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Alpha toxin has become the subject of research in recent years. The objective of this article was to review and summarize recent research on the molecular structure and biological function of the alpha toxin of Clostridium perfringens . This includes the work of our research team, as well as that of other researchers. Clostridium perfringens is an anaerobic, spore-forming, Gram-positive bacillus. It can cause various intestinal diseases, such as gas gangrene, food poisoning, non-foodborne diarrhea, and enteritis. Clostridium perfringens can be classified into 5 toxinotypes A, B, C, D, and E, based on the production of major toxins. Each type of C. perfringens produces alpha toxin, which is one of the most important lethal and dermonecrotic toxins and is considered a primary virulence factor. Alpha toxin is a multifunctional metalloenzyme with phospholipase C and sphingomyelinase activities that simultaneously hydrolyze phosphatidylcholine and sphingomyelin. It can therefore destroy the integrity of cell membranes and eventually cause cell lysis. The clinical effects of alpha toxins are characterized by cytotoxicity, hemolytic activity, lethality, skin necrosis, platelet aggregation, and increased vascular permeability. Future research will concentrate on the pathogenesis of a lpha toxin exposure, clarifying the interaction between alpha toxin and the cell membrane and investigating the mechanism of activating platelet function. This research will have substantial theoretical and practical value in controlling disease progression, identifying targeted therapeutic sites, and reducing the toxic effects of vaccines., Competing Interests: Conflict of interest: The authors declare that there is no conflict of interest associated with the publication of this article., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

Previously, we identified the human annexin A1 as a purification tag for column-free purification with gentler calcium-responsive precipitation. In this work, we used the annexin A1 tagged green fluorescent protein constructs for detecting extracellular production in Escherichia coli, Bacillus subtilis, and Pichia pastoris, and identified that the leaderless fusion protein was transported extracellularly in E. coli with supply of additives including Triton X-100. The coexpressed enzymes, culture compositions, and induction conditions in E. coli extracellular expression systems were optimized. With coexpression of phospholipase C from Bacillus cereus and addition of 0.2 % Triton X-100 after induction for 60 h at 28 °C, the annexin A1 tagged green fluorescent protein and 5-aminolevulinate dehydratase from E. coli were overexpressed and purified from lysogeny broth by precipitation with 20 mM Ca 2+ and redissolution with 25 mM EDTA with the acceptable protein purities and recoveries. The silica binding peptide was fused to the annexin A1 tagged fluorescent protein fusion for successive affinity precipitation and purification. With incubation of the specific protease, the released tag-free protein displayed higher purity via on-resin cleavage than that through cleavage of the free fusion protein. The tandem tag is applicable for two-step purification of small or large amounts of other fusion proteins in the culture and recovery of tag-free proteins at low cost., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Based on the specific binding of drug molecules to cell membrane receptors, a screening and separation method for active compounds of natural products was established by combining phospholipase C (PLC) sensitized hollow fiber microscreening by a solvent seal with high-performance liquid chromatography technology. In the process, the factors affecting the screening were optimized. Under the optimal screening conditions, we screened honokiol (HK), magnolol (MG), negative control drug carbamazepine, and positive control drug amentoflavone, the repeatability of the method was tested. The PLC activity was determined before and after the screening. Experimental results showed that the sensitization factors of PLC of HK and MG were 61.0 and 48.5, respectively, and amentoflavone was 15.0, carbamazepine could not bind to PLC. Moreover, the molecular docking results were consistent with this measurement, indicating that HK and MG could be combined with PLC, and they were potential interacting components with PLC. This method used organic solvent to seal the PLC greatly ensuring the activity, so this method had the advantage of integrating separation, and purification with screening, it not only exhibited good reproducibility and high sensitivity but was also suitable for screening the active components in natural products by various targets in vitro., (© 2024 Wiley‐VCH GmbH.)

Recently, phosphatidylglycerol (PG) focused on its important role in chloroplast photosynthesis, mitochondrial function of the sperm, an inhibitory effect on SARS-CoV-2 ability to infect naïve cells, and reducing lung inflammation caused by coronavirus disease 2019. To develop an enzymatic PG determination method as the high-throughput analysis of PG, a PG-specific phospholipase C (PG-PLC) was found in the culture supernatant of Amycolatopsis sp. NT115. PG-PLC (54 kDa by SDS-PAGE) achieved the maximal activity at pH 6.0 and 55 °C and was inhibited by detergents, such as Briji35, Tween 80, and sodium cholate, but not by EDTA and metal ions, except for Zn2+. The open reading frame of the PG-PLC gene consisted of 1620 bp encoding 515-amino-acid residues containing the preceding 25-amino-acid residues (Tat signal peptide sequence). The putative amino acid sequence of PG-PLC was highly similar to those of metallophosphoesterases; however, its substrate specificity was completely different from those of known PLCs., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

