N49 phospholipase A2, a unique subgroup of snake venom group II phospholipase A2

A novel phospholipase A₂ (PLA₂) with Asn at its site 49 was purified from the snake venom of Protobothrops mucrosquamatus by using SP-Sephadex C25, Superdex 75, Heparin-Sepharose (FF) and HPLC reverse-phage C₁₈ chromatography and designated as TM-N49. It showed a molecular mass of 13.875 kDa on MALDI-TOF. TM-N49 does not possess enzymatic, hemolytic and hemorrhagic activities. It fails to induce platelet aggregation by itself, and does not inhibit the platelet aggregation induced by ADP. However, it exhibits potent myotoxic activity causing inflammatory cell infiltration, severe myoedema, myonecrosis and myolysis in the gastrocnemius muscles of BALB/c mice. Phylogenetic analysis found that that TM-N49 combined with two phospholipase A₂s from Trimeresurus stejnegeri, TsR6 and CTs-R6 cluster into one group. Structural and functional analysis indicated that these phospholipase A₂s are distinct from the other subgroups (D49 PLA₂, S49 PLA₂ and K49 PLA₂) and represent a unique subgroup of snake venom group II PLA₂, named N49 PLA₂ subgroup.     

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.     

Diverse cellular transformation capability of overexpressed genes in human hepatocellular carcinoma

For isolation of novel cellular transforming genes that potentially participated in hepatocarcinogenesis, we conducted anchorage-independent growth (AIG) assays on 10 human liver cancer cell lines and observed strong AIG capabilities in PLC5 and Huh7 but negligible in Tong cells. After cloning of genes by differential subtractive chain reactions (DSC) from strong AIG to AIG negative cells, we sequenced 2304 clones and identified 245 genes. After four stringent criteria for selection of transforming genes among DSC clones, our results of quantitative RT-PCR analysis indicated that six genes, DDX3, EIF3S2, CLIC1, HDGF, GPC3, and HSPCA were overexpressed in 64%, 62%, 60%, 58%, 49%, and 47%, respectively, of 45 hepatocellular carcinoma (HCC) tissues. The results of cellular transformation capability by AIG assays indicated that the transfectants of EIF3S2 showed the strongest (> 100-fold), DDX3 and CLIC1 were moderate, GPC3 and HSPCA were weak, and HDGF was none in forming colonies in soft agar. Together, our results suggested that Tong is a suitable human cell line for screening of overexpressed and/or cellular transforming genes. In addition, our results suggested that diverse functions of cellular transforming genes in various biological pathways could transform human Tong cells and potentially reveal new targets for drug development of HCC.     

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.     

The expression of insulin-like growth factor binding proteins in human hepatocellular carcinoma

Insulin-like growth factors (IGF), IGF receptors and IGF binding proteins (IGFBPs) play an important role in cell growth and differentiation. The liver is the major source of IGF-1 and at least two IGFBPs (IGFBP-1 and IGFBP-3). IGFBPs most often serve to attenuate the effects of IGF at the receptor level and thereby limit IGF-induced cell growth and differentiation. Although changes in IGFBP expression have been described during controlled liver growth such as hepatic regeneration following partial hepatectomy, there is limited knowledge of IGFBPs gene expression in uncontrolled growth or hepatocellular carcinoma. In the present study, we employed Northern blotting techniques to document the expression of IGFBP-1, 3 and 4 in normal human livers, cirrhotic and hepatocellular carcinoma tissues. The results revealed no differences in IGFBP-1, 3 and 4 mRNA levels between normal and cirrhotic tissues. However, the expression of all three IGFBPs mRNA were significantly down regulated in hepatocellular carcinoma tissues. These findings are in keeping with IGFBPs playing an important inhibitory role in the development and/or growth of hepatocellular carcinoma in humans.     

Activation of phospholipase A2 by freshly added lysophospholipids

Reaction progress curves for the hydrolysis of dimyristoylphosphatidylcholine by pig pancreatic phospholipase A₂ exhibits a latency phase. Addition of 1-palmitoyllysophosphatidylcholine to the preformed vesicles reduces the latency phase and enhances the binding of phospholipase A₂ to the vesicles. In contrast, the binary codispersions prepared from diacylphospholipids premixed with lysophosphatidylcholine do not exhibit such enhanced susceptibility to the phospholipase. This effect appears to be due to organizational defects created by asymmetrical incorporation of lysophospholipid molecules into the outer monolayer of the vesicles, and the action of phospholipase is not observed when the additive is equilibrated in both the monolayers of the vesicles.     

