- Email: [email protected]
Pigmentation Genes: the Tyrosinase Gene Family and the pmel 17 Gene Family
MOLECULAR BIOLOGY OF THE TYROSINASE GENE
Pigmentation Genes: the Tyrosinase Gene Family ana the pmel17 Gene Family Byoung s. Kwon Department of Microbiology and Immunology, and Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana, U.S.A. We propose that at least two families of genes regulate the melanin biosynthesis. The first is the tyrosinase gene family, which is comprised of tyrosinase (c locus), gp75 (b locus) and DOPAchrome tautomerase (sit locus). The second is the pmel 17 gene family, which is composed of pmel 17 (puta tive si locus) and chicken melanosomal matrix protein
(MMP) 115. It appears that the tyrosinase gene family regu lates melanin synthesis in the proximal steps of the melanin biosynthetic pathway and the pmel 17 gene family might be important at distal steps of the pathway. ] Invest Dermatol
elanin pigments are classified into two types: cuta neous and neuronal melanin. Neuronal melanin is found at the substantia nigra and the locus ceru leus, and is synthesized in nerve cells. The func tion of melanin in the brain and its biosynthetic pathways are still unknown. Cutaneous melanin, which is found in the skin, hair follicles, and the eyes, is synthesized and secreted by melanocytes. Melanocytes in the skin and hair follicles and in the uveal components of the eye are differentiated from neural crest, whereas those in the retina are differentiated from the optic cup [1]. The melanin is synthesized in organelles called melanosomes, and is transferred to surrounding keratinocytes through the dendritic process. There are two major classes of cutaneous melanin: the black brown eumelanin and the yellow-red pheomelanin. The metabolic pathways of eumelanin and pheomelanin biosynthesis, as shown in Fig 1, was established by Raper et al in the 1920s [2], by Mason in the 1940s [3], and by Prota et al in the 1960s [4]. L-tyrosine is the initial substrate for the melanin pathway. The enzyme tyrosinase catalyzes the oxidation of L-tyrosine to L-DOPA (3.4-dihydroxyphenylalan ine), and the dehydrogenation of DOPA to DOPAquinone. DO PAquinone is a very reactive compound that quickly enters the pheomelanin or eumelanin pathway. In the classic Raper-Mason scheme of eumelanin synthesis, the steps in the eumelanin pathway are assumed to be spontaneous. Although this scheme of eumelanin synthesis in vitro represents a milestone in pigment cell research, it has led to some misconceptions and generalizations about melanin biosynthesis. Some of the major objections to this classic scheme have been reviewed [5,6]. The most serious limitation of the Raper Mason picture of eumelanin synthesis is the assertion that melanin is formed under conditions involving the sole interaction of tyrosinase
with the primary substrate, tyrosine. Logan and Weatherhead [7] provided the first indirect evidence for post-tyrosinase inhibition of melanogenesis in the hair follicles of Sibetian hamsters, suggesting a second regulatory site. In fact, recent work suggests that there are at least two other sites where enzymes tnight be involved [8,9]. Con trol of melanization by additional factors could be significant for the understanding of tyrosinase-positive albinisms as well as variations in skin and hair pigmentation. In pheomelanin synthesis [4], DOPAquinone reacts with sulfhy dryl compounds such as cysteine or glutathione, to form cysteinyl DOPA intermediate compounds, which undergo oxidative cycliza tion and polymerization to pheomelanin. No enzyme control of pheomelanin synthesis has been described, but the ability of gluta thione to form glutathione-DOPA and cysteinyl-DOPA suggests a reasonable expectation that these compounds could be enzymati cally regulated. Recendy, several genes that appear to regulate the melanin bio synthesis have been isolated and characterized. I propose these to be classified into 1) the tyrosinase gene family, and 2) the pmel 17 gene family. Members of the tyrosinase gene family may catalyze mela nin biosynthesis at the proximal steps of Mason-Raper pathway and those of the pmel 17 gene family may catalyze melanin biosynthesis at the distal steps. Some members of pmel 17 gene family may constitute the melanosomal matrix.
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Reprint requests to: Dr. Byoung S. Kwon, Department of Microbiology and Immunology, and Walther Oncology Center, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis. IN 46202. Abbreviations: AZ: amelanotic melanoma cells flMSH: p melanocyte-stimulating hormone DHICA: 5,6-dihydroxyindole carboxylic acid mMX: isobutylmethylxanthine MMP: melanosomal matrix protein NM: normal human melanocyte
100:134S-140S, 1993
TYROSINASE GENE FAMILY In 1986 at the Pan-American Pigment Cell Meeting, Tucson, Ari zona, we reported a full cDNA sequence corresponding to human tyrosinase. The human tyrosinase cDNA (pmel 34) was assigned to mouse c-Iocus on chromosome 7 and TYR locus on human chro mosome 11 [10,11]. Since then, we (Byoung S. Kwon, Asifa K. Haq, Gwan S. Kim, Seymour H. Pomerantz, and Ruth Halaban) and others have reported sequences of human and mouse cDNAs and gene structure of tyrosinase [12-15]. Shibahara et al [16] iso lated a melanocyte-specific gene that was assigned to the brown (b) locus on mouse chromosome 4 [17]. Houghton et al [18] showed that the mouse b-Iocus protein is homologous to a known human melanosomal protein gp75. We have mapped the human b-Iocus gene to chromosome 9 and named it CAS2 locus [19]. The latest addition of the tyrosinase fatnily is DOPAchrome tautomerase whose gene is located at slaty (sit) locus of mouse chromosome 14 [20,21]. The human homologue of the DOPAchrome tautomerase
0022-202X/93/S06.00 Copyright © 1993 by The Society for Investigative Dermatology, Inc. 134S
VOL lOll, NO.2, SUPPLEMENT,
TYROSINASE AND pmel
FEBRUARY 1993
17
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Table L
Properties of Tyrosinase Family
Similar in size (60-75 kD) Similar in structure Signal sequence Transmembrane domain Cysteine and histidine-rich Copper-containing enzymes Evolutionarily conserved Antigenically related
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Figure 3. Amino acid sequence analysis of the potential pmel17 protein. A. The deduced amino acid sequence of pmel 17. The signal region is under lined with a heavy line. The potential glycosylation sites are underlined with a thin line. The putative transmembrane region is doubly underlined. Three repeat motifs of 26 amino acids are indicated by an overline with vertical bars. Each bar indicates the start amino acid of the repeat. B. the hydropathicity pro61e of the deduced amino acid sequence of pmel17. Local hydropathicity values calculated by the method of Kyte and Doolittle were plotted versus amino acid residues. Positive values indicate hydrophobic regions. and nega tive values indicate hydrophilic regions. C. schematic representation of the potential pmel17 protein. The entire coding region is boxed. The potential signal peptide is hatched. The transmembrane segment is indicated by horizon tal lines. The central repeat motifs are stippled. Positions of histidines (H). cysteines (C). and possible N-linked glycosylation sites (CHO) are indi cated. The numbers 1-22 on the box indicate the positions of askrisks, which indicate the positions of Ser/Thr-Ser/Thr sequences.