As an essential enzyme for phospholipid degradation, phospholipase C (PLC) has been used for enzymatic degumming of vegetable oils and production of valuable phospholipid derivatives. In this study, rational engineering based on B-factor analysis and molecular dynamic simulation analysis were employed to rationally identify mutation candidates and a PLC double mutant F96R/Q153P was designed from Bacillus cereus HSL3. Compared to the wild-type PLC, F96R/Q153P exhibited significantly improved thermal properties, including higher temperature optima and better thermal stability. It showed the highest optimal reaction temperature (90 °C) reported so far. F96R/Q153P displayed 4.94 times kcat and 2.37 times k cat /K m as much as the wild-type, as well as improved substrate adaptability. Structural insights revealed that the mutations caused reduced proportion of random coil and constraint of certain loop fluctuations. These results demonstrated the great potential of knowledge-based rational design for improving the catalytic characteristics of industrial enzymes in the enzymatic degumming process., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Pleckstrin homology (PH) domains are common modules of ∼120 amino acids found in proteins involved in signalling, cytoskeletal organization, membrane transport, and modification of phospholipids. Previous live cell studies have involved the use of the green-fluorescent protein (GFP) labelling of PH-domain of phospholipase C δ1 (PLC δ1) to study the interactions of molecules at the membrane interface., Methods and Results: For this study, the aim was to construct and express the GFP-PH domain of PLC δ1 in the Saccharomyces cerevisiae BY4741. The transformants expressing GFP-PH domain of PLC δ1 displayed localised fluorescence to the cell periphery (plasma membrane) while the negative control expressed GFP within the cytoplasm only. No GFP was observed in the non-transformed yeast cells., Conclusions: Thus, this technique could be useful in future molecular interactions studies targeted specifically at the yeast cell membrane interface in live yeast cells., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Objectives: Clostridium perfringens is a common anaerobic pathogen causing enteritis/enterocolitis and wound infections in humans. We analyzed clonal diversity and toxin gene prevalence in C. perfringens clinical isolates from humans in northern Japan., Methods: Prevalence of nine toxin genes was analyzed for 585 C. perfringens isolates from patients collected for 20-month period between May 2019 and December 2020 by molecular methods. Sequence type (ST) based on multilocus sequence typing (Xiao's scheme) and alpha-toxin (PLC) sequence type were determined for a total of 124 isolates selected in the present study along with those in our previous study (2017-2018)., Results: Toxinotypes A (68.2%) was the most frequent, followed by F (31.6%), and G (0.2%), while additional toxin genes encoding binary enterotoxin (BEC/CPILE) and beta2 toxin were identified in one and six isolates, respectively. Among the 124 isolates with various toxin gene profiles, 62 STs including 53 novel types were identified, revealing the presence of six clonal complexes (CCs) consisting of 27 STs. Most of enterotoxin gene (cpe)-positive isolates belonged to CC36, CC41, and CC117. Based on 22 key amino acids in alpha toxin sequence, four PLC types (I-IV) including 21 subtypes were classified, and their relation to individual STs/CCs was clarified. Two isolates harboring bec/cpile belonged to different STs (ST95, ST131) and PLC types (If, IVb), indicating distribution of this toxin gene to distinct lineages., Conclusions: The present study revealed the diversity in C. perfringens clones of human origin with various toxin gene profiles represented by ST/CC and PLC type., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Many studies have confirmed the enzymatic activity of a mammalian phosphatidylcholine (PC) phospholipase C (PLC) (PC-PLC), which produces diacylglycerol (DAG) and phosphocholine through the hydrolysis of PC in the absence of ceramide. However, the protein(s) responsible for this activity have never yet been identified. Based on the fact that tricyclodecan-9-yl-potassium xanthate can inhibit both PC-PLC and sphingomyelin synthase (SMS) activities, and SMS1 and SMS2 have a conserved catalytic domain that could mediate a nucleophilic attack on the phosphodiester bond of PC, we hypothesized that both SMS1 and SMS2 might have PC-PLC activity. In the present study, we found that purified recombinant SMS1 and SMS2 but not SMS-related protein have PC-PLC activity. Moreover, we prepared liver-specific Sms1/global Sms2 double-KO mice. We found that liver PC-PLC activity was significantly reduced and steady-state levels of PC and DAG in the liver were regulated by the deficiency, in comparison with control mice. Using adenovirus, we expressed Sms1 and Sms2 genes in the liver of the double-KO mice, respectively, and found that expressed SMS1 and SMS2 can hydrolyze PC to produce DAG and phosphocholine. Thus, SMS1 and SMS2 exhibit PC-PLC activity in vitro and in vivo., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Environmental stimuli are primarily perceived at the plasma membrane. Stimuli perception leads to membrane disintegration and generation of molecules which trigger lipid signaling. In plants, lipid signaling regulates important biological functions however, the molecular mechanism involved is unclear. Phospholipases C (PLCs) are important lipid-modifying enzymes in eukaryotes. In animals, PLCs by hydrolyzing phospholipids, such as phosphatidylinositol-4,5-bisphosphate [PI(4,5)P 2 ] generate diacylglycerol (DAG) and inositol- 1,4,5-trisphosphate (IP 3 ). However, in plants their phosphorylated variants i.e., phosphatidic acid (PA) and inositol hexakisphosphate (IP 6 ) are proposed to mediate lipid signaling. Specific substrate preferences divide PLCs into phosphatidylinositol-PLC (PI-PLC) and non-specific PLCs (NPC). PLC activity is regulated by various cellular factors including, calcium (Ca 2+ ) concentration, phospholipid substrate, and post-translational modifications. Both PI-PLCs and NPCs are implicated in plants' response to stresses and development. Emerging evidences show that PLCs regulate structural and developmental features, like stomata movement, microtubule organization, membrane remodelling and root development under abiotic stresses. Thus, crucial insights are provided into PLC mediated regulatory mechanism of abiotic stress responses in plants. In this review, we describe the structure and regulation of plant PLCs. In addition, cellular and physiological roles of PLCs in abiotic stresses, phosphorus deficiency, aluminium toxicity, pollen tube growth, and root development are discussed., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Bioaugmentation is a promising method of the remediation of soils polluted by persistent organic pollutants (POP). Unfortunately, it happens frequently that the microorganisms inoculated into the soil die out due to the presence of enzymes secreted by autochthonous microorganisms. Especially destructive are here phospholipases C (PLC) and lipases which destruct the microorganism's cellular membrane. The composition of bacterial membranes differs between species, so it is highly possible that depending on the membrane constitution some bacteria are more resistant to PLCs and lipases than other. To shed light on these problems we applied phospholipid Langmuir monolayers as model microbial membranes and studied their interactions with α-toxin (model bacterial PLC) and the lipase isolated from soil fungus Candida rugosa. Membrane phospholipids differing in their headgroup (phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols and cardiolipins) and in their tail structure were applied. The monolayers were characterized by the Langmuir technique, visualized by Brewster angle microscopy, and the packing mode of the phospholipid molecules was verified by the application of the diffraction of synchrotron radiation. We also studied the mutual miscibility of diacylglycerols and the native phospholipids as their interaction is crucial for the understanding of the PLC and lipase activity. It turned out that all the investigated phospholipid classes can be hydrolyzed by PLC; however, they differ profoundly in the hydrolysis degree. Depending on the effects of the initial PLC action and the mutual organization of the diacylglycerol and phospholipid molecules the lipase can ruin the model membranes or can be completely neutral to them., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Listeria monocytogenes is an intracellular food-borne pathogen that causes listeriosis, a severe and potentially life-threatening disease. Listeria uses a number of virulence factors to proliferate and spread to various cells and tissues. In this process, three bacterial virulence factors, the pore-forming protein listeriolysin O and phospholipases PlcA and PlcB, play a crucial role. Listeriolysin O belongs to a family of cholesterol-dependent cytolysins that are mostly expressed by gram-positive bacteria. Its unique structural features in an otherwise conserved three-dimensional fold, such as the acidic triad and proline-glutamate-serine-threonine-like sequence, enable the regulation of its intracellular activity as well as distinct extracellular functions. The stability of listeriolysin O is pH- and temperature-dependent, and this provides another layer of control of its activity in cells. Moreover, many recent studies have demonstrated a unique mechanism of pore formation by listeriolysin O, i.e., the formation of arc-shaped oligomers that can subsequently fuse to form membrane defects of various shapes and sizes. During listerial invasion of host cells, these membrane defects can disrupt phagosome membranes, allowing bacteria to escape into the cytosol and rapidly multiply. The activity of listeriolysin O is profoundly dependent on the amount and accessibility of cholesterol in the lipid membrane, which can be modulated by the phospholipase PlcB. All these prominent features of listeriolysin O play a role during different stages of the L. monocytogenes life cycle by promoting the proliferation of the pathogen while mitigating excessive damage to its replicative niche in the cytosol of the host cell., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Phosphoinositides make up only a small fraction of cellular phospholipids yet control cell function in a fundamental manner. Through protein interactions, phosphoinositides define cellular organelle identity and regulate protein function and organization and recruitment at the cytosol-membrane interface. As a result, perturbations on phosphoinositide metabolism alter cell physiology and lead to a wide range of human diseases, including cancer and diabetes. Among seven phosphoinositide members, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 , also known as PI(4,5)P 2 or PIP 2 ) is abundant in the plasma membrane. Besides its role in the second messenger pathway of phospholipase C that cleaves PtdIns(4,5)P 2 to form diacylglycerol and inositol-1,4,5-trisphosphate (IP 3 ), PtdIns(4,5)P 2 regulates membrane trafficking and the function of the cytoskeleton, ion channels, and transporters. The nanoscale organization of PtdIns(4,5)P 2 in the plasma membrane becomes essential to understand cellular signaling specificity in time and space. Here, we describe a single-molecule method to visualize the nanoscale distribution of PtdIns(4,5)P 2 in the plasma membrane by using super-resolution microscopy and the dual-color fluorescent probes based on the PLC δ1 pleckstrin homology (PH) domain. This approach can be extended to image other phosphoinositides by changing the specific probes.

