5 S,15 S-Dihydroperoxyeicosatetraenoic Acid (5,15-diHpETE) as a Lipoxin Intermediate: Reactivity and Kinetics with Human Leukocyte 5-Lipoxygenase, Platelet 12-Lipoxygenase, and Reticulocyte 15-Lipoxygenase-1

Abstract

The reaction of 5 S,15 S-dihydroperoxyeicosatetraenoic acid (5,15-diHpETE) with human 5-lipoxygenase (LOX), human platelet 12-LOX, and human reticulocyte 15-LOX-1 was investigated to determine the reactivity and relative rates of producing lipoxins (LXs). 5-LOX does not react with 5,15-diHpETE, although it can produce LXA₄ when 15-HpETE is the substrate. In contrast, both 12-LOX and 15-LOX-1 react with 5,15-diHpETE, forming specifically LXB₄. For 12-LOX and 5,15-diHpETE, the kinetic parameters are k_cat = 0.17 s^-1 and k_cat/ K_M = 0.011 μM^-1 s^-1 [106- and 1600-fold lower than those for 12-LOX oxygenation of arachidonic acid (AA), respectively]. On the other hand, for 15-LOX-1 the equivalent parameters are k_cat = 4.6 s^-1 and k_cat/ K_M = 0.21 μM^-1 s^-1 (3-fold higher and similar to those for 12-HpETE formation by 15-LOX-1 from AA, respectively). This contrasts with the complete lack of reaction of 15-LOX-2 with 5,15-diHpETE [Green, A. R., et al. (2016) Biochemistry 55, 2832-2840]. Our data indicate that 12-LOX is markedly inferior to 15-LOX-1 in catalyzing the production of LXB₄ from 5,15-diHpETE. Platelet aggregation was inhibited by the addition of 5,15-diHpETE, with an IC₅₀ of 1.3 μM; however, LXB₄ did not significantly inhibit collagen-mediated platelet activation up to 10 μM. In summary, LXB₄ is the primary product of 12-LOX and 15-LOX-1 catalysis, if 5,15-diHpETE is the substrate, with 15-LOX-1 being 20-fold more efficient than 12-LOX. LXA₄ is the primary product with 5-LOX but only if 15-HpETE is the substrate. Approximately equal proportions of LXA₄ and LXB₄ are produced by 12-LOX but only if LTA₄ is the substrate, as described previously [Sheppard, K. A., et al. (1992) Biochim. Biophys. Acta 1133, 223-234].

Figure 1:. Biosynthetic pathways for LX production.

The carbon whose hydrogen atom is abstracted is listed next to the LOX isozyme, such as C7, C10 or C13. For the conversion of 5,15-DiHpETE to the two lipoxins, the LOX isozyme is not specified because that is the subject of this publication.

Figure 2:. UPLC-UV-Vis chromatogram of h12-LOX (A) and h15-LOX-1 (B) products from 5,15-diHpETE.

The wavelength of 302 nm is the absorbance maximum for lipoxins (Supplemental S1). At 302 nm, 11 peaks are observed in the unreduced (solid line) reaction that coalesce to 2 clusters of peaks upon reduction (dashed line). Based on the UV-Vis spectrum, retention times, parent masses, and MS/MS spectra (Supplemental S2), the identities of these peaks have been determined to be all-trans-LxB₄ and its 14S-epimer at 7.9 min, LxB₄ and its 14S-epimer at 8.6 min and 5,14,15-trihydroperoxy-LxB₄ at 13.6 min. The peaks at 9.5, 9.8, 10.0, 10.3 and 11.5 min are the monohydroperoxide products and the peaks at 11.3, 12.2, and 13.2 min are the dihydroperoxide products. *Note: although only one double bond stereochemistry is depicted for these compounds, the differing retention times suggest rearrangements. MS/MS spectra contain fragments that indicate the hydroperoxide moiety’s position may differ between these mono- and dihydroperoxy-HETEs.

Figure 3:. UPLC-UV-Vis chromatogram of h12-LOX products from 5,15-diHETE.

The wavelength of 302 nm is the absorbance maximum for lipoxins (Supplemental S1). At this wavelength, three peaks, at times 8.1, 8.8, and 9.8 min, are observed in the unreduced reaction (solid line) that coalesce to two peaks, 8.1 and 8.8 min, upon reduction (dashed line). Based on the UV-Vis spectrum, retention times, parent mass, and MS/MS spectra (Supplemental S2), the identities of these peaks have been determined to be all-trans-LxB₄ at 8.1, LxB₄ at 8.8 and 14-hydroperoxy-LxB₄ at 9.8 min.

Figure 4:. 5,15-diHpETE, but not LXB₄, inhibits collagen-mediated platelet aggregation.

Isolated human platelets were treated with increasing concentrations of either LXB₄ or 5,15-diHpETE for ten minutes and then stimulated with collagen (0.25 µg/mL). A) Representative tracings of 5,15-diHpETE or LXB₄ treated platelets stimulated with collagen. B) Data represents mean ± S.E.M. Statistical analysis was performed comparing oxylipin concentration to vehicle control using one-way ANOVA with Dunnett’s multiple comparison test. *P<0.05, **P<0.01, ***P<0.001.

Figure 5:. Platelets produce LxB4 from 5,15-diHPETE.

The amount of LxB₄ produced by platelets treated with 5, 10 or 20 µM of 5,15-diHPETE are plotted in this histogram. The standard deviation of each value is represented by error bars.

Figure 6:. Reaction scheme for LXA4 and LXB4 production.

The production of LXA4 and LXB4 is dependent on both the order of LOX reactivity and the nature of the substrate. The carbon atom from which the hydrogen atom is abstracted is indicated below the LOX isozyme label, such as C7, C10 or C13. The central pathway which involves 5(S),15(S)-diHpETE is the subject of this publication.