Yuan-Hao Hsu

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.

Localizing the membrane binding region of Group VIA Ca2+-independent phospholipase A2 using peptide amide hydrogen/deuterium exchange mass spectrometry.

The Group VIA-2 Ca2+-independent phospholipase A2 (GVIA-2 iPLA2) is composed of seven consecutive N-terminal ankyrin repeats, a linker region, and a C-terminal phospholipase catalytic domain. No structural information exists for this enzyme, and no information is known about the membrane binding surface. We carried out deuterium exchange experiments with the GVIA-2 iPLA2 in the presence of both phospholipid substrate and the covalent inhibitor methyl arachidonoyl fluorophosphonate and located regions in the protein that change upon lipid binding. No changes were seen in the presence of only methyl arachidonoyl fluorophosphonate. The region with the greatest change upon lipid binding was region 708–730, which showed a >70% decrease in deuteration levels at numerous time points. No decreases in exchange due to phospholipid binding were seen in the ankyrin repeat domain of the protein. To locate regions with changes in exchange on the enzyme, we constructed a computational homology model based on homologous structures. This model was validated by comparing the deuterium exchange results with the predicted structure. Our model combined with the deuterium exchange results in the presence of lipid substrate have allowed us to propose the first structural model of GVIA-2 iPLA2 as well as the interfacial lipid binding region.

Potent and selective fluoroketone inhibitors of group VIA calcium-independent phospholipase A2.

Group VIA calcium-independent phospholipase A(2) (GVIA iPLA(2)) has recently emerged as a novel pharmaceutical target. We have now explored the structure-activity relationship between fluoroketones and GVIA iPLA(2) inhibition. The presence of a naphthyl group proved to be of paramount importance. 1,1,1-Trifluoro-6-(naphthalen-2-yl)hexan-2-one (FKGK18) is the most potent inhibitor of GVIA iPLA(2) (X(I)(50) = 0.0002) ever reported. Being 195 and >455 times more potent for GVIA iPLA(2) than for GIVA cPLA(2) and GV sPLA(2), respectively, makes it a valuable tool to explore the role of GVIA iPLA(2) in cells and in vivo models. 1,1,1,2,2,3,3-Heptafluoro-8-(naphthalene-2-yl)octan-4-one inhibited GVIA iPLA(2) with a X(I)(50) value of 0.001 while inhibiting the other intracellular GIVA cPLA(2) and GV sPLA(2) at least 90 times less potently. Hexa- and octafluoro ketones were also found to be potent inhibitors of GVIA iPLA(2); however, they are not selective.

A Phospholipid Substrate Molecule Residing in the Membrane Surface Mediates Opening of the Lid Region in Group IVA Cytosolic Phospholipase A2

The Group IVA (GIVA) phospholipase A2 associates with natural membranes in response to an increase in intracellular Ca2+ along with increases in certain lipid mediators. This enzyme associates with the membrane surface as well as binding a single phospholipid molecule in the active site for catalysis. Employing deuterium exchange mass spectrometry, we have identified the regions of the protein binding the lipid surface and conformational changes upon a single phospholipid binding in the absence of a lipid surface. Experiments were carried out using natural palmitoyl arachidonyl phosphatidylcholine vesicles with the intact GIVA enzyme as well as the isolated C2 and catalytic domains. Lipid binding produced changes in deuterium exchange in eight different regions of the protein. The regions with decreased exchange included Ca2+ binding loop one, which has been proposed to penetrate the membrane surface, and a charged patch of residues, which may be important in interacting with the polar head groups of phospholipids. The regions with an increase in exchange are all located either in the hydrophobic core underneath the lid region or near the lid and hinge regions from 403 to 457. Using the GIVA phospholipase A2 irreversible inhibitor methyl-arachidonyl fluorophosphonate, we were able to isolate structural changes caused only by pseudo-substrate binding. This produced results that were very similar to natural lipid binding in the presence of a lipid interface with the exception of the C2 domain and region 466-470. This implies that most of the changes seen in the catalytic domain are due to a substrate-mediated, not interface-mediated, lid opening, which exposes the active site to water. Finally experiments carried out with inhibitor plus phospholipid vesicles showed decreases at the C2 domain as well as charged residues on the putative membrane binding surface of the catalytic domain revealing the binding sites of the enzyme to the lipid surface.