Mammalian phospholipase C (PLC) isozymes are major signaling nodes that regulate a wide range of cellular processes. Dysregulation of PLC activity has been associated with a growing list of human diseases such as cancer and Alzheimer's disease. However, methods to directly and continuously monitor PLC activity at membranes with high sensitivity and throughput are still lacking. We have developed XY-69, a fluorogenic PIP 2 analog, which can be efficiently hydrolyzed by PLC isozymes either in solution or at membranes. Here, we describe the optimized assay conditions and protocol to measure the activity of PLC-γ1 (D1165H) with XY-69 in lipid vesicles. The described protocol also applies to other PLC isozymes.

Phosphoinositide-specific phospholipase Cs (PLCs) are expressed in all mammalian cells and play critical roles in signal transduction. To obtain a comprehensive understanding of these enzymes in physiology and pathology, a detailed structural, biochemical, cell biological and genetic information is required. In this review, we cover all these aspects to summarize current knowledge of the entire superfamily. The families of PLCs have expanded from 13 enzymes to 16 with the identification of the atypical PLCs in the human genome. Recent structural insights highlight the common themes that cover not only the substrate catalysis but also the mechanisms of activation. This involves the release of autoinhibitory interactions that, in the absence of stimulation, maintain classical PLC enzymes in their inactive forms. Studies of individual PLCs provide a rich repertoire of PLC function in different physiologies. Furthermore, the genetic studies discovered numerous mutated and rare variants of PLC enzymes and their link to human disease development, greatly expanding our understanding of their roles in diverse pathologies. Notably, substantial evidence now supports involvement of different PLC isoforms in the development of specific cancer types, immune disorders and neurodegeneration. These advances will stimulate the generation of new drugs that target PLC enzymes, and will therefore open new possibilities for treatment of a number of diseases where current therapies remain ineffective., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In order to interpret the molecular structure and biological characteristics of Clostridium perfringens alpha-toxin (CPA), the CPA251-370 gene was cloned and the 120 amino acid carboxy terminal of CPA (CPA251-370) was obtained. The secondary and three-dimensional (3D) structures of CPA251-370 were predicted. The secondary structure of CPA251-370 consisted primarily of 35.48% β-sheets and 44.35% random coils. Compared with the CPA toxin consisting of 10 α-helices and eight β-sheets, the 3D structure of CPA251-370 only contained eight β-sheets. The circular dichroism (CD) spectrum detection showed that the CD spectrum of CPA251-370 changed slightly compared with the CD spectrum of CPA. Biological activity assays showed that CPA251-370 had lost the phospholipase C (PLC) activity and haemolytic activity of CPA. More importantly, the mice immunized with CPA251-370 were protected against a challenge with 1 MLD C. perfringens type A strain C57-1. This study laid a solid foundation for explaining the relationship between molecular structure and biological characteristics of CPA in the future. Our research also provides CPA251-370 as a candidate strains for genetic engineering subunit vaccines of C. perfringens type A., (© 2019 Blackwell Verlag GmbH.)

Background: Clostridium perfringens is the causative agent of several diseases and enteric infections in animals and humans. The virulence of C. perfringens is largely attributable to the production of numerous toxins; of these, the alpha toxin (CPA) plays a crucial role in histotoxic infections (gas gangrene). CPA toxin consists of two domains, i.e., the phospholipase C active site, which lies in the N-terminal domain amino acid (aa residues 1-250), and the C-terminal region (aa residues 251-370), which is responsible for the interaction of the toxin with membrane phospholipids in the presence of calcium ions. All currently produced clostridial vaccines contain toxoids derived from culture supernatants that are inactivated, mostly using formalin. The CPA is an immunogenic antigen; recently, it has been shown that mice that were immunized with the C-terminal domain of the toxin produced in E. coli were protected against C. perfringens infections and the anti-sera produced were able to inhibit the CPA activity. Monoclonal and polyclonal antibodies were produced only against full-length CPA and not against the truncated forms., Results: In the present study, we have reported for the first time; about the generation of a recombinant baculovirus capable of producing a deleted rCPA toxin (rBacCPA250-363H6) lacking the N-terminal domain and the 28 amino acids (aa) of the putative signal sequence. The insertion of the L21 consensus sequence upstream of the translational start codon ATG, drastically increases the yield of recombinant protein in the baculovirus-based expression system. The protein was purified by Ni-NTA affinity chromatography and the lack of toxicity in vitro was confirmed in CaCo-2 cells. Polyclonal antibodies and eight hybridoma-secreting Monoclonal antibodies were generated and tested to assess specificity and reactivity. The anti-sera obtained against the fragment rBacCPA250-363H6 neutralized the phospholipase C activity of full-length PLC., Conclusions: The L21 leader sequence enhanced the expression of atoxic C-terminal recombinant CPA protein produced in insect cells. The monoclonal and polyclonal antibodies obtained were specific and highly reactive. The availability of these biologicals could contribute to the development of diagnostic assays and/or new recombinant protein vaccines.

This study provides insights into dynamic nanostructural changes in phospholipid systems during hydrolysis with phospholipase C, the fate of the hydrolysis products, and the kinetics of lipolysis. The effect of lipid restructuring of the vesicle was investigated using small-angle X-ray scattering and cryogenic scanning electron microscopy. The rate and extent of phospholipid hydrolysis were quantified using nuclear magnetic resonance. Hydrolysis of two phospholipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), results in the cleavage of the molecular headgroup, causing two strikingly different changes in lipid self-assembly. The diacylglycerol product of PC escapes the lipid bilayer, whereas the diacylglycerol product adopts a different configuration within the lipid bilayer of the PE vesicles. These results are then discussed concerning the change of the lipid configuration upon the lipid membrane and its potential implications in vivo, which is of significant importance for the detailed understanding of the fate of lipidic particles and the rational design of enzyme-responsive lipid-based drug delivery systems.

To explore the biological activity of Clostridium welchii α-toxin (CPA), the Asp56 residue of CPA was mutated to glycine (CPA D56G) by site-directed mutagenesis, and the 250 amino acid amino-terminal phospholipase C (PLC)-containing domain of CPA (PLC1-250) was isolated. The secondary and three-dimensional (3D) structures of CPA D56G and PLC1-250 were predicted, and the results showed that the secondary structures of CPA D56G and PLC1-250 were composed of α-helices and random coils. The 3D structures of CPA D56G and PLC1-250 were similar to the 3D structures of CPA. The circular dichroism (CD) spectrum of CPA D56G differed from the CD spectrum of CPA, but the CD spectrum of PLC1-250 was similar to the CD spectrum of CPA. Biological activity assays showed that CPA D56G lost the PLC activity of CPA and that mice immunized with CPA D56G were protected against a challenge with 1 MLD C. welchii type A strain C57-1. In addition, PLC1-250 contained the PLC activity of CPA. This study laid a solid foundation for future studies on the relationship between the molecular structure and biological function of CPA and its molecular mechanism. Our study also provided CPA D56G as a candidate strain for engineering a CPA subunit vaccine for C. welchii type A.

