title: Novel 5-lipoxygenase isoforms affect the biosynthesis of 5-lipoxygenase products.
abstract: 5-Lipoxygenase (5-LO) is the essential enzyme for the biosynthesis of leukotrienes, important mediators of inflammation. This study investigated whether variants of 5-LO exist in human leukocytes. 5-LO mRNA isoforms that are consistent with alternative splicing were identified by RT-PCR in a cell line or cell type-specific pattern. All evaluated cells expressed mRNA containing all 14 exons of 5-LO with the expected splicing sites. Individual isoforms that retained intron 10 (α-10), lacked exon 13 (Δ-13), and lacked exons 10 and 13 (Δ-10,13) or that lacked the first 96 base pairs of exon 10 (Δ-p10) were identified. Immunoreactive bands coeluting with the cloned α-10 and Δ-13 isoforms were measured in primary neutrophils and in Raji cells. When expressed in HEK293 cells, alternative proteins were without catalytic activity. However, when coexpressed with the active full-length 5-LO, alternative isoforms significantly decreased the biosynthesis of 5-LO products by up to 44%, as assessed by reverse-phase HPLC analysis. Additionally, in stimulated neutrophils the full-length active 5-LO was detected by immunoblot in both nuclear and non-nuclear compartments, while the Δ-13 isoform was only detected in the nuclear fraction. These alternative 5-LO isoforms may represent a new mechanism for the regulation of the 5-LO pathway and lipid mediator biosynthesis.
authors.affiliation: Département de Chimie et Biochimie. Université de Moncton, Moncton, NB, E1A 3E9, Canada.
authors.forename: Luc H
authors.initials: LH
authors.lastname: Boudreau
authors.forename: Jonathan
authors.initials: J
authors.lastname: Bertin
authors.forename: Philippe P
authors.initials: PP
authors.lastname: Robichaud
authors.forename: Mark
authors.initials: M
authors.lastname: Laflamme
authors.forename: Rodney J
authors.initials: RJ
authors.lastname: Ouellette
authors.forename: Nicolas
authors.initials: N
authors.lastname: Flamand
authors.forename: Marc E
authors.initials: ME
authors.lastname: Surette
mesh_terms: D017398:Alternative Splicing / Q000502:physiology; D001094:Arachidonate 5-Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D001402:B-Lymphocytes / Q000166:cytology; D002051:Burkitt Lymphoma; D002461:Cell Line, Transformed; D015777:Eicosanoids / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D005091:Exons / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D057809:HEK293 Cells; D006801:Humans; D007249:Inflammation / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007948:Leukemia, Monocytic, Acute; D015289:Leukotrienes / Q000378:metabolism; D009504:Neutrophils / Q000166:cytology / Q000201:enzymology* / Q000276:immunology; D012333:RNA, Messenger / Q000235:genetics
chemical_list: D015777:Eicosanoids; D007527:Isoenzymes; D015289:Leukotrienes; D012333:RNA, Messenger; D001094:Arachidonate 5-Lipoxygenase