Efficient production of a novel alkaline cold-active phospholipase C from Aspergillus oryzae by molecular chaperon co-expression for crude oil degumming.

A novel alkaline cold-active phospholipase C (PLC) gene (AoPC) from Aspergillus oryzae was cloned. AoPC exhibited the highest sequence similarity of 32.5% with that of a PLC from Arabidopsis thaliana. The gene was co-expressed in Pichia pastoris with molecular chaperone PDI (protein disulfide isomerases), and the highest PLC activity of 82, 782 U mL-1 was achieved in a 5-L fermentor. The recombinant enzyme (AoPC) was most active at pH 8.0 and 25 °C, respectively, and it was stable over a broad pH range of 4.5-9.0 and up to 40 °C. It is the first fungal alkaline PLC. The application of AoPC (with 25% citric acid, w/w) in oil degumming process significantly reduced the phosphorus of crude soybean oil by 93.3% to a commercially acceptable level (<10 mg kg-1). Therefore, the relatively high yield and excellent properties of AoPC may possess it great potential in crude oil refining industry.

Recent research progress with phospholipase C from Bacillus cereus.

Phospholipase C (PLC) catalyzes the hydrolysis of phospholipids to produce phosphate monoesters and diacylglycerol. It has many applications in the enzymatic degumming of plant oils. PLC Bc , a bacterial PLC from Bacillus cereus, is an optimal choice for this activity in terms of its wide substrate spectrum, high activity, and approved safety. Unfortunately, its large-scale production and reliable high-throughput screening of PLC Bc remain challenging. Herein, we summarize the research progress regarding PLC Bc with emphasis on the screening methods, expression systems, catalytic mechanisms and inhibitor of PLC Bc . This review hopefully will inspire new achievements in related areas, to promote the sustainable development of PLC Bc and its application.

A novel phosphatidylcholine-hydrolyzing phospholipase C induced by phosphate starvation in Arabidopsis.

During phosphate starvation, it is known that phospholipids are degraded, and conversely, a nonphosphorus galactolipid digalactosyldiacylglycerol accumulates in the root plasma membrane of plants. We report a novel phospholipase C that hydrolyzes phosphatidylcholine and is greatly induced in response to phosphate deprivation in Arabidopsis. Since phosphatidylcholine-hydrolyzing activity by phospholipase C was highly up-regulated in phosphate-deprived plants, gene expression of some phospholipase C was expected to be induced during phosphate starvation. Based on amino acid sequence similarity to a bacterial phosphatidylcholine-hydrolyzing phospholipase C, six putative phospholipase Cs were identified in the Arabidopsis genome, one of which, NPC4, showed significant transcriptional activation upon phosphate limitation. Molecular cloning and functional expression of NPC4 confirmed that the NPC4 gene encoded a functional phosphatidylcholine-hydrolyzing phospholipase C that did not require Ca(2+) for its activity. Subcellular localization analysis showed that NPC4 protein was highly enriched in the plasma membrane. Analyses of transferred DNA-tagged npc4 mutants revealed that disruption of NPC4 severely reduces the phosphatidylcholine-hydrolyzing phospholipase C activity in response to phosphate starvation. These results suggest that NPC4 plays an important role in the supply of both inorganic phosphate and diacylglycerol from membrane-localized phospholipids that would be used for phosphate supplementation and the replacement of polar lipids in the root plasma membrane during phosphate deprivation.

Neuronal lineage-specific induction of phospholipase Cepsilon expression in the developing mouse brain.

Phospholipase C is a key enzyme of intracellular signal transduction in the central nervous system. We and others recently discovered a novel class of phospholipase C, phospholipase Cepsilon, which is regulated by Ras and Rap small GTPases. As a first step toward analysis of its function, we have examined the spatial and temporal expression patterns of phospholipase Cepsilon during mouse development by in situ hybridization and immunohistochemistry. Around embryonic day 10.5, abundant expression of phospholipase Cepsilon is observed specifically in the outermost layer of the neural tube. On embryonic day 12 and later, it is observed mainly in the marginal zone of developing brain and spinal cord as well as in other regions undergoing neuronal differentiation, such as the retina and olfactory epithelium. The phospholipase Cepsilon-expressing cells almost invariably express microtubule-associated protein 2, but hardly express nestin or glial fibrillary acidic protein, indicating that the expression of phospholipase Cepsilon is induced specifically in cells committed to the neuronal lineage. The expression of phospholipase Cepsilon persists in the terminally differentiated neurons and exhibits no regional specificity. Further, an in vitro culture system of neuroepithelial stem cells is employed to show that abundant expression of phospholipase Cepsilon occurs in parallel with the loss of nestin expression as well as with the induction of microtubule-associated protein 2 expression and neuronal morphology. Also, glial fibrillary acidic protein-positive glial lineage cells do not exhibit the high phospholipase Cepsilon expression. These results suggest that the induction of phospholipase Cepsilon expression may be a specific event associated with the commitment of the neural precursor cells to the neuronal lineage.