Background: Inorganic polyphosphate (polyP) is a fundamental and ubiquitous molecule in prokaryotes and eukaryotes. PolyP has been found in mammalian tissues with particularly high levels of long-chain polyP in bone and cartilage where critical questions remain as to its localization and function. Here, we investigated polyP presence and function in osteoblast-like SaOS-2 cells and cell-derived matrix vesicles (MVs), the initial sites of bone mineral formation., Methods: PolyP was quantified by 4',6-diamidino-2-phenylindole (DAPI) fluorescence and characterized by enzymatic methods coupled to urea polyacrylamide gel electrophoresis. Transmission electron microscopy and confocal microscopy were used to investigate polyP localization. A chicken embryo cartilage model was used to investigate the effect of polyP on mineralization., Results: PolyP increased in concentration as SaOS-2 cells matured and mineralized. Particularly high levels of polyP were observed in MVs. The average length of MV polyP was determined to be longer than 196 P i residues by gel chromatography. Electron micrographs of MVs, stained by two polyP-specific staining approaches, revealed polyP localization in the vicinity of the MV membrane. Additional extracellular polyP binds to MVs and inhibits MV-induced hydroxyapatite formation., Conclusion: PolyP is highly enriched in matrix vesicles and can inhibit apatite formation. PolyP may be hydrolysed to phosphate for further mineralization in the extracellular matrix., General Significance: PolyP is a unique yet underappreciated macromolecule which plays a critical role in extracellular mineralization in matrix vesicles., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Members of the G protein-coupled receptor and TMEM16 (transmembrane protein 16) protein families are phospholipid scramblases that facilitate rapid, bidirectional movement of phospholipids across a membrane bilayer in an ATP-independent manner. On reconstitution into large unilamellar vesicles, these proteins scramble more than 10,000 lipids/protein/s as measured with co-reconstituted fluorescent nitrobenzoxadiazole (NBD)-labeled phospholipids. Although NBD-labeled phospholipids are ubiquitously used as reporters of scramblase activity, it remains unclear whether the NBD modification influences the quantitative outcomes of the scramblase assay. We now report a refined biochemical approach for measuring the activity of scramblase proteins with radiolabeled natural phosphatidylinositol ([ 3 H]PI) and exploiting the hydrolytic activity of bacterial PI-specific phospholipase C (PI-PLC) to detect the transbilayer movement of PI. PI-PLC rapidly hydrolyzed 50% of [ 3 H]PI in large symmetric, unilamellar liposomes, corresponding to the lipid pool in the outer leaflet. On reconstitution of a crude preparation of yeast endoplasmic reticulum scramblase, purified bovine opsin, or purified Nectria haematococca TMEM16, the extent of [ 3 H]PI hydrolysis increased, indicating that [ 3 H]PI from the inner leaflet had been scrambled to the outer leaflet. Using transphosphatidylation, we synthesized acyl-NBD-PI and used it to compare our PI-PLC-based assay with conventional fluorescence-based methods. Our results revealed quantitative differences between the two assays that we attribute to the specific features of the assays themselves rather than to the nature of the phospholipid. In summary, we have developed an assay that measures scrambling of a chemically unmodified phospholipid by a reconstituted scramblase., (© 2018 Wang et al.)

Phospholipase C (PLC) enzymes produce second messengers that increase the intracellular Ca 2+ concentration and activate protein kinase C (PKC). These enzymes also share a highly conserved arrangement of core domains. However, the contributions of the individual domains to regulation are poorly understood, particularly in isoforms lacking high-resolution information, such as PLCϵ. Here, we used small-angle X-ray scattering (SAXS), EM, and functional assays to gain insights into the molecular architecture of PLCϵ, revealing that its PH domain is conformationally dynamic and essential for activity. We further demonstrate that the PH domain of PLCβ exhibits similar dynamics in solution that are substantially different from its conformation observed in multiple previously reported crystal structures. We propose that this conformational heterogeneity contributes to subfamily-specific differences in activity and regulation by extracellular signals.

PEGylation is one of the most promising and extensively studied strategies for improving the properties of proteins as well as enzymic physical and thermal stability. Phospholipase C, hydrolyzing the phospholipids offers tremendous applications in diverse fields. However, the poor thermal stability and higher cost of production have restricted its industrial application. This study focused on improving the stabilization of recombinant PLC by chemical modification with methoxypolyethylene glycol-Succinimidyl Succinate (SS-mPEG, MW 5000). PLC gene from isolate Bacillus cereus HSL3 was fused with SUMO, a novel small ubiquitin-related modifier expression vector and over expressed in Escherichia coli. The soluble fraction of SUMO-PLC reached 80% of the total recombinant protein. The enzyme exhibited maximum catalytic activity at 80 °C and was relatively thermostable at 40-70 °C. It showed extensive substrate specificity pattern and marked activity toward phosphatidylcholine, which made it a typical non-specific PLC for industrial purpose. SS-mPEG-PLC complex exhibited an enhanced thermal stability at 70-80 °C and the catalytic efficiency (K cat /K m ) had increased by 3.03 folds compared with free PLC. CD spectrum of SS-mPEG-PLC indicated a possible enzyme aggregation after chemical modification, which contributed to the higher thermostability of SS-mPEG-PLC. The increase of antiparallel β sheets in secondary structure also made it more stable than parallel β sheets. The presence of SS-mPEG chains on the enzyme molecule surface somewhat changed the binding rate of the substrates, leading to a significant improvement in catalytic efficiency. This study provided an insight into the addition of SS-mPEG for enhancing the industrial applications of phospholipase C at higher temperature., (Copyright © 2018 Elsevier B.V. All rights reserved.)

ApoB-100 and Phosphatidylcholine-specific phospholipase C (PC-PLC) are important contributors to atherosclerosis development. ApoB-100 is the main structural protein of LDL, being directly associated with atherosclerosis plaque generation. PC-PLC is highly expressed in atherosclerosis lesions and contributes to their progression. We show how phosphatidylcholine-coated nanomicelles can be used for specific characterisation of atherosclerosis plaque. Results show that ApoB-100 in the protein corona of the nanomicelle targets the particles to atherosclerotic areas in apolipoprotein E -/- mice. Furthermore, PC-PLC selectively removes the polar heads from the phospholipid coating of the nanomicelles leading to their accumulation. To fully characterise the behaviour of the nanomicelles, we developed multimodal probes using a nanoemulsion step. Hybrid imaging revealed plaque accumulation of the nanomicelles and colocalisation with PC-PLC expression and ApoB-100 in the plaque. This study shows how protein corona composition and enzyme-driven nanomaterial accumulation can be used for detection of atherosclerosis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Nonspecific phospholipase C (NPC), which belongs to a phospholipase C subtype, is a class of phospholipases that hydrolyzes the primary membrane phospholipids, such as phosphatidylcholine, to yield sn-1, 2-diacylglycerol and a phosphorylated head-group. NPC plays multiple physiological roles in lipid metabolism and signaling in plants. To fully understand the putative roles of NPC genes in upland cotton, we cloned NPC genes from Gossypium hirsutum and carried out structural, expression and evolutionary analysis., Results: Eleven NPC genes were cloned from G. hirsutum, which were found on chromosomes scaffold269.1, D03, A07, D07, A08, D11, and scaffold3511_A13. All GhNPCs had typical phosphoesterase domains and have hydrolase activity that acts on ester bonds. GhNPCs were annotated as phospholipase C, which was involved in glycerophospholipid metabolism, ether lipid metabolism, and biosynthesis of secondary metabolites. These GhNPCs showed differential expression patterns in distinct plant tissues and in response to various types of stress (low-phosphate, salt, drought, and abscisic acid). They also had different types and numbers of cis-element. GhNPCs could be classified into four subfamilies. Four pairs of GhNPCs were generated by whole-genome duplication and they underwent purifying selection., Conclusions: Our results suggested that GhNPCs are involved in regulating key abiotic stress responses and ABA signaling transduction, and they may have various functional roles for different members under complex abiotic stress conditions. Functional divergence may be the evolutionary driving force for the retention of four pairs of duplicate NPCs. Our analysis provides a solid foundation for the further functional characterization of the GhNPC gene family, and leads to potential applications in the genetic improvement of cotton cultivars.

