The Lipid Library

PHOSPHATIDIC ACID, LYSOPHOSPHATIDIC ACID AND RELATED LIPIDS

STRUCTURE, OCCURRENCE, BIOCHEMISTRY AND ANALYSIS

1.  Phosphatidic Acid

Phosphatidic acid is not an abundant lipid constituent of any living organism to my knowledge, but it is extremely important as an intermediate in the biosynthesis of triacylglycerols and phospholipids. Indeed, it is often over-estimated in tissues as it can arise by inadvertent enzymatic hydrolysis during inappropriate storage or extraction conditions during analysis. The molecule is acidic and bears a negative charge so requires a counter ion.

The main biosynthetic route in plant and animal tissues involves sequential acylation of α-glycerophosphate, derived from catabolism of glucose, by acyl-coA derivatives of fatty acids as illustrated. Specific acyltransferases catalyse first the acylation of position sn-1 to form lysophosphatidic acid (1-acyl-sn-glycerol-3-phosphate) and then of position sn-2 to yield phosphatidic acid.

Under some conditions, phosphatidic acid can be generated from 1,2-diacyl-sn-glycerols by the action of diacylglycerol kinases (see our webpage on diacylglycerols). However, a more important route in quantitative terms is via hydrolysis of other phospholipids, but especially phosphatidylcholine, by the enzyme phospholipase D (or by a family or related enzymes of this kind). This enzyme is present in most animal cell types, and its activity is regulated by phosphatidylinositol-4,5-bisphosphate and protein kinase C.

The subsequent steps in the utilization of phosphatidic acid in the biosynthesis of triacylglycerols and phospholipids are described in separate documents in this section of the website. In brief, hydrolysis of phosphatidic acid by the enzyme phosphatidate phosphatase is the source of sn-1,2-diacylglycerols (DAG), which are the precursors for the biosynthesis of triacylglycerols (TAG), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) via the so-called Kennedy pathway (also of monogalactosyldiacylglycerols in plants). Via reaction with cytidine triphosphate, phosphatidic acid is the precursor of cytidine diphosphate diacylglycerol, which is the key intermediate in the synthesis of phosphatidylglycerol (PG), and thence of cardiolipin, and of phosphatidylinositol (PI) and phosphatidylserine (PS). Depending on the organism and other factors, phosphatidylserine can be a precursor for phosphatidylethanolamine. Similarly, the latter can give rise to phosphatidylcholine by way of mono- and dimethylphosphatidylethanolamine intermediates.

The fatty acid composition of phosphatidic acid can resemble that of the eventual products, though in many instances this can be much altered by re-modelling after synthesis via deacylation-reacylation reactions.

The phosphatidic acid generated by the action of phospholipase D and by diacylglycerol kinases may have signalling functions as a second messenger, although it is not certain whether all the activities suggested by studies in vitro operate in vivo. Nonetheless, phosphatidic acid has been implicated in many aspects of animal cell biochemistry and physiology, including cell proliferation and differentiation, cell transformation, tumor progression and survival signalling. It appears to regulate some membrane trafficking events, and it is involved in activation of the enzyme NADPH oxidase, which operates as part of the defense mechanism against infection and tissue damage during inflammation. It may have a role in promoting phospholipase A₂ activity, and it appears to function in vesicle formation and transport within the cell. By binding to targeted proteins, including protein kinases, protein phosphatases and G-proteins, it may increase or inhibit their activities. For example in yeast, phosphatidic acid on the endoplasmic reticulum binds directly to a specific transcriptional repressor to keep it inactive outside the nucleus; when the lipid precursor inositol is added, this phosphatidic acid is rapidly depleted, releasing the transcriptional factor so that it can be translocated to the nucleus where it is able to repression target genes. The overall effect is a mechanism to control phospholipid synthesis. In addition, the murine phosphatidylinositol 4-phosphate 5-kinase does not appear to function unless phosphatidic acid is bound.

In relation to signalling activities, it should be noted that phosphatidic acid can be metabolized to sn-1,2-diacylglycerols or to lysophosphatidic acid (see next section), both of which have distinctive signalling functions in their own right. Conversely, both of these compounds can be in effect be de-activated by conversion back to phosphatidic acid.

In many cell types, vesicle trafficking, secretion and endocytosis may also require phosphatidic acid derived by the action of phospholipase D. For example, a specific form of this enzyme located on the outer surface of mitochondria acts upon cardiolipin to promote mitochondrial fusion.

Some of these effects may be explained simply by the physical properties of phosphatidic acid, which has a propensity to form a hexagonal II phase, especially in the presence of calcium ions. Thus, hydrolysis of phosphatidylcholine, a cylindrical, non-fusogenic lipid, converts it into cone-shaped, fusogenic phosphatidic acid, which promotes negative membrane curvature. It can effect membrane fusion in model systems, probably because of its ability to form non-bilayer phases. Also of relevance in this context is its overall negative charge, and it is not always clear whether some of the observed biological effects are specific to phosphatidic acid or simply to negatively charged phospholipids in general. However, it has been demonstrated that the positively charged lysine and arginine residues on proteins can bind with some specificity to phosphatidic acid through hydrogen bonding with the phosphate group, thus distinguishing it from other phospholipids.

