title: Identification and Characterization of a New Protein Isoform of Human 5-Lipoxygenase.
abstract: Leukotrienes (LTs) are inflammatory mediators that play a pivotal role in many diseases like asthma bronchiale, atherosclerosis and in various types of cancer. The key enzyme for generation of LTs is the 5-lipoxygenase (5-LO). Here, we present a novel putative protein isoform of human 5-LO that lacks exon 4, termed 5-LOΔ4, identified in cells of lymphoid origin, namely the Burkitt lymphoma cell lines Raji and BL41 as well as primary B and T cells. Deletion of exon 4 does not shift the reading frame and therefore the mRNA is not subjected to non-mediated mRNA decay (NMD). By eliminating exon 4, the amino acids Trp144 until Ala184 are omitted in the corresponding protein. Transfection of HEK293T cells with a 5-LOΔ4 expression plasmid led to expression of the corresponding protein which suggests that the 5-LOΔ4 isoform is a stable protein in eukaryotic cells. We were also able to obtain soluble protein after expression in E. coli and purification. The isoform itself lacks canonical enzymatic activity as it misses the non-heme iron but it still retains ATP-binding affinity. Differential scanning fluorimetric analysis shows two transitions, corresponding to the two domains of 5-LO. Whilst the catalytic domain of 5-LO WT is destabilized by calcium, addition of calcium has no influence on the catalytic domain of 5-LOΔ4. Furthermore, we investigated the influence of 5-LOΔ4 on the activity of 5-LO WT and proved that it stimulates 5-LO product formation at low protein concentrations. Therefore regulation of 5-LO by its isoform 5-LOΔ4 might represent a novel mechanism of controlling the biosynthesis of lipid mediators.
authors.affiliation: Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438, Frankfurt, Germany.
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.
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: Philipp
authors.initials: P
authors.lastname: Graab
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.|Department of Biology, Technical University of Darmstadt, 64287, Darmstadt, Germany.
authors.forename: Meike J
authors.initials: MJ
authors.lastname: Saul
authors.affiliation: Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438, Frankfurt, Germany.
authors.forename: Bettina
authors.initials: B
authors.lastname: Hofmann
authors.affiliation: Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438, Frankfurt, Germany.
authors.forename: Dieter
authors.initials: D
authors.lastname: Steinhilber
mesh_terms: D001094:Arachidonate 5-Lipoxygenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007501:Iron / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature
chemical_list: D007527:Isoenzymes; D011994:Recombinant Proteins; D007501:Iron; D001094:Arachidonate 5-Lipoxygenase