Chemical characterization of pheomelanogenesis starting from dihydroxyphenylalanine or tyrosine and cysteine.

Abstract

Two types of melanin pigment are produced in mammals; the brown-to-black eumelanins and the yellow-to-reddish-brown pheomelanins. The switch from one type of melanin to the other appears to be regulated by the levels of tyrosinase and thiols, such as cysteine and glutathione. This study examines the process of pheomelanin formation starting from dihydroxyphenylalanine (dopa) or tyrosine and cysteine. We prepared pheomelanins by tyrosinase oxidation of dopa or tyrosine in the presence of cysteine. Experimental variables were reaction time, tyrosinase concentration, and dopa or tyrosine to cysteine ratio. Following the reactions, we measured concentrations of tyrosine, dopa, cysteine and cysteinyldopas, amounts of total melanin (TM) by Soluene-350 solubilization and aminohydroxyphenylalanine (AHP), a specific indicator of pheomelanin, formed by hydriodic acid hydrolysis, and absorbance ratio, A₆₅₀/A₅₀₀. It was found that (1) mixed melanogenesis is a heterogeneous process in which pheomelanogenesis proceeds first, followed by eumelanogenesis, as shown by changes in the tyrosine and cysteinyldopa concentrations, the AHP/TM ratio, and the A₆₅₀/A₅₀₀ ratio during the course of melanogenesis and (2) lower tyrosinase concentration favors pheomelanogenesis even when the availability of cysteine is limited, as shown by AHP/TM ratios that were higher than the corresponding tyrosine to cysteine ratios. These results indicate that the switch from eumelanogenesis to pheomelanogenesis can be achieved by lowering the tyrosinase activity, which conforms to our proposal that tyrosinase activity is the major factor controlling the course of melanogenesis.

Introduction

Mammalian melanocytes produce two chemically distinct types of melanin pigments, the black-to-brown eumelanins and the yellow-to-reddish-brown pheomelanins 1, 2. Both types of melanins are synthesized from the common precursor, dopaquinone, which is produced from tyrosine by the action of tyrosinase (EC 1.14.18.1) in the melanosome, the specialized organelle of the melanocyte. Dopaquinone undergoes a series of redox reactions leading to the formation of eumelanins or, alternatively, in the presence of thiols such as cysteine or glutathione, it can act as the precursor of pheomelanins (Fig. 1). A switch in the synthesis of one type of melanin to the other is mediated by certain chemicals, such as α-melanocyte stimulating hormone (α-MSH) and agouti signal protein 3, 4, 5. This switch also occurs in nature, as seen in the agouti mouse, which is characterized by a yellow striped band against a black background on each hair shaft 3, 4, 5.

Extensive chemical and enzymic studies have led to the general concept that eumelanins are derived from oxidative polymerization of 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid 1, 2. Although much less is known about the structure of pheomelanins [6], it is generally accepted that pheomelanins consist of 1,4-benzothiazine units formed from cysteinyldopas (CDs), the products of an addition reaction of cysteine to dopaquinone 1, 2, 7. In reality, most mammalian melanin pigments are mixed and consist of eumelanins and pheomelanins, either as copolymers or as mixtures 8, 9, 10, 11.

The genetics and molecular biology of melanogenesis in follicular melanocytes have been extensively studied 3, 12, 13, 14. More than 10 genes involved directly or indirectly in melanogenesis have now been identified and cloned. The albino, brown, and slaty genes encode tyrosinase, tyrosinase-related protein-1 (TRP1) and tyrosinase-related protein-2 (TRP2), respectively. Tyrosinase catalyzes the hydroxylation of the amino acid tyrosine to dihydroxyphenylalanine (dopa; actually in the form of dopaquinone) and the oxidation of dopa to dopaquinone 15, 16. TRP1 and TRP2 have been shown to regulate eumelanogenesis catalytically at steps distal to tyrosinase. Thus, TRP1 functions as 5,6-dihydroxyindole-2-carboxylic acid oxidase [17], while TRP2 functions as dopachrome tautomerase [18]. Several other proteins have been shown to function in melanogenesis, including the silver-locus encoded protein [19]and the product of the pink-eyed dilution locus [20]. Expression of these proteins, however, is not required during pheomelanogenesis 21, 22, 23.

When the level of cysteine is higher than the level of dopaquinone being produced, CDs are produced exclusively, thus leading to pheomelanogenesis 11, 24. The switch between eu- and pheomelanogenesis appears to be regulated primarily at the level of tyrosinase: During pheomelanogenesis, the activity and expression of tyrosinase is lower than that found during eumelanogenesis 9, 10, 11, 25. The activity and expression of tyrosinase is regulated by many factors, including α-MSH acting via the melanocortin receptor-1, MC1R, and agouti signal protein, which antagonizes this action 5, 26.

We previously developed a microanalytical high-performance liquid chromatography (HPLC) method to quantitate pheomelanins that was based on the formation of the specific degradation product, aminohydroxyphenylalanine (AHP) [27]. We also introduced a spectrophotometric method that measures follicular melanin in hair and wool following their solubilization in hot Soluene-350 plus water 28, 29. With that spectrophotometric method, we have recently shown that (1) the absorbance at 500 nm (A₅₀₀ value) reflects the total combined amount of eu- and pheomelanins (total melanin, TM) and (2) the absorbance ratio between 650 and 500 nm (A₆₅₀/A₅₀₀ ratio) is correlated to the relative proportion of eumelanin to total melanin [30]. The AHP/TM ratio has also been shown to be useful in characterizing natural pheomelanins 28, 29, 30. Using these methods to examine the process of pheomelanogenesis starting from dopa or tyrosine and cysteine, we now report that pheomelanogenesis proceeds preferentially during the early phase of melanogenesis under conditions of low tyrosinase activity and high cysteine concentration.