Development of analytical methods fort he quantitative analysis of the enzyme abundance and activity in the arachidonic acid cascade

The arachidonic acid (ARA) cascade is a key regulatory pathway where eicosanoids and other oxylipins are formed via the oxidation of polyunsaturated fatty acids. These oxylipins are involved in controlling physiological functions such as vascular tone, blood clotting and immune regulation. Changes in the oxylipin pattern can be caused by direct enzyme inhibition or modulated gene expression. In order to understand the effect mechanisms modulating the ARA cascade it is essential to not only investigate the metabolite, i.e. oxylipin, concentrations but also the abundances of the enzymes involved in their formation. For this reason, a quantitative targeted proteomics method for the ARA cascade was developed within this thesis. A detailed workflow for method development was established where enzyme/protein levels are measured via sequence-specific, representative peptides chosen on the basis of several criteria. With liquid chromatography-tandem mass spectrometry-based analysis, the cyclooxygenase (COX) pathway of the ARA cascade was investigated in human colon carcinoma cell lines and primary macrophages where strong correlations were found between the oxylipins formed via the COX pathway and the COX-2 abundance. The modulating effects of polyphenols on the 5-lipoxygenase (LOX) pathway of the ARA cascade were investigated in cell-free enzyme assays and human neutrophils. Resveratrol, its dimer ɛ-viniferin and a resveratrol imine analogue directly inhibited the 5-LOX with micromolar potencies and modulated the total oxylipin pattern in the neutrophils with remarkably different effects. Genistein did not act as a 5-LOX inhibitor in the cell-free assay but the modulated oxylipin pattern in the cells suggests a global cellular interference. The enzymes of the LOX pathway were included in the targeted proteomics method which together with an extended targeted oxylipin metabolomics platform enables a sensitive multi- omics analysis of the ARA cascade in a single sample. The comparison of the multiple reaction monitoring (MRM) detection and the MRM^-based approach with an additional fragmentation step demonstrated that MRM is more favorable in targeted proteomics due to its higher sensitivity, greater linear range and higher multiplexing capacities. The analysis with the multi- omics approach showed that the 5 LOX protein is induced together with its product formation during the differentiation of human monocytic THP 1 cells to macrophages. M1-like human primary macrophages were also characterized by the abundance of 5-LOX and its activating protein (FLAP), while 15-LOX and 15-LOX-2 dominated the protein pattern of the M2 like macrophages accompanied by high levels of the oxylipins formed via these enzymes. The developed methodology allows mechanistic investigation of a modulation of the ARA cascade as demonstrated by lipo-poly-saccharide stimulation as well as pharmaceutical treatment of the cells. Overall, with this thesis, a new quantitative proteomics strategy for the enzymes of the ARA cascade is established which can be easily extended to further proteins. The use of this technique in combination with targeted oxylipin metabolomics has provided new insights into the mechanisms involved in the modulation of the ARA cascade in human immune cells.     

白鲢鱼肌肉脂肪氧合酶的分离纯化与鉴定

采用硫酸氨沉淀和羟基磷灰石柱层析法分离白鲢鱼肌肉脂肪氧合酶(lipoxygenase,LOX),同时采用反相高效液相(RP-HPLC)检测其反应产物,对鲢鱼肌肉LOX 进行鉴定,并研究鲢鱼肌肉LOX 的底物特异性。结果表明：白鲢鱼肌肉LOX 催化花生四烯酸主要形成12- 氢过氧化物,12-LOX 是鲢鱼肌肉中主要的LOX;白鲢鱼LOX的最适底物是亚麻酸。     

Flocculation,cell surface hydrophobicity and 3‐OH oxylipins in the SMA strain of Saccharomyces pastorianus

Three‐hydroxy‐oxylipins (3‐OH oxylipins) have been previously detected in brewing yeast production strains at flocculation onset. In this work, the SMA strain of Saccharomyces pastorianus was characterized during growth in a miniature fermentation assay by measuring flocculation and cell surface hydrophobicity (CSH). Proportions of 3‐OH oxylipin were also measured concurrently during growth in the miniature fermentation assay and a defined 3‐OH oxylipin extraction protocol using ethyl acetate is presented along with a novel derivatization and gas chromatography–mass spectrometry (GC‐MS) detection approach. When the SMA strain was grown in the assay, near maximal CSH and flocculation levels were achieved by a 36 h fermentation time. Under the same culture conditions, the oxylipin 3‐OH decanoic acid (3‐OH 10:0) was identified. This oxylipin could not be detected early in the fermentation, but elevated relative levels of 3‐OH 10:0 were reached by 36 h, coinciding with increased CSH levels. It was previously presumed that the formation of 3‐OH oxylipins at flocculation onset might increase the CSH. However, results from this study suggest that 3‐OH 10:0 may not contribute to cell wall hydrophobicity. The flocculation behaviour of the SMA strain was also monitored in the presence of 3‐OH 10:0, but exposure to this oxylipin did not impact the sedimentation of this yeast, suggesting that 3‐OH oxylipins may not act as mediators of quorum sensing in this strain. Copyright © 2015 The Institute of Brewing & Distilling     

