Glycosylation of tyrosinase is a determinant of melanin production in cultured melanoma cells

Abstract

Abstract. The majority of malignant melanoma cell types are able to produce melanin and the degree of melanin synthesis in various types of cultured cell line differs. In this study, we evaluated three types of cultured cell line, MNT-1, HM3KO and G-361, with differing melanin production levels. The level was greatest in the MNT-1 cells, lower in the HM3KO cells and lowest in the G-361 cells. In addition, a positive correlation between melanin production and tyrosinase activity was observed. The molecular masses of tyrosinases from HM3KO and G-361 cells were marginally lower than those from MNT-1 cells. Glycosylation inhibitor treatment on MNT-1 cells caused decreases in the molecular mass of tyrosinase, its activity and melanin production. An immunoprecipitation assay using anti-tyrosinase indicated that the immature glycosylated tyrosinases were associated with a type of chaperone, Hsp70. The interaction between tyrosinase and Hsp70 was also detected in HM3KO and G-361 cells. The results indicated that the immature glycosylation of tyrosinase has a critical effect on the melanin-producing ability of melanoma cells. IntroductionMelanin is important in protecting the skin from the harmful effects of ultraviolet irradiation and absorbing toxic drugs and chemicals. Melanin is synthesized in two predominant forms, black-brown pigments (eumelanin) and red-yellow pigments (pheomelanin), within the melanosomes of melanocytes by at least three melanogenic enzymes. Tyrosinase (EC1.14.18.1) is one of the predominant enzymes mediated by the melanin production process in melanosomes (1). The enzyme catalyzes the rate-limiting step of the hydroxylation of tyrosine to dihydroxyphenylalanine (DOPA) and its further oxidation to DOPA-quinone (2). Melanin synthesis is maintained by a number of regulatory processes that act at various steps during the synthesis of the protein in the endoplasmic reticulum (ER), Golgi body and melanosomes, and disorders in any of these processes lead to abnormal pigmentation.Malignant melanoma (MM) cells are derived from epidermal melanocytes and nevi (3), and numerous cultured melanoma cell lines have been established from human and mouse MMs. The majority of MMs produce melanin, and the degree of melanin synthesis differs for each type of cultured cell line (4-6). For example, MNT-1 cells were demonstrated to be highly pigmented MMs (4); however, SK-Mel-28 cells were not able to produce melanin due to a mutation in the endocytic pathway that affected the endosomal trafficking of tyrosinase (5). In addition, B16F10 cells were pigmented; whereas amelanotic melanoma was not pigmented, due to the increased expression of proteasome subunit p27 (6).In this study, three cultured cell lines, MNT-1, HM3KO and G-361, were observed. These cell lines differed in their degrees of melanin synthesis, and thus, the determinants of the degree of melanin synthesis were investigated. Materials and methodsCell culture. The MNT-1 (from Dr VJ Hearing, Laboratory of Cell Biology, National Cancer Institute, National Ins...

CMT-308, a Nonantimicrobial Chemically-Modified Tetracycline, Exhibits Anti-Melanogenic Activity by Suppression of Melanosome Export

“…As tyrosinase is a metalloenzyme with two copper ions; chelators that can sequester copper have also shown promise as a target for pigmentation disorders [ 14 , 15 , 16 ]. The intracellular transport of tyrosinase is regulated by copper uptake and the N-glycosylation process [ 17 ]; the role of the enzyme α-glucosidase has been implicated in this glycosylation process in previous reports [ 18 , 19 ]. Consequently, certain antidiabetic drugs such as voglibose [ 20 ] and acarbose [ 21 ], which are known to inhibit α-glucosidase activity, also suppress melanogenesis.…”

The consequences of deglycosylation of recombinant intra-melanosomal domain of human tyrosinase

“…In the cell, N-glycosylation plays an essential role in the transfer of tyrosinase from ER to the cytoplasm and in maintaining its folding, stability, and enzymatic activity (Imokawa and Mishima, 1984; Branza-Nichita et al, 2000; Wang and Hebert, 2006). The glycosylation of tyrosinase might regulate the melanin production in different types of cultured melanoma cells (Mikami et al, 2013). It has been proven in mouse melanoma cells, that inhibition of the early steps of the N-glycosylation process strongly affects tyrosinase activity, thereby changing melanin synthesis.…”

Histidine residues at the copper-binding site in human tyrosinase are essential for its catalytic activities

“…In this study, recombinant wild-type and mutant human tyrosinase enzymes were overexpressed in E. coli BL21 (DE3) and utilised to analyse the catalytic activity. It has been reported that N-glycosylation is necessary for human tyrosinase stability and activity in vivo [29][30][31] . However, our previous studies and those of another group have also shown that recombinant human tyrosinase from E. coli could exhibit activity without phosphorylation or glycosylation 18,24,32 .…”