Recombinant expression, characterization, and application of a phospholipase B from Fusarium oxysporum.

In this study, a gene encoding a putative lipase from Fusarium oxysporum was optimized via codon optimization and expressed in Pichia pastoris KM71. The gene product was identified as a phospholipase B (PLB). The engineered P. pastoris was further cultured in a 3.6-L bioreactor. After optimization of the induction conditions, this system produced 6.6mgmL-1 protein and 6503.8UmL-1 PLB activity in the culture medium. Efficient expression of this PLB in P. pastoris should reduce the costs of production and application. The purified enzyme, with a specific activity of 1170Umg-1, was optimally active at pH 5.0 and 55°C. The results of a degumming experiment performed using the recombinant PLB showed that the phosphorus content of a test oil was decreased from 75.88ppm to 3.3ppm in 2h under optimal reaction conditions. This study provides a basis for the industrial use of F. oxysporum PLB in oil degumming applications.

Stage-specific expression, immunolocalization of Clonorchis sinensis lysophospholipase and its potential role in hepatic fibrosis.

Lysophospholipase, belonging to the complex family of phospholipases, is supposed to play a vital role in virulence and pathogenesis of parasites and fungi. In the current study, the potential role of Clonorchis sinensis lysophospholipase (CslysoPLA) in hepatic fibrosis induced by C. sinensis was explored for the first time. In the liver of the cat infected with C. sinensis, CslysoPLA was recognized in the lumen between adult worms and surrounding bile duct epithelia together with some inside the cells by means of immunolocalization. Both Cell Counting Kit-8 (CCK-8 assay) and cell cycle analysis of human hepatic stellate cell line LX-2 showed that a higher percentage of cells were at proliferation phase after incubation with lower concentrations of recombinant CslysoPLA (rCslysoPLA). Quantitative real-time polymerase chain reaction (RT-PCR) demonstrated an upregulation in fibrogenic genes of smooth muscle α-actin, collagen III, matrix metalloproteinase 2 and tissue inhibitors of metalloproteinase II in LX-2 treated with rCslysoPLA. Moreover, human biliary epithelial cell line 5100 proliferated significantly in response to rCslysoPLA. Notably, CslysoPLA was localized in the adenomatoid hyperplastic tissue within the intrahepatic bile duct of experimentally infected rats by immunolocalization analysis. In addition, quantitative RT-PCR implied that CslysoPLA was differentially expressed at the developmental stages of C. sinensis (metacercariae, adult worms and eggs), with the highest level at metacercariae stage. Immunolocalization analysis showed that CslysoPLA was distributed in the intestine, vitelline gland, tegument and eggs in the adult worms and in the tegument and vitelline gland in the metacercariae, respectively. Collectively, it suggests that CslysoPLA might be involved in the initiation and promotion of C. sinensis-related human hepatic fibrosis and advance future studies on its promotion to C. sinensis-induced cholangiocarcinogenesis.

High level expression and characterization of a thermostable lysophospholipase from Thermococcus kodakarensis KOD1.

Phospholipases can catalyze the hydrolysis of one or more ester and phosphodiester bonds and have a considerable interest in the food, oil leather and pharmaceutical industries. In this report, a lysophospholipase gene from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (LysoPL-tk) was cloned. The gene of 783 bp encodes a 260-amino acid protein with a molecular mass of 29 kDa. LysoPL-tk has a consensus motif (GxSxG) and a catalytic triad (S, D, H) of esterases in the deduced amino acid sequence. LysoPL-tk was expressed in Escherichia coli and purified to homogeneity. The enzyme can degrade substrates with both short and long acyl chain lengths. The apparent K (m) value for p-nitrophenyl butyrate was 607.1 µM with V (max) values of 95.5 U/mg. The enzyme was active at a broad range of pH (5-8) and temperatures (70-95 °C) with the optimum pH and temperature being 8.0 and 85 °C, respectively. The high yield, broad substrate range along with its thermo-stability indicates that LysoPL-tk is a potential enzyme in industrial application.

Fatty acid production in genetically modified cyanobacteria.

