Investigation of the intracellular transport of tyrosinase and tyrosinase related protein (TRP)-1. The effect of endoplasmic reticulum (ER)-glucosidases inhibition.

Melanin biosynthesis is completely inhibited in the B16 melanoma cells following their incubation with inhibitors of the two ER glucosidases. This is primarily due to the inactivation of tyrosinase. Under the same conditions, the DOPA-oxidase activity of TRP-1 was only partially affected. In this report we investigate the effects of the perturbation of N-glycan processing in ER on the transport and activation of tyrosinase and TRP-1. We have localized the DOPA-oxidase activity in normal and inhibited cells and suggest that the first DOPA-reactive compartment of the secretory pathway (trans Golgi network) is also the site of tyrosinase activation. The inhibition of N-glycan processing does not affect the intracellular trafficking of the two melanogenic enzymes that are correctly transported to melanosomes. Immunoprecipitation experiments followed by analysis in SDS-PAGE under non-reducing conditions suggest that in inhibited cells, both tyrosinase and TRP-1 are synthesized in a modified conformation as compared to the normal proteins. These data suggest that the inhibition of melanin synthesis is not due to a defective transport but rather to conformational changes induced in the structure of tyrosinase and TRP-1 during their transit through the ER.

Inhibition of N-glycan processing in B16 melanoma cells results in inactivation of tyrosinase but does not prevent its transport to the melanosome.

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.