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Temperature-sensitive Glycosaminoglycan Biosynthesis in a Chinese Hamster Ovary Cell Mutant Containing a Point Mutation in Glucuronyltransferase I.
- Source :
-
Journal of Biological Chemistry . 2/13/2004, Vol. 279 Issue 7, p5693-5698. 6p. 10 Color Photographs, 3 Diagrams, 5 Graphs. - Publication Year :
- 2004
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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 (GlcAβl,3Galβl,3Galβ1, 4Xylβ-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 GIcATI 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 proteintagged 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 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. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 00219258
- Volume :
- 279
- Issue :
- 7
- Database :
- Academic Search Index
- Journal :
- Journal of Biological Chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 12523452
- Full Text :
- https://doi.org/10.1074/jbc.M311621200