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Molecular mechanisms of biosynthesis and degradation of yolk proteins of the silkworm,Bombyx mori

家蚕卵黄蛋白生物合成与降解的分子机制

基本信息

批准号：
62480047
负责人：
YAMASHITA Okitsugu
金额：
$ 4.03万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

Yolk proteins the silkworm, Bombyx mori consist of three major proteins and among them, egg-specific (ESP) behaves differently from others. The present study was carried out to elucidate the molecular mechanisms of biosynthesis and degradation of this protein. From the genomic library, ESP gene was isolated and the partial structure was carified. cDNA of ESP was constracted and sequenced. The complete amino acid sequence was deduced from the nucleotids sequence. The in vitro translation experiment concluded that esp was translated as a nascent peptide-aving segnal sequence. The post-translational modification occurred sequentially through signal cleavage, glycosylation, phosphorylation. ESP contains appreciable amounts of zinc, which suggest that ESP interacts with nucleic acids.During embryogenesis, ESP is degraded sequentially into three peptides by a unique protease. This protease had the high specificity to ESP without atacking to the other yolk proteins. Cleavage sites were identified by analysing N-termina amino acid sequence as at the carboxyl site of lysine^<114> and argine^<210>. The activity of this protease chenged temporaly according to the embryogenesis. This developmental profile in activity well corresponded to the changes in mRNA activity. The in vitro translation experiment showed that the enzyme protein is synthesized as a nascent peptide and then processed into a mature form of protein through signal cleavage. The nascent peptide showed the biological activity which was comparable to the processed proteins. This is the first report of the biologically active products of translation.

家蚕（Bombyx mori）卵黄蛋白主要由三种蛋白组成，其中卵特异性蛋白（ESP）的行为与其他蛋白不同。本研究旨在阐明该蛋白的生物合成和降解的分子机制。从基因组文库中分离出ESP基因，并对其部分结构进行了鉴定。构建了ESP的cDNA序列。由核苷酸序列推导出完整的氨基酸序列。体外翻译实验表明esp被翻译为一个新生的保肽片段序列。翻译后修饰依次发生在信号切割、糖基化、磷酸化过程中。电潜泵含有相当数量的锌，这表明电潜泵与核酸相互作用。在胚胎发生过程中，ESP被一种独特的蛋白酶依次降解为三种肽。该蛋白酶对ESP具有高特异性，且不攻击其他蛋黄蛋白。通过分析n端氨基酸序列确定裂解位点为赖氨酸^<114>和精氨酸^<210>的羧基位点。这种蛋白酶的活性根据胚胎发生的不同而有暂时的变化。这种发育特征与mRNA活性的变化相吻合。体外翻译实验表明，该酶蛋白是作为新生肽合成的，然后通过信号切割加工成成熟的蛋白质形式。新生肽显示出与加工蛋白相当的生物活性。这是首次报道具有生物活性的翻译产物。