Specific amino acids affecting Drosophila melanogaster prophenoloxidase activity in vitro
Specific amino acids affecting Drosophila melanogaster prophenoloxidase activity in vitro
复制标题
影响果蝇酚氧化酶体外活性的特定氨基酸
DOI：
10.1016/j.dci.2012.04.007
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发表时间：
2012-09-01
影响因子：
2.9
通讯作者：
Ling, Erjun
中科院分区：
文献类型：
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作者：
Chen, Yang;Liu, Fei;Ling, Erjun
昆虫酚氧化酶原（PPO）是一种重要的酶，它能诱导入侵病原体周围和伤口处的黑化，以防止进一步感染。果蝇有三个PPO基因，它们在S2细胞中过量表达后具有不同的生化特性。如通过S2细胞的自动黑化所示，重组PPO 3（rPPO 3）在添加Cu 2+后被激活（Cu 2+辅助的细胞黑化，而没有乙醇激活和底物添加：+Cu 2 +; DOPA，乙醇）。造成这种现象的确切原因尚不清楚。在本研究中，使用定点突变和过表达的方法，我们发现位置保持器，两个独立的氨基酸，（等于六叶曼陀罗氨基酸残基：MsPPO 1中的F218和5393，M5 PPO 2中的F224和E395）和缺失片段（类似于M5 PPO 1中的（565）RPGDPGT（571）和M5 PPO 2中的（571）QGSDPRR（577）），影响rPPO 3-S2细胞Cu ~（2+）辅助的自黑化。一些突变几乎拯救了rPPO 3 Cu 2+辅助的自激活，这表明野生型rPPO 3的自激活不是由于丝氨酸蛋白酶的切割。我们还发现，在活性位点口袋中的相应的氨基酸对PPO 1和PPO 3具有类似的作用。PPO 1染色活性（PPO转染期间添加或不添加Cu 2 +;乙醇活化和底物添加后细胞变黑：+/-Cu 2 +; +DOPA，+乙醇）与活性位点口袋大小呈正相关，rPPO 3也是如此。与PPO 3 C端缺失片段相对应的rPPO 1片段缺失后对rPPO 1染色活性无影响。然而，rPPO 2突变体的染色活性下降后，删除这些相应的氨基酸序列。将PPO 1或PPO 2的相应片段插入到PPO 3中，突变体rPPO 3对染色活性没有影响，但Cu ~（2+）辅助的自激活作用显著降低。因此，我们发现，一些氨基酸是重要的rPPO 3铜+辅助自激活以及PPO染色活性在体外。(C)2012爱思唯尔有限公司保留所有权利。
Insect prophenoloxidase (PPO) is a key enzyme that induces melanization around invading pathogens and at wounds to prevent further infection. Drosophila melanogaster has three PPO genes which have different biochemical properties following over-expression in S2 cells. As shown by automatic melanization of S2 cells, recombinant PPO3 (rPPO3) became activated upon Cu2+ addition (Cu2+-aided cells melanization without ethanol activation and substrate addition: +Cu2+; DOPA, Ethanol). The exact reasons for this phenomenon are still unknown. In this study, using site-directed mutagenesis and over-expression methods, we found that the place holder, two independent amino acids (equal to Manduca sexta amino acid residues: F218 and 5393 in MsPPO1, F224 and E395 in M5PPO2) in the active site pocket and a missing fragment (similar to (565)RPGDPGT(571) in M5PPO1 and (571)QGSDPRR(577) in M5PPO2) at the C-terminus of PPO3, affect rPPO3-S2 cells Cu2+-aided auto-melanization. Some mutations nearly rescued rPPO3 Cu2+-aided auto-activation, which suggests that the auto-activation of wild type rPPO3 was not due to cleavage by serine proteases. We also found that the corresponding amino acids in the active site pocket have similar effect on PPO1 as on PPO3. PPO1 staining activity (Cu2+ added or not during PPO transfection; cells melanized after ethanol activation and substrate addition: +/- Cu2+; +DOPA, +Ethanol) has a positive relationship with the active site pocket size as does rPPO3. The fragment of rPPO1 corresponding to the one missing from the C-terminus of PPO3 has no influence on rPPO1 staining activity after it is deleted. However, the staining activities of rPPO2 mutants decreased after deletion of those corresponding amino acid sequences. When the corresponding fragments from PPO1 or PPO2 were inserted into PPO3, the mutant rPPO3 had no influence on staining activity, but had a significantly lowered Cu2+-taided auto-activation. Thus, we found that some amino acids are important for rPPO3 Cu2+-aided auto-activation as well as PPO staining activity in vitro. (C) 2012 Elsevier Ltd. All rights reserved.