Inhibition of N-Glycan Processing in B16 Melanoma Cells Results in Inactivation of Tyrosinase but Does Not Prevent Its Transport to the Melanosome

Abstract

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 ␣-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 (Glc 3 Man 7-9 ) predominated. Removal of the glucose residues with ␣-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.

Sialylated Core 1 O-Glycans Influence the Sorting of Pmel17/gp100 and Determine Its Capacity to Form Fibrils

“…This constitutes an advantage to study the dynamics of iPmel17, which was previously and incorrectly proposed to represent an ER form of Pmel17 based only on its sensitivity to EndoH. Interestingly, Pmel17 is not the only melanosomal protein with this type of sensitivity to EndoH digestion, because similar patterns have been described for correctly processed, although inactive, tyrosinase (56) and for DOPAchrome tautomerase (57). Fig.…”

Hepatitis B virus (HBV) envelope glycoproteins vary drastically in their sensitivity to glycan processing: Evidence that alteration of a single N-linked glycosylation site can regulate HBV secretion

“…However, in contrast to HBV, enveloped HIV virus is formed and secreted (although it is noninfectious). Another example in which the inhibition of glycan processing with NB-DNJ resulted in regional misfolding is that of tyrosinase, an enzyme involved in the production of melanin (25). In this case, the hyperglucosylated enzyme is correctly transported to melanosomes but is inactive as a result of being unable to bind the copper required for activity (25).…”

“…Another example in which the inhibition of glycan processing with NB-DNJ resulted in regional misfolding is that of tyrosinase, an enzyme involved in the production of melanin (25). In this case, the hyperglucosylated enzyme is correctly transported to melanosomes but is inactive as a result of being unable to bind the copper required for activity (25). In the case of the HBV M protein, the most likely effect of NB-DNJ would be that the M protein is severely misfolded.…”

Mechanisms of Copper Incorporation into Human Ceruloplasmin

“…While further studies will be needed to directly test this hypothesis, support is found in recent experiments examining copper incorporation into tyrosinase. Those experiments demonstrate specific mutations that abrogate copper incorporation but do not affect trafficking of the apoprotein to the melanosome (25).…”