Differential characteristics of human 15‐lipoxygenase isozymes and a novel splice variant of 15S‐lipoxygenase

Abstract

The lipoxygenases (LOs) are a family of nonheme iron dioxygenases that catalyse the insertion of molecular oxygen into polyunsaturated fatty acids. Five members of this gene family have been described in man, 5-LO, 12S-LO, 12R-LO, 15-LO and 15S-LO. Using partially purified recombinant 15S-LO enzyme and cells constitutively expressing this protein, we have compared the activity, substrate specificity, kinetic characteristics and regulation of this enzyme to that previously reported for 15-LO. 15S-LO has a threefold higher K m , similar V max and increased specificity of oxygenation for arachidonic acid, and a similar K m but decreased V max for linoleic acid in comparison to 15-LO. Unlike 15-LO, 15S-LO is not suicide inactivated by the products of fatty acid oxygenation. However, in common with other LOs, 15S-LO activity is regulated through calcium-dependent association of the enzyme with the membrane fraction of cells.In addition, whilst independently cloning the recently described 15S-LO, we identified a splice variant containing an in-frame 87-bp deletion corresponding to amino acids 401±429 inclusive. Modelling of the 15S-LO and subsequent studies with partially purified recombinant protein suggest that the deleted region comprises a complete a-helix flanking the active site of the enzyme resulting in decreased specificity of oxygenation and affinity for fatty acid substrates.Alternative splicing of 15S-LO would therefore provide a further level of regulation of fatty acid metabolism. These results demonstrate that there are substantial differences in the enzyme characteristics and regulation of the 15-LO isozymes which may reflect differing roles for the proteins in vivo. Although the LOs produce specific hydroxyeicosatetraenoic (HETE) enantiomers, the enzymes do not all display absolute positional specificity with respect to oxygenation of AA. For example, while 15-LOb exclusively oxygenates C15 of AA [8], 15-LOa oxygenates AA at both C15 and C12 [9]. In addition, the degree of homology between members of the LO family does not define the product profile. For example, amino acid sequence alignments demonstrate that 15-LOb is more closely related to 12R-LO than 15-LOa, suggesting complex evolutionary paths in the LO family.The LOs are responsible for the synthesis of a number of inflammatory mediators, including leukotrienes and lipoxins, which are implicated in inflammatory disorders such as bronchial asthma [10]. Lipoxygenase metabolites have also been implicated in a number of noninflammatory biological processes. For example, 12-HETE and 15-HETE have been implicated in tumour metastases and the formation of atherosclerotic plaques, respectively [11,12]. The important biological roles of these LO metabolites and the complexity of substrate utilization by members of the LO family emphasize the importance of understanding factors which affect the activity and regulation of these enzymes.Overexpression and characterization of recombinant 5-LO, 12-LO and 15-LOa have been carried out allowing kinetic analysis...

Oxidative metabolism of lipoamino acids and vanilloids by lipoxygenases and cyclooxygenases

“…The kinetic parameters determined for each enzyme with arachidonic acid were in agreement with values reported in the literature [2,[35][36][37]. Notably pl12-LO exhibited very similar kinetic parameters for the lipaomino acids and arachidonate.…”

Cell-autonomous induction of functional tumor suppressor 15-lipoxygenase 2 (15-LOX2) contributes to replicative senescence of human prostate progenitor cells

“…Cells were processed for 15-LOX2 staining 5 weeks after infection. Representative images from one clone each of Kilty et al, 1999) or completely lack (e.g., 15-LOX2sv-b and 15-LOX2sv-c;Bhatia et al, 2003), the AA-metabolizing activity and do not produce appreciable amounts of 15(S)-HETE. Fourth, just as 15-LOX2sv-b possesses tumor-inhibitory effects (Bhatia et al, 2003), enforced expression of 15-LOX2sv-b in young NHP or PCa cells also induces cell-cycle arrest and a senescence-like phenotype.…”

“…Previously, we observed that primary NHP cells that expressed 15-LOX2, a molecule with a limited tissue distribution (i.e., prostate, lung, hair root, and cornea) and most abundantly expressed in adult prostate (Brash et al, 1997;Kilty et al, 1999), were generally big, flat, and cell-cycle arrested (Tang et al, 2002), raising the possibility that the 15-LOX2 may be associated with the NHP cell senescence. To test this possibility, we carried out triple staining for 15-LOX2, SA-bgal, and BrdU.…”

“…15-LOX2 preferentially metabolizes arachidonic acid (AA) to generate a fatty acid, 15(S)-HETE (Brash et al, 1997;Kilty et al, 1999). To determine whether the upregulated 15-LOX2 in NHP cells is enzymatically active, we measured 15(S)-HETE levels in cultured NHP6 cells in the presence of AA.…”

Evidence that Sp1 positively and Sp3 negatively regulate and androgen does not directly regulate functional tumor suppressor 15-lipoxygenase 2 (15-LOX2) gene expression in normal human prostate epithelial cells

“…Introduction 15-Lipoxygenase 2 (15-LOX2) shows the highest homology to murine 8-LOX, has at least three splice variants (termed 15-LOX2sv-a/b/c (15-LOX2 splice variant a, b or c)), mainly metabolizes arachidonic acid (AA) to 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE], and is primarily expressed in prostate, lung, skin, and cornea (Brash et al, 1997;Kilty et al, 1999;Tang et al, 2002). 15-LOX2 expression and activity are decreased in highgrade prostate intraepithelial neoplasia and prostate cancer (PCa) (Shappell et al, 1999).…”