Pigment Cell Research Suppl. 2: 84-89 (1992)
The Nature of Tyrosinase Isozymes KATSUHIKO TSLJKAMOTO’, MERCEDES J m N E Z ’ AND VINCENT J. HEARING Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 ‘Department of Dermatology, Yamanashi Medical College, 1 110 Shimokato, Tamaho, Nakakoma, Yamanashi, Japan 409-38; 2Unidad Docente de Biologia, Facultad de Veterinaria, Universidad de Murcia, Espinardo, Murcia, Spain
Tyrosinase [EC 1.14.18.11, the enzyme critical to the formation of melanin, occurs in vivo as a number of isozymic forms. These isozymic forms have generally been considered to arise as the rgmlt of various post-translational modifications of a single protein precursor. It has been shown by a number of studies that tyrosinase is synthesized de mvo on ribosomes, transported through the smooth endoplasmic reticulum and processed (i.e. glycosylated and sialylated) in the Gold complex. The enzyme is then transported via coated vesicles to premelanosomes and incorporatedwithin the melanosomalmembrane, whereupon melanin synthesis can proceed (cf ref 1 for review and relevant citations). However, with the advent of gene cloning techniques, a number of tyrosinase-related proteins have now been identified (2-6, reviewed in 7), and the question remains as to whether these other gene products are, at least in part, responsible for observed tyrosinase activity and its isozymic heterogeneity. Further, the pathway for the formation of eu- and pheomelanins has now been shown to be under much greater regulatory control than previously thought. In addition to tyrosinase, it has been shown that other enzymes such as DOPAchrome isomerase (8-12, alternatively termed DOPAchrome tautomerase), peroxidases (13) and/or catalases (14), glutamine metabolic enzymes (15,16), metal cations (17,18) and melanogenic inhibitors (19.20) might play a role in the regulation of melanogenesis in mammals. Thus the entire question of the identity of the various tyrosinase isozymes and the catalytic function of each of them has received renewed interest. Two of the candidate clones for tyrosinase have been mapped to the ulbino and brown loci of mice (4,6). Each of those genes would be expected to encode a protein which can modulate melanin production since the albino locus regulates whether melanin will be formed while the
brown locus regulates the type of melanin produced (black versus brown eumelanin). These two genes have been cloned from murine and from human systems (2,4,5,21), sequenced and found to be highly homologous at several areas thought to be critical to their catalytic function, although they have extensive variation at their amino and carboxyl termini. Both of those clones have many predicted characteristics that would be expected of tyrosinase, including size (-60 .kD, processed to 70 kD), melanocyte specific expression, two putative copper binding sites, potential glycosylation sites and a stable nature (by virtue of cysteine-rich domains). In addition to the brown and ulbino genes, two other homologous genes which map to different chromosomes have also been identified. The clone 5A originally thought to be identical to the brown locus has now been found to encode a distinct gene (termed TRP-2)on chromosome 14 (I Jackson, personal communication), while Pmell7-1 has been mapped to chromosome 10. Both TRP-2 (6) and Pmell7-1 (3) have significant sequence homology to the brown and albino genes, and immunological crossreactivity of their gene products, although their full sequences have not yet been published. It is an interesting fact that many laboratories have set out to clone the tyrosinase gene by different strategies, but have ended up cloning at least four different genes which are so highly homologous.
-
Our goal has been to identify the products of those genes and characterizetheir functions and interactions. Our approach has been to prepare synthetic peptides which correspond to the primary sequences predicted by the cloned DNA sequences and to raise specific antibodies against those peptides which can be used in various protocols to identify, isolate and characterize the gene products. It is important to note that when we consider the brown or dbino locus-encoded proteins in this paper, we
K. Tsukamoto et al.
85
are discussing the proteins encoded by those genes in the wild-type (that is, functionally normal) and not from the mutations. We have previously described the peptides we have synthesized,the specificitiesof the antibodiesgeneratedand the patterns of products identified by metabolic labeling and immunoprecipitation (22.23). In sum, those studies have shown that the antibodies specifically recognize the gene products as expected and that there is no cross-reactivity between them, i.e. they are specific for the peptide against which they were raised. The de now proteins recognized with short pulse labeling (-30 min) is approximately 59 kD (as predicted by the gene sequences) and, following longer labeling times, the glycosylated nature of the proteins is evident. Following such processing, the size of each protein is increased to 70 kD,suggesting that added &hydrates account for this difference. Isoelectric focusing resolves two bands for each of these proteins with PI'S at pH 6.5 and pH 5.8; there is an equal distribution between these two forms, and the lower PI of the two corresponds well with the predicted PI of these proteins @H 5.98). Comparison of the catalytic activity and the isozymic banding pattern identified by the peptide antibodies and Western immunoblotting of extracts of melanacytes also demonstratesthe multiple isoforms of the enzymes, with the major one correlating in mobility to the brown and ufbinoproteins (Figure 1). The critical point
-
A) Enzymatic Activity
B) Western lmmuooblotting
*-
*
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Tyroiinr W P A hydrwylau' oxidaw.
normal
(I
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a albino protein Igti
m
brown protein
LI
albino protcin
.