Phosphatidic acid in plants. Phospholipase D activity and the phosphatidic acid produced may be even more significant in plants. They have long been recognized as of importance during germination and senescence, and they appear to have a role in response to stress damage and pathogen attack. A high content of phosphatidic acid induced by phospholipase D action during wounding or senescence brings about a loss of the membrane bilayer phase, as a consequence of the conical shape of this phospholipid in comparison to the cylindrical shape of structural phospholipids. As a result, cells lose their viability. The phosphatidic acid generated in this way is broken down further by phosphatases, acyl-hydrolases and lipoxygenases into fatty acids and other small molecules, which are subsequently absorbed and recycled. In addition, phosphatidic acid is important in the response to other forms of stress, including osmotic stress (salinity or drought), cold, and oxidation, although much remains to be learned of the mechanism by which it exerts its effects.

Phosphatidic acid is of considerable importance in cellular signalling in plants, for example in promoting pollen-tube growth, decreasing peroxide-induced cell death, and mediating the signalling processes that lead to responses to the plant hormone abscisic acid. Thus in the 'model' plant Arabidopsis, which contains twelve distinct members of the phospholipase D family, phosphatidic acid generated by the action of the enzymes interacts with a protein phosphatase to signal the closure of stomata promoted by abscisic acid; it interacts also with a further enzyme to mediate the inhibition of stomatal opening effected by abscisic acid. Together these reactions constitute a signalling pathway that regulates water loss from plants.

2.  Lysophosphatidic Acid

Lysophosphatidic acid or 1-acyl-sn-glycerol-3-phosphate differs from phosphatidic acid in having only one mole of fatty acid per mole of lipid. As such, it is the simplest possible glycerophospholipid. It is the biosynthetic precursor of phosphatidic acid. Although it is present at very low levels only in animal tissues, it is extremely important biologically, influencing many biochemical processes. These activities seem to be shared by the 1-alkyl- and alkenyl-ether forms.

In particular, lysophosphatidic acid is an intercellular lipid mediator with growth factor-like activities, and is rapidly produced and released from activated platelets to influence target cells. However, a more important source is the activity of a specific lysophospholipase D (‘autotaxin’), part of the blood-clotting process, on lysophosphatidylcholine, which yields lysophosphatidic acid in an albumin-bound form. This is more abundant in serum (1-5 μM) than in plasma, where it accounts for much of the biological activity.

Further studies have established that it is produced by a wide variety of cell types, both by the action of autotoxin and by that of a phosphatidic acid-selective phospholipase A₁, and that most mammalian cells express receptors for lysophosphatidic acid. It may initiate signalling in the cells in which it is produced, as well as affecting neighbouring cells.

In the last few years, the characterization of cloned lysophosphatidic acid receptors in combination with strategies of molecular genetics has allowed determination of both signalling and biological effects that are dependent on receptor mechanisms. Experimental activation of these receptors has shown that a range of downstream signalling cascades mediate lysophosphatidic acid signalling. These include activation of protein kinases, adenyl cyclase and phospholipase C, release of arachidonic acid, and much more. There is evidence that lysophosphatidic acid is involved in cell survival in some circumstances, and in programmed cell death in others.

There is particular interest in the activity of lysophosphatidic acid in various disease states, where intervention in its metabolism has the potential for beneficial health effects. For example, a finding that lysophosphatidic acid is markedly elevated in the plasma of ovarian cancer patients, compared to healthy controls may be especially significant. In particular, elevated plasma levels were found in patients in the first stage of ovarian cancer, suggesting that it may represent a useful marker for the early detection of the disease. Lysophosphatidic acid is believed to stimulate DNA synthesis and the proliferation of ovarian cancer cells, and it may induce cell migration. Therefore, it is a target of the pharmaceutical industry for cancer therapy.

In addition, lysophosphatidic acid generated by the action of a lysophospholipase D is believed to play an important role in reproductive biology. Under certain conditions, it can become athero- and thrombogenic and might aggravate cardiovascular disease. This may be especially important in cancer patients. Lysophosphatidic acid has also been found in saliva in significant amounts, and it has been suggested that it is involved in wound healing in the upper digestive organs such as the mouth, pharynx, and esophagus. It has similar effects when applied topically to skin wounds, probably by stimulating proliferation of new cells to seal the wound.

Catabolic deactivation of lysophosphatidic acid is accomplished by dephosphorylation to monoacylglycerol by a family of three lipid phosphate phosphatases, which also dephosphorylate sphingosine-1-phosphate, phosphatidic acid and ceramide 1-phosphate in a non-specific manner.

Other lysophospholipids and especially the sphingolipid analogue, sphingosine-1-phosphate, show a related range of activities.

