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Polysorbates (PS) are surfactants commonly added in a therapeutic protein drug product as excipients to protect proteins from denaturation and aggregation during storage, transportation, and delivery. Significant degradation of PS in drug products could lead to shortened drug shelf lives and PS-degrading activity in drug products must be minimized. Identification of lipases that degrade PS could lead to better process control in drug manufacturing. In 2016, phospholipase B-like 2 (PLBD2) was proposed as a residual host cell protein responsible for degrading PS20 in a drug formulation. We have carried out a series of studies to verify the role of PLBD2 in degrading polysorbates in drug products purified from recombinant Chinese Hamster Ovary (CHO) cells. Genetic knock-out and immuno-depletion results showed that when PLBD2 was removed or depleted, the degradation of PS20 or PS80 was neither diminished nor reduced. In addition, a quantitative analysis of PLBD2 and PS20 degradation in multiple formulated mAb products did not establish a correlation between the amount of PLBD2 and the level of PS20 degradation. Collectively these results suggest that PLBD2 is not the primary cause of polysorbate degradation in formulated drug products purified using standard Protein A and ion exchange chromatography.

The intraspecific geographical venom variations of Calloselasma rhodostoma from Malaysia (CR-M), Indonesia (CR-I), Thailand (CR-T) and Vietnam (CR-V) were investigated through 1D SDS-PAGE and nano-ESI-LCMS/MS. The venom antigenicity, procoagulant activities and neutralization using Thai C. rhodostoma Monovalent Antivenom (CRMAV) were also investigated. SDS-PAGE patterns of the venoms were relatively similar with minor variations. Proteomic analysis revealed that snake venom metalloproteinases (SVMPs, particularly P–I class), serine proteases (SVSPs) and snaclecs dominated the venom protein composition (68.96–81.80%), followed by L-amino acid oxidase (LAAO) and phospholipase A2 (PLA2) (7.37–11.08% and 5.18–13.81%, respectively), corroborating C. rhodostoma envenoming effects (hemorrhage, consumptive coagulopathy, thrombocytopenia and local tissue necrosis). Other proteins of lower abundances (2.82–9.13%) identified include cysteine-rich secretory proteins (CRISP), phospholipase B, phosphodiesterase, nerve growth factor, 5′-nucleotidase, aminopeptidase and hyaluronidase. All four venoms exhibited strong procoagulant effects which were neutralized by CRMAV to different extents. CRMAV immunoreactivity was high toward venoms of CR-M, CR-I and CR-T but relatively low for CR-V venom. Among the venom samples from different locales, CR-V venom proteome has the smallest SVMP composition while SVSP, PLA2 and phosphodiesterase were more abundant in the venom. These variations in C. rhodostoma venom protein composition could partly explain the differences seen in immunoreactivity. (198 words).

The genus Porthidium includes nine pitviper species inhabiting Mexico, Central America, and northern South America. Porthidium porrasi is a species endemic to the Southwest of Costa Rica, for which no information on its venom was available. In this study, the proteomic composition and functional activities of P. porrasi venom are described. The most abundant venom proteins were identified as metalloproteinases (36.5%). In descending order of abundance, proteins belonging to the disintegrin, phospholipase A2, serine proteinase, C-type lectin/ lectin-like, vascular endothelial growth factor, Cysteine-rich secretory protein, L-amino acid oxidase, phospholipase B, and phosphodiesterase families were also identified. P. porrasi venom showed a weak lethal potency in mice (10 μg/g body weight by intraperitoneal route), induced marked hemorrhage and edema, and weak myotoxic effect. These in vivo activities, as well as those assayed in vitro (proteolytic and phospholipase A2 activities) correlated with compositional data. A comparison of P. porrasi venom with those of three other Porthidium species studied to date reveals a generally conserved compositional and functional pattern in this pitviper genus. Importantly, the lethal effect of P. porrasi venom in mice was adequately cross-neutralized by a heterospecific polyvalent antivenom, supporting its use in the treatment of eventual envenomings by this species. Universidad de Costa Rica/[741-B7-608]/UCR/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP) UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Biología

Phospholipase-B-like (SVPLB-like) enzymes are present in relatively small amounts in a number of venoms, however, their biological function and mechanisms of action are un-clear. A three-dimensional model of the SVPLB-like enzyme from Crotalus adamanteus was generated by homology modeling based on the crystal structures of bovine Ntn-hydrolyases and the modeled protein possesses conserved domains characteristic of Ntn-hydrolases. Molecular dynamics simulations indicate that activation by autocatalytic cleavage results in the removal of 25 amino acids which increases accessibility to the active site. SVPLB-like enzymes possess a highly reactive cysteine and are hence amidases that to belong to the N-terminal nucleophile (Ntn) hydrolase family. The Ntn-hydrolases (N-terminal nucleophile) form a superfamily of diverse enzymes that are activated autocatalytically; wherein the N-terminal catalytic nucleophile is implicated in the cleavage of the amide bond.

Hypnale hypnale (hump-nosed pit viper) is considered to be one among the medically important venomous snake species of India and Sri Lanka. In the present study, venom proteome profiling of a single Hypnale hypnale from Western Ghats of India was achieved using SDS-PAGE based protein separation followed by LC-MS/MS analysis. The identities of the proteins that were not established using the Mascot search were determined through de novo sequencing tools such as Novor followed by MS-BLAST based sequence similarity search algorithm and PEAKS proteomics software. The combined proteomics analysis revealed a total of 37 proteins belonging to nine different snake venom families, in which 7 proteins were exclusively identified through de novo strategies. The enzymatic and non-enzymatic venom protein families identified include serine proteases, metalloproteases, phospholipase A2, thrombin-like enzymes, phospholipase B, C-type lectins/snaclecs, disintegrins, cysteine rich secretory proteins and nerve growth factor. Among these, disintegrins, nerve growth factor, phospholipase B and cysteine rich secretory protein families were identified for the first time in HPV venom. This could possibly explain the regiospecific venom variation seen across snake species. Taken together, the venom proteome profiling on Indian Hypnale hypnale venom correlates with the clinical manifestations often seen in the envenomed victims.

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 6.6 mg mL−1 protein and 6503.8 U mL−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 1170 U mg−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 75.88 ppm to 3.3 ppm in 2 h under optimal reaction conditions. This study provides a basis for the industrial use of F. oxysporum PLB in oil degumming applications.

Classical lipases are well known for being enzymes hydrolysing triacylglycérols as substrate, except the porcine pancreatic lipase (PPL) which was able to hydrolyze phosphatidylcholine. Amino acid sequence alignments revealed that Valine 260 residue in PPL lid, postulated to be responsible for the PPL phospholipase activity, was present in the Turkey pancreatic lipase (TPL). The importance of Val 260 in the phospholipase activities expression has been reported. To confirm this fact, Val 260 was mutated to Alanine in the TPL lid. Mutated protein has conserved its phospholipase activity as well as the non mutated TPL. Therefore, Valine 260 residue in the lid is not involved in the pancreatic lipases phospholipase activity. The rTPL phospholipase activity was also studied using monolayer technique. This avian pancreatic lipase has shown phospholipase activity toward differently charged phospholipids. The highest phospholipase activity was found on phosphatidylglycerol (negatively charged substrate) at a surface pressure of 20mN/m, but when a zwitterionic substrate was used (DLPC), a lower activity was found at a surface pressure of 10mN/m. However, it is worth noticing that the TPL phospholipase activity is about 100 fold lower than its lipase activity. GC chromatography analyses of the released fatty acids from the hydrolysis of 1,2-POPC have shown that rTPL hydrolyses esters bonds at the sn-1 as well as the sn-2 position of phospholipids. Hence, rTPL shows a low phospholipase activity in comparison to its activity toward triacylglycerols.

