Thematic Review Series: Phospholipases: Central Role in Lipid Signaling and Disease: A new era of secreted phospholipase A2

Among more than 30 members of the phospholipase A₂ (PLA₂) superfamily, secreted PLA₂ (sPLA₂) enzymes represent the largest family, being Ca²⁺-dependent low-molecular-weight enzymes with a His-Asp catalytic dyad. Individual sPLA₂s exhibit unique tissue and cellular distributions and enzymatic properties, suggesting their distinct biological roles. Recent studies using transgenic and knockout mice for nearly a full set of sPLA₂ subtypes, in combination with sophisticated lipidomics as well as biochemical and cell biological studies, have revealed distinct contributions of individual sPLA₂s to various pathophysiological events, including production of pro- and anti-inflammatory lipid mediators, regulation of membrane remodeling, degradation of foreign phospholipids in microbes or food, or modification of extracellular noncellular lipid components. In this review, we highlight the current understanding of the in vivo functions of sPLA₂s and the underlying lipid pathways as revealed by a series of studies over the last decade.     

Emerging roles of secreted phospholipase A2 enzymes: An update

Phospholipase A₂ (PLA₂) enzymes catalyze the hydrolysis of the sn-2 position of glycerophospholipids to produce free fatty acids and lysophospholipids. More than one third of the mammalian PLA₂ enzymes belong to the secreted PLA₂ (sPLA₂) family, which consists of low molecular mass, Ca²⁺-requiring enzymes with a His–Asp catalytic dyad. Individual sPLA₂ enzymes exhibit unique tissue and cellular localizations and specific enzymatic properties, suggesting their distinct biological roles. The past decade has met a new era of the sPLA₂ research field toward deciphering their in vivo functions by developing several specific tools and methods. These include i) the production of transgenic and knockout mouse lines for several sPLA₂s, ii) the development of specific analytical tools including the production of large amounts of recombinant sPLA₂ proteins, and iii) applying mass spectrometry lipidomics to unveil their specific enzymatic properties occurring in vivo. It is now obvious that individual sPLA₂s are involved in diverse biological events through lipid mediator-dependent and -independent processes, act redundantly or non-redundantly in the context of physiology and pathophysiology, and may represent potential drug targets or novel bioactive molecules in certain situations. In this review, we will highlight the newest understanding of the biological roles of sPLA₂s in the past few years.     

Recent progress in phospholipase A₂ research: from cells to animals to humans

Mammalian genomes encode genes for more than 30 phospholipase A₂s (PLA₂s) or related enzymes, which are subdivided into several classes including low-molecular-weight secreted PLA₂s (sPLA₂s), Ca²⁺-dependent cytosolic PLA₂s (cPLA₂s), Ca²⁺-independent PLA₂s (iPLA₂s), platelet-activating factor acetylhydrolases (PAF-AHs), lysosomal PLA₂s, and a recently identified adipose-specific PLA. Of these, the intracellular cPLA₂ and iPLA₂ families and the extracellular sPLA₂ family are recognized as the “big three”. From a general viewpoint, cPLA₂α (the prototypic cPLA₂) plays a major role in the initiation of arachidonic acid metabolism, the iPLA₂ family contributes to membrane homeostasis and energy metabolism, and the sPLA₂ family affects various biological events by modulating the extracellular phospholipid milieus. The cPLA₂ family evolved along with eicosanoid receptors when vertebrates first appeared, whereas the diverse branching of the iPLA₂ and sPLA₂ families during earlier eukaryote development suggests that they play fundamental roles in life-related processes. During the past decade, data concerning the unexplored roles of various PLA₂ enzymes in pathophysiology have emerged on the basis of studies using knockout and transgenic mice, the use of specific inhibitors, and information obtained from analysis of human diseases caused by mutations in PLA₂ genes. This review focuses on current understanding of the emerging biological functions of PLA₂s and related enzymes.     

