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 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-diam...

Heparanases are endoglycosidases that cleave the heparan sulfate glycosaminoglycans from proteoglycan core proteins and degrade them to small oligosaccharides. Inside cells, these enzymes are important for the normal catabolism of heparan sulfate proteoglycans (HSPGs), generating glycosaminoglycan fragments that are then transported to lysosomes and completely degraded. When secreted, heparanases are thought to degrade basement membrane HSPGs at sites of injury or inflammation, allowing extravasion of immune cells into nonvascular spaces and releasing factors that regulate cell proliferation and angiogenesis. Heparanases have been described in a wide variety of tissues and cells, but because of difficulties in developing simple assays to follow activity, very little has been known about enzyme diversity until recently. Within the last 10 years, heparanases have been purified from platelets, placenta, and Chinese hamster ovary cells. Characterization of the enzymes suggests there may be a family of heparanase proteins with different substrate specificities and potential functions.

Heparan sulfate proteoglycans on Chinese hamster ovary (CHO) cell surfaces can bind and internalize basic fibroblast growth factor (bFGF). We have investigated whether this interaction affects heparan sulfate catabolism in vitro by measuring the ability of partially purified CHO heparanase activities to degrade 35 S-labeled heparan sulfate glycosaminoglycans in the absence or presence of bFGF. Our studies show that the presence of the growth factor prevents partially purified heparanases from degrading the nascent 81-kDa chains to short 6-kDa products, whether the glycosaminoglycan is free in solution or covalently bound to core proteins. A 30 -60 molar excess of the growth factor is required to inhibit completely chain degradation by heparanases, implying that multiple bFGF molecules must be bound to the glycosaminoglycan to prevent heparanase-catalyzed catabolism. This hypothesis is supported by protection studies indicating that nascent CHO heparan sulfate glycosaminoglycans have at least four to eight bFGF binding sites/chain. It does not appear, however, that the growth factor inhibits heparanase-catalyzed degradation of the glycosaminoglycan by binding to the sequence cleaved by the enzyme. Both the nascent and short chains bind bFGF with similar affinity (K d values of 27.0 ؎ 3.5 and 38.9 ؎ 5.1 nM, respectively), indicating that heparanase activities do not destroy the bFGF binding sites. Rather, our results suggest that the growth factor interferes sterically with heparanase action by binding the heparan sulfate chain at a sequence next to the cleavage site or at a secondary site recognized by the enzyme.Heparan sulfate proteoglycans (HSPGs), 1 molecules composed of heparan sulfate (HS) glycosaminoglycan chains covalently linked to a protein core, are ubiquitously present on cell surfaces and in extracellular matrix and basement membranes (1-3). Their expression appears to be developmentally regulated (4, 5) and cell-specific (6). Proteoglycans are implicated in a number of cellular processes, including cell adhesion, migration, differentiation, and proliferation (for review, see Refs. 7 and 8). Most of the identified proteoglycan functions are attributed to the interaction of the glycosaminoglycan chain with a protein ligand, such as lipoprotein lipase, fibronectin, or various members of heparin-binding growth factor family (9). The last has received a great deal of attention after the presence of heparin or heparan sulfate had been shown to be prerequisite for high affinity binding of basic fibroblast growth factor (bFGF) to its cell surface fibroblast growth factor receptor (FGFR) (10 -12). It was thought originally that binding to HS might change the growth factor conformation so that it can be recognized by the FGFR; however, cocrystallization of bFGF with heparin oligosaccharides demonstrated no structural changes in the growth factor upon the binding (13). Since heparin can form a ternary complex with bFGF and FGFR (14 -16), it has been proposed that the proteoglycans function as a coreceptor for ...

The Chinese hamster ovary cell mutant, pgsE-606, synthesizes undersulphated heparan sulphate glycosaminoglycans because of a deficiency in N-sulphotransferase activity [Bame and Esko (1989) J. Biol. Chem. 264, 8059-8065]. We compared the heparan sulphate proteoglycans synthesized by mutant and wild-type cells to determine what effect the undersulphation defect had on proteoglycan structure. The majority of heparan sulphate proteoglycans synthesized by pgsE-606 were undersulphated, but the mutant also synthesized a population of proteoglycans that were sulphated to the same extent as wild-type molecules. Anion-exchange analysis of the glycosaminoglycans in each proteoglycan population showed that they were all modified in the same way. The length of the glycosaminoglycans in each proteoglycan population were similar, suggesting that N-sulphation does not affect chain polymerization. To examine whether the sulphation state of the attached heparan sulphate glycosaminoglycans was dependent on the protein core, we purified syndecan-1 from mutant and wild-type cells using antibodies against the core protein. As with the unfractionated heparan sulphate proteoglycans, pgsE-606 synthesized both undersulphated and sulphated syndecan-1. Each pool contained either undersulphated or sulphated glycosaminoglycan chains respectively. Thus the modification of all heparan sulphate chains on a core protein occurs on a proteoglycan-wide basis (i.e. to the same extent).