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Catalytic and non-catalytic functions of human IIA phospholipase A2
Review
Catalytic and non-catalytic functions of human IIA phospholipase A2 Charles N. Birts, C. Howard Barton and David C. Wilton School of Biological Sciences, University of Southampton, Boldrewood Campus, Southampton, SO16 7PX, United Kingdom
Group IIA phospholipase A2 (PLA2) is a low-molecularmass secreted PLA2 enzyme that has been identified as an acute phase protein with a role in the inflammatory response to infection and trauma. The protein is possibly unique in being highly cationic and having a global distribution of surface arginine and lysine residues. This structure supports two functions of the protein. (1) An anti-bacterial role where the enzyme is targeted to the anionic cell membrane of Gram-positive bacteria and phospholipid hydrolysis assists in bacterial killing. (2) A proposed non-catalytic role in which the protein forms supramolecular aggregates with anionic phospholipid vesicles or debris. These aggregates are then internalized via interactions with cell surface heparin sulphate proteoglycans and macropinocytosis for disposal by macrophages. Introduction to the history, structure and function of human group IIA PLA2 Group IIA phospholipase A2 (gIIA PLA2) (Figure 1) is one of a family of low-molecular-mass, secreted PLA2 enzymes [1,2] originally found in snake venoms, and as a digestive enzyme in the pancreas. This enzyme was the first nonpancreatic mammalian PLA2 to be identified, being present at high concentrations in platelets and in the synovial fluid of patients with rheumatoid arthritis [3]. It was originally implicated as the enzyme responsible for arachidonic release during the inflammatory response of the body, and thus a potential target in the development of new anti-inflammatory drugs. However, although secreted PLA2s have been implicated [1], this arachidonic acid releasing role is now primarily ascribed to the group IVA cytosolic PLA2 [4]. The potentially central role of the IIA enzyme in inflammation at the time of its discovery provided the necessary impetus for producing crystal structures for the human enzyme [5,6]. At an early stage, the enzyme was characterized as an acute phase protein under the transcriptional control of pro-inflammatory cytokine signalling [7], with serum levels dramatically elevated as a result of tissue trauma and infection. Such a large increase (up to 1000-fold, with concentrations approaching 1 mM) in serum protein concentration suggests functions involving ‘‘bulk’’ interactions with other intracellular structures and not a ‘‘more subtle’’ role in generating signalling intermediates. Along with other secreted PLA2s, the enzyme demonstrates minimal phospholipid substrate specificity at the active site, both in terms of acyl chain and headgroup preference; the enzyme Corresponding author: Wilton, D.C. ([email protected]).
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has no preference for arachidonic acid. Naturally occurring phospholipid will always present as aggregates such as the membrane bilayer under physiological condition, and only after binding to the aggregate (interfacial binding) will the enzyme be able to access a phospholipid substrate molecule. Thus, a lack of interfacial binding will mean that there is no enzyme activity, and an interfacial binding step (E ! E*) is a requirement for overall catalysis, according to Figure 2. This means that apparent phospholipid specificity can be achieved as a result of selective interfacial binding. The interfacial binding step will be most affected by the surface structure of the secreted PLA2, and in particular it is the interfacial binding surface of the enzyme (i-face) that directly interacts with the phospholipid bilayer interface [1,8]. Because the overall core structure of most secreted PLA2s are similar, without major differences in substrate specificity, it could be argued that it is differences in the specific surface properties of individual enzymes rather Glossary Acute phase proteins: these proteins are released primarily from the liver after trauma and infection, and are involved in helping to repair tissue damage and fighting infection. They include blood clotting proteins, complement factors, Creactive protein and serum amyloid P. Angiogenesis: the process of new blood vessel formation. In addition to a central involvement in mammalian embryo development, this process plays a crucial role in tumour growth and metastasis. Gram-positive and Gram-negative bacteria: Gram-negative bacteria possess a membrane, and a lipopolysaccharide coat external to the cell wall. Grampositive bacteria lack a lipopolysaccharide coat and are limited by the cell wall outside of the cell membrane. Interfacial binding and catalysis: because the critical micelle concentration (CMC) of natural phospholipids is very low (<1010 M), it is necessary for the enzyme to first bind a phospholipid aggregate, such as the bilayer, to access its phospholipid substrate. Thus, interfacial binding is a compulsory additional step in overall phospholipid hydrolysis. Macropinocytosis: a form of bulk uptake of fluid and solid cargo into cytoplasmic vacuoles, called macropinosomes. Microparticles and microvesicles: small phospholipid particles or blebs released from cells or platelets during activation or cell death that are characteristically anionic, with phosphatidylserine exposed on the surface of the particles. Phorbol 12-myristate 13-acetate (PMA): a cell-permeable chemical analogue of diacylglycerol (DAG). DAG is released from membrane phospholipid by the action of PLC and is an activator of protein kinase C. Phospholipase A2s: a family of enzymes that are able to hydrolyze the sn2 position of a glycerophospholipid to release fatty acid and the corresponding lysophospholipid. Secreted phospholipase A2s: a group of 14 kDa enzymes that were originally identified in snake venoms and the pancreas. A number of other mammalian non-pancreatic secreted PLA2s have been identified, including the group IIC, IID, IIE, IIF, III, V, X and XIIA enzymes [2], which brings the total number of mouse gene products to 10, but only 9 for humans where IIC exists as a pseudogene [1]. In addition, two gene products are structurally related to sPLA2s, but lack enzyme activity [1]. Like the other sPLA2s, the human IIA enzyme has an N-terminal signal sequence allowing secretion from the cell into the extracellular environment that must be its primary site of action.
0968-0004/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2009.08.003 Available online 8 October 2009
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Figure 1. Sites of phospholipid hydrolysis by phospholipases. Arrows indicate the position of phospholipase-catalysed hydrolysis of a glycerol-based phospholipid. R1 and R2 represent the fatty acyl chains and X represents the alcohol head group. PLA1, PLA2, PLC and PLD refer to phospholipase A1, A2, C and D respectively. The carbon atoms (1, 2 or 3) of the glycerol backbone of the phospholipid are indicated in terms of the stereochemical numbering (sn).
than catalytic properties that reflect physiological function. Indeed, some secreted PLA2s express such low catalytic activity with natural phospholipids that a noncatalytic role cannot be ruled out [1]. In this review we focus on the surface properties of the human IIA PLA2 and how these properties are linked to function. The structural and physiological properties of the family of secreted PLA2s including roles in cardiovascular disease, inflammation and many types of cancer have been comprehensively reviewed [1,9–13]. Structure and catalytic properties of gIIA PLA2 The crystal structure of the human IIA PLA2 [5,6] established that this enzyme had the same basic structural elements as the secreted venom and pancreatic PLA2s. In particular, there was an active site His-Asp dyad in which a water molecule replaces the serine found in the classical protease/lipase catalytic triad mechanism [1]. Because of the crucial role of the active site histidine, site directed mutagenesis has produced proteins, H48Q and H48N, with greatly reduced catalytic activity, whereas the crystal structure of H48Q confirmed the structural integrity of this mutant [14]. The H48N mutant has < 0. 5% of the catalytic activity of the wild type enzyme [14] and has been used recently to investigate the non-catalytic roles of the human group IIA enzyme [15]. The human group IIA protein is highly basic, with a calculated pI of 9.4 and a net charge of +19 (+27; 8), assuming that the cationic residues are His, Arg and Lys and the anionic groups are Asp and Glu [16]. This positive charge is globally distributed over the protein surface [16] (Figure 3), a possibly unique feature of this protein. This feature results in the ability of the protein to form supramolecular aggregates in the presence of anionic phospholipid vesicles [17,18], a characteristic not shared by other secreted PLA2s. Another structural characteristic is that
Figure 2. Schematic illustration of interfacial catalysis. (a) The free enzyme (E) binds the phospholipid aggregate to produce the E* form. Substrate binding at the active site can now produce E*S, which, following catalysis, will generate the E*P complex. Following product release, the enzyme remains bound to the interface and continues through multiple catalytic cycles (scooting mode catalysis). (b) The overall catalytic reaction for one catalytic cycle is summarized. Adapted from [67].
