Alterations in lipid-linked oligosaccharide metabolism in human melanoma cells concomitant with induction of stress proteins.
Challenge of human A375 melanoma cells with sodium arsenite induced the synthesis of stress proteins and stimulated [3H]mannose incorporation into a novel component migrating on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular mass of 14 kDa (designated M14). Enhanced M14 expression was elicited by heavy metals (zinc, copper, cadmium, and nickel), thiol-reactive agents (iodoacetamide and auranofin), and hyperthermia. The kinetics of M14 induction and recovery from stress were similar to those of the stress proteins, but M14 half-life was only 15 min. Incorporation of [3H]mannose into M14 was inhibited by tunicamycin but not by cycloheximide or actinomycin D. M14 was metabolically labeled with [32P]orthophosphate but not by [35S] methionine or [3H]asparagine. Further studies revealed that M14 was selectively soluble in chloroform/methanol/water (10:10:3) and sensitive to both endo-beta-N-acetylglucosaminidase H digestion and mild acid hydrolysis. The latter released a water-soluble mannose-labeled moiety which eluted from Bio-Gel P-6 in a manner similar to Glc3Man9GlcNAc2. Together, these data suggest that M14 is a lipid-oligosaccharide intermediate of N-linked protein glycosylation and that enhanced expression of this class of molecule in response to chemical insults and hyperthermia is a newly described cellular reaction to stress.
Niewiarowska
A
A
Department of Cell Biology, Smith Kline & French Laboratories, Philadelphia, Pennsylvania 19101.
Caltabiano
M M
MM
Bailey
D S
DS
Poste
G
G
Greig
R G
RG
D001151:Arsenic / Q000494:pharmacology; D018053:Arsenites; D002850:Chromatography, Gel; D006031:Glycosylation; D006360:Heat-Shock Proteins / Q000096:biosynthesis*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D050356:Lipid Metabolism; D008358:Mannose / Q000378:metabolism; D008545:Melanoma / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism*; D012995:Solubility; D014407:Tumor Cells, Cultured / Q000378:metabolism
D018053:Arsenites; D006360:Heat-Shock Proteins; D009844:Oligosaccharides; C015001:arsenite; D001151:Arsenic; D008358:Mannose