An Animal Cell Mutant Defective in Heparan Sulfate Hexuronic Acid 2- -Sulfation

The interaction of heparan sulfate with protein ligands depends on unique oligosaccharide sequences containing iduronic acid (IdUA), N-sulfated glucosamine residues, and O-sulfated sugars. To study the role of O-sulfation in greater detail, we isolated a Chinese hamster ovary cell mutant defective in 2-O-sulfation of iduronic acid. The mutant, pgsF-17, was identified by a colony blotting assay in which colonies of mutagen-treated cells were replica plated to two disks of polyester cloth. One disk was blotted with 125I-labeled basic fibroblast growth factor (bFGF) to measure binding to cell surface proteoglycans. The other disk was incubated with 35SO4 to measure proteoglycan biosynthesis. Autoradiography revealed a colony that did not bind 125I-bFGF, but incorporated 35SO4 normally (mutant pgsF-17). Complete deaminative cleavage of heparan sulfate revealed that material from pgsF-17 lacked IdUA(2OSO3)-GlcNSO3 and IdUA(2OSO3)-GlcNSO3(6OSO3), but contained a higher proportion of glucuronic acid GlcUA-GlcNSO3(6OSO3) and IdUA-GlcNSO3(6OSO3). Assay of the 2-O-sulfotransferase that acts on IdUA residues showed that mutant 17 lacked enzyme activity. Interestingly, the alteration resulted in accumulation of GlcNSO3 groups, suggesting that under normal conditions 2-O-sulfation decreases GlcNAc N-deacetylation/N-sulfation, and that the reactions occur simultaneously. The formation of IdUA and 6-O-sulfated glucosaminyl residues appears to be independent of 2-O-sulfation. pgsF-17 also lacks 2-O-sulfated GlcUA residues, suggesting that the same enzyme is responsible for 2-O-sulfation of IdUA and GlcUA residues. Mutant 17 provides a useful tool for studying the regulation of heparan sulfate biosynthesis and the relationship of heparan sulfate fine structure to its biological function. The interaction of heparan sulfate with protein ligands depends on unique oligosaccharide sequences containing iduronic acid (IdUA), N-sulfated glucosamine residues, and O-sulfated sugars. To study the role of O-sulfation in greater detail, we isolated a Chinese hamster ovary cell mutant defective in 2-O-sulfation of iduronic acid. The mutant, pgsF-17, was identified by a colony blotting assay in which colonies of mutagen-treated cells were replica plated to two disks of polyester cloth. One disk was blotted with 125I-labeled basic fibroblast growth factor (bFGF) to measure binding to cell surface proteoglycans. The other disk was incubated with 35SO4 to measure proteoglycan biosynthesis. Autoradiography revealed a colony that did not bind 125I-bFGF, but incorporated 35SO4 normally (mutant pgsF-17). Complete deaminative cleavage of heparan sulfate revealed that material from pgsF-17 lacked IdUA(2OSO3)-GlcNSO3 and IdUA(2OSO3)-GlcNSO3(6OSO3), but contained a higher proportion of glucuronic acid GlcUA-GlcNSO3(6OSO3) and IdUA-GlcNSO3(6OSO3). Assay of the 2-O-sulfotransferase that acts on IdUA residues showed that mutant 17 lacked enzyme activity. Interestingly, the alteration resulted in accumulation of GlcNSO3 groups, suggesting that under normal conditions 2-O-sulfation decreases GlcNAc N-deacetylation/N-sulfation, and that the reactions occur simultaneously. The formation of IdUA and 6-O-sulfated glucosaminyl residues appears to be independent of 2-O-sulfation. pgsF-17 also lacks 2-O-sulfated GlcUA residues, suggesting that the same enzyme is responsible for 2-O-sulfation of IdUA and GlcUA residues. Mutant 17 provides a useful tool for studying the regulation of heparan sulfate biosynthesis and the relationship of heparan sulfate fine structure to its biological function.