Eicosanoid Diversity of Stony Corals

Oxylipins are well-established lipid mediators in plants and animals. In mammals, arachidonic acid (AA)-derived eicosanoids control inflammation, fever, blood coagulation, pain perception and labor, and, accordingly, are used as drugs, while lipoxygenases (LOX), as well as cyclooxygenases (COX) serve as therapeutic targets for drug development. In soft corals, eicosanoids are synthesized on demand from AA by LOX, COX, and catalase-related allene oxide synthase-lipoxygenase (cAOS-LOX) and hydroperoxide lyase-lipoxygenase (cHPL-LOX) fusion proteins. Reef-building stony corals are used as model organisms for the stress-related genomic studies of corals. Yet, the eicosanoid synthesis capability and AA-derived lipid mediator profiles of stony corals have not been determined. In the current study, the genomic and transcriptomic data about stony coral LOXs, AOS-LOXs, and COXs were analyzed and the eicosanoid profiles and AA metabolites of three stony corals, Acropora millepora, A. cervicornis, and Galaxea fascicularis, were determined by reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with MS-MS and a radiometric detector. Our results confirm that the active LOX and AOS-LOX pathways are present in Acropora sp., which correspond to the genomic/sequence data reported earlier. In addition, LOX, AOS-LOX, and COX products were detected in the closely related species G. fascicularis. In conclusion, the functional 8R-LOX and/or AOS-LOX pathways are abundant among corals, while COXs are restricted to certain soft and stony coral lineages

The differences between the residues of coral cAOSs and <i>C</i>. <i>imbricata</i> cHPL.

<p><b>A</b>–alignment of amino acid sequences of coral cAOSs and <i>C</i>. <i>imbricata</i> cHPL presenting the conserved (blue) and distinct (grey vs red, respectively) residues in the substrate channel. The following amino acid sequences were compared: <i>Gersemia fruticosa</i> cAOS (NCBI ID: EU082210.1); <i>P</i>. <i>homomalla</i> cAOS (NCBI ID: AF003692.1); <i>Clavularia viridis</i> cAOS (NCBI ID: AB188528.1); <i>C</i>. <i>imbricata</i> cAOS (NCBI ID: KF000373.1); <i>C</i>. <i>imbricata</i> cHPL (NCBI ID: KF000374.1). <b>B–</b>the crystal structure of <i>P</i>. <i>homomalla</i> cAOS (blue) superpositioned with the model of <i>C</i>. <i>imbricata</i> cHPL (red) highlighting the main differences in the substrate pocket. The difference in the SSSAGE155-160PVKEGD fragments is not shown due to the illustrative purposes. The conserved amino acids between cAOS and cHPL are presented as a white and grey backbone, respectively. The heme is presented in yellow and the heme iron in orange.</p

SDS-PAGE analysis of purified wt cHPL and corresponding mutants.

<p>Protein samples with an equal heme concentration (0.4 μM) were compared.</p

The oligomerization analysis of <i>C</i>. <i>imbricata</i> protein samples.

<p><b>A</b>—wt cHPL; <b>B</b>—cHPL YS176-177NL. Similar oligomerization states were determined also for cHPL ME59-60LK, P65A, F150L. <b>C</b>—cHPL I357V; <b>D</b>—cHPL R56G; <b>E</b>—wt cHPL-LOX; <b>F</b>—wt cAOS; <b>G</b>—cAOS L150F. Oligomers and monomers of wt cHPL and mutants eluted at 8 and 16 min, respectively.</p

The docking analysis of <i>P</i>. <i>homomalla</i> cAOS with 8<i>R</i>-HpETE and AO.

<p><b>A</b>– 8<i>R</i>-HpETE located in the substrate pocket; <b>B</b>–the rotated view of 8<i>R</i>-HpETE in the substrate pocket with a hydrophobic surface; <b>C</b>–AO in the substrate pocket; <b>D–</b>an alternative positioning of AO in the substrate pocket. The colors used in the figure are presented as follows: heme–yellow; heme iron–orange; ligands–green; the interacting residues of <i>P</i>. <i>homomalla</i> cAOS–blue; the interacting residues of <i>C</i>. <i>imbricata</i> cHPL–cyan; residues supporting the coordination of ligand or heme–gray; oxygen atoms–red; nitrogen atoms–blue. For clarity, the distances between the ligand and selected residues are given in the text.</p

Up-regulated expression of AOS-LOXa and increased eicosanoid synthesis in response to coral wounding.

