Regulation of 20-hydroxyecdysone on the larval pigmentation and the expression of melanin synthesis enzymes and yellow gene of the swallowtail butterfly, Papilio xuthus

The swallowtail butterfly, Papilio xuthus, changes its larval body pattern dramatically during the fourth ecdysis. Cuticular pigmentation occurs with precise timing just before ecdysis. We previously found that the cuticular pigmentation was regulated by three melanin synthesis genes, tyrosine hydroxylase (TH), dopa decarboxylase (DDC), and ebony. We discovered that yellow is strongly expressed in the presumptive black markings earlier than TH and DDC. Because the ecdysis is triggered by 20-hydroxyecdysone (20E), the effects of 20E on the pigmentation and expression of the melanin synthesis genes were examined. Here, we established a method for the topical application of 20E to molting specimens, so that 20E has only a partial effect, resulting in successful ecdysis. When we applied 20E during the mid-phase of the molting period, when the 20E titer is declining, cuticular pigmentation was completely inhibited. The cessation of hormonal treatments caused delayed pigmentation. yellow expression was promoted by a high titer of 20E, whereas the expression of TH, DDC, and ebony was suppressed, suggesting that a decline in the 20E concentration is necessary for the induction of the expression of the latter three genes. These results indicate that cuticular pigmentation is controlled by the exposure to 20E and its removal.     

BmPAH Catalyzes the Initial Melanin Biosynthetic Step in Bombyx mori

Pigmentation during insect development is a primal adaptive requirement. In the silkworm, melanin is the primary component of larval pigments. The rate limiting substrate in melanin synthesis is tyrosine, which is converted from phenylalanine by the rate-limiting enzyme phenylalanine hydroxylase (PAH). While the role of tyrosine, derived from phenylalanine, in the synthesis of fiber proteins has long been known, the role of PAH in melanin synthesis is still unknown in silkworm. To define the importance of PAH, we cloned the cDNA sequence of BmPAH and expressed its complete coding sequence using the Bac-to-Bac baculovirus expression system. Purified recombinant protein had high PAH activity, some tryptophan hydroxylase activity, but no tyrosine hydroxylase activity, which are typical properties of PAH in invertebrates. Because melanin synthesis is most robust during the embryonic stage and larval integument recoloring stage, we injected BmPAH dsRNA into silkworm eggs and observed that decreasing BmPAH mRNA reduced neonatal larval tyrosine and caused insect coloration to fail. In vitro cultures and injection of 4^th instar larval integuments with PAH inhibitor revealed that PAH activity was essential for larval marking coloration. These data show that BmPAH is necessary for melanin synthesis and we propose that conversion of phenylalanine to tyrosine by PAH is the first step in the melanin biosynthetic pathway in the silkworm.     

Regulation of pteridine-requiring enzymes by the cofactor tetrahydrobiopterin

Tetrahydrobiopterin (BH4) is synthesized from guanosine triphosphate (GTP) by GTP cyclohydrolase I (GCH), 6-pyruvoyltetrahydropterin synthase (PTS), and sepiapterin reductase (SPD). GCH is the rate-limiting enzyme. BH4 is a cofactor for three pteridine-requiring monooxygenases that hydroxylate aromatic L-amino acids, i.e., tyrosine hydroxylase (TH), tryptophan hydroxylase (TPH), and phenylalanine hydroxylase (PAH), as well as for nitric oxide synthase (NOS). The intracellular concentrations of BH4, which are mainly determined by GCH activity, may regulate the activity of TH (an enzyme-synthesizing catecholamines from tyrosine), TPH (an enzyme-synthesizing serotonin and melatonin from tryptophan), PAH (an enzyme required for complete degradation of phenylalanine to tyrosine, finally to CO2 + H2O), and also the activity of NOS (an enzyme forming NO from arginine), Dominantly inherited hereditary progressive dystonia (HPD), also termed DOPA-responsive dystonia (DRD) or Segawa's disease, is a dopamine deficiency in the nigrostriatal dopamine neurons, and is caused by mutations of one allele of the GCH gene. GCH activity and BH4 concentrations in HPD/DRD are estimated to be 2-20% of the normal value. By contrast, recessively inherited GCH deficiency is caused by mutations of both alleles of the GCH gene, and the GCH activity and BH4 concentrations are undetectable. The phenotypes of recessive GCH deficiency are severe and complex, such as hyperphenylalaninemia, muscle hypotonia, epilepsy, and fever episode, and may be caused by deficiencies of various neurotransmitters, including dopamine, norepinephrine, serotonin, and NO. The biosynthesis of dopamine, norepinephrine, epinephrine, serotonin, melatonin, and probably NO by individual pteridine-requiring enzymes may be differentially regulated by the intracellular concentration of BH4, which is mainly determined by GCH activity. Dopamine biosynthesis in different groups of dopamine neurons may be differentially regulated by TH activity, depending on intracellular BH4 concentrations and GCH activity. The nigrostriatal dopamine neurons may be most susceptible to a partial decrease in BH4, causing dopamine deficiency in the striatum and the HPD/DRD phenotype.     