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quences of human and mouse tyrosinase, human and mouse gp75, and mouse DOPAchrome tautomerase. The overall amino acid identity among the members of the tyrosinase family is approxi mately 45%. Fourteen cysteines are aligned throughout the pro teins. Six histidines that are potentially associated with copper are all conserved. Most of the tryptophans are aligned. Characteristics of these proteins are listed in Table I and the chromosomal location of the genes in the tyrosinase family is summarized in Table II.
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VOL. 100. NO. 2. SUPPLEMENT. FEBRUARY 1993
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17 GENE FAMILIES
Figure 5 Comparison of the primary structure of pmd 17 with that of MMP 115. The putative signal sequence (PJJd). positions of histidine residues (H). positions of cysteine residues (C). possible N-linked glycosylation sites (CHO). and putative transmembrane regions (II) are indicated. The three repeats of 20-26 amino acid motif are indicated (0). The percentage of identical residues in a given segment are indicated between the two proteins. The numbers in parentheses in each segment indicate the similarity between the proteins when chemically similar amino acids are taken into account. •.
Pmel 17 GENE FAMILY Pmel 17 cDNA was isolated from a human melanocyte cDNA library in a Agt 11 vector by screening the library with anti-tyrosin ase antibodies. We have recently published the deduced amino acid sequence of human pmel 17 gene and its chromosomal loca tion in mouse and man [22). Figure 3A shows the amino acid se quence deduced from the longest open reading frame of pmel 17. The open reading frame encodes 668 amino acids with an M.. of 70,944. The first 23 amino acids show characteristics of a signal peptide of secretory or membrane-associated proteins and fit the -1, -3 rule [23). Inclusion of a histidine in a signal peptide is unusual but not unknown [23). Thus, the protein backbone of pro cessed pmel 17 would be composed of 645 amino acids with an M.. of 68.600. Five potential asparagine-linked glycosylation sites are located at amino acid positions 81, 106, 111, 321, and 568 as indicated in Fig 3A,C. There is a stretch of 26 amino acids that constitutes a hydro phobic domain toward the C terminus of the protein (amino acids 598-623). This hydrophobic region is bordered by charged residues at either end (Glu597 and Arg624-626), consistent with a transmem brane segment that makes a single helical span. One of the striking features of the protein is the relatively high percentage of serines (9.3%) and threonines (9.3%) and their peculiar arrangements. In addition, leucine, valine, and glycine are other amino acid residues present at high levels in this protein (11.9%, 10.0%, and 9.3%, respectively). There are three sets of 26-amino acid motif in the middle of the molecule (amino acids 315-392) (Fig 3A,C). Each starts with four identical amino acids (Pro-Thr-Ala-Glu) and has a hydrophobicity pattern (Fig 3B) similar to the others. The first two sets contain three Thr-Thr or Ser-Thr sequences. The third set contains one Ser-Thr and one Thr-Thr sequence. There is a histidine-rich region in front of the three 26-amino acid repeats and a cysteine-rich re gion following the repeat motif near the transmembrane domain (Fig 3C). The significance of such an arrangement of amino acids in this protein remains to be determined. Human pmel 17 gene has been assigned to 12pter - q21 on chromosome 12. An extensive mapping study has been performed on the mouse chromosome. We have used a panel of mouse-ham ster somatic cell hybrids, progeny of an intersubspecies backcross and an interspecies backcross for restriction fragment length poly morphisms. We found that the mouse pmel 17 gene was mapped to mouse chromosome 10. When we aligned the interspecific link-
Figure 6. Sequence comparison of pmd 17 family. The sequences of human pmd 17 -deduced protein (top row) and chicken MMP lIS-deduced protein aligned to maximize amino acid similarity. Identical amino acids are indicated by two bars and amino acids with chemical similarity by two dots. Gaps (-) were introduced for maximal alignment.
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age map with the conventional composite linkage map on mouse chromosome 10, this alignment placed the mouse pmel 17 near the si. We have compared the amino acid sequence of pmel 17 with that of human tyrosinase and gp75. As shown in Fig 4, we found a limited degree of amino acid similarities among the three proteins. There are five short regions of similarity in the three proteins: one in N-terminal portion, three in the middle, and one in the C terminus of the three molecules. H owever, the similarity between pmel 17deduced protein and members of the tyrosinase family were far less than that within members of the tyrosinase family. Clearly, the primary structure of pmel 17 was different from the members of the tyrosinase family. Especially, the alignment of cysteines and histidines is poor. Therefore, I propose that pmel 17 does not belong to the tyrosinase gene family and forms its own gene family.