Fish nocardiosis is a chronic systemic granulomatous disease, and Nocardia seriolae is the main pathogen that causes it. The pathogenesis and virulence factors of N. seriolae are not fully understood. A phospholipase C (PLC), which is likely to be a secreted protein targeting host cell mitochondria, was found by a bioinformatics analysis of the whole genome sequence of N. seriolae. In order to determine the subcellular localization and study the preliminary function of PLC from N. seriolae (NsPLC), in this study gene cloning, secreted protein identification, subcellular localization in host cells and apoptosis detection of NsPLC were carried out. Mass spectrometry analysis of extracellular products from N. seriolae showed that NsPLC was a secreted protein. Subcellular localization of NsPLC-GFP fusion protein in fathead minnow (FHM) cells revealed that the green fluorescence exhibited a punctate distribution near the nucleus and did not co-localize with mitochondria. In addition, an apoptosis assay suggested that apoptosis was induced in FHM cells by the overexpression of NsPLC. This study may lay the foundations for further studies on the function of NsPLC and promote the understanding of the virulence factors and pathogenic mechanism of N. seriolae., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Apolipophorin III (apoLp-III) is an insect apolipoprotein (18kDa) that comprises a single five-helix bundle domain. In contrast, human apolipoprotein A-I (apoA-I) is a 28kDa two-domain protein: an α-helical N-terminal domain (residues 1-189) and a less structured C-terminal domain (residues 190-243). To better understand the apolipoprotein domain organization, a novel chimeric protein was engineered by attaching residues 179 to 243 of apoA-I to the C-terminal end of apoLp-III. The apoLp-III/apoA-I chimera was successfully expressed and purified in E. coli. Western blot analysis and mass spectrometry confirmed the presence of the C-terminal domain of apoA-I within the chimera. While parent apoLp-III did not self-associate, the chimera formed oligomers similar to apoA-I. The chimera displayed a lower α-helical content, but the stability remained similar compared to apoLp-III, consistent with the addition of a less structured domain. The chimera was able to solubilize phospholipid vesicles at a significantly higher rate compared to apoLp-III, approaching that of apoA-I. The chimera was more effective in protecting phospholipase C-treated low density lipoprotein from aggregation compared to apoLp-III. In addition, binding interaction of the chimera with phosphatidylglycerol vesicles and lipopolysaccharides was considerably improved compared to apoLp-III. Thus, addition of the C-terminal domain of apoA-I to apoLp-III created a two-domain protein, with self-association, lipid and lipopolysaccharide binding properties similar to apoA-I. The apoA-I like behavior of the chimera indicate that these properties are independent from residues residing in the N-terminal domain of apoA-I, and that they can be transferred from apoA-I to apoLp-III., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Clostridium perfringens alpha-toxin (ATX) is considered as a prototype of cytotoxic bacterial phospholipases C, and is the major virulence factor in C. perfringens-induced gas gangrene. It is known that, depending on the dose, ATX causes membrane disruption and cytolysis or only limited hydrolysis of its substrates. In the latter case, toxin activity leads to the unregulated generation of bioactive lipids that can ultimately induce cell death. We have characterized apoptosis and necrosis in highly ATX-sensitive, ganglioside-deficient cells exposed to different concentrations of ATX and we have studied the lipidomic profile of cells treated with ATX as compared to native cells to detect the main changes in the lipidomic profile and the possible involvement of lipid signals in cell death. ATX causes both apoptosis and necrosis, depending on dose and time. ATX activates cell death, stimulating the release of cytochrome C from mitochondria and the consequent activation of caspases-3. Moreover GM95 cells treated with ATX showed important lipidomic alterations, among them we detected a general decrease in several phospholipid species and important changes in lipids involved in programmed cell death e.g. ceramide. The data suggest two different mechanisms of cell death caused by ATX, one leading to (mainly saturated) glycerophospholipid hydrolysis related to an increase in diacylglycerols and associated to membrane damage and necrosis, and a second mechanism involving chiefly sphingomyelin hydrolysis and generation of proapoptotic lipidic mediators such as ceramide, N-acylethanolamine and saturated non-esterified fatty acids., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Phospholipase C (PLC) 1 is known to help the pathogen B. cereus entry to the host cell and human PLC is over expressed in multiple cancers. Knowledge of dynamic activity of the enzyme PLC while in action on membrane lipids is essential and helpful to drug design and delivery. In view of this, interactions of PLC with liposome of various lipid compositions have been visualized by testing enzyme activity and microenvironments around the intrinsic fluorophores of the enzyme. Overall change of the protein's conformation has been monitored by fluorescence spectroscopy and circular dichroism (CD). Liposome aggregation and fusion were predicted by increase in turbidity and vesicle size. PLC in solution has high fluorescence and exhibit appreciable shift in its emission maxima, upon gradual change in excitation wavelength towards the red edge of the absorption band. REES fluorescence studies indicated that certain Trp fluorophores of inactive PLC are in motionally restricted compact/rigid environments in solution conformation. PLC fluorescence decreased in association with liposome and Trps loosed rigidity where liposome aggregation and fusion occurred. We argue that the structural flexibility is the cause of decrease of fluorescence, mostly to gain optimum conformation for maximum activity of the enzyme PLC. Further studies deciphered that the enzyme PLC undergoes change of conformation when mixed to LUVs prepared with specific lipids. CD data at the far-UV and near-UV regions of PLC in solution are in excellent agreement with the previous reports. CD analyses of PLC with LUVs, showed significant reduction of α-helices, increase of β-sheets; and confirmed dramatic change of orientations of Trps. In case of liposome composed of lipid raft like composition, the enzyme binds very fast, hydrolyze PC with higher rate, exhibit highest structural flexibility and promote vesicle fusion. These data strongly suggest marked differences in conformation transition induced PLC activation and liposome fusion on the lipid composition., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objective: In this study, we constructed recombinant Kluyveromyces lactis strains to produce phospholipase C (PLC) of Bacillus cereus. The recombinant enzymes were purified and characterized., Methods: We cloned the PLC encoding gene bcplc of Bacillus cereus. And the amplified fragments were inserted into pKLAC1 to obtain expression plasmids. K. lactis harboring the above plasmids was cultivated to express PLC that was purified by HisTrapTM affinity chromatography and characterized., Results: PLC of B. cereus was cloned and expressed in K. lactis. The recombinant enzyme had shown activity of 19251 U/mg when using p-nitrophenyl phosphorycholine as substrate. Purified PLC exhibited optimum temperature at 80 °C and optimal pH at 9.0. The recombinant enzyme was stable below 40 °C and pH between 7.0 and 8.0. Cu2+ and Co2+ inhibited its activity whereas Zn2+, Mn2+, Ca2+ and Mg2+ stimulated its activity., Conclusion: It is the first time to express and characterize the PLC gene in K. lactis. These research results provide reference for the study of recombinant PLC.

