Berger M, Schwarz K, Thiele H, Reimann I, Huth A, Borngräber S, Kühn H, Thiele B J
Institute of Biochemistry, University Clinics Charité, Hessische Str. 3-4, Humboldt-University Berlin, Germany.
J Mol Biol. 1998 May 22;278(5):935-48. doi: 10.1006/jmbi.1998.1737.
In rabbit reticulocytes an arachidonic acid 15-lipoxygenase (15-LOX) is expressed at high yield. Rescreening a rabbit reticulocyte cDNA library for alternative 15-LOX transcripts, a full length cDNA which encodes a novel lipoxygenase was isolated. The predicted amino acid sequence of this enzyme shared a high degree (99%) of identity with the reticulocyte-type 15-lipoxygenase. Among the six amino acid residues different in both enzymes a Phe-Leu exchange was detected at position 353. Recently, site-directed mutagenesis studies have revealed that this amino acid exchange converts a 15-lipoxygenase to a 12-lipoxygenase. In fact, when the novel enzyme was expressed in Escherichia coli, mainly 12-lipoxygenation of arachidonic acid was observed. The recombinant enzyme exhibited a rather broad substrate specificity. Various C-18 and C-20 polyenoic fatty acids and even complex substrates such as biomembranes were effectively oxygenated. Thus, the novel enzyme may be classified as leukocyte-type 12-lipoxygenase. Genomic polymerase chain reaction of the 3' region of the leukocyte-type 12-lipoxygenase gene indicated that introns 10 to 13 differed to about 10% from the corresponding sequences of the 15-lipoxygenase gene although their size and the intron-exon organization were very similar. In the 3'-untranslated region of the novel mRNA a C+U-rich, 20-fold repetitive element was found which appears to be highly related to the differentiation control element of the 15-lipoxygenase mRNA. Activity assays with a variety of cells and tissues prepared from normal rabbits suggested that only peripheral monocytes abundantly express the enzyme, suggesting a tissue-specific regulation of gene expression. These data indicate for the first time the co-expression of two separate genes for a reticulocyte-type 15-lipoxygenase and for a leukocyte-type 12-lipoxygenase in one species. This is of importance for the implication of both enzymes in red blood cell development and atherogenesis.
在兔网织红细胞中，花生四烯酸15-脂氧合酶（15-LOX）高表达。通过重新筛选兔网织红细胞cDNA文库以寻找替代性15-LOX转录本，分离出了一个编码新型脂氧合酶的全长cDNA。该酶的预测氨基酸序列与网织红细胞型15-脂氧合酶具有高度（99%）的同一性。在两种酶不同的六个氨基酸残基中，在第353位检测到苯丙氨酸-亮氨酸的交换。最近，定点诱变研究表明，这种氨基酸交换可将15-脂氧合酶转变为12-脂氧合酶。事实上，当该新型酶在大肠杆菌中表达时，主要观察到花生四烯酸的12-脂氧合作用。重组酶表现出相当广泛的底物特异性。各种C-18和C-20多烯脂肪酸甚至复杂底物如生物膜都能被有效氧化。因此，该新型酶可归类为白细胞型12-脂氧合酶。白细胞型12-脂氧合酶基因3'区域的基因组聚合酶链反应表明，内含子10至13与15-脂氧合酶基因的相应序列相差约10%，尽管它们的大小和内含子-外显子组织非常相似。在新型mRNA的3'-非翻译区发现了一个富含C+U的、20倍重复的元件，它似乎与15-脂氧合酶mRNA的分化控制元件高度相关。对正常兔制备的多种细胞和组织进行的活性测定表明，只有外周单核细胞大量表达该酶，提示基因表达存在组织特异性调控。这些数据首次表明在一个物种中同时表达两种独立的基因，分别编码网织红细胞型15-脂氧合酶和白细胞型12-脂氧合酶。这对于这两种酶在红细胞发育和动脉粥样硬化形成中的作用具有重要意义。