3.  Cyclic Phosphatidic Acid

Cyclic phosphatidic acid (sometimes termed ‘cyclic lysophosphatidic acid’) was isolated originally from a slime mould, but has now been detected in a wide range of organisms including humans, especially in the brain but also in serum (at a concentration of 10^-7M). It has a cyclic phosphate at the sn-2 and sn-3 positions of the glycerol carbons, and this structure is absolutely necessary for its activities. In particular, it is found in tissues subject to injury, and while it may have some similar signalling functions to lysophosphatidic acid per se, it also has some quite distinct biological activities. For example, cyclic phosphatidic acid is known to be a specific inhibitor of DNA polymerase alpha. It has an appreciable effect on the inhibition of cancer cell invasion and metastasis, a finding that is currently attracting great pharmacological interest.

Studies of the biosynthesis of cyclic phosphatidic acid in fetal bovine serum suggest that it is the product of an enzyme related to the human enzyme autotaxin, a serum lysophospholipase D that produces lysophosphatidic acid. This enzyme appears to produce cyclic phosphatidic acid in serum by an intramolecular transphosphatidylation reaction. However, it can also be formed artefactually by the addition of strong acid to serum.

4.  Pyrophosphatidic Acid

Pyrophosphatidic acid or sn-1,2-diacylglycero-3-pyrophosphate is an unusual and little known phospholipid that was first identified as a minor component in yeasts, and is also know to be present in mushrooms and higher plants as a product of the enzyme phosphatidic acid kinase.

It is rapidly metabolized back to phosphatidic acid by a specific phosphatase and thence to diacylglycerols, and it may have a function in the phospholipase C and D signalling cascades in plants. Pyrophosphatidic acid is barely detectable in non-stimulated plant cells but its concentration increases very rapidly in response to stress situations, including osmotic stress and attack by pathogens. Such findings add to the belief that it is an important signalling molecule in plants under stress. In yeasts, it may have a role in the regulation of the synthesis and metabolism of phospholipids, especially phosphatidylserine.

5.  Analysis

Phosphatidic acid and related lipids are not the easiest to analyse. On adsorption chromatography, retention times tend to be variable and may be dependent to some extent on the nature of the cations associated with the acidic lipids. However, two-dimensional TLC can give good results. Phosphatidic acid, lysobisphosphatidic acid and pyrophosphatidic acid are never easy to distinguish, and the best hope for success appears to lie with modern liquid chromatography-mass spectrometric methods.

Recommended Reading

• Bargmann, B.O.R. and Munnik, T. The role of phospholipase D in plant stress responses. Current Opinion Plant Biology, 9, 515-522 (2006).
• Christie, W.W. Lipid Analysis (3rd edition). (Oily Press, Bridgwater) (2003).
• Haucke, V. and Di Paolo, G. Lipids and lipid modifications in the regulation of membrane traffic. Current Opinion in Cell Biology, 19, 426-435 (2007).
• Ishii, I., Fukushima, N., Ye, X. and Chun, J. Lysophospholipid receptors: signaling and biology. Annu. Rev. Biochem., 73, 321-354 (2004).
• Murakami-Murofushi, K., Uchiyama, A., Fujiwara, Y., Kobayashi, T., Kobayashi, S., Mukai, M., Murofushi, H. and Tigyi, G. Biological functions of a novel lipid mediator, cyclic phosphatidic acid. Biochim. Biophys. Acta, 1582, 1-7 (2002).
• Pyne, S. Lysolipids: sphingosine-1-phosphate and lysophosphatidic acid. In: Bioactive Lipids. pp. 37-61. (edited by A. Nicolaou and G. Kokotos, The Oily Press, Bridgwater) (2004)
• Stace, C.L. and Ktistakis, N.T. Phosphatidic acid- and phosphatidylserine-binding proteins. Biochim. Biophys. Acta, 1761, 913-926 (2006).
• Tokumura, A. Physiological and pathophysiological roles of lysophosphatidic acids produced by secretory lysophospholipase D in body fluids. Biochim. Biophys. Acta, 1582, 18-25 (2002) - and various other articles in this volume.
• Vance, D.E. and Vance, J. (editors) Biochemistry of Lipids, Lipoproteins and Membranes (4th Edition). (Elsevier Science, Amsterdam) (2002) - several chapters.
• van Schooten, B., Testerink, C. and Munnik, T. Signalling diacylglycerol pyrophosphate, a new phosphatidic acid metabolite. Biochim. Biophys. Acta, 1761, 151-159 (2006).
• Wang, X., Devaiah, S.P., Zhang, W. and Welti, R. Signaling functions of phosphatidic acid. Prog. Lipid Res., 45, 250-278 (2006).

Updated: 7/3/2008 Credits/disclaimer	Scottish Crop Research Institute (and MRS Lipid Analysis Unit), Invergowrie, Dundee (DD2 5DA), Scotland