Phospholipases are ubiquitous in nature and the target of significant research aiming at both their physiological roles and technical applications in e.g. the food industry. In the search for sensitive and selective phospholipase assays, we have focused on synthetic FRET (Förster resonance energy transfer) substrates. This has led to the development of a facile, easily scalable and low cost synthesis of fluorogenic phospholipids featuring the dansyl/dabcyl fluorophore/quencher-pair on the fatty acid ω-position and on the phosphatidylethanolamine head group, respectively. Hence, the two substrates lyso-(dansyl-FA)-GPE-dabcyl (6) and (dansyl-FA) 2 -GPE-dabcyl (7) were synthesized by a chemoenzymatic strategy, in which preparation of (6) further included a novel selective enzymatic esterification step. As proof of concept, activity of a handful of phospholipases, one from each of the PLA1, PLA2, PLC and PLD classes, were assayed using substrates (6) and (7), and the kinetic parameter k cat /K M was determined. The PLA1 (Lecitase Ultra™) was found to be highly active on both substrates, whereas the PLD (from white cabbage) had no activity, presumably due to steric effects associated with the dabcyl-functionalization of the head group. It was further substantiated that the substrates are specific towards phospholipase activity as the tested lipase (Lipolase™) showed close to zero activity., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Phospholipase C (PLC) is an important enzyme of signal transduction pathways by generation of second messengers from membrane lipids. PLCs are also indicated to cleave glycosylphosphatidylinositol (GPI)-anchors of surface proteins thus releasing these into the environment. However, it remains unknown whether this enzymatic activity on the surface is due to distinct PLC isoforms in higher eukaryotes. Ciliates have, in contrast to other unicellular eukaryotes, multiple PLC isoforms as mammals do. Thus, Paramecium represents a perfect model to study subcellular distribution and potential surface activity of PLC isoforms. We have identified distinct subcellular localizations of four PLC isoforms indicating functional specialization. The association with different calcium release channels (CRCs) argues for distinct subcellular functions. They may serve as PI-PLCs in microdomains for local second messenger responses rather than free floating IP 3 . In addition, all isoforms can be found on the cell surface and they are found together with GPI-cleaved surface proteins in salt/ethanol washes of cells. We can moreover show them in medium supernatants of living cells where they have access to GPI-anchored surface proteins. Among the isoforms we cannot assign GPI-PLC activity to specific PLC isoforms; rather each PLC is potentially responsible for the release of GPI-anchored proteins from the surface., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Trueperella pyogenes and Clostridium perfringens are two kinds of conditional pathogens frequently associated with wound infections and succeeding lethal complications in various economic livestock. Pyolysin (PLO) and phospholipase C (PLC) are the key virulence factors of these two pathogens, respectively. In our study, a chimeric protein called rPC-PD4, which is composed of the binding regions of PLO and PLC, was synthesized. The toxicity of rPC-PD4 was evaluated. Results revealed that rPC-PD4 is a safe chimeric molecule that can be used to develop vaccines. Immunizing BALB/c mice with rPC-PD4 induced high titers of serum antibodies that could efficiently neutralize the hemolytic activity of recombinant PLO and PLC. After the challenge with T. pyogenes or C. perfringens was performed through the intraperitoneal route, we observed that rPC-PD4 immunization could provide partial immunoprotection and reduce lung, intestine, and liver tissue damage to mice. This work demonstrated the efficacy of the rationally designed rPC-PD4 chimeric protein as a potential vaccine candidate against C. perfringens and T. pyogenes., (Copyright © 2016. Published by Elsevier Ltd.)

As a mode of reproduction in plants, apomixis leads to the generation of clones via seeds. Apomictic plants form viable diploid female gametes without meiosis (apomeiosis) and produce embryos without fertilization (parthenogenesis). Apomeiosis, as a major component of apomixis, has recently been reported in some Arabidopsis thaliana mutants; dyad mutants of SWI1 showed developmental processes common to apomeiosis, such as producing functional diploid gametes. However, the orthologs of SWI1 genes in natural apomicts has not been previously reported. To identify the relationship between the SWI1 gene and the apomeiosis process, we isolated and sequenced SWI1 orthologs from Boechera species, including apomictic and sexual species. Boechera species are close relatives of A. thaliana and thus are advantageous model species for apomixis research. The SWI1 cDNAs were obtained by RT-PCR from apomictic and sexual Boechera young flower buds. We sequenced partial SWI1 transcripts that were 650bp for B. holboellii and 684bp for B. stricta. These SWI1-like sequences showed 86% similarity for B. holboellii and 92% for B. stricta to the A. thaliana SWI1 transcript. We also used available genome data and amplified genomic sequences for SWI1 orthologs in B. holboellii and B. stricta. The predicted proteins contain a phospholipase C domain and a nuclear localization signal. Sequence analysis did not show significant mutations related to apomixis, and phylogenetic analysis showed that SWI1-like sequences were common across plant families, regardless of the presence of a sexual or apomictic reproduction system. We also investigated the expression levels of SWI1 mRNA in the B. holboellii and B. stricta young unopened flower buds and found that relatively high levels of expression occurred in apomicts., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

RAS is a molecular switch that regulates a large number of pathways through interactions with many effector proteins. Most RAS/effector complexes are short-lived, demonstrating fast association and fast dissociation rate and Kds ranging from 10(-8)-10(-5) M, compatible with the signaling function of these interactions in the cell. RAS effectors share little sequence homology but all contain an RAS binding domain that exhibits ubiquitin fold. All effectors bind to the same epitope on RAS by forming an intermolecular beta sheet and creating a number of favorable hydrogen bonds and salt bridges across the binding interface. Several hot-spots on both RAS and effector molecules constitute a general recognition mode. RAS/effector interactions occur only when RAS is found in the active, GTP-bound state, and are disrupted upon GTP hydrolysis, most probably due to increased flexibility of the RAS molecule. Recent NMR studies demonstrate how in the presence of multiple binding partners, RAS prefers certain effectors to others. The hierarchy of these interactions could be altered for RAS oncogenic mutants, thus perturbing the network of the downstream signaling. Insights obtained through biophysical and structural studies of effectors interacting with RAS and its mutants establish the basic principles that could be used for designing drugs in RAS-associated diseases.

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.