Overexpression of phospholipase Cbeta-1 protects NIH3T3 cells from oxidative stress-induced cell death.

Oxidative stress has been implicated in a wide range of cellular damage which includes DNA oxidation, membrane lipid peroxidation, and apoptosis. In our study, we found that overexpression of PLC-beta1 in NIH3T3 fibroblasts protected them from cell death occuring in response to oxidative stress. Cell death caused by treatment with prooxidant tert-butylhydroperoxide (TBH), H2O2, or CdCl2 was considerably suppressed in PLC-beta1 overexpressed NIH/beta1-14 cells in comparison to control NIH/neo cells. However, overexpression of PLC-beta1 failed to protect the cells from toxicity by diamide or KCN. In addition, while accumulation of c-fos mRNA was observed within 30 min of TBH treatment in vector transfected NIH/neo cells, TBH-induced c-fos mRNA generation was completely suppressed in NIH/beta1-14 cells, while that of c-jun and GAPDH was not affected. These findings suggest that PLC-beta1 may play a role in process that can protect cells from oxidative stress-induced cell death.

Role of phospholipase Cgamma at fertilization and during mitosis in sea urchin eggs and embryos.

It is well known that stimulation of egg metabolism after fertilization is due to a rise in intracellular free calcium concentration. In sea urchin eggs, this first calcium signal is followed by other calcium transients that allow progression through mitotic control points of the cell cycle of the early embryo. How sperm induces these calcium transients is still far from being understood. In sea urchin eggs, both InsP3 and ryanodine receptors contribute to generate the fertilization calcium transient, while the InsP3 receptor generates the subsequent mitotic calcium transients. The identity of the mechanisms that generate InsP3 after fertilization remains an enigma. In order to determine whether PLCgamma might be the origin of the peaks of InsP3 production that punctuate the first mitotic cell cycles of the fertilized sea urchin egg, we have amplified by RT-PCR several fragments of sea urchin PLCgamma containing the two SH2 domains. The sequence shares similarities with SH2 domains of PLCgamma from mammals. One fragment was subcloned into a bacterial expression plasmid and a GST-fusion protein was produced and purified. Antibodies raised to the GST fusion protein demonstrate the presence of PLCgamma protein in eggs. Microinjection of the fragment into embryos interferes with mitosis. A related construct made from bovine PLCgamma also delayed or prevented entry into mitosis and blocked or prolonged metaphase. The bovine construct also blocked the calcium transient at fertilization, in contrast to a tandem SH2 control construct which did not inhibit either fertilization or mitosis. Our data indicate that PLCgamma plays a key role during fertilization and early development.

Genetic characterization of a phospholipase C gene from Candida albicans: presence of homologous sequences in Candida species other than Candida albicans.

