Identification and Characterization of a New Protein Isoform of Human 5-Lipoxygenase
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Identification and Characterization of a New Protein Isoform of Human 5-Lipoxygenase. / Häfner, Ann-Kathrin; Beilstein, Kim; Graab, Philipp; Ball, Ann-Katrin; Saul, Meike J; Hofmann, Bettina; Steinhilber, Dieter.
in: PLOS ONE, Jahrgang 11, Nr. 11, 2016, S. e0166591.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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TY - JOUR
T1 - Identification and Characterization of a New Protein Isoform of Human 5-Lipoxygenase
AU - Häfner, Ann-Kathrin
AU - Beilstein, Kim
AU - Graab, Philipp
AU - Ball, Ann-Katrin
AU - Saul, Meike J
AU - Hofmann, Bettina
AU - Steinhilber, Dieter
PY - 2016
Y1 - 2016
N2 - 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.
AB - 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.
KW - Arachidonate 5-Lipoxygenase/chemistry
KW - Cell Line, Tumor
KW - Chromatography, High Pressure Liquid
KW - Enzyme Stability
KW - Escherichia coli/metabolism
KW - HEK293 Cells
KW - Humans
KW - Iron/metabolism
KW - Isoenzymes/chemistry
KW - Models, Molecular
KW - Recombinant Proteins/metabolism
KW - Temperature
U2 - 10.1371/journal.pone.0166591
DO - 10.1371/journal.pone.0166591
M3 - SCORING: Journal article
C2 - 27855198
VL - 11
SP - e0166591
JO - PLOS ONE
JF - PLOS ONE
SN - 1932-6203
IS - 11
ER -