Abstract
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 35SO4 into glycosaminoglycans in the mutant is temperature-sensitive, with greater synthesis occurring at 33 degrees C compared with 37 degrees 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 degrees C. At 37 degrees 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.
Highlights
Proteoglycans play important roles as regulators in many biologic processes, such as cell adhesion, proliferation, differentiation, and cytokine and growth factor action [1, 2]
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
This oligosaccharide serves as a primer for chain elongation, forming either heparan sulfate or chondroitin sulfate depending upon the addition of a GlcNAc or GalNAc residue, respectively [3, 4]
Summary
Proteoglycans play important roles as regulators in many biologic processes, such as cell adhesion, proliferation, differentiation, and cytokine and growth factor action [1, 2]. These data suggest that pgsG cells contain a labile form of GlcATI that causes conditional expression of glycosaminoglycans dependent on temperature. We described previously a set of Chinese hamster ovary cell mutants designated pgsG that lack GlcATI enzyme activity and fail to make both heparan sulfate and chondroitin sulfate [20].
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