To avoid costly biomass recovery in photosynthetic microbial biofuel production, we genetically modified cyanobacteria to produce and secrete fatty acids. Starting with introducing an acyl-acyl carrier protein thioesterase gene, we made six successive generations of genetic modifications of cyanobacterium Synechocystis sp. PCC6803 wild type (SD100). The fatty acid secretion yield was increased to 197 ± 14 mg/L of culture in one improved strain at a cell density of 1.0 × 10(9) cells/mL by adding codon-optimized thioesterase genes and weakening polar cell wall layers. Although these strains exhibited damaged cell membranes at low cell densities, they grew more rapidly at high cell densities in late exponential and stationary phase and exhibited less cell damage than cells in wild-type cultures. Our results suggest that fatty acid secreting cyanobacteria are a promising technology for renewable biofuel production.

Loss of detectability of Charcot-Leyden crystal protein transcripts in blood cells after treatment with dimethyl sulfoxide.

Charcot-Leyden crystal protein (CLC) is a major secretory effector protein of eosinophils. In addition, CLC has been identified as marker for regulatory T-cells and differential expression of CLC has been described under diverse pathological conditions. By analysis of DNA microarray data from peripheral blood mononuclear cells (PBMC) we found differences for the expression of CLC between PBMC that had been cryopreserved or had been used for RNA isolation immediately after cell separation. Reverse transcriptase-polymerase chain reaction (RT-PCR) of separated cell populations indicated that contaminating granulocytes were the main source of CLC transcripts in PBMC. CLC was only detectable in fresh PBMC and not in cryopreserved material. Transcripts corresponding to CLC were also detectable by RT-PCR only in fresh PBMC and eosinophils. Loss of CLC transcripts in PBMC was induced by a short pulse with dimethyl sulfoxide (DMSO), indicating that the freezing medium was responsible for this phenomenon. We conclude that CLC transcripts are lost during cryopreservation in the presence of DMSO and can never be identified as differentially expressed in cryopreserved samples.

Expression, purification, refolding and characterization of a putative lysophospholipase from Pyrococcus furiosus: retention of structure and lipase/esterase activity in the presence of water-miscible organic solvents at high temperatures.

A putative lysophospholipase (PF0480) encoded by the Pyrococcus furiosus genome has previously been cloned and expressed in Escherichia coli. Studies involving crude extracts established the enzyme to be an esterase; however, owing presumably to its tendency to precipitate into inclusion bodies, purification and characterization have thus far not been reported. Here, we report the overexpression and successful recovery and refolding of the enzyme from inclusion bodies. Dynamic light scattering suggests that the enzyme is a dimer, or trimer, in aqueous solution. Circular dichroism and fluorescence spectroscopy show, respectively, that it has mixed beta/alpha structure and well-buried tryptophan residues. Conformational changes are negligible over the temperature range of 30-80 degrees C, and over the concentration range of 0-50% (v/v) of water mixtures with organic solvents such as methanol, ethanol and acetonitrile. The enzyme is confirmed to be an esterase (hydrolyzing p-NP-acetate and p-NP-butyrate) and also shown to be a lipase (hydrolyzing p-NP-palmitate), with lipolytic activity being overall about 18- to 20-fold lower than esterase activity. Against p-NP-palmitate the enzyme displays optimally activity at pH 7.0 and 70 degrees C. Remarkably, over 50% activity is retained at 70 degrees C in the presence of 25% acetonitrile. The high organic solvent stability and thermal stability suggest that this enzyme may have useful biodiesel-related applications, or applications in the pharmaceutical industry, once yields are optimized.

Identification of an insulin-regulated lysophospholipase with homology to neuropathy target esterase.

Neuropathy target esterase (NTE) is a member of the family of patatin domain-containing proteins and exhibits phospholipase activity in brain and cultured cells. NTE was originally identified as target enzyme for organophosphorus compounds that cause a delayed paralyzing syndrome with degeneration of nerve axons. Here we show that the structurally related murine protein NTE-related esterase (NRE) is a potent lysophospholipase. The enzyme efficiently hydrolyzes sn-1 esters in lysophosphatidylcholine and lysophosphatidic acid. No lipase activity was observed when triacylglycerols, cholesteryl esters, retinyl esters, phosphatidylcholine, or monoacylglycerol were used as substrates. Although NTE is predominantly expressed in the nervous system, we found the highest NRE mRNA levels in testes, skeletal muscle, cardiac muscle, and adipose tissue. Induction of NRE mRNA concentrations in these tissues during fasting suggested a nutritional regulation of enzyme expression and, in accordance with this observation, insulin reduced NRE mRNA levels in a dose-dependent manner in 3T3-L1 adipocytes. A green fluorescent protein-NRE fusion protein colocalized to the endoplasmic reticulum and lipid droplets. Thus, NRE is a previously unrecognized ER- and lipid droplet-associated lysophospholipase. Regulation of enzyme expression by the nutritional status and insulin suggests a role of NRE in the catabolism of lipid precursors and/or mediators that affect energy metabolism in mammals.

