Calcium-dependent translocation of cytosolic phospholipase A2 in pancreatic beta-cells

Cytosolic phospholipase A(2)(cPLA(2)), an enzyme responsible for the generation of arachidonic acid, is located in the cytosolic compartment in most tissues and it translocates to membrane compartments when activated. We found that cPLA(2) distribution in pancreatic beta-cells is different from that of most other mammalian cells: it is evenly distributed throughout the beta-cell, in both cytoplasmic and nuclear compartments. Agents that increased intracellular Ca(2+) in the MIN6 beta-cell line also stimulated a redistribution of cPLA(2) immunoreactivity such that the majority of the enzyme moved from the nucleus to the cytoplasm. The time course of events was compatible with the elevation in Ca(2+) being responsible for translocation of cPLA(2). These observations suggest that cPLA(2) may be compartmentalised in unstimulated beta-cells, perhaps to limit its access to substrate prior to elevations in intracellular Ca(2+).     

Toll-like receptor 9-mediated cytosolic phospholipase A2 activation regulates expression of inducible nitric oxide synthase

Although CpG containing DNA is an important regulator of innate immune responses via toll-like receptor 9 (TLR9), excessive activation of this receptor is detrimental to the host. Here, we show that cytosolic phospholipase A₂ (cPLA₂) activation is important for TLR9-mediated inducible nitric oxide synthase (iNOS) expression. Activation of TLR9 signaling by CpG induces iNOS expression and NO production. Inhibition of TLR9 blocked the iNOS expression and NO production. The CpG also stimulates cPLA₂-hydrolyzed arachidonic acid (AA) release. Inhibition of cPLA₂ activity by inhibitor attenuated the iNOS expression by CpG response. Additionally, knockdown of cPLA₂ protein by miRNA also suppressed the CpG-induced iNOS expression. Furthermore, the CpG rapidly phosphorylates three MAPKs and Akt. A potent inhibitor for p38 MAPK or Akt blocked the CpG-induced AA release and iNOS expression. These results suggest that TLR9 activation stimulates cPLA₂ activity via p38 or Akt pathways and mediates iNOS expression.     

Reversible activation of secretory phospholipase A2 by sulfhydryl reagents

Secretory phospholipase A(2)s (sPLA(2)s) have been implicated in physiological and pathological events, but the regulatory mechanism(s) of their activities in cells remains to be solved. Previously, we reported that phenylarsine oxide (PAO), a sulfhydryl reagent, stimulated arachidonic acid (AA) release in rat pheochromocytoma PC12 cells. In this study, we examined the effects of thimerosal, another sulfhydryl reagent, to clarify the sulfhydryl modification and activation of sPLA(2) molecules in cells. Like PAO, thimerosal-stimulated AA release in an irreversible manner and the responses were not additive. Dithiol compounds such as dithiothreitol inhibited AA release from both the thimerosal- and the PAO-treated cells, and monothiol compounds (l-Cys and glutathione) decreased the thimerosal response. Both sulfhydryl reagents stimulated AA release from the HEK293T cells expressing human sPLA(2)X, and stimulated the sPLA(2) activities of bee venom sPLA(2) and the soluble fraction of sPLA(2)X-expressing cells. Our results suggest that the sPLA(2)s in cells are inactive and modification of disulfide bonds in the molecules can be a trigger of sPLA(2) activation in cells. Sulfhydryl reagents are useful tools for studying the regulatory mechanism(s) of sPLA(2) activity in cells.     

Secretory phospholipase A(2) inhibits epidermal growth factor-induced receptor activation

Secretory phospholipase A(2) (sPLA(2)) plays important roles in mediating various cellular processes, including cell proliferation, differentiation, apoptosis, and inflammatory response. In this study, we demonstrated that a basic sPLA(2) inhibits epidermal growth factor (EGF)-induced EGF receptor activation, as determined by autophosphorylation of EGF receptor, EGF-activated phospholipase D (PLD) activity, and phospholipase C-gamma(1) (PLC-gamma(1)) tyrosine phosphorylation in a human epidermoid carcinoma cell line, A-431. Treatment of cells with exogenous neutral sphingomyelinase (SMase) or a cell permeable ceramide analog, C(2)-ceramide, also caused similar inhibitory effects on EGF-induced activation of EGF receptor, tyrosine phosphorylation of PLC-gamma(1), and the activation of PLD. sPLA(2)-induced inhibition of EGF receptor was associated with arachidonic acid release, which was followed by an increase in intracellular ceramide formation. Both sPLA(2) and exogenous C(2)-ceramide are able to inhibit the proliferation of A-431. The data presented indicate for the first time that sPLA(2) downregulates the EGF receptor-mediated intracellular signal transduction that may be mediated by arachidonic acid and/or ceramide.     

