Rapid and reversible inhibition of tyrosinase activity by glucosidase inhibitors in human melanoma cells.
The dependence of constitutively expressed tyrosinase (dopa oxidase) activity on glycosylation in lightly pigmented human melanoma cells (MM96E) was determined using tunicamycin (TM), which prevents transfer of oligosaccharide chains to nascent protein (core glycosylation), the glucosidase inhibitors castanospermine (CS) and deoxynojirimycin (dNM), and the mannosidase inhibitors deoxymannojirimycin (dMM) and swainsonine (SW). TM caused irreversible inhibition of tyrosinase activity and carbohydrate synthesis as judged by incorporation of 3H-fucose. Tyrosinase in CS- and dNM-treated cells showed 50% loss of activity within 5 h but recovered rapidly when the drugs were removed; dMN and SW had little effect. Expression of the tyrosinase 2B7 epitope and of an 80-kDa melanosomal antigen (B8G3) was inhibited by TM but not by CS, dNM, dMM, or SW. CS and dNM appeared to decrease the half-life of active tyrosinase. Overall, these results indicate that 1) in addition to the requirement for core glycosylation the removal of glucose residues plays a critical role in the formation of active human tyrosinase; 2) glucosidase inhibitors appear to cause an accumulation of inactive tyrosinase and increase the degradation rate of active enzyme; and 3) later stages in oligosaccharide processing are not required for maintaining tyrosinase activity.
Takahashi
H
H
Queensland Institute of Medical Research, Brisbane, Australia.
Parsons
P G
PG
D017485:1-Deoxynojirimycin; D000911:Antibodies, Monoclonal; D000941:Antigens / Q000378:metabolism; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D002846:Chromatography, Affinity; D005434:Flow Cytometry; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D005959:Glucosidases / Q000037:antagonists & inhibitors*; D006801:Humans; D007212:Indolizines / Q000494:pharmacology*; D008544:Melanocytes / Q000378:metabolism; D008546:Melanoma, Experimental / Q000201:enzymology* / Q000473:pathology; D008715:Methionine / Q000378:metabolism; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012878:Skin Neoplasms / Q000201:enzymology*; D013347:Subcellular Fractions / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014407:Tumor Cells, Cultured; D054836:Indolizidines
D017485:1-Deoxynojirimycin; D000911:Antibodies, Monoclonal; D000941:Antigens; D005944:Glucosamine; D005959:Glucosidases; D007212:Indolizines; D008715:Methionine; D014442:Monophenol Monooxygenase; D013462:Sulfur Radioisotopes; C037806:castanospermine; D054836:Indolizidines