Isolation and biochemical, functional and structural characterization of a novel L-amino acid oxidase from Lachesis muta snake venom
Isolation and biochemical, functional and structural characterization of a novel L-amino acid oxidase from Lachesis muta snake venom
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DOI：
10.1016/j.toxicon.2012.08.008
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发表时间：
2012-12-01
期刊：
影响因子：
2.8
通讯作者：
Arantes, Eliane Candiani
中科院分区：
文献类型：
--
作者：
Bregge-Silva, Cristiane;Nonato, Maria Cristina;Arantes, Eliane Candiani
本研究的目的是从Lachesis穆塔蛇毒中分离LAAO（LmLAAO）并对其进行生化、功能和结构鉴定。开发了两种不同的纯化方案，两者都提供了高度均一和活性的LmLAAO。它是一种同二聚体酶，非还原条件下摩尔质量约为120 kDa，SDS-PAGE中还原条件下摩尔质量约为60 kDa，质谱法测定摩尔质量约为60852 Da。从LmLAAO直接测序40个氨基酸残基，并从从毒腺获得的表达序列标签数据库中鉴定和表征其完整的cDNA。人工构建基于序列同源性的模型，以预测其三维结构。LmLAAO对疏水性氨基酸表现出催化偏好（与Leu的Km为0.97 mmol/L）。在注射100 μ g LmLAAO后，在小鼠中观察到组织学上的轻度肌坏死，并通过CK活性增加15倍证实。LmLAAO对AGS细胞系（胃腺癌，IC 50：22.7 μ g/mL）和MCF-7细胞系（乳腺癌，IC 50：1.41 μ g/mL）诱导细胞毒性。它对巴西利什曼原虫具有抗寄生虫活性（IC 50：2.22 μ g/mL），但克氏锥虫对LmLAAO具有抗性。总之，LmLAAO显示出较低的全身毒性，但具有重要的体外药理作用。(C)2012爱思唯尔有限公司保留所有权利。
The aim of this study was the isolation of the LAAO from Lachesis muta venom (LmLAAO) and its biochemical, functional and structural characterization. Two different purification protocols were developed and both provided highly homogeneous and active LmLAAO. It is a homodimeric enzyme with molar mass around 120 kDa under non-reducing conditions, 60 kDa under reducing conditions in SDS-PAGE and 60852 Da by mass spectrometry. Forty amino acid residues were directly sequenced from LmLAAO and its complete cDNA was identified and characterized from an Expressed Sequence Tags data bank obtained from a venom gland. A model based on sequence homology was manually built in order to predict its three-dimensional structure. LmLAAO showed a catalytic preference for hydrophobic amino acids (K-m of 0.97 mmol/L with Leu). A mild myonecrosis was observed histologically in mice after injection of 100 mu g of LmLAAO and confirmed by a 15-fold increase in CK activity. LmLAAO induced cytotoxicity on AGS cell line (gastric adenocarcinoma, IC50: 22.7 mu g/mL) and on MCF-7 cell line (breast adenocarcinoma, IC50:1.41 mu g/mL). It presents antiparasitic activity on Leishmania brasiliensis (IC50: 2.22 mu g/nnL), but Trypanosoma cruzi was resistant to LmLAAO. In conclusion, LmLAAO showed low systemic toxicity but important in vitro pharmacological actions. (C) 2012 Elsevier Ltd. All rights reserved.