In octocorals, a catalase-like allene oxide synthase (AOS) and an 8R-lipoxygenase (LOX) gene are fused together encoding for a single AOS-LOX fusion protein. Although the AOS-LOX pathway is central to the arachidonate metabolism in corals, its biological function in coral homeostasis is unclear. Using an acute incision wound model in the soft coral Capnella imbricata, we here test whether LOX pathway, similar to its role in plants, can contribute to the coral damage response and regeneration. Analysis of metabolites formed from exogenous arachidonate before and after fixed time intervals following wounding indicated a significant increase in AOS-LOX activity in response to mechanical injury. Two AOS-LOX isoforms, AOS-LOXa and AOS-LOXb, were cloned and expressed in bacterial expression system as active fusion proteins. Transcription levels of corresponding genes were measured in normal and stressed coral by qPCR. After wounding, AOS-LOXa was markedly up-regulated in both, the tissue adjacent to the incision and distal parts of a coral colony (with the maximum reached at 1 h and 6 h post wounding, respectively), while AOS-LOXb was stable. According to mRNA expression analysis, combined with detection of eicosanoid product formation for the first time, the AOS-LOX was identified as an early stress response gene which is induced by mechanical injury in coral

The products derived from the radiolabeled 8<i>R</i>-HpETE substrate by wt cHPL, selected cHPL mutants, and cAOS L150F.

<p><b>A</b>– C8-oxo acid formed by wt cHPL representing also the products formed by cHPL R56G, P65A, ME59-60LK, and SSSAGE155-160PVKEG; <b>B</b>– C8-oxo acid and α-ketol formed by cHPL F150L; <b>C</b>– C8-oxo acid, α-ketol and product 1 and product 2 formed by cHPL YS176-177NL; <b>D</b>–α-ketol formed by cAOS L150F. The product pattern is identical to wt cAOS’s (data not shown).</p

Structural and functional insights into the reaction specificity of catalase-related hydroperoxide lyase: A shift from lyase activity to allene oxide synthase by site-directed mutagenesis

<div><p>Two highly identical fusion proteins, an allene oxide synthase-lipoxygenase (AOS-LOX) and a hydroperoxide lyase-lipoxygenase (HPL-LOX), were identified in the soft coral <i>Capnella imbricata</i>. Both enzymes initially catalyze the formation of 8<i>R</i>-hydroperoxy-eicosatetraenoic acid (8<i>R</i>-HpETE) from arachidonic acid by the C-terminal lipoxygenase (LOX) domain. Despite the fact that the defined catalytically important residues of N-terminal catalase-related allene oxide synthase (cAOS) domain are also conserved in <i>C</i>. <i>imbricata</i> hydroperoxide lyase (cHPL), their reaction specificities differ. In the present study, we tested which of the amino acid substitutions around the active site of cHPL are responsible for a control in the reaction specificity. The possible candidates were determined via comparative sequence and structural analysis of the substrate channel and the heme region of coral cAOSs and <i>C</i>. <i>imbricata</i> cHPL. The amino acid replacements in cHPL—R56G, ME59-60LK, P65A, F150L, YS176-177NL, I357V, and SSSAGE155-160PVKEGD—with the corresponding residues of cAOS were conducted by site-directed mutagenesis. Although all these mutations influenced the catalytic efficiency of cHPL, only F150L and YS176-177NL substitutions caused a shift in the reaction specificity from HPL to AOS. The docking analysis of <i>P</i>. <i>homomalla</i> cAOS with 8<i>R</i>-HpETE substrate revealed that the Leu150 of cAOS interacts with the C5-C6 double bond and the Leu177 with the hydrophobic tail of 8<i>R</i>-HpETE. We propose that the corresponding residues in cHPL, Phe150 and Ser177, are involved in a proper coordination of the epoxy allylic radical intermediate necessary for aldehyde formation in the hydroperoxide lyase reaction.</p></div

Kinetic parameters of wt cHPL, wt cAOS and selected mutants with 8<i>R</i>-HpETE and H₂O₂.

<p>Kinetic parameters of wt cHPL, wt cAOS and selected mutants with 8<i>R</i>-HpETE and H₂O₂.</p

The proposed difference in reaction mechanisms between coral cAOS and cHPL.

<p>The hydrogen abstraction at C9 of the epoxy allylic carbocation is initiated by His67 of cAOS (blue). In cHPL-catalyzed reaction (red), no hydrogen abstraction occurs and instead, an unstable hemiacetal forms via the breakage of C8-C9 bond of the epoxide and the subsequent rebound of hydroxide.</p