Preparation of the Full Set of Recombinant Mouse- and Human-Secreted Phospholipases A2.

A family of 14-20kDa, disulfide-rich, calcium-dependent secreted phospholipases A2 (sPLA2s) that release fatty acids from the sn-2 position of glycerophospholipids can be found in mammals. They have a diverse array of tissue distribution and biological functions. In this chapter we provide detailed protocols for production of nearly all of the mouse and human sPLA2s mainly by expression in bacteria and in vitro refolding or by expression in insect cells. High-resolution mass spectrometry and enzymatic assays were, respectively, used to show that all disulfides are formed and that the enzymes are active, strongly suggesting that each sPLA2 was prepared in the structurally native form. The availability of these proteins has allowed kinetic studies to be carried out, to prepare highly selective antisera, to screen for selective inhibitors, to study receptor binding, and to study the action of each enzyme on mammalian cell membranes and their in vivo biological roles.

Role of the phospholipase A2 receptor in liposome drug delivery in prostate cancer cells.

The M-type phospholipase A2 receptor (PLA2R1) is a member of the C-type lectin superfamily and can internalize secreted phospholipase A2 (sPLA2) via endocytosis in non-cancer cells. sPLA2 itself was recently shown to be overexpressed in prostate tumors and to be a possible mediator of metastasis; however, little is known about the expression of PLA2R1 or its function in prostate cancers. Thus, we examined PLA2R1 expression in primary prostate cells (PCS-440-010) and human prostate cancer cells (LNCaP, DU-145, and PC-3), and we determined the effect of PLA2R1 knockdown on cytotoxicity induced by free or liposome-encapsulated chemotherapeutics. Immunoblot analysis demonstrated that the expression of PLA2R1 was higher in prostate cancer cells compared to that in primary prostate cells. Knockdown of PLA2R1 expression in PC-3 cells using shRNA increased cell proliferation and did not affect the toxicity of cisplatin, doxorubicin (Dox), and docetaxel. In contrast, PLA2R1 knockdown increased the in vitro toxicity of Dox encapsulated in sPLA2 responsive liposomes (SPRL) and correlated with increased Dox and SPRL uptake. Knockdown of PLA2R1 also increased the expression of Group IIA and X sPLA2. These data show the novel findings that PLA2R1 is expressed in prostate cancer cells, that PLA2R1 expression alters cell proliferation, and that PLA2R1 modulates the behavior of liposome-based nanoparticles. Furthermore, these studies suggest that PLA2R1 may represent a novel molecular target for controlling tumor growth or modulating delivery of lipid-based nanomedicines.

T-cell and antibody responses to phospholipase A2 from different species show distinct cross-reactivity patterns.

BACKGROUND: Secreted phospholipases A2 (sPLA2) represent antigens to which humans may be rarely or frequently exposed. Thus, the investigation of humoral and cellular immune responses to sPLA2s from different species can provide a suitable model in the study of antibody and T-cell cross-reactivity. METHODS: Specific IgE, IgG1, IgG4, and IgA antibodies were analyzed by ELISA against sPLA2s from pancreas of Bos taurus (BT), Apis mellifera (AM) bee venom, Daboia russellii (DR) and Naja mossambica (NM) snake venoms, and human group III (hGIII) sPLA2 using sera of nonallergic beekeepers, AM-allergic patients, and healthy controls. T-cell cross-reactivity was investigated in PBMC, and T-cell clones (TCC) are generated against AM sPLA2. RESULTS: Hyperimmune and allergic individuals showed high levels of sPLA2-specific IgG4 and significant IgG4 cross-reactivity between BT, DR, and NM sPLA2s. Furthermore, IgE, IgA, and IgG1 cross-reactivities against BT, DR, and NM sPLA2s were also detectable in the range of 22.2-44.8%. Allergic patients showed significant T-cell proliferative response to NM sPLA2 together with increased IFN-γ and IL-13 production even though they had never been exposed to cobra venom. Although nonallergic healthy controls show no cross-reactivity at T-cell level, they did have low levels of IgG4 and IgA against BT, DR, and NM sPLA2s. Human TCC spanning three major T-cell epitopes of AM sPLA2 showed minor proliferative response to NM and hGIII sPLA2s. CONCLUSIONS: This study shows that T cells and antibodies may show cross-reactivity between different species without being naturally exposed to sPLA2s.

