PLA₂

A Short Phospholipase Review

by Michael H. Gelb, Ph.D and Gerard Lambeau, Ph.D.

More than a century ago, human pancreatic juice and cobra venom were reported to contain a phospholipase A₂ (PLA₂) activity that hydrolyzes egg yolk lecithin to produce a hemolytic product that was called lysolecithin. The subsequent isolation of this secreted PLA₂ activity from a variety of snake venoms and mammalian exocrine glands revealed structurally-conserved enzymes that were small (14 kDa) and extensively cross-linked by cysteine disulfide bonds, and whose most obvious function was a digestive one. Later, the various sPLA₂s found in snake venoms were found to exert diverse toxic effects. A second type of mammalian sPLA₂ discovered in neutrophils was then proposed to play a major role in host defense, indicating that both venom and mammalian sPLA₂s are endowed with other functions aside from digestion. A new era was opened with the discovery of a novel structurally different intracellular PLA₂ in human neutrophils and platelets, now known as cPLA₂-α (group IVA).

It is now apparent that mammalian PLA₂s constitute a superfamily of distinct intracellular and secreted enzymes whose products are important for signal transduction processes, lipid mediator release, lipid metabolism and host defense. There are four distinct clades within the superfamily, including small, secreted sPLAs, large intracellular cPLAs, calcium-independent iPLAs, and PAF acetylhydrolases (PAF-AHs) (Figure 1 and Table 1). Translocation, secretion, and catalytic activation by a large number of different stimuli control and modulate phospholipases under both physiological and pathophysiological conditions.

Cytosolic Phospholipases (cPLA₂)

Most if not all mammalian cells contain the cytosolic phospholipase cPLA₂- that exists in the cytosol but translocates to perinuclear and Golgi membranes when the intracellular Ca²⁺ rises above about 0.5 M. This translocation is mediated by the Ca²⁺-dependent membrane binding domain, also known as the C2 domain. Intracellular PLA₂s are relatively large enzymes and display no sequence homology with sPLA₂s. The catalytic domain contains an active site serine nucleophile contained within an active site GXSXG motif. The catalytic serine hydroxyl attacks the substrate ester carbonyl group to form an acyl enzyme. These properties are distinct from sPLA₂s which use Ca²⁺ mainly as a catalytic cofactor and a water molecule as the nucleophile to hydrolyze substrate esters without formation of a covalent acyl enzyme intermediate. cPLA₂- displays a significant specificity for the release of arachidonic acid in preference to more saturated fatty acids from the sn-2 position of phospholipids. It is now well established in a number of mammalian cells that cPLA₂- liberates arachidonic acid from membrane phospholipids for the biosynthesis of the eicosanoids in reponse to fast-acting agonists. Examples include thrombin-stimulated blood platelets, and opsinized zymosan-stimulated neutrophils and macrophages. This has been established by studies with cPLA₂--deficient mice and with potent cPLA₂- inhibitors. Two paralogs of cPLA₂-, referred to as cPLA₂- and cPLA₂-, have recently been identified. cPLA₂- and—display little fatty acyl preference and thus may not be involved in arachidonic acid release. However, this remains to be proven, and the physiological functions of cPLA₂- and—are not known.

Calcium Independent Phospholipases (iPLA₂)

Mammalian cells also contain Ca²⁺-independent PLA₂s. Group IVA-1 (also known as iPLA₂) and its alternative splice variant VIA-2 that are serine-dependent enzymes displaying preference for saturated or mono-unsaturated sn-2 fatty acyl chains. Available evidence suggests that iPLA₂ is directly responsible for removing highly saturated fatty acids from phospholipids as a way to incorporate more unsaturated fatty acids such as arachidonic acid into cellular phospholipids (phospholipid acyl chain remodeling). This proposal is based on the demonstration that the potent iPLA₂ inhibitor bromoenol lactone (BEL) blocks incorporation of free arachidonic acid into phospholipids in a number of mammalian cells. At this point, it is difficult to judge whether iPLA₂ is mainly responsible for phospholipid remodeling in cells. Other proposed physiological functions of iPLA₂ remain to be substantiated. It should be noted that the potent cPLA₂- inhibitors AACOCF₃ and MAFP have been shown to also potently inhibit iPLA₂. Thus the use of these compounds cannot serve as the sole basis for establishing the role of cPLA₂- and iPLA₂ in cellular processes. Mammalian cells also contain a novel Ca²⁺-independent PLA₂, group VIB, that shares approximately 25% homology with iPLA₂. The function of this enzyme remains to be established. How iPLA₂s bind to membranes, and whether they cycle between the membrane and soluble fractions, remains to be determined.