41kD >
26kD >
M, STDS
normal a brownm albim nbbil p r a m prolein
I.&
IgG
IgG
normal o browno albino rabbll pnwein protcln lgti IgG lgG
mrml (I browno album rabbll pmcm prwm I& IzG IgG
Figure 2. Purity of Immune-Affinity Prepared Proteins. B16 melanoma cells ~ b b o l i c a l l ylabeledwith [3sS]-methi~ni~ and d i e d by dioimmunopmipitationwith the primary antibodies l i s d at the ,as detailed in (23). Immunoprecipitated proteins were then s u b j e c s t o a second round of precipitation with antibodies, as noted on the bottom, separated by SDS gel electrophoresis and visualized with autoradiography.
to note here is that both gene products have highly similar physical properties and can not be readily resolved by conventional biochemical techniques. In addition to demonstratingthat immunological reagents must be used to distinguish between these two proteins, it further suggests that previous studies which isolated and characterized tyrosinases from mammalian sources probably contained the various proteins from these multiple gene loci. This raises an interesting point as to whether tyrosinase is truly bi- or tri-functiod, or whether the various catalytic potentials of the enzyme as previously described resulted from the individual activities of proteins that were copurified in the ostensibly pure tyrosinase fractions. We have used immunoaffinity purification to isolate the proteins of inand characterize their catalytic function. It is, of course, critical to demonstratethe purity of the proteins thus isolated, and we have employed several different protocds to conclusively demonstrate this fact (one such is shown in Figure 2, others are shown in ref 23). All of those protocols have demonstrated that our preparations of brown and albino proteins are not crosscontaminated,although we can not at this time eliminatethe possibility that those fractions contain yet other tyrosinaserelated gene products of similar size and charge (e.g. TRP-2 or Pmell7-1).
Figure 1. Immunoreactivity of Peptide Antibodies. Proteins from an NP40 extmct of B16 melanoma cells (30pg/lane) were w t e d by SDS gel electrophoresis under nondenaturing cond$ions and transferred '9 nitrucellulose mem+nes. Enzyme activities were visualited wth (A) U-["CJ-tyros~ or DOPA as substrates.or (B) reactivi with antibodies wry detected by peroxldaseconjugad stxmnciary a o t L e s , as m (22).
An examination of the catalytic functions of the immune-affinity purified proteins showed that both proteins, i.e. the brown and albino locus encoded proteins, were able to carry out both catalytic functions ascribed to
K. Tsukamoto et al.
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W P A DOPAchrome Melanin ki~$&eOxuJase lsomerese Formetlon
Figure 3. Melanogenic Activities of Immune-Affinity Purified Proteins. Proteins from an NP40 extract of B16 melanoma cells growing in covalently bound to Protein A as detailed in (23); proteins specifi&eluted from either control IgG, or IgG which recognized the albino or brown locus encoded proteins were then tested by standard melanogenic assays. vivo were immune-purified over I
tyrosinase (Figure 3). Shown is the average recovery of melanogenic activities following a single passage of an NP40 extract of B16 melanoma cells over a 1 ml immuneaffinity column. While most (30-7096) of the melanogenic activities were bound to the column containing IgG recognizing the albino protein, a significant amount was bound to the column which purified the brown locus encoded protein (negligible amounts were recovered from the control IgG column). Both the brown and the albino protein were competent to utilize tyrosine or DOPA as a substrate and both elicited melanin formation, although neither could utilize DOPAchrome as a substrate, thus seeming to eliminate either from consideration as encoding DOPAchrome isomerase (tautomerase). It should be noted that while the recoveries seen from the crude extract would indicate that the albino locus encoded tyrosinase contains the majority of tyrosinase activities in the cell, the actual specific activity of the ulbino protein is at least 10-fold higher since it is present in much less quantity relative to the brown protein (cf below). Although it would actually be consistent with expectations if the catalytic activities of tyrosinases resided on separate gene products (e.g. tyrosine hydroxylase activity on the albino protein, DOPA oxidase activity on the brown protein), this has not been demonstrable in spite of our intense efforts to prove it to be true. Interestingly, although we were unsuccessful in our early attempts to demonstrate cooperative interaction between these two proteins (23). we have finally had some limited success in this regard. In our initial studies, we tried adding the proteins together and immediately assaying them for melanogenic activity; those studies resulted in insignificant increases in activity. However, we now find that if the proteins are given time to interact prior to assay,
~
they will show synergistic interactions (Figure 4). In this study, we have diluted the protein preparations to an equimolar concentration, and then added them together in varying ratios and allowed them to incubate overnight at 4°C. The very low level of the tyrosinase activity of the brown protein when compared on an equimolar basis (cf 1:1 in the Figure) to the albino protein can be seen readily in this experiment, as discussed above. The percent stimulation of activity (i.e. the amount of activity above what would be expected on an additive basis) is shown with the 3-dimensional (solid-black) horizontal line on the p p h . One can see that synergistic interaction increases towards 150% as the molar ratio is adjusted to 5:l (brown:albino), thus approximating the in vivo ratio (typically between 10: 1 and 20: 1). We have since had success in further increasing this stimulation to >20096 as we have optimized various environmental conditions, such as detergent concentration, time of incubation, temperature, etc. Our preliminary experiments on the kinetics responsible for this synergism suggests an increase in the binding constant of the enzyme for tyrosine, which is in turn consistent with a possible conformational (allosteric) change in the enzyme. Such a mechanism of action was proposed by our group long ago (24) in response to added DOPA cofactor, and it remains an interesting possibility that the interaction of the brown and albino proteins might provide for an initial conformational change in the enzyme to stimulate melanogenesis. Future studies will be directed towards examining the mechanics of this interaction, especially with a view towards establishing its relevance under physiological conditions, i.e. with limited substrate and cofactor, and at a more acidic pH. This might also provide a natural mechanism for delaying the onset of melanogenesis throughout intracellular processing, if the brown and albino proteins are processed independently and delivered to the melanosome in separate vesicles.
I
1:5 1:l 5 :1 BrowAbino Protein Ratio
Figure 4. Synergism of brown and albino Proteins. Proteins that bad been immune-affinity purified as detailed for Figure 3, were mixed in the molar ratios as shown at the bottom, for 16 hrs at 4OC, and then assayed for melanin synthesis using the ["C]-tyrosine assay. Results am reported as pmol melanin produced/ Fg protedhr.
K. Tsukamoto et a].
87
la o
s
s
10
a0
50
aoimimrm
Time oi incubation (min)
I
Figure 5. Peroxidase Assay of Melanogenic Proteins. Sam les as noted on the right were assayed for peroxidase activity using 8PD as substrate, under standard conditions, for various times as noted on the bottom of the p p h . Samples shown are horseradish peroxidase (0.1 U), B16 cell extPBCt (100 pg), dbino protein (1 pg), b m n protein ( I pg), and buffer only (as a control).
