Structure elucidation of prothoracicotropic hormone from the silkworm

家蚕促促胸腺激素的结构解析

• 批准号: 61470134
• 负责人: SUZUKI Akinori
• 金额: $ 3.52万
• 依托单位: Department of Agricultural Chemistry, The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1986
• 资助国家: 日本
• 起止时间: 1986 至 1987
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-61470134/
• 关键词: silkworm; prothoracicotropic hormone; bombyxin; insulin; determination of disulfide bridges; amino acid sequsnce; monoclonal antibody; 免疫組織化学; 脳ホルモン; プペチドホルモン; インスリン族ペプチド

Two kinds of prothoracicotropic hormones, 22K-PTTH and bombyxin(4K-PTTH), have been isolated from adult heads of the silkmoth, Bombyx mori. 22K-PTTH, that activates Bombyx prothoracic glands to induce synthesis and release of ecdysone, consists of many heterogeneous molecular species, one of which has been isolated and its N-terminal sequence of 13 amino acid residues determined.Bombyxin, that can activate prothoracic glands of another silkmoth, Samia cynthia ricini, consists of two peptide chains, A and B chains with 20 and 28 amino acid residues, respectively, which are connected by disulfide bonds to each other. It has a considerable sequence homology (40%) with vertebrate insulin. Thermolysin digestion of bombyxin-II, one of various molecular form of bombyxins, afforded two cystine peptides. Sequencing of the peptides revealed that one contained one cystine and the other two. Hte former established the connection fo A(20)-B(22). As to the latter cystine peptide, there were three possible structures with different disulfide bonds, which were all synthesized. They were separated on reverse phase HPLC, one of them was co-eluted with the natural thermolysin peptide. Thus, the connection of A(6)-A(11) and A(7)-B(10) was established. This mode of disulfide bridges is identical with that of insulin. Such homologous structure of bombyxin-II with insulin permitted us to construct a three dimensional structural model of bombyxin-II, which showed a considerable difference in the surface of the molecules. This difference might account for the difference of biological activity.Monoclonal antibldy was made against an N-terminal synthetic decapeptide of A chain coupled with bovine serum albumin. Immunohistochemistry using this antibody showed that bombyxin is produced in median 4 pairs of neurosecretory cells, transferred to corpora allata and released into the hemolymph.

从家蚕成虫头部中分离到两种促前胸腺激素，即22K-PTTH和4K-PTTH。22K-PTTH激活家蚕前胸腺，诱导蜕皮激素的合成和释放，由多种异相分子组成，其中一个已分离出来，并测定了其N端13个氨基酸残基的序列；激活另一种蚕蛾(Samia Cynthia Ricini)前胸腺的22K-PTTH由A和B两条肽链组成，A链和B链分别有20和28个氨基酸残基，它们之间通过二硫键相连。它与脊椎动物胰岛素有相当大的序列同源性(40%)。家蚕素-II是家蚕毒素的多种分子形式之一，用热分解法消化可得到两个半胱氨酸肽。对这些多肽进行了测序，发现其中一个含有一个半胱氨酸，另外两个。前者建立了A(20)-B(22)的连接。对于后者的半胱氨酸肽，有三个可能的二硫键结构，它们都是合成的。用反相高效液相色谱分离，其中一种与天然热裂解素肽共洗脱。由此，建立了A(6)-A(11)和A(7)-B(10)之间的联系。这种二硫键模式与胰岛素的模式相同。家蚕毒素-II与胰岛素的这种同源结构使我们能够构建家蚕毒素-II的三维结构模型，显示出分子表面的显著差异。这种差异可能是生物活性不同的原因。针对A链N端合成的十肽与牛血清白蛋白偶联，进行了单抗。使用该抗体的免疫组织化学显示，家蚕素在中间4对神经分泌细胞中产生，转移到鼻咽体，并释放到血淋巴中。

项目成果

H. Jhoti: "Prothoracicotropic hormone has an insulin-like teratiary structure" FEBS Letters. 219. 419-423 (1987)

H. Jhoti：“促胸腺激素具有类似胰岛素的三级结构”FEBS Letters。

Hiromichi Nagasawa: Proc. Natl. Acad. Sci. USA. 83. 5840-5843 (1986)

长泽宏道：Proc。

Hiroshi Kataoka: Agric. Biol. Chem.51. 1067-1076 (1987)

片冈宏：农业。

鈴木昭憲: 植物防疫. 40. 244-229 (1986)

铃木昭典：植物保护。40。244-229（1986）

Hiromichi Nagasawa: "Amino acid sequence of a prothoracicotropic hormone of the silkworm, Bombyx mori" Proc. Natl. Acad. Sci. USA. 83. 5840-5843 (1986)

Hiromichi Nagasawa：“蚕促胸腺激素的氨基酸序列，Bombyx mori”Proc。