Phospholipids also play a major role in maintaining the lipid droplet (LD) morphology. In our current study, deletion of LPL1 resulted in altered morphology of LDs and was confirmed by microscopic analysis. LPL1/YOR059c contains lipase specific motif GXSXG and acetate labeling in the LPL1 overexpressed strains depicted a decrease in glycerophospholipids and an increase in free fatty acids. The purified Lpl1p showed phospholipase activity with broader substrate specificity, acting on all glycerophospholipids primarily at sn-2 position and later at sn-1 position. Localization studies precisely revealed that Lpl1 is exclusively localized in the LD at the stationary phase. Site directed mutagenesis experiments clearly demonstrated that the lipase motif is vital for the phospholipase activity. In summary, our results demonstrate that yeast Lpl1 exerts phospholipase activity, plays a vital role in LD morphology, and its absence results in altered LD size. Based on the localization and enzyme activity we renamed YOR059c as LPL1 (LD phospholipase 1).

Phospholipase B from Pseudomonas fluorescens BIT-18 can cleave acyl chains at the sn-1 and sn-2 positions of a phospholipid and has been successfully used to degum vegetable oils in our previous work. This study focused on the heterologous overexpression of phospholipase B (Pf-PLB-P) in Pichia pastoris to investigate its characteristics and application in degumming vegetable oils. After optimizing the fermentation conditions, the maximum achieved enzyme activity was 65 U/ml, which was twice the enzyme activity of wild-strain P. fluorescens BIT-18. Purified Pf-PLB-P was obtained by ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration. The kinetic constants Km and Vmax were determined to be 4.75 mM and 98.67 mmol/(L min), respectively. Pf-PLB-P enzyme activity was detected at 25–55 °C and pH 4.5–9.5, and the temperature range was observed to be slightly broadened than that of the wild type. Based on these characteristics, Pf-PLB-P was also successfully used to degum soybean and peanut oils, whose phosphorus contents decreased from 125.1 mg/kg to 4.96 mg/kg and 96 mg/kg to 3.54 mg/kg, respectively. These results indicate that Pf-PLB-P produced by P. pastoris has potential industrial use.

Despite a strict requirement for sterol removal for sperm to undergo acrosome exocytosis (AE), the mechanisms by which changes in membrane sterols are transduced into changes in sperm fertilization competence are poorly understood. We have previously shown in live murine sperm that the plasma membrane overlying the acrosome (APM) contains several types of microdomains known as membrane rafts. When characterizing the membrane raft-associated proteomes, we identified phospholipase B (PLB), a calcium-independent enzyme exhibiting multiple activities. Here, we show that sperm surface PLB is activated in response to sterol removal. Both biochemical activity assays and immunoblots of subcellular fractions of sperm incubated with the sterol acceptor 2-hydroxypropyl-β-cyclodextrin (2-OHCD) confirmed the release of an active PLB fragment. Specific protease inhibitors prevented PLB activation, revealing a mechanistic requirement for proteolytic cleavage. Competitive inhibitors of PLB reduced the ability of sperm both to undergo AE and to fertilize oocytes in vitro, suggesting an important role in fertilization. This was reinforced by our finding that incubation either with protein concentrate released from 2-OHCD-treated sperm or with recombinant PLB peptide corresponding to the catalytic domain was able to induce AE in the absence of other stimuli. Together, these results lead us to propose a novel mechanism by which sterol removal promotes membrane fusogenicity and AE, helping confer fertilization competence. Importantly, this mechanism provides a basis for the newly emerging model of AE in which membrane fusions occur during capacitation/transit through the cumulus, prior to any physical contact between the sperm and the oocyte's zona pellucida.

A gene from Pseudomonas fluorescens BIT-18 encoding a protein with phospholipase B activity (Pf-PLB) was cloned in E. coli BL21 (DE3). The open reading frame consists of 1272 bp and potentially encodes a protein of 423 amino acid residues with a calculated molecular mass of 45.8 kDa. The nucleotide sequence of Pf-PLB is 45%, 42%, 41%, 40%, 33%, and 31% identical to that of Bifidobacterium animals, Mycobacterium parascrofulaceum, Acidobacterium capsulatum, Lactobacillus johnsonii, Moraxella bovis, and Moraxella catarrhalis, respectively. The His-tagged protein was purified by affinity chromatography and the eluted protein hydrolyzed both the 1- and 2-ester bond of phosphatidylcholine. The recombinant Pf-PLB had optimal activity at pH 6.0 and 30 °C, and it showed 20.1% higher efficiency in the conversion rate of the phosphorus content than the wild-type.

A phospholipase A₂ was identified from MDCK cell homogenates with broad specificity toward glycerophospholipids including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol. The phospholipase has the unique ability to transacylate short chain ceramides. This phospholipase is calcium-independent, localized to lysosomes, and has an acidic pH optimum. The enzyme was purified from bovine brain and found to be a water-soluble glycoprotein consisting of a single peptide chain with a molecular weight of 45 kDa. The primary structure deduced from the DNA sequences is highly conserved between chordates. The enzyme was named lysosomal phospholipase A₂ (LPLA₂) and subsequently designated group XV phospholipase A₂. LPLA₂ has 49% of amino acid sequence identity to lecithin-cholesterol acyltransferase and is a member of the αβ-hydrolase superfamily. LPLA₂ is highly expressed in alveolar macrophages. A marked accumulation of glycerophospholipids and extensive lamellar inclusion bodies, a hallmark of cellular phospholipidosis, is observed in alveolar macrophages in LPLA₂(-/-) mice. This defect can also be reproduced in macrophages that are exposed to cationic amphiphilic drugs such as amiodarone. In addition, older LPLA₂(-/-) mice develop a phenotype similar to human autoimmune disease. These observations indicate that LPLA₂ may play a primary role in phospholipid homeostasis, drug toxicity, and host defense.

As a phospholipase B, neuropathy target esterase (NTE) is responsible for the conversion of phosphatidylcholine (PC) to glycerophosphocholine (GPC). We examined the role of cAMP in the regulation of NTE in mammalian cells. Endogenous NTE activity was increased by cAMP-elevating chemicals, including dibutyryl cAMP, forskolin and forskolin plus 1-isobutyl-3-methylxanthine (IBMX), but decreased by the adenyl cyclase inhibitor SQ22536 which can reduce intracellular cAMP levels. Exogenous GFP-tagged NTE activity was not affected by changes in intracellular cAMP. NTE protein levels were up-regulated by the cAMP-elevating reagents and down-regulated by the inhibitor. The effect of the adenyl cyclase activator forskolin on NTE protein and mRNA levels was blocked by pretreatment with the protein kinase A (PKA) activity inhibitor H89. In addition, we found that changes in GPC, but not PC, levels were correlated with cAMP induced changes in NTE activity. These results are the first evidence that cAMP/PKA signals regulate NTE expression and GPC content in mammalian cells.

