Edward A. Dennis

THE EXPANDING SUPERFAMILY OF PHOSPHOLIPASE A(2) ENZYMES: CLASSIFICATION AND CHARACTERIZATION

The phospholipase A(2) (PLA(2)) superfamily consists of a broad range of enzymes defined by their ability to catalyze the hydrolysis of the middle (sn-2) ester bond of substrate phospholipids. The hydrolysis products of this reaction, free fatty acid and lysophospholipid, have many important downstream roles, and are derived from the activity of a diverse and growing superfamily of PLA(2) enzymes. This review updates the classification of the various PLA(2)s now described in the literature. Four criteria have been employed to classify these proteins into one of the 11 Groups (I-XI) of PLA(2)s. First, the enzyme must catalyze the hydrolysis of the sn-2 ester bond of a natural phospholipid substrate, such as long fatty acid chain phospholipids, platelet activating factor, or short fatty acid chain oxidized phospholipids. Second, the complete amino acid sequence of the mature protein must be known. Third, each PLA(2) Group should include all of those enzymes that have readily identifiable sequence homology. If more than one homologous PLA(2) gene exists within a species, then each paralog should be assigned a Subgroup letter, as in the case of Groups IVA, IVB, and IVC PLA(2). Homologs from different species should be classified within the same Subgroup wherever such assignments are possible as is the case with zebra fish and human Group IVA PLA(2) orthologs. The current classification scheme does allow for historical exceptions of the highly homologous Groups I, II, V, and X PLA(2)s. Fourth, catalytically active splice variants of the same gene are classified as the same Group and Subgroup, but distinguished using Arabic numbers, such as for Group VIA-1 PLA(2) and VIA-2 PLA(2)s. These four criteria have led to the expansion or realignment of Groups VI, VII and VIII, as well as the addition of Group XI PLA(2) from plants.

Lipidomics reveals a remarkable diversity of lipids in human plasma

The focus of the present study was to define the human plasma lipidome and to establish novel analytical methodologies to quantify the large spectrum of plasma lipids. Partial lipid analysis is now a regular part of every patients blood test and physicians readily and regularly prescribe drugs that alter the levels of major plasma lipids such as cholesterol and triglycerides. Plasma contains many thousands of distinct lipid molecular species that fall into six main categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterols, and prenols. The physiological contributions of these diverse lipids and how their levels change in response to therapy remain largely unknown. As a first step toward answering these questions, we provide herein an in-depth lipidomics analysis of a pooled human plasma obtained from healthy individuals after overnight fasting and with a gender balance and an ethnic distribution that is representative of the US population. In total, we quantitatively assessed the levels of over 500 distinct molecular species distributed among the main lipid categories. As more information is obtained regarding the roles of individual lipids in health and disease, it seems likely that future blood tests will include an ever increasing number of these lipid molecules.

Solubilization of phospholipids by detergents. Structural and kinetic aspects.

Most amphiphiles in biological membranes including phospholipids, steroids, and membrane proteins are insoluble amphiphiles and would form liquid crystals or insoluble precipitates alone in aqueous media. Detergents are soluble amphiphiles and above a critical concentration and temperature form micelles of various sizes and shapes. Much of the recent progress in studying the insoluble amphiphiles is due to the formation of thermodynamically stable isotropic solutions of these compounds in the presence of detergents. This process, which is commonly denoted as solubilization," involves transformation of lamellar structures into mixed micelles. The information available to date on the solubilization of phospholipids, which constitute the lipid skeleton of biomembranes, by the common detergents is discussed in this review, both with respect to the kinetics of this process and the structure of the various phospholipid-detergent mixed micelles formed. It is hoped that this discussion will lead to somewhat more useful, although still necessarily fairly empirical, approaches to the solubilization of phospholipids by detergents.

Phospholipase A2 Enzymes: Physical Structure, Biological Function, Disease Implication, Chemical Inhibition, and Therapeutic Intervention