the IIA enzyme lacks tryptophan. Tryptophan residues on the interfacial binding surface of peripheral membrane proteins are particularly effective in promoting interfacial binding to neutral phospholipid surfaces [19–21]. As a result, the human group IIA enzyme shows low affinity for zwitterionic interfaces [18], and in the absence of interfacial binding membrane hydrolysis is not possible (Figure 2). Insertion of specific tryptophan residues into the i-face of the IIA enzyme enhances interfacial binding to zwitterionic phospholipid vesicles and phospholipid hydrolysis [21]. The outer leaflet of mammalian cell membranes is normally zwitterionic. This zwitterionic property makes these cells refractory to hydrolysis when exposed to the IIA enzyme during infection and trauma when serum levels are substantially elevated. Particularly noteworthy is the exceptionally high concentration in human tears at 30 mg/ml (2 mM) [22]; yet human corneal epithelial cells are highly resistant to hydrolysis by added human group IIA enzyme [18]. By contrast, mammalian cell membranes would be rapidly hydrolyzed after the addition of a low concentration of secreted PLA2s derived from snake venoms [21], as these enzymes can bind productively to the zwitterionic surface of the outer monolayer of the plasma membrane. 29
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Figure 3. The crystal structure of human IIA PLA2 showing the surface distribution of arginine and lysine (cationic) residues. The crystal structure is taken from [5]. The cationic residues are shown in blue, and the active site His-48 is shown in green. The interfacial binding surface is shown toward the viewer (a), and away from the viewer (b). The global distribution of cationic residues is readily seen.
The presence of numerous arginine and lysine residues on the surface of the protein is consistent with the ability of this secreted PLA2 to bind to heparin and heparan sulphate proteoglycans (HSPGs). This electrostatic binding, however, is of only moderate affinity [23] and atypical compared with those proteins that have a basic consensus sequence and are known to bind heparin and HSPGs with high affinity. In fact, charge reversal mutagenesis of individual arginine or lysine residues failed to identify localized regions of positive charge on the protein surface that were specifically involved in binding to heparin and heparan sulphate [23]. Moreover, such binding did not correlate with the (minimal) ability of the enzyme to hydrolyze the plasma membranes of cells in culture, whereas two mutant cells lines, one lacking heparan sulphates and one lacking glycosoaminoglycans were hydrolysed at a similar rate to normal cells [23]. Overall, despite many interesting studies indicating a possible involvement of HSPGs in the physiological role of the extra-cellular IIA enzyme [24,25], a direct physiological role of such interactions involving membrane hydrolysis remains to be established. A role of HSPGs in intracellular trafficking of the enzyme has been proposed [26], but other studies do not support such an involvement [27]. The distribution of human gIIA PLA2 Two factors that provide the basis for the physiological function of a protein are protein structure and tissue distribution. The expression and distribution of the IIA enzyme is particularly revealing. The serum concentration of the enzyme increases up to 1000-fold after trauma and infection, yet the cellular origins of this extracellular enzyme remain unclear. Many cells are able to secrete the IIA enzyme and include mesangial cells, smooth muscle cells, endothelial cells, mast cells, neutrophils, macrophages and hepatic cells, as well as platelets [28,29]. The recognition of the IIA enzyme as an acute phase protein [7] that is induced in HepG2 cells over several days by IL-1, IL-6 and TNF indicated that the hepatocyte could be a likely candidate as a source of the elevated enzyme levels in serum seen under inflammatory conditions. Thus, liver biopsy of patients with acute pan30
creatitis and elevated serum IIA PLA2 levels revealed enhanced levels of gene expression in hepatocytes compared with control studies [30]. In one patient with highly elevated serum IIA levels the need for a liver transplant resulted in a dramatic fall in IIA levels on the first day post transplantation [31]. Physiological functions of human gIIA PLA2 linked to the global cationic nature of the protein The structure of human group IIA PLA2 is unusual because of the highly cationic nature of the protein, having a global distribution of positively-charged arginine and lysine residues over its surface (Figure 3). This structure provides the basis for two physiological roles, a well established antibacterial role and a proposed new role in the clearance of cell-derived anionic phospholipid microparticles/microvesicles. Antibacterial activity The locations of the enzyme are consistent with its antibacterial role within innate immunity. In addition to the high serum concentrations observed in septicaemia, IIA PLA2 is highly expressed in Paneth cells of the small intestine [32–35], in lacrimal cells and human tears [22,36] and in prostate cells and human seminal plasma [37]. These locations reflect possible sites of bacterial invasion into externally-exposed body cavities. The very high concentration of IIA PLA2 seen in human tears, where it is the primary antibacterial agent against Gram-positive bacteria, is significant [22]. The antibacterial properties of secreted PLA2s, particularly against Gram-positive bacteria, has been reviewed previously [38,39], so the discussion here will focus primarily on how the structure of the human group IIA enzyme provides the basis for this antibacterial activity, and also how target organisms can generate resistance to this enzyme by neutralization of the anionic charge of the bacterial cell wall and cell membrane. Ultimately, the antibacterial properties depend on the ability of the enzyme to target and hydrolyse phospholipids in the anionic cell membrane. In the case of Staphylococcus aureus four basic steps have been identified [40]: binding
Review of the enzyme to the anionic bacterial cell surface; penetration of the enzyme through the anionic peptidoglycan layer of the cell wall; hydrolysis of anionic cell membrane phospholipid; and activation of bacterial autolysins (cell-walldegrading enzymes). Historically, an important observation was the ability of bactericidal/permeability-increasing protein (BPI) to enhance the membrane phospholipid hydrolysing properties of certain secreted PLA2s, including rabbit IIA PLA2 against the Gram-negative bacterium Escherichia coli [41]. However, this enhanced hydrolytic activity in the presence of BPI was only seen with highly cationic enzymes including some venom enzymes [42]. It was subsequently shown that human IIA enzyme was able to hydrolyse E. coli phospholipid in the presence of BPI [43]. The role of BPI might be to permeabilize the lipopolysaccharide coat (outer membrane) of the Gram-negative bacterium [44,45] allowing the PLA2 to subsequently penetrate the bacterial cell wall and hydrolyse the anionic bacterial cell membrane. The importance of the highly cationic nature of the human IIA enzyme in allowing bacterial cell wall penetration was highlighted by studies with particular Grampositive bacteria where the cell wall could be selectively permeabilized by enzyme degradation. In these studies a fluorescence displacement assay [46] was used where it was possible to monitor PLA2-catalysed cell membrane hydrolysis using bacterial cell suspensions as substrates [47]. Suspensions of Micrococcus luteus were readily hydrolysed by the human group IIA enzyme, but not by other 15kDa secreted PLA2s including the porcine pancreatic enzyme and Naja naja snake venom enzyme. This selectivity stems from the ability of the highly cationic human IIA enzyme to penetrate the anionic bacterial cell wall. Removal of this permeability barrier, as a result of hydrolysis of the cell wall with lysozyme, allowed all three PLA2s to display similar activity. Cell membrane degradation by the IIA enzyme resulted in at least 50% phospholipid degradation as judged by electrospray mass spectrometry, and identified phosphatidylglycerol as the major phospholipid substrate [47]. However, the bactericidal activity of the human enzyme under these conditions was low, highlighting the capacity of the organism to recover from significant phospholipid hydrolysis. In the case of S. aureus this survival is due to enhanced phospholipid synthesis by both de novo and salvage pathways [48]. It would be anticipated that in innate immunity, bacterial killing will be achieved by the synergistic action of an arsenal of functionally distinct antibacterial peptides and proteins. The role of the surface positive charge in facilitating access to and hydrolysis of the bacterial cell membranes has been investigated using a strategy of charge reversal mutagenesis in which surface arginine or lysine residues of the human IIA enzyme were substituted for glutamate or aspartate residues by site directed mutagenesis. Studies using M. luteus as a model substrate showed that the reduction in membrane hydrolysis and bacterial killing correlated with loss of positive charge on the enzyme surface [49]. Mutagenesis did not affect the catalytic activity of the enzyme, as full enzyme activity was displayed using bacterial cell suspensions in which the cell wall had been
