title: Characterization and cellular localization of human 5-lipoxygenase and its protein isoforms 5-LOΔ13, 5-LOΔ4 and 5-LOp12.
abstract: Human 5-lipoxygenase (5-LO-WT) initiates the leukotriene (LT) biosynthesis. LTs play an important role in diseases like asthma, atherosclerosis and in many types of cancer. In this study, we investigated the 5-LO isoforms 5-LO∆13, 5-LO∆4 and 5-LOp12, lacking the exons 13, 4 or a part of exon 12, respectively. We were able to detect the mRNA of the isoforms 5-LO∆13 and 5-LOp12 in B and T cell lines as well as in primary B and T cells and monocytes. Furthermore, we found that expression of 5-LO and particularly of the 5-LO∆13 and 5-LOp12 isoforms is increased in monocytes from patients with rheumatoid arthritis and sepsis. Confocal microscopy of HEK293T cells stably transfected with tagged 5-LO-WT and/or the isoforms revealed that 5-LO-WT is localized in the nucleus whereas all isoforms are located in the cytosol. Additionally, all isoforms are catalytically inactive and do not seem to influence the specific activity of 5-LO-WT. S271A mutation in 5-LO-WT and treatment of the cells with sorbitol or KN-93/SB203580 changes the localization of the WT enzyme to the cytosol. Despite colocalization with the S271A mutant, the isoforms did not affect LT biosynthesis. Analysis of the phosphorylation pattern of 5-LO-WT and all the isoforms revealed that 5-LOp12 and 5-LO∆13 are highly phosphorylated at Ser271 and 5-LOp12 at Ser523. Furthermore, coexpression of the isoforms inhibited or stimulated 5-LO-WT expression in transiently and stably transfected HEK293T cells suggesting that the isoforms have other functions than canonical LT biosynthesis.
authors.affiliation: Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany.
authors.forename: Ann-Katrin
authors.initials: AK
authors.lastname: Ball
authors.affiliation: Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany.
authors.forename: Kim
authors.initials: K
authors.lastname: Beilstein
authors.affiliation: Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Wittmann
authors.affiliation: Department of Molecular Hematology, Goethe University Medical School, 60590 Frankfurt am Main, Germany.
authors.forename: Duran
authors.initials: D
authors.lastname: Sürün
authors.affiliation: Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany.
authors.forename: Meike J
authors.initials: MJ
authors.lastname: Saul
authors.affiliation: Department of Molecular Hematology, Goethe University Medical School, 60590 Frankfurt am Main, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Schnütgen
authors.affiliation: Centre de recherche de l'IUCPQ, Département de Médecine et Faculté de Médecine, Université Laval, Québec, QC G1V 4G5, Canada.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Flamand
authors.affiliation: Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Capelo
authors.affiliation: Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany.
authors.forename: Astrid S
authors.initials: AS
authors.lastname: Kahnt
authors.affiliation: General Pharmacology and Toxicology, Goethe-University Frankfurt, Theodor-Stern Kai 7, 60590 Frankfurt, Germany.
authors.forename: Helena
authors.initials: H
authors.lastname: Frey
authors.affiliation: General Pharmacology and Toxicology, Goethe-University Frankfurt, Theodor-Stern Kai 7, 60590 Frankfurt, Germany.
authors.forename: Liliana
authors.initials: L
authors.lastname: Schaefer
authors.affiliation: Institute of Pharmaceutical Biology, Goethe-University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany.
authors.forename: Rolf
authors.initials: R
authors.lastname: Marschalek
authors.affiliation: Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany. Electronic address: haefner@pharmchem.uni-frankfurt.de.
authors.forename: Ann-Kathrin
authors.initials: AK
authors.lastname: Häfner
authors.affiliation: Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany. Electronic address: steinhilber@em.uni-frankfurt.de.
authors.forename: Dieter
authors.initials: D
authors.lastname: Steinhilber
mesh_terms: D001094:Arachidonate 5-Lipoxygenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure*; D003600:Cytosol / Q000378:metabolism / Q000648:ultrastructure*; D015971:Gene Expression Regulation, Enzymologic; D057809:HEK293 Cells; D006801:Humans; D015289:Leukotrienes / Q000096:biosynthesis; D009504:Neutrophils / Q000378:metabolism / Q000648:ultrastructure; D010766:Phosphorylation; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D015289:Leukotrienes; D020033:Protein Isoforms; D001094:Arachidonate 5-Lipoxygenase
keywords: 5-Lipoxygenase; Alternative splicing; Confocal microscopy; Inflammation; Protein isoforms