1、彭章川 112012331002074 家蚕基因组生物学国家重点实验室昆虫的复杂性表皮色素沉着Manickam Sugumarane-mail: manickam.sugumaranumb.edu保护软体动物的外骨骼昆虫的角质层在所有的昆虫的生物学和生理学中起着至关重要的作用。角质层不仅符合作为保护层用来抵抗寄生虫和致病菌的入侵，同时还具有支持身体各部位，防止动物脱水，允许气态交流，并为废物处理提供一个场所，以及多种其他功能。昆虫体壁在颜色和硬度上表现出了巨大的变化，不仅存在不同种类的昆虫中，而且还存在同种昆虫不同生命阶段中。大量的研究已经从生物化学，分子生物学，遗传学和免疫学对昆虫和其他节肢
2、动物的色素生成展开了研究。然而，我们对表皮色素的结构以及相关的分子遗传方面所知甚少。昆虫作为动物世界中群体最大的动物，拥有有各种各样的颜色和颜色模式，这些颜色和颜色模式是由一套复杂的酶，传播途径，控制元件和遗传特征调节的。昆虫表皮的颜色从很浅到乌黑，在高等动物，体壁颜色很大程度上是因为黑色素的沉着。在脊椎动物尤其是在人类的情况下，不仅黑色素基因多态性的结构基础已经清楚明确，而且在这些差异下的分子遗传学机制的不同已经在一定程度上被清楚地认定。褐黑素和黑色素不同含量的组合和不同的分布导致了哺乳动物的皮肤颜色的一般性变化(Ito and Wakamatsu, 2008; Figure 1 boxed
3、)。化学、分子生物学、病理学与这些色素生成途径的联系在很大程度上也很清楚了。对于昆虫而言（如黑腹果蝇）尤其是机体内的色素积淀和表皮硬化的发育遗传学已经取得了一些进展。科学家开始为不同酶和基因对表皮色素沉着和硬化的作用提供有价值的意见（Sugumaran， 1998 年，2002 年 Wittkopp 和 Beldade，2009 年） 。昆虫表皮色素沉着的图示如图 1 所示，除了可以从多巴和多巴胺生成褐黑素和真黑素外，昆虫也通过多巴胺的衍生物产生额外的色斑。乙酰化的多巴胺生成 N-乙酰多巴胺（ NADA） ，这在昆虫表皮硬化过程中起着总要重要作用。酚氧化酶（PO）氧化 N-乙酰多巴胺（NADA
4、）生成 N-乙酰多巴胺醌（NADA 醌） ，NADA 醌醌异构酶作用于 NADA 醌生成甲基化 N-乙酰多巴胺醌，随后被另一个异构酶作用而生成 1,2 - 脱氢 NADA。该化合物进一步被酚氧化酶（PO）氧化为甲基化亚胺酰胺醌。表皮蛋白质和几丁质聚合物的醌、甲基醌和甲基化亚胺酰胺醌的反应为表皮硬化提供了足够的表皮硬化必须的加和和胶链。硬化（骨骼化）途径和所有动物的黑化作用途径概述非常类似（Sugumaran，1998 年，2002 年;图 2A，B） 。昆虫表皮骨骼化途径也为表皮提供了不同的着色。在一般情况下，NADA 骨质似乎是略带色或几乎无色。多巴胺与 -丙氨酸结合生成昆虫表皮骨骼化的又一
5、个重要前体物质，N- -丙氨酰多巴胺 (NBAD)，它可以由同一组的反应转换为 NADA。结果 NBAD 骨质产生黄色至棕色角质层，在昆虫的翅膀中，可能会有其他的色素积淀和色素出现，比如：蝶啶、凤蝶色素（NBAD和犬尿氨酸的氧化缩合物） 。犬尿氨酸也能生成昆虫的眼睛色素-眼色素。这种复杂的色素积淀产生昆虫表皮如此鲜艳多样的颜色。图一：昆虫中的一般色素产生反应。虚线所覆盖的区域是脊椎动物所使用的主要途径。整个图画接示昆虫使用更复杂的途径。类胡萝卜素和其它色素的生成途径是不包括在该图中的。箭头并不总是表示一个单一的反应。PO：酚氧化酶，其中包括酪氨酸酶和漆酶。DDC：多巴脱羧酶； ADC：天门冬氨
6、酸脱羧酶；NADA ：N-乙酰多巴胺；NBAD：N- 丙氨酰多巴胺。图 2：（A）简单的黑色素生成机制。酪氨酸酶通过羟基化酪氨酸生成多巴和氧化多巴生成多巴醌启动黑色素的生成。 （半胱氨酸多加上多巴醌进一步氧化聚合半胱氨酰多巴而生成黄红色的褐黑素。 ）多巴醌容易在分子内的被环化和氧化生成多巴色素。多巴色素互变异构酶（Tyrp2）通过瞬态醌甲基化物中间体把多巴色素转换成向 5,6 - 二羟基吲哚-2 - 羧酸（DHICA） 。DHICA 被 DHICA 氧化酶氧化生成褐色至黑色的真黑素色素，5,6 - 二羟基氧化聚合物是通过脱羧转变为多巴色素生成的 5,6 - 二羟基氧化聚合的。反应 1-4 所示