PAF Acetylhydrolases (PAF-AH)

A number of PLA₂s that act preferentially on platelet activating factor (PAF) have been reported over the past decade. PAF is an unusual phosphatidylcholine analog that contains a short sn-2 acetyl group and has potent pro-inflammatory activity. The plasma PAF-AH (group VIIA) is the most extensively studied enzyme of this type. A clear role of this enzyme in the degradation of PAF is demonstrated by the prolonged half-life of plasma PAF in humans that lack group VIIA PAF-AH. This enzyme shows poor activity towards phospholipids with naturally-occuring, long-chain sn-2 fatty acids. In addition to PAF, group VIIA PAF-AH hydrolyzes a number of phospholipids with oxidatively truncated fatty acyl chains. Recent studies demonstrate that this enzyme is not an interfacial enzyme and also suggest that it may have a broad substrate specificity as an esterase in addition to being a phospholipase A₂. Mammalian cells also contain an intracellular PAF-AH (group VIIB) that is homologous to the plasma enzyme. The physiological function of group VIIB PLA₂ remains to be established. Finally, other PAF acetylhydrolases have been detected, again based on their ability to hydrolyze PAF in preference to long-chain phospholipids. Group VIIIA and VIIIB share 62% sequence homology. They constitute the catalytic subunits of a heterotrimeric complex, the third subunit being the regulatory component which is the product of causative gene for Miller-Deicker lissencephaly, a rare neurodegenerative sydrome. The function of group VIIIA and VIIIB PAF acetylhydrolases in the brain remains to be determined, but it may be noted that PAF has been proposed as a retrograde messenger in long term potentiation. Among the PAF acetylhydrolases, potent inhibitors have been described only for the plasma form .

Secreted Phospholipases (sPLA₂)

The mammalian family of PLA₂ also includes a growing number of secreted enzymes (Fig. 1 and 2). Except for the secreted PAF acetyl hydrolase (PAF-AH), sPLA₂s are disulfide-rich proteins that display little sn-2 fatty acyl chain specificity and utilize a catalytic histidine and Ca²⁺ as an essential cofactor. sPLA₂s also contain an interfacial recognition site made up of hydrophobic and basic residues that surround the buried active site and allow attachment of the enzyme to the membrane surface .Twelve sPLA₂s have been identified in mice (mGIB, mGIIA, mGIIC, mGIID, mGIIE, mGIIF, mGIII, mGV, mGX, mGXIIA, mGXIIB, and mOtoconin-95). Humans contain all of these except IIC, which occurs as a pseudogene. Many of these sPLA₂s were identified in the past few years, and studies are under way to determine their physiological functions. Based on their structure, the mammalian sPLA₂s can be divided into three main structural collections called I/II/V/X, III, and XII. Interestingly, in both mouse and humans, the genes for six of the 8 sPLA₂s in the I/II/V/X collection occur as a gene cluster (Fig. 2) suggesting recent duplication events. However, the different sPLA₂ paralogs are not closely related isoforms because the amino acid identity between any two is in the range of <15–51% (see the table in Fig. 2 for the most conserved sPLA₂s of the I/II/V/X collection). These sPLA₂s have also dramatically different enzymatic properties. For instance, their specific activities on pure phosphatidylglycerol or phosphatidylcholine vesicles are extremely different (Fig. 3). Group X sPLA₂ is in fact the sole enzyme that can readily hydrolyze phosphatidylcholine and release AA from live cells, when added exogenously (Fig. 3 and ). These properties plus the fact that each sPLA₂ displays a distinct tissue distribution pattern argue for distinct physiological functions of these enzymes. Furthermore, the tissue distribution and regulation of expression under pathological conditions of the human sPLA₂s are often different from those of the mouse orthologs, suggesting that the function of a particular sPLA₂ in the mouse may be distinct from that of its human ortholog. sPLA₂s can also be distinguished by their different affinities for known sPLA₂ inhibitors. Indole analogs including indoxam appear at the moment as the most potent inhibitors of many but not all of the sPLA₂s. Interestingly, these inhibitors have therapeutic potential in several animal models of human diseases. Specific inhibitors for each of the sPLA₂s remain to be established. Pyrazole-1 appears so far as the most specific inhibitor for group IIA sPLA₂.