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R ecently Mochii et al [24] published a cDNA sequence of a melanosomal matrix protein called MMP 115 from chicken melano cytes. The deduced protein structure has a number of similarities to pmel 17 -deduced protein structure. As shown in Figs 5 and 6, both proteins contain three repeats of 20 to 26 amino acid motif in the middle of the protein. The overall amino acid identity be tween the two proteins is approximately 43%. There are two re gions of higher homology that are located immediately upstream and downstream of the three repeats; it seems that two stretches of amino acids that are highly homologous to one another between pmel 17 and MMP 115 bracketed the three sets of 20-26 amino acid motif. The lowest homology between the two proteins lies in the repeat motif. The amino acid identity in this region is approxi mately 28%, whereas the amino acid identity of the bracket regions is 55%. It is intriguing that the homology is very low in the re gions containing the three repeat motif. Perhaps these regions de termine the functions of the protein, indicating that the two pro teins play a role in the melanogenesis in a related but distinct man ner. We believe there will be more members of the pmel 17 gene family. We have tested whether the expression of pmel 17 mRNA was correlated with the level of melanization in melanocytes [25].First, we determined the abundance of the pmel 17 mRNA in three lines of human melanocytes that expressed different levels of melaniza tion. We compared the melanin content, tyrosinase activity, level of pmel 17 mRNA, and level of tyrosinase mRNA in the normal human melanocytes (NM), melanotic (LG), and amelanotic (AZ) melanoma cells. For technical convenience, the comparison is presented only between NM and LG. The melanin content ofNM was 4.3 times higher than that of LG. The tyrosinase activity and the level of tyrosinase mRNA ofNM (Fig 7) was approximately 1.5 times and 2.5 times higher than those ofLG, respectively. In con trast, the expression of the pmel 17 mRNA was approximately fourfold higher in NM than in LG (Fig 8). These data indicate that pmel 17 gene expression is more closely correlated with the level of melanin content than is tyrosinase expression, which suggests that pmel 17 may function as a catalyst of melanin biosynthesis at a step distal to tyrosinase. We investigated further the possible involvement of pmel 17 in the process of melanization. PMSH and isobutylmethylxanthine (IBMX) are known to stimulate melanin synthesis [26]. When Cloudman S-91 cells or human melanoma LG cells were treated with PMSH and IBMX they expressed elevated melanin content. The effects of PMSH and/or IBMX on the expression of the pmel 17 mRNA are shown in Fig 9A,B. When the cells were treated with the agents, there were two- to fourfold increases in the expres sion of the pmel 17 mRNA in both cell lines. These data indicate t at pmel 17 might be a positive regulator of melanin biosynthe
�
16.6
S1S.
3
2
7.8
A NM
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Figure 7. Expression of tyrosinase mRNA (Pmel 34) in human melano cytes with different levels of me1anization. The amount of 4 p.g of poly(A)+ mRNA from normal melanocytes (NM), melanotic melanoma (LG), and amelanotic melanoma cells (AZ) was run on a 1.2% denaturing agarose gel, blotted and hybridized to our human tyrosinase probe (Pmel34). The inten sity of bands was measured by a densitometer at 500 nm. The numbers in the graph at right indicate the integrated areas of the scanning densitometer for each band.
The biochemical reactions associated with the members of the tyrosinase gene family are fairly well characterized. Perhaps they may form a multienzyme complex in the melanosomal membrane [27] and regulate the quantity and quality of melanin by catalyzing reactions up to the synthesis ofIndole 5.6-quinone or 5,6-dihydroxy indole carboxylic acid ( DHICA). We do not know what other biochemical reactions are involved in the conversion of Indole 5.6-quinone or DHICA to melanochrome and of melanochrome to melanin. Perhaps the initial portion of these distal processes occurs in association with membrane and further reactions occur in melano somal matrix. This motion reconciles the observation that pmel 17 contains a membrane-spanning domain but MMP 115 does not. I propose that the members of pmel 17 gene family are involved in the distal step of melanin biosynthesis, ie, those steps after Indole 5.6-quinone and DHICA formation.
This work was supported by grants from the National Institutes of Health (AR.40248) and the March ofDimf5 Birth Dejects Foundation.
TYROSINASE AND
VOL. 100, NO. 2, SUPPLEMENT, FEBRUARY 1993
17 GENE FAMILIES
1398
36.0
5
..
Figure 8. Expression of pmel 17 mRNA in human melanocytes with diff"erent levels of melanization. The same blot used for Fig 7 (tyrosinase) was hybridized to the Pmel17-1 probe after removal of the Pmel 34 probe. The in tensity ofbands was measured by a densi tometer at 500 nm. The numbers in the graph at right indicate the integrated areas of the scanning densitometer for each band. Pme114-2 is a control probe, a cDNA clone isolated from a human melanocyte cDNA library, whose RNA was detectable in almost equal amounts in all human and mouse cells tested.
8.3
3
- 28S
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2
....-- pmel17-1 -18S 0.5
..