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permeabilized by lysozyme treatment [49]. The same overall cell membrane and antibacterial activity of the human IIA enzyme and charge reversal mutants was observed using suspensions of S. aureus [49,50] and Listeria innocua [49]. The importance of the negative charge of the peptidoglycan cell wall and cell membrane in the antibacterial action of the IIA enzyme has been highlighted in studies involving mutants of S. aureus [51]. The negative charge of the bacterial cell wall is due to the presence of structural polyanions, in particular, wall teichoic acid and lipotechoic acid that contain repeating units of alditol (ribitol or glycerol) phosphate [52]. S. aureus is able to reduce this negative charge by esterification of the alditol with Dalanine, thus leaving the alanine residue with a net positive charge. This modification reduces the antibacterial effectiveness of the IIA enzyme. Mutants that are unable to carry out the alanylation are 30-100-fold more sensitive to killing by IIA PLA2 [50]. In another bacterial process the addition of L-lysine to the headgroup of both phosphatidylglycerol (PG) and cardiolipin results in the addition of two amino groups into the anionic cell membrane phospholipid. In this case, however, membrane hydrolysis is reduced by less than 3-fold compared with mutants that lack the ability to form lysylphosphatidylglycerol [50]. This observation could reflect the fact that less than 50% of the PG is modified with lysine [53], still allowing binding of the IIA enzyme to the cell membrane, and hence phospholipid hydrolysis. Additional support for the role of the IIA PLA2 in bacterial killing comes from studies with transgenic mice over-expressing the human IIA enzyme. These mice are resistant to various bacterial infections [54–56]. Moreover, phospholipid hydrolysis of S. aureus membranes by added IIA enzyme is enhanced in the presence of human neutrophils (which do not contain the IIA enzyme), and leads to synergistic bactericidal activity where a role for the NADPH oxidase system of the neutrophil has been suggested [57]. A model to explain the ability of the IIA enzyme to help kill Gram-positive bacteria is shown in Figure 4. The model highlights the importance of the global positive charge on the surface of the IIA enzyme in allowing penetration of the anionic bacterial cell wall, and then high-affinity binding to the anionic cell membrane, thus allowing membrane phospholipid hydrolysis to proceed. High concentrations of the enzyme reported during infection are unable to hydrolyse the zwitterionic host cell membrane, thus protecting host cells and tissues from hydrolysis by this extracellular enzyme. Cellular uptake of gIIA PLA2 and the removal of cell debris The effect of the exogenous IIA PLA2 on host cell function has been the subject of numerous studies, but remains unclear and might be the result of cell-specific responses [1,13]. A particular focus has been the involvement of this enzyme in arachidonic release and the subsequent production of signalling molecules, such as the prostaglandins as part of the inflammatory response. This area of investigation has been discussed in detail elsewhere [1] and 31
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Figure 4. A model of bacterial cell wall penetration and phospholipid hydrolysis by human group IIA secreted PLA2 (gIIA sPLA2). The global distribution of the positive charge on this PLA2 has been proposed to allow the enzyme to move through the negatively charged cell wall, mediated by the continuous making and breaking of electrostatic bonds. For simplicity, the bacterial cell membrane is shown as containing only negatively charged phospholipids (shown in blue), but will contain some zwitterionic phospholipids, such as phosphatidylethanolamine. Adapted from [38].
remains controversial. A fundamental problem in defining a role for the IIA PLA2 in vivo is the almost zero catalytic activity of this particular secreted PLA2 within the extracellular host environment where available phospholipid surfaces (membranes and lipoproteins) are normally zwitterionic. Thus, functions linked to phospholipid hydrolysis with the release of fatty acids (arachidonic acid) and lysophospholipids by this enzyme are difficult to substantiate, although the ability of the enzyme to participate in intracellular phospholipid hydrolysis has been demonstrated [27]. The possible binding and uptake of recombinant IIA enzyme into cells has been investigated recently, using a fluorescently-labelled protein that allowed analysis by FACS and fluorescent microscopy [15]. It was possible to bioengineer fluorescent IIA protein molecules that were still fully functional by judicious attachment of chemical fluorophores to specific parts of the protein surface. An extensive study using human monocyte-derived THP-1 cells revealed uptake of the enzyme, but only after the cells had been differentiated by phorbol 12-myristate 13acetate (PMA) to produce a macrophage-like cell phenotype. This treatment correlated with an increase in the expression of cell surface HSPGs [58] and IIA PLA2 uptake was shown to be dependent on interaction of the cationic IIA PLA2 with these anionic polymers. Uptake was energydependent and led to nuclear localization of the enzyme, a characteristic observed with many cationic proteins and peptides [59]. The observation that IIA PLA2 uptake into THP-1 cells did not require the enzyme to be catalytically active was of particular interest. This conclusion was based on the crucial observation that all observed trafficking phenomena for the fluorescent IIA PLA2 were replicated with a fluor32
escent catalytically-inactive mutant, H48N, in which the catalytic histidine is replaced by glutamine. Moreover, protein uptake was accompanied by significant cell swelling due to fluid uptake, a typical characteristic of macropinocytosis. As this IIA PLA2 is uniquely able to form supra-molecular aggregates with anionic phospholipid vesicles [15,17,18] a role for the clearance of such aggregates (cell debris and anionic microparticles) was studied. Using fluorescently-labelled anionic phospholipid vesicles, it was possible to demonstrate that the cellular uptake of these vesicles into PMA-differentiated (but not non-differentiated) THP-1 cells was considerably enhanced (over 10fold) by the presence of the IIA PLA2, a phenomenon that also did not require the enzyme to be catalytically active [15]. Overall, a hitherto unrealised function of the IIA enzyme was proposed as part of the inflammatory response, namely the removal of cell debris and microparticles (Figure 5) resulting from tissue trauma in a process that did not require a catalytic role for the enzyme. This proposal, of a novel role for the IIA PLA2, allows some interesting correlations. For example, the synovial fluid of patients with rheumatoid arthritis contains high concentrations of microparticles and it is in this fluid that the IIA enzyme was first discovered at high concentrations [3]. Such microparticles have been proposed to be inflammatory [60], and hence the presence of the II PLA2 could be to facilitate removal of such particles and help resolve inflammation. In another remarkable correlation, higher expression of the human IIA enzyme has been uniquely linked to reduced metastasis and mortality in patients with gastric cancer [61], and high levels of serum microparticles in such patients is associated with increased morbidity and mortality [62]. The role of microparticles in angiogenesis, a crucial process in cardiovascular disease
Review
Figure 5. A model for the IIA PLA2 uptake into cells, and its role in the uptake of anionic particles. In this model, the globally cationic IIA PLA2 interacts with anionic microparticles to produce coated particles or larger aggregates. Following uptake by macropinocytosis, acidification and possibly other endosomal events should allow IIA PLA2 release and nuclear accumulation. The nature of the aggregate will depend on the size and charge density of the particles and this might also determine the endocytic pathway. Other endocytic routes have been considered [15].
