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J. Biol. Chem., Vol. 279, Issue 7, 5693-5698, February 13, 2004

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Temperature-sensitive Glycosaminoglycan Biosynthesis in a Chinese Hamster Ovary Cell Mutant Containing a Point Mutation in Glucuronyltransferase I^*

Ge Wei, Xiaomei Bai, and Jeffrey D. Esko

From the Department of Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California, San Diego, La Jolla, California 92093-0687

In previous studies, we reported the isolation and characterization of a Chinese hamster ovary cell mutant (pgsG) defective in glucuronyltransferase I (GlcATI). This enzyme adds the terminal GlcA residue in the core protein-linkage tetrasaccharide (GlcA1,3Gal1,3Gal1, 4Xyl-O-) on which glycosaminoglycan assembly occurs (Bai, X. M., Wei, G., Sinha, A., and Esko, J. D. (1999) J. Biol. Chem. 274, 13017–13024; Wei, G., Bai, X. M., Sarkar, A. K., and Esko, J. D. (1999) J. Biol. Chem. 274, 7857–7864). Here we show that incorporation of ³⁵SO₄ into glycosaminoglycans in the mutant is temperature-sensitive, with greater synthesis occurring at 33 °C compared with 37 °C. Wild-type cells show the opposite thermal dependence. Rabbit antiserum to hamster GlcATI failed to detect cross-reactive material in pgsG cells by immunofluorescence and Western blotting. Furthermore, expression of chimeric proteins composed of mutant GlcATI fused to IgG binding domain of protein A or to green fluorescent protein did not yield the proteins at the expected mass. The green fluorescent protein-tagged version appeared as a truncated protein, and immunofluorescence showed large perinuclear bodies at 30 °C. At 37 °C, the fusion protein was not readily detectable. Sequencing cDNAs from mutant and wild-type cells revealed a single base transition (G331A) in the open reading frame in pgsG cells, which resulted in a Val-111 Met substitution. These data suggest that pgsG cells contain a labile form of GlcATI that causes conditional expression of glycosaminoglycans dependent on temperature.

Received for publication, October 23, 2003

^* This work was supported by National Institutes of Health Grant R37GM33063 (to J. D. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work. Present address: Neose West, 6330 Nancy Ridge Rd., Ste. 102-130, San Diego, CA 92121.

To whom correspondence should be addressed: Dept. of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Dr., CMM-East 1055, La Jolla, CA 92093-0687. Tel.: 858-822-1100; Fax: 858-534-5611; E-mail: jesko{at}ucsd.edu.

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