There has been a recent suggestion that the brown locus encoded product functions as a melanosomal-specific catalase (14). and we have examined the ability of our affinity purified proteins to so function. It has also been suggested that peroxidase might function in the melanogenic pathway (13). Using various substrates (OPD is shown in Figure 5). we can demonstrate that there is a p e r o x i k activity in the original extract; however, that activity does not copurify with either the brown or the ufbino proteins. On the other hand, the ability of these melanogenic proteins to reduce levels of hydrogen peroxide during melanogenesis can be seen in Figure 6. In those experiments, we incubated known amounts of the protein fractions in the presence of H,O,. and following 5 hrs of reaction, assayed for the hydrogen peroxide concentration remaining with a standard dilution of horseradish peroxidase (which requires H202 for its activity and is an extremely sensitive idcator). Again, the original melanocyte extract has considerable levels of a catalase-like activity, but here the activity copurifies with both of the melanogenic proteins. It is impossible to state at this point whether the activity is truly catalase in nature; it has been reported that Hz% participates in the cleavage of the phenyl ring of indole-quinones (25) and it might easily be that this is the manner in which the Hz% level is decreased. A final determination of the nature of this activity will have to await the complete characterization of the substrates and products involved. However, it is obvious that this activity, whatever it might be, is present on both proteins and is not specific to the brown locus-encoded protein. If the brown protein contains tyrosinase activity. why then don’t dbim melanocytes have at least some sort
11
19.
1:1
dilution
Figure 6. Hz02 Use by Melanogenic Proteins. Samples as detailed for Figure 5 were incubated for 5 hr in the resence of 0.01%hydrogen peroxide. following which 0.1 pU Lrseradish peroxidase was added to each well and activity WBS visualized by OPD.
rudimentary melanogenic function ? There are actually quite a few previous reports showing the presence of active tyrosinase in albino mutations (reviewed in 7) which supports the concept of alternative tyrosinases. The explanation of why melanin is not pr@uced may actually be quite trivial: There are a host of melanogenic factors critical to the expression of melanin synthesis; primary among them are melanogenic inhibitors. The regulation of melanin production in vivo may actually depend on a dynamic equilibrium between active enzyme and its inhibitors. If one then reduces the amount of active tyrosinase (e.g. by a mutation at the ulbino locus) then you may reduce the overall level of tyrosinase to that which can be totally repressed by the endogenous inhibitor. This model would.then predict that the brown mutation should also result in a reduction in tyrosinase activity; previous reports (26,27) have stated that tyrosinase activities in brown mutations are si@icantly increased. In those reports however, crude extracts of skin andor eyes were used, thus resulting in significant contamination of the extracts with nonmelanocytic cell types. The results in Figure 7 show that when pure melanocytes growing in culture are assayed for tyrosinase activity, that the melanogenic activities of the brown mutants are significantly reduced compared to the black, as we would predict. Note too that the ability to use DOPAchrome as a substrate is not lost in albino or brown mutation cells, further supporting the fact neither of these genes encodes that melanogenic activity, as previously reported (28). From the sum of these and previous studies, we feel that it is obvious that melanin production depends on multiple enzymes derived from distinct genetic loci. Tyrosinase from the albino locus is critical of course, but other tyrosinase-related enzymes, peroxidase, glutathione rcductasc, DOPAchrome isomerase, and even metal ions,
88
K. Tsukarnoto et al. ACKNOWLEDGEMENTS The authors wish to express their thanks to Dr. Dorothy Bennett for the mutant melanocyte lines used in this study, and to Dr. Ian Jackson for sharing unpublished information regarding his TRP-2 gene. We also thank Mr. Paul Montague and Wilfred Vieira for their expert technical assistance. REFERENCES 1 Hearing VJ, Jimtnez, M. Mammalian tyrosinase: the critical regulatory control point in melanocyte pigmentation. Int J Biochem 1987:19:1141-7.
Figure 7. Melanogenic Activities of Pigment Mutations. Melan-a (black), melan-b (brown mutant) and melanc (albino mutant) cell lines were cultured as described (29), then solubilie with NP40 and assayed for melanogenic activities by standard techtuques. Data is reported as pmol productllhdpg protein in the extract.
must interact to regulate the quantity and quality of melanin produced. It is now clear that tyrosinases belong to a gene family, of which the brown and albino loci have been definitively mapped; TRP-2 has been localized to chromosome 14 and Pmell7-1 to chromosome 10, but the exact loci of those two genes are not yet known. The albino and brown genes in humans have now been cloned, although mutations of the brown locus have not yet been identified in humans. The interactions between the brown and albino gene products, as well as other tyrosinase-relatedproteins, in determining various patterns of melankation are not yet clear. However, these data are consistent with previous studies (cf Ref 1) which demonstratedlesser but significant levels of tyrosinase activities in albino mutations and thus argued against the albino locus being the structural locus for the enzyme. It is now obvious that the albino locus is the structural locus for tyrosinase, but it is not the only one. Both the brown and the albino locus encoded tyrosinases are catalytically bifunctional, although the specific catalytic activity of the latter is -20-fold higher. Certainly, the albino locus encoded tyrosinase has been shown to be absolutely essential for melanin production, but its activity is somehow modulated by the product of the brown locus, and perhaps by the products of the other tyrosinase-related loci. It is also obvious that the disperse heterogeneity of demonstrabletyrosinase isozymes must be, at least in part, due to the presence of these catalytically active products from multiple loci. Future studieswill have to be directed at more fully establishing the substrate specificities and product formation, as well as the working relationships of these different gene products in the regulation of mammalian melanogenesis.