Aims Neuropathy target esterase (NTE) was proposed as the initial target during the process of organophosphate-induced delayed neuropathy (OPIDN) in humans and some sensitive animals. NTE was recently identified as a novel phospholipase B that is anchored to the cytoplasmic side of the endoplasmic reticulum. However, little is known about the degradation of NTE. In this study, we have investigated the role of the macroautophagic-lysosomal pathway in NTE degradation in neuronal and non-neuronal cells. Main methods Macroautophagy inhibitors and activators were used to interrupt the lysosomal pathway, and NTE protein level was followed using western blotting analysis. A fluorescent microscopy assay was used to determine the co-localization of NTE and lysosomes. Key findings Western blotting analysis showed that the macroautophagy inhibitors 3-methyladenine and ammonium chloride increased the levels of a heterologously expressed NTE-GFP fusion protein as well as endogenous NTE. Starvation had the opposite effect. The role of macroautophagy in NTE degradation was further supported by the co-localization of exogenous NTE with lysosomes in starved COS7 cells. Furthermore, the contribution of NTE activity and protein domains to the degradation of NTE by macroautophagy was investigated, showing that both the transmembrane and regulatory domains played a role in the degradation of NTE and that the catalytic domain, and thus NTE activity, was not involved. Significance Our findings clearly demonstrate, for the first time, that the macroautophagy/lysosome pathway plays a role in controlling NTE quantity, providing a further understanding of the function of NTE.

Little is known regarding early biochemical events in organophosphate-induced delayed neurotoxicity (OPIDN) except for the essential inhibition of neuropathy target esterase (NTE). We hypothesized that the homeostasis of lysophosphatidylcholine (LPC) and/or phosphatidylcholine (PC) in nervous tissues might be disrupted after exposure to the organophosphates (OP) which participates in the progression of OPIDN because new clues to possible mechanisms of OPIDN have recently been discovered that NTE acts as lysophospholipase (LysoPLA) in mice and phospholipase B (PLB) in cultured mammalian cells. To bioassay for such phospholipids, we induced OPIDN in hens using tri-o-cresyl phosphate (TOCP) as an inducer with phenylmethylsulfonyl fluoride (PMSF) as a negative control; and the effects on the activities of NTE, LysoPLA and PLB, the levels of PC, LPC, and glycerophosphocholine (GPC), and the aging of NTE enzyme in the brain, spinal cord, and sciatic nerves were examined. The results demonstrated that the activities of NTE, NTE-LysoPLA, LysoPLA, NTE-PLB and PLB were significantly inhibited in both TOCP- and PMSF-treated hens. The inhibition of NTE and NTE-LysoPLA or NTE-PLB showed a high correlation coefficient in the nervous tissues. Moreover, the NTE inhibited by TOCP was of the aged type, while nearly all of the NTE inhibited by PMSF was of the unaged type. No significant change in PC or LPC levels was observed, while the GPC level was significantly decreased. However, there is no relationship found between the GPC level and the delayed symptoms or aging of NTE. All results suggested that LPC and/or PC homeostasis disruption may not be a mechanism for OPIDN because the PC and LPC homeostasis was not disrupted after exposure to the neuropathic OP, although NTE, LysoPLA, and PLB were significantly inhibited and the GPC level was remarkably decreased.

Phospholipase B (Plb1) is secreted by pathogenic fungi and is a proven virulence determinant in Cryptococcus neoformans. Cell-associated Plb1 is presumptively involved in fungal membrane biogenesis and remodelling. We have also identified it in cryptococcal cell walls. Motif scanning programs predict that Plb1 is attached to cryptococcal membranes via a glycosylphosphatidylinositol (GPI) anchor, which could regulate Plb1 export and secretion. A functional GPI anchor was identified in cell-associated Plb1 by (G)PI-specific phospholipase C (PLC)-induced release of Plb1 from strain H99 membrane rafts and inhibition of GPI anchor synthesis by YW3548, which prevented Plb1 secretion and transport to membranes and cell walls. Plb1 containing beta-1,6-linked glucan was released from H99 (wild-type strain) cell walls by beta-1,3 glucanase, consistent with covalent attachment of Plb1 via beta-1,6-linked glucans to beta-1,3-linked glucan in the central scaffold of the wall. Naturally secreted Plb1 also contained beta-1,6-linked glucan, confirming that it originated from the cell wall. Plb1 maintains cell wall integrity because a H99 deletion mutant, DeltaPLB1, exhibited a morphological defect and was more susceptible than H99 to cell wall disruption by SDS and Congo red. Growth of DeltaPLB1 was unaffected by caffeine, excluding an effect of Plb1 on cell wall biogenesis-related signaling pathways. Environmental (heat) stress caused Plb1 accumulation in cell walls, with loss from membranes and reduced secretion, further supporting the importance of Plb1 in cell wall integrity. This is the first demonstration that Plb1 contributes to fungal survival by maintaining cell wall integrity and that the cell wall is a source of secreted enzyme.

Organophosphorus compounds (OP) such as phenyl saligenin phosphate (PSP) and mipafox (MPX) which cause delayed neuropathy, inhibit neuropathy target esterase (NTE), while OPs such as paraoxon (PXN) react more readily with acetylcholinesterase. In yeast and mammalian cell lines, NTE has been shown to have phospholipase B (PLB) activity which deacylates intracellular phosphatidylcholine to glycerophosphocholine (GroPCho) and can be detected by metabolic labeling with [(14)C]choline. Here we investigated PLB activity in primary cultures of mouse neural cells. In cortical and cerebellar granule neurons and astrocytes, [(14)C]GroPCho labeling was inhibited by PSP and MPX: phenyl dipentylphosphinate (PDPP), a non-neuropathic NTE inhibitor, was more potent, while PXN, was substantially less so. In all three cell types, conversion of [(14)C]phosphatidylcholine to [(14)C]GroPCho over 24 h was relatively small (2.3-14%). Consequently, even with >80% inhibition of [(14)C]GroPCho production, increased [(14)C]phosphatidylcholine was not detected. At concentrations of 1-10 microM, only PSP was cytotoxic to cortical and cerebellar granule neurons after 24-h exposure. Moreover, dramatic changes in glial cell morphology were induced by PSP, but not PDPP or MPX, with rapid (2-3 h) rounding up of astrocytes and of Schwann cells in cultures of dissociated mouse dorsal root ganglia. We conclude that PLB activity is present in a variety of cultured mouse neural cell types but that acute loss of this activity is not cytotoxic. Conversely, the rapid toxic effects of PSP in vitro suggest that a serine hydrolase distinct from NTE is required continuously by neurons and glia.