普通烟草脂氧合酶基因家族鉴定及表达模式分析

脂氧合酶（LOX）是脂肪酸代谢途径的关键酶,在植物的生长发育及多种逆境胁迫响应过程中发挥重要作用。本研究对烟草基因组中的LOX家族成员进行鉴定,并利用进化分析、共线性分析和表达分析等方法解析LOX家族成员的潜在生物学功能。结果表明,在普通烟草中共鉴定得到18个LOX基因,其中12个LOX基因被锚定在染色体上。系统进化分析表明,普通烟草中新鉴定的LOX家族成员被分为9-LOX和13-LOX两个亚家族,其中13-LOX又可被分为Type I和Type II两个亚类。共线性分析表明,烟草NtLOX07、NtLOX10基因分别与拟南芥AtLOX05、AtLOX01基因形成同源基因对,并且大部分同源基因之间具有相似的表达模式。表达模式分析发现,普通烟草LOX基因表达具有一定的组织特异性,并且NtLOX06等基因能够被机械损伤和茉莉酸甲脂（MeJA）处理显著诱导。因此,普通烟草LOX家族成员可能在植物胁迫响应过程中发挥着重要的作用。     

姜黄素与猪脂肪氧合酶相互作用及对蛋白质结构的影响

利用分光光度计法、荧光光谱法以及圆二色谱法(CD)研究姜黄素与猪12-脂肪氧合酶(12-Lipoxygenase,12-LOX)的相互作用。结果表明,姜黄素能够抑制猪12-LOX酶活,并且浓度越高,抑制作用越强,姜黄素抑制12-LOX的IC₅₀值为2.156 μg/mL。荧光光谱结果表明姜黄素对猪12-LOX有较强的荧光猝灭作用,猝灭机制为静态猝灭,两者之间的作用力主要是范德华力和氢键。同步荧光光谱研究表明,姜黄素与猪12-LOX的结合位点更接近于色氨酸残基。圆二色谱结果表明,姜黄素能够与猪12-LOX相互作用,并使LOX二级结构发生变化。     

15-Lipoxygenase is a component of the mammalian sperm cytoplasmic droplet

Lipoxygenases (LOXs) are a family of enzymes capable of peroxidizing phospholipids. A member of the LOX family of enzymes, 15-LOX, participates in the degradation of mitochondria and other organelles within differentiating red blood cells, the reticulocytes. The present study provides biochemical and immunocytochemical evidence for the presence of 15-LOX in the sperm cytoplasmic droplet (CD). Testicular, epididymal and ejaculated spermatozoa were evaluated for the presence of 15-LOX using an affinity-purified immune serum raised against a synthetic peptide corresponding to the C-terminal sequence of rabbit reticulocyte 15-LOX. Western blotting revealed an appropriate single band of approximately 81 kDa in boar spermatozoa but not in boar seminal plasma. When ejaculated boar spermatozoa were subjected to separation on a 45/90% Percoll gradient, 15-LOX co-migrated with the immotile sperm and cellular debris/CD fractions, but not with the motile sperm fraction containing morphologically normal spermatozoa without CDs. Varied levels of 15-LOX were expressed in ejaculated sperm samples from boars with varied semen quality. By immunofluorescence, prominent 15-LOX immunoreactivity was found within the residual body in the testis and within the CDs from caput, corpus and cauda epididymal and ejaculated spermatozoa. Components of the ubiquitin-dependent proteolytic pathway, which is thought to facilitate both spermiogenesis and reticulocyte organelle degradation, were also detected in the sperm CD. These included ubiquitin, the ubiquitin-conjugating enzyme E2, the ubiquitin C-terminal hydrolase PGP 9.5, and various 20S proteasomal core subunits of the alpha- and beta-type. The 15-LOX and various components of the ubiquitin-proteasome pathway were also detected in sperm CDs of other mammalian species, including the human, mouse, stallion and wild babirusa boar. We conclude that 15-LOX is prominently present in the mammalian sperm CD and thus may contribute to spermiogenesis, CD function or CD removal.     