2 Shibahara S, Tomita Y, Sakakura T, Nager C, Chaudhuri B, Muller R. Cloning and expression of cDNA encoding mouse tyrosinase. Nucl Acids Res, 1986:14:2413-27.
3 Kwon BS, Halaban R, Kim GS. Usack L, Pomerantz SH, Haq AK. A melanocyte-specific complementary DNA clone whose expression is inducible by melanotropin and isobutylmethyl xanthine. Mol Biol Med 1987~41339-55. 4 Kwon BS, Haq AK, Pomerantz SH, Halaban R. Isolation and sequence of a cDNA locus for human tyrosinase that maps at the mouse c-albino locus. Proc Natl Acad Sci USA 1987:84:7473-7. 5 Yamamoto H, Takeuchi S, Kudo T, Makino K, Nakata A, Shinoda T, Takeuchi T. Cloning and sequencing of
mouse tyrosinase cDNA. Jpn J Gen 1987:62:271-4. 6 Jackson IJ. A cDNA encoding tyrosinase-related protein maps to the brown locus in mice. Proc Natl Acad Sci USA 1988:85:4392-6. 7 Hearing VJ, Jimtnez M. Analysis of mammalian pigmentation at the molecular level. Pigment Cell Res 1989:2:75-85.
8 Pawelek J, K6rner A, Bergsmm A, Bologna J. New regulators of melanin biosynthesis and the autodestruction of melanoma cells. Nature 1980~286: 617-9.
.
9 Barber JI, Townsend D, Olds DP, King RA. DOPAchrome oxidoreductase: a new enzyme in the pigment pathway. J Invest Dermatol 1984:83:145-9. 10 Leonard W, Townsend D, King RA. Function of DOPAchrome oxidoductase and metal ions in DOPAchrome conversion in the eumelanin pathway. Biochemistry 1988:27:6 156-9. 11 Pawelek JM. DOPAchrome conversion factor functions as an isomerase. Biochem Biophys Res 1990:166:1328-33. COIUIUU~
K. Tsukamoto et al. 12 A r m P, Garcia-Borron JC. Solano F, Lozano JA. Regulation of distal mammalian melanogenesis. I. partial purification and characterization of a DOPAchrome converting factor: DOPAchrome tautomerase. Biochim BiophysActa 1990:1035:266-75. 13 d’Ischia M, Napolitano A, Prota G. Peroxidase as an alternativeto tyrosinase in the oxidative polymerization of 5,6-dihydroxyindoles to melanin@). Biochim Biophys Acta 1991:(in press). 14 Halaban R, Moellmann G. Murine and human b locus pigmentation genes encode a glycoprotein (gp75) with catalase activity. Proc Natl Acad Sci USA 1990:87:48O9-4813. 15 Mojamdar M, Ichihashi M, Mishima Y. y-Glutamyl transpeptidase, tyrosinase and 5-S-cysteinyldopa production in melanoma cells. J Invest Dermatol 1983~81: 119-21. 16 Prota G. Cysteine and glutathione in mammalian pigmentation. In: Cavallini D, GauU GE, Zappia V, eds. Natural Sulfur Compounds. New York: Plenum Press, 19801391-8. 17 Palumbo A, d’Ischia M. Misuraca G, Prota G, Schultz TM. Structural modifications in biosynthetic melanins induced by metal ions. Biochim Biophys Acta 1988~964: 193-9. 18 Palumbo A, d’Ischia M, Misuraca G, C a r r a ~ L, Prota G. Activation of mammalian tyrosinase by ferrous ions. Biochim Biophys Acta 1990:1033:256-60. 19 Kameyama K, Montague PM, Hearing VJ. The expression of melanocyte stimulating hormone receptors correlates with mammalian pigmentation and can be modulated by interferons. J Cell Physiol 1988:137:35-44. 20 Kameyama K, Jimtnez M, Muller J, Ishida Y, Hearing VJ. Regulation of mammalian melanogenesis by tyrosinase inhibition.Differentiation 1989:42:28-36. 21 Vijayasaradhi S. Bouchard B, Houghton AN. The melanoma antigen gp75 is the human homologue of the mouse b (brown) locus gene product. J Exp Med 1990:171:1375-80.
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22 Jimtnez M. Maloy WL. Hearing VJ. Specific identification of an authentic tyrosinase clone. J Biol Chem 1989:264:3397-3403. 23 Jimtnez M. Tsukamoto K, Hearing VJ. Tyrosinases from two different loci are expressed by normal and by transformed melanocytes. J Biol Chem 1991: 266: 1147-56. 24 Hearing VJ, Eke1 TM. Montague PM, Hearing ED. Nicholson JM. Mammalian tyrosinase: Isolation by a simple new procedure and characterizationof its steric requirements for cofacto: activity. Arch Biochem Biophys 1978:185:4O7-18. 25 Korytowski W, Sarna T. Bleaching of melanin pigments. Role of copper ions and hydrogen peroxide in autooxidation and photooxidation of synthetic DOPA-melanin. J Biol Chem 1990:265: 12410-6. 26 Coleman DL. Effect of genic substitution on the incorporation of tyrosine into melanin of mouse skin. Arch Biochem Biophys 1962:69:562-8. 27 Tamate HB, Hirobe T, Wakamatsu K. It0 S,Shibahara S, lshikawa K. Levels of tyrosinase and its mRNA in coat-color mutants of C57EiWlOJ congenic mice: effects of genic substitution at the agouti. brown, albino, ddute and pink-eyed dilution loci. J Exp Zoo1 1989:250:304-11. 28 Lamoreux ML. Woolley C, Pendergast P. Genetic controls over activities of tyrosinase and DOPAchrome conversion factor in murine melanocytes. Genetics 1986:113:967-84. 29 Bennett DC, Cooper PJ, Dexter TH, Devlin LM, Heasman J, Nester B. Cloned mouse melanocyte lines carrying germline mutations albino and brown: complementation in culture. Development 1989: 1051379-85.