Phosphatidylcholine (PtdCho) is the major phospholipid component of eukaryotic membranes and deciphering the molecular mechanisms regulating PtdCho homeostasis is necessary to fully understand many pathophysiological situations where PtdCho metabolism is altered. This concept is illustrated in this review by summarizing recent evidence on Nte1p, a yeast endoplasmic reticulum resident phospholipase B that deacylates PtdCho producing intracellular glycerophosphocholine. The mammalian and Drosophila homologues, neuropathy target esterase and swiss cheese, respectively, have been implicated in normal brain development with increased intracytoplasmic vesicularization and multilayered membrane stacks as cytological signatures of their absence. Consistent with a role in lipid and membrane homeostasis, Nte1p-mediated PtdCho deacylation is strongly affected by Sec14p, a component of the yeast secretory machinery characterized by its ability to interface between lipid metabolism and vesicular trafficking. The preference of Nte1p toward PtdCho produced through the CDP–choline pathway and the downstream production of choline by the Gde1p glycerophosphodiesterase for resynthesis of PtdCho by the CDP–choline pathway are also highlighted.

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.

Secreted phospholipase B enzymes (PLB1) with high levels of N-linked glycosylation are proven fungal virulence determinants. We demonstrated that removal of N-linked glycans from secreted cryptococcal PLB1 leads to loss of enzyme activity. To determine if individual N-glycan attachment sites affect secretion of active enzyme, we altered three along the entire length of the protein, by site-directed mutagenesis, namely Asn56, Asn430 and Asn550 to Ala, in wild-type PLB1 (full length) and a glycosylphosphatidylinositol (GPI) anchorless version (PLB1GPI−) that is hypersecreted due to lack of membrane association. Alteration of Asn56 and Asn550 in both PLB1 and PLB1GPI− abolished enzyme secretion while alteration of Asn430 reduced secretion by 60%, following expression in Saccharomyces cerevisiae. Reduced secretion coincided with reduced enzyme in membranes and cell walls confirming a reduction in the rate of PLB1 transport to the cell surface. Deglycosylation of cryptococcal PLB1 increased its susceptibility to proteolysis suggesting that the absence of full glycosylation status leads to degradation of unstable PLB1, resulting in reduced traffic through the secretory pathway. We conclude that individual N-linked glycans are required for optimal transport of PLB1 to the cell surface and optimal secretion of both PLB1 and PLB1GPI−.

N-Acyl ethanolamines (NAEs) are a large class of signaling lipids implicated in diverse physiological processes, including nociception, cognition, anxiety, appetite, and inflammation. It has been proposed that NAEs are biosynthesized from their corresponding N-acyl phosphatidylethanolamines (NAPEs) in a single enzymatic step catalyzed by a phospholipase D (NAPE-PLD). The recent generation of NAPE-PLD(-/-) mice has revealed that these animals possess lower brain levels of saturated NAEs but essentially unchanged concentrations of polyunsaturated NAEs, including the endogenous cannabinoid anandamide. These findings suggest the existence of additional enzymatic routes for the production of NAEs in vivo. Here, we report evidence for an alternative pathway for NAE biosynthesis that proceeds through the serine hydrolase-catalyzed double-deacylation of NAPE to generate glycerophospho-NAE, followed by the phosphodiesterase-mediated cleavage of this intermediate to liberate NAE. Furthermore, we describe the functional proteomic isolation and identification of a heretofore uncharacterized enzyme alpha/beta-hydrolase 4 (Abh4) as a lysophospholipase/phospholipase B that selectively hydrolyzes NAPEs and lysoNAPEs. Abh4 accepts lysoNAPEs bearing both saturated and polyunsaturated N-acyl chains as substrates and displays a distribution that closely mirrors lysoNAPE-lipase activity in mouse tissues. These results support the existence of an NAPE-PLD-independent route for NAE biosynthesis and suggest that Abh4 plays a role in this metabolic pathway by acting as a (lyso)NAPE-selective lipase.

Secreted phospholipase B (PLB1), which contains three enzyme activities in the one protein, is necessary for the initiation of pulmonary infection by Cryptococcus neoformans and for dissemination from the lung via the lymphatics and blood. Adhesion to lung epithelium is the first step in this process, therefore we investigated the role of PLB1 in adhesion to a human lung epithelial cell line, A549, using C. neoformans var. grubii wild-type strain H99, a PLB1 deletion mutant (deltaplb1), and a reconstituted strain (deltaplb1rec). Adhesion of H99 and deltaplb1rec was approximately 69% greater than deltaplb1 at 4 h. Adhesion of deltaplb1 significantly increased after killing by chemicals or heat, and Fourier-transformed analysis by FTIR spectroscopy indicated this was due to changes in capsular and/or cell wall polysaccharides and proteins. Inhibition by specific PLB1 antibodies, or inhibitors of phospholipase B (PLB), but not lysophospholipase (LPL) or lysophospholipase transacylase (LPTA) activities decreased the adhesion of H99 and deltaplb1rec by 33-58%. Growth under conditions of osmotic stress and high glucose concentration increased both PLB secretion and subsequent cryptococcal adhesion. Dose-dependent increases (to 67%) in adhesion of live deltaplb1 were observed in the presence of 0.1-2 mM palmitic acid. We conclude that PLB1 plays a role in the binding of C. neoformans to host lung epithelial cells, possibly due to production of fatty acids from plasma membranes and/or surfactant by PLB activity.

We previously demonstrated that secretory phospholipase A2 (sPLA2) and lysophosphatidylcholine (LPC) exhibit neurotrophin-like neuritogenic activity in the rat pheochromocytoma cell line PC12. In this study, we further analyzed the mechanism whereby sPLA2 displays neurite-inducing activity. Exogenously added mammalian group X sPLA2 (sPLA2-X), but not group IB and IIA sPLA2s, induced neuritogenesis, which correlated with the ability of sPLA2-X to liberate LPC into the culture media. In accordance, blocking the effect of LPC by supplementation of bovine serum albumin or phospholipase B attenuated neuritogenesis by sPLA2 or LPC. Overproduction or suppression of G2A, a G-protein-coupled receptor involved in LPC signaling, resulted in the enhancement or reduction of neuritogenesis induced by sPLA2 treatment. These results indicate that the neuritogenic effect of sPLA2 is mediated by generation of LPC and subsequent activation of G2A.

Larvae of the gregarious ectoparasitoid, Euplectrus separatae, a species that parasitizes Pseudaletia separata, migrate from the dorsal to the ventral side of the host larva for pupation 7 days after parasitization. The parasitized host larvae die after the migration. The body mass of the parasitoid larvae increases while that of the host larva drastically decreases. Most of the tissue in the dead host larvae completely collapses. In this study, we examined the cause of host death and how the tissues collapse. Artificial removal of all parasitoid larvae before their migration on day 7 rescued the host larvae, but removal after parasitoid migration did not rescue the hosts. Tissues of the dead host larvae were completely liquefied. Injection of saliva from day 7 parasitoid larvae into host larvae killed the host larvae. High activity of a trypsin-like enzyme was detected in the saliva of day 7 parasitoids. Though phospholipase B and hyaluronidase were also detected in the saliva, commercial phospholipase B and hyaluronidase did not kill the hosts, whereas an injection of commercial trypsin was lethal. The trypsin-injected hosts showed the same tissue collapse as noted in parasitized and saliva-injected hosts. Leupeptin, a trypsin inhibitor, reduced mortality when injected into day 7 hosts (parasitoids were removal following migration). These observations suggest that the day 7 parasitoid larvae release saliva containing a trypsin-like enzyme to digest the host tissues following migration.

