Research into Cytotoxicity of Eosinophil Granule Proteins

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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