7、的硬化的平行反应在图 2B 中。 （B ）昆虫表皮硬化统一的机制。 N-乙酰多巴胺醌（酰基可以是乙酰基= NADA 或 b-丙氨酰= NBAD）被酚氧化酶氧化成其相应的醌，可以参与在醌鞣反应。苯醌异构酶（B）转换成醌醌甲基化物，并为他们提供醌甲基化物鞣革。喹啉醌的甲基化物也由醌甲基化物的异构酶（C）异构化为 1,2 - 脱氢-N-acyldopamines 。脱氢化合物的氧化产生的醌甲基化物亚胺酰胺侧链形成加合物和交叉链接（D = 非酶反应）反应。反应 1-4 类似的指定反应 1-4 发生在 eumelanization（图 2A） 。最近，Arakane 等（2009 年）描述了赤拟谷盗中天
8、门冬氨酸脱羧酶（ADC）和多巴脱羧酶（DDC）基因的特征，他们对这两个基因进行 RNAi 实验，以评估其在表皮色素的发展模式中的作用。注射 ADC 双链 RNA 引起多巴胺的沉积， NBAD 的不足导致成年甲虫表皮出现异常的暗色，黑色着色突变甲虫也表现出相同的特征。注射 -丙氨酸（DC 的反应的产物）改变了这种效果。显然，不能通过天门冬氨酸脱羧产生 -丙氨酸会造成 N-乙酰多巴胺（NADA）的减少，因此在表皮硬化之前通过多巴脱羧生成多巴胺是深色突变体有黑色色素产生的原因，ADC 双链 RNA 干涉实验改变了这些突变个体的黑色状态。在 DDC双链 RNA 注入幼虫的情况下会产生致死表型的蛹，而经
9、过 dsDDC 管理的蛹没有表现出这样的致死性但表现出异常深褐色成年体色。在这些甲虫，累积异常高浓度的多巴由于多巴脱羧酶的失去功能。dsRNA 处理表皮也表现出改变的机械性能和减少交联，从而暗示NBAD 在硬化过程中发挥关键作用。虽然这样的观测先前已经被遗传和抑制研究，这是第一次用 dsRNA 来确定 ADC 和 DDC 在表皮的硬化和着色方面的作用。但研究同时也提出了更多的问题。为什么是多余的多巴胺不转换为 NADA 作为骨质前体替代资源呢？为什么在多巴胺积累动物和多巴积累动物之间会有很大的颜色差异？在昆虫中的色素的问题也进一步证明一个事实，即有多个酚氧化酶提供多种功能，而不是说人类只有一个
10、酪氨酸酶。此外，昆虫具有独特的多巴色素异构酶，它能够被归类为一个异构酶，因为它主要将多巴色素转换成醌甲基化物的同分异构体，但是会引起所得的醌甲基化物的脱羧生成 5,6 - 二羟基吲哚（Sugumaran ，2002 年） 。这与哺乳动物的多巴色素异构酶形成鲜明对比（通常称为多巴色素互变异构酶） ，该酶通常进行多巴色素转向 5,6 - 二羟基吲哚-2 - 羧酸的异构反应。由于只有最近我们已经开始使用先进的分子生物学工具提出这些问题，答案可能会会在不久的将来出现。Complexities of cuticular pigmentation in insectsManickam Sugumarane
11、-mail: manickam.sugumaranumb.eduInsect cuticle, the exoskeleton that protects the soft-bodied animals, plays a crucial role in the biology and physiology of all insects. The cuticle serves not only as the protective covering against invading parasites and pathogens, but also supports the body parts,
12、prevents dehydration of the animals,allows gaseous exchanges and provides a venue for waste disposal, and a variety of other functions. Insect cuticle exhibits tremendous variations in its color and hardness, not only among different insects, but also among different life stages of the same insect.