Group IID,IIE, IIF and III secreted phospholipase A2s

Among the 11 members of the secreted phospholipase A₂ (sPLA₂) family, group IID, IIE, IIF and III sPLA₂s (sPLA₂-IID, -IIE, -IIF and -III, respectively) are “new” isoforms in the history of sPLA₂ research. Relative to the better characterized sPLA₂s (sPLA₂-IB, -IIA, -V and -X), the enzymatic properties, distributions, and functions of these “new” sPLA₂s have remained obscure until recently. Our current studies using knockout and transgenic mice for a nearly full set of sPLA₂s, in combination with comprehensive lipidomics, have revealed unique and distinct roles of these “new” sPLA₂s in specific biological events. Thus, sPLA₂-IID is involved in immune suppression, sPLA₂-IIE in metabolic regulation and hair follicle homeostasis, sPLA₂-IIF in epidermal hyperplasia, and sPLA₂-III in male reproduction, anaphylaxis, colonic diseases, and possibly atherosclerosis. In this article, we overview current understanding of the properties and functions of these sPLA₂s and their underlying lipid pathways in vivo.     

Emerging roles for phospholipase A2 enzymes in cancer

Phospholipase A₂ (PLA₂) enzymes (EC3.1.4.4) regulate the release of biologically active fatty acids and lysophospholipids from membrane phospholipid pools. These lipids are also substrates for intracellular biochemical pathways that generate potent autocrine and paracrine lipid mediators such as the eicosanoids and platelet activating factor. These factors, in turn, regulate cell proliferation, survival, differentiation, motility, tissue vascularisation, and immune surveillance in virtually all tissues, functions that are subverted by cancer cells for tumour growth and metastasis. Thus the relevance of PLA₂-dependent pathways to the genesis and progression of cancer has been of interest since their discovery and with recent technological advances, their role in tumourigenesis has become more tractable experimentally. Limited human genetic studies have not yet identified PLA₂ enzymes as classical mutated oncogenes or tumour suppressor genes. However, there is strong evidence that of the 22 identified human PLA₂ enzymes, ten of which have been studied in cancer to date, most are aberrantly expressed in a proportion of tumours derived from diverse organs. Correlative and functional studies implicate the expression of some secreted enzymes (sPLA₂s), particularly the best studied enzyme Group IIA sPLA₂ in either tumour promotion or inhibition, depending on the organ involved and the biochemical microenvironment of tumours. As in immune-mediated inflammatory pathologies, genetic deletion studies in mice, supported by limited studies with human cells and tissues, have identified an important role for Group IVA PLA₂ in regulating certain cancers. Pharmacological intervention studies in prostate cancer suggest that hGIIA-dependent tumour growth is dependent on indirect regulation of Group IVA PLA₂. Group VI calcium-independent PLA₂ enzymes have also been recently implicated in tumourigenesis with in vitro studies suggesting multiple possible roles for these enzymes. Though apparently complex, further characterization of the regulatory relationships amongst PLA₂ enzymes, lipid mediator biosynthetic enzymes and the lipid mediators they produce during tumour progression is required to define the biochemical context in which the enzymes modulate cancer growth and development.     

Phospholipase A2-like activity of human bocavirus VP1 unique region

Human bocavirus (HBoV) is a new parvovirus first discovered in 2005, which is associated with acute respiratory infection. Analysis of sequence homology has revealed that a putative phospholipase A₂ (PLA₂) motif exists in the VP1 unique region of HBoV. However, little is known about whether the VP1 unique region of HBoV has PLA₂ enzymatic activity and how these critical residues contribute to its PLA₂ activity. To address these issues, the VP1 unique region protein and four of its mutants, were expressed in Eschericha coli. The purified VP1 unique protein (VP1U) showed a typical Ca²⁺-dependent secreted PLA₂-like (sPLA₂) activity, which was inhibited by sPLA₂-specific inhibitors in a time-dependent manner. Mutation of one of the amino acids (21Pro, 41His, 42Asp or 63Asp) in VP1U almost eliminated the sPLA₂ activity of HBoV VP1U. These data indicate that VP1U of HBoV has sPLA₂-like enzymatic activity, and these residues are crucial for its sPLA₂-like activity. Potentially, VP1U may be a target for the development of anti-viral drugs for HBoV.     

