Mutant alleles at the brown locus encoding tyrosinase-related protein-1 (TRP-1) affect proliferation of mouse melanocytes in culture

Tyrosinase related protein-1 (TRP-1) is a melanocyte-specific gene product involved in eumelanin synthesis. Mutation in the Tyrp1 gene is associated with brown pelage in mouse and oculocutaneous albinism Type 3 in humans (OCA3). It has been demonstrated that TRP-1 expresses DHICA oxidase activity in the murine system. However, its actual function in the human system is still unclear. The study was designed to determine the effects of mutation at two Typr1 alleles, namely the Tyrp1b (brown) and Tyrp1b-cj (cordovan) compared with wild type Tyrp1B (black) on melanocyte function and melanin biosynthesis. The most significant finding was that both of the Tyrp1 mutations (i.e. brown expressing a point mutation and cordovan expressing decreased amount of TRP-1 protein) resulted in attenuation of cell proliferation rates. Neither necrosis nor apoptosis was responsible for the observed decrease in cell proliferation rates of the brown and cordovan melanocytes. Ultrastructural evaluation by electron microscopic analysis revealed that both mutations in Tyrp1 affected melanosome maturation without affecting its structure. These observations demonstrate that mutation in Tyrp1 compromised tyrosinase activity within the organelle. DOPA histochemistry revealed differences in melanosomal stages between black and brown melanocytes but not between black and cordovan melanocytes. There were no significant differences in tyrosine hydroxylase activities of tyrosinase and TRP-1 in wild type black, brown and cordovan melanocyte cell lysates. We conclude that mutations in Tyrp1 compromise cell proliferation and melanosomal maturation in mouse melanocyte cultures.     

Melanocytes and pigmentation are affected in dopachrome tautomerase knockout mice

The tyrosinase family comprises three members, tyrosinase (Tyr), tyrosinase-related protein 1 (Tyrp1), and dopachrome tautomerase (Dct). Null mutations and deletions at the Tyr and Tyrp1 loci are known and phenotypically affect coat color due to the absence of enzyme or intracellular mislocalization. At the Dct locus, three mutations are known that lead to pigmentation phenotype. However, these mutations are not null mutations, and we therefore set out to generate a null allele at the Dct gene locus by removing exon 1 of the mouse Dct gene. Mice deficient in Dct [Dct(tm1(Cre)Bee)] lack Dct mRNA and dopachrome tautomerase protein. They are viable and do not show any abnormalities in Dct-expressing sites such as skin, retinal pigment epithelium, or brain. However, the mice show a diluted coat color phenotype, which is due to reduced melanin content in hair. Primary melanocytes from Dct knockout mice are viable in culture and show a normal distribution of tyrosinase and tyrosinase-related protein 1. In comparison to the knockout, the slaty mutation (Dct(slt)/Dct(slt)) has less melanin and affects growth of primary melanocytes severely. In summary, we have generated a knockout of the Dct gene in mice with effects restricted to pigment production and coat color.     

Mutant Alleles at the Brown Locus Encoding Tyrosinase‐Related Protein‐1 (TRP‐1) Affect Proliferation of Mouse Melanocytes in Culture *

