Chinese hamster ovary cell mutants defective in glycosaminoglycan assembly and glucuronosyltransferase I

The proteoglycans of animal cells typically contain one or more heparan sulfate or chondroitin sulfate chains. These glycosaminoglycans assemble on a tetrasaccharide primer, -GlcA beta 1,3Gal beta 1,3Gal beta 1,4Xyl beta-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 (xylosyl-transferase 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 (SO4)-S-35 and [6-H-3]GlcN into glycosaminoglycan chains. Incubation of these cells with naphthol-beta-D-xyloside (Xyl beta-O-Np) resulted in accumulation of linkage region intermediates containing 1 or 2 mol of galactose (Gal beta 1, 4Xyl beta-O-Np and Gal beta 1, 3Gal beta 1, 4Xyl beta-O-Np) and sialic acid (Sia alpha 2,3Gal beta 1, 3Gal beta 1, 4Xyl beta-O-Np) but not any GlcA-containing oligosaccharides. Extracts of the mutants completely lacked UDP-glucluronic acid:Gal beta 1,3Gal-R glucuronosyltransferase (GlcAT-I) activity, as measured by the transfer of GlcA from UDP-GlcA to Gal beta 1,3Gal beta-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.