Distinct expression pattern of the full set of secreted phospholipases A2 in human colorectal adenocarcinomas: sPLA2-III as a biomarker candidate.

Recent studies suggest that secreted phospholipases A2 (sPLA2s) represent attractive potential tumour biomarkers and therapeutic targets for various cancers. As a first step to address this issue in human colorectal cancer, we examined the expression of the full set of sPLA2s in sporadic adenocarcinomas and normal matched mucosa from 21 patients by quantitative PCR and immunohistochemistry. In normal colon, PLA2G2A and PLA2G12A were expressed at high levels, PLA2G2D, PLA2G5, PLA2G10 and PLA2G12B at moderate levels, and PLA2G1B, PLA2G2F and PLA2G3 at low levels. In adenocarcinomas from left and right colon, the expression of PLA2G3 was increased by up to 40-fold, while that of PLA2G2D and PLA2G5 was decreased by up to 23- and 14-fold. The variations of expression for sPLA2-IID, sPLA2-III and sPLA2-V were confirmed at the protein level. The expression pattern of these sPLA2s appeared to be linked respectively to the overexpression of interleukin-8, defensin alpha6, survivin and matrilysin, and downregulation of SFRP-1 and RLPA-1, all these genes being associated to colon cancer. This original sPLA2 profile observed in adenocarcinomas highlights the potential role of certain sPLA2s in colon cancer and suggests that sPLA2-III might be a good candidate as a novel biomarker for both left and right colon cancers.

Hydrolysis of minor glycerophospholipids of plasma lipoproteins by human group IIA, V and X secretory phospholipases A2.

We investigated the hydrolysis of the minor glycerophospholipids of human HDL(3), total HDL and LDL using human group IIA, V and X secretory phospholipases A(2) (sPLA(2)s). For this purpose we employed the enzyme and substrate concentrations and incubation times optimized for hydrolysis of phosphatidylcholine (PtdCho), the major glycerophospholipid of plasma lipoproteins. In contrast to PtdCho, which was readily hydrolyzed by group V and X sPLA(2)s, and to a lesser extent by group IIA sPLA(2), the minor ethanolamine, inositol and serine glycerophospholipids exhibited marked resistance to hydrolysis by all three sPLA(2)s. Thus, when PtdCho was hydrolyzed about 80%, the ethanolamine and inositol glycerophospholipids reached a maximum of 40% hydrolysis. The hydrolysis of phosphatidylserine (PtdSer), which was examined to a more limited extent, showed similar resistance to group IIA, V and X sPLA(2)s, although the group V sPLA(2) attacked it more readily than group X sPLA(2) (52% versus 39% hydrolysis, respectively). Surprisingly, the group IIA sPLA(2) hydrolysis remained minimal at 10-15% for all minor glycerophospholipids, and was of the order seen for the PtdCho hydrolysis by group IIA sPLA(2) at the 4-h digestion time. All three enzymes attacked the oligo- and polyenoic species in proportion to their mole percentage in the lipoproteins, although there were exceptions. There was evidence of a more rapid destruction of the palmitoyl compared to the stearoyl arachidonoyl glycerophospholipids. Overall, the characteristics of hydrolysis of the molecular species of the lipoprotein-bound diradyl GroPEtn, GroPIns and GroPSer by group V and X sPLA(2)s differed significantly from those observed with lipoprotein-bound PtdCho. As a result, the acidic inositol and serine glycerophospholipids accumulated in the digestion residues of both LDL and HDL, and presumably increased the acidity of the residual particles. An accumulation of the ethanolamine glycerophospholipids in the sPLA(2) digestion residues also had not been previously reported. These results further emphasize the diversity in the enzymatic activity of the group IIA, V and X sPLA(2)s. Since these sPLA(2)s possess comparable tissue distribution, their combined activity may exacerbate their known proinflammatory and proatherosclerotic function.

