Research into Cytotoxicity of Eosinophil Granule Proteins

嗜酸性粒细胞颗粒蛋白被认为与过敏性疾病如支气管哮喘有关。然而，要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变应性疾病中的作用是困难的。因此，为了获得重组嗜酸性粒细胞颗粒蛋白，我们以大鼠骨髓细胞的mRNA为模板，采用RT-PCR和cDNA末端快速扩增法，分别克隆了大鼠嗜酸性粒细胞主要碱性蛋白（MBP）和嗜酸性粒细胞阳离子蛋白（ECP）的cDNA，结果表明，大鼠MBP的mRNA编码大鼠MBP的前体，其分子量为25.2kDa。推导的大鼠MBP的氨基酸序列表明，大鼠MBP的前原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，成熟MBP与人和豚鼠MBP一样是高度阳离子化的蛋白质。从编码大鼠ECP的cDNA推导的氨基酸序列表明，大鼠MBP的分子量为1000 - 10000 kDa，与人和豚鼠MBP的分子量相同。 ...更多信息 ht为15.5kDa，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同源性为51 - 54%，与小鼠嗜酸性粒细胞相关核糖核酸酶的氨基酸同源性为65%。大鼠嗜酸性粒细胞颗粒蛋白具有RNase活性，因为RNase活性所需的氨基酸是高度保守的。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 μ g/ml时显示出有效的杀菌活性。这些发现有力地表明，嗜酸性粒细胞颗粒蛋白，包括MBP和ECP是高毒性的细菌。嗜酸性粒细胞浸润于支气管哮喘患者的支气管腔内，其主要功能是杀灭吸入的细菌。结论支气管哮喘患者嗜酸性粒细胞对支气管上皮细胞有损伤作用。少

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