The Nature of Tyrosinase Isozymes KATSUHIKO TSLJKAMOTO’, MERCEDES J m N E Z ’ AND VINCENT J. HEARING Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 ‘Department of Dermatology, Yamanashi Medical College, 1 110 Shimokato, Tamaho, Nakakoma, Yamanashi, Japan 409-38; 2Unidad Docente de Biologia, Facultad de Veterinaria, Universidad de Murcia, Espinardo, Murcia, Spain
Tyrosinase [EC 1.14.18.11, the enzyme critical to the formation of melanin, occurs in vivo as a number of isozymic forms. These isozymic forms have generally been considered to arise as the rgmlt of various post-translational modifications of a single protein precursor. It has been shown by a number of studies that tyrosinase is synthesized de mvo on ribosomes, transported through the smooth endoplasmic reticulum and processed (i.e. glycosylated and sialylated) in the Gold complex. The enzyme is then transported via coated vesicles to premelanosomes and incorporatedwithin the melanosomalmembrane, whereupon melanin synthesis can proceed (cf ref 1 for review and relevant citations). However, with the advent of gene cloning techniques, a number of tyrosinase-related proteins have now been identified (2-6, reviewed in 7), and the question remains as to whether these other gene products are, at least in part, responsible for observed tyrosinase activity and its isozymic heterogeneity. Further, the pathway for the formation of eu- and pheomelanins has now been shown to be under much greater regulatory control than previously thought. In addition to tyrosinase, it has been shown that other enzymes such as DOPAchrome isomerase (8-12, alternatively termed DOPAchrome tautomerase), peroxidases (13) and/or catalases (14), glutamine metabolic enzymes (15,16), metal cations (17,18) and melanogenic inhibitors (19.20) might play a role in the regulation of melanogenesis in mammals. Thus the entire question of the identity of the various tyrosinase isozymes and the catalytic function of each of them has received renewed interest. Two of the candidate clones for tyrosinase have been mapped to the ulbino and brown loci of mice (4,6). Each of those genes would be expected to encode a protein which can modulate melanin production since the albino locus regulates whether melanin will be formed while the
brown locus regulates the type of melanin produced (black versus brown eumelanin). These two genes have been cloned from murine and from human systems (2,4,5,21), sequenced and found to be highly homologous at several areas thought to be critical to their catalytic function, although they have extensive variation at their amino and carboxyl termini. Both of those clones have many predicted characteristics that would be expected of tyrosinase, including size (-60 .kD, processed to 70 kD), melanocyte specific expression, two putative copper binding sites, potential glycosylation sites and a stable nature (by virtue of cysteine-rich domains). In addition to the brown and ulbino genes, two other homologous genes which map to different chromosomes have also been identified. The clone 5A originally thought to be identical to the brown locus has now been found to encode a distinct gene (termed TRP-2)on chromosome 14 (I Jackson, personal communication), while Pmell7-1 has been mapped to chromosome 10. Both TRP-2 (6) and Pmell7-1 (3) have significant sequence homology to the brown and albino genes, and immunological crossreactivity of their gene products, although their full sequences have not yet been published. It is an interesting fact that many laboratories have set out to clone the tyrosinase gene by different strategies, but have ended up cloning at least four different genes which are so highly homologous.
-
Our goal has been to identify the products of those genes and characterizetheir functions and interactions. Our approach has been to prepare synthetic peptides which correspond to the primary sequences predicted by the cloned DNA sequences and to raise specific antibodies against those peptides which can be used in various protocols to identify, isolate and characterize the gene products. It is important to note that when we consider the brown or dbino locus-encoded proteins in this paper, we
K. Tsukamoto et al.
85
are discussing the proteins encoded by those genes in the wild-type (that is, functionally normal) and not from the mutations. We have previously described the peptides we have synthesized,the specificitiesof the antibodiesgeneratedand the patterns of products identified by metabolic labeling and immunoprecipitation (22.23). In sum, those studies have shown that the antibodies specifically recognize the gene products as expected and that there is no cross-reactivity between them, i.e. they are specific for the peptide against which they were raised. The de now proteins recognized with short pulse labeling (-30 min) is approximately 59 kD (as predicted by the gene sequences) and, following longer labeling times, the glycosylated nature of the proteins is evident. Following such processing, the size of each protein is increased to 70 kD,suggesting that added &hydrates account for this difference. Isoelectric focusing resolves two bands for each of these proteins with PI'S at pH 6.5 and pH 5.8; there is an equal distribution between these two forms, and the lower PI of the two corresponds well with the predicted PI of these proteins @H 5.98). Comparison of the catalytic activity and the isozymic banding pattern identified by the peptide antibodies and Western immunoblotting of extracts of melanacytes also demonstratesthe multiple isoforms of the enzymes, with the major one correlating in mobility to the brown and ufbinoproteins (Figure 1). The critical point
-
A) Enzymatic Activity
B) Western lmmuooblotting
*-
*
4.1 kn >
?hhD > M, S'I'US
Tyroiinr W P A hydrwylau' oxidaw.
normal
(I
bmun
rabbi1
protein
lgti
IgG
a albino protein Igti
m
brown protein
LI
albino protcin
.