Novel functions of phospholipase A2s: Overview

The phospholipase A₂ (PLA₂) family comprises a group of lipolytic enzymes that typically hydrolyze the sn-2 position of (glycerol) phospholipids to give rise to fatty acids and lysophospholipids. The mammalian genome encodes more than 30 (even 50) PLA₂s or related enzymes, which are classified into several subfamilies on the basis of their structures and functions. The PLA₂ family has been implicated not only in signal transduction by producing lipid mediators, but also in membrane homeostasis, energy production, and barrier function. Disturbance of PLA₂-regulated lipid pathways often hampers tissue and cellular homeostasis and can be linked to various diseases. This special issue overviews the current state of understanding of the classification, enzymatic properties, and physiological functions of various enzymes belonging to the PLA₂ family. This article is part of a Special Issue entitled Novel functions of phospholipase A₂ Guest Editors: Makoto Murakami and Gerard Lambeau.     

Secreted Phospholipases A2 - not just Enzymes: Revisited

Secreted phospholipases A₂ (sPLA₂s) participate in a very broad spectrum of biological processes through their enzymatic activity and as ligands for membrane and soluble receptors. The physiological roles of sPLA₂s as enzymes have been very well described, while their functions as ligands are still poorly known. Since the last overview of sPLA₂-binding proteins (sPLA₂-BPs) 10 years ago, several important discoveries have occurred in this area. New and more sensitive analytical tools have enabled the discovery of additional sPLA₂-BPs, which are presented and critically discussed here. The structural diversity of sPLA₂-BPs reveals sPLA₂s as very promiscuous proteins, and we offer some structural explanations for this nature that makes these proteins evolutionarily highly advantageous. Three areas of physiological engagement of sPLA₂-BPs have appeared most clearly: cellular transport and signalling, and regulation of the enzymatic activity of sPLA₂s. Due to the multifunctionality of sPLA₂s, they appear to be exceptional pharmacological targets. We reveal the potential to exploit interactions of sPLA₂s with other proteins in medical terms, for the development of original diagnostic and therapeutic procedures. We conclude this survey by suggesting the priority questions that need to be answered.     

Phospholipase A2 in astrocytes

Astrocytes comprise the major cell type in the central nervous system (CNS) and they are essential for support of neuronal functions by providing nutrients and regulating cell-to-cell communication. Astrocytes also are immune-like cells that become reactive in response to neuronal injury. Phospholipases A₂ (PLA ₂) are a family of ubiquitous enzymes that degrade membrane phospholipids and produce lipid mediators for regulating cellular functions. Three major classes of PLA ₂ are expressed in astrocytes: group IV calcium-dependent cytosolic PLA ₂ (cPLA₂), group VI calcium-independent PLA ₂ (iPLA₂), and group II secretory PLA ₂ (sPLA₂). Upregulation of PLA ₂ in reactive astrocytes has been shown to occur in a number of neurodegenerative diseases, including stroke and Alzheimer’s disease. This review focuses on describing the effects of oxidative stress, inflammation, and activation of G protein-coupled receptors on PLA ₂ activation, arachidonic acid (AA) release, and production of prostanoids in astrocytes.     

Expression of phospholipases A2 in primary human lung macrophages: Role of cytosolic phospholipase A2-α in arachidonic acid release and platelet activating factor synthesis

Macrophages are a major source of lipid mediators in the human lung. Expression and contribution of cytosolic (cPLA₂) and secreted phospholipases A₂ (sPLA₂) to the generation of lipid mediators in human macrophages are unclear. We investigated the expression and role of different PLA₂s in the production of lipid mediators in primary human lung macrophages. Macrophages express the alpha, but not the zeta isoform of group IV and group VIA cPLA₂ (iPLA₂). Two structurally-divergent inhibitors of group IV cPLA₂ completely block arachidonic acid release by macrophages in response to non-physiological (Ca²⁺ ionophores and phorbol esters) and physiological agonists (lipopolysaccharide and Mycobacterium protein derivative). These inhibitors also reduce by 70% the synthesis of platelet-activating factor by activated macrophages. Among the full set of human sPLA₂s, macrophages express group IIA, IID, IIE, IIF, V, X and XIIA, but not group IB and III enzymes. Me-Indoxam, a potent and cell impermeable inhibitor of several sPLA₂s, has no effect on arachidonate release or platelet-activating factor production. Agonist-induced exocytosis is not influenced by cPLA₂ inhibitors at concentrations that block arachidonic acid release. Our results indicate that human macrophages express cPLA₂-alpha, iPLA₂ and several sPLA₂s. Cytosolic PLA₂-alpha is the major enzyme responsible for lipid mediator production in human macrophages.     

