Human Reticulocyte 15-lipoxygenase Research Articles

DDX6 recruits translational silenced human reticulocyte 15-lipoxygenase mRNA to RNP granules.

Erythroid precursor cells lose the capacity for mRNA synthesis due to exclusion of the nucleus during maturation. Therefore, the stability and translation of mRNAs that code for specific proteins, which function in late stages of maturation when reticulocytes become erythrocytes, are controlled tightly. Reticulocyte 15-lipoxygenase (r15-LOX) initiates the breakdown of mitochondria in mature reticulocytes. Through the temporal restriction of mRNA translation, the synthesis of r15-LOX is prevented in premature cells. The enzyme is synthesized only in mature reticulocytes, although r15-LOX mRNA is already present in erythroid precursor cells. Translation of r15-LOX mRNA is inhibited by hnRNP K and hnRNP E1, which bind to the differentiation control element (DICE) in its 3' untranslated region (3'UTR). The hnRNP K/E1-DICE complex interferes with the joining of the 60S ribosomal subunit to the 40S subunit at the AUG. We took advantage of the inducible human erythroid K562 cell system that fully recapitulates this process to identify so far unknown factors, which are critical for DICE-dependent translational regulation. Applying RNA chromatography with the DICE as bait combined with hnRNP K immunoprecipitation, we specifically purified the DEAD-box RNA helicase 6 (DDX6) that interacts with hnRNP K and hnRNP E1 in a DICE-dependent manner. Employing RNA interference and fluorescence in situ hybridization, we show that DDX6 colocalizes with endogenous human (h)r15-LOX mRNA to P-body-like RNP granules, from which 60S ribosomal subunits are excluded. Our data suggest that in premature erythroid cells translational silencing of hr15-LOX mRNA is maintained by DDX6 mediated storage in these RNP granules.

Substrate Specificity Changes for Human Reticulocyte and Epithelial 15-Lipoxygenases Reveal Allosteric Product Regulation

Human reticulocyte 15-lipoxygenase (15-hLO-1) and epithelial 15-lipoxygenase (15-hLO-2) have been implicated in a number of human diseases, with differences in their substrate specificity potentially playing a central role. In this paper, we present a novel method for accurately measuring the substrate specificity of the two 15-hLO isozymes and demonstrate that both cholate and specific LO products affect substrate specificity. The linoleic acid (LA) product, 13-hydroperoxyoctadienoic acid (13-HPODE), changes the ( k cat/ K m) (AA)/( k cat/ K m) (LA) ratio more than 5-fold for 15-hLO-1 and 3-fold for 15-hLO-2, while the arachidonic acid (AA) product, 12-( S)-hydroperoxyeicosatetraenoic acid (12-HPETE), affects only the ratio of 15-hLO-1 (more than 5-fold). In addition, the reduced products, 13-( S)-hydroxyoctadecadienoic acid (13-HODE) and 12-( S)-hydroxyeicosatetraenoic acid (12-HETE), also affect substrate specificity, indicating that iron oxidation is not responsible for the change in the ( k cat/ K m) (AA)/( k cat/ K m) (LA) ratio. These results, coupled with the dependence of the 15-hLO-1 k cat/ K m kinetic isotope effect ( (D) k cat/ K m) on the presence of 12-HPETE and 12-HETE, indicate that the allosteric site, previously identified in 15-hLO-1 [Mogul, R., Johansen, E., and Holman, T. R. (1999) Biochemistry 39, 4801-4807], is responsible for the change in substrate specificity. The ability of LO products to regulate substrate specificity may be relevant with respect to cancer progression and warrants further investigation into the role of this product-feedback loop in the cell.

