PLA₂: A Short Phospholipase Review

by Kirk Maxey, M.D. and Jim MacDonald

The first characterization of lipases which are selective for the phospholipid sn-2 acyl group followed the observation that human pancreatic juice and snake venom could hydrolyze egg lecithin, liberating free fatty acids. Isolation of this activity revealed enzymes that were small and extensively crosslinked by cysteine disulfide bonds, making them relatively stable, rigid, water-soluble proteins. The high degree of homology between enzymes of such divergent species is less surprising when one considers that venoms are the products of modified salivary glands, and both salivary and pancreatic glands share functional and embryonic kinship. Based on the presence or absence of short sequences flanking an internal cysteine and the Cterminus (i.e., the carboxy extension, the elapid loop, and the pancreatic loop, see Figure 2), the enzymes were placed into groups I and II, each subdivided into subgroups A and B.

Recently at least 4 new types of small, calcium-dependent phospholipases have been characterized. In addition, several larger, calcium-independent A₂ phospholipases with completely different active site chemistry have also been described. These new members were added sequentially to the early members of the family with corresponding roman numerals which now reach at least X. New and unique functions accompanied the new enzymes creating an expanded family which now includes:

• Small, indiscriminate enzymes designed to digest food and kill or injure prey.
• Small, more selective enzymes which release specific fatty acids during signal transduction and inflammation.
• Mostly large, highly selective enzymes that function as initiators of signal transduction and modulate the fatty acid composition of membranes.
• Medium-sized signal-terminators specific for short sn-2 acyl groups which inactivate PAF and remove oxidized phospholipids.

The complete spectrum of PLA₂ activities and homology is given in Figure 1. There are essentially two large subgroups within the phospholipase family. In one group are enzymes that are all small, extensively disulfide-crosslinked and usually secreted; they are the sPLA₂ group. This group requires mM calcium for activity, and has a calcium ion in the active site participating in the hydrolysis reaction. The prototype for this group is the platelet sPLA₂ IIA (see Figure 2). In the second group are mechanistically distinct esterase-like enzymes which utilize a serine nucleophile contained within an active site G-X-S-X-G motif. Enzymatic function is calcium independent, and most are inhibited by suicide substrates like bromoenol lactone (BEL) and methylarachidonyl fluorophosphonate (MAFP). This calcium independent, iPLA₂ group includes the well-studied 85 kDa cytosolic PLA₂ as well as the PAF acetyl hydrolases. (While the Type IV enzyme requires µM calcium for membrane binding and full activity, this is unrelated to the active site chemistry.) The term “cytosolic” has been used to designate PLA₂ subgroups, but enzymes from both the His-Asp and the serine-lipase families are found on both sides of the plasma membrane, making this designation somewhat confusing. The term cPLA₂ is best used to designate strictly the 85 kDa, Type IV enzyme which is ubiquitous in vertebrate cells. A brief description of the key attributes of fifteen well-described A₂ phospholipases is given below.

The Small, Secretory His-Asp PLA₂ Enzymes

Venoms

IA - ≈14 kDa; found in cobra and krait venom. Has elapid loop, lacks pancreatic loop and carboxyl extension. Non-selective for sn-2 acyl residue or polar head group. His-Asp active site residues; requires mM calcium.

IIB - ≈14 kDa found in gaboon viper. Has carboxyl extension, elapid loop, but lacks pancreatic loop. His-Asp active site residues; requires mM calcium.

III - ≈14 kDa, relatively divergent enzyme from bee and lizard venom. Contains active site His-Asp but displaced 10-12 residues toward the N-terminus - elapid and pancreatic loops are not defined. Only 8 cysteine residues compared with 14 in most other sPLA₂s.

IX - ≈14 kDa, divergent venom PLA₂ from predatory marine cone snail. His-Asp doublet is at residue 36-37, 12 cysteine residues, low phospholipid selectivity.