Modeled ligand-protein complexes elucidate the origin of substrate specificity and provide insight into catalytic mechanisms of phenylalanine hydroxylase and tyrosine hydroxylase.

NMR spectroscopy and X-ray crystallography have provided important insight into structural features of phenylalanine hydroxylase (PAH) and tyrosine hydroxylase (TH). Nevertheless, significant problems such as the substrate specificity of PAH and the different susceptibility of TH to feedback inhibition by l-3,4-dihydroxyphenylalanine (l-DOPA) compared with dopamine (DA) remain unresolved. Based on the crystal structures 5pah for PAH and 2toh for TH (Protein Data Bank), we have used molecular docking to model the binding of 6(R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and the substrates phenylalanine and tyrosine to the catalytic domains of PAH and TH. The amino acid substrates were placed in positions common to both enzymes. The productive position of tyrosine in TH.BH4 was stabilized by a hydrogen bond with BH4. Despite favorable energy scores, tyrosine in a position trans to PAH residue His290 or TH residue His336 interferes with the access of the essential cofactor dioxygen to the catalytic center, thereby blocking the enzymatic reaction. DA and l-DOPA were directly coordinated to the active site iron via the hydroxyl residues of their catechol groups. Two alternative conformations, rotated 180 degrees around an imaginary iron-catecholamine axis, were found for DA and l-DOPA in PAH and for DA in TH. Electrostatic forces play a key role in hindering the bidentate binding of the immediate reaction product l-DOPA to TH, thereby saving the enzyme from direct feedback inhibition.     

Catecholamines and serotonin are differently regulated by tetrahydrobiopterin. A study from 6-pyruvoyltetrahydropterin synthase knockout mice

(6R)-L-erythro-5,6,7,8-Tetrahydrobiopterin (BH4) is an essential cofactor for tyrosine hydroxylase (TH), tryptophan hydroxylase, phenylalanine hydroxylase, and nitric-oxide synthase. These enzymes synthesize neurotransmitters, e.g. catecholamines, serotonin, and nitric oxide (NO). We established mice unable to synthesize BH4 by disruption of the 6-pyruvoyltetrahydropterin synthase gene, the encoded protein of which catalyzes the second step of BH4 biosynthesis. Homozygous mice were born at the almost expected Mendelian ratio, but died within 48 h after birth. In the brain of homozygous mutant neonates, levels of biopterin, catecholamines, and serotonin were extremely low. The number of TH molecules was highly dependent on the intracellular concentration of BH4 at nerve terminals. Alteration of the TH protein level by modulation of the BH4 content is a novel regulatory mechanism. Our data showing that catecholaminergic, serotonergic, and NO systems were differently affected by BH4 starvation suggest the possible involvement of BH4 synthesis in the etiology of monoamine-based neurological and neuropsychiatric disorders.     