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3
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•
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4
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B
Figure 9. The eff"ect of ,BMSH and mMX on the level of pmel 17 mRNA in Cloudman S-91 cells. Two independent experiments are shown. I, Cloudman S-91 cells were studied alone (lane 1) or were stimulated by incubation with ,BMSH (50 nM) (lane 2), mMX (0.02 rnM) (lane 3) or with both agents for 4 d (lane 4).5 Jlg of poly(A)+mRNA was loaded in each lane. Hybridization probes were pmel l7-1 (upper arrow) and pmel l4-2 (bottom arrow). II, Left: poly(A)+ mRNA from unstimulated (5 Jlg) and mMX plus ,BMSH-stimulated (4 Jlg) Cloudman S-91 cells. We did not strip off" the pmel l7-1 probe prior to the hybridization with pmel l4-2 DNA. Therefore, the upper band represents RNA homologous to pmel17. We intentionally loaded about 20% more RNA of unstimulated cells compared with that of stimulated cells to make sure that the elevation of pmel 17 mRNA in stimulated cells was not due to diff"erences in the amount of RNA loaded on the gel. Right: the intensity of the bands at the 22 S position was measured by a densitometer at 500 nm. Numbers indicate the integrated areas of the bands. I, Unstimulated Cloudman melanoma. II, Stimulated Cloudman melanoma.
1408
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
KWON
REFERENCES
16.
1.
Rawles ME: Origin of melanophores and their role in development of color patterns in vertebrates. Physiol Rev 28:383-408,1948
2.
Raper HS: The aerobic oxidases. Physiol
3.
Mason HS: The chemistry of melanin. III. Mechanism of oxidation of dihydroxyphenylalanine by tyrosinase. J BioI Chem 172:83-99, 1948
Rev 8:245-282,1928
Shibahara S,Tomita Y,Sakakura T,Nager C,Chaudhuri B,Muller R: Cloning and expression of cDNA encoding mouse tyrosinase. Nu cleic Acids Res 14:2413-2427,1986
17. Jackson IJ: A cDNA encoding tyrosinase-related protein maps to the brown locus in mouse. Proc Nad Acad Sci USA 85:4392-4396, 1988 18.
Houghton A,Bouchard B,Vijayasaradhi S: Differentiation antigens of normal and transformed human melanocytes. Presented at the meet ing of PanAmerican Society for Pigment Cell Research,Bethesda, MD, April 23-26,1989
19.
Chintamaneni C, Ramsay M, Colman MA, Fox MF, Pickard RT, Kwon BS: Ma�ing the human CAS2 gene,the homologue of the mouse brown (b) locus,to human chromosome qp22-pter. Biochem Biophys Res Commun 178:227-235,1991
20.
Jackson IJ,Chambers DM,Tsukamoto K,Copeland NG,Gilbert DJ, Jenkins NA,Hearing VJ: A second tyrosinase-related protein,TRP2,maps to and is mutated at the mouse slaty locus. EMBO J 11:527535,1992
4.
Prota G, Nicolaus RA: On the biogenesis of phaeomelanins. In: Mon tagna W,Hu F (eds.). Advances in Biology of Skin,Vol 8. Pergamon Press,New York,1966,pp 323-328
5.
Swan GA: Structure,chemistry and biosynthesis of the melanins. In: Hen W,Griseback H,Kirby GW (eds.). Fortschrihe der Chemie Orgnischer Naturstoffe, Vol 31. Springer-Verlag, New York, pp 522-528
6.
Prota GC: Pigment cell metabolism: chemical and enzymatic control. In: Veronesi U,Cascinelli N,Santinami M (eds.). Cutaneous Mela noma in 1985. Academic Press,London,1987,pp 233-241
7.
Logan A,Weatherhead B: Post-tyrosinase inhibition of melanogenesis by melatonin in hair follicles ill vitro. J Invest Dermatol 74:47-50, 1980
21.
8.
Komer A,PawelekJ: Mammalian tyrosinase catalyzes three reactions in the biosynthesis of melanins. Science 217:1163-1165,1982
Tsukamoto K,Jackson Ij, Urabe K, Montague PM, Hearing VJ: A second tyrosinase-related protein,TrP-2,is a melanogenic enzyme termed DOPAchrome tautomera5e. EMBO J 11:519-526,1991
22.
9.
Barber JI, Townsend D, Olds DP, King RA: Dopachrome oxidore ductase: a new enzyme in the pigment pathway. J Invest Dermatol 83:145-149,1984
Kwon BS, Chintamaneni C, Kozak CA, Copeland NG,Gilbert DJ, Jenkins N,Barton D,Francke U,Kobayashi Y,Kim K-K: A melano cyte-specific gene,Pme117,maps near the silver coat color locus on mouse chromosome 10 and is in a syntenic region on human chro mosome 12. Proc Nad Acad Sci USA 88:9228-9232,1991
10.
Kwon BS,Jaq AK,Pomerantz SH,Halaban R: Isolation and sequence of a cDNA clone for human tyrosinase that maps at the mouse c-albino locus. Proc Nad Acad Sci USA 84:7473-7477,1987
23.
Von Heijne G: Patterns of amino acids near signal-sequence cleavage sites. EurJ Biochem 133:17-21,1983
11.
Barton DE,Kwon BS,Francke U: Human tyrosinase gene,mapped to chromosome 11 (q14 -+ q21),defines second region of homology with mouse chromosome 7. Genomics 3:17-24,1988
24.
Mochii M,Agata K, Eguchi G: Complete sequence and expression of a eDNA encoding a chicken 115-leDa melanosomal matrix protein. Pigment Cell Res 4:41-47,1991
12.
Yamamoto H,Takeuchi S,Kudo T,Makino K,Nakata A,Shinoda T, Takeuchi T: Cloning and sequencing of mouse tyrosinase cDNA. JpnJ Genet 62:271-274,1987
25.
Kwon BS,Halaban R,Kim GS,Pomerantz S,Haq AI{: A melanocyte specific eDNA clone whose expression is inducible by MSH and mMX. Molecular Bioi Med 4:339-356,1987
13.
Kwon BS,Wakulchik M,Haq AK, Halaban R,Kesder D: Sequence analysis of mouse tyrosinase cDNA. Biochem Biophys Res Com mun 153:1301-1309,1988
26.