Inhibition of secretory phospholipase A2 enzyme by bilirubin: A new role as endogenous anti-inflammatory molecule

Bilirubin is a powerful antioxidant that suppresses the inflammatory process. However its interaction with proinflammatory PLA₂ enzyme is not known. Inhibition of several secretory phospholipase A₂ (sPLA₂) enzyme activities by bilirubin was studied using ¹⁴C-oleate labeled Escherichia coli as substrate. Bilirubin inhibits purified sPLA₂ enzyme from Vipera russellii and Naja naja venom and partially purified sPLA₂ enzymes from human ascitic fluid, pleural fluid and normal serum in a dose dependent manner. IC₅₀ values calculated for these enzymes ranges from 1.75 to 10.5 μM. Inflammatory human sPLA₂ enzymes are more sensitive to inhibition by bilirubin than snake venom sPLA₂s. Inhibition of sPLA₂ activity by bilirubin is independent of calcium concentration. Increasing substrate concentration (upto 180 nmol) did not relieve the inhibition of sPLA₂ by bilirubin and it is irreversible. Bilirubin quenched the relative fluorescence intensity of sPLA₂ in a dose dependent manner in the same concentration range at which in vitro sPLA₂ inhibition was observed. In the presence of bilirubin, apparent shift in the far UV-CD spectra of sPLA₂ was observed, indicating a direct interaction with the enzyme. Inhibition of sPLA₂ induced mouse paw edema by bilirubin confirms its sPLA₂ inhibitory activity in vivo also. These findings indicate that inhibition of sPLA₂ by bilirubin is mediated by direct interaction with the enzyme and bilirubin may act as an endogenous regulator of sPLA₂ enzyme activity.     

In silico and in vitro characterization of phospholipase A2 isoforms from soybean (Glycine max)

At the present, no secreted phospholipase A₂ (sPLA₂) from soybean (Glycine max) was investigated in detail. In this work we identified five sequences of putative secreted sPLA₂ from soybean after a BLAST search in G. max database. Sequence analysis showed a conserved PA2c domain bearing the Ca²⁺ binding loop and the active site motif. All the five mature proteins contain 12 cysteine residues, which are commonly conserved in plant sPLA₂s. We propose a phylogenetic tree based on sequence alignment of reported plant sPLA₂s including the novel enzymes from G. max. According to PLA₂ superfamily, two of G. max sPLA₂s are grouped as XIA and the rest of sequences as XIB, on the basis of differences found in their molecular weights and deviating sequences especially in the N- and C-terminal regions of the isoenzymes. Furthermore, we report the cloning, expression and purification of one of the putative isoenzyme denoted as GmsPLA₂-XIA-1. We demonstrate that this mature sPLA₂ of 114 residues had PLA₂ activity on Triton:phospholipid mixed micelles and determine the kinetic parameters for this system. We generate a model based on the known crystal structure of sPLA₂ from rice (isoform II), giving first insights into the three-dimensional structure of folded GmsPLA₂-XIA-1. Besides describing the spatial arrangement of highly conserved pair HIS-49/ASP-50 and the Ca⁺² loop domains, we propose the putative amino acids involved in the interfacial recognition surface. Additionally, molecular dynamics simulations indicate that calcium ion, besides its key function in the catalytic cycle, plays an important role in the overall stability of GmsPLA₂-XIA-1 structure.     

