Protein cross-linking Reactions for Insect Cuticle Sclerotization

昆虫角质层硬化的蛋白质交联反应

• 批准号: 0236039
• 负责人: Michael Kanost
• 金额: $ 73.25万
• 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: 2003
• 资助国家: 美国
• 起止时间: 2003-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0236039&HistoricalAwards=false
• 关键词: Protein; cross; linking; Reactions; Insect

This project will address how insects use structural proteins, catechols, oxidative enzymes, and chitin to stabilize their exoskeletons. The exoskeleton is primarily composed of two structural polymers, protein and chitin. The cuticle proteins are cross-linked by quinones derived from catechols by a process called sclerotization, a vital metabolic step that occurs during each stage of insect development. This interdisciplinary project will investigate the interactions of quinonoid metabolites of catecholic precursors with cuticular proteins from the tobacco hornworm, Manduca sexta. The research will test a hypothesis that sclerotization involves the formation of carbon-nitrogen cross-links between the nucleophilic imidazole nitrogens of histidine and the ring or side-chain carbons of o-quinones and p-quinone methides derived from catechols. A second hypothesis to be tested is that differences in hardness and flexibility from distinct regions of an exoskeleton are due to the occurrence of different sets of proteins and cross-linking agents expressed by the underlying epidermis. The first objective is to identify and characterize proteins expressed in cuticles with different physical properties. The cuticle of the M. sexta larval abdomen is soft and flexible, whereas the pupal abdomen is rigid and the larval head capsule quite hard. cDNAs obtained for proteins that are expressed in pupal abdominal cuticles, larval abdominal cuticle, and head capsule will be used to study the pattern of expression of the corresponding genes with regard to development and anatomy. The second objective is to investigate the structure and properties of two M. sexta laccases. Laccases oxidize catechols to quinones or quinone methides. Although laccases have been proposed to be important in insect cuticle sclerotization, they have received little study. cDNAs for two M. sexta laccases have been isolated, and their expression patterns characterized. The insect laccases are composed of a catalytic domain similar to those of fungal and plant laccases, as well as a unique amino-terminal domain not present in other known proteins. M. sexta laccases 1 and 2 will be expressed as recombinant proteins for characterization of their enzymatic properties, investigation of the function of the amino-terminal and catalytic domains, and for use in model sclerotization reactions described in objective 3. The third objective is to investigate biochemical and biophysical properties of cuticle using model sclerotization reactions. Selected proteins hypothesized to be responsible for formation of rigid or flexible cuticle will be expressed as recombinant proteins and used in model sclerotization reactions including a catechol, a phenoloxidase, and in the presence or absence of chitin or a chitin derivative. Protein-protein cross-links, protein-carbohydrate cross-links, and the effects of different catechols and cuticle proteins on the physical properties of cross-linked protein hydrogels and chitin-protein films produced in vitro will be investigated and compared with cuticles from different anatomical regions of M. sexta. Site directed mutagenesis experiments will test a hypothesis that histidine residues are involved in protein cross-linking during sclerotization. The exoskeleton of insects is composed of cuticle proteins and a polysaccharide called chitin. Each time an insect molts, these components of the exoskeleton are synthesized and then they are chemically cross-linked in a process called sclerotization. Sclerotization results in a light-weight material with unusual strength. This project investigates the chemistry of sclerotization, which is not yet well understood. Model sclerotization reactions will be carried out to determine how the cuticle proteins are linked to each other and to chitin. The biochemistry of insect cuticle sclerotization may be a useful target for design of novel methods for controlling insect pests of agriculture and insects that are vectors of diseases. New types of cross-linked biopolymers, based on the chemistry of cuticle sclerotization, may have potential uses in medicine and industry.

该项目将研究昆虫如何利用结构蛋白、儿茶酚、氧化酶和几丁质来稳定其外骨骼。外骨骼主要由两种结构聚合物组成：蛋白质和几丁质。角质层蛋白通过一种称为硬化的过程与儿茶酚衍生的醌交联，这是昆虫发育每个阶段发生的重要代谢步骤。这个跨学科项目将研究儿茶酚前体的醌类代谢物与烟草天蛾 Manduca sexta 的角质层蛋白的相互作用。该研究将检验一个假设，即硬化作用涉及组氨酸的亲核咪唑氮与邻苯二酚和对醌甲基化物的环或侧链碳之间形成碳氮交联，所述邻醌和对醌甲基化物源自儿茶酚。要测试的第二个假设是，外骨骼不同区域的硬度和柔韧性差异是由于底层表皮表达的不同组蛋白质和交联剂的出现造成的。 第一个目标是识别和表征具有不同物理特性的角质层中表达的蛋白质。 M. sexta 幼虫腹部的角质层柔软而有弹性，而蛹腹部坚硬，幼虫头囊相当坚硬。获得的在蛹腹部角质层、幼虫腹部角质层和头囊中表达的蛋白质的cDNA将用于研究相应基因在发育和解剖学方面的表达模式。第二个目标是研究两种 M. sexta 漆酶的结构和性质。漆酶将儿茶酚氧化成醌或醌甲基化物。 尽管漆酶被认为在昆虫角质层硬化中很重要，但对其的研究却很少。两种 M. sexta 漆酶的 cDNA 已被分离出来，并表征了它们的表达模式。昆虫漆酶由类似于真菌和植物漆酶的催化结构域以及其他已知蛋白质中不存在的独特氨基末端结构域组成。 M. sexta 漆酶 1 和 2 将表达为重组蛋白，用于表征其酶特性、研究氨基末端和催化结构域的功能，并用于目标 3 中描述的模型硬化反应。第三个目标是使用模型硬化反应研究角质层的生化和生物物理特性。假设负责形成刚性或柔性角质层的选定蛋白质将被表达为重组蛋白，并用于模型硬化反应，包括儿茶酚、酚氧化酶，并且在存在或不存在几丁质或几丁质衍生物的情况下。将研究蛋白质-蛋白质交联、蛋白质-碳水化合物交联以及不同儿茶酚和角质层蛋白质对体外产生的交联蛋白质水凝胶和几丁质蛋白质膜物理性质的影响，并与来自 M. sexta 不同解剖区域的角质层进行比较。 定点诱变实验将检验组氨酸残基参与硬化过程中蛋白质交联的假设。昆虫的外骨骼由角质层蛋白和一种称为几丁质的多糖组成。 昆虫每次蜕皮时，都会合成外骨骼的这些成分，然后在称为硬化的过程中进行化学交联。硬化作用产生具有异常强度的轻质材料。该项目研究了硬化化学，目前尚不清楚。 将进行模型硬化反应以确定角质层蛋白如何彼此连接以及与几丁质连接。 昆虫角质层硬化的生物化学可能是设计控制农业害虫和作为疾病媒介的昆虫的新方法的有用目标。 基于角质层硬化化学的新型交联生物聚合物可能在医学和工业中具有潜在用途。