Identification and characterization of a cyclooxygenase-like enzyme from Entamoeba histolytica
Identification and characterization of a cyclooxygenase-like enzyme from Entamoeba histolytica
复制标题
DOI：
10.1073/pnas.1835863100
复制
发表时间：
2003-11-11
影响因子：
11.1
通讯作者：
Chadee, K
中科院分区：
文献类型：
--
作者：
Dey, I;Keller, K;Chadee, K
肠道原生动物寄生虫溶组织内阿米巴（Entamoeba histolytica）仍然是世界范围内发病率和死亡率的重要原因。然而，几乎没有什么是已知的寄生虫分泌的分子，调节宿主免疫反应或上皮屏障功能的结肠。在此，我们描述了在大肠杆菌中的环加氧酶（考克斯）样酶的分离和表征。溶组织素，其负责前列腺素（PG）E-2的生物合成。PGE（2）的产生是组成型的，但对外源花生四烯酸底物有很强的依赖性。COX样活性和免疫反应蛋白定位于E.溶组织剂COX样蛋白（72 kDa）进行微测序，并通过逆转录酶PCR克隆。阿米巴考克斯与不同种属的考克斯-1/2酶在核苷酸和氨基酸水平上同源性很低。令人惊讶的是，花生四烯酸结合结构域和血红素协调和催化位点，这是保守的其他物种，在阿米巴缺席。在大肠杆菌中表达的阿米巴考克斯在体外表现出COX样酶活性，可将花生四烯酸转化为PGE（2），但不转化为PGD（2）或PGF（2 α）。考克斯活性被1 mM阿司匹林抑制，但不被吲哚美辛或考克斯-1/2特异性抑制剂抑制。综合起来，这些研究表明，E。溶组织菌通过以前未描述的祖先考克斯样酶产生PGE（2），这可能在发病机制和免疫逃避中发挥重要作用。
The intestinal protozoan parasite Entamoeba histolytica remains a significant cause of morbidity and mortality worldwide. However, almost nothing is known about the molecules secreted by the parasite that modulate host immune responses or epithelial barrier function in the colon. Herein, we describe the isolation and characterization of a cyclooxygenase (COX)-like enzyme in E. histolytica, that is responsible for the biosynthesis of prostaglandin (PG)E-2. PGE(2) produced by ameba was constitutive but highly dependent on exogenous arachidonic acid substrate. COX-like activity and the immunoreactive protein were localized to the nuclear fraction of E. histolytica. The COX-like protein (72 kDa) was microsequenced and cloned by reverse transcriptase PCR. Ameba COX showed little homology with COX-1/2 enzymes from different species at the nucleotide and amino acid levels. Surprisingly, the arachidonate-binding domain and heme-coordinating and catalytic sites, which are conserved in other species, were absent in ameba. Ameba COX expressed in Escherichia coli demonstrated COX-like enzyme activity in vitro by converting arachidonic acid into PGE(2) but not into PGD(2) or PGF(2alpha). COX activity was inhibited with 1 mM aspirin but not with indomethacin or COX-1/2-specific inhibitors. Taken together, these studies reveal that E. histolytica produces PGE(2), by means of a previously undescribed ancestral COX-like enzyme, which could play a major role in pathogenesis and immune evasion.