Work in progress. Occurence of phospholipase A1 and A2 in human decidua

Phospholipase A₂, an enzyme which may regulate the formation of polyunsaturated fatty acids utilized for prostaglandin synthesis, was found to have significant higher activity in decidual than in myometrial tissue. The major part of phospholipase A₂ in the decidua had an acid pH optimum, which indicates that most of the enzyme is stored in the lysosomes of this tissue. These findings, together with previous observations, lend further support to the view that lysosomal phospholipase A₂ released within decidual cells might be a trigger of abortion and parturition.     

Distribution of reaction products in phospholipase A2 hydrolysis

We have monitored the composition of supported phospholipid bilayers during phospholipase A₂ hydrolysis using specular neutron reflection and ellipsometry. Porcine pancreatic PLA₂ shows a long lag phase of several hours during which the enzyme binds to the bilayer surface, but only 5 ± 3% of the lipids react before the onset of rapid hydrolysis. The amount of PLA₂, which resides in a 21 ± 1 Å thick layer at the water-bilayer interface, as well as its depth of penetration into the membrane, increase during the lag phase, the length of which is also proportional to the enzyme concentration. Hydrolysis of a single-chain deuterium labelled d₃₁-POPC reveals for the first time that there is a significant asymmetry in the distribution of the reaction products between the membrane and the aqueous environment. The lyso-lipid leaves the membrane while the number of PLA₂ molecules bound to the interface increases with increasing fatty acid content. These results constitute the first direct measurement of the membrane structure and composition, including the location and amount of the enzyme during hydrolysis. These are discussed in terms of a model of fatty-acid mediated activation of PLA₂.     

Up-regulation of the expressions of phospholipase A2 inhibitors in the liver of a venomous snake by its own venom phospholipase A2

Venomous snakes such as Gloydius brevicaudus have three distinct types of phospholipase A₂ inhibitors (PLIα, PLIβ, and PLIγ) in their blood so as to protect themselves from their own venom phospholipases A₂ (PLA₂s). Expressions of these PLIs in G. brevicaudus liver were found to be enhanced by the intramuscular injection of its own venom. The enhancement of gene expressions of PLIα and PLIβ in the liver was also found to be induced by acidic PLA₂ contained in this venom. Furthermore, these effects of acidic PLA₂ on gene expression of PLIs were shown to be unrelated to its enzymatic activity. These results suggest that these venomous snakes have developed the self-protective system against their own venom, by which the venom components up-regulate the expression of anti-venom proteins in their liver.     

Mapping of suramin binding sites on the group IIA human secreted phospholipase A2

Suramin is a polysulphonated napthylurea used as an antiprotozoal/anthelminitic drug, which also inhibits a broad range of enzymes. Suramin binding to recombinant human secreted group IIA phospholipase A₂ (hsPLA₂GIIA) was investigated by molecular dynamics simulations (MD) and isothermal titration calorimetry (ITC). MD indicated two possible bound suramin conformations mediated by hydrophobic and electrostatic interactions with amino-acids in three regions of the protein, namely the active-site and residues located in the N- and C-termini, respectively. All three binding sites are located on the phospholipid membrane recognition surface, suggesting that suramin may inhibit the enzyme, and indeed a 90% reduction in hydrolytic activity was observed in the presence of 100 nM suramin. These results correlated with ITC data, which demonstrated 2.7 suramin binding sites on the hsPLA₂GIIA, and indicates that suramin represents a novel class of phospholipase A₂ inhibitor.     