Enzymatic oil degumming (removal of phospholipids) using phospholipase C (PLC) is a well-established and environmentally friendly process for vegetable oil refining. In this work, we report the production of recombinant Bacillus cereus PLC in Corynebacterium glutamicum ATCC 13869 in a high cell density fermentation process and its performance in soybean oil degumming. A final concentration of 5.5g/L of the recombinant enzyme was achieved when the respective gene was expressed from the tac promoter in a semi-defined medium. After treatment with trypsin to cleave the propeptide, the mature enzyme completely hydrolyzed phosphatidylcholine and phosphatidylethanolamine, which represent 70% of the phospholipids present in soybean oil. The results presented here show the feasibility of using B. cereus PLC for oil degumming and provide a manufacturing process for the cost effective production of this enzyme., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Clostridium perfringens alpha-toxin is a key mediator of gas gangrene, which is a life-threatening infection that manifests as fever, pain, edema, myonecrosis, and gas production. Alpha-toxin possesses phospholipase C and sphingomyelinase activities. The toxin is composed of an N-terminal domain (1-250 aa, N-domain), which is the catalytic site, and a C-terminal domain (251-370 aa, C-domain), which is the membrane-binding site. Immunization of mice with the C-domain of alpha-toxin prevents the gas gangrene caused by C. perfringens, whereas immunization with the N-domain has no effect. The central loop domain (55-93 aa), especially H….SW(84)Y(85)….G, plays an important role in the interaction with ganglioside GM1a. The toxin binds to lipid rafts in the presence of a GM1a/TrkA complex, and metabolites from phosphatidylcholine to diacylglycerol through the enzymatic activity of alpha-toxin itself. These membrane dynamics leads to the activation of endogenous PLCγ-1 via TrkA. In addition, treatment with alpha-toxin leads to the formation of diacylglycerol at membrane rafts in ganglioside-deficient DonQ cells; this in turn triggers endocytosis and cell death. This article summarizes the current the membrane-binding mechanism of alpha-toxin in detail.

The effects of Clostridium perfringensα-toxin on host cells have previously been studied extensively but the biophysical processes associated with toxicity are poorly understood. The work reported here shows that the initial interaction between the toxin and lipid membrane leads to measurable changes in the physical properties and morphology of the membrane. A Langmuir monolayer technique was used to assess the response of different lipid species to toxin. Sphingomyelin and unsaturated phosphatidylcholine showed the highest susceptibility to toxin lypolitic action, with a two stage response to the toxin (an initial, rapid hydrolysis stage followed by the insertion and/or reorganisation of material in the monolayer). Fluorescence confocal microscopy on unsaturated phosphatidylcholine vesicles shows that the toxin initially aggregates at discrete sites followed by the formation of localised "droplets" accumulating the hydrolysis products. This process is accompanied by local increases in the membrane dipole potential by about 50 (±42) mV. In contrast, red blood cells incubated with the toxin suffered a decrease of the membrane dipole potential by 50 (±40) mV in areas of high toxin activity (equivalent to a change in electric field strength of 10(7) V m(-1)) which is sufficient to affect the functioning of the cell membrane. Changes in erythrocyte morphology caused by the toxin are presented, and the early stages of interaction between toxin and membrane are characterised using thermal shape fluctuation analysis of red cells which revealed two distinct regimes of membrane-toxin interaction.

Activation at fertilization of the vertebrate egg is triggered by Ca(2+) waves. Recent studies suggest the phospholipase C zeta (PLCζ), a sperm-specific protein, triggers egg activation by an IP3-mediated Ca(2+) release and allow Ca(2+) waves at fertilization. In the present study we cloned, characterized, and phylogenetically positioned the European eel PLCζ (PLCζ1). It is 1521 bp long, with 10 exons encoding an open reading frame of 506 amino acids. The amino acid sequence contains an EF-hand domain, X and Y catalytic domains, and a carboxy-terminal C2 domain, all typical of other PLCζ orthologous. The tissue distribution was studied, and the gene expression was determined in testis during induced sexual maturation at three different thermal regimes. Also, brain and pituitary expression was studied through sex maturation at constant temperature. plcζ1 was expressed in brain of male and female, in testis but not in ovaries. By first time in vertebrates, it is reported plcζ1 expression in the pituitary gland. Testis plcζ1 expression increased through spermatogenesis under all the thermal regimes, but being significantly elevated at lower temperatures. It was very low when testis contained only spermatogonia or spermatocytes, while maximum expression was found during spermiogenesis. These results support the hypothesis for an eel sperm-specific PLCζ1 inducing egg activation, similarly to mammals and some teleosts, but different from some other teleost species, which express this protein in ovaries, but not in testes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A new, fast, selective, and reliable capillary electrophoresis method has been developed for analysis of selected phosphoesters (phosphoserine, phosphoethanolamine, phosphoglycerol) and phosphate. The method is based on separation of specific phosphate containing headgroups (phosphoesters) which are cleaved from the glycerol skeleton of a phospholipid by a regioselective enzyme (phospholipase C). Analysis of intact phospholipids with the same polar headgroup but different fatty acids shows that fatty acid composition has a high impact on separation of phospholipids, so analysis of separated polar headgroups, which avoids this influence, represents a much more suitable approach for phospholipid class research. Optimization of method parameters results in running buffers of relatively narrow pH interval (pH about 10) where all phosphoesters are separated. Further method validation has shown that direct UV detection has a sufficient detection limit for all analytes to perform suitable analyses of cell membrane lipids. The optimized method was tested on the lysate of cell membrane of Bacillus subtilis, where all analytes were determined.

Phosphatidylinositol (PtdIns) is a major phospholipid in eukaryotic cells. Many studies have revealed that the phosphoinositide (PI) signaling pathway plays an important role in plant growth and development. Phospholipase C (PLC) is reported to have a crucial role in the PI pathway. This work focuses on the isolation and investigation of PLC in response to abiotic stress factors in green gram. The PLC cDNA, designated VrPLC, encoding a protein of 591 amino acids was cloned and expressed in E. coli. The predicted isoelectric point (pI) and molecular weight were 5.96 and 67.3 kDa, respectively. The tertiary structure of the PLC was also predicted and found to be mainly composed of random coils. In addition, VrPLC expression analysis was performed under environmental stress and the results showed that the expression of VrPLC was rapidly induced in an abscisic acid independent manner in response to drought and salt stress. PLC expression was found to be up-regulated by SA and down-regulated by wound in leaf tissues; however, there was no significant difference in the expression of PLC in plants subjected to high temperature and H2O2. Our results suggest that a close link/relationship between PLC expression and stress responses in green gram.

Polyphosphoinositides (PPIn) are an important family of phospholipids located on the cytoplasmic leaflet of eukaryotic cell membranes. Collectively, they are critical for the regulation of many aspects of membrane homeostasis and signaling, with notable relevance to human physiology and disease. This regulation is achieved through the selective interaction of these lipids with hundreds of cellular proteins, and thus the capability to study these localized interactions is crucial to understanding their functions. In this review, we discuss current knowledge of the principle types of PPIn-protein interactions, focusing on specific lipid-binding domains. We then discuss how these domains have been re-tasked by biologists as molecular probes for these lipids in living cells. Finally, we describe how the knowledge gained with these probes, when combined with other techniques, has led to the current view of the lipids' localization and function in eukaryotes, focusing mainly on animal cells. This article is part of a Special Issue entitled Phosphoinositides., (Published by Elsevier B.V.)