Lipoxygenase mRNA in cultured human epidermal and oral keratinocytes

Although 15-lipoxygenase has not been purified from cultured human keratinocytes nor has cDNA coding for the protein been isolated from this source, this enzyme activity is implied by the finding of its stereospecific product in in vitro experiments. Based on two primer pairs derived from human reticulocyte 15-lipoxygenase cDNA, we detected approximately 260 bp and approximately 370 bp cDNA fragments that were indistinguishable by gel electrophoresis and Southern hybridization from those derived from reticulocyte 15-lipoxygenase cDNA. The approximately 260 bp polymerase chain reaction (PCR) fragment from a keratinocyte plasmid cDNA library was cloned and determined, by sequencing, to contain 262 bp that were 100% identical to the corresponding reticulocyte 15-lipoxygenase cDNA. These two reticulocyte-type 15-lipoxygenase PCR fragments were also detected from oral keratinocytes. Using platelet-type 12-lipoxygenase cDNA primers, we derived a 264 bp cDNA fragment from keratinocyte mRNA. By sequence analysis, this fragment was determined to be 99.6% identical to that from platelet-type 12-lipoxygenase cDNA. The same fragment was also observed from two amplified keratinocyte cDNA libraries, and from oral keratinocyte mRNA. This is the first demonstration of reticulocyte-type 15-lipoxygenase cDNA derived from the mRNA of cultured human keratinocytes.

Lipoxygenases in Corneal Epithelia of Man and Cynomolgus Monkey

Human and monkey corneal epithelia were investigated for lipoxygenases. The low-speed supernatant of human corneal epithelium converted arachidonic acid to one prominent metabolite, which was identified as 15-hydroxyeicosatetraenoic acid by gas chromarography mass spectrometry and by ultra violet analyses. Eicosatetraynoic acid, a lipoxygenase inhibitor, reduced the formation of 15-hydroxyeicosatetraenoic acid, by 90% at 30 μM, while esculetin (30 μM) and diclofenac (100 μM) did not inhibit the biosynthesis. Immunohistochemical analysis of human cornea using a polyclonal antibody against recombinant human reticulocyte 15-lipoxygenase showed staining of the epithelium, but not the stroma. The cytosolic fraction of epithelial cells of the monkey, Macaca fascicularis, converted [1- 14C]arachidonic acid to two major metabolites, which were identified as 15S-hydroxyeicosatetraenoic and 12S-hydroxyeicosatetraenoic acids by cochromatography with these compounds on reverse-phase, straight-phase and chiral high-performance liquid chromatography. Lipoxygenase activity could not be detected in the microsomal fraction. The results provide biochemical, pharmacological and immunohistological evidence of a 15-lipoxygenase in human corneal epithelium and suggest that monkey corneal epithelium contains cytosolic 12- and 15-lipoxygenases.

Cloning of the cDNA for human 12-lipoxygenase.

A full-length cDNA clone encoding 12-lipoxygenase (arachidonate:oxygen 12-oxidoreductase, EC 1.13.11.31) was isolated from a human platelet cDNA library by using a cDNA for human reticulocyte 15-lipoxygenase as probe for the initial screening. The cDNA had an open reading frame encoding 662 amino acid residues with a calculated molecular weight of 75,590. Three independent clones revealed minor heterogeneities in their DNA sequences. Thus, in three positions of the deduced amino acid sequence, there is a choice between two different amino acids. The deduced sequence from the clone plT3 showed 65% identity with human reticulocyte 15-lipoxygenase and 42% identity with human leukocyte 5-lipoxygenase. The 12-lipoxygenase cDNA recognized a 3.0-kilobase mRNA species in platelets and human erythroleukemia cells (HEL cells). Phorbol 12-tetradecanoyl 13-acetate induced megakaryocytic differentiation of HEL cells and 12-lipoxygenase activity and increased mRNA for 12-lipoxygenase. The identity of the cloned 12-lipoxygenase was assured by expression in a mammalian cell line (COS cells). Human platelet 12-lipoxygenase has been difficult to purify to homogeneity. The cloning of this cDNA will increase the possibilities to elucidate the structure and function of this enzyme.

Molecular cloning, primary structure, and expression of the human platelet/erythroleukemia cell 12-lipoxygenase.

