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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 shown that these reactions are catalyzed by a copolymerase encoded by EXT1 and EXT2, members of the exostosin family of putative tumor suppressors linked...

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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
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container_issue 36
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creator Wei, G
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Esko, J D
description 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.
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ispartof The Journal of biological chemistry, 2000-09, Vol.275 (36), p.27733-27740
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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
title Location of the Glucuronosyltransferase Domain in the Heparan Sulfate Copolymerase EXT1 by Analysis of Chinese Hamster Ovary Cell Mutants
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