1.1. Discovery of the Phospholipase A2 Superfamily Phospholipases represent one of the earliest enzyme activities to be identified and studied and the phospholipase A2 (PLA2) superfamily (see defining specificity1 in Figure 1) traces its roots to the identification of lytic actions of snake venom at the end of the 19th century. The enzyme was first purified and characterized from cobra venom and later from rattlesnake venom. As protein sequencing methodologies advanced in the 1970’s, it became apparent that these enzymes had an unusually large number of cysteines (over 10% of the amino acids) and as secreted enzymes, that they were all in the form of disulfide bonds. It was further recognized that in the case of PLA2, cobras and rattlesnakes had six disulfides in common, but one disulfide bond is located in distinctly different locations. This led to the designation of Type 1 and Type 2 for cobras (old world snakes) and rattlesnakes (new world snakes), respectively.2 During that same period, studies on the porcine pancreatic digestive enzyme that hydrolyzes phospholipids led to the determination that this mammalian enzyme (and also the human pancreatic enzyme) had the same disulfide bonding pattern as cobras and hence the designation as IB with the cobra enzyme as IA. Figure 1 The specific reaction catalyzed by phospholipase A2 at the sn-2 position of the glycerol backbone is shown. X, any of a number of polar headgroups; R1, fatty acids, or alkyl, or alkenyl groups and R2, fatty acids or acyl moieties. A dramatic change in the phospholipase A2 field that attracted the attention of the broader scientific community occurred in July, 1988 when at the first FASEB Summer Conference on Phospholipases, Jeffery J. Seilhamer and Lorin K. Johnson from California Biotechnology Inc.3 and Ruth M. Kramer from Biogen Research Corporation4 independently and with much fanfare and excitement reported the purification, sequencing and cloning of the first human non-pancreatic secreted PLA2 which they each had isolated from the human synovial fluid of arthritic knee joints. Since the sequence revealed that the disulfide bond pattern was more like the rattlesnake than the human pancreatic enzyme, this new form of PLA2 was designated IIA. All of these enzymes then became known as secreted or sPLA2s. It wasn’t until the late 1980’s that PLA2-like activities were reported in mammalian cells in contrast to extracellular secreted activities from venom and pancreas. In July, 1992, at the second FASEB Summer Conference on Phospholipases, James D. Clark from the Genetics Institute5 and Ruth M. Kramer (who had moved to Lilly Research Laboratories)6 independently reported the purification, sequencing, and cloning of the first human cytosolic PLA2 (cPLA2) from the U937 macrophage cell line. The sequence was unrelated to those of the secreted enzymes. To track this new enzyme and potentially additional PLA2s, a Group Numbering System7 was established utilizing the preexisting venom designation of I and II and expanding them to include subgroups IA, IB, and IIA (GIA, GIB, GIIA); adding Group III (GIII) for the clearly different PLA2 which had been purified from bee venom; and establishing the Group IV (GIV) designation for the new cytosolic PLA2 (cPLA2). This was fortuitous because soon thereafter a new form of secreted PLA2 was discovered. It was produced by macrophages and it had the same six disulfide bonds as Group I and Group II, but lacked the seventh disulfide bond entirely. To make clear that this sPLA2 was neither GI nor GII, this enzyme was designated as Group V (GV). At the Third FASEB Summer Conference on Phospholipases held in July, 1995, Edward A. Dennis from the University of California, San Diego8 reported on another cystosolic PLA2 purified from macrophages that differed from Group IV cPLA2 in that its activity was not dependent on Ca2+ and Simon S. Jones from the Genetics Institute9 reported that the cloned form from CHO cells had a very different sequence than cPLA2. This new Ca2+-independent PLA2 (iPLA2) was designated as Group VI PLA2 (GVI).10 Earlier, investigators from the University of Utah11 had isolated an enzyme from human plasma which hydrolyzed platelet activating factor (PAF), a phosphatidylcholine containing an acetate at the sn-2 position, and in 1995 Larry W. Tjoelker from ICOS12 reported its cloning. This enzyme and other related PAF acetyl hydrolases (PAF-AH) were later recognized more broadly as PLA2s with a specificity for a short acyl chain on the sn-2 position and for the plasma one for oxidized lipids for which the same enzyme was independently named lipoproteinassociated phospholipase A2 (Lp-PLA2). These enzymes were designated Group VII and VIII (GVII and GVIII).13 As additional specific PLA2s were discovered, they were either designated by letters as subgroups of the original Groups indicated above or as additional Groups. Especially noteworthy was the discovery of a number of additional sPla2s in which the sequence and/or disulfide bonding pattern varied significantly from the traditional Groups I, II, III, and V sPla2s. These new forms led to the additional Groups IX, X, XI XII,XIII, and XIV sPla2s representing new human forms (especially Group X, which may have important functions) as well unique enzymes from snail venom, rice shoots, parvovirus, and fungi/bacteria. The only new type of PLA2 reported that did not naturally fit in the four types discussed above (secreted, cytosolic, Ca2+-independent, PAF acetylhydrolases) is the lysosomal PLA2 (LPLA2) which was designated as Group XV (GXV).14 Recently, a new PLA2 was isolated from adipose tissue and designated as Group XVI (GXVI);15 it appears to be a new type of PLA2 called adipose-PLA2 (AdPLA). The current designations are summarized in Table 1. Table 1 The Phospholipase A2 Superfamily In this review, we will discuss in turn each of the six types of PLA2. For each, we will first discuss the various forms, in terms of groups, subgroups and mechanism of action, their structure and interaction with membranes, their biological activities and role in disease, and the development of selective inhibitors. Of course the commonly used type designation has little meaning today since as we have learned more about these enzymes, it has been recognized that secreted, cytosolic, Ca2+-independent, PAF-AH, and lysosomal make little sense since all four of the later categories are actually intracellular (cytosolic) enzymes, that the secreted ones may occur intracellularly in various vesicles, and that the PAF-AHs, lysosomal and some forms of cPLA2 are also Ca2+-independent. Thus the Group Numbering System designation provides an unambiguous definition of each enzyme form. Over the years, numerous excellent reviews on either the broad family of PLA2s16 or specific types including sPLA2s,17 cPLA2s,18 iPLA2s,19 PAF-AHs20 and LPLA221 have appeared as well as several review articles summarizing the classes of PLA2 inhibitors and their potential role for the treatment of inflammatory diseases.22 We have employed all of these prior reviews heavily in preparing this up-to-date and comprehensive single review covering all aspects of the entire phospholipase A2 superfamily.

