title: Temperature-sensitive glycosaminoglycan biosynthesis in a Chinese hamster ovary cell mutant containing a point mutation in glucuronyltransferase I.
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.
authors: Department of Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California, San Diego, La Jolla, California 92093-0687, USA.
authors: Ge
authors: G
authors: Wei
authors: Xiaomei
authors: X
authors: Bai
authors: Jeffrey D
authors: JD
authors: Esko
mesh_terms: D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000235:genetics*; D006025:Glycosaminoglycans / Q000096:biosynthesis / Q000737:chemistry*; D049452:Green Fluorescent Proteins; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000737:chemistry; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism; D008715:Methionine / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D017354:Point Mutation; D011134:Polysaccharides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013696:Temperature; D013997:Time Factors; D014633:Valine / Q000737:chemistry
chemical_list: D018076:DNA, Complementary; D006025:Glycosaminoglycans; D007074:Immunoglobulin G; D008164:Luminescent Proteins; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D008715:Methionine; C111364:galactosylgalactoylxylosylprotein 3-beta-glucuronosyltransferase; D014453:Glucuronosyltransferase; D014633:Valine