Melanin-synthesis enzymes coregulate stage-specific larval cuticular markings in the swallowtail butterfly, <Emphasis Type="Italic">Papilio xuthus</Emphasis>

Like the adult wing, butterfly larvae are unique in their coloring. However, the molecular mechanisms underlying the formation of insect larval color patterns are largely unknown. The larva of the swallowtail butterfly Papilio xuthus changes its color pattern markedly during the 4th ecdysis. We investigated its cuticular color pattern, which is thought to be composed of melanin and related pigments derived from tyrosine. We cloned three enzymes involved in the melanin-synthesis pathway in P. xuthus: tyrosine hydroxylase (TH), dopa decarboxylase (DDC), and ebony. Whole-mount in situ hybridization showed that the expression of both TH and DDC is strongly correlated with the black markings. ebony is strongly expressed only in the reddish-brown area. The expression pattern of each enzyme coincides with the cuticular color pattern of the subsequent instar. We also investigated the uptake of melanin precursors into cultured integument. Inhibition of either TH or DDC activity prevents in vitro pigmentation completely. Addition of dopamine to integuments in the presence of TH inhibitor causes overall darkening without specific markings. From these results, specific larval cuticular color patterns are regulated by stage-specific colocalization of enzymes in epidermal cells rather than by the differential uptake of melanin precursors into individual epidermal cells. Epidermal cells expressing TH and DDC, but not ebony, produce the black cuticle, and epidermal cells expressing TH, DDC, and ebony produce the reddish-brown cuticle.     

The role of tetrahydrobiopterin and catecholamines in the developmental regulation of tyrosine hydroxylase level in the brain

Tyrosine hydroxylase (TH) is a rate‐limiting enzyme for dopamine synthesis and requires tetrahydrobiopterin (BH4) as an essential cofactor. BH4 deficiency leads to the loss of TH protein in the brain, although the underlying mechanism is poorly understood. To give insight into the role of BH4 in the developmental regulation of TH protein level, in this study, we investigated the effects of acute and subchronic administrations of BH4 or dopa on the TH protein content in BH4‐deficient mice lacking sepiapterin reductase. We found that BH4 administration persistently elevated the BH4 and dopamine levels in the brain and fully restored the loss of TH protein caused by the BH4 deficiency in infants. On the other hand, dopa administration less persistently increased the dopamine content and only partially but significantly restored the TH protein level in infant BH4‐deficient mice. We also found that the effects of BH4 or dopa administration on the TH protein content were attenuated in young adulthood. Our data demonstrate that BH4 and catecholamines are required for the post‐natal augmentation of TH protein in the brain, and suggest that BH4 availability in early post‐natal period is critical for the developmental regulation of TH protein level.     

Mechanism of oxygen activation by tyrosine hydroxylase

The mechanism by which the tetrahydropterin-requiring enzyme tyrosine hydroxylase (TH) activates dioxygen for substrate hydroxylation was explored. TH contains one ferrous iron per subunit and catalyzes the conversion of its tetrahydropterin cofactor to a 4a-carbinolamine concomitant with substrate hydroxylation. These results are in accord with shared mechanisms of oxygen activation by TH and the more commonly studied tetrahydropterin-dependent enzyme phenylalanine hydroxylase (PAH) and strongly suggest that a peroxytetrahydropterin is the hydroxylating species generated during TH turnover. In addition, TH can also utilize H2O2 as a cofactor for substrate hydroxylation, a result not previously established for PAH. A detailed mechanism for the reaction is proposed. While the overall pattern of tetrahydropterin-dependent oxygen activation by TH and PAH is similar, the H2O2-dependent hydroxylation performed by TH provides an indication that subtle differences in the Fe ligand field exist between the two enzymes. The mechanistic ramifications of these results are briefly discussed.     

