Temperature-sensitive glycosaminoglycan biosynthesis in a Chinese hamster ovary cell mutant containing a point mutation in glucuronyltransferase I

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 (GlcAbeta1,3Galbeta1,3Galbeta1, 4Xylbeta-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 (SO4)-S-35 into glycosaminoglycans in the mutant is temperature-sensitive, with greater synthesis occurring at 33degreesC compared with 37degreesC. 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 30degreesC. At 37degreesC, the fusion protein was not readily detectable. Sequencing cDNAs from mutant and wildtype 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.