Interfacial activation of porcine pancreatic phospholipase A(2) studied with 7-nitrobenz-2-oxa-1,3-diazol-4-yl-labeled lipids

The interfacial activation of porcine pancreatic phospholipase A(2) (PLA(2)) during the hydrolysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine liposomes at different temperatures has been monitored by fluorescence changes of the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) lipid derivatives 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (C(12)-NBD-PC) and 12-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)]dodecanoic acid (C(12)-NBD-FA) inserted in the substrate vesicles. These long-chain monitors, in contrast to the previously used C(6)-NBD-PC, detect latency times of PLA(2) action, similar to those measured by the classic titrimetric, pH-stat method. Interestingly, hydrolysis of the host vesicles results in a decrease in fluorescence not only of C(12)-NBD-PC, a substrate analog, but also of product derivative C(12)-NBD-FA. Ultrafiltration experiments show that C(12)-NBD-FA does not migrate to the aqueous phase upon hydrolysis of the host liposomes. Besides, in a simulated hydrolysis experiment in which increasing proportions of palmitic acid and 1-palmitoyl-sn-glycero-3-phosphocholine were cosonicated with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, C(12)-NBD-PC fluorescence was insensitive to products, whereas C(12)-NBD-FA did show a decreased emission intensity as in the actual hydrolysis experiments. The phenomenon is triggered above a critical concentration of products (10 mol%) suggesting that cosegregation of NBD-FA (either added as such or generated by hydrolysis of C(12)-NBD-PC) and products may be related to the decrease in fluorescence. Phase separation should create microdomains of increased C(12)-NBD-FA surface density and cause concentration quenching. In addition, and taking into account that the NBD group may be located near the interfacial region, it is possible that in segregating with products, the fluorescent moiety of C(12)-NBD-FA becomes exposed to microenvironments of higher surface polarity, which further decreases its quantum yield.     

Trace amounts of enhancing factor/phospholipase A2 in mouse peritoneal exudate cells

Enhancing factor (EF), a mouse phospholipase A₂ (PLA₂), has been purified from the small intestines, based on its ability to increase the binding of epidermal growth factor in a radioreceptor assay. EF/PLA₂ was found to be localized predominantly in the Paneth cells in the small intestines. Whether mouse intestinal EF/PLA₂ is identical/similar to mouse secretory PLA₂ was to be determined. Phospholipases are known to play a crucial role in the process of inflammation. This paper reports the presence of trace amounts of EF/PLA₂ in the peritoneal exudate cells. Western blot analysis of the acid extracts showed the presence of a 14 kDa immunologically cross-reactive protein. RT-PCR analysis using EF specific primers amplified a ∼700 bp product which was further confirmed to be EF-specific by nested PCR analysis and sequencing. Presence of EF in the peritoneal exudate cells could be a unique mode of transport of growth factor modulator to the site of injury to aid in regeneration/cell proliferation of damaged tissue.     

Clustering of sialylated glycosylphosphatidylinositol anchors mediates PrP-induced activation of cytoplasmic phospholipase A2 and synapse damage

Precisely how the accumulation of PrP^Sc causes the neuronal degeneration that leads to the clinical symptoms of prion diseases is poorly understood. Our recent paper showed that the clustering of specific glycosylphosphatidylinositol (GPI) anchors attached to PrP proteins triggered synapse damage in cultured neurons. First, we demonstrated that small, soluble PrP^Sc oligomers caused synapse damage via a GPI-dependent process. Our hypothesis, that the clustering of specific GPIs caused synapse damage, was supported by observations that cross-linkage of PrP^C, either chemically or by monoclonal antibodies, also triggered synapse damage. Synapse damage was preceded by an increase in the cholesterol content of synapses and activation of cytoplasmic phospholipase A₂ (cPLA₂). The presence of a terminal sialic acid moiety, a rare modification of mammalian GPI anchors, was essential in the activation of cPLA₂ and synapse damage induced by cross-linked PrP^C. We conclude that the sialic acid modifies local membrane microenvironments (rafts) surrounding clustered PrP molecules resulting in aberrant activation of cPLA₂ and synapse damage. A recent observation, that toxic amyloid-β assemblies cross-link PrP^C, suggests that synapse damage in prion and Alzheimer diseases is mediated via a common molecular mechanism, and raises the possibility that the pharmacological modification of GPI anchors might constitute a novel therapeutic approach to these diseases.     

