Strict Regiospecificity of Human Epithelial 15-Lipoxygenase-2 Delineates Its Transcellular Synthesis Potential

Abstract

Lipoxins are an important class of lipid mediators that induce the resolution of inflammation and arise from transcellular exchange of arachidonic acid (AA)-derived lipoxygenase products. Human epithelial 15-lipoxygenase-2 (h15-LOX-2), the major lipoxygenase in macrophages, has exhibited strict regiospecificity, catalyzing only the hydroperoxidation of carbon 15 of AA. To determine the catalytic potential of h15-LOX-2 in transcellular synthesis events, we reacted it with the three lipoxygenase-derived monohydroperoxy-eicosatetraenoic acids (HPETE) in humans: 5-HPETE, 12-HPETE, and 15-HPETE. Only 5-HPETE was a substrate for h15-LOX-2, and the steady-state catalytic efficiency (kcat/Km) of this reaction was 31% of the kcat/Km of AA. The only major product of h15-LOX-2's reaction with 5-HPETE was the proposed lipoxin intermediate, 5,15-dihydroperoxy-eicosatetraenoic acid (5,15-diHPETE). However, h15-LOX-2 did not react further with 5,15-diHPETE to produce lipoxins. This result is consistent with the specificity of h15-LOX-2 despite the increased reactivity of 5,15-diHPETE. Density functional theory calculations determined that the radical, after abstracting the C10 hydrogen atom from 5,15-diHPETE, had an energy 5.4 kJ/mol lower than that of the same radical generated from AA, demonstrating the facility of 5,15-diHPETE to form lipoxins. Interestingly, h15-LOX-2 does react with 5S,6R-diHETE, forming LipoxinA4, indicating the gemdiol does not prohibit h15-LOX-2 reactivity. Taken together, these results demonstrate the strict regiospecificity of h15-LOX-2 that circumscribes its role in transcellular synthesis.

Figure 1

h15-LOX-2 converts 5-HPETE to 5,15-diHPETE. (A) Total ion count (TIC) chromatogram of h15-LOX-2’s reaction with 5-HPETE, which displays ions with a parent m/z of 335.2. The large peak has a retention time of 8.9 min and a λ_max value of 245 nm, as seen in the UV–vis spectra of the peak (inset), which matches the 5,15-diHETE standard. (B) Mass spectrum of the peak at 8.9 min. The diagnostic peaks for 5,15-diHETE are bolded and boxed.

Figure 2

Structures of in silico models and dehydrogenation mechanisms. (1) Model m1 represents arachidonic acid’s conjugated system and the two hydrogen atom abstractions A and B that result in two radical structures with similar energies. (2) Model m2 represents 5-HPETE and 15-HPETE’s conjugated system and the two hydrogen atom abstractions A and B that result in two radical structures with different energies. (3) Model m3 represents 5,15-diHPETE and the resulting low energy radical structure from a C10 hydrogen atom abstraction.

Figure 3

h15-LOX-2 cannot synthesize lipoxins from the 5,15-diHPETE intermediate. A TIC chromatogram of the reactions of h15-LOX-2 (solid line) and h15-LOX-1 (dashed line) with 5,15-diHPETE, displaying ions with a parent m/z of 351.2. In the h15-LOX-1 reactions, LipoxinB₄ and -A₄ peaks were confirmed with retention times, UV–vis spectra, and MS spectra as compared to the standards. The tiny peak at the retention time of LxA₄ in the h15-LOX-2 reaction was not LxA₄, as determined by its MS² spectra. Thus, no lipoxin peaks were seen for h15-LOX-2.

Figure 4

h15-LOX-2 converts 5S,6R-diHETE to LipoxinA₄. (A) A TIC chromatogram of h15-LOX-2’s reaction with 5S,6R-diHETE, displaying ions with a parent m/z of 351.2. The large peak has a retention time of 3.9 min and a λ_max value of 302 nm, as seen in the UV–vis spectrum of the peak (inset), which matches the LipoxinA₄ standard. (B) Mass spectrum of the peak at 3.9 min. The diagnostic peaks for LipoxinA₄ are bolded and boxed.

Scheme 1. Biosynthetic Routes to Lipoxins^a

^aLipoxinA₄, LipoxinB₄, and their respective isomers can arise from arachidonic acid (AA) through the pathways laid out in this scheme. The names of major products are boxed, such as LTA₄ and LxB₄. Enzymes are listed above the arrows of the reactions performed. The arrows with no enzyme listed are hydrolysis reactions that either are catalyzed by soluble epoxide hydrolase or are nonenzymatic, as indicated by the products. The numerous products observed that arise from nonenzymatic hydrolysis, indicated by a single asterisk, are listed in Figure S2. Reactions that h15-LOX-2 can perform are indicated by two asterisks; however, these reactions can also be performed by h15-LOX-1. Please note that this scheme has been simplified. For example, the required reductions of the hydroperoxy moieties to hydroxyl moieties are typically performed by glutathione peroxidase. These reactions, along with the 14R-oxygenase and 6R-oxygenase activities of h12-LOX and h5-LOX, have been excluded for clarity.

Scheme 2. Positional Specificity of 15-Lipoxygenases^a

^a(A) h15-LOX-2 has demonstrated the ability to only abstract the hydrogen atom at C13 and allow oxygen attack at C15 (solid arrows) of arachidonic acid. (B) h15-LOX-1 can abstract a hydrogen atom from C13 and oxygenate at C15 as well but also abstracts the C10 hydrogen atom and facilitates oxygen attack at the C12 position (dashed arrows).