Upregulation of group IB secreted phospholipase A(2) and its M-type receptor in rat ANTI-THY-1 glomerulonephritis.

Treatment of rat glomerular mesangial cell (GMC) cultures with pancreatic secreted phospholipase A(2) (sPLA(2)-IB) results in an enhanced expression of sPLA(2)-IIA and COX-2, possibly via binding to its specific M-type sPLA(2) receptor. In the current study, we have investigated the expression and regulation of sPLA(2)-IB and its receptor during glomerulonephritis (GN). In vivo we used the well-established rat model of anti-Thy 1.1 GN (anti-Thy 1.1-GN) to study the expression of sPLA(2)-IB and the M-type sPLA(2) receptor by immunohistochemistry. In addition, in vitro we determined the interkeukin (IL)-1beta-regulated mRNA and protein expression in primary rat glomerular mesangial and endothelial cells as well as in rat peripheral blood leukocytes (PBLs). Shortly after induction of anti-Thy 1.1-GN, sPLA(2)-IB expression was markedly upregulated in the kidney at 6-24 h. Within glomeruli, the strongest sPLA(2)-IB protein expression was detected on infiltrated granulocytes and monocytes. However, at the same time, the M-type receptor was also markedly upregulated on resident glomerular cells. In vitro, the most prominent cytokine-stimulated secretion of sPLA(2)-IB was observed in monocytes isolated from rat PBLs. Treating glomerular endothelial cells (GECs) with cytokines elicited only weak sPLA(2)-IB expression, but treatment of these cells with exogenous sPLA(2)-IB resulted in a marked expression of the endogenous sPLA(2)-IB. Mesangial cells did not express sPLA(2)-IB at all. The M-type sPLA(2) receptor protein was markedly upregulated on cytokine-stimulated mesangial and endothelial cells as well as on lymphocytes and granulocytes. During anti-Thy 1.1 rat GN, sPLA(2)-IB and the M-type sPLA(2) receptor are induced as primary downstream genes stimulated by inflammatory cytokines. Subsequently, both sPLA(2)-IB and the M-type sPLA(2) receptor are involved in the autocrine and paracrine amplification of the inflammatory process in different resident and infiltrating cells.

A peptide derived from bee venom-secreted phospholipase A2 inhibits replication of T-cell tropic HIV-1 strains via interaction with the CXCR4 chemokine receptor.

We have previously shown that secreted phospholipases A2 (sPLA2) from bee and snake venoms have potent anti-human immunodeficiency virus (HIV) activity. These sPLA2s block HIV-1 entry into host cells through a mechanism linked to sPLA2 binding to cells. In this study, 12 synthetic peptides derived from bee venom sPLA2 (bvPLA2) have been tested for inhibition of HIV-1 infection. The p3bv peptide (amino acids 21 to 35 of bvPLA2) was found to inhibit the replication of T-lymphotropic (T-tropic) HIV-1 isolates (ID(50) = 2 microM) but was without effect on monocytotropic (M-tropic) HIV-1 isolates. p3bv was also found capable of preventing the cell-cell fusion process mediated by T-tropic HIV-1 envelope. Finally, p3bv can inhibit the binding of radiolabeled stromal cell-derived factor (SDF)-1alpha, the natural ligand of CXCR4, and the binding of 12G5, an anti-CXCR4 monoclonal antibody. Taken together, these results indicate that p3bv blocks the replication of T-tropic HIV-1 strains by interacting with CXCR4. Its mechanism of action however appears distinct from that of bvPLA2 because the latter inhibits replication of both T-tropic and M-tropic isolates and does not compete with SDF-1alpha and 12G5 binding to CXCR4.

Cloning and cDNA sequence analysis of Lys(49) and Asp(49) basic phospholipase A(2) myotoxin isoforms from Bothrops asper.

