Analysis of Carbohydrate Properties Essential for Melanogenesis in Tyrosinases of Cultured Malignant Melanoma Cells by Differential Carbohydrate Processing Inhibition

Abstract

In order to clarify the biologic significance of carbohydrate processing in tyrosinases for melanogenesis, we have studied the effect of differential carbohydrate processing inhibitors on the recovery process of interrupted melanization which occurs after exposure of cultured B-16 melanoma cells to the inhibitor of core carbohydrate synthesis, glucosamine (Glc). Treatment of this glycosylation-dependent repigmentation process with the early-stage carbohydrate processing inhibitors deoxynojirimycin (dNM), castanospermine (CS), and monensin (MS) at 0.8 mM, 40 micrograms/ml, and 30 nM, respectively, in the presence of 2 mM theophylline (Tp) almost completely inhibits the reappearance of the pigment 48-72 h after removal of Glc. In contrast, treatment with the later stage carbohydrate processing inhibitor swaisonine (SW) at 40-80 micrograms/ml does not interrupt the repigmentation process. Electrophoretic analysis of tyrosinases in the soluble fractions of these melanoma cells demonstrates that the alteration of soluble tyrosinase isozymes by all the processing inhibitors is associated with a dose-dependent loss of sialic acid-rich T1 tyrosinase and the concomitant appearance or increase of sialic acid-poor tyrosinases. In the large granule fraction, a recovery of membrane-bound tyrosinase (T3) is seen following both MS and SW treatments, whereas dNM treatment results in the substantial loss of T3 tyrosinase. At the electron microscopic level, a translocation of tyrosinase from GERL and coated vesicles to many unmelanized vacuolar premelanosomes occurs in MS-treated cells in contrast to its predominant distribution in the GERL-coated vesicle system of dNM-treated cells, which contain many unmelanized premelanosomes. The present evidence for differential effects on intracellular tyrosinase transfer and melanization by different stages of carbohydrate processing inhibition suggests that asparagine-linked oligosaccharides, relating to the first mannose-trimming stages, determine the function of tyrosinase transfer as well as melanization through a specific intracellular recognition process in pigment cells.