Sigal E, Sloane D L, Conrad D J
Cardiovascular Research Institute, University of California, San Francisco 94143-0911.
J Lipid Mediat. 1993 Mar-Apr;6(1-3):75-88.
Arachidonate 15-lipoxygenase (15-lipoxygenase) is a lipid-peroxidizing enzyme associated with specific inflammatory cells seen in asthma and atherosclerosis. In atherosclerosis, 15-lipoxygenase is induced in the macrophages of human and rabbit lesions and has been implicated in foam cell formation. In human lung, 15-lipoxygenase is preferentially expressed in airway epithelial cells and eosinophils. Our studies have focused both on the regulation of expression and on the structure-function relationships of the enzyme. To determine factors that could regulate expression, peripheral blood monocytes were purified and cultured with combinations of 18 factors. Only interleukin-4 (60 pM) induced 15-lipoxygenase mRNA, protein and enzymatic activity. Interferon-gamma (100 pM) inhibited the interleukin-4 dependent induction of 15-lipoxygenase. Results with cultured human airway cells were similar. These data suggest that expression of 15-lipoxygenase is regulated by interleukin-4, and that 15-lipoxygenase is a potential downstream effector molecule for this potent cytokine. In parallel studies, we have investigated determinants of positional specificity using site-directed mutagenesis and bacterial expression of human 15-lipoxygenase. Hypotheses for mutagenesis were derived from an analysis of conserved differences among multiple lipoxygenase sequences. Switching four amino acids in 15-lipoxygenase to their counterparts in 12-lipoxygenase resulted in a variant enzyme that produced equal 12- and 15-lipoxygenation. Further analysis has identified two amino acids that completely control the positional specificity of 15-lipoxygenase. These data have led to a preliminary model of the enzyme's active site region.
花生四烯酸15 -脂氧合酶（15 - lipoxygenase）是一种与哮喘和动脉粥样硬化中特定炎症细胞相关的脂质过氧化酶。在动脉粥样硬化中，15 -脂氧合酶在人和兔病变的巨噬细胞中被诱导表达，并与泡沫细胞的形成有关。在人肺中，15 -脂氧合酶优先在气道上皮细胞和嗜酸性粒细胞中表达。我们的研究集中在该酶的表达调控及其结构 - 功能关系上。为了确定可能调控表达的因素，我们纯化了外周血单核细胞并用18种因子的组合进行培养。只有白细胞介素 - 4（60 pM）能诱导15 -脂氧合酶mRNA、蛋白质和酶活性。干扰素 - γ（100 pM）抑制白细胞介素 - 4依赖性的15 -脂氧合酶诱导。培养的人气道细胞的结果与之相似。这些数据表明15 -脂氧合酶的表达受白细胞介素 - 4调控，并且15 -脂氧合酶是这种强效细胞因子的潜在下游效应分子。在平行研究中，我们利用定点诱变和人15 -脂氧合酶的细菌表达来研究位置特异性的决定因素。诱变的假设来源于对多种脂氧合酶序列保守差异的分析。将15 -脂氧合酶中的四个氨基酸替换为12 -脂氧合酶中的对应氨基酸，产生了一种能产生等量12 -和15 -脂氧化产物的变体酶。进一步分析确定了两个完全控制15 -脂氧合酶位置特异性的氨基酸。这些数据已得出该酶活性位点区域的初步模型。