Tyrosinase related protein‐1 (TRP‐1) is a melanocyte‐specific gene product involved in eumelanin synthesis. Mutation in the Tyrp1 gene is associated with brown pelage in mouse and oculocutaneous albinism Type 3 in humans (OCA3). It has been demonstrated that TRP‐1 expresses DHICA oxidase activity in the murine system. However, its actual function in the human system is still unclear. The study was designed to determine the effects of mutation at two Typr1 alleles, namely the Tyrp1^b (brown) and Tyrp1^b‐cj (cordovan) compared with wild type Tyrp1^B (black) on melanocyte function and melanin biosynthesis. The most significant finding was that both of the Tyrp1 mutations (i.e. brown expressing a point mutation and cordovan expressing decreased amount of TRP‐1 protein) resulted in attenuation of cell proliferation rates. Neither necrosis nor apoptosis was responsible for the observed decrease in cell proliferation rates of the brown and cordovan melanocytes. Ultrastructural evaluation by electron microscopic analysis revealed that both mutations in Tyrp1 affected melanosome maturation without affecting its structure. These observations demonstrate that mutation in Tyrp1 compromised tyrosinase activity within the organelle. DOPA histochemistry revealed differences in melanosomal stages between black and brown melanocytes but not between black and cordovan melanocytes. There were no significant differences in tyrosine hydroxylase activities of tyrosinase and TRP‐1 in wild type black, brown and cordovan melanocyte cell lysates. We conclude that mutations in Tyrp1 compromise cell proliferation and melanosomal maturation in mouse melanocyte cultures.     

Epitope mapping of the melanosomal matrix protein gp100 (PMEL17): rapid processing in the endoplasmic reticulum and glycosylation in the early Golgi compartment

Melanosomes, specific organelles produced only by melanocytes, undergo a unique maturation process that involves their transition form amorphous rounded vesicles to fibrillar ellipsoid organelles, during which they move from the perinuclear to the distal areas of the cells. This depends upon the trafficking and processing of gp100 (also known as Pmel17 and the silver protein), a protein of great interest, because it elicits immune responses in melanoma patients but in which specific function(s) remains elusive. In this study, we have used biochemical and immunochemical approaches to more critically assess the synthesis, processing, glycosylation, and trafficking of gp100. We now report that gp100 is processed and sorted in a manner distinct from other melanosomal proteins (such as tyrosinase, Tyrp1 and Dct) and is predominantly delivered directly to immature melanosomes following its rapid processing in the endoplasmic reticulum and cis-Golgi. Following its arrival, gp100 is cleaved at the amino and at the carboxyl termini in a series of specific steps that result in the reorganization of immature melanosomes to the fibrillar mature melanosomes. Once this structural reorganization occurs, melanogenic enzymes begin to be targeted to the melanosomes, which are then competent to synthesize melanin pigment.     

A dominant mutation in tyrp1A leads to melanophore death in zebrafish

Melanin biosynthesis in vertebrates depends on the function of three enzymes of the tyrosinase family, tyrosinase (Tyr), tyrosinase‐related protein 1 (Tyrp1), and dopachrome tautomerase (Dct or Tyrp2). Tyrp1 might play an additional role in the survival and proliferation of melanocytes. Here, we describe a mutation in tyrp1A, one of the two tyrp1 paralogs in zebrafish, which causes melanophore death leading to a semi‐dominant phenotype. The mutation, an Arg‐>Cys change in the amino‐terminal part of the protein, is similar to mutations in humans and mice where they lead to blond hair (in melanesians) or dark hair with white bases, respectively. We demonstrate that the phenotype in zebrafish depends on the presence of the mutant protein and on melanin synthesis. Ultrastructural analysis shows that the melanosome morphology and pigment content are altered in the mutants. These structural changes might be the underlying cause for the observed cell death, which, surprisingly, does not result in patterning defects.     

MART-1 is required for the function of the melanosomal matrix protein PMEL17/GP100 and the maturation of melanosomes

More than 125 genes that regulate pigmentation have been identified to date. Of those, MART-1 has been widely studied as a melanoma-specific antigen and as a melanosome-specific marker. Whereas the functions of other melanosomal proteins, such as tyrosinase, tyrosinase-related protein-1, dopachrome tautomerase, and Pmel17, are known, the function of MART-1 in melanogenesis, is unclear. A role for MART-1 in pigmentation is expected because its expression pattern and subcellular distribution is quite similar to the other melanosomal proteins and usually correlates with melanin content. We investigated the function of MART-1 using a multidisciplinary approach, including the use of siRNA to inhibit MART-1 function and the use of transfection to re-express MART-1 in MART-1-negative cells. We show that MART-1 forms a complex with Pmel17 and affects its expression, stability, trafficking, and the processing which is required for melanosome structure and maturation. We conclude that MART-1 is indispensable for Pmel17 function and thus plays an important role in regulating mammalian pigmentation.     