14.
Muller G, Ruppert S, Schmid E, Schutz G: Functional analysis of alternatively spliced tyrosinase gene transcripts. EMBO J 7:27232730,1988
Lener AB, Moellmann G,VargaJM,Halaban R,PawelekJ: Action of melanocyte-stimulating hormone on pigment cells. In: Hormones and Cell Culture. Cold Spring Harbor Conference on Cell Prolifera tion 6,Cold Spring Harbor Laboratory Press,Cold Spring Harbor, NY,1979,pp 187-197
27.
15.
Ruppert S,Muller G,Kwon BS,Axhur F: Multiple transcripts of the mouse tyrosinase gene are generated by alternative splicing. EMBO J 7:2715-2722,1988
PawelekJM,Chakraborty AK, Orlow SJ: Purification of high molecu lar weight melanogenic complex containing MSH receptors,DO PAchrome isomerase,gp 75,and tyrosinase. J Clin Invest (in press)
Pigmentation Genes: the Tyrosinase Gene Family ana the pmel17 Gene Family Byoung s. Kwon Department of Microbiology and Immunology, and Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana, U.S.A. We propose that at least two families of genes regulate the melanin biosynthesis. The first is the tyrosinase gene family, which is comprised of tyrosinase (c locus), gp75 (b locus) and DOPAchrome tautomerase (sit locus). The second is the pmel 17 gene family, which is composed of pmel 17 (puta tive si locus) and chicken melanosomal matrix protein
(MMP) 115. It appears that the tyrosinase gene family regu lates melanin synthesis in the proximal steps of the melanin biosynthetic pathway and the pmel 17 gene family might be important at distal steps of the pathway. ] Invest Dermatol
elanin pigments are classified into two types: cuta neous and neuronal melanin. Neuronal melanin is found at the substantia nigra and the locus ceru leus, and is synthesized in nerve cells. The func tion of melanin in the brain and its biosynthetic pathways are still unknown. Cutaneous melanin, which is found in the skin, hair follicles, and the eyes, is synthesized and secreted by melanocytes. Melanocytes in the skin and hair follicles and in the uveal components of the eye are differentiated from neural crest, whereas those in the retina are differentiated from the optic cup [1]. The melanin is synthesized in organelles called melanosomes, and is transferred to surrounding keratinocytes through the dendritic process. There are two major classes of cutaneous melanin: the black brown eumelanin and the yellow-red pheomelanin. The metabolic pathways of eumelanin and pheomelanin biosynthesis, as shown in Fig 1, was established by Raper et al in the 1920s [2], by Mason in the 1940s [3], and by Prota et al in the 1960s [4]. L-tyrosine is the initial substrate for the melanin pathway. The enzyme tyrosinase catalyzes the oxidation of L-tyrosine to L-DOPA (3.4-dihydroxyphenylalan ine), and the dehydrogenation of DOPA to DOPAquinone. DO PAquinone is a very reactive compound that quickly enters the pheomelanin or eumelanin pathway. In the classic Raper-Mason scheme of eumelanin synthesis, the steps in the eumelanin pathway are assumed to be spontaneous. Although this scheme of eumelanin synthesis in vitro represents a milestone in pigment cell research, it has led to some misconceptions and generalizations about melanin biosynthesis. Some of the major objections to this classic scheme have been reviewed [5,6]. The most serious limitation of the Raper Mason picture of eumelanin synthesis is the assertion that melanin is formed under conditions involving the sole interaction of tyrosinase
with the primary substrate, tyrosine. Logan and Weatherhead [7] provided the first indirect evidence for post-tyrosinase inhibition of melanogenesis in the hair follicles of Sibetian hamsters, suggesting a second regulatory site. In fact, recent work suggests that there are at least two other sites where enzymes tnight be involved [8,9]. Con trol of melanization by additional factors could be significant for the understanding of tyrosinase-positive albinisms as well as variations in skin and hair pigmentation. In pheomelanin synthesis [4], DOPAquinone reacts with sulfhy dryl compounds such as cysteine or glutathione, to form cysteinyl DOPA intermediate compounds, which undergo oxidative cycliza tion and polymerization to pheomelanin. No enzyme control of pheomelanin synthesis has been described, but the ability of gluta thione to form glutathione-DOPA and cysteinyl-DOPA suggests a reasonable expectation that these compounds could be enzymati cally regulated. Recendy, several genes that appear to regulate the melanin bio synthesis have been isolated and characterized. I propose these to be classified into 1) the tyrosinase gene family, and 2) the pmel 17 gene family. Members of the tyrosinase gene family may catalyze mela nin biosynthesis at the proximal steps of Mason-Raper pathway and those of the pmel 17 gene family may catalyze melanin biosynthesis at the distal steps. Some members of pmel 17 gene family may constitute the melanosomal matrix.