Although the lepidopteran larva Pseudaletia separata is attacked by the gregarious ectoparasitoid Euplectrus separatae, it continues to feed and grow. Lipid concentration in the hemolymph of the parasitized host was higher than that of the nonparasitized host from 3 to 8 days after parasitization. Artificial injection of parasitoid venom also elevated lipid concentration in the host hemolymph. One day after venom injection the host's fat body contained many lipid particles, but most of the lipid particles disappeared 7 days later. Light microscopy and transmission electron microscopy showed the lipid particles leaving the fat body cells as a result of the lysis of the fat body cells. These results suggest that the venom elevated the lipid concentration in the host hemolymph by provoking the release of lipid particles from the fat body. Though most of the lipid particles were freely floating in the host hemolymph, a portion of the released lipid particles were phagocytized by hemocytes. The amount of lipid that was loaded to lipophorin in the hemolymph of the venom-injected host was measured, but it was not sufficient to explain the high lipid titer in the hemolymph of parasitized and venom-injected host larvae. The fact that parasitoid larva consumed many hemocytes as evidenced by their presence in the midgut supported the hypothesis that the parasitoid larvae fed on the host hemolymph containing the free lipid particles, the hemocytes phagocytizing the lipid particles, and the lipid-loaded lipophorin. The possibility of the venom contribution to the disruption of the intercellular matrix was examined. The venom showed high activity of matrix metalloproteinase (MMP), especially when it was mixed with the hemolymph of non-parasitized 5th instar larvae. We suggest that the MMP in the venom was activated by some components of the host hemolymph. On the other hand, the venom mixed with hemolymph could not decompose gelatin on zymography, suggesting that the venom-MMP is a different type from gelatinase. Activity of phospholipases A(2), B, C and hyaluronidase were measured with agar plates. High activities of phospholipase B and hyaluronidase were detected. These results suggest that the venom-MMP initially attacked the specific site of the intercellular-matrix of the fat body, and then the hyaluronidase and the phospholipase B cause lysis of the fat body cell, allowing lipid particles to be released into the host hemolymph.

c-Fos, a transcription factor, associates to endoplasmic reticulum and modulates phospholipid biosynthesis. Its surface thermodynamic properties allow it to differentially interact with phospholipid monolayers with a selective dependence on the lipid polar head group and the lateral surface pressure. We explored the c-Fos ability to modulate phospholipid degradation by phospholipases (ppPLA2, Bacillus cereus PLC, and sphingomyelinase) using the monolayer technique. Experiments conducted under constant packing conditions show that c-Fos modulates phospholipase activity in a finely tuned way, depending on the membrane intermolecular packing. Surface lateral pressures above 12-16 mN/m induce c-Fos to activate phospholipase A2 and sphingomyelinase, and abolish phospholipase C activity. The effects of c-Fos on other steps of the catalytic process, lag-time and extent, are synergic with those on activity. We show for the first time that c-Fos participates in modulating phospholipid degradation and that it can affect the formation of lipid second messenger products by PLA2, PLC, and sphingomyelinase.

Two species of giant hornet phospholipase B (PLB), alpha and beta, were purified from the venom of Vespa mandarinia. The purification procedure was simplified by two steps of column chromatographies, Sephadex G-100 and SP-Sepharose. The molecular sizes of PLB alpha and beta were 29.5 and 26.0 kDa, respectively. The isoelectric point of alpha and beta enzymes were pH 10.6 and 10.7, respectively. The temperature optimum for egg yolk lecithin was a broad peak at 40-60 degrees C for both enzymes. Amino acid compositions of both enzymes were high contents of aspartic acid, glycine, leucine, lysine and other aliphatic amino acids. Cystine was similar amounts to other species of phospholipases (PLs). The K(m) values of alpha and beta enzymes were 8.29 and 7.53 mg/ml for egg yolk lecithin, respectively. In the catalytic specificity for L-alpha-phosphatidylcholine-beta-oleoil-gamma-palmitoil, the K(m) values of alpha enzyme for gamma-palmitoil and beta-oleoil residues were 0.528 and 1.392 mM, respectively. While the K(m) values of beta enzyme for gamma-palmitoil and beta-oleoil residues were 7.91 and 2. 68 mM, respectively. Both alpha and beta enzymes were inhibited strongly by cepharanthine. The lecithin hydrolysis of alpha enzyme was competitively inhibited, but beta enzyme was uncompetitive. Cepharanthine also inhibited noncompetitively PLA(2)s of bovine pancreas, bee venom and Naja mossambica mossambica.

The phospholipase A2 (PLA2) superfamily consists of a broad range of enzymes defined by their ability to catalyze the hydrolysis of the middle (sn-2) ester bond of substrate phospholipids. The hydrolysis products of this reaction, free fatty acid and lysophospholipid, have many important downstream roles, and are derived from the activity of a diverse and growing superfamily of PLA2 enzymes. This review updates the classification of the various PLA2’s now described in the literature. Four criteria have been employed to classify these proteins into one of the 11 Groups (I^XI) of PLA2’s. First, the enzyme must catalyze the hydrolysis of the sn-2 ester bond of a natural phospholipid substrate, such as long fatty acid chain phospholipids, platelet activating factor, or short fatty acid chain oxidized phospholipids. Second, the complete amino acid sequence of the mature protein must be known. Third, each PLA2 Group should include all of those enzymes that have readily identifiable sequence homology. If more than one homologous PLA2 gene exists within a species, then each paralog should be assigned a Subgroup letter, as in the case of Groups IVA, IVB, and IVC PLA2. Homologs from different species should be classified within the same Subgroup wherever such assignments are possible as is the case with zebra fish and human Group IVA PLA2 orthologs. The current classification scheme does allow for historical exceptions of the highly homologous Groups I, II, V, and X PLA2’s. Fourth, catalytically active splice variants of the same gene are classified as the same Group and Subgroup, but distinguished using Arabic numbers, such as for Group VIA-1 PLA2 and VIA-2 PLA2’s. These four criteria have led to the expansion or realignment of Groups VI, VII and VIII, as well as the addition of Group XI PLA2 from plants. fl 2000 Elsevier Science B.V. All rights reserved.

Lipid peroxidation of membrane phospholipids can proceed both enzymatically via the mammalian 15-lipoxygenase-1 or the NADPH-cytochrome P-450 reductase system and non-enzymatically. In some cells, such as reticulocytes, this process is biologically programmed, whereas in the majority of biological systems lipid peroxidation is a deleterious process that has to be repaired via a deacylation-reacylation cycle of phospholipid metabolism. Several reports in the literature pinpoint a stimulation by lipid peroxidation of the activity of secretory phospholipase A(2)s (mainly pancreatic and snake venom enzymes) which was originally interpreted as a repair function. However, recent experiments from our laboratory have demonstrated that in mixtures of lipoxygenated and native phospholipids the former are not preferably cleaved by either secretory or cytosolic phospholipase A(2)s. We propose that the platelet activating factor (PAF) acetylhydrolases of type II, which cleave preferentially peroxidised or lipoxygenated phospholipids, are competent for the phospholipid repair, irrespective of their role in PAF metabolism. A corresponding role of Ca(2+)-independent phospholipase A(2), which has been proposed to be involved in phospholipid remodelling in biomembranes, has not been addressed so far. Direct and indirect 15-lipoxygenation of phospholipids in biomembranes modulates cell signalling by several ways. The stimulation of phospholipase A(2)-mediated arachidonic acid release may constitute an alternative route of the arachidonic acid cascade. Thus, 15-lipoxygenase-mediated oxygenation of membrane phospholipids and its interaction with phospholipase A(2)s may play a crucial role in the pathogenesis of diseases, such as bronchial asthma and atherosclerosis.

