Chinese Hamster Ovary Cell Mutants Defective in Glycosaminoglycan Assembly and Glucuronosyltransferase I

doi:10.1074/jbc.274.19.13017

Chinese Hamster Ovary Cell Mutants Defective in Glycosaminoglycan Assembly and Glucuronosyltransferase I*

From the Division of Cellular and Molecular Medicine, Glycobiology Program, University of California, San Diego, La Jolla, California 92093-0687

Abstract

The proteoglycans of animal cells typically contain one or more heparan sulfate or chondroitin sulfate chains. These glycosaminoglycans assemble on a tetrasaccharide primer, -GlcAβ1,3Galβ1,3Galβ1,4Xylβ-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-β-d-xyloside (Xylβ-O-Np) resulted in accumulation of linkage region intermediates containing 1 or 2 mol of galactose (Galβ1, 4Xylβ-O-Np and Galβ1, 3Galβ1, 4Xylβ-O-Np) and sialic acid (Siaα2,3Galβ1, 3Galβ1, 4Xylβ-O-Np) but not any GlcA-containing oligosaccharides. Extracts of the mutants completely lacked UDP-glucuronic acid:Galβ1,3Gal-R glucuronosyltransferase (GlcAT-I) activity, as measured by the transfer of GlcA from UDP-GlcA to Galβ1,3Galβ-O-naphthalenemethanol (<0.2versus 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.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ On leave from the Dept. of Biochemistry and Molecular Genetics, University of Alabama, Birmingham, AL 35294.
↵§ To whom correspondence should be addressed: Division of Cellular and Molecular Medicine, Dept. of Medicine, The Glycobiology Program, University of California, San Diego, 9500 Gilman Dr., CMM East 1055, La Jolla, CA 92093-0687. Tel.: 619-822-1100; Fax: 619-534-5611; E-mail: jesko{at}ucsd.edu.
↵2 K. Kimata, personal communication.
↵3 The relative amount of charged material was dependent on the concentration of xyloside in the growth medium; ∼50% of the oligosaccharides in wild-type cells and ∼20% in the mutant were charged when the xyloside concentration was increased to ≥10 μm.
Abbreviations:

GAG

glycosaminoglycan

CHO

Chinese hamster ovary

PBS

phosphate-buffered saline

FGF2-SAP

basic fibroblast growth factor-saporin-6 chimera

GlcAT-I

UDP-glucuronic acid:Galβ1,3Gal-R glucuronosyltransferase

GlcAT-P

UDP-glucuronic acid:glycoprotein glucuronosyltransferase

MOPS

3-[N-morpholino]propanesulfonic acid

XylT

xylosyltransferase

GalT-I

galactosyltransferase I
- Received January 19, 1999.
- Revision received February 22, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.