M
Reprint requests to: Dr. Byoung S. Kwon, Department of Microbiology and Immunology, and Walther Oncology Center, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis. IN 46202. Abbreviations: AZ: amelanotic melanoma cells flMSH: p melanocyte-stimulating hormone DHICA: 5,6-dihydroxyindole carboxylic acid mMX: isobutylmethylxanthine MMP: melanosomal matrix protein NM: normal human melanocyte
100:134S-140S, 1993
TYROSINASE GENE FAMILY In 1986 at the Pan-American Pigment Cell Meeting, Tucson, Ari zona, we reported a full cDNA sequence corresponding to human tyrosinase. The human tyrosinase cDNA (pmel 34) was assigned to mouse c-Iocus on chromosome 7 and TYR locus on human chro mosome 11 [10,11]. Since then, we (Byoung S. Kwon, Asifa K. Haq, Gwan S. Kim, Seymour H. Pomerantz, and Ruth Halaban) and others have reported sequences of human and mouse cDNAs and gene structure of tyrosinase [12-15]. Shibahara et al [16] iso lated a melanocyte-specific gene that was assigned to the brown (b) locus on mouse chromosome 4 [17]. Houghton et al [18] showed that the mouse b-Iocus protein is homologous to a known human melanosomal protein gp75. We have mapped the human b-Iocus gene to chromosome 9 and named it CAS2 locus [19]. The latest addition of the tyrosinase fatnily is DOPAchrome tautomerase whose gene is located at slaty (sit) locus of mouse chromosome 14 [20,21]. The human homologue of the DOPAchrome tautomerase
0022-202X/93/S06.00 Copyright © 1993 by The Society for Investigative Dermatology, Inc. 134S
VOL lOll, NO.2, SUPPLEMENT,
TYROSINASE AND pmel
FEBRUARY 1993
17
GENE
1358
FAMILlES
TYROSINE
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Table L
Properties of Tyrosinase Family
Similar in size (60-75 kD) Similar in structure Signal sequence Transmembrane domain Cysteine and histidine-rich Copper-containing enzymes Evolutionarily conserved Antigenically related
TableD. Tyrosinase gp75 TRP-2
Chromosomal Assignment of Tyrosinase Gene Family
Human Mouse Human Mouse Human Mouse
TYR
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KWON
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
1
MDLVLKRCLL HLAVIGALLA VGATKVPRNQ DWLGVSRQLR TKAWNRQLYP
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Figure 3. Amino acid sequence analysis of the potential pmel17 protein. A. The deduced amino acid sequence of pmel 17. The signal region is under lined with a heavy line. The potential glycosylation sites are underlined with a thin line. The putative transmembrane region is doubly underlined. Three repeat motifs of 26 amino acids are indicated by an overline with vertical bars. Each bar indicates the start amino acid of the repeat. B. the hydropathicity pro61e of the deduced amino acid sequence of pmel17. Local hydropathicity values calculated by the method of Kyte and Doolittle were plotted versus amino acid residues. Positive values indicate hydrophobic regions. and nega tive values indicate hydrophilic regions. C. schematic representation of the potential pmel17 protein. The entire coding region is boxed. The potential signal peptide is hatched. The transmembrane segment is indicated by horizon tal lines. The central repeat motifs are stippled. Positions of histidines (H). cysteines (C). and possible N-linked glycosylation sites (CHO) are indi cated. The numbers 1-22 on the box indicate the positions of askrisks, which indicate the positions of Ser/Thr-Ser/Thr sequences.
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gene has not been isolated and has not been assigned to a human chromosome. Consequently. the tyrosinase gene family consists of tyrosinase, gp7S, and DOPAchrome tautomerase. The amino acid sequences and overall makeup of the primary structure of these genes are very similar. They all contain signal sequences and trans membrane domain, which indicates that they all exist as a melano somal membrane-bound form. Figure 2 shows the comparison of the potential amino acid se-
Hunan pile I17P
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quences of human and mouse tyrosinase, human and mouse gp75, and mouse DOPAchrome tautomerase. The overall amino acid identity among the members of the tyrosinase family is approxi mately 45%. Fourteen cysteines are aligned throughout the pro teins. Six histidines that are potentially associated with copper are all conserved. Most of the tryptophans are aligned. Characteristics of these proteins are listed in Table I and the chromosomal location of the genes in the tyrosinase family is summarized in Table II.
rn � � � ! � � V V � "�" iV � V -� L� � � V '�V � V D u LG
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Figure 4. Alignment of human Pmel 17 protein (human pmel 17P), human tyrosinase. and human CAS2 protein (human CAS2P) in homologous regions. Numbers indicate amino acid positions of the homologous regions, counting from the initiation residue. methionine, and those in parentheses indicate amino acid positions of potential mature proteins. The amino acids that are shared are boxed. Gaps (-) are introduced to allow maximal alignment.
VOL. 100. NO. 2. SUPPLEMENT. FEBRUARY 1993
I
I
200
TYROSINASE AND
pmel
100
100
400
1378
17 GENE FAMILIES
Figure 5 Comparison of the primary structure of pmd 17 with that of MMP 115. The putative signal sequence (PJJd). positions of histidine residues (H). positions of cysteine residues (C). possible N-linked glycosylation sites (CHO). and putative transmembrane regions (II) are indicated. The three repeats of 20-26 amino acid motif are indicated (0). The percentage of identical residues in a given segment are indicated between the two proteins. The numbers in parentheses in each segment indicate the similarity between the proteins when chemically similar amino acids are taken into account. •.