Oxylipin Associated Co‐Flocculation in Yeasts

According to the lectin‐theory, the yeast Schizosaccharomyces pombe lacks the specific receptors (α‐mannans) necessary to facilitate co‐flocculation with Saccharomyces cerevisiae species. In this study we demonstrate oxylipin associated co‐flocculation between Sacch. cerevisiae and S. pombe strains using differential cell staining, immunofluoresence and ultrastructural studies. Using a 3‐hydroxy (OH) oxylipin specific antibody coupled to a fluorescing compound, 3‐OH oxylipins were found to be present on the cell surfaces of Sacch. cerevisiae and S. pombe. The presence of 3‐OH oxylipins was confirmed using gas chromatography‐mass spectrometry. Strikingly, when acetylsalicylic acid (aspirin), a 3‐OH oxylipins inhibitor, was added to Sacch. cerevisiae which was then mixed with S. pombe strains grown in complex media, co‐flocculation was significantly inhibited. We conclude that aspirin‐sensitive 3‐OH 8:0 is probably involved in co‐flocculation.     

Packaging Influenced Total Chlorophyll, Soluble Protein, Fatty Acid Composition and Lipoxygenase Activity in Broccoli Florets

Polyunsaturated fatty acid (PUFA) degradation by lipoxygenase (LOX) may contribute to postharvest deterioration of vegetables and fruits. In broccoli, modified atmosphere packaging (MAP), automatic misting (AM) and vent packaging (VP) may reduce postharvest deterioration. MAP storage resulted in increased chlorophyll and C-18 polyunsaturated fatty acids (PUFA) in broccoli florets by 96 hr. Reduced chlorophyll, PUFA and soluble protein were observed in nonpackaged (NP) samples. Chlorophyll remained near initial levels in vent packaged (VP) products. AM resulted in increased relative PUFA and reduced losses of soluble protein. VP reduced PUFA and soluble protein. Optimal pH of broccoli floret LOX was 5.5-6.0. Water-soluble LOX activity in MAP and NP samples followed a trend similar to PUFA changes in both samples. No differences were found among treatments when enzyme activity was expressed on soluble protein basis.     

Quantitative profiling of oxylipins through comprehensive LC-MS/MS analysis of Fusarium verticillioides and maize kernels

Fusarium verticillioides is one of the most important fungal pathogens causing ear and stalk rot in maize, even if frequently asymptomatic, producing a harmful series of compounds named fumonisins. Plant and fungal oxylipins play a crucial role in determining the outcome of the interaction between the pathogen and its host. Moreover, oxylipins result as signals able to modulate the secondary metabolism in fungi. In keeping with this, a novel, quantitative LC-MS/MS method was designed to quantify up to 17 different oxylipins produced by F. verticillioides and maize kernels. By applying this method, we were able to quantify oxylipin production in vitro – F. verticillioides grown into Czapek–Dox/yeast extract medium amended with 0.2% w/v of cracked maize – and in vivo, i.e. during its growth on detached mature maize ears. This study pinpoints the role of oxylipins in a plant pathogen such as F. verticillioides and sets up a novel tool aimed at understanding the role oxylipins play in mycotoxigenic pathogens during their interactions with respective hosts.     

Peroxidase and Lipoxygenase Influence on Stability of Polyunsaturated Fatty Acids in Sweet Corn (Zea mays L.) during Frozen Storage

The effect of blanching treatments and packaging materials on lipoxygenase (LOX) and peroxidase (POD) activity and fatty acid stability of two cultivars of sweet corn (Jubilee and GH-2684) were evaluated during 9 mo storage at ?20°C. Complete inactivation of LOX and POD was obtained with 9 and 15 min of steam blanching, respectively. Relative fatty acid content revealed no change in fatty acid composition during storage. Control of degradation of polyunsaturated fatty acids (PUFA) did not depend on oxygen permeability of different packaging materials. Blanching had little effect on PUFA degradation after 9 mo storage.     

Recombinant Lipoxygenases and Oxylipin Metabolism in Relation to Food Quality

Lipoxygenases catalyze the conversion of polyunsaturated fatty acids into hydroperoxides, that are in turn converted to oxylipins, which play important roles in defence reactions in plants and animals. This review describes the distribution of lipoxygenases in Nature, their diversity in terms of structure and catalytic activity, and their significance for food biotechnology. The last includes the production of flavors and aromas, the destruction of vitamins, pigments and other anti-oxidants, the improvement of dough rheology during baking, and the potential of recombinant lipoxygenases, and other enzymes of oxylipin metabolism, for food biotechnology.     