Thermodynamic characterization of cytosolic phospholipase A2 alpha inhibitors

Cytosolic phospholipase A₂ alpha (cPLA₂α, type IVA phospholipase) acts at the membrane surface to release free arachidonic acid, which is metabolized into inflammatory mediators, including leukotrienes and prostaglandins. Thus, specific cPLA₂α inhibitors are predicted to have antiinflammatory properties. However, a key criterion in the identification and development of such inhibitors is to distinguish between compounds that bind stoichiometrically to cPLA₂α and nonspecific membrane perturbants. In the current study, we developed a method employing isothermal titration calorimetry (ITC) to characterize the binding of several distinct classes of cPLA₂α inhibitors. Thermodynamic parameters and the binding constants were obtained following titration of the inhibitor to the protein at 30 °C and pH 7.4. The compounds tested bound cPLA₂α with a 1:1 stoichiometry, and the dissociation constant K_d of the inhibitors calculated from the ITC experiments correlated well with the IC₅₀ values obtained from enzymatic assays. Interestingly, binding was observed only in the presence of a micellar surface, even for soluble compounds. The site of binding of these inhibitors within cPLA₂α was analyzed by testing for binding in the presence of methyl arachidonyl fluorophosphonate (MAFP), an irreversible active site inhibitor of cPLA₂α. Lack of binding of inhibitors in the presence of MAFP suggested that the compounds tested bound specifically at or near the active site of the protein. Furthermore, the effect of various detergents on the binding of certain inhibitors to cPLA₂α was also tested. The results are discussed with reference to thermodynamic parameters such as changes in enthalpy (ΔH), entropy (ΔS), and free energy (ΔG). The data obtained from these studies provide not only structure-activity relationships for compounds but also important information regarding mechanism of binding. This is the first example of ITC used for studying inhibitors of enzymes with interfacial kinetics.     

Clustering of sialylated glycosylphosphatidylinositol anchors mediates PrP-induced activation of cytoplasmic phospholipase A2 and synapse damage

Precisely how the accumulation of PrP^Sc causes the neuronal degeneration that leads to the clinical symptoms of prion diseases is poorly understood. Our recent paper showed that the clustering of specific glycosylphosphatidylinositol (GPI) anchors attached to PrP proteins triggered synapse damage in cultured neurons. First, we demonstrated that small, soluble PrP^Sc oligomers caused synapse damage via a GPI-dependent process. Our hypothesis, that the clustering of specific GPIs caused synapse damage, was supported by observations that cross-linkage of PrP^C, either chemically or by monoclonal antibodies, also triggered synapse damage. Synapse damage was preceded by an increase in the cholesterol content of synapses and activation of cytoplasmic phospholipase A₂ (cPLA₂). The presence of a terminal sialic acid moiety, a rare modification of mammalian GPI anchors, was essential in the activation of cPLA₂ and synapse damage induced by cross-linked PrP^C. We conclude that the sialic acid modifies local membrane microenvironments (rafts) surrounding clustered PrP molecules resulting in aberrant activation of cPLA₂ and synapse damage. A recent observation, that toxic amyloid-β assemblies cross-link PrP^C, suggests that synapse damage in prion and Alzheimer diseases is mediated via a common molecular mechanism, and raises the possibility that the pharmacological modification of GPI anchors might constitute a novel therapeutic approach to these diseases.     

A role for G-proteins in the epidermal growth factor stimulation of phospholipase A2 in rat kidney mesangial cells

We have previously demonstrated phospholipase C (PLC) independent activation of phospholipase A₂(PLA₂) by epidermal growth factor (EGF) in glomerular mesangial cells in culture. In the current study using glass beads to permeabilize [³H]- or [¹⁴C]-arachidonate labelled mesangial cells we demonstrate that guanine nucleotides modulate the EGF-mediated stimulation of arachidonic acid release (75% inhibition with 100 M GDPS and 108% augmentation with 100 M GTPS). GTPS alone stimulated both the release of free arachidonic acid and production of diacylglycerol (DAG), while EGF itself neither stimulated DAG nor augmented the DAG response to GTPS. These findings suggest the intermediacy of a G-protein in PLC-independent stimulation of PLA₂ by a growth factor, and provide a model system for determining the relationship between G-protein intermediacy and the intrinsic tyrosine kinase activity of the growth factor receptor.Abbreviations EGF Epidermal Growth Factor - PLC phospholipase C - PLA₂ phospholipase A₂ - DAG Diacylglycerol - NEFA non-esterified fatty acid - GTPS guanosine-5-0-[3-thio]triphosphate - GDP\S guanosine-5-0-[2-thio]diphosphate     

