The Rat Leukocyte-type 12-Lipoxygenase Exhibits an Intrinsic Hepoxilin A3 Synthase Activity

Abstract

Hepoxilins are biologically relevant eicosanoids formed via the 12-lipoxygenase pathway of the arachidonic acid cascade. Although these eicosanoids exhibit a myriad of biological activities, their biosynthetic mechanism has not been investigated in detail. We examined the arachidonic acid metabolism of RINm5F rat insulinoma cells and found that they constitutively express a leukocyte-type 12S-lipoxygenase. Moreover, we observed that RINm5F cells exhibit an active hepoxilin A 3 synthase that converts exogenous 12S-HpETE (12S-5Z,8-Z,10E,14Z-12-hydro(pero)xy-eicosa-5,8,10,14-tetraenoic acid) or arachidonic acid predominantly to hepoxilin A 3 . 12S-lipoxygenase and hepoxilin A 3 synthase activities were co-localized in the cytosol; immunoprecipitation with an anti-12S-lipoxygenase antibody co-precipitated the two catalytic activities. These data suggested that hepoxilin A 3 synthase activity may be considered an intrinsic catalytic property of the leukocyte-type 12S-lipoxygenase. To test this hypothesis we cloned the leukocyte-type 12S-LOX from RINm5F cells, expressed it in Pichia pastoris, and found that the recombinant enzyme exhibited both 12S-lipoxygenase and hepoxilin A 3 synthase activities. The recombinant human platelet-type 12S-lipoxygenase and the porcine leukocyte-type 12S-lipoxygenase also exhibited hepoxilin A 3 synthase activity. In contrast, the native rabbit reticulocyte-type 15S-lipoxygenase did not convert 12S-HpETE to hepoxilin isomers. These data suggest that the positional specificity of lipoxygenases may be crucial for this catalytic function. This hypothesis was confirmed by site-directed mutagenesis studies that altered the positional specificity of the rat leukocyte-type 12S-and the rabbit reticulocyte-type 15-lipoxygenase. In summary, it may be concluded that naturally occurring 12S-lipoxygenases exhibit an intrinsic hepoxilin A 3 synthase activity that is minimal in lipoxygenase isoforms with different positional specificity.

8R-Lipoxygenase-catalyzed synthesis of a prominent cis-epoxyalcohol from dihomo-γ-linolenic acid: a distinctive transformation compared with S-lipoxygenases

“…Although heretofore only trans -epoxyalcohols have been reported from lipoxygenase catalysis (e.g., 23,28,[39][40][41], other enzymes can make the cis -epoxides. The majority of these are mechanistically quite distinct, however, because the epoxide is formed via oxygen transfer.…”

Hepoxilin A3 (HXA3) synthase deficiency is causative of a novel ichthyosis form

“…3B). This activity is impaired when positional specificity of AA was altered [9]. In HXA 3 synthase amino acid Leu353 was found to be crucial sequence determinant for AA oxygenation [12], and not the conventional sequence determinants Met419 and Ile418 as shown for porcine leukocyte-type 12S-LOX, human platelet-type 12S-LOX or rabbit leukocyte-type 15S-LOX-1 [13].…”

“…Recombinant rat HXA 3 synthase (30 nM) was incubated with 50 lg 12R-HpETE in 500 ll PBS at 37°C for 30 min. Lipids were extracted, purified by HPLC and assayed Me-TMS derivatives by GC-MS as described earlier [9]. obtained through 12R-LOX pathway.…”

“…We therefore hypothesized that in addition to 12R-LOX the HXA 3 synthase, that converts 12S-HpETE to 8S-HXA 3 , is inevitably required to maintain epidermal permeability barrier function. HXA 3 synthase was recently identified by us as an intrinsic activity in rat leukocyte-type and platelettype 12S-LOXs, but not in porcine leukocyte-type 12S-LOX or rabbit leukocyte-type 15S-LOX [9]. Taking this for granted, mutations in any of the gene products 12R-LOX, eLOX3 or HXA 3 synthase would de facto lead to HXALI and/or other ARCIs.…”

“…For purification see details in [9]. After Ni-agarose column step the enzyme was enriched to 90% of the activity.…”