G protein beta2 subunit interacts directly with neuropathy target esterase and regulates its activity.

Neuropathy target esterase (NTE) was identified as the primary target of organophosphate compounds that cause a delayed neuropathy with degeneration of nerve axons. NTE is a novel phospholipase B anchored to the cytoplasmic face of endoplasmic reticulum and essential for embryonic and nervous development. However, little is known about the regulation of NTE. A human fetal brain cDNA library was screened for proteins that interact with NTE, Gbeta2 and Gbeta2-like I subunits were found to be able to bind the C-terminal of NTE in yeast. The interaction of Gbeta2 and NTE was confirmed by in vivo co-immunoprecipitation analysis in COS7 cells. Furthermore, depletion of Gbeta2 by RNA interference down regulated the activity of NTE but not its expression level. In addition, the activity of NTE was down regulated by the G protein signal pathway influencing factor, pertussis toxin, treatment in vivo. These findings suggest that Gbeta2 may play a significant role in maintaining the activity of NTE.

A specific human lysophospholipase: cDNA cloning, tissue distribution and kinetic characterization.

Lysophospholipases are critical enzymes that act on biological membranes to regulate the multifunctional lysophospholipids; increased levels of lysophospholipids are associated with a host of diseases. Herein we report the cDNA cloning of a human brain 25 kDa lysophospholipid-specific lysophospholipase (hLysoPLA). The enzyme (at both mRNA and protein levels) is widely distributed in tissues, but with quite different abundances. The hLysoPLA hydrolyzes lysophosphatidylcholine in both monomeric and micellar forms, and exhibits apparent cooperativity and surface dilution kinetics, but not interfacial activation. Detailed kinetic analysis indicates that the hLysoPLA binds first to the micellar surface and then to the substrate presented on the surface. The kinetic parameters associated with this surface dilution kinetic model are reported, and it is concluded that hLysoPLA has a single substrate binding site and a surface recognition site. The apparent cooperativity observed is likely due to the change of substrate presentation. In contrast to many non-specific lipolytic enzymes that exhibit lysophospholipase activity, hLysoPLA hydrolyzes only lysophospholipids and has no other significant enzymatic activity. Of special interest, hLysoPLA does not act on plasmenylcholine. Of the several inhibitors tested, only methyl arachidonyl fluorophosphonate (MAFP) potently and irreversibly inhibits the enzymatic activity. The inhibition by MAFP is consistent with the catalytic mechanism proposed for the enzyme - a serine hydrolase with a catalytic triad composed of Ser-119, Asp-174 and His-208.

Sequence, expression in Escherichia coli, and characterization of lysophospholipase II.

Here we report the sequence, expression in Escherichia coli cells, and characterization of a new small-form lysophospholipase named lysophospholipase II from mouse embryo. The cDNA clone was found and identified among mouse expressed sequence tags in the database search for the homologue of lysophospholipase I previously cloned from rat liver (H. Sugimoto et al., J. Biol. Chem. 271 (1996) 7705-7711). The predicted amino acids sequence contained 231 residues with a calculated molecular weight of 24794, and showed 64% identity to that of lysophospholipase I with the Gly-X-Ser-X-Gly esterase/lipase consensus. The lacZ fusion protein expressed in E. coli cells exhibited lysophospholipase activity and reacted with antibody raised against previously purified pig gastric lysophospholipase II (H. Sunaga et al., Biochem. J. 308 (1995) 551-557), but not with antibody against rat liver lysophospholipase I. The expressed enzyme was purified to a specific activity of 0.15 micromol/min per mg by DEAE-Sepharose A-500 chromatography. The enzyme preferentially utilized zwitterionic lysophospholipids in the order of lysophosphatidylcholine>lysophosphatidylethanolamine, but poorly acidic lysophospholipids, such as lysophosphatidylserine, lysophosphatidylinositol, and lysophosphatidic acid. Not only the 1-acyl isomer, but also the 2-acyl isomer were deacylated. Northern blot analysis and reverse transcription-polymerase chain reaction revealed that lysophospholipase II transcript as well as lysophospholipase I transcript was widely distributed in mouse tissues.

