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Biochemical analyses for the sclerotization of in sect cuticle

科内角质层硬化的生化分析

基本信息

批准号：
14540632
负责人：
IZUMI Susumu
金额：
$ 2.3万
依托单位：
Tokyo Metropolitan University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-14540632/
关键词：
insect cuticle sclerotization phenoloxidase laccase silkworm cDNA expression 構造解析表皮細胞精製

项目摘要

Insect cuticle is an extracellular matrix composed of proteins, chitin and lipids, and functions as an exoskeleton. Its stiffness is important properties for the function of cuticle. It is thought that the cross-linking of cuticular components after the ecdysis brings the stiffness of the cuticle. This hardening process is called quinone tanning, which is catalyzed by laccase-type phenoloxidase (laccase). We purified activated laccase from pupal cuticles of silkworm, Bombyx mori, just after larval-pupal ecdysis. After digestion of the cuticle with trypsin, solubilized proteins were fractionated by a combination of column chromatography. Finally, laccase was eluted from Mono Q column as a single peak on FPLC. Molecular weight of the typsin-activated laccase was estimated about 70k by SDS-PAGE. Western bound with other cuticle components. We could reproduce quinone tanning using isolated pupal cuticle proteins and dopamin in vitro. The result proved that laccase plays an important role in cross-linking of cuticular components. The amino acid sequences of some peptides prepared from purified laccase indicated high similarity to Manduca laccase like protein. Further, a database search showed that the nucleotide sequence of cDNA for Manduca laccase like protein is similar to a B.mori EST lone, ce2359. Nucleotide sequence of the clone encodes a protein of 764 amino acid residues. The primary structure deduced from the nucleotide sequence the partial amino acid sequences of B.mori laccase. Northen blot analysis revealed that m RNA begins to appear in epidermal cells during larval-pupal molting.

昆虫角质层是由蛋白质、几丁质和脂类组成的细胞外基质，具有外骨骼的功能。它的硬度是角质层功能的重要特性。据认为，角质层组分在蜕皮后的交联带来了角质层的硬度。这种硬化过程被称为醌鞣，它是由漆酶型酚氧化酶（漆酶）催化的。从家蚕幼虫蜕皮后的蛹壳中分离纯化了活性漆酶。用胰蛋白酶消化角质层后，通过柱层析的组合分级分离溶解的蛋白质。最后，漆酶从Mono Q柱上洗脱，在FPLC上为单峰。SDS-PAGE测得酶的分子量约为70 k。与其他角质层成分结合。我们可以复制醌鞣分离蛹角质层蛋白质和多巴胺在体外。结果表明，漆酶对表皮组分的交联起着重要作用。部分漆酶多肽的氨基酸序列与Manduca漆酶样蛋白的氨基酸序列高度相似。此外，数据库检索显示，Manduca漆酶样蛋白的cDNA的核苷酸序列与B.mori EST lone，ce 2359相似。该克隆的核苷酸序列编码764个氨基酸残基的蛋白质。根据B.mori漆酶的核苷酸序列和部分氨基酸序列推测其一级结构. Northen杂交分析表明，在幼虫-蛹蜕皮过程中，表皮细胞中开始出现mRNA。