Antibacterial properties of recombinant human non-pancreatic secretory phospholipase A2

Human non-pancreatic secretory phospholipase A₂ (hnpsPLA₂) is a group IIA phospholipase A₂ which plays an important role in the innate immune response. This enzyme was found to exhibit bactericidal activity toward Gram-positive bacteria, but not Gram-negative ones. Though native hnpsPLA₂ is active over a broad pH range, it is only highly active at alkaline conditions with the optimum activity pH of about 8.5. In order to make it highly active at neutral pH, we have obtained two hnpsPLA₂ mutants, Glu89Lys and Arg100Glu that work better at neutral pH in a previous study. In the present study, we tested the bactericidal effects of the native hnpsPLA₂ and the two mutants. Both native hnpsPLA₂ and the two mutants exhibit bactericidal activity toward Gram-positive bacteria. Furthermore, they can also kill Escherichia coli, a Gram-negative bacterium. The two mutants showed better bactericidal activity for E. coli at neutral pH than the native enzyme, which is consistent with the enzyme activities. As hnpsPLA₂ is highly stable and biocompatible, it may provide a promising therapy for bacteria infection treatment or other bactericidal applications.     

Identification of novel phospholipase A2 group IX members in metazoans

Sequence homologues of the bacterium Streptomyces violaceoruber and sea anemone Nematostella vectensis PLA₂ pfam09056 members were identified in several bacteria, fungi and metazoans illustrating the evolution of this PLA₂ sub-family. Comparison of their molecular structures revealed that bacteria and fungi members are part of the GXIV of PLA₂s while metazoan representatives are similar with GIX PLA₂ of the marine snail Conus magus. Members of GXIV and GIX PLA₂s show modest overall sequence similarity (21–35%) but considerable motif conservation within the putative Ca²⁺-binding, catalytic sites and cysteine residue positions which are essential for enzyme function. GXIV PLA₂s of bacteria and fungi typically contain four cysteine residues composing two intramolecular disulphide bonds. GIX PLA₂ homologues were identified in cnidarians and molluscs and in a single tunicate but appear to be absent from other metazoan genomes. The mature GIX PLA₂ deduced peptides contain up to ten cysteine residues capable of forming five putative disulphide bonds. Three disulphide bonds were identified in GIX PLA₂s, two of which correspond to those localized in GXIV PLA₂s. Phylogenetic analysis demonstrates that metazoan GIX PLA₂s cluster separate from the bacterial and fungal GXIV PLA₂s and both pfam09056 members form a group separate from the prokaryote and eukaryote GXIIA PLA₂ pfam06951. Duplicate PLA₂ pfam09056 genes were identified in the genomes of sea anemone N. vectensis and oyster Crassostrea gigas suggest that members of this family evolved via species-specific duplication events. These observations indicate that the newly identified metazoan pfam09056 members may be classified as GIX PLA₂s and support the idea of the common evolutionary origin of GXIV and GIX PLA₂ pfam09056 members, which emerged early in bacteria and were maintained in the genomes of fungi and selected extant metazoan taxa.     

Microcapillary-like structures prompted by phospholipase A2 activation in endothelial cells and pericytes co-cultures on a polyhydroxymethylsiloxane thin film

A thin film of poly(hydroxymethylsiloxane) (PHMS) has been deposited on glass dishes and tested as artificial support material for vascularization from mixed cultures of endothelial cells (EC) and pericytes (PC). The EC/PC co-cultures adhered massively on PHMS, with the formation of net-like microcapillary structures. Such evidence was not found on control glass substrates in the same co-culture conditions neither on PHMS for EC and PC in monocultures. The physicochemical characterization of PHMS and control glass surface by time-of-flight secondary ion mass spectrometry, X-ray photoelectron spectroscopy, water contact angle and atomic force microscopy, pointed to the main role of the polymer hydrophobilicy to explain the observed cellular behavior. Moreover, enhanced intercellular cross-talk was evidenced by the up-regulation and activation of cytoplasmic and Ca(2+)-independent phospholipase A(2) (cPLA(2) and iPLA(2)) expression and cPLA(2) phosphorylation, leading to the cell proliferation and microcapillary formation on the PHMS surface, as evidenced by confocal microscopy analyses. Co-cultures, established with growth-arrested PCs by treatment with mitomycin C, showed an increase in EC proliferation on PHMS. AACOCF(3) or co-transfection with cPLA(2) and iPLA(2)siRNA reduced cell proliferation. The results highlight the major role played by EC/PC cross-talk as well as the hydrophobic character of the substrate surface, to promote microcapillary formation. Our findings suggest an attractive strategy for vascular tissue engineering and provide new details on the interplay of artificial substrates and capillary formation.     