Pmel 17 GENE FAMILY Pmel 17 cDNA was isolated from a human melanocyte cDNA library in a Agt 11 vector by screening the library with anti-tyrosin ase antibodies. We have recently published the deduced amino acid sequence of human pmel 17 gene and its chromosomal loca tion in mouse and man [22). Figure 3A shows the amino acid se quence deduced from the longest open reading frame of pmel 17. The open reading frame encodes 668 amino acids with an M.. of 70,944. The first 23 amino acids show characteristics of a signal peptide of secretory or membrane-associated proteins and fit the -1, -3 rule [23). Inclusion of a histidine in a signal peptide is unusual but not unknown [23). Thus, the protein backbone of pro cessed pmel 17 would be composed of 645 amino acids with an M.. of 68.600. Five potential asparagine-linked glycosylation sites are located at amino acid positions 81, 106, 111, 321, and 568 as indicated in Fig 3A,C. There is a stretch of 26 amino acids that constitutes a hydro phobic domain toward the C terminus of the protein (amino acids 598-623). This hydrophobic region is bordered by charged residues at either end (Glu597 and Arg624-626), consistent with a transmem brane segment that makes a single helical span. One of the striking features of the protein is the relatively high percentage of serines (9.3%) and threonines (9.3%) and their peculiar arrangements. In addition, leucine, valine, and glycine are other amino acid residues present at high levels in this protein (11.9%, 10.0%, and 9.3%, respectively). There are three sets of 26-amino acid motif in the middle of the molecule (amino acids 315-392) (Fig 3A,C). Each starts with four identical amino acids (Pro-Thr-Ala-Glu) and has a hydrophobicity pattern (Fig 3B) similar to the others. The first two sets contain three Thr-Thr or Ser-Thr sequences. The third set contains one Ser-Thr and one Thr-Thr sequence. There is a histidine-rich region in front of the three 26-amino acid repeats and a cysteine-rich re gion following the repeat motif near the transmembrane domain (Fig 3C). The significance of such an arrangement of amino acids in this protein remains to be determined. Human pmel 17 gene has been assigned to 12pter - q21 on chromosome 12. An extensive mapping study has been performed on the mouse chromosome. We have used a panel of mouse-ham ster somatic cell hybrids, progeny of an intersubspecies backcross and an interspecies backcross for restriction fragment length poly morphisms. We found that the mouse pmel 17 gene was mapped to mouse chromosome 10. When we aligned the interspecific link-
Figure 6. Sequence comparison of pmd 17 family. The sequences of human pmd 17 -deduced protein (top row) and chicken MMP lIS-deduced protein aligned to maximize amino acid similarity. Identical amino acids are indicated by two bars and amino acids with chemical similarity by two dots. Gaps (-) were introduced for maximal alignment.
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THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
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age map with the conventional composite linkage map on mouse chromosome 10, this alignment placed the mouse pmel 17 near the si. We have compared the amino acid sequence of pmel 17 with that of human tyrosinase and gp75. As shown in Fig 4, we found a limited degree of amino acid similarities among the three proteins. There are five short regions of similarity in the three proteins: one in N-terminal portion, three in the middle, and one in the C terminus of the three molecules. H owever, the similarity between pmel 17deduced protein and members of the tyrosinase family were far less than that within members of the tyrosinase family. Clearly, the primary structure of pmel 17 was different from the members of the tyrosinase family. Especially, the alignment of cysteines and histidines is poor. Therefore, I propose that pmel 17 does not belong to the tyrosinase gene family and forms its own gene family.
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R ecently Mochii et al [24] published a cDNA sequence of a melanosomal matrix protein called MMP 115 from chicken melano cytes. The deduced protein structure has a number of similarities to pmel 17 -deduced protein structure. As shown in Figs 5 and 6, both proteins contain three repeats of 20 to 26 amino acid motif in the middle of the protein. The overall amino acid identity be tween the two proteins is approximately 43%. There are two re gions of higher homology that are located immediately upstream and downstream of the three repeats; it seems that two stretches of amino acids that are highly homologous to one another between pmel 17 and MMP 115 bracketed the three sets of 20-26 amino acid motif. The lowest homology between the two proteins lies in the repeat motif. The amino acid identity in this region is approxi mately 28%, whereas the amino acid identity of the bracket regions is 55%. It is intriguing that the homology is very low in the re gions containing the three repeat motif. Perhaps these regions de termine the functions of the protein, indicating that the two pro teins play a role in the melanogenesis in a related but distinct man ner. We believe there will be more members of the pmel 17 gene family. We have tested whether the expression of pmel 17 mRNA was correlated with the level of melanization in melanocytes [25].First, we determined the abundance of the pmel 17 mRNA in three lines of human melanocytes that expressed different levels of melaniza tion. We compared the melanin content, tyrosinase activity, level of pmel 17 mRNA, and level of tyrosinase mRNA in the normal human melanocytes (NM), melanotic (LG), and amelanotic (AZ) melanoma cells. For technical convenience, the comparison is presented only between NM and LG. The melanin content ofNM was 4.3 times higher than that of LG. The tyrosinase activity and the level of tyrosinase mRNA ofNM (Fig 7) was approximately 1.5 times and 2.5 times higher than those ofLG, respectively. In con trast, the expression of the pmel 17 mRNA was approximately fourfold higher in NM than in LG (Fig 8). These data indicate that pmel 17 gene expression is more closely correlated with the level of melanin content than is tyrosinase expression, which suggests that pmel 17 may function as a catalyst of melanin biosynthesis at a step distal to tyrosinase. We investigated further the possible involvement of pmel 17 in the process of melanization. PMSH and isobutylmethylxanthine (IBMX) are known to stimulate melanin synthesis [26]. When Cloudman S-91 cells or human melanoma LG cells were treated with PMSH and IBMX they expressed elevated melanin content. The effects of PMSH and/or IBMX on the expression of the pmel 17 mRNA are shown in Fig 9A,B. When the cells were treated with the agents, there were two- to fourfold increases in the expres sion of the pmel 17 mRNA in both cell lines. These data indicate t at pmel 17 might be a positive regulator of melanin biosynthe
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Figure 7. Expression of tyrosinase mRNA (Pmel 34) in human melano cytes with different levels of me1anization. The amount of 4 p.g of poly(A)+ mRNA from normal melanocytes (NM), melanotic melanoma (LG), and amelanotic melanoma cells (AZ) was run on a 1.2% denaturing agarose gel, blotted and hybridized to our human tyrosinase probe (Pmel34). The inten sity of bands was measured by a densitometer at 500 nm. The numbers in the graph at right indicate the integrated areas of the scanning densitometer for each band.