and cancer metastasis, has been reviewed [63]. We would argue the IIA PLA2 concentration could affect the turnover of microparticles in extracellular fluids. Microparticle hydrolysis by IIA PLA2 has previously been demonstrated to produce the lipid mediator lysophosphatidic acid [64]. Concluding remarks The IIA PLA2 enzyme has remarkable structural characteristics and is abundantly expressed in certain locations and under certain pathological conditions. This extracellular enzyme is unable to hydrolyze the plasma membrane of host cells as a result of the very low affinity of the enzyme for the essentially zwitterionic surface of the outer monolayer of the plasma membrane. This characteristic means that such cells are not adversely affected (lysed) by the enzyme, and is a very different situation than that seen with other secreted PLA2s. For example, when certain snake venom PLA2s are added to mammalian cells, they can bind the membrane surface with high affinity and execute phospholipid hydrolysis and cell lysis. By contrast, the human IIA enzyme can target and hydrolyse membranes rich in anionic phospholipids, such as bacterial cell
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membranes. Moreover, the globally-cationic structure of the enzyme allows it to penetrate the anionic cell wall of Gram-positive bacteria, allowing the enzyme to access the anionic bacterial cell membrane where subsequent hydrolysis facilitates bacterial killing. This same global cationic nature of the IIA enzyme allows it to form aggregates with anionic phospholipid particles (cell debris). The resulting cationic supra-molecular aggregates can be taken up by activated macrophages, such as human THP-cells, in a process involving HSPGs and macropinocytosis, allowing debris removal and subsequent destruction. Phospholipid hydrolysis is not required for this process. Overall, the process would be part of the acute phase response to facilitate the removal of damaged cells and cell debris. Such a role has been proposed previously for the IIA PLA2 in conjunction with C-reactive protein, but in a process that involved phospholipid hydrolysis [65]. The accumulation and/or targeting of IIA PLA2 to the nucleus might suggest modulatory effects on gene regulation, and under appropriate conditions increased expression of COX2 has been observed [15]. The physiological process of the uptake and nuclear targeting of cationic supra-molecular aggregates involving the IIA protein may be utilizing the pathways of non-viral gene transfection that use this process for entry into cells of DNA coated with cationic reagents. The probable complexity of the physiological action of this enzyme and our lack of complete understanding of many features of its effects on cells has been critically reviewed [1]. In addition, the recent description of the binding of the IIA PLA2 to integrins and the proliferation of monocytic cells is another example of a possible non-catalytic and pro-inflammatory role for this protein [66]. In summary, this review highlights the potential role of the IIA PLA2 in resolving inflammatory situations involving both catalytic (antibacterial) and non-catalytic (debris removal) functions. In this context, the IIA PLA2 is not seen as pro-inflammatory as originally assumed when the enzyme was first isolated, but could have primarily an antiinflammatory (resolving) role in the inflammatory response. There is still much to be learned about the role of this enzyme in disease processes, and how its role might depend on the target cell and the state of activation of that cell (Box 1).
Box 1. Outstanding Questions What effects does the accumulation of the human IIA PLA2 in the nucleus have on gene expression? Is human IIA PLA2 taken up into activated cells in the absence of cell debris? What is the effect of over-expression/lack of expression of IIA PLA2 on microparticle clearance? Is there extracellular microparticle hydrolysis by IIA PLA2, and how does this affect microparticle clearance? Is there a specific receptor in humans that can bind human IIA PLA2 [1]? Overall, does the IIA PLA2 have diverse interactions and effects (pro- and anti-inflammatory), depending on the type of target cell and the activation status of the cell?
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Trends in Biochemical Sciences Vol.35 No.1 25 Boilard, E. et al. (2003) Interaction of low molecular weight group IIA phospholipase A(2) with apoptotic human T cells: role of heparan sulfate proteoglycans. FASEB J. 17, 1068–1080 26 Murakami, M. et al. (2001) Distinct arachidonate-releasing functions of mammalian secreted phospholipase A(2)s in human embryonic kidney 293 and rat mastocytoma RBL-2H3 cells through heparan sulfate shuttling and external plasma membrane mechanisms. J. Biol. Chem. 276, 10083–10096 27 Mounier, C.M. et al. (2004) Arachidonic acid release from mammalian cells transfected with human groups IIA and X secreted phospholipase A(2) occurs predominantly during the secretory process and with the involvement of cytosolic phospholipase A(2)-alpha. J. Biol. Chem. 279, 25024–25038 28 Jaross, W. et al. (2002) Biological effects of secretory phospholipase A(2) group IIA on lipoproteins and in atherogenesis. Eur. J. Clin. Inves. 32, 383–393 29 Niessen, H.W.M. et al. (2003) Type II secretory phospholipase A2 in cardiovascular disease: a mediator in atherosclerosis and ischemic damage to cardiomyocytes? Cardiovasc. Res. 60, 68–77 30 Talvinen, K.A. et al. (2001) Expression of group II phospholipase A2 in the liver in acute pancreatitis. Scand. J. Gastroenterol. 36, 1217–1221 31 Nevalainen, T.J. et al. (1996) Origin of circulating group II phospholipase A2 in hepatocytes in a patient with epitheloid hemangioendothelioma of the liver. Lab. Invest. 74, 585–591 32 Harwig, S.S.L. et al. (1995) Bactericidal properties of murine intestinal phospholipase A(2). J. Clin. Invest. 95, 603–610 33 Nevalainen, T.J. et al. (1995) Expression of group-ll phospholipase-A2 in the human gastrointestinal-tract. Lab. Invest. 72, 201–208 34 Qu, X.D. et al. (1996) Secretion of type II phospholipase A(2) and cryptdin by rat small intestinal paneth cells. Infect. Immun. 64, 5161–5165 35 Yoshikawa, T. et al. (2001) Cellular localization of group IIA phospholipase A(2) in rats. J. Histochem. Cytochem. 49, 777–782 36 Nevalainen, T.J. et al. (1994) Secretion of group 2 phospholipase A-2 by lacrimal glands. Invest. Ophthalmol. Vis. Sci. 35, 417–421 37 Nevalainen, T.J. et al. (1993) Synovial-type (group -II) phospholipaseA2 human seminal plasma. Andrologia 25, 355–358 38 Buckland, A.G. and Wilton, D.C. (2000) The antibacterial properties of secreted phospholipases A(2). Biochim. Biophys. Acta 1488, 71–82 39 Nevalainen, T.J. et al. (2008) Antibacterial actions of secreted phospholipases A(2). Review. Biochim. Biophys. Acta 1781, 1–9 40 Foreman-Wykert, A.K. et al. (1999) Cell-wall determinants of the bactericidal action of group IIA phospholipase A(2) against Grampositive bacteria. J. Clin. Inves. 