A clonal derivative of tunicamycin‐resistant Chinese hamster ovary cells with increased N‐acetylglucosamine‐phosphate transferase activity has altered asparagine‐linked glycosylation

Abstract

A population of Chinese hamster ovary (CHO) cells resistant to the antibiotic tunicamycin (TM) had previously been isolated (Criscuolo, B.A., and Krag, S.S. (1982) J. Cell Biol. 94:586-591) by a stepwise selection procedure using progressive increments of TM added to the medium. TM inhibits asparagine-linked glycoprotein biosynthesis by blocking the transfer of N-acetylglucosamine-1-phosphate from the sugar nucleotide UDP-N-acetylglucosamine to the isoprenoid lipid carrier, dolichyl phosphate. Four clonal derivatives were isolated from the TM-resistant population in the presence of 27 micrograms TM/ml and were found to overproduce the N-acetylglucosamine-phosphate transferase activity to the same extent (approximately 15-fold compared to wild-type cells). One of these clones, 3E11, was greater than 550-fold more resistant to TM than wild-type cells. The resistance phenotype remained during at least 2.5 months of growth in the absence of TM. 3E11 cells exhibited chromosomal translocations, but no homogeneously staining regions (HSR) or double minute chromosomes. The N-acetylglucosamine-phosphate transferase activity in 3E11 cells was membrane-associated and was inhibited by TM. A 140,000-dalton membrane protein and at least four other membrane proteins were enriched in 3E11 cells. Mannosylphosphoryldolichol synthase and glucosylphosphoryldolichol synthase activities were not elevated in membranes prepared from 3E11 cells. Asparagine-linked glycosylation was altered such that 3E11 cells synthesized primarily a truncated oligosaccharide, Man5GlcNAc2, perhaps due to the reduced amount of mannosylphosphoryldolichol relative to wild-type cells.

p185HER2 monoclonal antibody has antiproliferative effects in vitro and sensitizes human breast tumor cells to tumor necrosis factor.

“…To determine the nature of the epitope recognized by 4D5, NIH 3T3/HER2-3400 cells were treated with tunicamycin, which prevents addition of N-linked oligosaccharides to proteins (15,41). Cells treated with this antibiotic for 5.5 h contained two proteins which were immunoprecipitated by a polyclonal antibody against the carboxy-terminal peptide of p185LIER2 (Fig.…”

Overexpression of a Gene That Encodes the First Enzyme in the Biosynthesis of Asparagine-Linked Glycans Makes Plants Resistant to Tunicamycin and Obviates the Tunicamycin-Induced Unfolded Protein Response

“…the second enzyme in the pathway). Tunicamycin is cytotoxic, but experiments with animal (Waldman et al, 1987;Lehrman et al, 1988) and plant (Zeng and Elbein, 1995) cells show that it is possible to select tissue culture cells that are resistant to tunicamycin. In such resistant cells GPT is highly expressed as a result of an increase in the copy number of the gene caused by amplification.…”

Control of carbohydrate processing: increased beta-1,6 branching in N-linked carbohydrates of Lec9 CHO mutants appears to arise from a defect in oligosaccharide-dolichol biosynthesis.

“…radioactivity incorporated into water-soluble and chloroform-soluble fractions was determined ( Table 3). The aqueous fraction (containing water-soluble intermediates such as mannose, mannose-phosphate and GDP-mannose [17]) contained similar amounts of label in each cell line to those in the lipid fraction (containing monoglycosylated lipids such as mannosylphosphoryldolichol [32,35]). However, the chloroform-methanol-water fraction (containing oligosaccharide-lipid intermediates [32]) possessed 11 times more label when obtained from parental cells than from Lec9 cells, and the tryptic peptides were ca.…”