Phospholipase A2

Phospholipase A2 (PLA2) designates a class of enzymes that hydrolyze the sn-2 ester of glycerophospholipids to produce a fatty acid and a lysophospholipid. It has become clear that some of these enzymes liberate arachidonic acid in mammalian cells for the biosynthesis of eicosanoids, and thus there has been considerable interest in developing PLA2 inhibitors. Based on amino acid sequences, there are now more than 12 distinct groups of mammalian PLA2s, as well as many non-mammalian forms, all of which have been classified into 14 distinct groups with many subgroups.

Since naturally occurring phospholipids have virtually no solubility in water, PLA2 must bind to the lipid-water interface to access its substrate, and it is clear that catalysis occurs at the interface (interfacial catalysis). One has to be careful with the interpretation of kinetic data including inhibition data, and in fact many of the previously reported PLA2 inhibitors work by non-specific mechanisms. One of the problems is that the substrate forms an interface, and the inhibitor can potentially partition into the membrane phase. Thus, inhibition of a PLA2 by micromolar amounts of a compound using micromolar amounts of substrate may occur because a large fraction of the interface could be occupied by inhibitor. This may change the physical nature of the interface, causing the enzyme to desorb from the membrane into the aqueous phase, resulting in non-specific inhibition. Inhibitors that work by this non-specific mechanism are clearly not useful for studying the role of PLA2s in complex cellular processes. Among those inhibitors that bind tightly to the active site of PLA2s, and thus operate by a specific mechanism, the issue of PLA2 group specificity is important.

To date, 10 groups of mammalian secreted PLA2s, known as s-PLA2s, have been identified. Group IB PLA2, also known as pancreatic PLA2, is found not only as part of the digestive fluid where it functions to hydrolyze dietary phospholipids, but also in non-digestive tissues including spleen where it has unknown functions. Group IIA PLA2 was the first non-pancreatic mammalian PLA2 to be identified as a component of synovial fluid and platelets. This enzyme is pro-inflammatory, displays potent bactericial properties and is a target for the development of anti-inflammatory agents including anti-sepsis agents. Groups IIC, IID, IIE, IIF, III, X, XIIA and XIIB secreted PLA2 were discovered by recombinant DNA techniques. The group IIC gene is functional in mice but occurs as a pseudogene in humans. Group XIIB is best designated as a secreted PLA2-like protein since it has a natural mutation of a key catalytic residue that renders this protein devoid of phospholipase activity. The function of these recently discovered secreted PLA2s are unknown. Group V PLA2, also discovered at the DNA level, is an active enzyme secreted from macrophages and probably a variety of other cells. Recent gene disruption studies implicate a role of this enzyme in arachidonic acid release in stimulated macrophages. All of these secreted PLA2s have similar size, three-dimensional structure, and active site residues (except for group XIIB as noted above). They require submillimolar amounts of calcium for catalytic activity.

Mammalian cells also contain two intracellular enzymes that act on long-chain phospholipids. Group IVA PLA2 translocates from the cytosol to internal membranes in response to micromolar calcium, and shows specificity for arachidonyl-containing phospholipids. A wide variety of studies have shown that this enzyme, also known as cPLA2a, releases arachidonic acid from membrane phospholipids for the biosynthesis of eicosanoids. Recent paralogs of cPLA2a have been identified in the genome, but their functions are not known. It has been proposed that the calcium-independent group VI PLA2 may be involved in phospholipid remodeling, insulin secretion from b cells and in stores-operated calcium entry. Groups VII and VIII PLA2s are highly specific for phospholipids with short sn-2 chains and are thought to terminate the action of platelet activation factor by hydrolyzing the sn-2 ester and to act on oxidized phospholipids.

 

The Table below contains accepted modulators and additional information. For a list of additional products, see the "Similar Products" section below.

 