Pancreas

IB - ≈14 kDa, has His-Asp active site residues; requires mM calcium; 14 cysteines and most residues of both the elapid loop and pancreatic loop - lacks the carboxyl extension. Non-selective toward phospholipid substrate.

Venoms, Platelets, Synoviocytes & Other Cells

IIA - ≈14 kDa, has His-Asp active site residues; requires mM calcium; 14 cysteines and most residues of the elapid loop but not pancreatic loop; has the carboxyl extension. Released at inflammatory sites and liberates free arachidonic acid; transcriptional regulation by proinflammatory mediators.

IIC - ≈14 kDa, has His-Asp active site residues; requires mM calcium; 14 cysteines and most residues of both the elapid loop and pancreatic loop - lacks the carboxyl extension. Inactive pseudogene in humans, expressed in mice and rats. Part of a tightly linked family that includes IIA and V on human chromosome 1.

V - ≈14 kDa, has His-Asp active site residues; requires mM calcium; 14 cysteines and most residues of both the elapid loop and pancreatic loop - lacks the carboxyl extension. Present in mast cells, macrophages where it functions in inflammation and signal transduction. Part of the IIA, IIC linkage group.

X - ≈14 kDa, has His-Asp active site carboxyl extension, elapid loop, 16 cysteine residues. Cloned from lung and expressed prominently in spleen, thymus and leukocytes.

The Large, Serine Esterase-type PLA₂ Enzymes

IV - The 85 kDa, cytosolic enzyme which is selective for arachidonate at sn-2 and is tightly coupled to signal transduction. Activated by MAPK phosphorylation of serine 505; translocates from cytosol to nuclear envelope. CalB domain; requires µM Ca⁺⁺ for membrane association and full activity. Ubiquitous expression in all cells. G-X-S-G-L active site serine irreversibly inhibited by arachidonyl trifluoromethyl ketone (AACOCF₃) and MAFP.

VI - 80-85 kDa homotetramer with multiple ankyrin repeats. G-X-S-X-G active site serine irreversibly inhibited by BEL, AACOCF₃, and MAFP. Mobilizes arachidonate in stimulated P388D1 macrophages.

VII - 45 kDa enzyme associated mainly with the human plasma LDL fraction. GX-S-X-G active site and catalytic triad composed of ²⁷³Ser, ²⁹⁶Asp, and ³⁵¹His. Named plasma PAF-acetylhydrolase (PAF-AH) for its preference for short and/or oxidized sn-2 acyl groups.

VIII - Intracellular heterotrimer composed of 29, 30, and 40 kDa subunits. The 29 and 30 kDa subunits are highly homologous and both act as specific PAFacetylhydrolases; both contain the active site GDS. Also called PAF-AH isomer IB. Only accepts PAF and not oxidized phospholipids as substrate.

(XI) - Intracellular 40 kDa monomeric PAF-AH has high (41%) homology to PLA₂ VII (plasma PAF-AH), both serine and cysteine active site residues, and accepts short and/or oxidized sn-2 acyl groups.

(XII) - 40 kDa calcium-independent PLA₂ isolated from myocardium, inhibited by BEL. Prefers sn-2 arachidonyl plasmalogen substrates, and is regulated by Ca⁺⁺/calmodulin inhibition.

(XIII) - 28 kDa calcium-independent PLA₂ isolated from rabbit kidney, inhibited by BEL. Selectively releases arachidonic acid from plasmalogen substrates

