Polymerization of 5,6-dihydroxyindole-2-carboxylic acid to melanin by the pmel 17 silver locus protein

Recent advances in melanogenesis have focused on the role of dihydroxyindole-2-carboxylic acid [(HO)(2)IndCOOH]. For example, it has been shown that formation of (HO)(2)IndCOOH from dopachrome is catalyzed by dopachrome tautomerase, that the melanogenic protein tyrosinase-related protein (TRP)-1 can oxidize (HO)(2)IndCOOH to its indole quinone, that (HO)(2)IndCOOH-melanins can be synthesized chemically, that mammalian melanins are naturally rich in (HO)(2)IndCOOH subunits, and that (HO)(2)Ind- COOH is incorporated into melanins of melanomas in mice. The question thus emerges as to the mechanism(s) by which (HO)(2)IndCOOH and other precursors become incorporated into melanins in vivo. Accordingly, an activity was partially purified that catalyzed melanin formation with (HO)(2)IndCOOH as a substrate. Analyses of the (HO)(2)IndCOOH polymerization factor from Cloudman melanoma cells revealed the following: it was proteinaceous in that it was heat labile and destroyed by proteinase K; it was a glycoprotein in that it adhered to wheat germ agglutinin and was eluted with N-acetyl glucosamine; it was located predominantly in the melanosomal fraction of cell homogenates; the activity was reduced by exposure to the metal chelators EDTA and EGTA, but not by phenylthiourea, a tyrosinase inhibitor; the (HO)(2)IndCOOH polymerization reaction was inhibited by superoxide dismutase. In addition, the activity was found with the mouse pmel 17/silver locus protein immunopurified from human melanoma cells, and was significantly reduced in extracts of mouse melanocytes cultured from silver (si/si) mice compared to extracts from Si/Si melanocytes. in summary, an activity has been identified in human and mouse melanoma cells that catalyzes the superoxide-dependent polymerization of (HO)(2)IndCOOH to melanin in vitro, and appears to be a function of the pmel 17/silver protein of the human pmel 17 gene and the mouse silver locus.