Variants of 5‐lipoxygenase from human neutrophils impact on leukotriene biosynthesis in HEK293 cells

5‐lipoxygenase (5‐LO) catalyzes the oxygenation of arachidonic acid and is an essential enzyme forleukotriene (LT) biosynthesis. Human neutrophils (PMN) are important producers of LTB 4 , a potent chemoattractant involved in the inflammatory response. In this study, we evaluated whether alternative forms of 5‐LO mRNA exist in human PMN and if they are implicated in the biosynthesis of LTB 4 . RT − PCR analysis of total mRNA from human PMN revealed several potential variants of 5‐LO mRNA. Molecular cloning and sequencing experiments showed that at least three isoforms are present in human PMN. One containing all 14 exons with the expected splicing sites (full 5‐LO) producing a known protein of 78 kDa. Another isoform retained intron 10 (alpha‐10) resulting in a shift of the open reading frame and produces a protein of expected molecular weight of 56 kDa. A third isoform lacked the first 96 nucleotides of exon 10 (delta‐p10) and produces a protein of expected molecular weight of 74 kDa. HEK293 cells transfected with the full 5‐LO produced LTB 4 and 5‐hydroxyeicosatetraenoic acid (5‐HETE) following ionophore stimulation; however, cells transfected with either alternative isoform produced no 5‐LO products. HEK293 cells transfected with the full 5‐LO and co‐transfected with the alpha‐10 or the delta‐p10 isoform produced 25±2% (mean±sem) and 49±7% (mean±sem) less 5‐LO products, respectively, compared to cells expressing only the full 5‐LO. These alternative isoforms of 5‐LO may represent a new mechanism for the regulation of LT biosynthesis in inflammatory cells. (Supported by the Canadian Institutes of Health Research and the Canada Research Chairs Program).