Research into Cytotoxicity of Eosinophil Granule Proteins

嗜酸性粒细胞颗粒蛋白的细胞毒性研究

• 批准号: 07557163
• 负责人: OHUCHI Kazuo
• 金额: $ 0.83万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557163/
• 关键词: rat eosinophils; eosinophil granule proteins; major basic protein; eosinophil cationic protein; ribonuclease; cDNA; cytotoxicity; eosinophil-derived neurotoxin; ラットMBP; ラットMBP cDNA

Eosinophil granule proteins are supposed to be associated with allergic diseases such as bronchial sathma. However, it is difficult to obtain a large amount of eosinophil granule proteins to investigate roles of eosinophil granule proteins in allergic diseases. Therefore, to produce recombinant eosinophil granule proteins, we cloned cDNA for rat major basic protein (MBP) and rat eosinophil cationic protein (ECP) by RT-PCR and rapid amplification of cDNA ends procedure using mRNA in rat bone marrow cells as templates.It is indicated that mRNA for rat MBP encodes prepro-form of rat MBP,of which molecular weight is 25.2 kDa. The deduced amino acid sequence of rat MBP revealed that the rat prepro-MBP has three functional domains, namely, the signal peptide, the acidic peptide that contains numerous acidic amino acids, and the mature MBP that is highly cationic protein, as in human and guinea pig MBP.The amino acid sequence deduced from cDNA encoding rat ECP revealed that the molecular weig … More ht is 15.5 kDa and isoelectric point is 9.85, indicating that rat ECP is also highly cationic. The homology of amino acid sequence between rat ECP and human eosinophil ribonucleases are 51-54%, and that between rat ECP and murine eosinophil-associated ribonucleases are 65%. Rat ECP is supposed to have RNase activity because the amino acids required for RNase activity are highly conserved.We also investigated whether rat eosinophil granule proteins have cytotoxic activity against mammalian cells and bacteria. Granule proteins extracted from 10^6 rat eosinophils showed no cytotoxic activity against rat bronchial epithelial cells and rat fibroblasts. However, purified rat MBP and ECP showed potent bactericidal activities at 3 mug/ml. These findings strongly indicate that eosinophil granule proteins including MBP and ECP are highly toxic for bacteria. Eosinophils infiltrated into the bronchial lumen in bronchial asthmatic patients might primarily function as killers of bacteria inhaled. The possibility that eosinophils damage bronchial epithelial cells in bronchial asthmatic patients might be ruled out. Less

嗜酸性粒细胞颗粒蛋白被认为与支气管哮喘等过敏性疾病有关。然而，很难获得大量的嗜酸性粒细胞颗粒蛋白来研究嗜酸性粒细胞颗粒蛋白在过敏性疾病中的作用。因此，为了生产重组嗜酸性粒细胞蛋白，我们通过RT-PCR克隆了大鼠主要碱性蛋白（MBP）和大鼠嗜酸性粒细胞阳离子蛋白（ECP）的cDNA，并以大鼠骨髓细胞中的mRNA为模板快速扩增cDNA末端。结果表明，大鼠MBP的mRNA编码大鼠MBP的前原形式，其分子量为 25.2 kDa。推导的大鼠MBP氨基酸序列显示，大鼠前MBP前体具有三个功能结构域，即信号肽、含有大量酸性氨基酸的酸性肽和成熟MBP，其为高阳离子蛋白，与人和豚鼠MBP一样。从编码大鼠ECP的cDNA推导的氨基酸序列显示，分子重量为 15.5 kDa，等电点为9.85，表明大鼠ECP也是高度阳离子的。大鼠ECP与人嗜酸性粒细胞核糖核酸酶的氨基酸序列同源性为51-54%，大鼠ECP与鼠嗜酸性粒细胞相关核糖核酸酶的氨基酸序列同源性为65%。大鼠ECP被认为具有RNase活性，因为RNase活性所需的氨基酸高度保守。我们还研究了大鼠嗜酸性粒细胞颗粒蛋白是否对哺乳动物细胞和细菌具有细胞毒活性。从10^6大鼠嗜酸性粒细胞中提取的颗粒蛋白对大鼠支气管上皮细胞和大鼠成纤维细胞没有细胞毒活性。然而，纯化的大鼠 MBP 和 ECP 在 3 微克/毫升时显示出有效的杀菌活性。这些发现强烈表明，包括 MBP 和 ECP 在内的嗜酸性粒细胞颗粒蛋白对细菌具有剧毒。浸润到支气管哮喘患者支气管腔内的嗜酸性粒细胞可能主要起到杀死吸入细菌的作用。可以排除嗜酸性粒细胞损害支气管哮喘患者的支气管上皮细胞的可能性。较少的

项目成果

Nittoh, T., Hirakata, M., Mue, S.and Ohuchi, K.: "Identification of cDNA encoding rat eosinophil cationic protein/eosinophil-associated ribonuclease." Biochim.Biophys.Acta.(in press). (1997)

Nitoh, T.、Hirakata, M.、Mue, S. 和 Ohuchi, K.：“编码大鼠嗜酸性粒细胞阳离子蛋白/嗜酸性粒细胞相关核糖核酸酶的 cDNA 的鉴定”。

Nittoh, T., Arii, M., Suzuki, R., Kitoh, A., Watanabe, M., Mue, S.and Ohuchi, K.: "Increase in eosinophilic cell population in bone marrow of rats by immunization with Ascaris suum antigen." Immunol.Invest.(in press). (1997)

Nitoh, T.、Arii, M.、Suzuki, R.、Kitoh, A.、Watanabe, M.、Mue, S. 和 Ohuchi, K.：“通过蛔虫免疫增加大鼠骨髓中的嗜酸性细胞群

Kazuo Ohuchi et al.: "Identification of cDNA encoding rat eosinophil cationic protein/eosinophil-associated ribonuclease." Biochim. Biophys. Acta. (in press). (1997)

Kazuo Ohuchi 等人：“编码大鼠嗜酸性粒细胞阳离子蛋白/嗜酸性粒细胞相关核糖核酸酶的 cDNA 的鉴定”。

Kazuo Ohuchi et al.: "Characterization of leukocyte-derived neutrophil chemotactic factor-2 and its possible roles in neutrophil infiltration in allergic inflammation in rats" Int.Arch.Allergy Immunol.108. 148-157 (1995)

Kazuo Ohuchi 等人：“白细胞衍生的中性粒细胞趋化因子 2 的特征及其在大鼠过敏性炎症中中性粒细胞浸润中的可能作用”Int.Arch.Allergy Immunol.108。

Kazuo Ohuchi et al.: "Cloning of cDNA for rat eosinophil major basic protein." Biochim.Biophys.Acta,. 1264. 261-264 (1995)

Kazuo Ohuchi 等人：“大鼠嗜酸性粒细胞主要碱性蛋白 cDNA 的克隆。”