Location of the Glucuronosyltransferase Domain in the Heparan Sulfate Copolymerase EXT1 by Analysis of Chinese Hamster Ovary Cell Mutants

• Published in: The Journal of biological chemistry 2000-09, Vol.275 (36), p.27733-27740
• Main Authors: Wei, G, Bai, X, Gabb, M M, Bame, K J, Koshy, T I, Spear, P G, Esko, J D
• Format: Article
• Language: English
• Subjects: Acetylglucosamine - metabolism; Amino Acid Sequence; Animals; CHO Cells; Cloning, Molecular; Cricetinae; Exostoses, Multiple Hereditary; Glucuronosyltransferase - metabolism; Heparitin Sulfate - genetics; Humans; Kinetics; Mice; Molecular Sequence Data; Mutation, Missense; N-Acetylglucosaminyltransferases - chemistry; N-Acetylglucosaminyltransferases - genetics; N-Acetylglucosaminyltransferases - metabolism; Proteins - metabolism; Recombinant Proteins - metabolism; Sequence Alignment; Sequence Homology, Amino Acid
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M002990200

Heparan sulfate formation occurs by the copolymerization of glucuronic acid (GlcA) and N- acetylglucosamine (GlcNAc) residues. Recent studies have shown 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 sulfate (Lidholt, K., Weinke, J. L., Kiser, C. S., Lugemwa, F. N., Bame, K. J., Cheifetz, S., Massagué, 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 corrects the deficiencies in the mutants, whereas EXT2 and the related EXT-like cDNAs do not. Analysis of the EXT1 mutant alleles revealed clustered missense 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 provide insight into the location of the GlcA transferase subdomain of the enzyme and indicate that loss of the GlcA transferase domain may be sufficient to cause hereditary multiple exostoses.