Farideh Ghomashchi

Interfacial Kinetic and Binding Properties of the Complete Set of Human and Mouse Groups I, II, V, X, and XII Secreted Phospholipases A2

Expression of the full set of human and mouse groups I, II, V, X, and XII secreted phospholipases A2 (sPLA2s) in Escherichia coli and insect cells has provided pure recombinant enzymes for detailed comparative interfacial kinetic and binding studies. The set of mammalian sPLA2s display dramatically different sensitivity to dithiothreitol. The specific activity for the hydrolysis of vesicles of differing phospholipid composition by these enzymes varies by up to 4 orders of magnitude, and yet all enzymes display similar catalytic site specificity toward phospholipids with different polar head groups. Discrimination between sn-2 polyunsaturated versus saturated fatty acyl chains is <6-fold. These enzymes display apparent dissociation constants for activation by calcium in the 1–225 μm range, depending on the phospholipid substrate. Analysis of the inhibition by a set of 12 active site-directed, competitive inhibitors reveals a large variation in the potency among the mammalian sPLA2s, with Me-Indoxam being the most generally potent sPLA2 inhibitor. A dramatic correlation exists between the ability of the sPLA2s to hydrolyze phosphatidylcholine-rich vesicles efficiently in vitro and the ability to release arachidonic acid when added exogenously to mammalian cells; the group V and X sPLA2s are uniquely efficient in this regard.

Groups IV, V, and X Phospholipases A2s in Human Neutrophils ROLE IN EICOSANOID PRODUCTION AND GRAM-NEGATIVE BACTERIAL PHOSPHOLIPID HYDROLYSIS

The bacterial tripeptide formyl-Met-Leu-Phe (fMLP) induces the secretion of enzyme(s) with phospholipase A2 (PLA2) activity from human neutrophils. We show that circulating human neutrophils express groups V and X sPLA2 (GV and GX sPLA2) mRNA and contain GV and GX sPLA2 proteins, whereas GIB, GIIA, GIID, GIIE, GIIF, GIII, and GXII sPLA2s are undetectable. GV sPLA2 is a component of both azurophilic and specific granules, whereas GX sPLA2 is confined to azurophilic granules. Exposure to fMLP or opsonized zymosan results in the release of GV but not GX sPLA2 and most, if not all, of the PLA2 activity in the extracellular fluid of fMLP-stimulated neutrophils is due to GV sPLA2. GV sPLA2 does not contribute to fMLP-stimulated leukotriene B4 production but may support the anti-bacterial properties of the neutrophil, because 10–100 ng per ml concentrations of this enzyme lead to Gram-negative bacterial membrane phospholipid hydrolysis in the presence of human serum. By use of a recently described and specific inhibitor of cytosolic PLA2-α (group IV PLA2α), we show that this enzyme produces virtually all of the arachidonic acid used for the biosynthesis of leukotriene B4 in fMLP- and opsonized zymosan-stimulated neutrophils, the major eicosanoid produced by these pro-inflammatory cells.

On the Diversity of Secreted Phospholipases A2 CLONING, TISSUE DISTRIBUTION, AND FUNCTIONAL EXPRESSION OF TWO NOVEL MOUSE GROUP II ENZYMES

Over the last decade, an expanding diversity of secreted phospholipases A2 (sPLA2s) has been identified in mammals. Here, we report the cloning in mice of three additional sPLA2s called mouse group IIE (mGIIE), IIF (mGIIF), and X (mGX) sPLA2s, thus giving rise to eight distinct sPLA2s in this species. Both mGIIE and mGIIF sPLA2s contain the typical cysteines of group II sPLA2s, but have relatively low levels of identity (less than 51%) with other mouse sPLA2s, indicating that these enzymes are novel group II sPLA2s. However, a unique feature of mGIIF sPLA2 is the presence of a C-terminal extension of 23 amino acids containing a single cysteine. mGX sPLA2 has 72% identity with the previously cloned human group X (hGX) sPLA2 and displays similar structural features, making it likely that mGX sPLA2 is the ortholog of hGX sPLA2. Genes for mGIIE and mGIIF sPLA2s are located on chromosome 4, and that of mGX sPLA2 on chromosome 16. Northern and dot blot experiments with 22 tissues indicate that all eight mouse sPLA2s have different tissue distributions, suggesting specific functions for each. mGIIE sPLA2 is highly expressed in uterus, and at lower levels in various other tissues. mGIIF sPLA2 is strongly expressed during embryogenesis and in adult testis. mGX sPLA2 is mostly expressed in adult testis and stomach. When the cDNAs for the eight mouse sPLA2s were transiently transfected in COS cells, sPLA2 activity was found to accumulate in cell medium, indicating that each enzyme is secreted and catalytically active. Using COS cell medium as a source of enzymes, pH rate profile and phospholipid headgroup specificity of the novel sPLA2s were analyzed and compared with the other mouse sPLA2s.