Well established physiological functions for mammalian sPLA₂s include the following. Pancreatic sPLA₂ (group IB) has a well known role in the digestion of dietary phospholipids, but other sPLA₂s are probably also involved in phospholipid degradation in the gastrointestinal track and may also be present in pancreatic juice. The first non-pancreatic mammalian sPLA₂ to be identified was the group IIA enzyme, which is expressed at high levels during inflammation and is the principal bactericidal agent against Gram-positive bacteria in human tears, and also works in concert with neutrophils as a bactericidal agent. Groups IIA, V and presumably X sPLA₂s are involved in liberation of arachidonic acid from phospholipids, for example in endothelial cells and macrophages, for the biosynthesis of eicosanoids. sPLA₂s IIA and X may also play roles in cancer. One or more keratinocyte sPLA₂s is involved in the generation of free fatty acids, which are one of the main constituents of the permeability barrier of the outermost layer of skin. Physiological functions for groups IIC, IID, IIE, IIF, III, and XII sPLA₂s have not yet been reported, although overexpression of groups IID, IIE, IIF, and III in HEK293 cells results in arachidonic acid release, which can be converted into prostaglandins.

sPLA₂ Receptors

Mammals also contain a collection of proteins that bind sPLA₂s tightly. Two types of sPLA₂ receptor (M- and N-type), the cell surface proteoglycan glypican, and soluble sPLA₂-binding proteins have been identified and may play a role in the physiological functions of mammalian sPLA₂s. These proteins may also be involved in the toxicity of a wide variety of myotoxic and neurotoxic sPLA₂s found in reptile and invertebrate venoms. The fact that several sPLA₂s display extremely low specific activities on different substrates (Fig. 3) suggest that their biological functions may not only be related to their enzymatic activity, but rather to their binding properties to a variety of specific binding proteins and receptors. For example, groups IID, IIIE, XIIA and XIIB sPLA₂s have very low or no catalytic activity. A number of reports has shown that sPLA₂s can trigger signaling events that can not be simply explained by their catalytic activity, reinforcing the idea that sPLA₂s would essentially function as ligands, and not as enzymes (and references therein). This view is in agreement with the model proposed some time ago for venom sPLA₂s. Finally, because sPLA₂s can also hydrolyze non cellular phospholipids including those from microvesicles, LDL, and lung surfactant, sPLA₂s may generate their different biological effects through a variety of molecular pathways as illustrated in figure 4. In conclusion, mammalian PLA₂s clearly constitute a superfamily of both intracellular and secreted enzymes. This is in marked contrast with the phospholipase C and D families that occur only intracellularly. Understanding the biological functions of the different PLA₂s, especially the secreted types, is now a very challenging area. The elucidation of the function of each PLA₂ member and the development of specific inhibitors will open new directions for the treatment of inflammatory disorders and cancer diseases.

The authors wish to express their thanks to people who have contributed to this work, and to Franck Aguila for creating the illustrations. Because of space limitations, we regret that only a limited number of references could be cited.

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