Albino (al) is a tetrahydrobiopterin (BH4)-deficient mutant of the silkworm Bombyx mori

Albino (al) is a lethal mutant of Bombyx mori that exhibits a colourless cuticle after the first ecdysis and dies without feeding on mulberry. Previous studies have indicated that sclerotisation was insufficient because of defective phenylalanine and tyrosine metabolism in albino larvae. However, the genetic mechanism underlying the albino phenotype has not been determined. Dopamine plays a central role in insect cuticle colouration and sclerotisation. The pathway for dopamine biosynthesis from phenylalanine involves phenylalanine hydroxylase (PAH; EC 1.14.16.1) and tyrosine hydroxylase (TH; EC 1.14.16.2). Tetrahydrobiopterin (BH4) is an essential cofactor of aromatic amino acid hydroxylases, including PAH and TH. Thus, BH4 is indispensable for cuticle colouration and sclerotisation. Here we report on identifying mutations in the gene that encodes for the Bombyx homolog of 6-pyruvoyl-tetrahydropterin synthase (PTS) which is involved in the biosynthesis of BH4, in 2 strains with different al alleles. In strain a60 (al), a transposable element was inserted in exon 2 of BmPTS. In strain a61 (al²), an 11-bp deletion was identified in the exon 2 region of BmPTS. After oral administration of BH4 to the al² larvae, the survival rate was effectively increased and the larval integument was pigmented. These results indicated that BmPTS was responsible for the albino mutants of B. mori. We conclude that (i) a mutation in BmPTS leads to an insufficient supply of BH4 and results in defective dopamine biosynthesis and (ii) lack of dopamine results in cuticle colouration and sclerotisation failure. Lemon (lem) is a BH4-deficient mutant. It has been reported that de novo synthesis of zygotic BH4 was indispensable for viability of the embryo in eggs laid by lem (lem/lem^l) females. We found that lem/lem, al²/al² larvae produced by lem (lem/lem) females were viable during the first instar stage, suggesting that al²/al² embryo could synthesis BH4 by using maternally transmitted BmPTS.     

The AP-1 repressor, JDP2, is a bona fide substrate for the c-Jun N-terminal kinase

Phenylalanine hydroxylase (PAH) is activated by its substrate phenylalanine and inhibited by its cofactor tetrahydrobiopterin (BH(4)). The crystal structure of PAH revealed that the N-terminal sequence of the enzyme (residues 19-29) partially covered the enzyme active site, and suggested its involvement in regulation. We show that the protein lacking this N-terminal sequence does not require activation by phenylalanine, shows an altered structural response to phenylalanine, and is not inhibited by BH(4). Our data support the model where the N-terminal sequence of PAH acts as an intrasteric autoregulatory sequence, responsible for transmitting the effect of phenylalanine activation to the active site.     

Molecular Biology of Catecholamine-Related Enzymes in Relation to Parkinson's Disease

1. Catecholamine (dopamine, norepinephrine, and epinephrine) biosynthesis is regulated by tyrosine hydroxylase (TH). TH activity is regulated by the concentration of the cofactor tetrahydrobiopterin (BH4), whose level is regulated by GTP cyclohydrolase I (GCH) activity. Thus, GCH activity indirectly regulates TH activity and catecholamine levels.2. TH activity in the nigrostriatal dopaminergic neurons is most sensitive to the decrease in BH4.3. Mutations of GCH result in reductions in GCH activity, BH4, TH activity, and dopamine, causing either recessively inherited GCH deficiency or dominantly inherited hereditary progressive dystonia [HPD; Segawa's disease; also called dopa-responsive dystonia (DRD)].4. In juvenile parkinsonism and Parkinson's disease, which have dopamine deficiency in the basal ganglia as HPD/DRD, the GCH gene may be normal, and the molecular mechanism of the dopamine deficiency in the basal ganglia is different from that in HPD/DRD.     

A review of recent advances on the regulation of pigmentation in the human epidermis.

