Importance of Glycoproteins in the Initiation of Melanogenesis: An Electron Microscopic Study of b-16 Melanoma Cells After Release from Inhibition of Glycosylation

Specific inhibition of core carbohydrate synthesis has been found to induce selective aberration and melanin loss in melanosomes, accompanied by alteration of the carbohydrate moiety in functioning glycoproteins, tyrosinases. In order to further clarify the biologic significance of glycoproteins in the initiation of melanization, initial melanogenesis which occurs during the recovery period following interrupted melanogenesis induced by glycosylation inhibitors, has been electron microscopically investigated. Changes in the tyrosinase activity of the corresponding melanogenic subcellular compartments have also been studied electron cytochemically. Removal of glycosylation inhibition was carried out after exposure of B-16 melanotic melanoma cell to the inhibitors for 10-20 culture days resulting in the loss of their melanization. Recovery of melanization begins visibly 48h later, thereafter almost attaining the previous normal level by 72h. At the electron microscopic level, re-formation of melanosomal matrix with periodicity is observed within premelanosomes 48h after removal of glucosamine, soon followed by deposition of apparent melanin particles along their periodicity by 72h. In tunicamycin experiments, melanization within premelanosomes starts after the concentration of the fine threadlike interior becomes less distinct followed by a prominence of multiple accumulation of microvesicles within the interior. Electron microscopic dopa reaction shows that the deposition of dopa melanin is prominent in Golgi-associated endoplasmic reticulum of lysosome and coated vesicles up to 24h after the removal. Thereafter, predominant localization of dopa finally becomes almost uniform. These observations suggest 2 possible mechanisms for the involvement of glycosylation in melanogenesis: first, the translocation of tyrosinases may be regulated by the presence of specific carbohydrate moieties; second, melanosomalmatrix proteins contain carbohydrates which may contribute to the tyrosinase-accepting function or in vivo melanizing function of tyrosinases after forming particle-bound T3 tyrosinase