Differential contributions of protein kinase C isoforms in the regulation of group IIA secreted phospholipase A2 expression in cytokine-stimulated rat fibroblasts

Protein kinase C (PKC) is a family of serine/threonine kinases involved in various signal transduction pathways. We investigated the roles of PKC in the regulation of group IIA secreted phospholipase A₂ (sPLA₂-IIA) expression in cytokine-stimulated rat fibroblastic 3Y1 cells. Here we show that the induction of sPLA₂-IIA by proinflammatory cytokines was under the control of both classical cPKCα and atypical aPKCλ/ι pathways by using PKC inhibitors, a PKC activator, and PKC knockdowns. Treatment of 3Y1 cells with PKC selective inhibitors having broad specificity, such as chelerythrine chloride and GF109203X, blocked IL-1β/TNFα-dependent induction of sPLA₂-IIA protein in a dose-dependent manner. Treatment with the PKC activator phorbol 12-myristate 13-acetate (PMA), which activates cPKC and novel nPKC isoforms, markedly attenuated the cytokine-dependent induction of sPLA₂-IIA expression. In comparison, 24-h pretreatment with PMA, which down-regulates these PKC isoforms, markedly enhanced sPLA₂-IIA expression. Results with short hairpin RNA (shRNA)-mediated knockdown of PKC isoforms revealed that the cytokine-induced sPLA₂-IIA expression was markedly enhanced in cPKCα knockdown cells compared to those in replicate control cells. In contrast, knockdown of the aPKCλ/ι isoform reduced the cytokine-induced expression of sPLA₂-IIA. These results suggest that the aPKCλ/ι pathway is required for the induction of sPLA₂-IIA expression and that the cPKCα pathway acts as a negative regulator of sPLA₂-IIA expression in cytokine-stimulated rat fibroblasts.     

Emerging roles for phospholipase A2 enzymes in cancer

Phospholipase A₂ (PLA₂) enzymes (EC3.1.4.4) regulate the release of biologically active fatty acids and lysophospholipids from membrane phospholipid pools. These lipids are also substrates for intracellular biochemical pathways that generate potent autocrine and paracrine lipid mediators such as the eicosanoids and platelet activating factor. These factors, in turn, regulate cell proliferation, survival, differentiation, motility, tissue vascularisation, and immune surveillance in virtually all tissues, functions that are subverted by cancer cells for tumour growth and metastasis. Thus the relevance of PLA₂-dependent pathways to the genesis and progression of cancer has been of interest since their discovery and with recent technological advances, their role in tumourigenesis has become more tractable experimentally. Limited human genetic studies have not yet identified PLA₂ enzymes as classical mutated oncogenes or tumour suppressor genes. However, there is strong evidence that of the 22 identified human PLA₂ enzymes, ten of which have been studied in cancer to date, most are aberrantly expressed in a proportion of tumours derived from diverse organs. Correlative and functional studies implicate the expression of some secreted enzymes (sPLA₂s), particularly the best studied enzyme Group IIA sPLA₂ in either tumour promotion or inhibition, depending on the organ involved and the biochemical microenvironment of tumours. As in immune-mediated inflammatory pathologies, genetic deletion studies in mice, supported by limited studies with human cells and tissues, have identified an important role for Group IVA PLA₂ in regulating certain cancers. Pharmacological intervention studies in prostate cancer suggest that hGIIA-dependent tumour growth is dependent on indirect regulation of Group IVA PLA₂. Group VI calcium-independent PLA₂ enzymes have also been recently implicated in tumourigenesis with in vitro studies suggesting multiple possible roles for these enzymes. Though apparently complex, further characterization of the regulatory relationships amongst PLA₂ enzymes, lipid mediator biosynthetic enzymes and the lipid mediators they produce during tumour progression is required to define the biochemical context in which the enzymes modulate cancer growth and development.     

Uncoupling protein 2 mediates resistance to gemcitabine-induced apoptosis in hepatocellular carcinoma cell lines

Oxidative stress induction is a common effector pathway for commonly used chemotherapeutic agents like gemcitabine (GEM) in hepatocellular carcinoma (HCC) patients. However, GEM alone or in combination with oxiplatin hardly renders any survival benefits to HCC patients. Mitochondrial uncoupling protein 2 (UCP2) is known to suppress mitochondrial reactive oxygen species (ROS) generation, thus mitigating oxidative stress-induced apoptosis. We demonstrate in the present study, using a panel of HCC cell lines that sensitivity to GEM in HCC well correlate with the endogenous level of UCP2 protein expression. Moreover, ectopic overexpression of UCP2 in a HCC cell line with low endogenous UCP2 expression, HLE, significantly decreased mitochondrial superoxide induction by the anti-cancer drug GEM. Conversely, UCP2 mRNA silencing by RNA interference in HCC cell lines with high endogenous UCP2 expression significantly enhanced GEM-induced mitochondrial superoxide generation and apoptosis. Cumulatively, our results suggest a critical role for mitochondrial uncoupling in GEM resistance in HCC cell lines. Hence, synergistic targeting of UCP2 in combination with other chemotherapeutic agents might be more potent in HCC patients.     