A pyrrolidine-based specific inhibitor of cytosolic phospholipase A2α blocks arachidonic acid release in a variety of mammalian cells

We analyzed a recently reported (K. Seno, T. Okuno, K. Nishi, Y. Murakami, F. Watanabe, T. Matsuur, M. Wada, Y. Fujii, M. Yamada, T. Ogawa, T. Okada, H. Hashizume, M. Kii, S.-H. Hara, S. Hagishita, S. Nakamoto, J. Med. Chem. 43 (2000)) pyrrolidine-based inhibitor, pyrrolidine-1, against the human group IV cytosolic phospholipase A(2) alpha-isoform (cPLA(2)alpha). Pyrrolidine-1 inhibits cPLA(2)alpha by 50% when present at approx. 0.002 mole fraction in the interface in a number of in vitro assays. It is much less potent on the cPLA(2)gamma isoform, calcium-independent group VI PLA(2) and groups IIA, X, and V secreted PLA(2)s. Pyrrolidine-1 blocked all of the arachidonic acid released in Ca(2+) ionophore-stimulated CHO cells stably transfected with cPLA(2)alpha, in zymosan- and okadaic acid-stimulated mouse peritoneal macrophages, and in ATP- and Ca(2+) ionophore-stimulated MDCK cells.

Localization of Structural Elements of Bee Venom Phospholipase A2 Involved in N-type Receptor Binding and Neurotoxicity

We have shown previously that neurotoxic venom secretory phospholipases A2 (sPLA2s) have specific receptors in brain membranes called N-type receptors that are likely to play a role in the molecular events leading to neurotoxicity of these proteins. The sPLA2 found in honey bee venom is neurotoxic and binds to this receptor with high affinity. In this paper, we have used a number of mutants of bee venom sPLA2 produced in Escherichia coli to determine the structural elements of this protein that are involved in its binding to N-type receptors. Mutations in the interfacial binding surface, in the Ca2+-binding loop and in the hydrophobic channel lead to a dramatic decrease in binding to N-type receptors, whereas mutations of surface residues localized in other parts of the sPLA2 structure do not significantly modify the binding properties. Neurotoxicity experiments show that mutants with low affinity for N-type receptors are devoid of neurotoxic properties, even though some of them retain high enzymatic activity. These results provide further evidence for the involvement of N-type receptors in neurotoxic processes associated with venom sPLA2s and identify the surface region surrounding the hydrophobic channel of bee venom sPLA2 as the N-type receptor recognition domain.

A novel anti-inflammatory role for secretory phospholipase A2 in immune complex-mediated arthritis

Phospholipase A2 (PLA2) catalyses the release of arachidonic acid for generation of lipid mediators of inflammation and is crucial in diverse inflammatory processes. The functions of the secretory PLA2 enzymes (sPLA2), numbering nine members in humans, are poorly understood, though they have been shown to participate in lipid mediator generation and the associated inflammation. To further understand the roles of sPLA2 in disease, we quantified the expression of these enzymes in the synovial fluid in rheumatoid arthritis and used gene‐deleted mice to examine their contribution in a mouse model of autoimmune erosive inflammatory arthritis. Contrary to expectation, we find that the group V sPLA2 isoform plays a novel anti‐inflammatory role that opposes the pro‐inflammatory activity of group IIA sPLA2. Mechanistically, group V sPLA2 counter‐regulation includes promotion of immune complex clearance by regulating cysteinyl leukotriene synthesis. These observations identify a novel anti‐inflammatory function for a PLA2 and identify group V sPLA2 as a potential biotherapeutic for treatment of immune‐complex‐mediated inflammation.

Crystal Structure of Human Group X Secreted Phospholipase A2 ELECTROSTATICALLY NEUTRAL INTERFACIAL BINDING SURFACE TARGETS ZWITTERIONIC MEMBRANES

The crystal structure of human group X (hGX) secreted phospholipase A2 (sPLA2) has been solved to a resolution of 1.97 Å. As expected the protein fold is similar to previously reported sPLA2 structures. The active site architecture, including the positions of the catalytic residues and the first and second shell water around the Ca2+ cofactor, are highly conserved and remarkably similar to the group IB and group IIA enzymes. Differences are seen in the structures following the (1–12)-N-terminal helix and at the C terminus. These regions are proposed to interact with the substrate membrane surface. The opening to the active site slot is considerably larger in hGX than in human group IIA sPLA2. Furthermore, the electrostatic surface potential of the hGX interfacial-binding surface does not resemble that of the human group IIA sPLA2; the former is highly neutral, whereas the latter is highly cationic. The cationic residues on this face of group IB and IIA enzymes have been implicated in membrane binding and ink cat * allostery. In contrast, hGX does not show activation by the anionic charge at the lipid interface when acting on phospholipid vesicles or short-chain phospholipid micelles. Together, the crystal structure and kinetic results of hGX supports the conclusion that it is as active on zwitterionic as on anionic interfaces, and thus it is predicted to target the zwitterionic membrane surfaces of mammalian cells.