Membrane-Associated Transporter Protein (MATP) Regulates Melanosomal pH and Influences Tyrosinase Activity

The SLC45A2 gene encodes a Membrane-Associated Transporter Protein (MATP). Mutations of this gene cause oculocutaneous albinism type 4 (OCA4). However, the molecular mechanism of its action in melanogenesis has not been elucidated. Here, we discuss the role of MATP in melanin production. The SLC45A2 gene is highly enriched in human melanocytes and melanoma cell lines, and its protein, MATP, is located in melanosomes. The knockdown of MATP using siRNAs reduced melanin content and tyrosinase activity without any morphological change in melanosomes or the expression of melanogenesis-related proteins. Interestingly, the knockdown of MATP significantly lowered the melanosomal pH, as verified through DAMP analysis, suggesting that MATP regulates melanosomal pH and therefore affects tyrosinase activity. Finally, we found that the reduction of tyrosinase activity associated with the knockdown of MATP was readily recovered by copper treatment in the in vitro L-DOPA oxidase activity assay of tyrosinase. Considering that copper is an important element for tyrosinase activity and that its binding to tyrosinase depends on melanosomal pH, MATP may play an important role in regulating tyrosinase activity via controlling melanosomal pH.     

Varp Is a Novel Rab32/38-binding Protein That Regulates Tyrp1 Trafficking in Melanocytes

Two small GTPase Rabs, Rab32 and Rab38, have recently been proposed to regulate trafficking of melanogenic enzymes to melanosomes in mammalian epidermal melanocytes; however, the exact molecular mechanism of Rab32/38-mediated transport of melanogenic enzymes has never been clarified, because no Rab32/38-specific effector has ever been identified. In this study, we screened for a Rab32/38-specific effector by a yeast two-hybrid assay using a guanosine triphosphate (GTP)-locked Rab32/38 as bait and found that VPS9-ankyrin-repeat protein (Varp)/Ankrd27, characterized previously as a guanine nucleotide exchange factor (GEF) for Rab21, functions as a specific Rab32/38-binding protein in mouse melanocyte cell line melan-a. Deletion analysis showed that the first ankyrin-repeat (ANKR1) domain functions as a GTP-dependent Rab32/38-binding domain, but that the N-terminal VPS9 domain (i.e., Rab21-GEF domain) does not. Small interfering RNA-mediated knockdown of endogenous Varp in melan-a cells caused a dramatic reduction in Tyrp1 (tyrosinase-related protein 1) signals from melanosomes but did not cause any reduction in Pmel17 signals. Furthermore, expression of the ANKR1 domain in melan-a cells also caused a dramatic reduction of Tyrp1 signals, whereas the VPS9 domain had no effect. Based on these findings, we propose that Varp functions as the Rab32/38 effector that controls trafficking of Tyrp1 in melanocytes.     

Human pigmentation genes: identification, structure and consequences of polymorphic variation

The synthesis of the visible pigment melanin by the melanocyte cell is the basis of the human pigmentary system, those genes directing the formation, transport and distribution of the specialised melanosome organelle in which melanin accumulates can legitimately be called pigmentation genes. The genes involved in this process have been identified through comparative genomic studies of mouse coat colour mutations and by the molecular characterisation of human hypopigmentary genetic diseases such as OCA1 and OCA2. The melanocyte responds to the peptide hormones alpha-MSH or ACTH through the MC1R G-protein coupled receptor to stimulate melanin production through induced maturation or switching of melanin type. The pheomelanosome, containing the key enzyme of the pathway tyrosinase, produces light red/yellowish melanin, whereas the eumelanosome produces darker melanins via induction of additional TYRP1, TYRP2, SILV enzymes, and the P-protein. Intramelanosomal pH governed by the P-protein may act as a critical determinant of tyrosinase enzyme activity to control the initial step in melanin synthesis or TYRP complex formation to facilitate melanogenesis and melanosomal maturation. The search for genetic variation in these candidate human pigmentation genes in various human populations has revealed high levels of polymorphism in the MC1R locus, with over 30 variant alleles so far identified. Functional correlation of MC1R alleles with skin and hair colour provides evidence that this receptor molecule is a principle component underlying normal human pigment variation.     