103, 715–721 41 Weiss, J. et al. (1979) Determinant of the action of phospholipases A on the envelope phospholipids of Echerichia coli. J. Biol. Chem. 254, 11010–11014 42 Forst, S. et al. (1982) The role of phospholipase-A2 lysines in phospholipolysis of Escherichia coli killed by a membrane active neutrophil protein. J. Biol. Chem. 257, 4055–4057 43 Weiss, J. et al. (1994) Structural determinants of the action against Escherichia coli of a human inflammatory fluid phospholipase A(2) in concert with polymorphonuclear leukocytes. J. Biol. Chem. 269, 26331–26337 44 Wiese, A. et al. (1997) Mechanisms of action of the bactericidal/ permeability-increasing protein BPI on endotoxin and phospholipid monolayers and aggregates. Biochemistry 36, 10301–10310 45 Wiese, A. et al. (1997) Mechanisms of action of bactericidal/ permeability-increasing protein BPI on reconstituted outer membranes of gram-negative bacteria. Biochemistry 36, 10311– 10319 46 Wilton, D.C. (1990) A continuous fluorescence displacement assay for the measurement of phospholipase-A2 and other lipases that release long-chain fatty acids. Biochem. J. 266, 435–439 47 Buckland, A.G. et al. (2000) Bacterial cell membrane hydrolysis by secreted phospholipases A2: a major physiological role of human group IIa sPLA2 involving both bacterial cell wall penetration and interfacial catalysis. Biochim. Biophys. Acta 1484, 195–206 48 Foreman-Wykert, A.K. et al. (2000) Phospholipid synthesis by Staphylococcus aureus during (sub)lethal attack by mammalian 14kilodalton group IIA phospholipase A2. Infect. Immun. 68, 1259–1264 49 Beers, S.A. et al. (2002) The antibacterial properties of secreted phospholipases A(2) - A major physiological role for the group IIA
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enzyme that depends on the very high pI of the enzyme to allow penetration of the bacterial cell wall. J. Biol. Chem. 277, 1788–1793 Koprivnjak, T. et al. (2002) Role of charge properties of bacterial envelope in bactericidal action of human group IIA phospholipase A(2) against Staphylococcus aureus. J. Biol. Chem. 277, 47636–47644 Peschel, A. (2002) How do bacteria resist human antimicrobial peptides? Trends Microbiol. 10, 179–186 Neuhaus, F.C. and Baddiley, J. (2003) A continuum of anionic charge: structures and functions of D-alanyl-teichoic acids in gram-positive bacteria. Microbiol. Mol. Biol. Rev. 67, 686–723 Peschel, A. et al. (2001) Staphylococcus aureus resistance to human defensins and evasion of neutrophil killing via the novel virulence factor MprF is based on modification of membrane lipids with L-lysine. J. Exp. Med. 193, 1067–1076 Laine, V.O. et al. (1999) Protection by group II phospholipase A(2) against Staphylococcus aureus. J. Immunol. 162, 7402–7408 Laine, V.J.O. et al. (2000) Resistance of transgenic mice expressing human group II phospholipase A2 to Escherichia coli infection. Infect. Immun. 68, 87–92 Piris-Gimenez, A. et al. (2005) In vivo protective role of human group IIA phospholipase A(2) against experimental anthrax. J. Immunol. 175, 6786–6791 Femling, J.K. et al. (2005) Synergy between extracellular group IIA phospholipase A(2) and phagocyte NADPH oxidase in digestion of phospholipids of Staphylococcus aureus ingested by human neutrophils. J. Immunol. 175, 4653–4661 Edwards, I.J. et al. (1995) Differentiated macrophages synthesize a hepapan-sulphate proteoglycan and an oversulfated chondroitin sulfate proteoglycan that bind lipoprotein-lipase. Arterioscler. Thromb. Vasc. Biol. 15, 400–409
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59 Melikov, K. and Chernomordik, L.V. (2005) Arginine-rich cell penetrating peptides: From endosomal uptake to nuclear delivery. Cell. Mol. Life Sci. 62, 2739–2749 60 Distler, J.H.W. et al. (2005) The induction of matrix metalloproteinase and cytokine expression in synovial fibroblasts stimulated with immune cell microparticles. Proc. Nat. Acad. Sci. 102, 2892–2897 61 Leung, S.Y. et al. (2002) Phospholipase A2 group IIA expression in gastric adenocarcinoma is associated with prolonged survival and less frequent metastasis. Proc. Nat. Acad. Sci. U. S. A. 99, 16203– 16208 62 Kim, H.K. et al. (2003) Elevated levels of circulating platelet microparticles, VEGF, IL-6 and RANTES in patients with gastric cancer: possible role of a metastasis predictor. Eur. J. Cancer 39, 184–191 63 Mostefai, H.A. et al. (2008) Plasma membrane microparticles in angiogenesis: Role in ischemic diseases and in cancer. Physiol. Res. 57, 311–320 64 Fourcade, O. et al. (1995) Secretory phospholipase a(2) generates the novel lipid mediator lysophosphatidic acid in membrane microvesicles shed from activated cells. Cell 80, 919–927 65 Hack, C.E. et al. (1997) A role for secretory phospholipase A(2) and Creactive protein in the removal of injured cells. Immunology Today 18, 111–115 66 Saegusa, J. et al. (2008) Pro-inflammatory secretory phospholipase a2 type IIA binds to integrins alpha v beta 3 and alpha 4 beta 1 and induces proliferation of monocytic cells in an integrin-dependent manner. J. Biol. Chem. 283, 26107–26115 67 Bahnson, B.J. (2005) Structure, function and interfacial allosterism in phospholipase A2: insight from the anion-assisted dimer. Arch. Biochem. Biophys. 433, 96–106
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Catalytic and non-catalytic functions of human IIA phospholipase A2 Charles N. Birts, C. Howard Barton and David C. Wilton School of Biological Sciences, University of Southampton, Boldrewood Campus, Southampton, SO16 7PX, United Kingdom
Group IIA phospholipase A2 (PLA2) is a low-molecularmass secreted PLA2 enzyme that has been identified as an acute phase protein with a role in the inflammatory response to infection and trauma. The protein is possibly unique in being highly cationic and having a global distribution of surface arginine and lysine residues. This structure supports two functions of the protein. (1) An anti-bacterial role where the enzyme is targeted to the anionic cell membrane of Gram-positive bacteria and phospholipid hydrolysis assists in bacterial killing. (2) A proposed non-catalytic role in which the protein forms supramolecular aggregates with anionic phospholipid vesicles or debris. These aggregates are then internalized via interactions with cell surface heparin sulphate proteoglycans and macropinocytosis for disposal by macrophages. Introduction to the history, structure and function of human group IIA PLA2 Group IIA phospholipase A2 (gIIA PLA2) (Figure 1) is one of a family of low-molecular-mass, secreted PLA2 enzymes [1,2] originally found in snake venoms, and as a digestive enzyme in the pancreas. This enzyme was the first nonpancreatic mammalian PLA2 to be identified, being present at high concentrations in platelets and in the synovial fluid of patients with rheumatoid arthritis [3]. It was originally implicated as the enzyme responsible for arachidonic release during the inflammatory response of the body, and thus a potential target in the development of new anti-inflammatory drugs. However, although secreted PLA2s have been implicated [1], this arachidonic acid releasing role is now primarily ascribed to the group IVA cytosolic PLA2 [4]. The potentially central role of the IIA enzyme in inflammation at the time of its discovery provided the necessary impetus for producing crystal structures for the human enzyme [5,6]. At an early stage, the enzyme was characterized as an acute phase protein under the transcriptional control of pro-inflammatory cytokine signalling [7], with serum levels dramatically elevated as a result of tissue trauma and infection. Such a large increase (up to 1000-fold, with concentrations approaching 1 mM) in serum protein concentration suggests functions involving ‘‘bulk’’ interactions with other intracellular structures and not a ‘‘more subtle’’ role in generating signalling intermediates. Along with other secreted PLA2s, the enzyme demonstrates minimal phospholipid substrate specificity at the active site, both in terms of acyl chain and headgroup preference; the enzyme Corresponding author: Wilton, D.C. ([email protected]).
