Abstract
The proteoglycans of animal cells typically contain one or more heparan sulfate or chondroitin sulfate chains. These glycosaminoglycans assemble on a tetrasaccharide primer, -GlcAbeta1, 3Galbeta1,3Galbeta1,4Xylbeta-O-, attached to specific serine residues in the core protein. Studies of Chinese hamster ovary cell mutants defective in the first or second enzymes of the pathway (xylosyltransferase and galactosyltransferase I) show that the assembly of the primer occurs by sequential transfer of single monosaccharide residues from the corresponding high energy nucleotide sugar donor to the non-reducing end of the growing chain. In order to study the other reactions involved in linkage tetrasaccharide assembly, we have devised a powerful selection method based on induced resistance to a mitotoxin composed of basic fibroblast growth factor-saporin. One class of mutants does not incorporate 35SO4 and [6-3H]GlcN into glycosaminoglycan chains. Incubation of these cells with naphthol-beta-D-xyloside (Xylbeta-O-Np) resulted in accumulation of linkage region intermediates containing 1 or 2 mol of galactose (Galbeta1, 4Xylbeta-O-Np and Galbeta1, 3Galbeta1, 4Xylbeta-O-Np) and sialic acid (Siaalpha2,3Galbeta1, 3Galbeta1, 4Xylbeta-O-Np) but not any GlcA-containing oligosaccharides. Extracts of the mutants completely lacked UDP-glucuronic acid:Galbeta1,3Gal-R glucuronosyltransferase (GlcAT-I) activity, as measured by the transfer of GlcA from UDP-GlcA to Galbeta1,3Galbeta-O-naphthalenemethanol (<0.2 versus 3.6 pmol/min/mg). The mutation most likely lies in the structural gene encoding GlcAT-I since transfection of the mutant with a cDNA for GlcAT-I completely restored enzyme activity and glycosaminoglycan synthesis. These findings suggest that a single GlcAT effects the biosynthesis of common linkage region of both heparan sulfate and chondroitin sulfate in Chinese hamster ovary cells.
Highlights
The assembly of the glycosaminoglycans (GAGs),1 heparan sulfate and chondroitin sulfate, initiates by the transfer of xylose to specific serine residues in core proteins, which gives rise to the so-called linkage tetrasaccharide, GlcA1,3Gal1,3Gal1,4Xyl-O-Ser [1]
The linkage tetrasaccharide serves as a primer for the formation of heparan sulfate and chondroitin sulfate chains, which initiate by the attachment of either GlcNAc or GalNAc, respectively [14, 15]
Selection of Glycosaminoglycan-deficient Mutants—We have identified previously a number of CHO cell mutants altered in GAG biosynthesis using a “brute force” replica plating technique [43]
Summary
Cell Cultures—Chinese hamster ovary cells (CHO-K1) were obtained from the American Type Culture Collection (CCL-61, Rockville, MD). Charged materials were treated with (i) 10 milliunits of sialidase (Arthrobacter ureafaciens, Oxford Glycosystems) for 16 h in 100 mM sodium acetate (pH 5.0); some samples were desialylated by boiling in 10 mM HCl for 30 min; (ii) 100 units of -glucuronidase (bovine liver, Sigma) overnight in 100 mM sodium acetate (pH 5.0) buffer containing 0.3 M NaCl and 8 mM D-galactonic acid ␥-lactone to inhibit contaminating -galactosidase activity; and (iii) 1 milliunit of ␣-N-acetylgalactosaminidase (chicken liver, Oxford Glycosystems) overnight in 0.1 M citrate/phosphate buffer (pH 4.0). The sample was diluted with 30 ml of 20 mM HEPES buffer (pH 7.4) containing 0.5 M NaCl and 0.2% bovine serum albumin and loaded onto a 1-ml column of heparin-Sepharose CL-6B (Amersham Pharmacia Biotech). The column was washed with 30 ml of buffer and eluted with 2.5 ml of solution adjusted to 3 M NaCl. The sample was desalted on a PD-10 column (Amersham Pharmacia Biotech) equilibrated with 20 mM HEPES buffer (pH 7.4) containing 0.2% bovine serum albumin. The donor, UDP[1-3H]glucuronic acid, and the substrate, Gal1,3Gal-O-naphthalenemethanol, were synthesized as described [49]
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