Identification and characterization of mouse Rab32 by mRNA and protein expression analysis

Rab proteins, a subfamily of the ras superfamily, are low molecular weight GTPases involved in the regulation of intracellular vesicular transport. Cloning of human RAB32 was recently described. Presently, we report the cloning and characterization of the mouse homologue of Rab32. We show that murine Rab32 exhibits a ubiquitous expression pattern, with tissue-specific variation in expression level. Three cell types with highly specialized organelles, melanocytes, platelets and mast cells, exhibit relatively high level of Rab32. We show that in murine amelanotic in vitro transformed melanocytes as well as in human amelanotic metastatic melanoma cell lines, the expression of Rab32 is markedly reduced or absent, in parallel with the loss of expression of two key enzymes for the production of melanin, tyrosinase and Tyrp1. Therefore, in both mouse and human systems, the expression of Rab32 correlates with the expression of genes involved in pigment production. However, in melanoma samples, amelanotic due to a mutation in the tyrosinase gene, the expression of Rab32 remains at levels comparable to those observed in pigmented melanoma samples. Finally, we observed co-localization of Rab32 and the melanosomal proteins, Tyrp1 and Dct, indicating an association of Rab32 with melanosomes. Based on these data, we propose the inclusion of Rab32 to the so-called melanocyte/platelet family of Rab proteins.     

Production of Melanocyte‐Specific Antibodies to Human Melanosomal Proteins: Expression Patterns in Normal Human Skin and in Cutaneous Pigmented Lesions

Multiple factors affect skin pigmentation, including those that regulate melanocyte and/or keratinocyte function. Such factors, particularly those that operate at the level of the melanosome, are relatively well characterized in mice, but the expression and function of structural and enzymatic proteins in melanocytes in human skin are not as well known. Some years ago, we generated peptide‐specific antibodies to murine melanosomal proteins that proved to be instrumental in elucidating melanocyte development and differentiation in mice, but cross‐reactivity of those antibodies with the corresponding human proteins often was weak or absent. In an effort to characterize the roles of melanosomal proteins in human skin pigmentation, and to understand the underlying mechanism(s) of abnormal skin pigmentation, we have now generated polyclonal antibodies against the human melanocyte‐specific markers, tyrosinase, tyrosinase‐related protein 1 (TYRP1), Dopachrome tautomerase (DCT) and Pmel17 (SILV, also known as GP100). We used these antibodies to determine the distribution and function of melanosomal proteins in normal human skin (adult and newborn) and in various cutaneous pigmented lesions, such as intradermal nevi, lentigo simplex, solar lentigines and malignant melanomas. We also examined cytokeratin expression in these same samples to assess keratinocyte distribution and function. Immunohistochemical staining reveals distinct patterns of melanocyte distribution and function in normal skin and in various types of cutaneous pigmented lesions. Those differences in the expression patterns of melanocyte markers provide important clues to the roles of melanocytes in normal and in disrupted skin pigmentation.     