Role of phosphorylation sites and the C2 domain in regulation of cytosolic phospholipase A2.

Cytosolic phospholipase A2 (cPLA2) mediates agonist-induced arachidonic acid release, the first step in eicosanoid production. cPLA2 is regulated by phosphorylation and by calcium, which binds to a C2 domain and induces its translocation to membrane. The functional roles of phosphorylation sites and the C2 domain of cPLA2 were investigated. In Sf9 insect cells expressing cPLA2, okadaic acid, and the calcium-mobilizing agonists A23187 and CryIC toxin induce arachidonic acid release and translocation of green fluorescent protein (GFP)-cPLA2 to the nuclear envelope. cPLA2 is phosphorylated on multiple sites in Sf9 cells; however, only S505 phosphorylation partially contributes to cPLA2 activation. Although okadaic acid does not increase calcium, mutating the calcium-binding residues D43 and D93 prevents arachidonic acid release and translocation of cPLA2, demonstrating the requirement for a functional C2 domain. However, the D93N mutant is fully functional with A23187, whereas the D43N mutant is nearly inactive. The C2 domain of cPLA2 linked to GFP translocates to the nuclear envelope with calcium-mobilizing agonists but not with okadaic acid. Consequently, the C2 domain is necessary and sufficient for translocation of cPLA2 to the nuclear envelope when calcium is increased; however, it is required but not sufficient with okadaic acid.     

Role of membrane associated serine esterase in the activation of phospholipase A2 by calcium ionophore (A23187) in pulmonary arterial smooth muscle cells

Exposure of rabbit pulmonary arterial smooth muscle cells to 10 M of the calcium ionophore A23187 dramatically stimulates cell membrane-associated phospholipase A₂ activity and arachidonic acid release. In addition, A23187 also enhances cell membrane-associated serine esterase activity. Serine esterase inhibitors phenylmethylsulfonylfuoride and diisopropyl fluorophosphate prevent the increase in serine esterase and phospholipase A₂ activities and arachidonic acid release caused by A23187. A23187 still stimulated serine esterase and phospholipase A₂ activities and arachidonic acid release in cells pretreated with nominal Ca²⁺ free buffer. Treatment of the cell membrane with A23187 does not cause any appreciable change in serine esterase and phospholipase A₂ activities. Pretreatment of the cells with actinomycin D or cycloheximide did not prevent the increase in the cell membrane associated serine esterase and phospholipase A₂ activities, and arachidonic acid release caused by A23187. These results suggest that (i) a membrane-associated serine esterase plays an important role in stimulating the smooth muscle cell membrane associated phospholipase A₂ activity (ii) in addition to the presence of extracellular Ca²⁺, release of Ca²⁺ from intracellular storage site(s) by A23187 also appears to play a role in stimulating the cell membrane-associated serine esterase and phospholipase A₂ activities, and (iii) the increase in the cell membrane-associated serine esterase and phospholipase A₂ activities does not appear to require new RNA or protein synthesis.Abbreviations A23187 calcium ionophore - AA arachidonic acid - PMSF phenylmethyl sulfonylfuoride - DFP diisopropyl-fluorophosphate - DMEM Dulbecco's modified Eagles medium - FCS fetal calf serum - PBS phosphate buffered saline - HBPS Hank's buffered physiological saline - PLA₂ phospholipase A₂     

Compounds stimulating cytosolic phospholipase A2 activity with a combinational action mode

The screening of small synthetic compound libraries is a useful means of identifying molecules that modulate various cellular responses. We screened more than 10,000 different small compounds and identified three synthetic compounds that stimulate arachidonic acid (AA) release in a combinational manner in neutrophil-like differentiated HL60 cells. These three compounds were designated as AARIC-1, -2, and -3, representing AA release inducing compounds-1, -2, and -3. Although AA release was not induced by any single one of these compounds, it was dramatically stimulated by the three compounds in combination. Moreover, the effect of combined treatment by these compounds on AA release was completely abolished by MAFP and AACOCF(3), specific cytosolic phospholipase A(2) inhibitors. Furthermore, we found that AARIC-3 stimulates cytosolic calcium influx, while AARIC-1 induces ERK activation. Taken together, we demonstrate a useful approach to the study of complicated and nonlinear intracellular signaling networks using small synthetic compounds in combination.     

