Abstract
5-Lipoxygenase of mouse macrophages and bone marrow-derived mast cells (BMMC) was investigated. Indirect immunocytofluorescence combined with confocal microscopy provided evidence for distinct intracellular expression patterns and trafficking of 5-lipoxygenase upon cellular activation. In resting BMMC, 5-lipoxygenase was found within the nucleus co-localizing with the nuclear stain Yo-Pro-1. When BMMC were IgE/antigen-activated the 5-lipoxygenase immunofluorescence pattern was changed from nuclear to perinuclear. The absence of divalent cations in the incubation medium, or calcium ionophore A23187 challenge, altered the predominantly nuclear expression pattern to new sites both cytosolic and intranuclear. The cDNA for murine macrophage 5-lipoxygenase was cloned by the polymerase chain reaction and would predict a 674 amino acid protein. Using control cells obtained from 5-lipoxygenase-deficient mice it was determined that a single isoform accounts for both soluble and membrane-bound and nuclear and cytosolic-localized enzyme in macrophages and BMMC. A mutation at amino acid 672 (Val-->Met) introduced serendipitously during the cloning process was found to completely abolish 5-lipoxygenase enzyme activity when the enzyme was expressed in human embryonic kidney 293 cells. This subtle change is proposed to affect the ability of the COOH-terminal isoleucine to coordinate the essential non-heme iron atom. In macrophages and BMMC obtained from 5-lipoxygenase-deficient mice, compensatory changes in expression of genes involved in the biosynthesis of leukotriene B4 were investigated. 5-Lipoxygenase-activating protein expression was reduced by 50%, while leukotriene A4 hydrolase expression was unaltered. The 5-lipoxygenase gene was mapped to the central region of mouse chromosome 6 in a region that shares homology with human chromosome 10 by interspecific backcross analysis. These studies provide a global picture of the murine 5-lipoxygenase system and raise questions about the role of 5-lipoxygenase and leukotrienes within the nucleus.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM I EMBL Data Bank with accession number(s) L42198
5-lipoxygenase undergoes a Ca2+-dependent translocation from the cytosol to a membrane site which appears to be the nuclear envelope [3, 4]. 5-Lipoxygenase activating protein (FLAP), an 18-kDa membrane protein found in the nuclear envelope, acts apparently as an arachidonate-binding protein to facilitate the concerted formation of LTA4 [5, 6]
Our studies have focused primarily on bone marrow-derived mast cells (BMMC) and
Summary
5-H(P)ETE, 5-hydro(pero)xy-eicosatetraenoic acid; LT, leukotriene; FLAP, 5-lipoxygenase-activating protein; BMMC, bone marrow-derived mast cell; PCR, polymerase chain reaction; HEK, human embryonic kidney; RACE, rapid amplification of eDNA ends; DNP-BSA, dinitrophenyl bovine serum albumin; 13H(P)ODE, 13-hydro(pero)xy-octadecadienoic acid; RP-HPLC, reversed phase-high performance liquid chromatography; kb, kilobase(s). We demonstrate the importance of the amino acid residue two positions upstream of the carboxyl terminus for 5-lipoxygenase activity, the chromosomal location of the murine 5-lipoxygenase gene, and studies with macrophages and BMMC of 5-lipoxygenase-deficient mice designed to examine compensatory expression of other proteins key to the formation ofleukotrienes (FLAP and LTA4 hydrolase)
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