Human Neutrophils produce alternatively spliced variants of 5‐Lipoxygenase mRNA

Abstract

5-lipoxygenase (5-LO) catalyzes the oxygenation of the 5th carbon on arachidonic acid and is, therefore, an essential enzyme for leukotriene biosynthesis. Activation of the 5-LO pathway following the stimulation of human neutrophils (PMN) leads to the formation of LTB4, a potent chemoattractant involved in the inflammatory response. Our studies focus on the mechanisms by which the biosynthesis of 5-LO products is controlled. In this study, we evaluated whether alternative forms of 5-LO mRNA exist in human PMN. Following reverse transcription and PCR analyses of total mRNA extracted from PMN isolated from peripheral blood, several potential variants of 5-LO mRNA were identified. Molecular cloning and sequencing experiments revealed that at least two isoforms are present in human PMN: one containing all 14 exons with the expected splicing sites and one that results from the inclusion of intron 10. Approximately 20% of generated clones contained this variant isoform. Treatment of human PMN with granulocyte-macrophage colony stimulating factor (GM-CSF) did not result in the appearance of new isoforms. The retention of intron 10 in the mRNA produces a frame shift in the open reading frame and the immediate appearance of the stop codon TGA. Experiments are underway to determine whether the translation product of the alternative isoform impacts on the capacity for the biosynthesis of 5-LO products. (Supported by the Canadian Institutes of Health Research and the Canada Research Chairs Program)