Role of phospholipase in generating lipid second messengers in signal transduction.

Many lipids or lipid‐derived products generated by phospholipases acting on phospholipids in membranes are implicated as mediators and second messengers in signal transduction. Our current understanding of the primary sequence relationships within the class of extracellular phospholipase A2s and among the numerous forms of the mammalian phosphatidylinositol‐specific phospholipase Cs is reviewed. New results suggesting roles for these phospholipases as well as other phospholipases such as phospholipase C and D acting on phosphatidlycholine in generating arachidonic acid for eicosanoid biosynthesis, inositol phosphates for Ca2+ mobilization, and diglyceride for protein kinase C activation through receptor‐mediated processes, are discussed. In addition, the possible role of phospholipases acting on sphingolipids such as sphinglomyelinase in generating lipid mediators is considered.—Dennis, E. A.; Rhee, S. G.; Billah, M. M.; Hannun, Y. A. Role of phospholipases in generating lipid second messengers in signal transduction. FASEB J. 5: 2068–2077; 1991.

LMSD: LIPID MAPS structure database

The LIPID MAPS Structure Database (LMSD) is a relational database encompassing structures and annotations of biologically relevant lipids. Structures of lipids in the database come from four sources: (i) LIPID MAPS Consortiums core laboratories and partners; (ii) lipids identified by LIPID MAPS experiments; (iii) computationally generated structures for appropriate lipid classes; (iv) biologically relevant lipids manually curated from LIPID BANK, LIPIDAT and other public sources. All the lipid structures in LMSD are drawn in a consistent fashion. In addition to a classification-based retrieval of lipids, users can search LMSD using either text-based or structure-based search options. The text-based search implementation supports data retrieval by any combination of these data fields: LIPID MAPS ID, systematic or common name, mass, formula, category, main class, and subclass data fields. The structure-based search, in conjunction with optional data fields, provides the capability to perform a substructure search or exact match for the structure drawn by the user. Search results, in addition to structure and annotations, also include relevant links to external databases. The LMSD is publicly available at

Phospholipase A2 regulation of arachidonic acid mobilization

Phospholipase A2 (PLA2) constitutes a growing superfamily of lipolytic enzymes, and to date, at least 19 distinct enzymes have been found in mammals. This class of enzymes has attracted considerable interest as a pharmacological target in view of its role in lipid signaling and its involvement in a variety of inflammatory conditions. PLA2s hydrolyze the sn‐2 ester bond of cellular phospholipids, producing a free fatty acid and a lysophospholipid, both of which are lipid signaling molecules. The free fatty acid produced is frequently arachidonic acid (AA, 5,8,11,14‐eicosatetraenoic acid), the precursor of the eicosanoid family of potent inflammatory mediators that includes prostaglandins, thromboxanes, leukotrienes and lipoxins. Multiple PLA2 enzymes are active within and surrounding the cell and these enzymes have distinct, but interconnected roles in AA release.

Function and inhibition of intracellular calcium-independent phospholipase A2.