KINETIC BEHAVIOR OF SOYBEAN LIPOXYGENASE: A COMPARATIVE STUDY OF THE FREE ENZYME AND THE ENZYME IMMOBILIZED IN AN ALGINATE SILICA SOL‐GEL MATRIX1

Lipoxygenase (LOX) is an enzyme that regioselectively introduces a hydroperoxide into polyunsaturated fatty acids (PUFA). We recently reported a procedure that immobilizes soybean LOX within an alginate sol‐gel matrix. In this study, the kinetic profile of free LOX was compared with that of the sol‐gel immobilized LOX. The temperature dependent activity profile of free LOX was optimal at 25C whereas immobilized LOX had optimal activity over the temperature range of 25–35C. Enzyme activity, measured in aqueous buffer, for both the free and immobilized LOX preparations had K_m values of 2.5 and 1.40 mmoles/L, respectively, and V_max values of 0.056 and 0.02 μmol/min, respectively. The relative rates of oxidation of linoleic acid and acylgfycerols containing linoleoyl residues catalyzed by free and immobilized LOX also were determined The results showed that both free and immobilized LOX favor linoleic acid as a substrate. Relative substrate preference for free LOX was linoleic acid >1‐monolinolein > 1,3‐dilinolein >trilinolein, and for immobilized LOX was linoleic acid >l, 3‐dilinolein >1‐monolinolein >trilinolein. In general, LOX immobilized in alginate silica sol‐gel matrix retained the physical and chemical characteristics of free LOX.     

微生物多不饱和脂肪酸的生物合成、调控和利用

论述了微生物油脂及其多不饱和脂肪酸(PUFA)的重要性、来源、生物合成、生产技术及下游加工等。特别强调运用高新生物技术如突变、基因工程、脱饱和酶抑制剂、酶促生物转化等技术来增加新的PUFA种类,提高其产品价值,从而使其能与其他来源的PUFA相竞争。     

Bioactive Oxylipins in Saccharomyces cerevisiae

Some strains of Saccharomyces cerevisiae (including strains used in fermentation processes) produce short chain (mainly 8 carbon) oxylipins and not potent inflammatory long chain (20 carbon) oxylipins such as prostaglandins. When acetylsalicylic acid (aspirin) was added to cultures of Sacch. cerevisiae UOFS Y‐2330, flocculation was significantly inhibited as well as the production of 3‐hydroxy 8:0 thereby linking flocculation and this oxylipin. Furthermore, no traces of 3‐hydroxy 8:0 could be detected at the start of flocculation in this yeast. This research is based on (i) reports that yeasts in general can produce bioactive prostaglandins, (ii) findings suggesting a link between aspirin‐sensitive prostaglandins and biofilm formation by Candida albicans, (iii) the discovery that the addition of low concentrations of aspirin abolish yeast biofilm formation and sexual cell aggregation and (iv) the recent discovery of a novel potent aspirin‐sensitive pro‐inflammatory 3‐hydroxy prostaglandin E₂ synthesized by Candida albicans in conjunction with mammalian cells probably during candidiasis.     

The role of lipoxygenase-isoforms in atherogenesis

Lipoxygenases (LOXs) form a heterogeneous family of lipid-peroxidizing enzymes, and several LOX-isoforms (12/15-LOX, 5-LOX) have been implicated in atherogenesis. However, the precise role of these enzymes is still a matter of discussion. 12/15-LOXs are capable of oxidizing lipoproteins (low-density lipoprotein (LDL), high-density lipoprotein (HDL)) to atherogenic forms, and functional inactivation of this enzyme in murine atherosclerosis models slows down lesion formation. In contrast, rabbits that overexpress this enzyme were protected from lesion formation when fed a lipid-rich diet. To contribute to this discussion, we recently investigated the impact of 12/15-LOX overexpression on in vitro foam cell formation. When 12/15-LOX-transfected J774 cells were incubated in culture with modified LDL, we found that intracellular lipid deposition was reduced in the transfected cells when compared with the corresponding control transfectants. This paper briefly summarizes the current status of knowledge on the biological activity of different LOX-isoforms in atherogenesis and will also provide novel experimental data characterizing the role of 12/15-LOX in cellular LDL modification and for in vitro foam cell formation.     