The major pathway of arachidonic acid metabolism in human platelets proceeds via a 12-lipoxygenase enzyme; however, the biological role of the product of this reaction, 12-hydro(pero)xyeicosatetraenoic acid [12-H(P)ETE], is unknown. Using a combination of the polymerase chain reaction and conventional screening procedures, we have isolated cDNA clones encoding the human platelet/human erythroleukemia (HEL) cell 12-lipoxygenase. From the deduced primary structure, human platelet/HEL 12-lipoxygenase would encode a Mr 75,000 protein consisting of 663 amino acids. The cDNA encoding the full-length protein (pCDNA-121x) under the control of the cytomegalovirus promoter was expressed in simian COS-M6 cells. Intact cells and lysed-cell supernatants were able to synthesize 12-H(P)ETE from arachidonic acid, whereas no 12-H(P)ETE synthesis was detected in mock-transfected cells. A single 2.4-kilobase mRNA was detected in erythroleukemia cells but not in several other tissues and cell lines evaluated by Northern blot analysis. Comparison of the human platelet/HEL 12-lipoxygenase sequence with that of porcine leukocyte 12-lipoxygenase and human reticulocyte 15-lipoxygenase revealed 65% amino acid identity to both enzymes. By contrast, the leukocyte 12-lipoxygenase is 86% identical to human reticulocyte 15-lipoxygenase. Sequence data and previously demonstrated immunochemical and biochemical evidence support the existence of distinct 12-lipoxygenase isoforms. The availability of cDNA probes for human platelet/HEL cell 12-lipoxygenase should facilitate elucidation of the biological role of this pathway.

Cloning and sequence analysis of the cDNA for arachidonate 12-lipoxygenase of porcine leukocytes.

The complete amino acid sequence of arachidonate 12-lipoxygenase (EC 1.13.11.31) of porcine leukocytes was deduced by cloning and sequence analysis of DNA complementary to its mRNA. The sequence was confirmed by automated Edman degradation of the N-terminal regions of the native enzyme and its proteolytic fragments. The cDNA had an open reading frame encoding 662 amino acid residues with a calculated molecular weight of 74,911. Amino acid residues 533-545, Cys-(Xaa)3-Cys-(Xaa)3-His-(Xaa)3-His, showed significant homology to the short cysteine- or histidine-containing sequences proposed as the metal-binding domains of transcription factors and various metal-containing proteins [Berg, J. M. (1986) Science 232, 485-487]. The amino acid sequence of 12-lipoxygenase exhibited 86% identity with human reticulocyte 15-lipoxygenase and showed 41% identity with human leukocyte 5-lipoxygenase. The 12-lipoxygenase cDNA recognized a 3.4-kilobase mRNA species in various porcine cell types, with the largest amount in leukocytes, followed by pituitary, lung, jejunum, and spleen.

Expression of cloned human reticulocyte 15-lipoxygenase and immunological evidence that 15-lipoxygenases of different cell types are related.

Cloned 15-lipoxygenase has been expressed for the first time in eukaryotic and prokaryotic cells. Transfection of osteosarcoma cells with a mammalian expression plasmid containing the cDNA for human reticulocyte 15-lipoxygenase resulted in cell lines that were capable of oxidizing body arachidonic acid and linoleic acid. The lipoxygenase metabolites were identified by reverse-phase and straight-phase high pressure liquid chromatography, ultraviolet spectroscopy, and direct mass spectrometry, verifying that the cDNA for 15-lipoxygenase encodes an enzyme with authentic 15-lipoxygenase activity. Incubation of the transformed cells with arachidonic acid generated 15-hydroxyeicosatetraenoic acid (HETE) and 12-HETE in a ratio of 8.6 to 1, demonstrating that 15-lipoxygenase can also perform 12-lipoxygenation. Lesser amounts of 15-keto-ETE, four isomers of 8,15-diHETE, and one isomer of 14,15-diHETE were observed. Incubation with linoleic acid generated predominantly 13-hydroxy linoleic acid. The reaction was inhibited by eicosatetraynoic acid but not by indomethacin. Antibodies to a peptide corresponding to a unique region of the predicted amino acid sequence were generated and shown to react with one major band of 70 kDa on immunoblots of human leukocyte 15-lipoxygenase. To obtain antibodies to the full length enzyme, the cDNA was subcloned into a bacterial expression vector and was expressed as a fusion with the CheY protein. The overexpressed protein was readily purified from bacteria and was shown to be immunoreactive to the peptide-derived antibody. Antibodies raised to this recombinant enzyme did not cross-react with human leukocyte 5-lipoxygenase but did identify 15-lipoxygenase in rabbit reticulocytes, human leukocytes, and tracheal epithelial cells, suggesting that the 15-lipoxygenases from these different cell types are structurally related.