Snake venom myotoxic phospholipases A(2) contribute to much of the tissue damage observed during envenomation by Bothrops asper, the major cause of snake bites in Central America. Several myotoxic PLA(2)s have been identified, but their mechanism of myotoxicity is still unclear. To aid in the molecular characterization of these venom toxins, the complete open reading frames encoding two Lys(49) and one Asp(49) basic PLA(2) myotoxins from the Central American snake B. asper (terciopelo) were obtained by cDNA cloning from venom gland poly-adenylated RNA. The amino acid sequence deduced from the myotoxins II and III open reading frames corresponded in each case to one of the reported amino acid sequence isoforms. The sequence of a new myotoxin IV-like sequence (MT-IVa) contains conservative Val-->Leu(18) and Ala-->Val(23) substitutions when compared with the reported N-terminus of the native myotoxin IV, suggesting minor isoform variations among specimens of a single species. Sequence alignment studies indicated significant (>75% sequence identity) identities with other crotalid venom Lys(49) PLA(2)s, particularly bothropstoxin I/Ia isoforms of B. jararacussu and myotoxin II of B. asper.

Characterization of group X phospholipase A(2) as the major enzyme secreted by human keratinocytes and its regulation by the phorbol ester TPA.

HaCaT as well as human primary keratinocytes constitutively expressed mRNA of the human secreted phospholipase A(2) subtype groups X, V, IIA, and IID. A similar expression pattern was also found in human skin biopsies. Protein analysis showed that under serum-free conditions only group X secreted phospholipase A(2) is secreted into cell culture supernatants of HaCaT as well as human primary keratinocytes, whereas the other secreted phospholipases A(2) were not detectable at protein level. HaCaT keratinocytes constitutively released secreted phospholipase A(2) activity into the cell culture supernatant, being reflected by a constant release of fatty acids. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate, which is a potent inducer of inflammation in skin, drastically reduced the mRNA level of group X secreted phospholipase A(2) and other secreted phospholipase A(2) subtypes as well as secreted phospholipase A(2) activity in cell culture supernatants. This suggests that inhibition of secreted phospholipase A(2) expression and activity as well as of fatty acid release by 12-O-tetradecanoylphorbol-13-acetate treatment might be a critical step impairing the integrity of the epidermis during phorbol-ester-induced pathologic processes in skin. The results show that group X secreted phospholipase A(2) represents the major secreted phospholipase A(2) subtype in human keratinocytes and thus may indicate a physiologic role for this enzyme in epidermis in vivo.

Distinct arachidonate-releasing functions of mammalian secreted phospholipase A2s in human embryonic kidney 293 and rat mastocytoma RBL-2H3 cells through heparan sulfate shuttling and external plasma membrane mechanisms.

We analyzed the ability of a diverse set of mammalian secreted phospholipase A(2) (sPLA(2)) to release arachidonate for lipid mediator generation in two transfected cell lines. In human embryonic kidney 293 cells, the heparin-binding enzymes sPLA(2)-IIA, -IID, and -V promote stimulus-dependent arachidonic acid release and prostaglandin E(2) production in a manner dependent on the heparan sulfate proteoglycan glypican. In contrast, sPLA(2)-IB, -IIC, and -IIE, which bind weakly or not at all to heparanoids, fail to elicit arachidonate release, and addition of a heparin binding site to sPLA(2)-IIC allows it to release arachidonate. Heparin nonbinding sPLA(2)-X liberates arachidonic acid most likely from the phosphatidylcholine-rich outer plasma membrane in a glypican-independent manner. In rat mastocytoma RBL-2H3 cells that lack glypican, sPLA(2)-V and -X, which are unique among sPLA(2)s in being able to hydrolyze phosphatidylcholine-rich membranes, act most likely on the extracellular face of the plasma membrane to markedly augment IgE-dependent immediate production of leukotriene C(4) and platelet-activating factor. sPLA(2)-IB, -IIA, -IIC, -IID, and -IIE exert minimal effects in RBL-2H3 cells. These results are also supported by studies with sPLA(2) mutants and immunocytostaining and reveal that sPLA(2)-dependent lipid mediator generation occur by distinct (heparanoid-dependent and -independent) mechanisms in HEK293 and RBL-2H3 cells.