Time-dependent changes in the brain arachidonic acid cascade during cuprizone-induced demyelination and remyelination

Phospholipases A₂ (PLA₂) are the enzymatic keys for the activation of the arachidonic acid (AA) cascade and the subsequent synthesis of pro-inflammatory prostanoids (prostaglandins and tromboxanes). Prostanoids play critical roles in the initiation and modulation of inflammation and their levels have been reported increased in several neurological and neurodegenerative disorders, including multiple sclerosis (MS).Here, we aimed to determine whether brain expression PLA₂ enzymes and the terminal prostagland in levels are changed during cuprizone-induced demyelination and in the subsequent remyelination phase.Mice were given the neurotoxicant cuprizone through the diet for six weeks to induce brain demyelination. Then, cuprizone was withdrawn and mice were returned to a normal diet for 6 weeks to allow spontaneous remyelination.We found that after 4-6 weeks of cuprizone, sPLA₂(V) and cPLA₂, but not iPLA₂(VI), gene expression was upregulated in the cortex, concomitant with an increase in the expression of astrocyte and microglia markers. Cyclooxygenase (COX)-2 gene expression was consistently upregulated during all the demyelination period, whereas COX-1 sporadically increased only at week 5 of cuprizone exposure. However, we found that at the protein level only sPLA₂(V) and COX-1 were elevated during demyelination, with COX-1 selectively expressed by activated and infiltrated microglia/macrophages and astrocytes. Levels of PGE₂, PGD₂, PGI₂ and TXB₂ were also increased during demyelination. During remyelination, none of the PLA₂ isoforms was significantly changed, whereas COX-1 and -2 were sporadically upregulated only at the gene expression level. PGE₂, PGI₂ and PGD₂ levels returned to normal, whereas TXB₂ was still upregulated after 3 weeks of cuprizone withdrawal.Our study characterizes for the first time time-dependent changes in the AA metabolic pathway during cuprizone-induced demyelination and the subsequent remyelination and suggests that sPLA₂(V) is the major isoform contributing to AA release.     

Suppression of murine endotoxic shock by sPLA2 inhibitor,indoxam, through group IIA sPLA2-independent mechanisms

Endotoxic shock is a systemic inflammatory process, involving a variety of proinflammatory mediators. Two types of secretory phospholipase A₂ (sPLA₂) have been implicated in this process. Group IB sPLA₂ (PLA₂-IB) binds to the PLA₂ receptor (PLA₂R), and PLA₂R-deficient mice exhibit resistance to endotoxin-induced lethality with reduced plasma levels of proinflammatory cytokines, such as TNF-α. Group IIA sPLA₂ (PLA₂-IIA) is found in many tissues and cell types, and local and systemic levels are elevated under numerous inflammatory conditions including sepsis. In this study, we investigated the effect of a specific sPLA₂ inhibitor, indoxam, on murine endotoxic shock. Indoxam suppressed the elevation of plasma TNF-α with a similar potency in PLA₂-IIA-expressing and PLA₂-IIA-deficient mice after LPS challenge. In PLA₂-IIA-deficient mice, indoxam also suppressed the elevation of plasma IL-1β, IL-6 and NO, and prolonged survival after LPS challenge. Indoxam was found to block the PLA₂-IB binding to murine PLA₂R with a high potency (K_i=30 nM). The inhibitory effects of indoxam on the LPS-induced elevation of plasma TNF-α levels could not be observed in mice deficient in PLA₂R. These findings suggest that indoxam blocks the production of proinflammatory cytokines during endotoxemia through PLA₂-IIA-independent mechanisms, possibly via blockade of the PLA₂R function.     

Effects of the propeptide of group X secreted phospholipase A2 on substrate specificity and interfacial activity on phospholipid monolayers