Phospholipase C (PLC) enzymes are essential in regulating several important cellular functions in eukaryotes, including yeasts. In this study, PCR was used to identify a gene encoding PLC activity in Candida albicans, using oligonucleotide primers complementary to sequences encoding highly conserved amino acid regions within the X domains of previously characterized eukaryotic phospholipase C genes. The nucleotide sequence of the C. albicans gene, CAPLC1 (2997 bp), was determined from a recombinant clone containing C. albicans 132A genomic DNA; it encoded a polypeptide of 1099 amino acids with a predicted molecular mass of 124.6 kDa. The deduced amino acid sequence of this polypeptide (CAPLC1) exhibited many of the features common to previously characterized PLCs, including specific X and Y catalytic domains. The CAPLC1 protein also exhibited several unique features, including a novel stretch of 18-19 amino acid residues within the X domain and an unusually long N-terminus which did not contain a recognizable EF-hand Ca(2+)-binding domain. An overall amino acid homology of more than 27% with PLCs previously characterized from Saccharomyces cerevisiae and Schizosaccharomyces pombe suggested that the CAPLC1 protein is a delta-form of phosphoinositide-specific PLC (PI-PLC). PLC activity was detected in cell-free extracts of both yeast and hyphal forms of C. albicans 132A following 7 h and 24 h growth using the PLC-specific substrate p-nitrophenylphosphorylcholine (p-NPPC). In addition, CAPLC1 mRNA was detected by reverse transcriptase PCR in both yeast and hyphal forms of C. albicans 132A at the same time intervals. Expression of CAPLC1 activity was also detected in extracts of Escherichia coli DH5 alpha harbouring plasmids which contained portions of the CAPLC1 gene lacking sequences encoding part of the N-terminus. Southern hybridization and PCR analyses revealed that all C. albicans and Candida dubliniensis isolates examined possessed sequences homologous to CAPLC1. Sequences related to CAPLC1 were detected in some but not all isolates of Candida tropicalis, Candida glabrata and Candida parapsilosis tested, but not in the isolates of Candida krusei, Candida kefyr, Candida guillermondii and Candida lusitaniae examined. This paper reports the first description of the cloning and sequencing of a PLC gene from a pathogenic yeast species.

Identification of a phospholipase C beta2 region that interacts with Gbeta-gamma.

To delineate the phospholipase C (PLC; EC 3.1.4.3) beta2 sequences involved in interactions with the beta-gamma subunits of G proteins, we prepared a number of mammalian expression plasmids encoding a series of PLC beta2 segments that span the region from the beginning of the X box to the end of the Y box. We found the sequence extending from residue Glu-435 to residue Val-641 inhibited Gbeta-gamma-mediated activation of PLC beta2 in transfected COS-7 cells. This PLC beta2 sequence also inhibited ligand-induced activation of PLC in COS-7 cells cotransfected with cDNAs encoding the complement component C5a receptor and PLC beta2 but not in cells transfected with the alpha1B-adrenergic receptor, suggesting that the PLC beta2 residues (Glu-435 to Val-641) inhibit the Gbeta-gamma-mediated but not the Galpha-mediated effect. The inhibitory effect on Gbeta-gamma-mediated activation of PLC beta2 may be the result of the interaction between Gbeta-gamma and the PLC beta2 fragment. This idea was confirmed by the observation that a fusion protein comprising these residues (Glu-435 to Val-641) of PLC beta2 and glutathione S-transferase (GST) bound to Gbeta-gamma in an in vitro binding assay. The Gbeta-gamma-binding region was further narrowed down to 62 amino acids (residues Leu-580 to Val-641) by testing fusion proteins comprising various PLC beta2 sequences and GST in the in vitro binding assay.

Cloning and identification of amino acid residues of human phospholipase C delta 1 essential for catalysis.

In vitro single point mutagenesis, inositol phospholipid hydrolysis, and substrate protection experiments were used to identify catalytic residues of human phosphatidylinositide-specific phospholipase C delta 1 (PLC delta 1) isolated from a human aorta cDNA library. Invariant amino acid residues containing a functional side chain in the highly conserved X region were changed by in vitro mutagenesis. Most of the mutant enzymes were still able to hydrolyze inositol phospholipid with activity ranging from 10 to 100% of levels in the wild type enzyme. Exceptions were mutants with the conversion of Arg338 to Leu (R338L), Glu341 to Gly (E341G), or His356 to Leu (H356L), which made the enzyme severely defective in hydrolyzing inositol phospholipid. Phospholipid vesicle binding experiments showed that these three cleavage-defective mutant forms of PLC delta 1 could specifically bind to phosphatidylinositol 4,5-bisphosphate (PIP2) with an affinity similar to that of wild type enzyme. Western blotting analysis of trypsin-treated enzyme-PIP2 complexes revealed that a 67-kDa major protein fragment survived trypsin digestion if the wild type enzyme, E341G, or H356L mutant PLC delta 1 was preincubated with 7.5 microM PIP2, whereas if it was preincubated with 80 microM PIP2, the size of major protein surviving was comparable to that of intact enzyme. However, mutant enzyme R338L was not protected from trypsin degradation by PIP2 binding. These observations suggest that PLC delta 1 can recognize PIP2 through a high affinity and a low affinity binding site and that residues Glu341 and His356 are not involved in either high affinity or low affinity PIP2 binding but rather are essential for the Ca(2+)-dependent cleavage activity of PLC.

