissues (1-3). To a large extent, their chemical and Clone, Logan, UT). Growth medium lacking sulfate was pre- physical properties are determined by the glycosamino- pared from individual components (10), substituting chloride glycan chains attached glycosidically to serine residues of salts for sulfate salts and omitting streptomycin sulfate. De- core proteins (Fig. 1). For example, in cartilage, the chon- fined medium was supplemented with 10% (vol/vol) fetal bo- droitin sulfate proteoglycans produced by chondrocytes pro- vine serum dialyzed 106-fold against phosphate-buffered sa- vide a hydrated, viscous gel of high compressibility (1). In line (P1/NaCI) (11). other tissues, cells are surrounded by an extracellular matrix Mutageneses were performed with ethylmethane sulfonate composed of proteoglycans, collagen, and other macromol- as described (8). After outgrowth at 33°C and storage in liq- ecules, which together may influence cell adhesion, shape, uid nitrogen, an aliquot of cells was revived and tested for and possibly locomotion (4-6). In hepatocytes, membrane- the incidence of strains resistant to 1 mM ouabain. Mutagen- intercalated heparan sulfate proteoglycans are thought to treated stocks in which the frequency of ouabain-resistant bridge the extracellular matrix and the intracellular cytoskel- strains was greater than 1/104 viable cells were used for the eton, whereas proteoglycans bound to endothelial cell plas- mutant screenings described below. ma membranes facilitate the binding of circulating macro- Polyester Cloning. The immobilization of CHO cells on 17- molecules (4-6). The presence of proteoglycans in secretory ,um polyester discs was performed as described (8, 9), with granules and lysosomes (4) suggests that they may also play two layers of cloth per dish. The isolation of the mutants is a role in fundamental processes of the cell, such as secretion described in detail in the legend of Fig. 2. Glass beads and and endocytosis. polyester cloth were prepared as described (8, 9). Tissues and cells vary tremendously in the array of pro- Xylosyltransferase Assay. Measurement of xylosyltransfer- teoglycans present (7), and the arrangement of repeating di- ase activity using silk fibroin as substrate (12) was performed saccharide units containing ester sulfate, N-sulfate, N-acetyl in extracts prepared from cells grown at 37°C in normal groups, D-glucuronic acid, and L-iduronic acid suggests that growth medium. Typically, cells in the exponential phase of enormous molecular heterogeneity of the glycosaminogly- growth were harvested in 0.25 M sucrose containing 20 mM cans may exist (Fig. 1). Undoubtedly, the composition of Tris HCl (pH 7.4), 1 ,g of leupeptin per ml, 0.5 ug of pepsta- glycosaminoglycans is determined biosynthetically (2), but tin A per ml, 20 ,uM phenylmethylsulfonyl fluoride, and 10 the factors that control their assembly and distribution re- mM sodium azide by scraping with a rubber policeman. The mains unclear. An important approach to these problems is cell suspensions were centrifuged at 1100 x g at 4°C for 5 the isolation of mutants altered in specific biosynthetic min and resuspended at a protein concentration of 7-10 steps. mg/ml as measured by the method of Lowry et al. (13). After In this report we demonstrate a screening technique (8, 9) brief sonication, the extracts were stored at -20°C. Assay that permits the identification of Chinese hamster ovary conditions optimized for CHO cells are provided in the leg- (CHO) cell mutants bearing mutations in different stages of end of Table 4. Enzyme activity was not detectable in the supernatant of the low-speed centrifugation used to harvest The publication costs of this article were defrayed in part by page charge the cells. payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: CHO, Chinese hamster ovary. 3197 Downloaded by guest on September 26, 2021 3198 Biochemistry: Esko et aL Proc. Natl. Acad Sci. USA 82 (1985) CORE INITIATION POLYMER ELONGATION PROTEIN SYNTHESIS LINKAGE REGION SYNTHESIS & MODIFICATION A1 ± -f UDP-Gal UDP-GlcUA UDP-Xyl UDP-Gal UDP-GalNAc 'JDP-GlcUA * *..~~~~~~~~~~I SER - 0 - XYL - GAL - GAL - GLcUA - (GALNAC - GLCUA)N Io PAPS OV ~~~~~~~~~~~~So3 FIG. 1. Biosynthesis of a chondroitin sulfate proteoglycan in D 0 mammalian cells. PAPS, 3'-phosphoadenyl-5'-phosphosulfate. .04w 0 Composition of Radioactive Glycosaminoglycans in CHO Cells. CHO cells were labeled with 50 gCi of35S04 per ml for 3 days at 370C in growth medium lacking inorganic sulfate. The cells were harvested by adjusting the medium in the dish to 0.1 M NaOH. After neutralization with acetic acid, the mixture was digested at 40'C for 16 hr with 2 mg of Pronase FIG. 2. Visualization of mutants defective in "SO4 incorpo- (Calbiochem) per ml in 0.32 M NaCl/40 mM sodium acetate, ration by autoradiography. Mutagen-treated cells were placed in pH 5.5. The released glycosaminoglycans were collected in 100-mm (diameter) tissue culture dishes containing 15 ml of com- the presence of 2 mg of chondroitin sulfate (mixed isomers) plete growth medium to yield -300 colonies per plate at 33°C. After per ml by precipitation with cetylpyridinium chloride and 1 day, the attached cells were overlaid with two layers of polyester ethanol according to established procedures (14). Portions of cloth (17-pim pore diameter) fitted to the dish and glass beads were the radioactive material were treated for 8 hr at 40'C with 5 added (8, 9). The cells were incubated at 330C for another 16 days to milliunits of chondroitinase AC or ABC (15) or 0.5 unit of permit the formation of colonies. At this time the medium was aspi- heparitinase (16). Radioactive products were quantitated af- rated and the beads were decanted from the dish. The polyester ter treatment with 1% cetylpyridinium chloride in the pres- cloth was removed and placed at 330C in a 100-mm (diameter) bacte- rial culture dish containing 15 ml of growth medium lacking sulfate. ence of 0.32 M NaCl as described by Oldberg et al. (17). After 2 days, the discs were shifted to 400C for 16 hr. Next, they were placed in 5 ml of the same growth medium supplemented with "SO4 (10 ACi/ml). Four hours later, the discs were treated with 10%6 RESULTS trichloroacetic acid, which resulted in the precipitation of "5S-gly- cosaminoglycans linked to protein, while unincorporated radioac- Autoradiographic Detection of Mutant Colonies Defective in tive sulfate was removed by washing each disc in a Buchner funnel Sulfate Incorporation. CHO cells, like other mammalian cells under reduced pressure with -100 ml of 2% trichloroacetic acid. (3, 18), incorporate 35 O4 preferentially into glycosamino- After drying the discs at room temperature overnight, they were ex- glycans. Analysis of 35S-labeled glycosaminoglycans (35S- posed to Kodak XAR-5 x-ray film for -24 hr. The discs were then glycosaminoglycans) obtained from wild-type CHO cells re- stained with 0.05% Coomassie brilliant blue G in methanol/water/ vealed that 55% ± 5% of the radioactive material was sus- acetic acid, 45:45:10 (vol/vol), and destained in methanol/water/ ceptible to chondroitinase AC (or ABC) digestion, and 40% acetic acid as described (8, 9). Throughout these manipulations the ± 5% was cleaved by heparitinase (Experimental Proce- original master plate was stored at 280C under otherwise normal sulfated are soluble in growth conditions in medium supplemented with 10 units ofnystatin dures).