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Tyrosinase is the key enzyme in melanin biosynthesis, catalyzing multiple steps in this pathway. The mature glycoprotein is transported from the Golgi to the melanosome where melanin biosynthesis occurs. In this study, we have investigated the effects of inhibitors of N-glycan processing on the synthesis, transport, and catalytic activity of tyrosinase. When B16 mouse melanoma cells were cultured in the presence of N-butyldeoxynojirimycin, an inhibitor of the endoplasmic reticulum-processing enzymes alpha-glucosidases I and II, the enzyme was synthesized and transported to the melanosome but almost completely lacked catalytic activity. The cells contained only 2% of the melanin found in untreated cells. Structural analysis of the N-glycans from N-butyldeoxynojirimycin-treated B16 cells demonstrated that three oligosaccharide structures (Glc3Man7-9) predominated. Removal of the glucose residues with alpha-glucosidases I and II failed to restore enzymatic activity, suggesting that the glucosylated N-glycans do not sterically interfere with the enzyme's active sites. The mannosidase inhibitor deoxymannojirimycin had no effect on catalytic activity suggesting that the retention of glucosylated N-glycans results in the inactivation of this enzyme. The retention of glucosylated N-glycans does not therefore result in misfolding and degradation of the glycoprotein, as the enzyme is transported to the melanosome, but may cause conformational changes in its catalytic domains.

Original publication

DOI

10.1074/jbc.272.25.15796

Type

Journal article

Journal

J Biol Chem

Publication Date

20/06/1997

Volume

272

Pages

15796 - 15803

Keywords

1-Deoxynojirimycin, Animals, Biological Transport, Carbohydrate Sequence, Enzyme Inhibitors, Glycosylation, Kinetics, Melanins, Melanocytes, Melanoma, Mice, Molecular Sequence Data, Monophenol Monooxygenase, Oligosaccharides, Polysaccharides, Tumor Cells, Cultured