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.

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.

Secreted phospholipases A(2), a new class of HIV inhibitors that block virus entry into host cells.

Mammalian and venom secreted phospholipases A(2) (sPLA(2)s) have been associated with a variety of biological effects. Here we show that several sPLA(2)s protect human primary blood leukocytes from the replication of various macrophage and T cell-tropic HIV-1 strains. Inhibition by sPLA(2)s results neither from a virucidal effect nor from a cytotoxic effect on host cells, but it involves a more specific mechanism. sPLA(2)s have no effect on virus binding to cells nor on syncytia formation, but they prevent the intracellular release of the viral capsid protein, suggesting that sPLA(2)s block viral entry into cells before virion uncoating and independently of the coreceptor usage. Various inhibitors and catalytic products of sPLA(2) have no effect on HIV-1 infection, suggesting that sPLA(2) catalytic activity is not involved in the antiviral effect. Instead, the antiviral activity appears to involve a specific interaction of sPLA(2)s to host cells. Indeed, of 11 sPLA(2)s from venom and mammalian tissues assayed, 4 venom sPLA(2)s were found to be very potent HIV-1 inhibitors (ID(50) < 1 nM) and also to bind specifically to host cells with high affinities (K(0.5) < 1 nM). Although mammalian pancreatic group IB and inflammatory-type group IIA sPLA(2)s were inactive against HIV-1 replication, our results could be of physiological interest, as novel sPLA(2)s are being characterized in humans.

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.

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.

Receptors for a growing family of secreted phospholipases A2.

Phospholipases A2 (PLA2s) are enzymes that catalyse the hydrolysis of the sn-2 acyl bond of glycerophospholipids to produce free fatty acids and lysophospholipids. Numerous intracellular and secreted PLA2s (sPLA2s) have now been characterized. Because PLA2 products are important for cell signalling and the biosynthesis of biologically active lipids, including eicosanoids and platelet-activating factor, PLA2s are generally considered as key enzymes that control the release of lipid mediator precursors. However, the increasing number of mammalian sPLA2s and the recent identification of different membrane proteins that bind sPLA2s makes it likely that these enzymes also behave as ligands for receptors, and that their physiological function is not limited to their catalytic activity. Here, the current state of awareness regarding the different types of sPLA2-binding proteins is described. To date, five distinct mammalian sPLA2s and two main types (M and N) of sPLA2 receptors have been identified. Because most is known about the M-type receptor, particular attention will be paid to it, including a description of it molecular properties and of its possible biological roles with regard to sPLA2 function.

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.

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.

Identification of different receptor types for toxic phospholipases A2 in rabbit skeletal muscle.

Oxyuranus scutellatus scutellatus toxins 1 (OS1) and 2 (OS2) are two phospholipase A2S (PLA2) isolated from the venom of the Australian Taipan snake. Their iodinated derivatives have been used to characterize PLA2 binding sites on rabbit skeletal muscle. Competition and cross-linking experiments indicate that 125I-labelled OS2 binding sites in rabbit skeletal muscle in vivo are distributed into two classes of receptors. One class binds OS2 and OS1 and is insensitive to the bee venom PLA2. It is composed of a 180 kDa binding protein. This class of PLA2 receptor is expressed at a high level in rabbit myotube membranes. The other class of PLA2 receptor identified with 125I-OS2 also binds with high affinity the bee venom PLA2 but not OS1 and is composed of major polypeptides of 34, 48 and 82 kDa. This second class of receptor is similar to the one found in brain membranes. The density of the two classes of receptors varies during muscle development.

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.

Secretory phospholipase A2 potentiates glutamate-induced rat striatal neuronal cell death in vivo.