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has no preference for arachidonic acid. Naturally occurring phospholipid will always present as aggregates such as the membrane bilayer under physiological condition, and only after binding to the aggregate (interfacial binding) will the enzyme be able to access a phospholipid substrate molecule. Thus, a lack of interfacial binding will mean that there is no enzyme activity, and an interfacial binding step (E ! E*) is a requirement for overall catalysis, according to Figure 2. This means that apparent phospholipid specificity can be achieved as a result of selective interfacial binding. The interfacial binding step will be most affected by the surface structure of the secreted PLA2, and in particular it is the interfacial binding surface of the enzyme (i-face) that directly interacts with the phospholipid bilayer interface [1,8]. Because the overall core structure of most secreted PLA2s are similar, without major differences in substrate specificity, it could be argued that it is differences in the specific surface properties of individual enzymes rather Glossary Acute phase proteins: these proteins are released primarily from the liver after trauma and infection, and are involved in helping to repair tissue damage and fighting infection. They include blood clotting proteins, complement factors, Creactive protein and serum amyloid P. Angiogenesis: the process of new blood vessel formation. In addition to a central involvement in mammalian embryo development, this process plays a crucial role in tumour growth and metastasis. Gram-positive and Gram-negative bacteria: Gram-negative bacteria possess a membrane, and a lipopolysaccharide coat external to the cell wall. Grampositive bacteria lack a lipopolysaccharide coat and are limited by the cell wall outside of the cell membrane. Interfacial binding and catalysis: because the critical micelle concentration (CMC) of natural phospholipids is very low (<1010 M), it is necessary for the enzyme to first bind a phospholipid aggregate, such as the bilayer, to access its phospholipid substrate. Thus, interfacial binding is a compulsory additional step in overall phospholipid hydrolysis. Macropinocytosis: a form of bulk uptake of fluid and solid cargo into cytoplasmic vacuoles, called macropinosomes. Microparticles and microvesicles: small phospholipid particles or blebs released from cells or platelets during activation or cell death that are characteristically anionic, with phosphatidylserine exposed on the surface of the particles. Phorbol 12-myristate 13-acetate (PMA): a cell-permeable chemical analogue of diacylglycerol (DAG). DAG is released from membrane phospholipid by the action of PLC and is an activator of protein kinase C. Phospholipase A2s: a family of enzymes that are able to hydrolyze the sn2 position of a glycerophospholipid to release fatty acid and the corresponding lysophospholipid. Secreted phospholipase A2s: a group of 14 kDa enzymes that were originally identified in snake venoms and the pancreas. A number of other mammalian non-pancreatic secreted PLA2s have been identified, including the group IIC, IID, IIE, IIF, III, V, X and XIIA enzymes [2], which brings the total number of mouse gene products to 10, but only 9 for humans where IIC exists as a pseudogene [1]. In addition, two gene products are structurally related to sPLA2s, but lack enzyme activity [1]. Like the other sPLA2s, the human IIA enzyme has an N-terminal signal sequence allowing secretion from the cell into the extracellular environment that must be its primary site of action.
0968-0004/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2009.08.003 Available online 8 October 2009
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Figure 1. Sites of phospholipid hydrolysis by phospholipases. Arrows indicate the position of phospholipase-catalysed hydrolysis of a glycerol-based phospholipid. R1 and R2 represent the fatty acyl chains and X represents the alcohol head group. PLA1, PLA2, PLC and PLD refer to phospholipase A1, A2, C and D respectively. The carbon atoms (1, 2 or 3) of the glycerol backbone of the phospholipid are indicated in terms of the stereochemical numbering (sn).
than catalytic properties that reflect physiological function. Indeed, some secreted PLA2s express such low catalytic activity with natural phospholipids that a noncatalytic role cannot be ruled out [1]. In this review we focus on the surface properties of the human IIA PLA2 and how these properties are linked to function. The structural and physiological properties of the family of secreted PLA2s including roles in cardiovascular disease, inflammation and many types of cancer have been comprehensively reviewed [1,9–13]. Structure and catalytic properties of gIIA PLA2 The crystal structure of the human IIA PLA2 [5,6] established that this enzyme had the same basic structural elements as the secreted venom and pancreatic PLA2s. In particular, there was an active site His-Asp dyad in which a water molecule replaces the serine found in the classical protease/lipase catalytic triad mechanism [1]. Because of the crucial role of the active site histidine, site directed mutagenesis has produced proteins, H48Q and H48N, with greatly reduced catalytic activity, whereas the crystal structure of H48Q confirmed the structural integrity of this mutant [14]. The H48N mutant has < 0. 5% of the catalytic activity of the wild type enzyme [14] and has been used recently to investigate the non-catalytic roles of the human group IIA enzyme [15]. The human group IIA protein is highly basic, with a calculated pI of 9.4 and a net charge of +19 (+27; 8), assuming that the cationic residues are His, Arg and Lys and the anionic groups are Asp and Glu [16]. This positive charge is globally distributed over the protein surface [16] (Figure 3), a possibly unique feature of this protein. This feature results in the ability of the protein to form supramolecular aggregates in the presence of anionic phospholipid vesicles [17,18], a characteristic not shared by other secreted PLA2s. Another structural characteristic is that
Figure 2. Schematic illustration of interfacial catalysis. (a) The free enzyme (E) binds the phospholipid aggregate to produce the E* form. Substrate binding at the active site can now produce E*S, which, following catalysis, will generate the E*P complex. Following product release, the enzyme remains bound to the interface and continues through multiple catalytic cycles (scooting mode catalysis). (b) The overall catalytic reaction for one catalytic cycle is summarized. Adapted from [67].
the IIA enzyme lacks tryptophan. Tryptophan residues on the interfacial binding surface of peripheral membrane proteins are particularly effective in promoting interfacial binding to neutral phospholipid surfaces [19–21]. As a result, the human group IIA enzyme shows low affinity for zwitterionic interfaces [18], and in the absence of interfacial binding membrane hydrolysis is not possible (Figure 2). Insertion of specific tryptophan residues into the i-face of the IIA enzyme enhances interfacial binding to zwitterionic phospholipid vesicles and phospholipid hydrolysis [21]. The outer leaflet of mammalian cell membranes is normally zwitterionic. This zwitterionic property makes these cells refractory to hydrolysis when exposed to the IIA enzyme during infection and trauma when serum levels are substantially elevated. Particularly noteworthy is the exceptionally high concentration in human tears at 30 mg/ml (2 mM) [22]; yet human corneal epithelial cells are highly resistant to hydrolysis by added human group IIA enzyme [18]. By contrast, mammalian cell membranes would be rapidly hydrolyzed after the addition of a low concentration of secreted PLA2s derived from snake venoms [21], as these enzymes can bind productively to the zwitterionic surface of the outer monolayer of the plasma membrane. 29
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Figure 3. The crystal structure of human IIA PLA2 showing the surface distribution of arginine and lysine (cationic) residues. The crystal structure is taken from [5]. The cationic residues are shown in blue, and the active site His-48 is shown in green. The interfacial binding surface is shown toward the viewer (a), and away from the viewer (b). The global distribution of cationic residues is readily seen.