The biochemical reactions associated with the members of the tyrosinase gene family are fairly well characterized. Perhaps they may form a multienzyme complex in the melanosomal membrane [27] and regulate the quantity and quality of melanin by catalyzing reactions up to the synthesis ofIndole 5.6-quinone or 5,6-dihydroxy indole carboxylic acid ( DHICA). We do not know what other biochemical reactions are involved in the conversion of Indole 5.6-quinone or DHICA to melanochrome and of melanochrome to melanin. Perhaps the initial portion of these distal processes occurs in association with membrane and further reactions occur in melano somal matrix. This motion reconciles the observation that pmel 17 contains a membrane-spanning domain but MMP 115 does not. I propose that the members of pmel 17 gene family are involved in the distal step of melanin biosynthesis, ie, those steps after Indole 5.6-quinone and DHICA formation.
This work was supported by grants from the National Institutes of Health (AR.40248) and the March ofDimf5 Birth Dejects Foundation.
TYROSINASE AND
VOL. 100, NO. 2, SUPPLEMENT, FEBRUARY 1993
17 GENE FAMILIES
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Figure 8. Expression of pmel 17 mRNA in human melanocytes with diff"erent levels of melanization. The same blot used for Fig 7 (tyrosinase) was hybridized to the Pmel17-1 probe after removal of the Pmel 34 probe. The in tensity ofbands was measured by a densi tometer at 500 nm. The numbers in the graph at right indicate the integrated areas of the scanning densitometer for each band. Pme114-2 is a control probe, a cDNA clone isolated from a human melanocyte cDNA library, whose RNA was detectable in almost equal amounts in all human and mouse cells tested.
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Figure 9. The eff"ect of ,BMSH and mMX on the level of pmel 17 mRNA in Cloudman S-91 cells. Two independent experiments are shown. I, Cloudman S-91 cells were studied alone (lane 1) or were stimulated by incubation with ,BMSH (50 nM) (lane 2), mMX (0.02 rnM) (lane 3) or with both agents for 4 d (lane 4).5 Jlg of poly(A)+mRNA was loaded in each lane. Hybridization probes were pmel l7-1 (upper arrow) and pmel l4-2 (bottom arrow). II, Left: poly(A)+ mRNA from unstimulated (5 Jlg) and mMX plus ,BMSH-stimulated (4 Jlg) Cloudman S-91 cells. We did not strip off" the pmel l7-1 probe prior to the hybridization with pmel l4-2 DNA. Therefore, the upper band represents RNA homologous to pmel17. We intentionally loaded about 20% more RNA of unstimulated cells compared with that of stimulated cells to make sure that the elevation of pmel 17 mRNA in stimulated cells was not due to diff"erences in the amount of RNA loaded on the gel. Right: the intensity of the bands at the 22 S position was measured by a densitometer at 500 nm. Numbers indicate the integrated areas of the bands. I, Unstimulated Cloudman melanoma. II, Stimulated Cloudman melanoma.
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Jackson IJ,Chambers DM,Tsukamoto K,Copeland NG,Gilbert DJ, Jenkins NA,Hearing VJ: A second tyrosinase-related protein,TRP2,maps to and is mutated at the mouse slaty locus. EMBO J 11:527535,1992
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Komer A,PawelekJ: Mammalian tyrosinase catalyzes three reactions in the biosynthesis of melanins. Science 217:1163-1165,1982
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Barber JI, Townsend D, Olds DP, King RA: Dopachrome oxidore ductase: a new enzyme in the pigment pathway. J Invest Dermatol 83:145-149,1984
Kwon BS, Chintamaneni C, Kozak CA, Copeland NG,Gilbert DJ, Jenkins N,Barton D,Francke U,Kobayashi Y,Kim K-K: A melano cyte-specific gene,Pme117,maps near the silver coat color locus on mouse chromosome 10 and is in a syntenic region on human chro mosome 12. Proc Nad Acad Sci USA 88:9228-9232,1991
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Kwon BS,Jaq AK,Pomerantz SH,Halaban R: Isolation and sequence of a cDNA clone for human tyrosinase that maps at the mouse c-albino locus. Proc Nad Acad Sci USA 84:7473-7477,1987
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Von Heijne G: Patterns of amino acids near signal-sequence cleavage sites. EurJ Biochem 133:17-21,1983
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Barton DE,Kwon BS,Francke U: Human tyrosinase gene,mapped to chromosome 11 (q14 -+ q21),defines second region of homology with mouse chromosome 7. Genomics 3:17-24,1988
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Mochii M,Agata K, Eguchi G: Complete sequence and expression of a eDNA encoding a chicken 115-leDa melanosomal matrix protein. Pigment Cell Res 4:41-47,1991
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Yamamoto H,Takeuchi S,Kudo T,Makino K,Nakata A,Shinoda T, Takeuchi T: Cloning and sequencing of mouse tyrosinase cDNA. JpnJ Genet 62:271-274,1987
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Kwon BS,Halaban R,Kim GS,Pomerantz S,Haq AI{: A melanocyte specific eDNA clone whose expression is inducible by MSH and mMX. Molecular Bioi Med 4:339-356,1987
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Kwon BS,Wakulchik M,Haq AK, Halaban R,Kesder D: Sequence analysis of mouse tyrosinase cDNA. Biochem Biophys Res Com mun 153:1301-1309,1988
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Muller G, Ruppert S, Schmid E, Schutz G: Functional analysis of alternatively spliced tyrosinase gene transcripts. EMBO J 7:27232730,1988
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Ruppert S,Muller G,Kwon BS,Axhur F: Multiple transcripts of the mouse tyrosinase gene are generated by alternative splicing. EMBO J 7:2715-2722,1988
PawelekJM,Chakraborty AK, Orlow SJ: Purification of high molecu lar weight melanogenic complex containing MSH receptors,DO PAchrome isomerase,gp 75,and tyrosinase. J Clin Invest (in press)