Identification of functional domains of rat intestinal phospholipase B/lipase. Its cDNA cloning, expression, and tissue distribution.

A cDNA encoding a rat intestinal Ca(2+)-independent phospholipase B/lipase (PLB/LIP) was cloned from an ileac mucosa cDNA library using a probe amplified by polymerase chain reaction based on the purified enzyme's sequence. PLB/LIP consists of an NH2-terminal signal peptide, four tandem repeats of about 350 amino acids each, and a hydrophobic domain near the COOH terminus. The enzyme purified previously was found to be derived from the second repeat part. To examine the function of each domain, the full-length PLB/LIP, individual repeats, and a protein lacking the COOH-terminal hydrophobic stretch were expressed in COS-7 cells. The results showed that the second repeat, but not the other repeats, had all the activities (phospholipase A2, lysophospholipase, and lipase) found in the purified natural and expressed full-length enzymes, suggesting repeat 2 is a catalytic domain. The full-length enzyme was mainly present in membrane fractions and efficiently solubilized by treatment with 1% Triton X-100, but not with phosphatidylinositol-specific phospholipase C. Deletion of the COOH-terminal hydrophobic stretch caused the secretion of > 90% of synthesized PLB/LIP into culture media. These results suggest the hydrophobic domain is not replaced by a glycosylphosphatidylinositol anchor but serves as a membrane anchor directly. A message of the full-length PLB/LIP was abundantly expressed in the ileum and also, in a smaller, but significant amount, in the esophagus and testis. Immunohistochemistry showed that PLB/LIP is localized in brush border membranes of the absorptive cells, Paneth cells, and acrosomes of spermatid, suggesting its roles related and unrelated to intestinal digestion.

Identification of extracellular phospholipase B, lysophospholipase, and acyltransferase produced by Cryptococcus neoformans.

We recently identified phospholipase activity as a potential virulence factor of Cryptococcus neoformans. We have now defined the nature of the phospholipase activity produced by a clinical isolate of C. neoformans var. neoformans, under native conditions, by 1H and 31P nuclear magnetic resonance (NMR) spectroscopy and thin-layer chromatography (TLC) of radiolabelled substrates. Glycerophosphocholine was identified by NMR spectroscopy as the sole phospholipid degradation product of the reaction between substrate phosphatidylcholine (PC) and cryptococcal culture supernatants indicating the presence of phospholipase B (PLB). No lysophosphatidylcholine (lyso-PC) or products indicative of phospholipase C, phospholipase D, or other lipase activity were identified. Use of PC and lyso-PC containing radiolabelled acyl chains and separation of products by TLC confirmed the PLB and lysophospholipase (LPL) activities. Lysophospholipase transacylase (LPTA) activity was identified by the formation of radioactive PC from lyso-PC. Extracellular enzyme production was maximal after 6 to 10 h in fresh medium. Assay conditions were optimized for pH, linearity with time, enzyme concentration, and saturation by substrates to allow comparison with phospholipases from other organisms. LPL activity was 10- to 20-fold greater than PLB activity, with mean (+/- standard deviation) specific activities of 34.9 +/- 7.9 and 3.18 +/- 0.2 micromol of substrate hydrolyzed per min per mg of protein, respectively. The response of PLB to increasing substrate concentrations was bimodal, whereas inhibition of LPL and LPTA activities occurred at concentrations of substrate lyso-PC greater than 200 microM. Enzyme activities were stable at acid pH (3.8), with pH optima of 3.5 to 4.5. Activities were unchanged in the presence of exogenous serine protease inhibitors, divalent cations, and EDTA. We conclude that C. neoformans produces highly active extracellular PLB, LPL, and LPTA under native conditions.