Genomic structure and evolutionary conservation of the tyrosinase gene family from Fugu

The tyrosinase gene family encompasses three members, tyrosinase, tyrosinase-related protein 1 (Tyrp1) and dopachrome tautomerase (Dct), which encode for proteins implicated in melanin synthesis. In human and mouse, genomic organization is known for all three genes, revealing common features of regulatory elements and of exon/intron structure. We have set out to identify the complete family from a more primitive vertebrate, the pufferfish Fugu (Takifugu rubripes), which is characterized by a compact genome. We had recently isolated and characterized the Fugu tyrosinase gene (Genesis 28 (2000) 99-105). We now report the isolation and characterization of the two other members of the family, Tyrp1 and Dct. Regulatory sequences from these genes function in mouse pigment cells and are able to mediate reporter gene expression. Our results demonstrate the existence of all three tyrosinase family members in teleosts and underline the evolutionary conservation of the pigmentary system.     

Glycolipid-dependent sorting of melanosomal from lysosomal membrane proteins by lumenal determinants

Melanosomes are lysosome-related organelles that coexist with lysosomes in mammalian pigment cells. Melanosomal and lysosomal membrane proteins share similar sorting signals in their cytoplasmic tail, raising the question how they are segregated. We show that in control melanocytes, the melanosomal enzymes tyrosinase-related protein 1 (Tyrp1) and tyrosinase follow an intracellular Golgi to melanosome pathway, whereas in the absence of glycosphingolipids, they are observed to pass over the cell surface. Unexpectedly, the lysosome-associated membrane protein 1 (LAMP-1) and 2 behaved exactly opposite: they were found to travel through the cell surface in control melanocytes but followed an intracellular pathway in the absence of glycosphingolipids. Chimeric proteins having the cytoplasmic tail of Tyrp1 or tyrosinase were transported like lysosomal proteins, whereas a LAMP-1 construct containing the lumenal domain of Tyrp1 localized to melanosomes. In conclusion, the lumenal domain contains sorting information that guides Tyrp1 and probably tyrosinase to melanosomes by an intracellular route that excludes lysosomal proteins and requires glucosylceramide.     

A Tyrosinase missense mutation causes albinism in the Wistar rat

Tyrosinase serves as a key enzyme in the synthesis of melanin. In humans mutations in the TYR gene are associated with type 1 oculocutaneous albinism (OCA1) that leads to reduced or absent pigmentation of skin, hair and eye. Various mutations causing OCA in man, mouse, rabbit and cattle have been identified throughout the Tyrosinase gene including nonsense, missense, frameshift and splice site alterations. Here we report a missense substitution at codon R299H in exon 2 of the Tyr gene in the albino Wistar rat. As this very exchange has already been described in OCA patients, our findings reinforce the significance of this region for normal catalytic activity of tyrosinase protein.     

Correction of defective early tyrosinase processing by bafilomycin A1 and monensin in pink-eyed dilution melanocytes

Mutations in the human P gene result in oculocutaneous albinism type 2, the most common form of albinism. Mouse melan-p1 melanocytes, cultured from mice null at the homologous pink-eyed dilution (p) locus, exhibit defective melanin production. A variety of compounds including tyrosine, NH4Cl, bafilomycin A1, concanamycin, monensin, and nigericin are capable of restoring melanin synthesis in these cells. In the current study, we investigated the subcellular effects of bafilomycin A1 and monensin treatment of melan-p1 cells. Both agents play two roles in the processing of tyrosinase (Tyr) in melan-p1 cells. First, combined glycosidase digestion and immunoblotting analysis showed that these agents reduce levels of Tyr retained in the endoplasmic reticulum (ER) and facilitate the release of Tyr from the ER to the Golgi. Secondly, treatment with these compounds resulted in the stabilization of Tyr. Surprisingly, induction of melanin synthesis corresponds more closely with diminution of ER-retained Tyr, rather than the absolute amount of Tyr. Our results suggest that bafilomycin A1 and monensin induce melanin synthesis in melan-p1 cells mainly by facilitating Tyr processing from the ER to the Golgi by increasing the pH in either the ER or the ER-Golgi intermediate compartment.