Sun D, McDonnell M, Chen X S, Lakkis M M, Li H, Isaacs S N, Elsea S H, Patel P I, Funk C D
Center for Experimental Therapeutics, Departments of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 1998 Dec 11;273(50):33540-7. doi: 10.1074/jbc.273.50.33540.
Expressed sequence tag information was used to clone the full-length sequence for a new human lipoxygenase from the B cell line CCL-156. A related mouse sequence with 83% nucleotide identity to the human sequence was also cloned. The human lipoxygenase, when expressed via the baculovirus/insect cell system produced an approximately 80-kDa protein capable of metabolizing arachidonic acid to a product identified as 12-hydroxyeicosatetraenoic acid by mass spectrometry. Using chiral phase-high performance liquid chromatography, the product was identified as >98% 12(R)-hydroxyeicosatetraenoic acid as opposed to the S-stereoisomer formed by all other known mammalian lipoxygenases. The single copy human 12(R)-lipoxygenase gene was localized to the chromosome 17p13 region, the locus where most other lipoxygenase genes are known to reside. By reverse transcription-polymerase chain reaction, but not by Northern blot, analysis the 12(R)-lipoxygenase mRNA was detected in B cells and adult skin. However, the related mouse lipoxygenase mRNA was highly expressed in epidermis of newborn mice and to a lesser extent in adult brain cortex. By in situ hybridization the mouse lipoxygenase gene was demonstrated to be temporally and spatially regulated during embryogenesis. Expression was induced at embryonic day 15.5 in epidermis, nasal epithelium, and surface of the tongue. These results broaden the mammalian lipoxygenase family to include a 12(R)-lipoxygenase whose biological function remains to be determined.
表达序列标签信息被用于从B细胞系CCL - 156中克隆一种新的人脂氧合酶的全长序列。还克隆了一个与人类序列具有83%核苷酸同一性的相关小鼠序列。当通过杆状病毒/昆虫细胞系统表达时，人脂氧合酶产生一种约80 kDa的蛋白质，该蛋白质能够将花生四烯酸代谢为一种经质谱鉴定为12 - 羟基二十碳四烯酸的产物。使用手性相高效液相色谱法，该产物被鉴定为>98%的12(R)-羟基二十碳四烯酸，这与所有其他已知哺乳动物脂氧合酶形成的S - 立体异构体相反。单拷贝的人12(R)-脂氧合酶基因定位于染色体17p13区域，已知大多数其他脂氧合酶基因位于该位点。通过逆转录 - 聚合酶链反应分析（而非Northern印迹分析），在B细胞和成人皮肤中检测到了12(R)-脂氧合酶mRNA。然而，相关的小鼠脂氧合酶mRNA在新生小鼠的表皮中高度表达，在成年大脑皮层中的表达程度较低。通过原位杂交证明，小鼠脂氧合酶基因在胚胎发育过程中受到时间和空间上的调控。在胚胎第15.5天，在表皮、鼻上皮和舌表面诱导表达。这些结果扩展了哺乳动物脂氧合酶家族，使其包括一种生物学功能尚待确定的12(R)-脂氧合酶。