Phospholipase C-gamma1 is a tightly regulated, multidomain protein that generates the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. Kinetic analysis reveals that phospholipase C-gamma1 displays apparent allosteric behavior. A previous study determined that proteolytic cleavage of the SH domain region of phospholipase C-gamma1 yields an activated form of the enzyme (A. W. Fernald, G. A. Jones, and G. Carpenter Biochem. J. 302, 508, 1994). In this study, we show that activation of phospholipase C-gamma1 by proteolysis decreases both the cooperativity and the half-maximal value of the enzyme for substrate. Kinetic analysis revealed that the mole fraction of phosphatidylinositol 4,5-bisphosphate (PIP(2)) that resulted in half-maximal PIP(2) hydrolysis (S(0.5)) was lower for proteolyzed than uncleaved phospholipase C-gamma1 (0.08 mole fraction vs 0.18 mole fraction of PIP(2)). The cooperativity index was lower for proteolyzed than full-length phospholipase C-gamma1 (n = 2.5 vs n = 4). Kinetic analysis also revealed that the estimated dissociation constant was lower for phospholipase C-gamma1 that had been subjected to proteolysis (0.1 mM vs 1.0 mM PIP(2) for cleaved vs uncleaved phospholipase C-gamma1, respectively). It was previously hypothesized that activation of phospholipase C-gamma1 requires a conformational change that results in increased accessibility of substrate to the active site and that the SH domain of the enzyme is involved in the activation event. These experiments support the hypothesis that a portion of the protein covers the active site, allosterically inhibiting the enzyme, and that the removal of this "lid" domain activates the enzyme.

In the present study, we studied the involvement of A2 phospholipases (PLA2) in alpha2-adrenergic receptor-control of preadipocyte actin cytoskeleton. For that, various PLA2 inhibitors were tested on the ability of the selective alpha2-adrenergic agonist UK14304 to induce the spreading in alpha2AF2 preadipocytes. We observed that, whereas several Ca(2+)-dependent PLA2 blockers were ineffective, the Ca(2+)-independent phospholipase A2 (iPLA2) inhibitor, broenolactone (BEL), specifically blocked alpha2-adrenergic-dependent preadipocyte spreading without affecting the spreading activity of lysophosphatidic acid (LPA) or serum. BEL inhibition was completely restored by lysophosphatidic acid, but not by arachidonic acid or other fatty acids. The presence of the lysophospholipase (phospholipase B) suppressed the effect of LPA on preadipocyte spreading, but had no influence on alpha2-adrenergic-induced spreading. Thus, the extracellular production of LPA or fatty acids is not involved in iPLA2-dependent preadipocyte spreading. iPLA2 protein was found in preadipocytes but, conversely to cPLA2, did not exhibit any modification of its electrophoretic mobility after alpha2-adrenergic stimulation. We concluded that iPLA2 is involved in alpha2-adrenergic control of preadipocyte actin cytoskeleton.

The yeast genome contains two genes, designated as PLB2 and PLB3, that are 67% and 62% identical, respectively, to PLB1, which codes for a phospholipase B/lysophospholipase in yeast (Lee, S. K., Patton, J. L., Fido, M., Hines, L. K., Kohlwein, S. D., Paltauf, F., Henry, S. A., and Levin, D. E. (1994) J. Biol. Chem. 269, 19725-19730). Deletion and overexpression studies and in vivo and in vitro activity measurements suggest that both genes indeed code for phospholipases B/lysophospholipases. In cell free extracts of a plb1 plb2 plb3 triple mutant, no phospholipase B activity was detectable. Upon overexpression of PLB2 in a plb1 plb3 mutant background, phospholipase B activity was detectable in the plasma membrane, periplasmic space extracts and the culture supernatant. Similar to Plb1p, Plb2p appears to accept all major phospholipid classes, with a preference for acidic phospholipids including phosphatidylinositol 3',4'-bisphosphate and phosphatidic acid. Consistent with a function as an extracellular lysophospholipase, PLB2 overexpression conferred resistance to lyso-phosphatidylcholine. Deletion of Plb2p function had no effect on glycerophosphoinositol or glycerophosphocholine release in vivo, in contrast to a deletion of Plb3p function, which resulted in a 50% reduction of phosphatidylinositol breakdown and glycerophosphoinositol release from the cells. In vitro, Plb3p hydrolyzes only phosphatidylinositol and phosphatidylserine and, to a lesser extent, their lyso-analogs. Plb3p activity in a plb1 plb2 mutant background was observed in periplasmic space extracts. Both Plb3p and Plb2p display transacylase activity in vitro, in the presence or absence, respectively, of detergent.

A series of mechanism-based inhibitors of phospholipase A2 (SIBLINKS) were synthesized. These new SIBLINKS are phospholipid analogues that contain a para-substituted phenyl 3,3-dimethylglutaryl group in the place of the sn-2 acyl chain. The effect of the phenyl leaving group on inhibitory activity was studied by varying the electron-withdrawing ability of the para-substituted group. A strong correlation was observed between the leaving group potential of the suicide inhibitor and the inhibitory activity of the derivative toward cobra venom phospholipase A2.

Guinea pig intestinal phospholipase B is a calcium-independent phospholipase hydrolyzing sequentially the acyl ester bonds at sn-2 and sn-1 positions of glycerophospholipids, promoting the formation ofsn-glycero-3-phosphocholine from phosphatidylcholine. This 140-kDa glycoprotein from the brush border membrane of differentiated enterocytes contributes to lipid digestion as an ectoenzyme. The cDNA coding for guinea pig phospholipase B was revealed to be the homologue of AdRab-B, an mRNA appearing in rabbit upon intestine development. The sequence predicts a polypeptide of 1463 amino acids displaying four homologous repeats, two of them containing the lipase consensus sequence GXSXG. A 5-kilobase transcript was particularly abundant in mature ileal and jejunal enterocytes but was also detected in epididymis, where phospholipase B displayed a higher molecular mass (170 kDa versus 140 kDa in intestine), with no obvious evidence for enzyme activity. Trypsin treatment of phospholipase B immunoprecipitated from epididymal membranes reduced its size to 140 kDa, coinciding with the appearance of a significant phospholipase A2 activity. The same results were obtained in COS cells transfected with phospholipase B cDNA. Since sn-glycero-3-phosphocholine present at high concentrations in seminal plasma mainly stems from epididymis, this suggests a possible role of phospholipase B in male reproduction. This novel localization also unravels a mechanism of phospholipase B activation by limited proteolysis involving either trypsin in the intestinal lumen or a trypsin-like endopeptidase in the male reproductive tract.