CircPUM1 promotes hepatocellular carcinoma progression through the miR-1208/MAP3K2 axis

Hepatocellular carcinoma (HCC) is a common disease with a significant mortality, and there is no effective treatment for advanced patients. Growing evidence indicates that circRNAs are closely related to HCC progression, may be used as biomarkers and targets for the diagnosis and treatment of HCC. Recent researches have shown that circPUM1 may play an oncogene role in a variety of human cancers, but its role in HCC development has not been reported. Our study found that circPUM1 could promote the proliferation, migration and invasion of HCC cells in vitro. In addition, in vivo studies showed that circPUM1 could increase the development of HCC tumours and regulate the expression of EMT-related proteins. Furthermore, we demonstrated that circPUM1 could promote the development of HCC by up-regulating the expression of MAP3K2 via sponging miR-1208. Our study suggested that circPUM1 may be a potential therapeutic target for HCC.     

Production of egg yolk lysolecithin with immobilized phospholipase A2

Production of egg yolk lysolecithin was compared using free phospholipase A₂ (PLA₂) and immobilized PLA₂ in alginate-silicate sol-gel matrix. Choice of solvent, water content, calcium, and temperatures changed the activity of the free and immobilized PLA₂ a lot, owing to their effects on the catalytic properties of the enzyme as well as the conformational change of lecithin in ethanol-buffer mixture. Free PLA₂ shows typical microemulsion kinetics in ethanol-buffer system. The effect of the water content on the enzyme reaction was greatly influenced by the presence of calcium ion. In the absence of calcium ion, certain optimal water content for the production of lysolecithin always exists in the free PLA₂ reaction. However, with calcium ion, three distinctive regions were observed with free PLA₂ reactions. Initially, in the micro-aqueous region of the ethanol-buffer system with calcium ion, the hydrolysis activity of PLA₂ was proportional to the water content. Beyond the region, concave type of activity profiles were observed as the water content increases. As the water content increases further, the hydrolysis rate of the PLA₂ abruptly decreased by the phase separation. On the contrary, in case of immobilized enzyme, optimal water content for the production of lysolecithin exists regardless of the presence of calcium ion. The calcium ion was essential for achieving the maximum activity of both free and immobilized PLA₂. The addition of calcium ion not only affected the catalytic activity of the enzyme but also was necessary to improve the enzyme stability. As the immobilization of the enzyme remarkably increased thermal stability of the free enzyme, the immobilized PLA₂ is more desirable to be used in the production of various lysophospholipids. It was successfully reused over 250 h.     

Preparation of 2- and 3-substituted gibberellins A9 and A4 for bioassay

To test the effects of preventing enzymatic 2β- and 3β-hydroxylation on the biological activities of gibberellins, the preparation of the following compounds is described: 2β-methyl- and 2,2-dimethyl-gibberellins A₄ and A₉; 2α-fluoro-, 2β-fluoro- and 2β-methoxy-gibberellin A₉; and 3β-chloro-, 3β-fluoro-, 3β-methoxy- and 3-methylene A₉.     

Human C-apolipoproteins promote hydrolysis of dimyristoyl phosphatidylcholine by snake venom phospholipase A2

Concanavalin A-induced proliferation of rat T-lymphocytes is completely inhibited by 10^?5 M pyrazofurin, a potent inhibitor of pyrimidine de novo synthesis, as judged by cell viability and [³H]thymidine incorporation. Proliferation is completely restored by 5 × 10^?5 M uridine. Cytidine, deoxycytidine, deoxyuridine and thymidine 10 × 10^?5 M each, fail to re-establish proliferation but produce an isotropic dilution of [³H]thymidine uptake in DNA. Bases (cytosine, uracil and thymine) neither restore proliferation nor induce isotopic dilution. The unexpected inability of cytidine to reverse de novo pyrimidine synthesis inhibition suggests a lack of cytidine deaminase activity in rat T-lymphocytes. This is confirmed by a direct sensitive radioisotopic assay (<0.001 nmol.min^?1.10^?6 cells).