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‐melanms can be synthesized chemically, that mammalian melanins are naturally rich in (HO) 2 IndCOOH subunits, and that (HO) 2 IndCOOH 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.