Guinea pig intestinal phospholipase B: protein expression during enterocyte maturation and effects of N-oligosaccharide removal on enzymatic activities and protein stability.

Guinea pig phospholipase B (PLB) is an intestinal brush-border hydrolase displaying a broad substrate specificity towards various dietary lipids. PLB was detected by immunoblotting as a single 140-kDa polypeptide in all cell populations isolated from guinea pig intestinal mucosa, but increased in parallel to its activity from undifferentiated to mature cells, the specific activity of the enzyme remaining constant. Moreover, N-glycosylation, which contributed to 23% of the apparent molecular mass, was identical along the cell differentiation axis. In all cell fractions, N-linked sugar chains were of the complex type, since they were removed by N-glycosidase F, whereas PLB remained insensitive to endoglycosidase H. Moreover, lack of O-glycosylation was demonstrated by the insensitivity of PLB to O-glycosidase and by its failure to interact with Helix pomatia lectin after prior treatment with neuraminidase or alpha-fucosidase. Enzymatic removal of sugar chains reduced phospholipase A2, lysophospholipase and diacylglycerol lipase activities by 27-35%, kinetic analysis indicating a decrease in apparent Vmax values for the three enzymatic activities, whereas the Km remained unchanged. Finally, the carbohydrate-depleted form of PLB did not display gross changes in thermal stability, in contrast to PLB from microorganisms previously investigated. Our data indicate that the high level of PLB N-glycosylation is poorly related to its biological function. Whether carbohydrate chains are involved in proper targeting of the enzyme to the brush-border membrane remains to be established.

Purification, cDNA cloning, and regulation of lysophospholipase from rat liver.

A lysophospholipase was purified 506-fold from rat liver supernatant. The preparation gave a single 24-kDa protein band on SDS-polyacrylamide gel electrophoresis. The enzyme hydrolyzed lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylinositol, lysophosphatidylserine, and 1-oleoyl-2-acetyl-sn-glycero-3-phosphocholine at pH 6-8. The purified enzyme was used for the preparation of antibody and peptide sequencing. A cDNA clone was isolated by screening a rat liver lambda gt11 cDNA library with the antibody, followed by the selection of further extended clones from a lambda gt10 library. The isolated cDNA was 2,362 base pairs in length and contained an open reading frame encoding 230 amino acids with a Mr of 24,708. The peptide sequences determined were found in the reading frame. When the cDNA was expressed in Escherichia coli cells as the beta-galactosidase fusion, lysophosphatidylcholine-hydrolyzing activity was markedly increased. The deduced amino acid sequence showed significant similarity to Pseudomonas fluorescence esterase A and Spirulina platensis esterase. The three sequences contained the GXSXG consensus at similar positions. The transcript was found in various tissues with the following order of abundance: spleen, heart, kidney, brain, lung, stomach, and testis = liver. In contrast, the enzyme protein was abundant in the following order: testis, liver, kidney, heart, stomach, lung, brain, and spleen. Thus the mRNA abundance disagreed with the level of the enzyme protein in liver, testis, and spleen. When HL-60 cells were induced to differentiate into granulocytes with dimethyl sulfoxide, the 24-kDa lysophospholipase protein increased significantly, but the mRNA abundance remained essentially unchanged. Thus a posttranscriptional control mechanism is present for the regulation of 24-kDa lysophospholipase.

Possible implication of lysophosphatidylcholine in cell fusion accompanying implantation in rabbits.

Implantation in rabbits involves the cellular fusion of trophoblastic and uterine epithelial cells resulting in embryo penetration of the uterine endometrium. Since lysophospholipids, known to have fusigenic properties, could be responsible for this cell fusion, the metabolism of lysophospholipids was studied throughout gestation in blastocyst/yolk sac and extracoelic amnioallantoic fluids. Analysis of phospholipid composition revealed that lysophospholipids are present in blastocyst/yolk sac fluid. Their concentrations and haemolytic activity change during pregnancy. They increase and reach their highest values during days 7 to 9, the implantation days in rabbits. A clear correlation was observed between lysophosphatidylcholine concentrations in blastocyst/yolk sac fluid and haemolysis induced by this fluid. Phosphatidylcholine concentrations, phospholipase A2 activity, which generates lysophospholipids, and lysophospholipase A activity which hydrolyses lysophosphatidylcholine into fatty acid, were at their highest value at day 12. These data suggest that a transient accumulation of lysophospholipids could ensure local cell fusion. Moreover, we propose that the lysophospholipid concentrations in blastocyst/yolk sac fluid are dependent upon activities of phospholipase A2 and lysophospholipase.