Cloning and recombinant expression of human group IIF-secreted phospholipase A(2).

Mammalian-secreted phospholipases A(2) (sPLA(2)) form a diverse family of at least nine enzymes that hydrolyze phospholipids to release free fatty acids and lysophospholipids. We report here the cloning and characterization of human group IIF sPLA(2) (hGIIF sPLA(2)). The full-length cDNA codes for a signal peptide of 20 amino acid followed by a mature protein of 148 amino acids containing all of the structural features of catalytically active group II sPLA(2)s. hGIIF sPLA(2) gene is located on chromosome 1 and lies within a sPLA(2) gene cluster of about 300 kbp that also contains the genes for group IIA, IIC, IID, IIE, and V sPLA(2)s. In adult tissues, hGIIF is highly expressed in placenta, testis, thymus, liver, and kidney. Finally, recombinant expression of hGIIF sPLA(2) in Escherichia coli shows that the enzyme is Ca(2+)-dependent, maximally active at pH 7-8, and hydrolyzes phosphatidylglycerol versus phosphatidylcholine with a 15-fold preference.

Increasing molecular diversity of secreted phospholipases A(2) and their receptors and binding proteins.

Secreted phospholipases A(2) (sPLA(2)s) form a large family of structurally related enzymes which are widespread in nature. Snake venoms are known for decades to contain a tremendous molecular diversity of sPLA(2)s which can exert a myriad of toxic and pharmacological effects. Recent studies indicate that mammalian cells also express a variety of sPLA(2)s with ten distinct members identified so far, in addition to the various other intracellular PLA(2)s. Furthermore, scanning of nucleic acid databases fueled by the different genome projects indicates that several sPLA(2)s are also present in invertebrate animals like Drosophila melanogaster as well as in plants. All of these sPLA(2)s catalyze the hydrolysis of glycerophospholipids at the sn-2 position to release free fatty acids and lysophospholipids, and thus could be important for the biosynthesis of biologically active lipid mediators. However, the recent identification of a variety of membrane and soluble proteins that bind to sPLA(2)s suggests that the sPLA(2) enzymes could also function as high affinity ligands. So far, most of the binding data have been accumulated with venom sPLA(2)s and group IB and IIA mammalian sPLA(2)s. Collectively, venom sPLA(2)s have been shown to bind to membrane and soluble mammalian proteins of the C-type lectin superfamily (M-type sPLA(2) receptor and lung surfactant proteins), to pentraxin and reticulocalbin proteins, to factor Xa and to N-type receptors. Venom sPLA(2)s also associate with three distinct types of sPLA(2) inhibitors purified from snake serum that belong to the C-type lectin superfamily, to the three-finger protein superfamily and to proteins containing leucine-rich repeats. On the other hand, mammalian group IB and IIA sPLA(2)s can bind to the M-type receptor, and group IIA sPLA(2)s can associate with lung surfactant proteins, factor Xa and proteoglycans including glypican and decorin, a mammalian protein containing a leucine-rich repeat.

Secreted phospholipase A(2) induces vascular endothelial cell migration.

Secreted phospholipase A(2) (sPLA(2)) regulates a variety of cellular functions. The present investigation was undertaken to elucidate the potential role of sPLA(2) in endothelial cell (EC) migration. Bovine aortic endothelial cells (BAECs) exposed to sPLA(2) placed in the lower compartment of a modified Boyden chamber displayed increased migration compared to cells exposed to vehicle. The effect of sPLA(2) on EC migration was time and dose dependent. Migration of BAECs was observed at 30 minutes, increased over 1 to 2 hours, and declined thereafter. At 2 hours of stimulation, sPLA(2) (0.01-2 micromol/L) induced 1.2- to 3-fold increased cell migration compared with media alone. Among the different sPLA(2)s tested, bee venom, Naja naja, and porcine and human pancreatic PLA(2)s all evoked a migratory response in ECs. Moreover, human synovial fluid, obtained from patients with arthritis and containing sPLA(2) activity, induced EC migration. Migration of ECs was significantly reduced after exposure to a catalytic site mutant of pancreatic sPLA(2) with decreased lipolytic activity as compared to wild-type sPLA(2). Similarly, pretreatment of human synovial fluid with p-bromophenacyl bromide, an irreversible inhibitor of sPLA(2), markedly decreased the ability of human synovial fluid to stimulate EC migration. Moreover, migration of ECs was stimulated on exposure to hydrolytic products of sPLA(2) activity including arachidonic acid, lysophosphatidic acid, and lysophosphatidylcholine. These findings suggest that sPLA(2) plays a physiologic role in induction of EC migration. Moreover, the effects of sPLA(2) on EC migration are mediated, at least in part, by its catalytic activity. (Blood. 2000;96:3809-3815)