Group IB
(P8913, P6534)a
IIA, IIC, IID, IIE and IIF III IV V
Sourcesb Mammals Mammals
(IIC gene is functional in mice but a pseudogene in humans)
Mammals
Bee and wasp venoms
Mammals Mammals
Secreted or Cytosolic Secreted Secreted Secreted Cytosolic Secreted
Structural Information 133 aa (human)
Multiple disulfides
~130 aa
Multiple disulfides
IIF has a C-terminal extension
Mammalian enzyme has a ~16 kDa group III PLA2 domain and long N- and C-terminal extensions 749 aa (human) α-paralog
Several new paralogs have been recently identified
118 aa (human)
Multiple disulfides
Molecular Weight (kDa) 13 - 15 13 - 17 55 (mammalian)
18 (venom)
80-85 (α-paralog) 14
Cofactor µM –€“ mM Ca2+ required for active site substrate binding and catalysise µM –€“ mM Ca2+ required for active site substrate binding and catalysise µM –€“ mM Ca2+ required for active site substrate binding and catalysise Sub to low mM Ca2+ required for membrane binding
γ-paralog does not require Ca2+
µM –€“ mM Ca2+ required for active site substrate binding and catalysise
Tight Binding Inhibitorsc HK series sn-2-amide phospholipids
Indole analogsf
HK series sn-2-amide phospholipids
Indole analogse
HK series sn-2-amide phospholipids
Indole analogse
HK series sn-2-amide phospholipids
Indole analogsf
HK series sn-2-amide phospholipids
Indole analogse
Control Compounds Enantiomer of HK series
Some Indole analogs
Enantiomer of HK series
Some Indole analogs
Enantiomer of HK series
some Indole analogs
AACOCH3 Enantiomer of HK series
Some indole analogs
Inhibitor Specificity Many group IB inhibitors inhibit other secreted PLA2se Many group II inhibitors inhibit other secreted PLA2se Many group III inhibitors inhibit other secreted PLA2se AACOCF3 (A231) and MAFP also inhibit group VI PLA2S Many group V inhibitors inhibit other secreted PLA2se
Tissue Expressionb Pancreatic juice
Spleen
Lung
Several mammalian tissues
Tissue expression is partially overlapping
Kidney
Heart
Liver
Skeletal muscle
Most mammalian tissues Macrophages
Lung
Physiological Functiond Digestion of dietary phospholipids Bactericidal activity (IIA)
Functions of IIC, IID, IIE and IIF are unknown
Not Known Agonist-triggered arachidonic acid release for eicosanoid and platelet activator factor production (α-isoform)
Functions of other isoforms are unknown
Contributes to arachidonic acid release for eicosanoid production
Augmentation of cPLA2α function
Disease Relevanced Mouse IB deficient mice show resistance to obesity Sepsis and inflammation, possibly arthritis (IIA) Not Known Inflammation and allergy related to lipid mediator production Not Known

 

 

Group VI

VII VIII X XIIA
Sourcesb Mammals Human plasma Bovine brain Mammals Mammals
Secreted or Cytosolic Cytosolic Secreted Cytosolic Secreted Secreted
Structural Information iPLA2a isoform is 752 aa (human)
Several isoforms and splice forms are known
420 aa (human) 230 aa (bovine γ-subunit) 123 aa (human) 167 aa (human)
Multiple disulfides
Molecular Weight (kDa) iPLA2a isoform 80 - 85 45 29 14 19
Cofactor None
May be augmented by ATP
Not Known
Not Known
µM –€“ mM Ca2+ required for active site substrate binding and catalysise µM –€“ mM Ca2+ required for active site substrate binding and catalysise
Tight Binding Inhibitorsc BEL (B1552)
AACOCF3 (A231)
MAFP
Patented inhibitors from GSK Not Known AACOCF3 (A231)
MAFP
Pyrrophenone and related compounds
Not Known
Control Compounds Enantiomers BEL (B1552) have distinct specificity for the various iPLA2 isoforms Not Known
Not Known Enantiomer of HK series
Some indole analogs
Not Known
Inhibitor Specificity AACOCF3 (A231) and MAFP also inhibit cPLA Not Known
Not Known Many group IB inhibitors inhibit other secreted PLA2se Not Known
Tissue Expressionb Expressed in many mammalian tissues Human plasma
An isoform is expressed in mammalian cytosol
Brain Several mammalian tissues Several mammalian tissues
Physiological Functiond Stores operated calcium entry
Insulin secretion
Phospholipid acyl chain remodeling
Serum form degrades platelet activating factor and phospholipids with oxidized fatty acyl chains Not Known Not Known Not Known
Disease Relevanced Not Known
Not Known
Mutations cause Miller-Dieker lissencephaly Not Known Not Known

 

Footnotes

a) Not included in the table are a variety of other PLA2s from non-mammalian sources including PLA2s in snake and insect venoms, for example the group IA enzymes in cobra and krait venoms (P7778), rattlesnakes and bee venom (P9279).

b) The species and tissue distribution is only a partial listing.

c) Only those inhibitors that have been shown to bind specifically to the active site of the PLA2s have been listed (see text for more discussion); and the list is not necessarily comprehensive.

d) The list of physiological functions and disease relevance is only partial as the functions of PLA2s is under active investigation and is still unresolved in many cases.

e) See, Singer et al., J Biol. Chem., 277, 48535-49 (2002).

f) See, Smart, B.P., et al., Bioorg. Med. Chem., 12, 1737-1749 (2004).

 

Abbreviations

AACH(OH)CF3: 2-Hydroxy-1,1,1,-trifluoro-6,9,12,15-heneicosatetraene
AACOCF3: 2-oxo-1,1,1-Trifluoro-6,9-12,15-heneicosatetraene
AACOCH3: 2-oxo-6,9,12,15-Heneicosatetetraene
BEL: (E)-6-(Bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one
MAFP: 4,7,10,13-Nonadecatetraenyl fluorophosphonic acid methyl ester
SB-222657: N-[6-(4-Chlorophenyl)hexyl]-2-oxo-4-[(S)-(phenylmethyl)sulfinyl]-1-azetidineacetamide
SB-223777: N-[6-(4-Chlorophenyl)hexyl]-2-oxo-4-[(R)-(phenylmethyl)sulfinyl]-1-azetidineacetamide

 

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References