Cloning and expression of an airway epithelial 12-lipoxygenase

Abstract

Arachidonate 12-lipoxygenase generates metabolites that may regulate airway function. To further characterize this enzyme, we isolated a cDNA corresponding to 12-lipoxygenase from a bovine tracheal epithelium cDNA library using human reticulocyte 15-lipoxygenase cDNA as a probe. The resulting 2.9-kb cDNA, the identity of which was confirmed by expression of active catalytic function in Escherichia coli has a 2.0-kb open reading frame encoding a protein of 75,000 kDa and includes 5 bp of 5'-untranslated region and 0.9 kb of 3'-untranslated region. On Northern blots, the 12-lipoxygenase cDNA hybridized to one band (3.5 kb) of bovine tracheal epithelium RNA. Polyclonal antibodies that recognize human tracheal 15-lipoxygenase cross-reacted on immunoblots to the expressed bovine tracheal 12-lipoxygenase. Further, the deduced amino acid sequence is 86% identical (93% similar) to human 15-lipoxygenase but 64% identical to human platelet 12-lipoxygenase, suggesting that the bovine tracheal enzyme is the homologue of the human 15-lipoxygenase. This is the first sequence of an epithelial lipoxygenase from any species. A comparison of the bovine sequence with other lipoxygenase sequences shows that there are only four amino acids which are conserved differences between a 12-lipoxygenase and a 15-lipoxygenase. We hypothesize that these four amino acids may be responsible for the positional specificity of the enzyme.