What can venom phospholipases A(2) tell us about the functional diversity of mammalian secreted phospholipases A(2)?

Most venomous animals including snakes, bees and scorpions contain a variety of venom phospholipases A(2) (vPLA(2)s) which participate in both digestion of prey and venom toxicity. So far, more than 150 vPLA(2)s have been characterized. They all have a conserved fold with several disulfide bridges, can be catalytically active or not, and several of them can display a tremendous array of toxic effects including neurotoxicity and myotoxicity. Furthermore, the molecular diversity of vPLA(2)s found within a single snake venom can result from positive Darwinian selection. Over the last decade, receptors and binding proteins for vPLA(2)s have been identified in mammals, suggesting that vPLA(2)s can exert their toxicities through specific protein-protein interactions, besides their catalytic activity. The brain N-type receptors are involved in the neurotoxicity of vPLA(2)s, but are not yet cloned. The M-type receptor has been cloned from skeletal muscle, belongs to the superfamily of C-type lectins, and interestingly, has homology with vPLA(2) inhibitors purified from snake blood. The molecular diversity of vPLA(2)s and the presence of receptors for vPLA(2)s in mammals raises the possibility that there is also a diversity of mammalian secreted PLA(2)s (msPLA(2)s) which are the normal endogenous ligands of the vPLA(2) receptors. This view led us to clone five novel msPLA(2)s (IID, IIE, IIF, III, and X msPLA(2)s), which together with the previously cloned msPLA(2)s (IB, IIA, IIC, and V), indicate that mammals also express a large diversity of sPLA(2)s. M-type receptors can have IB and IIA msPLA(2)s as natural endogenous ligands, suggesting that msPLA(2)s, like vPLA(2)s, can function as both enzymes and ligands. msPLA(2)s were first implicated in lipid digestion, and more recently in host defense mechanisms including inflammation and antibacterial defense. The growing molecular diversity of msPLA(2)s, which all have a specific tissue distribution, and the presence of receptors suggest that msPLA(2)s, like vPLA(2)s, are endowed with a wide array of biological effects which remain to be discovered.

Redundant and segregated functions of granule-associated heparin-binding group II subfamily of secretory phospholipases A2 in the regulation of degranulation and prostaglandin D2 synthesis in mast cells.

We herein demonstrate that mast cells express all known members of the group II subfamily of secretory phospholipase A2 (sPLA2) isozymes, and those having heparin affinity markedly enhance the exocytotic response. Rat mastocytoma RBL-2H3 cells transfected with heparin-binding (sPLA2-IIA, -V, and -IID), but not heparin-nonbinding (sPLA2-IIC), enzymes released more granule-associated markers (beta-hexosaminidase and histamine) than mock- or cytosolic PLA2alpha (cPLA2alpha)-transfected cells after stimulation with IgE and Ag. Site-directed mutagenesis of sPLA2-IIA and -V revealed that both the catalytic and heparin-binding domains are essential for this function. Confocal laser and electron microscopic analyses revealed that sPLA2-IIA, which was stored in secretory granules in unstimulated cells, accumulated on the membranous sites where fusion between the plasma membrane and granule membranes occurred in activated cells. These results suggest that the heparin-binding sPLA2s bind to the perigranular membranes through their heparin-binding domain, and lysophospholipids produced in situ by their enzymatic action may facilitate the ongoing membrane fusion. In contrast to the redundant role of sPLA2-IIA, -IID, and -V in the regulation of degranulation, only sPLA2-V had the ability to markedly augment IgE/Ag-stimulated immediate PGD2 production, which reached a level comparable to that elicited by cPLA2alpha. The latter observation reveals an unexplored functional segregation among the three related isozymes expressed in the same cell population.