41kD >
26kD >
M, STDS
normal a brownm albim nbbil p r a m prolein
I.&
IgG
IgG
normal o browno albino rabbll pnwein protcln lgti IgG lgG
mrml (I browno album rabbll pmcm prwm I& IzG IgG
Figure 2. Purity of Immune-Affinity Prepared Proteins. B16 melanoma cells ~ b b o l i c a l l ylabeledwith [3sS]-methi~ni~ and d i e d by dioimmunopmipitationwith the primary antibodies l i s d at the ,as detailed in (23). Immunoprecipitated proteins were then s u b j e c s t o a second round of precipitation with antibodies, as noted on the bottom, separated by SDS gel electrophoresis and visualized with autoradiography.
to note here is that both gene products have highly similar physical properties and can not be readily resolved by conventional biochemical techniques. In addition to demonstratingthat immunological reagents must be used to distinguish between these two proteins, it further suggests that previous studies which isolated and characterized tyrosinases from mammalian sources probably contained the various proteins from these multiple gene loci. This raises an interesting point as to whether tyrosinase is truly bi- or tri-functiod, or whether the various catalytic potentials of the enzyme as previously described resulted from the individual activities of proteins that were copurified in the ostensibly pure tyrosinase fractions. We have used immunoaffinity purification to isolate the proteins of inand characterize their catalytic function. It is, of course, critical to demonstratethe purity of the proteins thus isolated, and we have employed several different protocds to conclusively demonstrate this fact (one such is shown in Figure 2, others are shown in ref 23). All of those protocols have demonstrated that our preparations of brown and albino proteins are not crosscontaminated,although we can not at this time eliminatethe possibility that those fractions contain yet other tyrosinaserelated gene products of similar size and charge (e.g. TRP-2 or Pmell7-1).
Figure 1. Immunoreactivity of Peptide Antibodies. Proteins from an NP40 extmct of B16 melanoma cells (30pg/lane) were w t e d by SDS gel electrophoresis under nondenaturing cond$ions and transferred '9 nitrucellulose mem+nes. Enzyme activities were visualited wth (A) U-["CJ-tyros~ or DOPA as substrates.or (B) reactivi with antibodies wry detected by peroxldaseconjugad stxmnciary a o t L e s , as m (22).
An examination of the catalytic functions of the immune-affinity purified proteins showed that both proteins, i.e. the brown and albino locus encoded proteins, were able to carry out both catalytic functions ascribed to
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W P A DOPAchrome Melanin ki~$&eOxuJase lsomerese Formetlon
Figure 3. Melanogenic Activities of Immune-Affinity Purified Proteins. Proteins from an NP40 extract of B16 melanoma cells growing in covalently bound to Protein A as detailed in (23); proteins specifi&eluted from either control IgG, or IgG which recognized the albino or brown locus encoded proteins were then tested by standard melanogenic assays. vivo were immune-purified over I
tyrosinase (Figure 3). Shown is the average recovery of melanogenic activities following a single passage of an NP40 extract of B16 melanoma cells over a 1 ml immuneaffinity column. While most (30-7096) of the melanogenic activities were bound to the column containing IgG recognizing the albino protein, a significant amount was bound to the column which purified the brown locus encoded protein (negligible amounts were recovered from the control IgG column). Both the brown and the albino protein were competent to utilize tyrosine or DOPA as a substrate and both elicited melanin formation, although neither could utilize DOPAchrome as a substrate, thus seeming to eliminate either from consideration as encoding DOPAchrome isomerase (tautomerase). It should be noted that while the recoveries seen from the crude extract would indicate that the albino locus encoded tyrosinase contains the majority of tyrosinase activities in the cell, the actual specific activity of the ulbino protein is at least 10-fold higher since it is present in much less quantity relative to the brown protein (cf below). Although it would actually be consistent with expectations if the catalytic activities of tyrosinases resided on separate gene products (e.g. tyrosine hydroxylase activity on the albino protein, DOPA oxidase activity on the brown protein), this has not been demonstrable in spite of our intense efforts to prove it to be true. Interestingly, although we were unsuccessful in our early attempts to demonstrate cooperative interaction between these two proteins (23). we have finally had some limited success in this regard. In our initial studies, we tried adding the proteins together and immediately assaying them for melanogenic activity; those studies resulted in insignificant increases in activity. However, we now find that if the proteins are given time to interact prior to assay,
~
they will show synergistic interactions (Figure 4). In this study, we have diluted the protein preparations to an equimolar concentration, and then added them together in varying ratios and allowed them to incubate overnight at 4°C. The very low level of the tyrosinase activity of the brown protein when compared on an equimolar basis (cf 1:1 in the Figure) to the albino protein can be seen readily in this experiment, as discussed above. The percent stimulation of activity (i.e. the amount of activity above what would be expected on an additive basis) is shown with the 3-dimensional (solid-black) horizontal line on the p p h . One can see that synergistic interaction increases towards 150% as the molar ratio is adjusted to 5:l (brown:albino), thus approximating the in vivo ratio (typically between 10: 1 and 20: 1). We have since had success in further increasing this stimulation to >20096 as we have optimized various environmental conditions, such as detergent concentration, time of incubation, temperature, etc. Our preliminary experiments on the kinetics responsible for this synergism suggests an increase in the binding constant of the enzyme for tyrosine, which is in turn consistent with a possible conformational (allosteric) change in the enzyme. Such a mechanism of action was proposed by our group long ago (24) in response to added DOPA cofactor, and it remains an interesting possibility that the interaction of the brown and albino proteins might provide for an initial conformational change in the enzyme to stimulate melanogenesis. Future studies will be directed towards examining the mechanics of this interaction, especially with a view towards establishing its relevance under physiological conditions, i.e. with limited substrate and cofactor, and at a more acidic pH. This might also provide a natural mechanism for delaying the onset of melanogenesis throughout intracellular processing, if the brown and albino proteins are processed independently and delivered to the melanosome in separate vesicles.
I
1:5 1:l 5 :1 BrowAbino Protein Ratio
Figure 4. Synergism of brown and albino Proteins. Proteins that bad been immune-affinity purified as detailed for Figure 3, were mixed in the molar ratios as shown at the bottom, for 16 hrs at 4OC, and then assayed for melanin synthesis using the ["C]-tyrosine assay. Results am reported as pmol melanin produced/ Fg protedhr.
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la o
s
s
10
a0
50
aoimimrm
Time oi incubation (min)
I
Figure 5. Peroxidase Assay of Melanogenic Proteins. Sam les as noted on the right were assayed for peroxidase activity using 8PD as substrate, under standard conditions, for various times as noted on the bottom of the p p h . Samples shown are horseradish peroxidase (0.1 U), B16 cell extPBCt (100 pg), dbino protein (1 pg), b m n protein ( I pg), and buffer only (as a control).
