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Biochemical characterization of three BLT receptors in zebrafish.

ABSTRACT: The leukotriene B4 (LTB4) receptor 1 (BLT1) is a high affinity receptor for LTB4, a chemotactic and inflammatory eicosanoid. The LTB4 receptor 2 (BLT2) was originally identified as a low affinity receptor for LTB4, and, more recently, as a high affinity receptor for 12-hydroxyheptadecatrienoic acid (12-HHT). The zebrafish BLT receptors have not been previously identified and the in vivo functions of these receptors have been unknown. In this paper, we describe one zebrafish BLT1-like receptor, Blt1, and two zebrafish BLT2-like receptors, Blt2a and Blt2b. Cells expressing Blt1 exhibited LTB4-induced intracellular [Ca2+] increases, inhibition of cAMP production, ligand-dependent [35S]GTP?S binding, and transforming growth factor-? (TGF?) shedding activity in a dose-dependent manner, similar to human BLT1. Cells expressing Blt2a and Blt2b exhibited 12-HHT- and LTB4-induced intracellular [Ca2+] increases, inhibition of cAMP production, [35S]GTP?S binding, and TGF? shedding activity, with a dose-dependency similar to human BLT2. Reverse transcription (RT)-PCR analysis and whole-mount in situ hybridization revealed that blt1, blt2a, blt2b, zebrafish LTA4 hydrolase (lta4h), and zebrafish 5-lipoxiganase (5lo) are expressed in zebrafish embryos. Knockdown of blt1 by morpholino antisense oligonucleotides resulted in delayed epiboly at gastrulation. Consistently, knockdown of lta4h, an enzyme mediating LTB4 production, induced a phenotype similar to knockdown of blt1. These results suggest that the LTB4-BLT1 axis is involved in epiboly in zebrafish development.

SUBMITTER: Okuno T

PROVIDER: S-EPMC4349892 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Biochemical characterization of three BLT receptors in zebrafish.

Okuno Toshiaki T Ishitani Tohru T Yokomizo Takehiko T

PloS one 20150304 3

The leukotriene B4 (LTB4) receptor 1 (BLT1) is a high affinity receptor for LTB4, a chemotactic and inflammatory eicosanoid. The LTB4 receptor 2 (BLT2) was originally identified as a low affinity receptor for LTB4, and, more recently, as a high affinity receptor for 12-hydroxyheptadecatrienoic acid (12-HHT). The zebrafish BLT receptors have not been previously identified and the in vivo functions of these receptors have been unknown. In this paper, we describe one zebrafish BLT1-like receptor, B ...[more]

PMID: 25738285

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Project description:Background:The CYP152 family member OleTJE from Jeotgalicoccus sp. ATCC 8456 has been well-known to catalyze the unusual one-step decarboxylation of free fatty acids towards the formation of terminal alkenes. Efforts to tune up its decarboxylation activity for better production of biological alkenes have been extensively explored via approaches such as site-directed mutagenesis and electron source engineering, but with limited success. To gain more insights into the decarboxylation mechanism and reaction bifurcation (decarboxylation versus hydroxylation), we turned to an alternative approach to explore the natural CYP152 resources for a better variety of enzyme candidates. Results:We biochemically characterized three new P450 fatty acid decarboxylases including OleTJH, OleTSQ and OleTSA, with respect to their substrate specificity, steady-state kinetics, and salt effects. These enzymes all act as an OleTJE-like fatty acid decarboxylase being able to decarboxylate a range of straight-chain saturated fatty acids (C8-C20) to various degrees. Site-directed mutagenesis analysis to the lower activity P450 enzyme OleTSA revealed a number of key amino acid residues within the substrate-binding pocket (T47F, I177L, V319A and L405I) that are important for delicate substrate positioning of different chain-length fatty acids and thus the decarboxylation versus hydroxylation chemoselectivity, in particular for the mid-chain fatty acids (C8-C12). In addition, the three new decarboxylases exhibited optimal catalytic activity and stability at a salt concentration of 0.5 M, and were thus classified as moderate halophilic enzymes. Conclusion:The P450 fatty acid decarboxylases OleTJE, OleTJH, OleTSQ and OleTSA belong to a novel group of moderate halophilic P450 enzymes. OleTJH from Jeotgalicoccus halophilus shows the decarboxylation activity, kinetic parameters, as well as salt tolerance and stability that are comparable to OleTJE. Site-directed mutagenesis of several key amino acid residues near substrate-binding pocket provides important guidance for further engineering of these P450 fatty acid decarboxylases that hold promising application potential for production of ?-olefin biohydrocarbons.

Project description:The methanogenic archaeon Methanosarcina barkeri encodes three distinct types of hydrogenase, whose functions vary depending on the growth substrate. These include the F420-dependent (Frh), methanophenazine-dependent (Vht), and ferredoxin-dependent (Ech) hydrogenases. To investigate their physiological roles, we characterized a series of mutants lacking each hydrogenase in various combinations. Mutants lacking Frh, Vht, or Ech in any combination failed to grow on H2-CO2, whereas only Vht and Ech were essential for growth on acetate. In contrast, a mutant lacking all three grew on methanol with a final growth yield similar to that of the wild type and produced methane and CO2 in the expected 3:1 ratio but had a ca. 33% lower growth rate. Thus, hydrogenases play a significant, but nonessential, role during growth on this substrate. As previously observed, mutants lacking Ech failed to grow on methanol-H2 unless they were supplemented with biosynthetic precursors. Interestingly, this phenotype was abolished in the ?ech ?frh and ?ech ?frh ?vht mutants, consistent with the idea that hydrogenases inhibit methanol oxidation in the presence of H2, which prevents production of the reducing equivalents needed for biosynthesis. Quantification of the methane and CO2 produced from methanol by resting cell suspensions of various mutants supported this conclusion. On the basis of the global transcriptional profiles, none of the hydrogenases were upregulated to compensate for the loss of the others. However, the transcript levels of the F420 dehydrogenase operon were significantly higher in all strains lacking frh, suggesting a mechanism to sense the redox state of F420 The roles of the hydrogenases in energy conservation during growth with each methanogenic pathway are discussed.IMPORTANCE Methanogenic archaea are key players in the global carbon cycle due to their ability to facilitate the remineralization of organic substrates in many anaerobic environments. The consequences of biological methanogenesis are far-reaching, with impacts on atmospheric methane and CO2 concentrations, agriculture, energy production, waste treatment, and human health. The data presented here clarify the in vivo function of hydrogenases during methanogenesis, which in turn deepens our understanding of this unique form of metabolism. This knowledge is critical for a variety of important issues ranging from atmospheric composition to human health.

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Biochemical characterization of three BLT receptors in zebrafish.

Publications

Biochemical characterization of three BLT receptors in zebrafish.

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