The presence of numerous arginine and lysine residues on the surface of the protein is consistent with the ability of this secreted PLA2 to bind to heparin and heparan sulphate proteoglycans (HSPGs). This electrostatic binding, however, is of only moderate affinity [23] and atypical compared with those proteins that have a basic consensus sequence and are known to bind heparin and HSPGs with high affinity. In fact, charge reversal mutagenesis of individual arginine or lysine residues failed to identify localized regions of positive charge on the protein surface that were specifically involved in binding to heparin and heparan sulphate [23]. Moreover, such binding did not correlate with the (minimal) ability of the enzyme to hydrolyze the plasma membranes of cells in culture, whereas two mutant cells lines, one lacking heparan sulphates and one lacking glycosoaminoglycans were hydrolysed at a similar rate to normal cells [23]. Overall, despite many interesting studies indicating a possible involvement of HSPGs in the physiological role of the extra-cellular IIA enzyme [24,25], a direct physiological role of such interactions involving membrane hydrolysis remains to be established. A role of HSPGs in intracellular trafficking of the enzyme has been proposed [26], but other studies do not support such an involvement [27]. The distribution of human gIIA PLA2 Two factors that provide the basis for the physiological function of a protein are protein structure and tissue distribution. The expression and distribution of the IIA enzyme is particularly revealing. The serum concentration of the enzyme increases up to 1000-fold after trauma and infection, yet the cellular origins of this extracellular enzyme remain unclear. Many cells are able to secrete the IIA enzyme and include mesangial cells, smooth muscle cells, endothelial cells, mast cells, neutrophils, macrophages and hepatic cells, as well as platelets [28,29]. The recognition of the IIA enzyme as an acute phase protein [7] that is induced in HepG2 cells over several days by IL-1, IL-6 and TNF indicated that the hepatocyte could be a likely candidate as a source of the elevated enzyme levels in serum seen under inflammatory conditions. Thus, liver biopsy of patients with acute pan30
creatitis and elevated serum IIA PLA2 levels revealed enhanced levels of gene expression in hepatocytes compared with control studies [30]. In one patient with highly elevated serum IIA levels the need for a liver transplant resulted in a dramatic fall in IIA levels on the first day post transplantation [31]. Physiological functions of human gIIA PLA2 linked to the global cationic nature of the protein The structure of human group IIA PLA2 is unusual because of the highly cationic nature of the protein, having a global distribution of positively-charged arginine and lysine residues over its surface (Figure 3). This structure provides the basis for two physiological roles, a well established antibacterial role and a proposed new role in the clearance of cell-derived anionic phospholipid microparticles/microvesicles. Antibacterial activity The locations of the enzyme are consistent with its antibacterial role within innate immunity. In addition to the high serum concentrations observed in septicaemia, IIA PLA2 is highly expressed in Paneth cells of the small intestine [32–35], in lacrimal cells and human tears [22,36] and in prostate cells and human seminal plasma [37]. These locations reflect possible sites of bacterial invasion into externally-exposed body cavities. The very high concentration of IIA PLA2 seen in human tears, where it is the primary antibacterial agent against Gram-positive bacteria, is significant [22]. The antibacterial properties of secreted PLA2s, particularly against Gram-positive bacteria, has been reviewed previously [38,39], so the discussion here will focus primarily on how the structure of the human group IIA enzyme provides the basis for this antibacterial activity, and also how target organisms can generate resistance to this enzyme by neutralization of the anionic charge of the bacterial cell wall and cell membrane. Ultimately, the antibacterial properties depend on the ability of the enzyme to target and hydrolyse phospholipids in the anionic cell membrane. In the case of Staphylococcus aureus four basic steps have been identified [40]: binding
Review of the enzyme to the anionic bacterial cell surface; penetration of the enzyme through the anionic peptidoglycan layer of the cell wall; hydrolysis of anionic cell membrane phospholipid; and activation of bacterial autolysins (cell-walldegrading enzymes). Historically, an important observation was the ability of bactericidal/permeability-increasing protein (BPI) to enhance the membrane phospholipid hydrolysing properties of certain secreted PLA2s, including rabbit IIA PLA2 against the Gram-negative bacterium Escherichia coli [41]. However, this enhanced hydrolytic activity in the presence of BPI was only seen with highly cationic enzymes including some venom enzymes [42]. It was subsequently shown that human IIA enzyme was able to hydrolyse E. coli phospholipid in the presence of BPI [43]. The role of BPI might be to permeabilize the lipopolysaccharide coat (outer membrane) of the Gram-negative bacterium [44,45] allowing the PLA2 to subsequently penetrate the bacterial cell wall and hydrolyse the anionic bacterial cell membrane. The importance of the highly cationic nature of the human IIA enzyme in allowing bacterial cell wall penetration was highlighted by studies with particular Grampositive bacteria where the cell wall could be selectively permeabilized by enzyme degradation. In these studies a fluorescence displacement assay [46] was used where it was possible to monitor PLA2-catalysed cell membrane hydrolysis using bacterial cell suspensions as substrates [47]. Suspensions of Micrococcus luteus were readily hydrolysed by the human group IIA enzyme, but not by other 15kDa secreted PLA2s including the porcine pancreatic enzyme and Naja naja snake venom enzyme. This selectivity stems from the ability of the highly cationic human IIA enzyme to penetrate the anionic bacterial cell wall. Removal of this permeability barrier, as a result of hydrolysis of the cell wall with lysozyme, allowed all three PLA2s to display similar activity. Cell membrane degradation by the IIA enzyme resulted in at least 50% phospholipid degradation as judged by electrospray mass spectrometry, and identified phosphatidylglycerol as the major phospholipid substrate [47]. However, the bactericidal activity of the human enzyme under these conditions was low, highlighting the capacity of the organism to recover from significant phospholipid hydrolysis. In the case of S. aureus this survival is due to enhanced phospholipid synthesis by both de novo and salvage pathways [48]. It would be anticipated that in innate immunity, bacterial killing will be achieved by the synergistic action of an arsenal of functionally distinct antibacterial peptides and proteins. The role of the surface positive charge in facilitating access to and hydrolysis of the bacterial cell membranes has been investigated using a strategy of charge reversal mutagenesis in which surface arginine or lysine residues of the human IIA enzyme were substituted for glutamate or aspartate residues by site directed mutagenesis. Studies using M. luteus as a model substrate showed that the reduction in membrane hydrolysis and bacterial killing correlated with loss of positive charge on the enzyme surface [49]. Mutagenesis did not affect the catalytic activity of the enzyme, as full enzyme activity was displayed using bacterial cell suspensions in which the cell wall had been
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permeabilized by lysozyme treatment [49]. The same overall cell membrane and antibacterial activity of the human IIA enzyme and charge reversal mutants was observed using suspensions of S. aureus [49,50] and Listeria innocua [49]. The importance of the negative charge of the peptidoglycan cell wall and cell membrane in the antibacterial action of the IIA enzyme has been highlighted in studies involving mutants of S. aureus [51]. The negative charge of the bacterial cell wall is due to the presence of structural polyanions, in particular, wall teichoic acid and lipotechoic acid that contain repeating units of alditol (ribitol or glycerol) phosphate [52]. S. aureus is able to reduce this negative charge by esterification of the alditol with Dalanine, thus leaving the alanine residue with a net positive charge. This modification reduces the antibacterial effectiveness of the IIA enzyme. Mutants that are unable to carry out the alanylation are 30-100-fold more sensitive to killing by IIA PLA2 [50]. In another bacterial process the addition of L-lysine to the headgroup of both phosphatidylglycerol (PG) and cardiolipin results in the addition of two amino groups into the anionic cell membrane phospholipid. In this case, however, membrane hydrolysis is reduced by less than 3-fold compared with mutants that lack the ability to form lysylphosphatidylglycerol [50]. This observation could reflect the fact that less than 50% of the PG is modified with lysine [53], still allowing binding of the IIA enzyme to the cell membrane, and hence phospholipid hydrolysis. Additional support for the role of the IIA PLA2 in bacterial killing comes from studies with transgenic mice over-expressing the human IIA enzyme. These mice are resistant to various bacterial infections [54–56]. Moreover, phospholipid hydrolysis of S. aureus membranes by added IIA enzyme is enhanced in the presence of human neutrophils (which do not contain the IIA enzyme), and leads to synergistic bactericidal activity where a role for the NADPH oxidase system of the neutrophil has been suggested [57]. A model to explain the ability of the IIA enzyme to help kill Gram-positive bacteria is shown in Figure 4. The model highlights the importance of the global positive charge on the surface of the IIA enzyme in allowing penetration of the anionic bacterial cell wall, and then high-affinity binding to the anionic cell membrane, thus allowing membrane phospholipid hydrolysis to proceed. High concentrations of the enzyme reported during infection are unable to hydrolyse the zwitterionic host cell membrane, thus protecting host cells and tissues from hydrolysis by this extracellular enzyme. Cellular uptake of gIIA PLA2 and the removal of cell debris The effect of the exogenous IIA PLA2 on host cell function has been the subject of numerous studies, but remains unclear and might be the result of cell-specific responses [1,13]. A particular focus has been the involvement of this enzyme in arachidonic release and the subsequent production of signalling molecules, such as the prostaglandins as part of the inflammatory response. This area of investigation has been discussed in detail elsewhere [1] and 31
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Figure 4. A model of bacterial cell wall penetration and phospholipid hydrolysis by human group IIA secreted PLA2 (gIIA sPLA2). The global distribution of the positive charge on this PLA2 has been proposed to allow the enzyme to move through the negatively charged cell wall, mediated by the continuous making and breaking of electrostatic bonds. For simplicity, the bacterial cell membrane is shown as containing only negatively charged phospholipids (shown in blue), but will contain some zwitterionic phospholipids, such as phosphatidylethanolamine. Adapted from [38].