A Ser48 phospholipase A 2 -homologue, ammodytin L, which is myotoxic in mammals and devoid of any phospholipase A 2 activity, completely inhibits the specific binding of the neurotoxic phospholipase A 2 , ammodytoxin C, to fish presynaptic membranes from Torpedo marmorata electric organ. In cross-linking experiments, 125 I-ammodytin L labels the same membrane proteins as 125 I-ammodytoxin C (70, 38.5-57.4 and 19.7 kDa). The formation of these adducts is completely prevented by the presence of ammodytoxin C but not of a non-toxic phospholipase A 2 , ammodytin I 2 . A chimeric phospholipase A 2 , constructed by associating the N-terminal half of ammodytoxin to the C-terminal half of ammodytin L, possesses a low, but significant phospholipase A 2 activity, however it is not toxic to mice, probably due to abolition of the specific neuronal acceptor binding in mammals. Nevertheless, the chimeric phospholipase A 2 is able to interact with the ammodytoxin acceptor in Torpedo marmorata electric organ. The existence of neuronal acceptors for ammodytin L and for the chimeric phospholipase A 2 suggests that they may act as neurotoxins in fish. As ammodytin L does not posses any enzymatic activity it, therefore, appears to be an excellent tool to investigate the mechanism of action of β-neurotoxins independently of their phospholipase A 2 activity.

3-(E)-Methoxycarbonyl-2,4,6-trienal compound A was stereoselectively synthesized and found to show strong inhibitory activity toward phospholipase A 2 (PLA 2 ) from bovine pancreas. As the inhibitory mechanism of PLA 2 by A , the irreversible formation of dihydropyridine resulting from the reaction of A with lysine residues in PLA 2 is proposed based on the model reactions.

A cDNA encoding a rat intestinal Ca2+-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.

A brush border membrane-associated phospholipase B/lipase was solubilized from the distal two-thirds of rat small intestine by autolysis during storage at -35 degrees C over 1 month, and then the enzyme was purified to homogeneity and characterized enzymatically and structurally. The purified enzyme exhibited broad substrate specificity including esterase, phospholipase A2, lysophospholipase, and lipase activities. SDS-gel electrophoretic and reverse-phase high performance liquid chromatographic analyses demonstrated that a single enzyme catalyzes these activities. It preferred hydrolysis at the sn-2 position of diacylphospholipid and diacylglycerol without strict stereoselectivity, whereas it apparently exhibited no positional specificity toward triacylglycerol. Diisopropyl fluorophosphate, an irreversible inhibitor of serine esterases and lipases inhibited purified enzyme. When the position of enzyme on SDS-gel electrophoresis under the non-reducing conditions was determined by assaying the activity eluted from sliced gels, brush border membrane-associated enzyme corresponded to a approximately 150-kDa protein; autolysis gave a 35-kDa product, in agreement with the results of immunoblot analysis. The purified 35-kDa enzyme consisted of a 14-kDa peptide and a glycosylated 21-kDa peptide. Their NH2-terminal amino acid sequences were determined and found in the second repeat of 161-kDa phospholipase B/lipase with 4-fold tandem repeats of approximately 38 kDa each, which we cloned and sequenced in the accompanying paper (Takemori, H., Zolotaryov, F., Ting, L., Urbain, T., Komatsubara, T., Hatano, O., Okamoto, M., and Tojo, H. (1988) J. Biol. Chem. 273, 2222-2231). These results indicate that the purified enzyme is the catalytic domain derived from the second repeat of brush border membrane-associated phospholipase B/lipase.

Cytosolic phospholipase A2 (cPLA2) hydrolyzes the sn-2-acyl ester bond of phospholipids and shows a preference for arachidonic acid-containing substrates. We found previously that Ser-228 is essential for enzyme activity and is likely to function as a nucleophile in the catalytic center of the enzyme (Sharp, J. D., White, D. L., Chiou, X. G., Goodson, T., Gamboa, G. C., McClure, D., Burgett, S., Hoskins, J., Skatrud, P. L., Sportsman, J. R., Becker, G. W., Kang, L. H., Roberts, E. F., and Kramer, R. M.(1991) J. Biol. Chem. 266, 14850-14853). cPLA2 contains a catalytic aspartic acid motif common to the subtilisin family of serine proteases. Substitution within this motif of Ala for Asp-549 completely inactivated the enzyme, and substitutions with either glutamic acid or asparagine reduced activity 2000- and 300-fold, respectively. Additionally, using mutants with cysteine replaced by alanine, we found that Cys-331 is responsible for the enzyme's sensitivity to N-ethylmaleimide. Surprisingly, substituting alanine for any of the 19 histidines did not produce inactive enzyme, demonstrating that a classical serine-histidine-aspartate mechanism does not operate in this hydrolase. We found that substituting alanine or histidine for Arg-200 did produce inactive enzyme, while substituting lysine reduced activity 200-fold. Results obtained with the lysine mutant (R200K) and a coumarin ester substrate suggest no specific interaction between Arg-200 and the phosphoryl group of the phospholipid substrate. Arg-200, Ser-228, and Asp-549 are conserved in cPLA2 from six species and also in four nonmammalian phospholipase B enzymes. Our results, supported by circular dichroism, provide evidence that Asp-549 and Arg-200 are critical to the enzyme's function and suggest that the cPLA2 catalytic center is novel.

Several lines of evidence indicate that the breakdown of plasmalogens in neural membranes during neurodegenerative diseases is a receptor-mediated process catalyzed by a plasmalogen-selective phospholipase A2. This enzyme has recently been purified from bovine brain. It does not require Ca2+ and is localized in cytosol. It has a molecular mass of 39 kDa and is strongly inhibited by glycosaminoglycans, with the pattern of inhibition being heparan sulfate0 > hyaluronic acid > chondroitin sulfate > heparin. This plasmalogen-selective phospholipase A2 is also inhibited by gangliosides and sialoglycoproteins. Substrate specificity and the effects of metal ions, detergents and inhibitors suggest that this phospholipase A2 is different from the well-known 85 kDa Ca2+-dependent cytosolic phospholipase A2 that has recently been cloned and is not plasmalogen-selective. The plasmalogen-selective phospholipase A2 may be regulated by glycosaminoglycans and sialoglycoconjugates and may be involved in the regulation of K+ channels. This enzyme, which plays a major role in the release of fatty acids during ischemic injury and reperfusion, shows promise as a major target for drug therapy.

Phospholipase A2 (EC 3.1.1.4: PLA2) is the group name of enzymes that cleave an acyl ester bond at the sn-2 position of glycerophospholipid (1). Mammalian PLA2s are classified into two types, secretory 14 kDa PLA2 and cytosolic higher molecular weight of PLA2 (2). Secretory PLA2 are further classified into two subgroups, group I and group II, on the basis of their characteristics in primary structure (3). Group II PLA2 (PLA2-II) is abundant at inflammatory loci and its expression is modulated by various inflammatory cytokines, and thus this type of PLA2 is thought to play some roles in the pathogenesis of inflammation (4,5). On the other hand, group I PLA2 (PLA2-I) is mainly secreted from the pancreas and the major physiological function of PLA2-I has long been thought to be digestion of phospholipids in nutrients (6). However, recent studies have revealed the presence of this type of PLA2 in several non-digestive organs such as lung and spleen (7). What is the biological role of PLA2-I in non-digestive organs? This question led us to initiate the series of studies on the PLA2 receptor.