Phospholipase C1 (PLC1), secretory phospholipase A2 (sPLA2) and Ca(2+)/H(+) exchanger proteins regulate calcium signaling and homeostasis in eukaryotes. In this study, we investigate functions for phospholipase C1 (plc-1), sPLA2 (splA2) and a Ca(2+)/H(+) exchanger (cpe-1) in the filamentous fungus Neurospora crassa. The Δplc-1, ΔsplA2, and Δcpe-1 mutants exhibited a growth defect on medium supplemented with the divalent ionophore A23187, suggesting that these genes might play a role in regulation of cytosolic free Ca(2+) concentration ([Ca(2+)](c)) in N. crassa. The strains lacking plc-1, splA2, and cpe-1 possessed higher carotenoid content than wild type at 8°C, 22°C, and 30°C, and showed increased ultraviolet (UV)-survival under conditions that induced carotenoid accumulation. Moreover, Δplc-1, ΔsplA2, and Δcpe-1 mutants showed reduced survival rate under hydrogen peroxide-induced oxidative stress and induced thermotolerance after exposure to heat shock temperatures. Thus, this study revealed multiple cellular roles for plc-1, splA2, and cpe-1 genes in regulation of [Ca(2+)](c), carotenoid accumulation, survival under stress conditions, and acquisition of thermotolerance induced by heat shock.

Objective: In this study, we constructed two recombinant Escherichia coli strains to produce phospholipase C (PLC) from Acinetobacter calcoaceticus. The recombinant enzymes were purified to homogeneity and characterized. [Methods] We cloned the PLC encoding gene plc1, plc2 from genome DNA of A. calcoaceticus ATCC17902. The amplified fragments were inserted into pET28a(+ to obtain expression plasmids. E. coli BL21 (DE3) harboring the above plasmids were cultivated and induced with isopropyl-beta-D-thiogalactopyranoside to express PLCs. The recombinant PLCs were purified by affinity chromatography and their catalytic properties were characterized., Results: Two PLCs from A. calcoaceticus were cloned and functional expressed in E. coli. The recombinant enzymes have activities of 31,160 +/- 418 U/mg for PLC1 and 13640 +/- 354 U/mg for PLC2, when using p-nitrophenyl phosphorycholine as substrate. The purified PLC1 and PLC2 exhibited optimum temperature at 65 degrees C and 50 degrees C, respectively. Their optimal pH were 8 and 7.5, respectively. PLC2 was stable under 40 degrees C and pH at 8, whereas the residual activity of PLC1 was less than 25% in the same condition. Mg2+ and Ca2+ stimulated two enzymes activity, whereas Zn2. stimulated PLC1 and inhibited PLC2. PLC1 and PLC2 hydrolyzed phosphatidylinositol., Conclusion: It is the first time to express and characterize the PLC gene from A. calcoaceticus ATCC17902. These research results provide reference for the study of food-safety microbiological PLC.

Toxicity of the extracellular products (ECPs) and the lethal attributes of phospholipase secreted by pathogenic Photobacterium damselae subsp. piscicida from cobia Rachycentron canadum was studied. An extracellular lethal toxin in the ECPs was partially purified by using Fast Protein Liquid Chromatography system. A protein band (27 kDa) exhibited phospholipase activity on Native-PAGE (by 0.3% egg yolk agar-overlay), was excised and eluted. The pI value of the purified phospholipase was determined as 3.65 and was determined as a phospholipase C by using the Amplex™ Red phosphatidylcholine -Specific phospholipase C Assay kit. The phospholipase showed maximum activity at temperature around 4-40 °C and maximal activity at pH between 8 and 9. The enzyme was inhibited by ethylenediamine-tetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS); but was activated by Ca(2+) and Mg(2+) and inactivated by Zn(2+) and Cu(2+) . Both the ECPs and phospholipase were hemolytic against erythrocytes of cobia and lethal to the fish with LD50 values of 3.25 and 0.91 µg protein g(-1) fish, respectively. In toxicity neutralization test, the rabbit antisera against the phospholipase could neutralize the toxicity of ECPs, indicating that the phospholipase is a major extracellular toxin produced by the bacterium., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A fluorous tagging strategy coupled with enzymatic synthesis is introduced to efficiently synthesize multiple phosphatidylinositides, which are then directly immobilized on a fluorous polytetrafluoroethylene (PTFE) membrane to probe protein-lipid interactions.

In order to proceed in detection and structural analysis of glycosphingolipids (GSLs) in crude lipid extracts, which still remains a challenge in glycosphingolipidomics, we developed a strategy to structurally characterize neutral GSLs in total lipid extracts prepared from in vitro propagated human monocytic THP-1 cells, which were used as a model cell line. The procedure divides into (1) extraction of total lipids from cellular material, (2) enzymatical disintegration of phospholipids by treatment of the crude lipid extract with phospholipase C, (3) subsequent multiple thin-layer chromatography (TLC) overlay detection of individual GSLs with a mixture of various anti-GSL antibodies, and (4) structural analysis of immunostained GSLs directly on the TLC plate using infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI-o-TOF MS) in combination with collision-induced dissociation (CID). Whereas GSLs were mostly undetectable in untreated crude lipid extracts, pretreatment with phospholipase C resulted in clear-cut mass spectra. MS(1) and MS(2) analysis gave similar results when compared to those obtained with a highly purified neutral GSL preparation of THP-1 cells, which served as a control. We could demonstrate in this study the feasibility of simultaneous multiple immunodetection of individual neutral GSLs in one and the same TLC run and their structural characterization in crude lipid extracts after phospholipase C treatment, thereby avoiding laborious and long-lasting sample purification. This powerful combinatorial technique allows for efficient structural characterization of GSLs in small tissue samples and takes a step forward in the emerging field of glycosphingolipidomics.

Plasma membrane Ca(2+) ATPases (PMCAs, also known as ATP2B1-ATP2B4) are known targets of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂], but if and how they control the PtdIns(4,5)P₂ pool has not been considered. We demonstrate here that PMCAs protect PtdIns(4,5)P₂ in the plasma membrane from hydrolysis by phospholipase C (PLC). Comparison of active and inactive PMCAs indicates that the protection operates by two mechanisms; one requiring active PMCAs, the other not. It appears that the mechanism requiring activity is the removal of the Ca(2+) required for sustained PLC activity, whereas the mechanism not requiring activity is PtdIns(4,5)P₂ binding. We show that in PMCA overexpressing cells, PtdIns(4,5)P₂ binding can lead to less inositol 1,4,5-triphosphate (InsP₃) and diminished Ca(2+) release from intracellular Ca(2+) pools. Inspection of a homology model of PMCA suggests that PMCAs have a conserved cluster of basic residues forming a 'blue collar' at the interface between the membrane core and the cytoplasmic domains. By molecular dynamics simulation, we found that the blue collar forms four binding pockets for the phosphorylated inositol head group of PtdIns(4,5)P₂; these pockets bind PtdIns(4,5)P₂ strongly and frequently. Our studies suggest that by having the ability to bind PtdIns(4,5)P₂, PMCAs can control the accessibility of PtdIns(4,5)P₂ for PLC and other PtdIns(4,5)P₂-mediated processes.