Calcium binding rigidifies the C2 domain and the intradomain interaction of GIVA phospholipase A2 as revealed by hydrogen/deuterium exchange mass spectrometry.

The GIVA phospholipase A2 (PLA2) contains two domains: a calcium-binding domain (C2) and a catalytic domain. These domains are linked via a flexible tether. GIVA PLA2 activity is Ca2+-dependent in that calcium binding promotes protein docking to the phospholipid membrane. In addition, the catalytic domain has a lid that covers the active site, presumably regulating GIVA PLA2 activity. We now present studies that explore the dynamics and conformational changes of this enzyme in solution utilizing peptide amide hydrogen/deuterium (H/D) exchange coupled with liquid chromatographymass spectrometry (DXMS) to probe the solvent accessibility and backbone flexibility of the C2 domain, the catalytic domain, and the intact GIVA PLA2. We also analyzed the changes in H/D exchange of the intact GIVA PLA2 upon Ca2+ binding. The DXMS results showed a fast H/D-exchanging lid and a slow exchanging central core. The C2 domain showed two distinct regions: a fast exchanging region facing away from the catalytic domain and a slow exchanging region present in the “cleft” region between the C2 and catalytic domains. The slow exchanging region of the C2 domain is in tight proximity to the catalytic domain. The effects of Ca2+ binding on GIVA PLA2 are localized in the C2 domain and suggest that binding of two distinct Ca2+ ions causes tightening up of the regions that surround the anion hole at the tip of the C2 domain. This conformational change may be the initial step in GIVA PLA2 activation.

Assessing Phospholipase A2 Activity toward Cardiolipin by Mass Spectrometry

Cardiolipin, a major component of mitochondria, is critical for mitochondrial functioning including the regulation of cytochrome c release during apoptosis and proper electron transport. Mitochondrial cardiolipin with its unique bulky amphipathic structure is a potential substrate for phospholipase A2 (PLA2) in vivo. We have developed mass spectrometric methodology for analyzing PLA2 activity toward various cardiolipin forms and demonstrate that cardiolipin is a substrate for sPLA2, cPLA2 and iPLA2, but not for Lp-PLA2. Our results also show that none of these PLA2s have significant PLA1 activities toward dilyso-cardiolipin. To understand the mechanism of cardiolipin hydrolysis by PLA2, we also quantified the release of monolyso-cardiolipin and dilyso-cardiolipin in the PLA2 assays. The sPLA2s caused an accumulation of dilyso-cardiolipin, in contrast to iPLA2 which caused an accumulation of monolyso-cardiolipin. Moreover, cardiolipin inhibits iPLA2 and cPLA2, and activates sPLA2 at low mol fractions in mixed micelles of Triton X-100 with the substrate 1-palmitoyl-2-arachidonyl-sn-phosphtidylcholine. Thus, cardiolipin functions as both a substrate and a regulator of PLA2 activity and the ability to assay the various forms of PLA2 is important in understanding its function.

Fluoroketone Inhibitionof Ca2+-Independent Phospholipase A2 through Binding Pocket Association Definedby Hydrogen/Deuterium Exchange and Molecular Dynamics

The mechanism of inhibition of group VIA Ca2+-independent phospholipase A2 (iPLA2) by fluoroketone (FK) ligands is examined by a combination of deuterium exchange mass spectrometry (DXMS) and molecular dynamics (MD). Models for iPLA2 were built by homology with the known structure of patatin and equilibrated by extensive MD simulations. Empty pockets were identified during the simulations and studied for their ability to accommodate FK inhibitors. Ligand docking techniques showed that the potent inhibitor 1,1,1,3-tetrafluoro-7-phenylheptan-2-one (PHFK) forms favorable interactions inside an active-site pocket, where it blocks the entrance of phospholipid substrates. The polar fluoroketone headgroup is stabilized by hydrogen bonds with residues Gly486, Gly487, and Ser519. The nonpolar aliphatic chain and aromatic group are stabilized by hydrophobic contacts with Met544, Val548, Phe549, Leu560, and Ala640. The binding mode is supported by DXMS experiments showing an important decrease of deuteration in the contact regions in the presence of the inhibitor. The discovery of the precise binding mode of FK ligands to the iPLA2 should greatly improve our ability to design new inhibitors with higher potency and selectivity.