A sensitive method for continuously monitoring the activity of the human cytosolic phospholipase A2 (cPLA2) is described. Recombinant cPLA2 efficiently hydrolyzes fatty acid esters of 7-hydroxycoumarin, producing the free fatty acid and the highly fluorescent 7-hydroxycoumarin. All of the observed 7-hydroxycoumarinyl ester hydrolase activity (7-HCEase) in a preparation of the purified recombinant cPLA2 was due to this enzyme since: (1) all of the ester hydrolase activity comigrated on nondenaturing polyacrylamide gel with a protein characterized as the cPLA2 by Western analysis; (2) the immunoreactive protein also possessed both phospholipase A2 and lysophospholipase activities; and (3) arachidonyl trifluoromethyl ketone, a potent inhibitor of the phospholipase A2 activity of cPLA2, also inhibited the 7-HCEase activity. A study of the 7-HCEase activity demonstrated that when 7-hydroxycoumarinyl gamma-linolenate was dispersed in a phospholipid matrix it was hydrolyzed by cPLA2 at a rate comparable to that of an arachidonyl-containing phospholipid substrate and with an identical reaction progress curve. In the presence of phospholipid vesicles, the cPLA2-catalyzed hydrolysis of hydrophobic 7-hydroxycoumarinyl esters was stimulated by submicromolar concentration of free calcium and showed a preference for polyunsaturated substrates. The cPLA2-catalyzed hydrolysis of the water-soluble substrate 7-hydroxycoumarinyl 6-heptenoate was catalyzed by cPLA2 in the absence of calcium and other lipids.

The Ca(2+)-sensitive cytosolic phospholipase A2 (cPLA2) displays both a phospholipase A2 and a lysophospholipase activity. Numerous hydrolases, including lipases, catalyze the hydrolysis of ester bonds by means of an active site triad of amino acids that includes a serine or a cysteine residue. We have examined whether human cPLA2 belongs to this class of enzymes by using site-directed mutagenesis. Although chemical inactivation of cPLA2 by the sulfhydryl reagent N-ethylmaleimide made it appear that cysteine(s) may be essential for catalysis, all 9 cysteine residues of cPLA2 proved dispensable, allowing near-normal enzyme activity when substituted by alanine. We noted that cPLA2 contains a 110-amino-acid region with sequence homology to phospholipase B (PLB) from Penicillium notatum. Interestingly, one of the conserved serines of cPLA2, Ser-228, within this domain aligns with the lipase consensus sequence Gly-X(Leu)-Ser(137)-X(Gly)-Gly of PLB. Replacement of Ser-228 by alanine (or threonine or cysteine) yielded catalytically inactive cPLA2, even though the native conformation was maintained as determined by CD spectroscopy. Likewise, the lysophospholipase activity was completely abolished by the Ser-228 mutations. In contrast, substitution by alanine of three different serines of cPLA2 (Ser-195, Ser-215, or Ser-577) that also aligned with the PLB sequence allowed for substantial enzymatic activity of cPLA2. Our findings provide evidence that 1) Ser-228 participates in the catalytic mechanism of cPLA2 and that 2) both the phospholipase A2 and the lysophospholipase activities of cPLA2 are catalyzed by the same active site residue(s).

The availability of purified G alpha 11 and the G-protein-regulated phospholipase C from turkey erythrocytes has allowed an examination of the direct effects of G-protein beta gamma-subunit on the components of the inositol lipid signaling system. Reconstitution of purified turkey erythrocyte or bovine brain beta gamma-subunit into phospholipid vesicles containing G alpha 11 inhibited AlF4- induced activation of phospholipase C. However, beta gamma-subunit at higher concentrations increased phospholipase C activity. This stimulatory effect of beta gamma-subunit on phospholipase C did not require the presence of the alpha-subunit. G alpha o had no effect on the catalytic activity of phospholipase C. However, coreconstitution of G alpha o and beta gamma-subunit shifted to the right the concentration-effect curve for beta gamma-subunit-promoted activation of phospholipase C. As was observed with G alpha 11, the increase in activity observed in the presence of beta gamma-subunit occurred as an increase in the maximal activity and with no change in the apparent affinity for Ca2+ for phospholipase C activation. The concentration dependence of G alpha 11 for activation of turkey erythrocyte phospholipase C and bovine brain phospholipase C-beta, as well as the concentration dependence of the two enzymes for activation by G alpha 11, were very similar. In contrast, beta gamma-subunit was a much less effective activator of bovine brain phospholipase C-beta than the turkey erythrocyte enzyme. The observation of direct effects of free beta gamma-subunit on phospholipase C extend the possibilities for receptor-mediated regulation of this signaling pathway.

In order to study the regulatory mechanisms of phospholipase C-gamma (PLC-gamma) via the intrinsic SH2/SH3 region (Z region), two recombinant Z proteins, rP45Z and rP38Z, derived from rat PLC-gamma 1 and PLC-gamma 2, respectively, were purified from the inclusion bodies of Escherichia coli. We examined their direct effects on phosphoinositide hydrolysis induced by four different PLC isoforms purified from bovine brain and thymus, and found that both of these Z proteins suppress the enzyme activity of all four PLC isoforms in a dose-dependent manner. This suppressive effect is very potent and stoichiometric. The kinetics studies indicate that the suppression is non-competitive. This suppression is eliminated by treatment with proteases but is not affected by heat treatment at 95 degrees C for 15 min, indicating that the primary structure might be important for the action of Z proteins. Comparative studies suggested that two Z proteins but not Src and phosphatidylinositol 3-kinase possess, adjacent to their SH2 and SH3 motifs, a phospholipase C inhibitor (PCI) region that strongly suppresses their phosphatidylinositol 4,5-bisphosphate (PIP2)-hydrolyzing activity. A series of synthetic peptides identical with the sequence of the proposed PCI region, including an octamer, YRKMRLRY, inhibited PIP2 hydrolysis induced by four different phospholipase C isoforms. These results demonstrate that both types of phospholipase C-gamma contain the PCI sequence which is responsible for the inhibition of PIP2 hydrolysis, indicating that phospholipase C-gamma is a self-regulating enzyme.

1. 1. Plasma membranes were treated with phospholipase A2, phospholipase C or phospholipase D. The phosphatidylethanolamine: ceramide-phosphorylethanolamine transferase was deactivated by phospholipase C treatment, whereas phospholipase A2 and phospholipase D did not affect the enzyme. 2. 2. Incorporation of phosphatidylethanolamine and phosphatidylglycerol into partially delipidated plasma membranes resulted in significant stimulation of the transferase, whereas inclusion of sphingomyelin and phosphatidylserine suppressed the enzyme activity. Our results suggest that phosphatidylserine is a regulator of sphingomyelin level in membranes. 3. 3. The activity of phosphatidylethanolamine xeramide-phosphorylethanolamine transferase was not influenced by the fluidity of its lipid environment.