Therapeutic application of natural inhibitors against snake venom phospholipase A(2)

Natural inhibitors occupy an important place in the potential to neutralize the toxic effects caused by snake venom proteins and enzymes. It has been well recognized for several years that animal sera, some of the plant and marine extracts are the most potent in neutralizing snake venom phospholipase A(2) (svPLA(2)). The implication of this review to update the latest research work which has been accomplished with svPLA(2) inhibitors from various natural sources like animal, marine organisms presents a compilation of research in this field over the past decade and revisiting the previous research report including those found in plants. In addition to that the bioactive compounds/inhibitor molecules from diverse sources like aristolochic alkaloid, flavonoids and neoflavonoids from plants, hydrocarbones -2, 4 dimethyl hexane, 2 methylnonane, and 2, 6 dimethyl heptane obtained from traditional medicinal plants Tragia involucrata (Euphorbiaceae) member of natural products involved for the inhibitory potential of phospholipase A(2) (PLA(2)) enzymes in vitro and also decrease both oedema induced by snake venom as well as human synovial fluid PLA(2). Besides marine natural products that inhibit PLA(2) are manoalide and its derivatives such as scalaradial and related compounds, pseudopterosins and vidalols, tetracylne from synthetic chemicals etc. There is an overview of the role of PLA(2) in inflammation that provides a rationale for seeking inhibitors of PLA(2) as anti-inflammatory agents. However, more studies should be considered to evaluate antivenom efficiency of sera and other agents against a variety of snake venoms found in various parts of the world. The implications of these new groups of svPLA(2) toxin inhibitors in the context of our current understanding of snake biology as well as in the development of new novel antivenoms therapeutics agents in the efficient treatment of snake envenomations are discussed.     

Fatty acid oxidation and myocardial phospholipase A2 activity

Summary Cis-unsaturated fatty acids, but not saturated fatty acids, inhibited phospholipase A₂ activity (PLA₂) in vitro, and may function as endogenous suppressors of lipolysis. To probe the possible role of lipid peroxidation in the regulation of myocardial lipid catabolism, a neutral-active and Ca²⁺-dependent PLA₂ was extracted from rat heart and was partially purified by sulfopropyl cation exchange chromatography. Myocardial PLA, activity was inhibited in a dose-dependent manner by oleic, linoleic, linolenic, and arachidonic acids; the IC₅₀ for arachidonic acid was approx 65 M. Palmitic acid was not inhibitory. When arachidonic acid was incubated at 37°C, exposed to air, there was a time- and pH-dependent peroxidation of the arachidonic acid as monitored by turbidity, thiobarbituric acid reactivity, and thin layer chromatography. Peroxidation was increased as the pH was lowered from 7.5 to 4.5, and was accompanied by a decrease in PLA₂ inhibitory potency. Thus, arachidonate incubated for 24 hours at pH's 4.5, 6.0 and 7.5 lost 84%, 32%, and 20% respectively, of its inhibitory potency. Therefore, in vitro acidosis promotes the oxidation of cis-unsaturated fatty acids and relieves their inhibitory or suppressive activity toward PLA₂s. Increased lipid peroxidation of unesterified unsaturated fatty acids during acidosis may therefore promote lipolysis observed during myocardial ischemia and reperfusion injury.     

Enhanced protein expression in mammalian cells using engineered SUMO fusions: secreted phospholipase A2

SUMOylation, the covalent attachment of SUMO (small ubiquitin-like modifier), is a eukaryotic post-translational event that has been demonstrated to play a critical role in several biological processes. When used as an N-terminal tag or fusion partner, SUMO has been shown to enhance functional protein production significantly by improving folding, solubility, and stability. We have engineered several SUMOs and, through their fusion, developed a system for enhancing the expression and secretion of complex proteins. To demonstrate the fidelity of this fusion technology, secreted phospholipase A(2) proteins (sPLA(2)) were produced using HEK-293T and CHO-K1 cells. Five mouse sPLA(2) homologs were expressed and secreted in mammalian cell cultures using SUMO or SUMO-derived, N-terminal fusion partners. Mean and median increases of 43- and 18-fold, respectively, were obtained using novel SUMO mutants that are resistant to digestion by endogenous deSUMOylases.