Group X secreted phospholipase A₂ (GX sPLA₂) plays important physiological roles in the gastrointestinal tract, in immune and sperm cells and is involved in several types of inflammatory diseases. It is secreted either as a mature enzyme or as a mixture of proenzyme (with a basic 11 amino acid propeptide) and mature enzyme. The role of the propeptide in the repression of sPLA₂ activity has been studied extensively using liposomes and micelles as model interfaces. These substrates are however not always suitable for detecting some fine tuning of lipolytic enzymes. In the present study, the monolayer technique is used to compare PLA₂ activity of recombinant mouse GX sPLA₂ (mGX) and its pro-form (PromGX) on monomolecular films of dilauroyl-phosphatidyl-ethanolamine (DLPE), -choline (DLPC) and -glycerol (DLPG). The PLA₂ activity and substrate specificity of mGX (PE ≈ PG > PC) were found to be surface pressure-dependent. mGX displayed a high activity on DLPE and DLPG but not on DLPC monolayers up to surface pressures corresponding to the lateral pressure of biological membranes (30–35 mN/m). Overall, the propeptide impaired the enzyme activity, particularly on DLPE whatever the surface pressure. However some conditions could be found where the propeptide had little effects on the repression of PLA₂ activity. In particular, both PromGX and mGX had similar activities on DLPG at a surface pressure of 30 mN/m. These findings show that PromGX can be potentially active depending on the presentation of the substrate (i.e., lipid packing) and one cannot exclude such an activity in a physiological context. A structural model of PromGX was built to investigate how the propeptide controls the activity of GX sPLA₂. This model shows that the propeptide is located within the interfacial binding site (i-face) and could disrupt both the interfacial binding of the enzyme and the access to the active site by steric hindrance.     

Genes encoding phospholipases A2 mediate insect nodulation reactions to bacterial challenge

We propose that expression of four genes encoding secretory phospholipases A₂ (sPLA₂) mediates insect nodulation responses to bacterial infection. Nodulation is the quantitatively predominant cellular defense reaction to bacterial infection. This reaction is mediated by eicosanoids, the biosynthesis of which depends on PLA₂-catalyzed hydrolysis of arachidonic acid (AA) from cellular phospholipids. Injecting late instar larvae of the red flour beetle, Tribolium castaneum, with the bacterium, Escherichia coli, stimulated nodulation reactions and sPLA₂ activity in time- and dose-related manners. Nodulation was inhibited by pharmaceutical inhibitors of enzymes involved in eicosanoid biosynthesis, and the inhibition was rescued by AA. We cloned five genes encoding sPLA₂ and expressed them in E. coli cells to demonstrate these genes encode catalytically active sPLA₂s. The recombinant sPLA₂s were inhibited by sPLA₂ inhibitors. Injecting larvae with double-stranded RNAs specific to each of the five genes led to reduced expression of the corresponding sPLA₂ genes and to reduced nodulation reactions to bacterial infections for four of the five genes. The reduced nodulation was rescued by AA, indicating that expression of four genes encoding sPLA₂s mediates nodulation reactions. A polyclonal antibody that reacted with all five sPLA₂s showed the presence of the sPLA₂ enzymes in hemocytes and revealed that the enzymes were more closely associated with hemocyte plasma membranes following infection. Identifying specific sPLA₂ genes that mediate nodulation reactions strongly supports our hypothesis that sPLA₂s are central enzymes in insect cellular immune reactions.     

Isozymes inhibited by active site blocking: versatility of calcium indifferent hesperidin binding to phospholipase A2 and its significance

sPLA₂ is released under inflammatory conditions from neutrophils, basophils and T-cells. They cleave the cellular phospholipids leading to the release of arachidonic acid and there by provide intermediates for biosynthesis of inflammatory mediators. The focus of this study is on the interaction of hesperidin, a natural flavonoid with Group IB, IIA, and V and X isozymes of sPLA₂. Affinity of hesperidin towards PLA₂ isozymes was analyzed through enzymatic studies and molecular modeling. The experiments showed that hesperidin competitively inhibited PLA₂ with IC₅₀ of 5.1?µM. Molecular modeling studies revealed the association of hesperidin with the docking scores ?6.90, ?9.53, ?5.63 and ?8.29?kcal for isozymes Group IB, IIA, V and X of PLA₂ respectively. Their binding energy values were calculated as ?20.25, ?21.63, ?21.66 and ?33.43?kcal for the Group IB, IIA, V and X respectively. Structural model for Group V was made by homology modeling since no structural coordinates were available. Molecular dynamics studies were carried out to evaluate the structural stability of protein ligand complex. The analyses showed that hesperidin blocked the entry of the substrate to the active site of PLA₂ and it was indifferent to the differences of the isozymes. Hence, hesperidin might serve as lead for designing highly specific anti-inflammatory drugs directed to the PLA₂ isozyme specific to various diseases, with IC₅₀ value of therapeutic significance.     

