SIMULTANEOUS EXPRESSION OF LEUKOCYTE-TYPE 12-LIPOXYGENASE AND RETICULOCYTE-TYPE 15-LIPOXYGENASE IN RABBITS

In rabbit reticulocytes an arachidonic acid 15-lipoxygenase (15-LOX) i s expressed at high yield. Rescreening a rabbit reticulocyte cDNA libr ary for alternative 15-LOX transcripts, a full length cDNA which encod es a novel lipoxygenase was isolated. The predicted amino acid sequenc e of this enzyme shared a high degree (99%) of identity with the retic ulocyte-type 15-lipoxygenase. Among the six amino acid residues differ ent in both enzymes a Phe-Leu exchange was detected at position 353. R ecently, site-directed mutagenesis studies have revealed that this ami no acid exchange converts a 15-lipoxygenase to a 12-lipoxygenase. Ln f act, when the novel enzyme was expressed in Escherichia coli, mainly 1 2-lipoxygenation of arachidonic acid was observed. The recombinant enz yme exhibited a rather broad substrate specificity. Various C-18 and C -20 polyenoic fatty acids and even complex substrates such as biomembr anes were effectively oxygenated. Thus, the novel enzyme may be classi fied as leukocyte-type 12-lipoxygenase. Genomic polymerase chain react ion of the 3' region of the leukocyte-type 12-lipoxygenase gene indica ted that introns 10 to 13 differed to about 10% from the corresponding sequences of the 15-lipoxygenase gene although their size and the int ron-exon organization were very similar. Ln 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 elem ent of the 15-lipoxygenase mRNA. Activity assays with a variety of cel ls and tissues prepared from normal rabbits suggested that only periph eral monocytes abundantly express the enzyme, suggesting a tissue-spec ific regulation of gene expression. These data indicate for the first time the co-expression of two separate genes for a reticulocyte-type 1 5-lipoxygenase and for a leukocyte-type 12-lipoxygenase in one species . This is of importance for the implication of both enzymes in red blo od cell development and atherogenesis. (C) 1998 Academic Press Limited .