There has been a recent suggestion that the brown locus encoded product functions as a melanosomal-specific catalase (14). and we have examined the ability of our affinity purified proteins to so function. It has also been suggested that peroxidase might function in the melanogenic pathway (13). Using various substrates (OPD is shown in Figure 5). we can demonstrate that there is a p e r o x i k activity in the original extract; however, that activity does not copurify with either the brown or the ufbino proteins. On the other hand, the ability of these melanogenic proteins to reduce levels of hydrogen peroxide during melanogenesis can be seen in Figure 6. In those experiments, we incubated known amounts of the protein fractions in the presence of H,O,. and following 5 hrs of reaction, assayed for the hydrogen peroxide concentration remaining with a standard dilution of horseradish peroxidase (which requires H202 for its activity and is an extremely sensitive idcator). Again, the original melanocyte extract has considerable levels of a catalase-like activity, but here the activity copurifies with both of the melanogenic proteins. It is impossible to state at this point whether the activity is truly catalase in nature; it has been reported that Hz% participates in the cleavage of the phenyl ring of indole-quinones (25) and it might easily be that this is the manner in which the Hz% level is decreased. A final determination of the nature of this activity will have to await the complete characterization of the substrates and products involved. However, it is obvious that this activity, whatever it might be, is present on both proteins and is not specific to the brown locus-encoded protein. If the brown protein contains tyrosinase activity. why then don’t dbim melanocytes have at least some sort
11
19.
1:1
dilution
Figure 6. Hz02 Use by Melanogenic Proteins. Samples as detailed for Figure 5 were incubated for 5 hr in the resence of 0.01%hydrogen peroxide. following which 0.1 pU Lrseradish peroxidase was added to each well and activity WBS visualized by OPD.
rudimentary melanogenic function ? There are actually quite a few previous reports showing the presence of active tyrosinase in albino mutations (reviewed in 7) which supports the concept of alternative tyrosinases. The explanation of why melanin is not pr@uced may actually be quite trivial: There are a host of melanogenic factors critical to the expression of melanin synthesis; primary among them are melanogenic inhibitors. The regulation of melanin production in vivo may actually depend on a dynamic equilibrium between active enzyme and its inhibitors. If one then reduces the amount of active tyrosinase (e.g. by a mutation at the ulbino locus) then you may reduce the overall level of tyrosinase to that which can be totally repressed by the endogenous inhibitor. This model would.then predict that the brown mutation should also result in a reduction in tyrosinase activity; previous reports (26,27) have stated that tyrosinase activities in brown mutations are si@icantly increased. In those reports however, crude extracts of skin andor eyes were used, thus resulting in significant contamination of the extracts with nonmelanocytic cell types. The results in Figure 7 show that when pure melanocytes growing in culture are assayed for tyrosinase activity, that the melanogenic activities of the brown mutants are significantly reduced compared to the black, as we would predict. Note too that the ability to use DOPAchrome as a substrate is not lost in albino or brown mutation cells, further supporting the fact neither of these genes encodes that melanogenic activity, as previously reported (28). From the sum of these and previous studies, we feel that it is obvious that melanin production depends on multiple enzymes derived from distinct genetic loci. Tyrosinase from the albino locus is critical of course, but other tyrosinase-related enzymes, peroxidase, glutathione rcductasc, DOPAchrome isomerase, and even metal ions,
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K. Tsukarnoto et al. ACKNOWLEDGEMENTS The authors wish to express their thanks to Dr. Dorothy Bennett for the mutant melanocyte lines used in this study, and to Dr. Ian Jackson for sharing unpublished information regarding his TRP-2 gene. We also thank Mr. Paul Montague and Wilfred Vieira for their expert technical assistance. REFERENCES 1 Hearing VJ, Jimtnez, M. Mammalian tyrosinase: the critical regulatory control point in melanocyte pigmentation. Int J Biochem 1987:19:1141-7.
Figure 7. Melanogenic Activities of Pigment Mutations. Melan-a (black), melan-b (brown mutant) and melanc (albino mutant) cell lines were cultured as described (29), then solubilie with NP40 and assayed for melanogenic activities by standard techtuques. Data is reported as pmol productllhdpg protein in the extract.
must interact to regulate the quantity and quality of melanin produced. It is now clear that tyrosinases belong to a gene family, of which the brown and albino loci have been definitively mapped; TRP-2 has been localized to chromosome 14 and Pmell7-1 to chromosome 10, but the exact loci of those two genes are not yet known. The albino and brown genes in humans have now been cloned, although mutations of the brown locus have not yet been identified in humans. The interactions between the brown and albino gene products, as well as other tyrosinase-relatedproteins, in determining various patterns of melankation are not yet clear. However, these data are consistent with previous studies (cf Ref 1) which demonstratedlesser but significant levels of tyrosinase activities in albino mutations and thus argued against the albino locus being the structural locus for the enzyme. It is now obvious that the albino locus is the structural locus for tyrosinase, but it is not the only one. Both the brown and the albino locus encoded tyrosinases are catalytically bifunctional, although the specific catalytic activity of the latter is -20-fold higher. Certainly, the albino locus encoded tyrosinase has been shown to be absolutely essential for melanin production, but its activity is somehow modulated by the product of the brown locus, and perhaps by the products of the other tyrosinase-related loci. It is also obvious that the disperse heterogeneity of demonstrabletyrosinase isozymes must be, at least in part, due to the presence of these catalytically active products from multiple loci. Future studieswill have to be directed at more fully establishing the substrate specificities and product formation, as well as the working relationships of these different gene products in the regulation of mammalian melanogenesis.
