Faculty Bibliography

Tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2, (TRP-2, dopachrome tautomerase) were shown by immunoblotting and enzyme assays to copurify from extracts of Cloudman S91 melanoma cells. Antibodies to TRP-1 and TRP-2 immunoprecipitated tyrosinase activity, suggesting a stable interaction (complex) among these proteins. The tyrosine hydroxylase activity of tyrosinase was reduced in the complexed form; treatment with Triton X-100 dissociated the complex and activated the tyrosinase present within it. To further study this complex, we employed sucrose gradient density centrifugation of extracts from cultured murine melanocytes. Tyrosinase, TRP-1, and TRP-2 all existed in high molecular weight 'multimers' of approximately 200 to > 700 kilodaltons. Extraction of cells with buffers containing the detergent CHAPS preserved the high molecular weight multimers; Triton X-100 caused their dissociation into monomers. Low pH, low ionic strength, and millimolar concentrations of calcium ions favored the maintenance of multimers. The results of this study demonstrate that the participation of tyrosinase, TRP-1, and TRP-2 in a multimeric complex could have important physiologic consequences, and raise the possibility that some of the well-known interactions between coat color genes may be explained by intermolecular interactions between the gene products

To identify a broad spectrum of melanosomal proteins, antisera were raised in rabbits against melanosomal protein fractions separated on the basis of their solubility in the nonionic detergent Triton X-114. Antisera against the different fractions each recognized a distinct set of bands when used for immunoblotting analysis with extracts of melanocytes cultured from wild-type black mice. Immunoblotting with antisera to whole melanosomes or to Triton X-114-soluble melanosomal proteins that segregated with the detergent phase gave identical patterns with protein extracts from melanocytes from wild-type mice and from mice homozygous for the si (silver) coat color mutation. By contrast, an antiserum against Triton X-114 soluble melanosomal proteins that segregated in the aqueous phase recognized an 85-kDa protein that was present in extracts from wild-type melanocytes but was absent from si melanocytes. This suggested that the protein was encoded at the si (silver) locus. This was confirmed by employing an antiserum directed against the carboxyl terminus of the predicted murine silver protein sequence. The detergent solubility, biochemical characteristics, and immunologic properties of the 85-kDa protein and of the authentic si gene product were identical. Further analysis demonstrated that this protein corresponds to a melanosomal matrix glycoprotein that we recently described. Our results suggest that employing polyclonal antisera to fractionated organelles such as melanosomes, to screen tissues from mutant mice, a technique that we call 'organelle scanning', can serve as a powerful means of identifying new organellar proteins and their respective genes

The silver mutation in mice causes progressive graying of hair due to the loss of functional follicular melanocytes. Recently the silver locus gene (called Pmel 17) has been cloned; its encoded product shares homology with a chick melanosomal matrix protein and a bovine retinal pigment epithelial protein. Although the sequence of the silver gene and the correlation of its expression with pigment production have been reported, its function in melanogenesis is still unknown. In an effort to characterize that function, we have synthesized the predicted carboxyl-terminal peptide of the mouse Pmel 17 protein and generated a rabbit polyclonal antibody (alpha PEP13) to it; that antibody recognized the silver protein specifically. The immunoaffinity-purified silver protein lacked all of the known melanogenic catalytic activities which other tyrosinase-related proteins (TRP) have, nor did it appear to modulate any of those TRP activities. Metabolic labeling experiments demonstrated that the silver protein disappears in vivo within a few hours, indicating that it is rapidly degraded, or quickly processed to lose its carboxyl terminus. Cross-reactivity experiments showed that a recently reported anti-melanosomal matrix protein antibody (alpha MX) also recognizes the silver protein, although at a different epitope from that of alpha PEP13. Using Western immunoblotting, we analyzed subcellular fractions isolated from B16 F10 melanoma cells and found that the silver protein was rich in the melanosome fraction but was absent from coated vesicles which deliver TRPs to melanosomes. These results suggest that the silver locus product is a melanosomal matrix protein which may contribute to melanogenesis as a structural protein, although the possibility remains that it also has a novel catalytic function in melanogenesis

The lines of Blaschko represent a pattern followed by many skin disorders. We review the clinical and histologic features of X-linked, congenital/nevoid, and acquired skin diseases that follow these lines. We also include cutaneous disorders that have a linear distribution but do not follow Blaschko's lines. Finally, we differentiate Blaschko's lines from other patterns on the skin such as dermatomes and Langer's lines

Melanocytic nevi have been reported in association with several congenital syndromes. This review describes the clinical and cutaneous manifestations of six syndromes associated with congenital melanocytic nevi, two associated with acquired nevi, and six associated with melanocytic nevi in which insufficient evidence exists to classify them as congenital or acquired. It is important to recognize these associations to evaluate and counsel patients with melanocytic nevi. Early recognition will also facilitate timely intervention

Antiserum raised in rabbits against the Triton X-100 insoluble fraction of melanosomes from mouse melanoma cells specifically decorates the internal matrix of melanosomes in immunoelectron microscopy. In metabolic labeling studies, the antiserum recognizes a protein of 94 kDa, which is processed to a band of 53 kDa. Whereas the precursor is relatively soluble in buffers containing Triton X-100, the processed protein requires the addition of sodium dodecyl sulfate for effective solubilization, as would be expected for a melanosomal matrix constituent. Tunicamycin reduces the Mr of the nascent protein to 75 kDa, but deoxymannojirimycin and swainsonine have no effect, suggesting that following initial glycosylation in the endoplasmic reticulum, the protein is not subject to processing by glycosidases in the Golgi apparatus or may bypass it entirely. Subcellular fractionation followed by immunoblotting confirms that the protein is present in the melanosome-rich, large granule fraction. Expression of the protein is regulated differently from that of the tyrosinase-related protein family. Conditions that greatly stimulate expression of tyrosinase-related proteins do not affect matrix protein expression, nor is the protein immunologically related to the tyrosinase-related protein family. Our results suggest that we have identified an authentic component of the mammalian melanosomal matrix, and that its characteristics lend support to a bipartite pathway for melanosomal biogenesis

The platinum mutation at the C (albino) locus in the mouse is a potential model for oculocutaneous albinism in humans other than type IA (tyrosinase-negative) albinism. Although tissues from mice homozygous for the mutation display substantial tyrosinase activity, cutaneous and ocular pigmentation is severely restricted in affected animals. Using specific antipeptide antisera, we demonstrate that ocular extracts from wild-type mice contain two isoforms of tyrosinase bearing either the amino-terminal PEP5 epitope or the carboxy-terminal PEP7 epitope. The latter isoform participates in a high-molecular-weight complex detectable on sucrose density gradients. In platinum mice, antiserum to the PEP7 epitope fails to recognize any protein species, and the high-molecular-weight form of tyrosinase is not detectable. Our results support a key role for this high-molecular-weight complex in melanogenesis, and suggest that mutations that interfere with the ability of tyrosinase to participate in a multimeric complex may be a cause of oculocutaneous albinism in people