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, -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.
Bai
X
X
Division of Cellular and Molecular Medicine, Glycobiology Program, University of California, San Diego, La Jolla, California 92093-0687, USA.
Wei
G
G
Sinha
A
A
Esko
J D
JD
D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D003412:Cricetulus; D014453:Glucuronosyltransferase / Q000235:genetics / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000378:metabolism*; D006027:Glycosides / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism
D006025:Glycosaminoglycans; D006027:Glycosides; D009844:Oligosaccharides; C010807:xylosides; C111364:galactosylgalactoylxylosylprotein 3-beta-glucuronosyltransferase; D014453:Glucuronosyltransferase