The secretory phospholipases A2 (sPLA2) OS2 (10, 20 and 50 pmol) or OS1, (50 pmol) purified from taipan snake Oxyuranus scutellatus scutellatus venom, and the excitatory amino acid glutamate (Glu) (2.5 and 5.0 micromol) were injected into the right striatum of male Wistar rats. Injection of 10 and 20 pmol OS2 caused no neurological abnormalities or tissue damage. OS2 (50 pmol) caused apathy and circling towards the injection side. Histology revealed an infarct at the injection site. Injection of 50 pmol OS1 showed very little or no signs of neurotoxicity. Injection of 2.5 micromol Glu caused no tissue damage or neurological abnormality. After injection of 5.0 micromol Glu, the animals initially circled towards the side of injection, and gradually developed generalized clonic convulsions. These animals showed a well demarcated striatal infarct. When non-toxic concentrations of 20 pmol OS2 and 2.5 micromol Glu were co-injected, a synergistic neurotoxicity was observed. Extensive histological damage occurred in the entire right hemisphere, and in several rats comprising part of the contralateral hemisphere. These animals were apathetic in the immediate hours following injection, with circling towards the side of injection in the following days. Thus, OS2 greatly potentiates glutamate excitoxicity in vivo.

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.

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.

Endocytic properties of the M-type 180-kDa receptor for secretory phospholipases A2.

Endocytic properties of the M-type 180-kDa receptor for secretory phospholipases A2 (sPLA2) were first investigated in rabbit myocytes that express it at high levels. Internalization of the receptor was shown to be clathrin-coated pit-mediated, rapid (ke = 0.1 min-1), and ligand-independent. The signal sequence for internalization was then identified upon transient and stable expression of various receptor constructs with mutated cytoplasmic sequences. Analysis of the internalization efficiency of the mutants suggested that the NSYY motif encodes the major endocytic signal, with the distal tyrosine residue playing the key role. Amino acid substitutions at the putative casein kinase II phosphorylation site of the receptor did not affect internalization. A chimeric protein composed of the extracellular and transmembrane domains of the rabbit sPLA2 receptor and of the cytoplasmic domain of the structurally homologous human macrophage mannose receptor retained the high affinity for sPLA2 and was internalization competent, exhibiting 50% endocytic activity of the M-type sPLA2 receptor. The results indicate the compatibility of the structural domains of the two parent proteins and provide evidence for the interchangeable character of their internalization signals.

Properties of receptors for neurotoxic phospholipases A2 in different tissues.

A radioiodinated derivative of OS2 (125I-OS2), a neurotoxic monochain phospholipase A2 isolated from taipan venom, was previously found to bind to a specific brain membrane receptor with very high affinity. 125I-OS2 is now used to identify the properties of neurotoxic phospholipase receptors in other tissues. Heart, skeletal muscle, kidney, lung, liver, pancreas, and smooth muscle membranes also contain high-affinity binding sites for toxic phospholipases A2. In most tissues, two different types of receptor sites have been characterized for 125I-OS2 with Kd1 and Kd2 values in the 1-5 pM and the 10-50 pM range respectively. Whereas all receptors are similar in the different tissues in terms of their affinity for 125I-OS2, maximal binding site capacities were very different, varying from 1.3 pmol/mg of protein in brain to 0.01 pmol/mg of protein in pancreas. In brain, heart, and skeletal muscle, receptor densities vary with in vivo development. Affinity labeling experiments have identified the subunit composition of OS2 receptors and indicated that these receptors do not have identical structures in the different tissues. Binding competition studies with OS2 and other toxic phospholipases showed tissue-dependent pharmacological profiles. All these results taken together suggest the existence of a family of receptors for neurotoxic phospholipases.

Multifunctional activity of the extracellular domain of the M-type (180 kDa) membrane receptor for secretory phospholipases A2.

M-type (180 kDa) receptors for secretory phospholipases A2 (sPLA2s) are thought to mediate some of the physiological effects of group I sPLA2, including smooth muscle contraction and cell proliferation. The M-type sPLA2 receptor is a large glycoprotein composed of several distinct extracellular domains which belongs to the C-type lectin superfamily. This receptor binds with high affinity both pancreatic group I and inflammatory group II sPLA2s as well as various sPLA2s purified from snake venoms. This paper shows that the rabbit M-type sPLA2 receptor is a multifunctional protein which is able to promote cell adhesion on type I and IV collagens most probably via its N-terminal fibronectin-like type II domain. It also shows that binding of sPLA2s to a recombinant soluble form of this receptor is associated with a noncompetitive inhibition of phospholipase A2 activity.

