Emmanuel Valentin

Increasing molecular diversity of secreted phospholipases A2 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.

On the Diversity of Secreted Phospholipases A2 CLONING, TISSUE DISTRIBUTION, AND FUNCTIONAL EXPRESSION OF TWO NOVEL MOUSE GROUP II ENZYMES

Over the last decade, an expanding diversity of secreted phospholipases A2 (sPLA2s) has been identified in mammals. Here, we report the cloning in mice of three additional sPLA2s called mouse group IIE (mGIIE), IIF (mGIIF), and X (mGX) sPLA2s, thus giving rise to eight distinct sPLA2s in this species. Both mGIIE and mGIIF sPLA2s contain the typical cysteines of group II sPLA2s, but have relatively low levels of identity (less than 51%) with other mouse sPLA2s, indicating that these enzymes are novel group II sPLA2s. However, a unique feature of mGIIF sPLA2 is the presence of a C-terminal extension of 23 amino acids containing a single cysteine. mGX sPLA2 has 72% identity with the previously cloned human group X (hGX) sPLA2 and displays similar structural features, making it likely that mGX sPLA2 is the ortholog of hGX sPLA2. Genes for mGIIE and mGIIF sPLA2s are located on chromosome 4, and that of mGX sPLA2 on chromosome 16. Northern and dot blot experiments with 22 tissues indicate that all eight mouse sPLA2s have different tissue distributions, suggesting specific functions for each. mGIIE sPLA2 is highly expressed in uterus, and at lower levels in various other tissues. mGIIF sPLA2 is strongly expressed during embryogenesis and in adult testis. mGX sPLA2 is mostly expressed in adult testis and stomach. When the cDNAs for the eight mouse sPLA2s were transiently transfected in COS cells, sPLA2 activity was found to accumulate in cell medium, indicating that each enzyme is secreted and catalytically active. Using COS cell medium as a source of enzymes, pH rate profile and phospholipid headgroup specificity of the novel sPLA2s were analyzed and compared with the other mouse sPLA2s.

What can venom phospholipases A2 tell us about the functional diversity of mammalian secreted phospholipases A2

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.

Both Group IB and Group IIA Secreted Phospholipases A2 Are Natural Ligands of the Mouse 180-kDa M-type Receptor

Snake venom and mammalian secreted phospholipases A2 (sPLA2s) have been associated with toxic (neurotoxicity, myotoxicity, etc.), pathological (inflammation, cancer, etc.), and physiological (proliferation, contraction, secretion, etc.) processes. Specific membrane receptors (M and N types) for sPLA2s have been initially identified with snake venom sPLA2s as ligands, and the M-type 180-kDa receptor was cloned from different animal species. This paper addresses the problem of the endogenous ligands of the M-type receptor. Recombinant group IB and group IIA sPLA2s from human and mouse species have been prepared and analyzed for their binding properties to M-type receptors from different animal species. Both mouse group IB and group IIA sPLA2s are high affinity ligands (in the 1–10 nm range) for the mouse M-type receptor. These two sPLA2s are expressed in the mouse tissues where the M-type receptor is also expressed, making it likely that both types of sPLA2s are physiological ligands of the mouse M-type receptor. This conclusion does not hold for human group IB and IIA sPLA2s and the cloned human M-type receptor. The two mouse sPLA2s have relatively high affinities for the mouse M-type receptor, but they can have much lower affinities for receptors from other animal species, indicating that species specificity exists for sPLA2 binding to M-type receptors. Caution should thus be exerted in avoiding mixing sPLA2s, cells, or tissues from different animal species in studies of the biological roles of mammalian sPLA2s associated with an action through their membrane receptors.

Cloning and recombinant expression of a novel mouse-secreted phospholipase A2.

Secreted phospholipases A2(sPLA2s) form a class of structurally related enzymes that are involved in a variety of physiological and pathological effects including inflammation and associated diseases, cell proliferation, cell adhesion, and cancer, and are now known to bind to specific membrane receptors. Here, we report the cloning and expression of a novel sPLA2 isolated from mouse thymus. Based on its structural features, this sPLA2 is most similar to the previously cloned mouse group IIA sPLA2 (mGIIA sPLA2). As for mGIIA sPLA2, the novel sPLA2 is made up of 125 amino acids with 14 cysteines, is basic (pI = 8.71) and its gene has been mapped to mouse chromosome 4. However, the novel sPLA2 has only 48% identity with mGIIA and displays similar levels of identity with the other mouse group IIC and V sPLA2s, indicating that the novel sPLA2 is not an isoform of mGIIA sPLA2. This novel sPLA2 has thus been called mouse group IID (mGIID) sPLA2. In further contrast with mGIIA, which is found mainly in intestine, transcripts coding for mGIID sPLA2 are found in several tissues including pancreas, spleen, thymus, skin, lung, and ovary, suggesting distinct functions for the two enzymes. Recombinant expression of mGIID sPLA2 in Escherichia coli indicates that the cloned sPLA2 is an active enzyme that has much lower specific activity than mGIIA and displays a distinct specificity for binding to various phospholipid vesicles. Finally, recombinant mGIID sPLA2 did not bind to the mouse M-type sPLA2 receptor, while mGIIA was previously found to bind to this receptor with high affinity.

Secreted phospholipases A2, a new class of HIV inhibitors that block virus entry into host cells

Mammalian and venom secreted phospholipases A2 (sPLA2s) have been associated with a variety of biological effects. Here we show that several sPLA2s protect human primary blood leukocytes from the replication of various macrophage and T cell–tropic HIV-1 strains. Inhibition by sPLA2s results neither from a virucidal effect nor from a cytotoxic effect on host cells, but it involves a more specific mechanism. sPLA2s 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 sPLA2s block viral entry into cells before virion uncoating and independently of the coreceptor usage. Various inhibitors and catalytic products of sPLA2 have no effect on HIV-1 infection, suggesting that sPLA2 catalytic activity is not involved in the antiviral effect. Instead, the antiviral activity appears to involve a specific interaction of sPLA2s to host cells. Indeed, of 11 sPLA2s from venom and mammalian tissues assayed, 4 venom sPLA2s were found to be very potent HIV-1 inhibitors (ID50 < 1 nM) and also to bind specifically to host cells with high affinities (K0.5 < 1 nM). Although mammalian pancreatic group IB and inflammatory-type group IIA sPLA2s were inactive against HIV-1 replication, our results could be of physiological interest, as novel sPLA2s are being characterized in humans.

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 (β-hexosaminidase and histamine) than mock- or cytosolic PLA2α (cPLA2α)-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 cPLA2α. The latter observation reveals an unexplored functional segregation among the three related isozymes expressed in the same cell population.

On the Functional Diversity of Secreted Phospholipases A2: Cloning of Novel Mammalian Enzymes and HIV-1 Antiviral Properties

Over the past decade, it has become clear that mammalian cells not only express a variety of intracellular phospholipases A2 (PLA2), but also a diverse set of secreted phospholipases A2 (sPLA2s). While PLA2s are generally considered as key enzymes which control the production of lipid mediators, the function of the 10 distinct sPLA2s cloned so far remains ill-defined. Using venom sPLA2s, two types of specific membrane receptors (N and M) have been identified in various mammalian tissues. Of physiological relevance, the M-type receptor can bind with high affinities to several mammalian sPLA2s, making it likely that mammalian sPLA2s are endogenous ligands of the receptors initially identified with venom sPLA2s, and that the physiological function of the mammalian sPLA2s is not limited to their catalytic activity.