Cloning and recombinant expression of a structurally novel human secreted phospholipase A2.

Mammals contain a diverse set of secreted phospholipases A(2) (sPLA(2)s) that liberate arachidonic acid from phospholipids for the production of eicosanoids and exert a variety of physiological and pathological effects. We report the cloning, recombinant expression, and kinetic properties of a novel human sPLA(2) that defines a new structural class of sPLA(2)s called group XII. The human group XII (hGXII) cDNA contains a putative signal peptide of 22 residues followed by a mature protein of 167 amino acids that displays homology to all known sPLA(2)s only over a short stretch of amino acids in the active site region. Northern blot and reverse transcription-polymerase chain reaction analyses show that the tissue distribution of hGXII is distinct from the other human sPLA(2)s with strong expression in heart, skeletal muscle, kidney, and pancreas and weaker expression in brain, liver, small intestine, lung, placenta, ovaries, testis, and prostate. Catalytically active hGXII was produced in Escherichia coli and shown to be Ca(2+)-dependent despite the fact that it is predicted to have an unusual Ca(2+)-binding loop. Similar to the previously characterized mouse group IIE sPLA(2)s, the specific activity of hGXII is low in comparison to that of other mammalian sPLA(2), suggesting that hGXII could have novel functions that are independent of its phospholipase A(2) activity.

Basic residues of human group IIA phospholipase A2 are important for binding to factor Xa and prothrombinase inhibition comparison with other mammalian secreted phospholipases A2.

Human secreted group IIA phospholipase A2 (hGIIA) was reported to inhibit prothrombinase activity because of binding to factor Xa. This study further shows that hGIIA and its catalytically inactive H48Q mutant prolong the lag time of thrombin generation in human platelet-rich plasma with similar efficiency, indicating that hGIIA exerts an anticoagulant effect independently of phospholipid hydrolysis under ex vivo conditions. Charge reversal of basic residues on the interfacial binding surface (IBS) of hGIIA leads to decreased ability to inhibit prothrombinase activity, which correlates with a reduced affinity for factor Xa, as determined by surface plasmon resonance. Mutation of other surface-exposed basic residues, hydrophobic residues on the IBS, and His48, does not affect the ability of hGIIA to inhibit prothrombinase activity and bind to factor Xa. Other basic, but not neutral or acidic, mammalian secreted phospholipases A2 (sPLA2s) exert a phospholipid-independent inhibitory effect on prothrombinase activity, suggesting that these basic sPLA2s also bind to factor Xa. In conclusion, this study demonstrates that the anticoagulant effect of hGIIA is independent of phospholipid hydrolysis and is based on its interaction with factor Xa, leading to prothrombinase inhibition, even under ex vivo conditions. This study also shows that such an interaction involves basic residues located on the IBS of hGIIA, and suggests that other basic mammalian sPLA2s may also inhibit blood coagulation by a similar mechanism to that described for hGIIA.

Two phospholipase A2 inhibitors from the plasma of Cerrophidion (Bothrops) godmani which selectively inhibit two different group-II phospholipase A2 myotoxins from its own venom: isolation, molecular cloning and biological properties.