remains controversial. A fundamental problem in defining a role for the IIA PLA2 in vivo is the almost zero catalytic activity of this particular secreted PLA2 within the extracellular host environment where available phospholipid surfaces (membranes and lipoproteins) are normally zwitterionic. Thus, functions linked to phospholipid hydrolysis with the release of fatty acids (arachidonic acid) and lysophospholipids by this enzyme are difficult to substantiate, although the ability of the enzyme to participate in intracellular phospholipid hydrolysis has been demonstrated [27]. The possible binding and uptake of recombinant IIA enzyme into cells has been investigated recently, using a fluorescently-labelled protein that allowed analysis by FACS and fluorescent microscopy [15]. It was possible to bioengineer fluorescent IIA protein molecules that were still fully functional by judicious attachment of chemical fluorophores to specific parts of the protein surface. An extensive study using human monocyte-derived THP-1 cells revealed uptake of the enzyme, but only after the cells had been differentiated by phorbol 12-myristate 13acetate (PMA) to produce a macrophage-like cell phenotype. This treatment correlated with an increase in the expression of cell surface HSPGs [58] and IIA PLA2 uptake was shown to be dependent on interaction of the cationic IIA PLA2 with these anionic polymers. Uptake was energydependent and led to nuclear localization of the enzyme, a characteristic observed with many cationic proteins and peptides [59]. The observation that IIA PLA2 uptake into THP-1 cells did not require the enzyme to be catalytically active was of particular interest. This conclusion was based on the crucial observation that all observed trafficking phenomena for the fluorescent IIA PLA2 were replicated with a fluor32
escent catalytically-inactive mutant, H48N, in which the catalytic histidine is replaced by glutamine. Moreover, protein uptake was accompanied by significant cell swelling due to fluid uptake, a typical characteristic of macropinocytosis. As this IIA PLA2 is uniquely able to form supra-molecular aggregates with anionic phospholipid vesicles [15,17,18] a role for the clearance of such aggregates (cell debris and anionic microparticles) was studied. Using fluorescently-labelled anionic phospholipid vesicles, it was possible to demonstrate that the cellular uptake of these vesicles into PMA-differentiated (but not non-differentiated) THP-1 cells was considerably enhanced (over 10fold) by the presence of the IIA PLA2, a phenomenon that also did not require the enzyme to be catalytically active [15]. Overall, a hitherto unrealised function of the IIA enzyme was proposed as part of the inflammatory response, namely the removal of cell debris and microparticles (Figure 5) resulting from tissue trauma in a process that did not require a catalytic role for the enzyme. This proposal, of a novel role for the IIA PLA2, allows some interesting correlations. For example, the synovial fluid of patients with rheumatoid arthritis contains high concentrations of microparticles and it is in this fluid that the IIA enzyme was first discovered at high concentrations [3]. Such microparticles have been proposed to be inflammatory [60], and hence the presence of the II PLA2 could be to facilitate removal of such particles and help resolve inflammation. In another remarkable correlation, higher expression of the human IIA enzyme has been uniquely linked to reduced metastasis and mortality in patients with gastric cancer [61], and high levels of serum microparticles in such patients is associated with increased morbidity and mortality [62]. The role of microparticles in angiogenesis, a crucial process in cardiovascular disease
Review
Figure 5. A model for the IIA PLA2 uptake into cells, and its role in the uptake of anionic particles. In this model, the globally cationic IIA PLA2 interacts with anionic microparticles to produce coated particles or larger aggregates. Following uptake by macropinocytosis, acidification and possibly other endosomal events should allow IIA PLA2 release and nuclear accumulation. The nature of the aggregate will depend on the size and charge density of the particles and this might also determine the endocytic pathway. Other endocytic routes have been considered [15].
and cancer metastasis, has been reviewed [63]. We would argue the IIA PLA2 concentration could affect the turnover of microparticles in extracellular fluids. Microparticle hydrolysis by IIA PLA2 has previously been demonstrated to produce the lipid mediator lysophosphatidic acid [64]. Concluding remarks The IIA PLA2 enzyme has remarkable structural characteristics and is abundantly expressed in certain locations and under certain pathological conditions. This extracellular enzyme is unable to hydrolyze the plasma membrane of host cells as a result of the very low affinity of the enzyme for the essentially zwitterionic surface of the outer monolayer of the plasma membrane. This characteristic means that such cells are not adversely affected (lysed) by the enzyme, and is a very different situation than that seen with other secreted PLA2s. For example, when certain snake venom PLA2s are added to mammalian cells, they can bind the membrane surface with high affinity and execute phospholipid hydrolysis and cell lysis. By contrast, the human IIA enzyme can target and hydrolyse membranes rich in anionic phospholipids, such as bacterial cell
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membranes. Moreover, the globally-cationic structure of the enzyme allows it to penetrate the anionic cell wall of Gram-positive bacteria, allowing the enzyme to access the anionic bacterial cell membrane where subsequent hydrolysis facilitates bacterial killing. This same global cationic nature of the IIA enzyme allows it to form aggregates with anionic phospholipid particles (cell debris). The resulting cationic supra-molecular aggregates can be taken up by activated macrophages, such as human THP-cells, in a process involving HSPGs and macropinocytosis, allowing debris removal and subsequent destruction. Phospholipid hydrolysis is not required for this process. Overall, the process would be part of the acute phase response to facilitate the removal of damaged cells and cell debris. Such a role has been proposed previously for the IIA PLA2 in conjunction with C-reactive protein, but in a process that involved phospholipid hydrolysis [65]. The accumulation and/or targeting of IIA PLA2 to the nucleus might suggest modulatory effects on gene regulation, and under appropriate conditions increased expression of COX2 has been observed [15]. The physiological process of the uptake and nuclear targeting of cationic supra-molecular aggregates involving the IIA protein may be utilizing the pathways of non-viral gene transfection that use this process for entry into cells of DNA coated with cationic reagents. The probable complexity of the physiological action of this enzyme and our lack of complete understanding of many features of its effects on cells has been critically reviewed [1]. In addition, the recent description of the binding of the IIA PLA2 to integrins and the proliferation of monocytic cells is another example of a possible non-catalytic and pro-inflammatory role for this protein [66]. In summary, this review highlights the potential role of the IIA PLA2 in resolving inflammatory situations involving both catalytic (antibacterial) and non-catalytic (debris removal) functions. In this context, the IIA PLA2 is not seen as pro-inflammatory as originally assumed when the enzyme was first isolated, but could have primarily an antiinflammatory (resolving) role in the inflammatory response. There is still much to be learned about the role of this enzyme in disease processes, and how its role might depend on the target cell and the state of activation of that cell (Box 1).
Box 1. Outstanding Questions What effects does the accumulation of the human IIA PLA2 in the nucleus have on gene expression? Is human IIA PLA2 taken up into activated cells in the absence of cell debris? What is the effect of over-expression/lack of expression of IIA PLA2 on microparticle clearance? Is there extracellular microparticle hydrolysis by IIA PLA2, and how does this affect microparticle clearance? Is there a specific receptor in humans that can bind human IIA PLA2 [1]? Overall, does the IIA PLA2 have diverse interactions and effects (pro- and anti-inflammatory), depending on the type of target cell and the activation status of the cell?
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