Functional Aspects

It is now appreciated that the phospholipases play many roles in cell growth, inflammation, and disease. Phospholipases perform mundane homeostatic chores such as processing nutrients for transport and grooming the plasma membrane. They supply the munitions for external and domestic attacks on invaders, and help to open and close the essential communication links between cells. PLA₂-IIA secreted by an activated platelet can bind to receptors on adjacent leukocyte or fibroblast membranes and release free arachidonate for PGH₂ or LTA₄ synthesis by any of several adjacent cells (See Figure 3). PLA₂-VII (PAF-AH) circulates through this same intracellular space, acting to terminate proinflammatory platelet activating factor and damping down the irritant signals of oxidatively deranged membrane phospholipids. Control over the small calcium dependent enzymes is exerted through synthesis as inactive proenzymes, sequestration in cell granules, and release on specific stimulation. Their synthesis is also transcriptionally regulated. The larger serine-esterase enzymes possess higher substrate selectivity and tend to be more tightly regulated. Both serine phosphorylation and calcium-mediated membrane translocation are required before PLA₂-IV will selectively release free arachidonate to a sequestered pool of pro-inflammatory enzymes. Recently discovered A₂ phospholipases in rabbit myocardium and kidney appear to release arachidonate selectively from plasmalogen phospholipids. The inhibition of the myocardial enzyme by Ca⁺⁺/calmodulin suggests that these new enzymes and their congeners represent an extension of the tightly regulated, functionally-specific phospholipases.

As more is learned about the intricate coupling of phospholipid metabolism and signal transduction, further discoveries and refinements will be made to our understanding of the esterases which hydrolize glycerophospholipids at the sn-2 position.

The authors wish to express their thanks to Linda Pobocik and Donna Barger for creating the illustrations, and to Leslie Crofford and Ed Dennis for thoughtful advice.

