The sulfated glycosaminoglycans, heparan sulfate and heparin, are increasingly implicated in cell-biological processes such as cytokine action, cell adhesion, and regulation of enzymic catalysis. These activities generally depend on interactions of the polysaccharides with proteins, mediated by distinct saccharide sequences, and expressed at various levels of specificity, selectivity, and molecular organization. The formation of heparin/ heparan sulfate in the cell requires an elaborate biosynthetic machinery, that is conceived in terms of a novel model of glycosaminoglycan assembly and processive modification. Recent advances in the identification and molecular analysis of the enzymes and other proteins involved in the biosynthesis provide novel tools to study the regulation of the process, presently poorly understood, at the subcellular and cellular levels. The potential medical importance of heparin-related compounds is likely to promote the biotechnological exploitation of components of the biosynthetic machinery.
Hereditary multiple exostoses, characterized by multiple cartilaginous tumors, is ascribed to mutations at three distinct loci, denoted EXT1-3. Here, we report the purification of a protein from bovine serum that harbored the D-glucuronyl (GlcA) and N-acetyl-D-glucosaminyl (GlcNAc) transferase activities required for biosynthesis of the glycosaminoglycan, heparan sulfate (HS). This protein was identified as EXT2. Expression of EXT2 yielded a protein with both glycosyltransferase activities. Moreover, EXT1, previously found to rescue defective HS biosynthesis
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...
The D-glucuronyltransferase and N-acetyl-D-glucosaminyltransferase reactions in heparan sulfate biosynthesis have been associated with two genes, EXT1 and EXT2, which are also implicated in the inherited bone disorder, multiple exostoses. Since the cell systems used to express recombinant EXT proteins synthesize endogenous heparan sulfate, and the EXT proteins tend to associate, it has not been possible to define the functional roles of the individual protein species. We therefore expressed EXT1 and EXT2 in yeast, which does not synthesize heparan sulfate. The recombinant EXT1 and EXT2 were both found to catalyze both glycosyltransferase reactions in vitro. Coexpression of the two proteins, but not mixing of separately expressed recombinant EXT1 and EXT2, yields hetero-oligomeric complexes in yeast and mammalian cells, with augmented glycosyltransferase activities. This stimulation does not depend on the membrane-bound state of the proteins.
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