Modification of Lys-6 and Lys-65 Affects the Structural Stability of Taiwan Cobra Phospholipase A2

To assess whether chemical modification of phospholipase A₂ (PLA₂) enzymes may affect their fine structure and consequently alter their enzymatic activity, the present study was carried out. Both Lys-6 and Lys-65 in the Taiwan cobra (Naja naja atra) PLA₂ were selectively modified with trinitrobenzene sulfonate and pyridoxal-5′-phosphate (PLP), respectively. Incorporation of either trinitrophenylated (TNP) or PLP groups on Lys-6 and Lys-65 caused a drop in PLA₂ activity, but the Ca²⁺-binding ability and global conformation of modified derivatives were not significantly different from that of native enzyme. A distinct enhancement of stability was observed with native PLA₂ when thermal unfolding was conducted in the presence of 20 mM Ca²⁺. Conformational transition induced by guanidine hydrochloride was also attenuated by the addition of Ca²⁺. Conversely, a marked decrease in the structural stability was noted with modified derivatives, and the enhancing effect of Ca²⁺ pronouncedly decreased. Together with the finding that the incorporated TNP and PLP groups did not equally affect enzymatic activity and structural stability of PLA₂, our data suggest that an alteration in the fine structure owing to the incorporated groups should contribute to the observed decrease in PLA₂ activity.     

Specific differential expression of phospholipase A2 subtypes in rat cerebellum

Phospholipase A₂ (PLA₂) is a family of enzymes playing diverse roles in lipid signaling in neurons and glia cells. In this study, we examined the expression of subtypes of PLA₂ in the cerebellum using immunolabeling and in situ hybridization methods. Two Ca²⁺-dependent cytosolic subtypes (cPLA₂α and cPLA₂β), one Ca²⁺-independent cytosolic subtype (iPLA₂), and two secretory subtypes (sPLA₂IIA and sPLA₂V) were detected in the cerebellum. cPLA₂α is present in somata and dendrites of Purkinje cells, while sPLA₂IIA is associated with the endoplasmic reticulum in perinuclear regions of Purkinje cell somata. iPLA₂ is present in granule cells, stellate cells and also in the nucleus of Purkinje cells. In addition, cPLA₂β is localized in granule cells, and sPLA₂V in Bergmann glia cells. These results provide an important basis for identifying functional roles of PLA₂s in the cerebellum.     

Proteolysis of Apolipoprotein A-I by Secretory Phospholipase A2: A NEW LINK BETWEEN INFLAMMATION AND ATHEROSCLEROSIS*

In the acute phase of the inflammatory response, secretory phospholipase A₂ (sPLA₂) reaches its maximum levels in plasma, where it is mostly associated with high density lipoproteins (HDL). Overexpression of human sPLA₂ in transgenic mice reduces both HDL cholesterol and apolipoprotein A-I (apoA-I) plasma levels through increased HDL catabolism by an unknown mechanism. To identify unknown PLA₂-mediated activities on the molecular components of HDL, we characterized the protein and lipid products of the PLA₂ reaction with HDL. Consistent with previous studies, hydrolysis of HDL phospholipids by PLA₂ reduced the particle size without changing its protein composition. However, when HDL was destabilized in the presence of PLA₂ by the action of cholesteryl ester transfer protein or by guanidine hydrochloride treatment, a fraction of apoA-I, but no other proteins, dissociated from the particle and was rapidly cleaved. Incubation of PLA₂ with lipid-free apoA-I produced similar protein fragments in the range of 6–15 kDa, suggesting specific and direct reaction of PLA₂ with apoA-I. Mass spectrometry analysis of isolated proteolytic fragments indicated at least two major cleavage sites at the C-terminal and the central domain of apoA-I. ApoA-I proteolysis by PLA₂ was Ca²⁺-independent, implicating a different mechanism from the Ca²⁺-dependent PLA₂-mediated phospholipid hydrolysis. Inhibition of proteolysis by benzamidine suggests that the proteolytic and lipolytic activities of PLA₂ proceed through different mechanisms. Our study identifies a previously unknown proteolytic activity of PLA₂ that is specific to apoA-I and may contribute to the enhanced catabolism of apoA-I in inflammation and atherosclerosis.