PLA₂ Reading List

1. Dennis, E.A. Phospholipases. Enzymes XVI, 307-353 (1983).
2. Dennis, E.A. Diversity of group types, regulation, and function of phospholipase A₂. J. Biol. Chem. 269, 1305713060 (1994).
3. Leslie, C.C. Properties and regulation of cytosolic phospholipase A₂. J. Biol. Chem. 272, 16709-16712 (1997).
4. Tischfield, J.A. A reassessment of the low molecular weight phospholipase A₂ Gene family in mammals. J. Biol.Chem. 272, 17247-17250 (1997).
5. Murakami, M., Nakatani, Y., Atsumi, G., et al. Regulatory functions of phospholipase A₂. Crit. Rev. Immunol. 17, 225-283 (1997).
6. Huang, M.Z., Gopalakrishnakone, P., Chung, M.C.M., et al. Complete amino acid sequence of an acidic, cardiotoxic phospholipase A₂ from the venom of Ophiophagus hannah (King cobra): A novel cobra venom enzyme with “pancreatic loop”. Arch. Biochem. Biophys. 338, 150-156 (1997).
7. Tang, J., Kriz, R.W., Wolfman, N., et al. A novel cytosolic calcium-independent phospholipase A₂ contains eight ankyrin motifs. J. Biol. Chem. 272, 8567-8575 (1997).
8. Kitadokoro, K., Hagishita, S., Sato, T., et al. Crystal structure of human secretory phospholipase A₂-IIA complex with the potent indolizine inhibitor 120-1032. J. Biochem. 123, 619-623 (1998).
9. Portilla, D. and Dai, G. Purification of a novel calciumindependent phospholipase A₂ from rabbit kidney. J. Biol. Chem. 271, 15451-15457 (1996).
10. Reddy, S.T., Winstead, M.V., Tischfield, J.A., et al. Analysis of the secretory phospholipase A₂ that mediates prostaglandin production in mast cells. J. Biol. Chem. 272, 13591-13596 (1997).
11. Tischfield, J.A., Xia, Y., Shih, D.M., et al. Low-molecular-weight, calcium-dependent phospholipase A₂ genes are linked and map to homologous chromosome regions in mouse and human. Genomics 32, 328-333 (1996).
12. Wolf, M.J. and Gross, R.W. The calcium-dependent association and functional coupling of calmodulin with myocardial phospholipase A₂. Implications for cardiac cycle-dependent alterations in phospholipolysis. J. Biol. Chem. 271, 20989-20992 (1996).
13. Ackermann, E.J., Kempner, E.S., and Dennis, E.A. Ca²⁺ independent cytosolic phospholipase A₂ from macrophage-like P388D1 cells. J. Biol. Chem. 269, 9227-9233 (1994).
14. Pickard, R.T., Chiou, X.G., Strifler, B.A., et al. Identification of essential residues for the catalytic function of 85 kDa cytosolic phospholipase A₂. Probing the role of histidine, aspartic acid, cysteine, and arginine. J. Biol. Chem. 271, 19225-19231 (1996).
15. Qiu, Z., Gijón, M.A., de Carvalho, M.S., et al. The role of calcium and phosphorylation of cytosolic phospholipase A₂ in regulating arachidonic acid release in macrophages. J. Biol. Chem. 273, 8203-8211 (1998).
16. Balboa, M.A., Balsinde, J., Winstead, M.V., et al. Novel group V phospholipase A₂ involved in arachidonic acid mobilization in murine P388D1 macrophages. J. Biol. Chem. 271, 32381-32384 (1996).
17. Balsinde, J. and Dennis, E.A. Distinct roles in signal transduction for each of the phospholipase A₂ enzymes present in P388D1 macrophages. J. Biol. Chem. 271, 6758-6765 (1996).
18. Reddy, S.T. and Herschman, H.R. Prostaglandin synthase-1 and prostaglandin synthase-2 are coupled to distinct phospholipases for the generation of prostaglandin D₂ in activated mast cells. J. Biol. Chem. 272, 3231-3237 (1997).
19. Balboa, M.A., Balsinde, J., Jones, S.S., et al. Identity between the Ca²⁺-independent phospholipase A₂ enzymes from P388D1 macrophages and Chinese hamster ovary cells. J. Biol. Chem. 272, 8576-8580 (1997).
20. Stafforini, D.M., McIntyre, T.M., Zimmerman, G.A., et al. Platelet-activating factor acetylhydrolases. J. Biol. Chem. 272, 17895-17898 (1997).
21. Stafforini, D.M., Prescott, S.M., Zimmerman, G.A., et al. Mammalian platelet-activating factor acetylhydrolases. Biochim. Biophys. Acta 1301, 161-173 (1996).
22. Balsinde, J., Bianco, I.D., Ackermann, E.J., et al. Inhibition of calcium-independent phospholipase A₂ prevents arachidonic acid incorporation and phospholipid remodeling in P388D1 macrophages. Proc. Natl. Acad. Sci. USA 92, 8527-8531 (1995).
23. Tjoelker, L.W., Eberhardt, C., Wilder, C., et al. Functional and structural features of plasma plateletactivating factor acetylhydrolase. Adv. Exp. Med. Bio. 416, 107-111 (1996).
24. Matsuzawa, A., Hattori, K., Aoki, J., et al. Protection against oxidative stress-induced cell death by intracellular platelet-activating factor-acetylhydrolase II. J. Biol. Chem. 272, 32315-32320 (1997).
25. Stafforini, D.M., Prescott, S.M., and McIntyre, T.M. Human plasma platelet-activating factor acetylhydrolase. J. Biol. Chem. 262, 4223-4230 (1987).
26. Miwa, M., Miyake, T., Yamanaka, T., et al. Characterization of serum platelet-activating factor (PAF) acetylhydrolase. Correlation between deficiency of serum PAF acetylhydrolase and respiratory symptoms in asthmatic children. J. Clin. Invest. 82, 1983-1991 (1988).
27. Cupillard, L., Koumanov, K., Mattéi, M., et al. Cloning, chromosomal mapping, and expression of a novel human secretory phospholipase A₂. J. Biol. Chem. 272, 1574515752 (1997).
28. Dennis, E.A. The growing phospholipase A₂ superfamily of signal transduction enzymes. Trends Biochem. Sci. 22, 1-2 (1997).