Differential coupling of G protein alpha subunits to seven-helix receptors expressed in Xenopus oocytes.

Xenopus oocytes were used to examine the coupling of the serotonin 1c (5HT1c) and thyrotropin-releasing hormone (TRH) receptors to both endogenous and heterologously expressed G protein alpha subunits. Expression of either G protein-coupled receptor resulted in agonist-induced, Ca(2+)-activated Cl- currents that were measured using a two-electrode voltage clamp. 5HT-induced Cl- currents were reduced 80% by incubating the injected oocytes with pertussis toxin (PTX) and inhibited 50-65% by injection of antisense oligonucleotides to the PTX-sensitive Go alpha subunit. TRH-induced Cl- currents were reduced only 20% by PTX treatment but were inhibited 60% by injection of antisense oligonucleotides to the PTX-insensitive Gq alpha subunit. Injection of antisense oligonucleotides to a novel Xenopus phospholipase C-beta inhibited the 5HT1c (and Go)-induced Cl- current with little effect on the TRH (and Gq)-induced current. These results suggest that receptor-activated Go and Gq interact with different effectors, most likely different isoforms of phospholipase C-beta. Co-expression of each receptor with seven different mammalian G protein alpha subunit cRNAs (Goa, Gob, Gq, G11, Gs, Golf, and Gt) was also examined. Co-expression of either receptor with the first four of these G alpha subunits resulted in a maximum 4-6-fold increase in Cl- currents; the increase depended on the amount of G alpha subunit cRNA injected. This increase was blocked by PTX for G alpha oa and G alpha ob co-expression but not for G alpha q or G alpha 11 co-expression. Co-expression of either receptor with Gs, Golf, or Gt had no effect on Ca(2+)-activated Cl- currents; furthermore, co-expression with Gs or Golf also failed to reveal 5HT- or TRH-induced changes in adenylyl cyclase as assessed by activation of the cystic fibrosis transmembrane conductance regulator Cl- channel. These results indicate that in oocytes, the 5HT1c and TRH receptors do the following: 1) preferentially couple to PTX-sensitive (Go) and PTX-insensitive (Gq) G proteins and that these G proteins act on different effectors, 2) couple within the same cell type to several different heterologously expressed G protein alpha subunits to activate the oocyte's endogenous Cl- current, and 3) fail to couple to G protein alpha subunits that activate cAMP or phosphodiesterase.

Cell-associated haemolytic activity of Helicobacter pylori.

Helicobacter pylori cells cultured on solid medium were quantitatively tested for haemolytic activity against erythrocytes of man, sheep, the guinea pig and rabbit. Using 4-day and 8-day cultures of two standard strains (ATCC 43504, IMMi 676), human erythrocytes were not lysed by 10% bacterial suspensions. Rabbit erythrocytes were the most sensitive to 8-day cultures. Hot-cold incubation yielded the highest haemolysis titres. The extent of haemolysis strongly correlated with the number of bacterial cells. Supplementation of the test medium (PBS, pH 7.4) with L-cysteine, dithiothreitol, MgCl2, EDTA, cholesterol, lecithin or sphingomyelin did not influence the haemolysis titres. They were significantly reduced in the presence of pronase E, human serum, bovine serum albumin or CaCl2, and by heat treatment of the bacteria. Supplementation of the test medium with cardiolipin strongly increased the haemolysis titres. Comparing the cell-associated haemolytic activity of 18 strains, the titres ranged from < 2 to 64, with a median titre of 16. No correlation was found between the haemolytic activity and phospholipase C activity of the cell suspensions. It was concluded that the formation of lysophosphatides and non-enzymatic factors rather than a sulphydryl-activated cytolysin or phospholipase C are responsible for the cell-associated haemolytic activity. This property may be involved in the pathogenicity and virulence of Helicobacter pylori.