Location of the glucuronosyltransferase domain in the heparan sulfate copolymerase EXT1 by analysis of Chinese hamster ovary cell mutants

Heparan sulfate formation occurs by the copolymerization of glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc) residues. Recent studies have show n that these reactions are catalyzed by a copolymerase encoded by EXT1 and EXT2, members of the exostosin family of putative tumor suppressors linked to hereditary multiple exostoses. Previously, we identified a collection of Chinese hamster ovary cell mutants (pgsD) that failed to make heparan sulf ate (Lidholt, K,, Weinke, J. L., Riser, C. S., Lugemwa, F. N., Bame, K. J., Cheifetz, S., Massague, J,, Lindahl, U., and Esko, J. D. (1992) Proc. Natl . Acad, Sci. U.S.A. 89, 2267-2271). Here, we show that pgsD mutants contain mutations that either alter GlcA transferase activity selectively or that affect both GlcNAc and GlcA transferase activities. Expression of EXT1 corr ects the deficiencies in the mutants, whereas EXT2 and the related EXT-like cDNAs do not. Analysis of the EXT1 mutant alleles revealed clustered misse nse mutations in a domain that included a (D/E)X(D/E) motif thought to bind the nucleotide sugar from studies of other transferases. These findings pr ovide insight into the location of the GlcA transferase subdomain of the en zyme and indicate that loss of the GlcA transferase domain may be sufficien t to cause hereditary multiple exostoses.