A lysophospholipase specific for exocrine pancreatic cells is stored in zymogen granules and secreted into pancreatic juice.

We separated by two-dimensional (2D) gel electrophoresis the content of isolated rat zymogen granules and from the gel excised a protein of apparent MW 77,500 and an isoelectric point of about 4.7. A rabbit antiserum against this previously uncharacterized rat zymogen granule protein recognized two cDNA clones in a rat pancreas expression library. The cDNA inserts of these two clones had sequences showing perfect homology to the published cDNA sequence of rat pancreatic lysophospholipase. The antiserum recognized only a single protein, lysophospholipase, on one and two-dimensional immunoblots of rat pancreas homogenates and isolated zymogen granules. The antiserum did not react with any protein in homogenates of rat liver, spleen, adrenal, parotid, and prostate tissue. The zymogen granule protein of the guinea pig, previously identified as Lipase 1, was recognized specifically by the antiserum against rat lysophospholipase. This guinea pig protein can now be regarded as lysophospholipase. The same protein was demonstrated in the transformed rat acinar cell line AR4-2J, where both the rate of total enzyme synthesized and the amount of mRNA increased following treatment with dexamethasone. Immunogold labeling established that pancreatic lysophospholipase is restricted exclusively to exocrine cells where it occurs only in compartments of the exocytotic pathway. It could also be detected in pancreatic juice in the ducts of the tissue. Finally, we have shown that lysophospholipase is not related to the zymogen granule membrane protein GP2. This work establishes that lysophospholipase is a normal member of the set of soluble enzymes and proenzymes that are stored in zymogen granules and secreted into pancreatic juice.

Increased extracellular secretion in fragile mutants of Saccharomyces cerevisiae.

Evidences are presented for a generally increased extracellular secretion capability of the fragile mutants of S. cerevisiae. Proteins secreted in wild type yeasts to the periplasmic space can not be retained by the defective cell wall of the fragile cells and are released into the medium. Changes in the structure of non-mannan components of the cell wall might be the reason for the extracellular release of the periplasmic proteins.

Synthesis of eosinophil-associated enzymes in HL-60 promyelocytic leukemia cells.

Eosinophils derived from HL-60 cells share many of the abnormalities of granule histochemistry and morphology frequently seen in eosinophils of patients with certain malignancies, especially those seen in acute myelomonocytic leukemia with abnormal eosinophils (FAB class M4eo). In order to understand the pathogenesis of these abnormalities, four enzymes, characteristic of the eosinophil, were studied in HL-60 promyelocytic leukemia cells at various stages of eosinophilic differentiation. Using biochemical and ultrahistochemical techniques, the following differences from normal eosinophil development were demonstrated. First, both myeloperoxidase and eosinophil peroxidase coexisted in the population of maturing HL-60 eosinophils. Second, the granules formed from the condensation of material in vacuoles which were derived from dilated segments of the endoplasmic reticulum; the role of the Golgi apparatus in processing of peroxidase appeared minimal. Third, low levels of lysophospholipase and arylsulfatase were present in the cells compared to normal eosinophils. Finally, crystallizations resembling precursor structures of Auer rods appeared in the granules of about 5% of the cells. These findings suggest that several disorders of the control of protein synthesis and processing exist in HL-60 eosinophils which may be responsible for the abnormal granule morphology and histochemistry.

Dextran sulfate-induced peritoneal lysophospholipase activity varies among mouse strains.

Lysophospholipase (LPL) activity resulting from the intraperitoneal injection of dextran sulfate (DS) was studied in different mouse strains. AKR/J and BALB/cByJ mouse strains showed decreased LPL levels when a low molecular weight DS was injected but increased LPL activity when high molecular weight DS was injected intraperitoneally. C57BL/6 mice had increased LPL activity with low molecular weight DS but decreased LPL activity with high molecular weight DS. All three mouse strains showed increased peritoneal-cell changes when injected with DS of a molecular weight of 79,000.