13、Extensive studies have been carried out on the biochemistry, molecular biology, genetics, and immunology of pigment production in insects and other arthropods. Yet,we know very little about structural aspects and the associated molecular genetic of the cuticular pigments.Being the largest group of a
14、nimals in the animal kingdom, insects come in a variety of colors and color patterns that are governed by a complex set of enzymes, pathways, control elements,and genetics. The color of insect cuticle varies from jet black to completely color-less. In higher animals, the body color is vastly due to
15、melanin pigment. In the case of vertebrate animals especially humans, not only the structural basis of melanin polymorphism has been clearly established, but even the molecular genetics mechanisms underlying these differences have been pinpointed clearly to certain extent. A combination of different
16、 amounts of pheomelanin and eumelanin and their differential distributions gives rise to the general variations in the skin color of mammals (Ito and Wakamatsu, 2008; Figure 1 boxed). The chemistry, molecular biology, and pathology associated with these pigment production pathways are also reasonabl
17、y well established. In the case of insects, some progress has been made on the developmental genetics of pigmentation and cuticular hardening especially in organisms such as Drosophila melanogaster. They are beginning to provide valuable insights on the role of different enzymes and genes on cuticul
18、ar pigmentation and hardening (Sugumaran, 1998, 2002; Wittkopp and Beldade, 2009). The general picture for insect cuticular pigmentation is shown in Figure 1. Apart from the pheomelanin and eumelanin that can be produced from dopa and dopamine, insects also produce additional pigmentation through de
19、rivatives of dopamine. Acetylation of dopamine generates N-acetyldopamine(NADA), which is a major cuticular hardening agent in insects. Phenoloxidase(PO) oxidizes NADA producing NADA quinone, NADA quinone is isomerized by quinone isomerase producing NADA quinone methide, which is subsequently conver
20、ted by another isomerase to 1,2-dehydro NADA. This compound is further oxidized by phenoloxidases to quinone methide imine amide. The reactions of quinones, quinone methides, and quinone methide imine amides with cuticular proteins and chitin polymer provide sufficient adducts and crosslinks necessa
21、ry for cuticular hardening. The hardening (= sclerotization) pathway is very similar to eumelanization pathway outlined in all animals (Sugumaran, 1998, 2002; Figure 2A, B). Sclerotization pathway also provides different coloration in insect cuticle. In general, NADA sclerotin seems to be slightly c
22、olored or nearly colorless. Conjugation of dopamine with b-alanine produces yet another major insect cuticular sclerotization precursor, N-b-alanyldopamine (NBAD), which can be converted by the same set of reactions outlined for NADA. The resultant NBAD sclerotin produces yellow to brown colored cut
23、icle. In insect wings, additional pigmentation such as pteridines, papiliochromes, (oxidative condensation products of NBAD and kynurenine) and other pigments may occur. Kynurenine is also converted to the insect eye pigment, ommochromes. This complexity of pigmentation gives rise to such vivid and
24、diverse color to insect cuticles.Figure 1. General pigment production reactions in insects. The area covered by broken line is the major pathway used by vertebrate animals. Insects seem to use more complex pathway indicted in the entire figure. Carotenoid and other pigment production pathways are no
25、t included in this figure. Arrows do not always indicate a single reaction. PO,phenoloxidase which include both tyrosinase and laccase; DDC, dopa decarboxylase; ADC, Aspartate decarboxylase; NADA, N-acetyldopamine;NBAD, N-b-alanyldopamine). Figure 2. (a) A simplified mechanism for eumelanogenesis. T
26、yrosinase initiates eumelanogenesis by hydroxylating tyrosine to dopa and oxidizing the resultant dopa to dopaquinone. (Cysteine addition to dopaquinone and further oxidative polymerization of cysteinyldopas leads to pheomelanin pigments that are yellow to red in color). Dopaquinone undergoes facile