Our previous Minireview (1) considered the three main kinds of phospholipase A2 (PLA2) : the well characterized Groups I, II, and III small Ca-dependent secretory phospholipase A2s (sPLA2), the 85-kDa Group IV Ca -dependent cytosolic phospholipase A2 (cPLA2), and the 80-kDa Ca -independent cytosolic phospholipase A2 (iPLA2). In the ensuing years, it has become clear that PLA2 represents a growing superfamily of enzymes with many additional sPLA2s (Groups IIC, V, and IX), further definition of the 80-kDa iPLA2 (Group VI), and two Ca-independent PLA2s specific for platelet-activating factor (PAF) (Groups VII and VIII) (2). All of the well studied sPLA2s appear to use a His-Asp catalytic mechanism and require Ca to be bound tightly in the active site of the enzyme. The well characterized iPLA2 appears to require a central Ser for catalysis and of course, no Ca. Interestingly, the Group IV cPLA2 does not use Ca 21 for catalysis, but rather the Ca dependence seems to relate to a calcium lipid-binding domain (CaLB or C-2 domain) at the N-terminal end responsible for association of the enzyme with the membrane. Thus, the catalytic mechanism and active site Ser do not involve Ca (3–5); therefore a mechanistic distinction between the Group IV cPLA2 and the iPLA2s may not be warranted at this time. This is relevant because most of the inhibitors that work on the Group IV cPLA2 also act on the Group VI iPLA2 (6, 7). Inhibitor specificity will be discussed in the next section. We (8) recently surveyed all of the reported Ca-independent PLA2 activities. While there exists a group of lysosomal iPLA2s and a group of characterized ectoenzymes with broad specificity, which may actually be general lipases (8), sequenced and well characterized intracellular iPLA2s are limited to the 80-kDa Group VI iPLA2 and the 29-kDa Group VIII enzyme, which is a PAF acetyl hydrolase (9). The latter hydrolyzes the acetyl chain present at the sn-2 position of PAF and perhaps acts on oxidized phospholipids as well but not on normal phospholipids carrying unoxidized long chain fatty acids at the sn-2 position (9). This enzyme and a secreted Group VII PAF acetyl hydrolase, both of which are really iPLA2s with a particular substrate specificity, have been considered elsewhere (2). The Group VI 80-kDa iPLA2 was first identified in P388D1 macrophages (10), purified (11), further characterized (12), and then cloned and sequenced by Jones and co-workers (13) from CHO cells. The CHO iPLA2 has been shown to represent a species variant of that present in P388D1 macrophage-like cells, where the iPLA2 has also been cloned and sequenced (14). The sequence of the Group VI iPLA2 reveals the presence of eight ankyrin-like domains and the G-X-S-X-G motif commonly found in other lipases. Interestingly, no known consensus sequences for posttranslational modification, such as phosphorylation sites, are apparent in the Group VI iPLA2 (13, 14). This is compatible with the possibility that the Group VI iPLA2 acts to remodel membrane phospholipids as a sort of housekeeping enzyme as will be discussed later.

Evolutionary relationships and implications for the regulation of phospholipase A2 from snake venom to human secreted forms

SummaryThe amino acid sequences of 40 secreted phospholipase A2s (PLA2) were aligned and a phylogenetic tree derived that has three main branches corresponding to elapid (group I), viperid (group II), and insect venom types of PLA2. The human pancreatic and recently determined nonpancreatic sequences in the comparison align with the elapid and viperid categories, repectively, indicating that at least two PLA2 genes existed in the vertebrate line before the divergence of reptiles and mammals about 200–300 million years ago. This allows resolution for the first time of major genetic events in the evolution of current PLA2s and the relationship of human PLA2s to those of snake venom, many of which are potent toxins. Implications for possible mechanisms of regulation of mammalian intra- and extracellular PLA2s are discussed, as well as issues relating to the search for the controlling enzymes in arachidonic acid release, prostaglandin generation, and signal transduction.

Regulated Accumulation of Desmosterol Integrates Macrophage Lipid Metabolism and Inflammatory Responses

Inflammation and macrophage foam cells are characteristic features of atherosclerotic lesions, but the mechanisms linking cholesterol accumulation to inflammation and LXR-dependent response pathways are poorly understood. To investigate this relationship, we utilized lipidomic and transcriptomic methods to evaluate the effect of diet and LDL receptor genotype on macrophage foam cell formation within the peritoneal cavities of mice. Foam cell formation was associated with significant changes in hundreds of lipid species and unexpected suppression, rather than activation, of inflammatory gene expression. We provide evidence that regulated accumulation of desmosterol underlies many of the homeostatic responses, including activation of LXR target genes, inhibition of SREBP target genes, selective reprogramming of fatty acid metabolism, and suppression of inflammatory-response genes, observed in macrophage foam cells. These observations suggest that macrophage activation in atherosclerotic lesions results from extrinsic, proinflammatory signals generated within the artery wall that suppress homeostatic and anti-inflammatory functions of desmosterol.