Dynamics of lipid droplets in the endometrium and fatty acids and oxylipins in the uterine lumen,blood, and milk of lactating cows during diestrus

Our objective was to investigate the lipid content of uterus, blood plasma, and milk at early, mid, and late diestrus. Lactating cows (n = 30) had the estrous cycle and ovulation synchronized by administration of exogenous hormones. Cows were blocked by parity and assigned randomly to receive transcervical uterine flushing and biopsy on d 5 (early diestrus), 10 (mid diestrus) or 15 (late diestrus) of the estrous cycle. Flushing and endometrial biopsy were performed in the uterine horn ipsilateral to the corpus luteum. The recovered flushing was used for analyses of lipid composition by liquid chromatography-tandem mass spectrometry and the biopsy was used for investigation of lipid droplet abundance in endometrial cryosections using a neutral lipid fluorescent dye. In addition, blood and milk samples were collected from all cows on d 5, 10, and 15. All blood samples were used to measure the concentration of progesterone in plasma, and all milk samples were used to determine milk composition. Subsamples of blood plasma and milk were also used to evaluate the composition of fatty acids and oxylipins using the same methodology used for uterine flushing samples. The abundance of lipid droplets in the endometrium increased 1.9-fold from d 5 to 10, and 2-fold from d 10 to 15. Concentration of long-chain fatty acids and oxylipins in uterine flushing were, on average, 2.2 and 2.5 times greater in samples collected on d 15 compared with those collected on d 5 and 10. These differences were not observed in blood and milk, suggesting that accumulation of fatty acids and oxylipins in the uterus is regulated locally. In addition to concentration, the profile of individual fatty acids and oxylipins in uterine lumen changed substantially during diestrus. The main categories with increased abundance at late diestrus were mono- and polyunsaturated fatty acids, and oxylipins derived from arachidonic acid, dihomo-γ-linolenic acid, and docosahexaenoic acid. In conclusion, fatty acids and oxylipins accumulate in the uterine lumen during diestrus and might work as a mechanism to supply these lipids to the developing conceptus at late diestrus, when the onset of elongation occurs and substantial synthesis of biomass and cell signaling by lipid mediators are required.     

人工加速老化对茶叶籽储藏特性的影响

研究了人工加速老化对茶叶籽含水率、含油率、可溶性蛋白、脂肪酶、脂肪氧化酶活性、酸价、过氧化值的影响及脂肪酸组分的变化。结果表明,人工加速老化促进茶叶籽老化进程,老化程度与老化强度成正比。随着人工加速老化时间的延长,茶叶籽含水率上升,可溶性蛋白含量下降;茶叶籽脂肪酶活力下降,脂肪氧化酶活力上升;茶叶籽油酸价和过氧化值增高;茶叶籽含油率逐渐升高,不饱和脂肪酸与饱和脂肪酸比值下降,单不饱和脂肪酸与多不饱和脂肪酸比值上升。通过对茶叶籽老化不同阶段储藏特性的测定可以预测茶叶籽劣变程度。     

CHARACTERIZATION OF LIPOXYGENASE FROM MACKEREL (SCOMBER SCOMBRUS) MUSCLE

The polyunsaturated fatty acids (PUFA) in fish render them potentially susceptible to enzymatic oxidation. Lipoxygenase (LOX) activity from mackerel (Scomber scombrus) was characterized with respect to pH stability and activity, temperature stability, buffer and substrate preferences and inhibition patterns. The partially purified enzyme was stable over the pH range 4.0–11.0 and had a pH-activity optimum pH of 5.6. Mackerel muscle LOX (mmLOX) was stable at 0–50C but lost activity at higher temperatures. Enzymatic assays using different buffers revealed that MES-NaOH and Bistris-HCl were the suitable buffers for reaction. Substrate preference was for unoleic acid compared to linolenic, arachidonic, docosahexaenoic oroleicacid. K_m values were 0.69 and 0.57 mM and V_max was 0.058 and 0.019 μmol mm^-1, for linoleic and linolenic acid, respectively. mmLOX was inhibited by classical LOX inhibitors such as BHA, BHT, NDGA, esculetin and also by the heme protein inhibitor KCN. Values for the IC₅₀ (concentration for half maximal inhibition) was between 0.02 μM and 41 μM.
Normal phase high performance liquid chromatography (HPLC) analyses revealed that both 13- and 9-hydroperoxides were formed during mmLOX catalyzed oxidation with Unoleic acid substrate although a higher proportion of the 13-isomer was formed. With enzymes from different mackerel tissues (skin, gills and muscle), mmLOX had the highest hydroperoxide forming ability.