A clonal derivative of tunicamycin-resistant Chinese hamster ovary cells with increased N-acetylglucosamine-phosphate transferase activity has altered asparagine-linked glycosylation.
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.
D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D002869:Chromosome Aberrations; D002999:Clone Cells / Q000187:drug effects / Q000201:enzymology; D006224:Cricetinae; D003412:Cricetulus / Q000235:genetics / Q000378:metabolism; D004351:Drug Resistance; D004721:Endoplasmic Reticulum / Q000648:ultrastructure; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology*; D005964:Glucosyltransferases / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis*; D008364:Mannosyltransferases / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D010053:Ovary; D010770:Phosphotransferases / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017855:Transferases (Other Substituted Phosphate Groups); D014415:Tunicamycin / Q000494:pharmacology*
D006023:Glycoproteins; D008565:Membrane Proteins; D014415:Tunicamycin; D001216:Asparagine; D005964:Glucosyltransferases; D008364:Mannosyltransferases; C033917:UDPglucose dolicholphosphate glucosyltransferase; C022755:dolichyl-phosphate beta-D-mannosyltransferase; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); C030012:UDP-N-acetylglucosamine-lysosomal-enzyme-N-acetylglucosaminephosphotransferase