Structure and Function of New Neurotransmitters in Brains in the Silkworm Bombyx Mori

1)家蚕在母蛹期由食道下神经节分泌一种内在的滞育激素，作用于受卵的卵巢和胚胎发育阻滞。AnyParP（从野蚕蛾Antheraea yamamai中分离得到的一种麻痹肽，命名为AnyParP）与该滞育激素具有相同的功能。即使AnyParP（23个残基，c端为羧酸）和滞育激素（24个残基，c端为酰胺）之间的氨基酸序列完全不同，这两种肽在胚胎滞育过程中也能发挥相同的功能。本文首次报道了一种不相关的昆虫肽对家蚕滞育激素的功能模仿。2)本研究从家蚕（Bomby mori）的蛹脑提取物中分离出一种蛋白质，并鉴定了其n端氨基酸序列。根据氨基酸序列合成退化寡脱氧核苷酸，并以其作为杂交探针筛选蛹脑cDNA文库。cDNA克隆序列分析表明，全长603 bp的开放阅读框编码含有15个氨基酸先导肽的201个氨基酸蛋白。作为脂质蛋白家族的一员，该蛋白与Galleria mellonella Gallerin具有高度的相似性。我们将这种来自家蚕中枢神经系统的新蛋白命名为“家蚕素”。免疫组化和Western blot分析表明，家蚕蛋白仅在中枢神经系统（CNS）中表达。3)首次在昆虫中枢神经系统中发现P2X_1样受体的免疫活性，利用P2X_1抗体对抗哺乳动物P2X_1受体382-399的肽段。家蚕5龄幼虫和蛹的大脑中分布着6对免疫反应体，它们与一种特异性的P2 x_1样蛋白存在发育阶段的关系。

1) In the case of the silkworm, an intrinsic diapause hormone secreted from subesophageal ganglion in the maternal-pupal stage, acts on ovaries and embryonic development arrests in oviposited eggs. AnyParP (a paralytic peptide isolated from the wild silkmoth, Antheraea yamamai, named as AnyParP) had the same function with this diapause hormone. Even if the sequence of amino acids between AnyParP (23 residues and C-terminal is carboxylic acid) and diapause hormone (24 residues and C-terminal is amide) is entirely different, both peptides can elicit the same function in embryonic diapause. This is the first report of a functional mimicry of the silkworm diapasue hormone by an unrelated insect peptide2) This study isolated a protein from pupal brain extract of the silkworm, Bomby mori using two chromatographic steps, and identified its N-terminal amino acid sequence. Based on the amino acid sequence, degenerate oligodeoxynucleotide was synthesized and used as a hybridization probe to screen a pupal brain cDNA library. The sequence analysis of the identified cDNA clones indicated that a 603 bp open reading frame encodes a 201-amino-acid protein containing a 15-amino-acid leader peptide. This protein has high similarity to the Galleria mellonella Gallerin as a member of the lipocalin family. We named this novel protein from the Bombyx central nervous system as Bombyrin. The immunohistochemistry and Western blot analysis indicated that Bombyrin is only expressed in the central nervous system (CNS) of Bombyx mori.3) The immunoactivity of P2X_1-like receptor was first found in the central nervous system of insects, using P2X_1 antibody against the peptide of mammalian P2X_1 receptor 382-399. Six pairs of immunoreactive somata were distributed in brains of 5th instar larvae and pupae of Bombyx mori, and they showed a relationship between developmental stages and a specific P2 X_1-like protein.