5S,15S-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 5,15-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 LXA 4 when 15-HpETE is the substrate. In contrast, both 12-LOX and 15-LOX-1 react with 5,15-diHpETE, forming specifically LXB 4. 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-1s-1 (106-fold and 1600-fold lower than for 12-LOX oxygenation of 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-1s-1 (3-fold higher and similar to that 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 (Biochemistry 55, 2832-2840, 2016). Our data indicate that 12-LOX is markedly inferior to 15-LOX-1 in catalyzing the production of LXB 4 from 5,15-diHpETE. Platelet aggregation was inhibited by the addition of 5,15-diHpETE, with an IC50 of 1.3 μM, however, LXB 4 did not significantly inhibit collagen-mediated platelet activation up to 10 μM. In summary, LXB 4 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. LXA 4 is the primary product with 5-LOX, but only if 15-HpETE is the substrate. Approximately equal proportions of LXA 4 and LXB 4 are produced by 12-LOX, but only if LTA 4 is the substrate, as described previously (Biochimica et Biophysica Acta 1133, 223-234, 1992).

Altered specificity of 15-LOX-1 in the biosynthesis of 7S,14S-diHDHA implicates 15-LOX-2 in biosynthesis of resolvin D5

“…Although changes in the positional specificity of LOXs have been demonstrated before (19), the finding that h15-LOX-1 primarily oxygenates C17 on DHA, but C14 on 7S-HDHA is surprising. In order to obtain a better understanding of this binding event, DHA and 7S-HDHA were docked to a model of h15-LOX-1.…”

“…Western blots were performed using rabbit anti-5-lipoxygenase polyclonal (Cayman chemicals) primary antibody diluted 1:500 and goat anti-rabbit- HpDHA (ε234nm = 25,000 M -1 cm -1 ) and 270 nm for 7S,14S-diHDHA (ε270nm = 40,000 M -1 cm -1 )(32, 81,82). 7S,17S-diHDHA has an absorbance max of 245 nm, however, due to overlap with the substrate peak at 234 nm formation of this product was measured at 254 nm using an extinction coefficient of 21,900 M -1 cm -1 to adjust for the decreased rate of absorbance change at this peak shoulder (19). KaleidaGraph (Synergy) was used to fit initial rates (at less than 20% turnover), as well as the second order derivatives In order to determine if 7S-HDHA was enzymatically converted to another chemical ex vivo, one mL of platelets (1.0 x10 9 platelets/mL) was incubated with either 10 µM 7S-HDHA, 10 µM 5S-HETE (positive control) or vehicle (DMSO) for 10 minutes at 37° C and then pelleted by centrifugation at 1000g for 2 minute.…”

“…In particular, 7S,14S-diHDHA appeared to be biosynthesized in a di-oxygenation fashion without an epoxide intermediate, distinct from the other analogues. Its structure suggests that it is synthesized through two sequential LOX oxygenation reactions at C7 and C14, without the requirement of a hydrolase to open the epoxide intermediate (19)(20)(21). Given these oxygenation sites, as well as the enzymatic preferences of specific LOX isozymes, it is reasonable to assume that the biosynthesis of 7S,14S-diHDHA involves h5-LOX oxidizing DHA at C7, with h12-LOX oxidizing at C14, but it is unclear which oxidation occurs first (Scheme 1, Pathway 2).…”

“…For example, h5-LOX produces primarily 5(S)-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5S-HpETE) from AA, but it produces multiple products when DHA is the substrate (35), presumably due to the length and unsaturation differences between AA and DHA, which presumably affects substrate positioning for hydrogen atom abstraction. LOX's also vary widely in their ability to tolerate oxygenated substrates, as reflected in their kinetic parameters (19). In addition, the specific hydrogen atom abstracted can be affected by the nature of the substrate (34).…”

<p>Eicosapentaenoic acid’s metabolism of 15-LOX-1 promotes the expression of miR-101 thus inhibits Cox2 pathway in colon cancer</p>

“…7 Most studies on 15-LOX-1 focus on linoleic acid metabolites formed by 15-LOX-1 catalysis, such as 13-HODE, 8 PGE2, 9 and LXA4. 10 Little is known about the role of resolvins in CC risk. 11 A recent study found that resolvins could affect cholangiocarcinoma cell proliferation, but the specific mechanism still requires further exploration.…”