Lipoprotein-Associated Phospholipase A2 Interacts with Phospholipid Vesicles via a Surface-Disposed Hydrophobic α-Helix

Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) plays important roles in both the inhibition and promotion of inflammation in human disease. It catalyzes the hydrolytic inactivation of plasma platelet activating factor (PAF) and is also known as PAF acetylhydrolase. High levels of PAF are implicated in a variety of inflammatory diseases such as asthma, necrotizing enterocolitis, and sepsis. Lp-PLA(2) also associates with lipoproteins in human plasma where it hydrolyzes oxidized phospholipids to produce pro-inflammatory lipid mediators that can promote inflammation and the development of atherosclerosis. Lp-PLA(2) plasma levels have recently been identified as a biomarker of vascular inflammation, atherosclerotic vulnerability, and future cardiovascular events. The enzyme is thus a prominent target for the development of inflammation and atherosclerosis-modulating therapeutics. While the crystallographically determined structure of the enzyme is known, the enzymes mechanism of interaction with PAF and the function-modulating lipids in lipoproteins is unknown. We have employed peptide amide hydrogen-deuterium exchange mass spectrometry (DXMS) to characterize the association of Lp-PLA(2) with dimyristoylphosphatidylcholine (DMPC) vesicles and found that specific residues 113-120 in one of the enzymes surface-disposed hydrophobic α-helices likely mediate liposome binding.

Structural basis of specific interactions of Lp-PLA2 with HDL revealed by hydrogen deuterium exchange mass spectrometry

Lipoprotein-associated phospholipase A2 (Lp-PLA2), specifically Group VIIA PLA2, is a member of the phospholipase A2 superfamily and is found mainly associated with LDL and HDL in human plasma. Lp-PLA2 is considered as a risk factor, a potential biomarker, a target for therapy in the treatment of cardiovascular disease, and evidence suggests that the level of Lp-PLA2 in plasma is associated with the risk of future cardiovascular and stroke events. The differential location of the enzyme in LDL/HDL lipoproteins has been suggested to affect Lp-PLA2 function and/or its physiological role and an abnormal distribution of the enzyme may correlate with diseases. Although a mutagenesis study suggested that a surface helix (residues 362–369) mediates the association between Lp-PLA2 and HDL, the molecular details and mechanism of association has remained unknown. We have now employed hydrogen deuterium exchange mass spectrometry to characterize the interaction between recombinant human Lp-PLA2 and human HDL. We have found that specific residues 113–120, 192–204, and 360–368 likely mediate HDL binding. In a previous study, we showed that residues 113–120 are important for Lp-PLA2-liposome interactions. We now find that residues 192–204 show a decreased deuteration level when Lp-PLA2 is exposed to apoA-I, but not apoA-II, the most abundant apoproteins in HDL, and additionally, residues 360–368 are only affected by HDL.The results suggest that apoA-I and phospholipid membranes play crucial roles in Lp-PLA2 localization to HDL.

Correction to “Fluoroketone Inhibition of Ca2+-Independent Phospholipase A2 through BindingPocket Association Defined by Hydrogen/Deuterium Exchange and MolecularDynamics”.

Page 1334. In the caption for Figure 6, the identification of the red circle and black square symbols is reversed. The correct caption should read as follows: Figure 6 Regions of H/D exchange most affected by PHFK binding. The deuteron number is shown for each fragment in the presence (red circles) and absence (black squares) of inhibitor. The black and red curves indicate the number of H/D exchanges at six time points ...