2 Shibahara S, Tomita Y, Sakakura T, Nager C, Chaudhuri B, Muller R. Cloning and expression of cDNA encoding mouse tyrosinase. Nucl Acids Res, 1986:14:2413-27.
3 Kwon BS, Halaban R, Kim GS. Usack L, Pomerantz SH, Haq AK. A melanocyte-specific complementary DNA clone whose expression is inducible by melanotropin and isobutylmethyl xanthine. Mol Biol Med 1987~41339-55. 4 Kwon BS, Haq AK, Pomerantz SH, Halaban R. Isolation and sequence of a cDNA locus for human tyrosinase that maps at the mouse c-albino locus. Proc Natl Acad Sci USA 1987:84:7473-7. 5 Yamamoto H, Takeuchi S, Kudo T, Makino K, Nakata A, Shinoda T, Takeuchi T. Cloning and sequencing of
mouse tyrosinase cDNA. Jpn J Gen 1987:62:271-4. 6 Jackson IJ. A cDNA encoding tyrosinase-related protein maps to the brown locus in mice. Proc Natl Acad Sci USA 1988:85:4392-6. 7 Hearing VJ, Jimtnez M. Analysis of mammalian pigmentation at the molecular level. Pigment Cell Res 1989:2:75-85.
8 Pawelek J, K6rner A, Bergsmm A, Bologna J. New regulators of melanin biosynthesis and the autodestruction of melanoma cells. Nature 1980~286: 617-9.
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9 Barber JI, Townsend D, Olds DP, King RA. DOPAchrome oxidoreductase: a new enzyme in the pigment pathway. J Invest Dermatol 1984:83:145-9. 10 Leonard W, Townsend D, King RA. Function of DOPAchrome oxidoductase and metal ions in DOPAchrome conversion in the eumelanin pathway. Biochemistry 1988:27:6 156-9. 11 Pawelek JM. DOPAchrome conversion factor functions as an isomerase. Biochem Biophys Res 1990:166:1328-33. COIUIUU~
K. Tsukamoto et al. 12 A r m P, Garcia-Borron JC. Solano F, Lozano JA. Regulation of distal mammalian melanogenesis. I. partial purification and characterization of a DOPAchrome converting factor: DOPAchrome tautomerase. Biochim BiophysActa 1990:1035:266-75. 13 d’Ischia M, Napolitano A, Prota G. Peroxidase as an alternativeto tyrosinase in the oxidative polymerization of 5,6-dihydroxyindoles to melanin@). Biochim Biophys Acta 1991:(in press). 14 Halaban R, Moellmann G. Murine and human b locus pigmentation genes encode a glycoprotein (gp75) with catalase activity. Proc Natl Acad Sci USA 1990:87:48O9-4813. 15 Mojamdar M, Ichihashi M, Mishima Y. y-Glutamyl transpeptidase, tyrosinase and 5-S-cysteinyldopa production in melanoma cells. J Invest Dermatol 1983~81: 119-21. 16 Prota G. Cysteine and glutathione in mammalian pigmentation. In: Cavallini D, GauU GE, Zappia V, eds. Natural Sulfur Compounds. New York: Plenum Press, 19801391-8. 17 Palumbo A, d’Ischia M. Misuraca G, Prota G, Schultz TM. Structural modifications in biosynthetic melanins induced by metal ions. Biochim Biophys Acta 1988~964: 193-9. 18 Palumbo A, d’Ischia M, Misuraca G, C a r r a ~ L, Prota G. Activation of mammalian tyrosinase by ferrous ions. Biochim Biophys Acta 1990:1033:256-60. 19 Kameyama K, Montague PM, Hearing VJ. The expression of melanocyte stimulating hormone receptors correlates with mammalian pigmentation and can be modulated by interferons. J Cell Physiol 1988:137:35-44. 20 Kameyama K, Jimtnez M, Muller J, Ishida Y, Hearing VJ. Regulation of mammalian melanogenesis by tyrosinase inhibition.Differentiation 1989:42:28-36. 21 Vijayasaradhi S. Bouchard B, Houghton AN. The melanoma antigen gp75 is the human homologue of the mouse b (brown) locus gene product. J Exp Med 1990:171:1375-80.
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22 Jimtnez M. Maloy WL. Hearing VJ. Specific identification of an authentic tyrosinase clone. J Biol Chem 1989:264:3397-3403. 23 Jimtnez M. Tsukamoto K, Hearing VJ. Tyrosinases from two different loci are expressed by normal and by transformed melanocytes. J Biol Chem 1991: 266: 1147-56. 24 Hearing VJ, Eke1 TM. Montague PM, Hearing ED. Nicholson JM. Mammalian tyrosinase: Isolation by a simple new procedure and characterizationof its steric requirements for cofacto: activity. Arch Biochem Biophys 1978:185:4O7-18. 25 Korytowski W, Sarna T. Bleaching of melanin pigments. Role of copper ions and hydrogen peroxide in autooxidation and photooxidation of synthetic DOPA-melanin. J Biol Chem 1990:265: 12410-6. 26 Coleman DL. Effect of genic substitution on the incorporation of tyrosine into melanin of mouse skin. Arch Biochem Biophys 1962:69:562-8. 27 Tamate HB, Hirobe T, Wakamatsu K. It0 S,Shibahara S, lshikawa K. Levels of tyrosinase and its mRNA in coat-color mutants of C57EiWlOJ congenic mice: effects of genic substitution at the agouti. brown, albino, ddute and pink-eyed dilution loci. J Exp Zoo1 1989:250:304-11. 28 Lamoreux ML. Woolley C, Pendergast P. Genetic controls over activities of tyrosinase and DOPAchrome conversion factor in murine melanocytes. Genetics 1986:113:967-84. 29 Bennett DC, Cooper PJ, Dexter TH, Devlin LM, Heasman J, Nester B. Cloned mouse melanocyte lines carrying germline mutations albino and brown: complementation in culture. Development 1989: 1051379-85.