Myotoxic phospholipases A(2) (PLA(2)s; group II) account for most of the muscle-tissue damage that results from envenomation by viperid snakes. In the venom of the Godman's viper (Cerrophidion godmani, formerly Bothrops godmani), an enzymically active PLA(2) (myotoxin I) and an inactive, Lys-49 variant (myotoxin II) induce extensive muscle damage and oedema. In this study, two distinct myotoxin inhibitor proteins of C. godmani, CgMIP-I and CgMIP-II, were purified directly from blood plasma by selective binding to affinity columns containing either myotoxin I or myotoxin II, respectively. Both proteins are glycosylated, acidic (pI=4) and composed of 20-25-kDa subunits that form oligomers of 110 kDa (CgMIP-I) or 180 kDa (CgMIP-II). In inhibition studies, CgMIP-I specifically neutralized the PLA(2) and the myotoxic, oedema-forming and cytolytic activities of myotoxins I, whereas CgMIP-II selectively inhibited the toxic properties of myotoxin II. N-terminal amino acid sequence analysis and sequencing of cDNAs encoding the two inhibitors revealed that CgMIP-I is similar to gamma-type inhibitors, which share a pattern of cysteine residues present in the Ly-6 superfamily of proteins, whereas CgMIP-II shares sequence identity with alpha-type inhibitors that contain carbohydrate-recognition-like domains, also found in C-type lectins and mammalian PLA(2) receptors. N-terminal sequencing of myotoxin I revealed a different primary structure from myotoxin II [De Sousa, Morhy, Arni, Ward, Díaz and Gutiérrez (1998) Biochim. Biophys. Acta 1384, 204-208], which provides insight into the nature of such pharmacological specificity.

Novel human secreted phospholipase A(2) with homology to the group III bee venom enzyme.

Venom and mammalian secreted phospholipases A(2) (sPLA(2)s) have been associated with numerous physiological, pathological, and toxic processes. So far, structurally related group I and II sPLA(2)s have been found in vertebrates such as mammals and snakes, whereas group III sPLA(2)s have mainly been found in venom from invertebrates such as bees and scorpions. Here we report the cloning and expression of a cDNA coding for a human group III (hGIII) sPLA(2). The full-length cDNA codes for a signal peptide of 19 residues followed by a protein of 490 amino acids made up of a central sPLA(2) domain (141 residues) flanked by large N- and C-terminal regions (130 and 219 residues, respectively). The sPLA(2) domain is 31% identical to bee venom sPLA(2) and displays all of the features of group III sPLA(2)s including 10 cysteines. The hGIII sPLA(2) gene consists of at least 7 exons and maps to chromosome 22q. By Northern blot analysis, a 4.4-kilobase hGIII transcript was found in kidney, heart, liver, and skeletal muscle. Transfection of hGIII sPLA(2) cDNA in COS cells led to accumulation of sPLA(2) activity in the culture medium, indicating that the cDNA codes for a secreted enzyme. Using small unilamellar vesicles as substrate, hGIII sPLA(2) was found to be a Ca(2+)-dependent enzyme showing an 11-fold preference for phosphatidylglycerol over phosphatidylcholine and optimal activity at pH 8.

Exogenously added human group X secreted phospholipase A(2) but not the group IB, IIA, and V enzymes efficiently release arachidonic acid from adherent mammalian cells.

Mammalian secreted phospholipases A(2) (sPLA2s) comprise a group of at least eight enzymes, including the recently identified group X sPLA2. A bacterial expression system was developed to produce human group X sPLA2 (hGX). Inhibition studies show that the sPLA2 inhibitor LY311727 binds modestly more tightly to human group IIA sPLA2 than to hGX and that a pyrazole-based inhibitor of group IIA sPLA2 is much less active against hGX. The phospholipid head group preference of vesicle-bound hGX was determined. hGX binds tightly to phosphatidylcholine vesicles, which is thought to be required to act efficiently on cells. Tryptophan 67 hGX makes a significant contribution to interfacial binding to zwitterionic vesicles. As little as 10 ng/ml hGX releases arachidonic acid for cyclooxygenase-2- dependent prostaglandin E(2) generation when added exogenously to adherent mammalian cells. In contrast, human group IIA, rat group V, and mouse group IB sPLA2s are virtually inactive at releasing arachidonate when added exogenously to adherent cells. Dislodging cells from the growth surface enhances the ability of all the sPLA2s to release fatty acids. Studies with CHO-K1 cell mutants show that binding of sPLA2s to glycosaminoglycans is not the basis for poor plasma membrane hydrolysis by group IB, IIA, and V sPLA2s.