27、 intramolecular cyclization and oxidation to generate dopachrome. Dopachrome tautomerase (Tyrp 2) converts dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) via a transient quinone methide intermediate. Oxidation of DHICA by DHICA oxidase produces yellow to brown eumelanins and oxidation p
28、olymerization of 5,6-dihydroxyindole produced by the decarboxylative transformation of dopachrome produces brown to black eumelanin pigments. Reactions 1-4 are parallel reactions in sclerotization shown in Figure 2B. (b) Unified mechanism for sclerotization insect cuticle. N-Acyldopamine quinones (a
29、cyl group could be acetyl = NADA or b-alanyl = NBAD) are oxidized by phenoloxidases (A) to their corresponding quinones that can participate in quinone tanning reactions. Quinone isomerase (B) converts the quinones to quinone methides and provides them for quinone methide tanning. Quinone methides a
30、re also isomerized to 1,2-dehydro-N-acyldopamines by quinone methide isomerase (C). Oxidation of the dehydro compounds yields the quinone methide imine amides that react with their side chain forming adducts and cross links(D = nonenzymatic reactions). Reactions 14 are similar to the designated reac
31、tions 14 occurring in eumelanization (Figure 2A).Recently, Arakane et al. (2009) characterized the aspartate decarboxylase(ADC) and dopa decarboxylase (DDC) genes from the red flour beetle, Tribolium castaneum and performed RNAi experiments to assess their role in the developmental pattern of cuticu
32、lar pigments. Injection of ADC dsRNA caused accumulation of dopamine, inadequate production of NBAD and abnormal dark coloration of adult cuticle in the beetles. Black colored mutant beetles also exhibited the same properties. Injection of -alanine, the product of ADC reaction, reversed these effect
33、s. Obviously,inability to produce b-alanine (by decarboxylation of aspartate) results in reduced NBAD production and hence dopamine produced by dopa decarboxylation prior to cuticular sclerotization is now diverted to darkly colored dopamine melanin pigments in the dark body colored mutants and ADC
34、dsRNA treated animals. In the case of DDC dsRNA injected larvae, a lethal pupal phenotype was observed, while dsDDC administered pupa exhibited no such mortality but showed only abnormally dark brown adult body color. In these beetles,abnormally high concentrations of dopa accumulate due to inabilit
35、y of dopa decarboxylase function. The dsRNA treated cuticle also exhibited altered mechanical properties and reduced crosslinking thereby indicating a crucial role for NBAD in the hardening process.Although such observations have been previously made by genetic and inhibition studies, this is the fi
36、rst time dsRNA has been used to confirm the roles of ADC and DDC in cuticular hardening and coloration. But the study also raises additional questions. Why is excess dopamine not converted to NADA and serve as an alternate source of sclerotin precursor? Why is there such a large color difference bet
37、ween dopamine accumulated animals and dopa accumulated animals? The pigment problem in insects is also compounded by the fact that there are multiple phenoloxidases serving multitude of functions as opposed to say humans where there is only one tyrosinase. Moreover, insects possess a unique dopachro
38、me isomerase, which even though it can be classified as an isomerase because it primarily converts dopachrome to an isomeric quinone methide, nonetheless causes decarboxylation of the resultant quinone methide generating 5,6-dihydroxyindole (Sugumaran, 2002). This is in sharp contrast to mammalian d
39、opachrome isomerase (usually called dopachrome tautomerase), which typically performs isomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid. Since only recently we have started using advanced molecular biology tools to ask such questions, it is likely an answer may come in the near fu
40、ture.ReferencesIto, S., and Wakamatsu, K. (2008). Chemistry of mixed melanogenesis: pivotal roles of dopaquinone. Photochem. Photobiol. 84, 582592.Sugumaran, M. (1998). Unified mechanism for sclerotization of insect cuticle.Adv. Insect Physiol. 27, 229334.Sugumaran, M. (2002). Comparative biochemist
41、ry of eumelanogenesis and the protective roles of phenoloxidase and melanin in insects. Pigment Cell Res. 15, 29.Wittkopp, P.J., and Beldade, P. (2009).Development and evolution of insect pigmentation: genetic mechanism and the potential consequences of pleiotropy. Seminars in Cell & Develop. Biol. 20, 6571