It has been recognised that the active transport of L-phenylalanine and its autocrine turnover to L-tyrosine via phenylalanine hydroxylase in the cytosol of epidermal melanocytes provides the majority of the L-tyrosine pool for melanogenesis. In this context, it has been shown that the cofactor 6(R)-L-erythro 5,6,7,8 tetrahydrobiopterin (6BH4) is produced de novo, recycled and regulated in both epidermal melanocytes and keratinocytes to control tyrosine hydroxylase, phenylalanine hydroxylase and tyrosinase activity. Inhibition of the enzymes by excessive 6BH4 levels is reversible with alpha-MSH by specific complex formation between 6BH4 and the hormone. This direct mechanism of alpha-MSH is supported by the presence of the entire POMC processing system in the melanosome indicating a receptor independent control of eumelanogenesis. Finally, the role of tyrosinase, TRP-1 and TRP2 is discussed in association with oxidative stress specifically related to hydrogen peroxide. These recent findings are based on detailed investigations of the depigmentation disorder vitiligo and Hermansky-Pudlák syndrome.     

Caterpillar color patterns are determined by a two‐phase melanin gene prepatterning process: new evidence from tan and laccase2

SUMMARY The larval color patterns in Lepidoptera exhibit splendid diversity, and identifying the genes responsible for pigment distribution is essential to understanding color‐pattern evolution. The swallowtail butterfly, Papilio xuthus, is a good candidate for analyzing marking‐associated genes because its body markings change dramatically at the final molt. Moreover, the silkworm Bombyx mori is most suitable for identification of lab‐generated color mutants because genome information and many color mutants are available. Here, we analyzed the expression pattern of 10 melanin‐related genes in P. xuthus, and analyzed whether these genes were responsible for Bombyx larval color mutants. We found that seven genes correlated strongly with the stage‐specific larval cuticular markings of P. xuthus, suggesting that, compared with Drosophila, more genes showed marking specificity in lepidopteran larvae. We newly found that the expression of both tan and laccase2 is strongly correlated with the larval black markings in both P. xuthus and B. mori. The results of F2 linkage analysis and mutant analysis strongly suggest that tan is the responsible gene for Bombyx larval color mutant rouge, and that tan is important in emphasizing black markings of lepidopteran larvae. Detailed comparison of temporal and spatial expression patterns showed that larval cuticular markings were regulated at two different phases. Marking‐specific expression of oxidizing enzymes preceded the marking‐specific expression of melanin synthesis enzymes at mRNA level, which is the reverse of the melanin synthesis step.     

Conformation of the substrate and pterin cofactor bound to human tryptophan hydroxylase. Important role of Phe313 in substrate specificity.

Tryptophan hydroxylase (TPH) carries out the 5-hydroxylation of L-Trp, which is the rate-limiting step in the synthesis of serotonin. We have prepared and characterized a stable N-terminally truncated form of human TPH that includes the catalytic domain (Delta90TPH). We have also determined the conformation and distances to the catalytic non-heme iron of both L-Trp and the tetrahydrobiopterin cofactor analogue L-erythro-7,8-dihydrobiopterin (BH2) bound to Delta90TPH by using 1H NMR spectroscopy. The bound conformers of the substrate and the pterin were then docked into the modeled three-dimensional structure of TPH. The resulting ternary TPH-BH2-L-Trp structure is very similar to that previously determined by the same methods for the complex of phenylalanine hydroxylase (PAH) with BH2 and L-Phe [Teigen, K., et al. (1999) J. Mol. Biol. 294, 807-823]. In the model, L-Trp binds to the enzyme through interactions with Arg257, Ser336, His272, Phe318, and Phe313, and the ring of BH2 interacts mainly with Phe241 and Glu273. The distances between the hydroxylation sites at C5 in L-Trp and C4a in the pterin, i.e., 6.1 +/- 0.4 A, and from each of these sites to the iron, i.e., 4.1 +/- 0.3 and 4.4 +/- 0.3 A, respectively, are also in agreement with the formation of a transient iron-4a-peroxytetrahydropterin in the reaction, as proposed for the other hydroxylases. The different conformation of the dihydroxypropyl chain of BH2 in PAH and TPH seems to be related to the presence of nonconserved residues, i.e., Tyr235 and Pro238 in TPH, at the cofactor binding site. Moreover, Phe313, which seems to interact with the substrate through ring stacking, corresponds to a Trp residue in both tyrosine hydroxylase and PAH (Trp326) and appears to be an important residue for influencing the substrate specificity in this family of enzymes. We show that the W326F mutation in PAH increases the relative preference for L-Trp as the substrate, while the F313W mutation in TPH increases the preference for L-Phe, possibly by a conserved active site volume effect.     

