Regulation of Protein Cross-Linking Reactions for Insect Cuticle Sclerotization

昆虫角质层硬化或鞣制是发生在每个发育阶段的重要过程，以硬化和稳定新分泌的角质层外骨骼。外骨骼具有多种功能，包括保护、运动、呼吸和交流，并具有独特的机械和化学特性来优化每种功能。结构聚合物、蛋白质和几丁质构成了角质层的主体。这些聚合物中的一种或两种与醌类鞣制剂的化学相互作用在很大程度上决定了外骨骼的最终性能。我们希望研究有助于稳定模式昆虫烟草角虫（Manduca sexta）蛹角质层的反应。硬化的主要假设包括形成碳-氮键，将表皮蛋白中的组氨酸侧链与邻醌和对醌衍生物的环或侧链碳交联。这项研究将探讨昆虫如何利用结构蛋白、儿茶酚和氧化酶来稳定它们的外骨骼。该项目的第一个目标是直接识别角质层中自然存在的化学物质，这些化学物质与结构蛋白交联。第二个目标是进一步表征鞣制酚氧化酶，漆酶，它在硬化过程中催化角质层中的类醌形成。我们将研究漆酶基因的表达调控和漆酶在被膜中的定位。第三个目标是克隆在硬化过程中交联的三种主要角质层蛋白的cdna，将其表达为重组蛋白，并将其用于体外模型角质层重建实验，其中，重组酚氧化酶应与儿茶酚一起产生通过儿茶酚交联的蛋白质。这些对昆虫外骨骼中儿茶酚和蛋白质氧化偶联的研究将为了解天然交联剂及其与表皮蛋白的共价相互作用提供新的基础信息。了解这种代谢生物化学如何通过蛋白质交联促进角质层硬化对昆虫生物学至关重要。对这一过程的特殊干扰将不利于昆虫的生长和发育，也可能促进选择性虫害防治新方法的发展。

Insect cuticle sclerotization or tanning is a vital process that occurs during each stage of development to harden and stabilize the newly secreted cuticular exoskeleton. The exoskeleton serves many functions including protection, locomotion, respiration and communication, and has unique mechanical and chemical properties to optimize each function. The structural polymers, protein and chitin, make up the bulk of the cuticle. Chemical interactions of one or both of these polymers with quinonoid tanning agents are largely responsible for the final properties of the exoskeleton. We wish to investigate the reactions which help to stabilize the pupal cuticle of the tobacco hornworm, Manduca sexta, our model insect. The main hypothesis for sclerotization involves the formation of carbon-nitrogen bonds to cross-link histidine side chains in cuticular proteins to the ring or side-chain carbons of o-quinones and p-quinone methides derived from catechols. This research will address how insects use structural proteins, catechols, and oxidative enzymes to stabilize their exoskeletons. The first objective of this project is to identify directly the naturally occurring chemicals in cuticle, which crosslink structural proteins. The second objective is to further characterize a tanning phenoloxidase, laccase, which catalyzes quinonoid formation in the cuticle during sclerotization. We will investigate the regulation of expression of the laccase gene and the localization of laccase in the integument. The third objective will be to clone the cDNAs for three of the major cuticular proteins that become cross-linked during sclerotization, express these as recombinant proteins, and use them for in vitro model cuticle reconstitution experiments, where, together with the catechols, the recombinant phenoloxidases should generate proteins cross-linked via catechols. These studies of the oxidative conjugation of catechols and proteins in the insect exoskeleton will provide new fundamental information about the natural cross-linking agents and their covalent interactions with cuticular proteins. Understanding how this metabolic biochemistry facilitates cuticle sclerotization via protein cross-linking is of vital importance to insect biology. Specific interference with this process would be detrimental to insect growth and development, and also may facilitate the development of new approaches to selective insect pest control.