The human 180-kDa receptor for secretory phospholipases A2. Molecular cloning, identification of a secreted soluble form, expression, and chromosomal localization.

Secretory phospholipases A2 (sPLA2) are structurally related enzymes found in mammals as well as in insect and snake venoms. They have been associated with several physiological, pathological, and toxic processes. Some of these effects are apparently linked to the existence of specific receptors for both venom and mammalian sPLA2s. We report here the molecular cloning and expression of one of these sPLA2 receptors from human kidney. Two transcripts were detected. One encodes for a transmembrane form of the sPLA2 receptor and the other one is an alternatively processed transcript, caused by polyadenylation occurring at a site within an intron in the C terminus part of the transcriptional unit. This transcript encodes for a shortened secreted soluble sPLA2 receptor lacking the coding region for the transmembrane segment. Quantitative polymerase chain reaction experiments indicate a 1.6:1 ratio between the levels of transcripts encoding for the membrane-bound and soluble forms of the receptor, respectively. Soluble and membrane-bound human sPLA2 receptors both bind sPLA2 with high affinities. However, the binding properties of the human receptors are different from those obtained with the rabbit membrane-bound sPLA2 receptor. The 180-kDa human sPLA2 receptor gene has been mapped in the q23-q24 bands of chromosome 2.

Cloning and expression of a membrane receptor for secretory phospholipases A2.

Snake venom and mammalian secretory phospholipases A2 are structurally related enzymes that have been associated with several toxic (neurotoxicity, myotoxicity, etc.), pathological (inflammation, hypersensitivity, etc.), or physiological (contraction, proliferation, etc.) processes. We have previously shown that snake venom PLA2s have specific high affinity receptors. Here, we report the molecular cloning of one of these PLA2 receptors (molecular mass approximately 180 kDa), previously purified from rabbit skeletal muscle. It is a membrane protein with a N-terminal cysteine-rich domain, a fibronectin type II domain, eight repeats of a carbohydrate recognition domain, a unique transmembrane domain, and a intracellular C-terminal domain. The 1458-residue PLA2 receptor, expressed in transfected cells, binds svPLA2 with very high affinities (Kd values approximately 10-20 pM). It also tightly binds the two structural types of msPLA2s, i.e. pancreatic PLA2 and synovial PLA2 (Kd approximately 1-10 nM). This receptor might have a key role in normal and pathological actions of secretory PLA2s.

Identification of the binding domain for secretory phospholipases A2 on their M-type 180-kDa membrane receptor.

The rabbit muscle (M)-type receptor for secretory phospholipases A2 (sPLA2s) has a large extracellular domain of 1394 amino acids, composed of an N-terminal cysteine-rich domain, a fibronectin-like type II domain, and eight carbohydrate recognition domains (CRDs). It is thought to mediate some of the physiological effects of mammalian sPLA2s, including vascular smooth muscle contraction and cell proliferation, and is able to internalize sPLA2s. Here, we show by site-directed mutagenesis that OS1, a snake venom sPLA2, binds to the receptor via its CRDs and that deletion of CRD 5 completely abolishes the binding of sPLA2s. Moreover, a receptor lacking all CRDs but CRD 5 was still able to bind OS1 although with a lower affinity. Deletion of CRDs 4 and 6, surrounding the CRD 5, slightly reduced the affinity for OS1, thus suggesting that these CRDs are also involved in the binding of OS1. The M-type sPLA2 receptor and the macrophage mannose receptor are homologous and are predicted to share the same tertiary structure. p-Aminophenyl-alpha-D-mannopyranoside bovine serum albumin, a known ligand of the macrophage mannose receptor, binds to the M-type sPLA2 receptor essentially via CRDs 3-6.