家蚕酪氨酸羟化酶基因BmTh的表达及功能

酪氨酸羟化酶作为儿茶酚胺合成的限速酶, 广泛存在于昆虫、哺乳动物和人类中, 是其新陈代谢不可缺少的酶类。在其他昆虫中, 酪氨酸羟化酶参与了黑色素的合成, 并在昆虫外骨骼的硬化过程中发挥关键作用。为了研究家蚕Bombyx mori酪氨酸羟化酶基因的生理生化功能, 本文对其基因结构、表达特征及功能进行了研究。基于家蚕基因组和基因芯片数据的生物信息学分析表明, BmTh位于家蚕1号染色体上, 含有8个外显子, 编码561个氨基酸。基因芯片数据显示在家蚕5龄第3天的头部和体壁组织中的表达量较高, RT-PCR验证结果与此一致。利用石蜡组织切片材料和RNA探针对BmTh进行表达定位, 原位杂交结果显示在家蚕头部边缘和体壁上有明显的杂交信号。在幼虫发育至熟蚕时注射酪氨酸羟化酶抑制剂3-indole-L-tyrosine （3-IT）, 20 mmol/L的浓度对幼虫几乎没有影响, 50 mmol/L的浓度导致幼虫变态不完全和化蛹困难, 100 mmol/L的浓度使幼虫致死且体色变黑。结果提示, BmTh对家蚕变态发育起重要作用, 是家蚕正常发育不可缺少的关键基因。     

Melanin pigmentation gives rise to black spots on the wings of the silkworm Bombyx mori

Several mutants of the silkworm Bombyx mori show body color variation at the larval and adult stages. The Wild wing spot (Ws) mutant exhibits a phenotype in which the moth has a spot on the apex of the forewing. In this study, we investigated this trait to elucidate the molecular mechanism underlying the color pattern. Microscopy of the black spot of Ws mutants showed that the pigment emerges in the scales of the wing, and accumulation of the pigment becomes strong just before eclosion. We next examined the relationship between the black spot of the Ws mutant and melanin. The spectrophotometry using alkaline extracts from the black spot in the wing showed the highest absorption intensity at 405 nm, which is the absorbance wavelength of melanin. Moreover, inhibition assays for enzymes implicated in melanin synthesis using 3-iodo-l-tyrosine (a tyrosine hydroxylase inhibitor) and L-α-methyl-DOPA (a dopa decarboxylase inhibitor) revealed that treatment with each inhibitor disrupted the pigmentation of the wing of the Ws mutant. On the basis of these results, we analyzed the expression pattern of five genes involved in melanin formation, and found that the expression levels of yellow and laccase2 were increased just before pigmentation, whereas those of DDC, tan, and TH were increased when the apex of the wing turned black. These results showed that melanin pigmentation gives rise to the black spot on the wing.     

Effects of melanin on tyrosine hydroxylase and phenylalanine hydroxylase.

Melanin inhibited rat liver phenylalanine hydroxylase, but activated tyrosine hydroxylase from rat brain (caudate nucleus), rat adrenal glands, and bovine adrenal medulla. Activation of tyrosine hydroxylase by melanin was demonstrated with the extensively dialyzed enzyme and in suboptimal concentrations of the substrate (tyrosine) and the cofactor (6-methyltetrahydropterin). Tyrosine hydroxylase from rat brain was activated by melanin more markedly than that from rat adrenal glands. Purified and extensively dialyzed bovine adrenal tyrosine hydroxylase had two Km values with 6-methyltetrahydropterin, depending upon its concentrations, but the melanin-activated tyrosine hydroxylase had a single Km value and showed the classical Michaelis-Menten kinetics.