Trifluoromethyl ketones and methyl fluorophosphonates as inhibitors of group IV and VI phospholipases A(2): structure-function studies with vesicle, micelle, and membrane assays.

A series of fatty alkyl trifluoromethyl ketones and methyl fluorophosphonates have been prepared and tested as inhibitors and inactivators of human groups IV and VI phospholipases A(2) (cPLA(2) and iPLA(2)). Compounds were analyzed with phospholipid vesicle-, detergent-phospholipid mixed-micelle-, and natural membrane-based assays, and, with few exceptions, the relative inhibitor potencies measured with the three assays were similar. Ph(CH(2))(4)COCF(3) and Ph(CH(2))(4)PO(OMe)F emerged as a potent inhibitor and inactivator, respectively, of iPLA(2), and both are poorly effective against cPLA(2). Of all 13 fatty alkyl trifluoromethyl ketones tested, the trifluoromethyl ketone analog of arachidonic acid is the most potent cPLA(2) inhibitor, and structurally similar compounds including the trifluoromethyl ketone analog of docosahexenoic acid are much poorer cPLA(2) inhibitors. Inactivation of cPLA(2) by fatty alkyl fluoromethylphosphonates is greatly promoted by binding of enzyme to the interface. The use of both vesicles and mixed micelles to assay phospholipase A(2) inhibitors and inactivators present at low mol fraction in the interface provides reliable rank order potencies of a series of compounds that correlate with their behavior in a natural membrane assay.

Role of phosphorylation and basic residues in the catalytic domain of cytosolic phospholipase A2α in regulating interfacial kinetics and binding and cellular function

Group IVA cytosolic phospholipase A2 (cPLA2α) is regulated by phosphorylation and calcium-induced translocation to membranes. Immortalized mouse lung fibroblasts lacking endogenous cPLA2α (IMLF-/-) were reconstituted with wild type and cPLA2α mutants to investigate how calcium, phosphorylation, and the putative phosphatidylinositol 4,5-bisphosphate (PIP2) binding site regulate translocation and arachidonic acid (AA) release. Agonists that elicit distinct modes of calcium mobilization were used. Serum induced cPLA2α translocation to Golgi within seconds that temporally paralleled the initial calcium transient. However, the subsequent influx of extracellular calcium was essential for stable binding of cPLA2α to Golgi and AA release. In contrast, phorbol 12-myristate 13-acetate induced low amplitude calcium oscillations, slower translocation of cPLA2α to Golgi, and much less AA release, which were blocked by chelating extracellular calcium. AA release from IMLF-/- expressing phosphorylation site (S505A) and PIP2 binding site (K488N/K543N/K544N) mutants was partially reduced compared with cells expressing wild type cPLA2α, but calcium-induced translocation was not impaired. Consistent with these results, Ser-505 phosphorylation did not change the calcium requirement for interfacial binding and catalysis in vitro but increased activity by 2-fold. Mutations in basic residues in the catalytic domain of cPLA2α reduced activation by PIP2 but did not affect the concentration of calcium required for interfacial binding or phospholipid hydrolysis. The results demonstrate that Ser-505 phosphorylation and basic residues in the catalytic domain principally act to regulate cPLA2α hydrolytic activity.

Function, Activity, and Membrane Targeting of Cytosolic Phospholipase A2ζ in Mouse Lung Fibroblasts

Group IVA cytosolic phospholipase A2 (cPLA2α) initiates eicosanoid production; however, this pathway is not completely ablated in cPLA2α–/– lung fibroblasts stimulated with A23187 or serum. cPLA2α+/+ fibroblasts preferentially released arachidonic acid, but A23187-stimulated cPLA2α–/– fibroblasts nonspecifically released multiple fatty acids. Arachidonic acid release from cPLA2 α–/– fibroblasts was inhibited by the cPLA2α inhibitors pyrrolidine-2 (IC50, 0.03 μm) and Wyeth-1 (IC50, 0.1 μm), implicating another C2 domain-containing group IV PLA2. cPLA2 α–/– fibroblasts contain cPLA2β and cPLA2ζ but not cPLA2ϵ or cPLA2δ. Purified cPLA2ζ exhibited much higher lysophospholipase and PLA2 activity than cPLA2β and was potently inhibited by pyrrolidine-2 and Wyeth-1, which did not inhibit cPLA2β. In contrast to cPLA2β, cPLA2ζ expressed in Sf9 cells mediated A23187-induced arachidonic acid release, which was inhibited by pyrrolidine-2 and Wyeth-1. cPLA2ζ exhibits specific activity, inhibitor sensitivity, and low micromolar calcium dependence similar to cPLA2α and has been identified as the PLA2 responsible for calcium-induced fatty acid release and prostaglandin E2 production from cPLA2 α–/– lung fibroblasts. In response to ionomycin, EGFP-cPLA2ζ translocated to ruffles and dynamic vesicular structures, whereas EGFP-cPLA2α translocated to the Golgi and endoplasmic reticulum, suggesting distinct mechanisms of regulation for the two enzymes.