Regulation of Protein Cross-Linking Reactions for Insect Cuticle Sclerotization

昆虫角质层硬化的蛋白质交联反应的调节

• 批准号: 9985959
• 负责人: Michael Kanost
• 金额: $ 37.5万
• 依托单位: Kansas State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2004-02-29
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9985959&HistoricalAwards=false
• 关键词: Regulation; Protein; Cross; Linking; Reactions

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.

昆虫角质层硬化或鞣制是发生在发育的每个阶段的重要过程，以硬化和稳定新分泌的角质外骨骼。 外骨骼具有许多功能，包括保护，运动，呼吸和通信，并且具有独特的机械和化学特性以优化每个功能。 结构聚合物，蛋白质和甲壳素，构成了角质层的主体。 这些聚合物中的一种或两种与醌鞣剂的化学相互作用在很大程度上决定了外骨骼的最终性质。我们希望研究的反应，这有助于稳定蛹角质层的烟草天蛾，天蛾，我们的模式昆虫。硬化的主要假设涉及形成碳-氮键，以将表皮蛋白中的组氨酸侧链交联至邻苯二酚衍生的邻醌和对苯醌甲基化物的环或侧链碳。 这项研究将解决昆虫如何使用结构蛋白，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天然存在的化学物质，这些化学物质交联结构蛋白。第二个目的是进一步表征鞣制酚氧化酶，漆酶，催化quinidazole形成在角质层硬化过程中。我们将研究漆酶基因的表达调控和漆酶在珠被中的定位。第三个目标是克隆硬化过程中交联的三种主要表皮蛋白的cDNA，将其表达为重组蛋白，并将其用于体外模型表皮重建实验，其中，与儿茶酚一起，重组酚氧化酶应产生通过儿茶酚交联的蛋白。这些研究的儿茶酚和蛋白质的氧化共轭在昆虫外骨骼将提供新的基础信息的天然交联剂和它们与角质层蛋白的共价相互作用。 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。对这一过程的特异性干扰将不利于昆虫的生长和发育，也可能有助于开发选择性害虫控制的新方法。