PHOSPHATIDIC ACID, LYSOPHOSPHATIDIC ACID AND RELATED LIPIDS

STRUCTURE, OCCURRENCE, BIOCHEMISTRY AND ANALYSIS

1. Phosphatidic Acid – Occurrence and Biosynthesis

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 and as a signalling molecule. Indeed, it is often over-estimated in tissues as it can arise by inadvertent enzymatic hydrolysis during inappropriate storage or extraction conditions during analysis. It is the simplest diacyl-glycerophospholipid, and the only one with a phosphomonoester as the head group. The molecule is acidic and carries a negative charge, i.e. it is an anionic lipid.

Structural formula of phosphatidic acid

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.

Biosynthesis of phosphatidic acid

In mammals, the glycerol-3-phosphate acyltransferase that catalyses the first step exists in four isoforms, two in mitochondria (designated GPAT1 and 2) and two in the endoplasmic reticulum (GPAT3 and 4). The activity in the endoplasmic reticulum predominates in adipose tissue, but the mitochondrial forms are responsible for half the activity in liver. All are membrane-bound enzymes, which are believed to span the membranes, but many questions remain regarding the regulation and function of the different isoforms. Similarly, at least two acyl-CoA:lysophosphatidic acid acyltransferases (LPAAT1 and LPAAT2) are known that catalyse the second step. Human LPAAT1 showed higher activity with 14:0-, 16:0- and 18:2-CoAs, while LPAAT2 prefers 20:4-CoA.

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 ). Such enzymes appear to be ubiquitous in nature, although those in bacteria and yeast are structurally different from the mammalian enzymes. The latter, at least ten isoforms of which exist exist with different sub-cellular locations and functions, use ATP as the phosphate donor. Aside from producing phosphatidic acid for phospholipid production or signalling, these enzymes may attenuate the signalling effects of diacylglycerols.

Biosynthesis of phosphatidic acid by diacylglycerol kinases

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.

Generation of phosphatidic acid by the action of phospholipase D

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.

phospholipid biosynthetic pathways

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.

2. Phosphatidic Acid - Biological Functions in Animals

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.

Scottish thistle 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.

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 differs from other anionic phospholipids in that its small anionic phosphomonoester head group lies very close to the hydrophobic interior of the lipid bilayer. Phosphatidic acid 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. An ‘electrostatic-hydrogen bond switch model’ has been proposed in which the head group of phosphatidic acid forms a hydrogen bond to a basic amino acid residue, leading to de-protonation of the head group, increasing its negative charge and thus enabling stronger interactions with further basic residues and tight docking with the membrane interacting protein.

3. Phosphatidic Acid – Biological Functions in Plants

The diacylglycerol signalling pathway is believed to be insignificant in plants. Instead, phosphatidic acid is the key plant lipid second messenger, which is rapidly and transiently generated in response to many different biotic and abiotic stresses. It can be produced from diacylglycerols, which are generated by the action of phospholipase C on membrane phospholipids, especially phosphatidylinositol; these are rapidly phosphorylated by a diacylglycerol kinase (there are 7 isoenzymes in Arabidopsis thaliana) to phosphatidic acid. Plants contain a large number of related enzymes of this type, 12 in Arabidopsis and 17 in rice, in comparison with two in humans and one in yeast. In plants, they can be into two groups, depending on their lipid-binding domains, with some homologous with the human and yeast enzymes, but most belonging to a second class that contains a characteristic ‘C2’(calcium- and lipid-binding) domain. Whether phosphatidic acid molecules from the two pathways elicit different responses is an open question.

Scottish thistle 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. As in mammalian cells, targets for such signalling include protein kinases and phosphatases, in addition to proteins involved in membrane trafficking and the organization of the cytoskeleton, and it can both activate or inhibit enzymes. It is involved in promoting the growth of pollen-tubes and root hairs, decreasing peroxide-induced cell death, and mediating the signalling processes that lead to responses to ethylene and to the plant hormone abscisic acid. Thus in the 'model' plant Arabidopsis, phosphatidic acid 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. A further difference from animal metabolism is that diacylglycerol pyrophosphate can be synthesised from phosphatidic acid in plants (see below).

4. Lysophosphatidic Acid

Structural formula of 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. Molecular species with both saturated and unsaturated fatty acid constituents occur in different tissues. 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. It is also important as the biosynthetic precursor of phosphatidic acid.

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 (200 μM in plasma) to yield 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.

Biosynthesis of lysophosphatidic acid

It is now established that it is produced by a wide variety of cell types by various mechanisms, including a phosphatidic acid-selective lysophospholipase A₁ producing an sn-2-acyl-lysophosphatidic acid, and almost certainly also by the action of a lysophospholipase A₂, monoacylglycerol kinase and glycerol-3-phosphate acyltransferase. A surprising recent finding is that the activity of the phosphatidic acid-selective lysophospholipase A₁ is essential for normal hair growth in humans. Autotaxin is the primary source of extracellular lysophosphatidic acid.

Although lysophospholipids are relatively small molecules, they carry a high content of information through the nature of the phosphate head group, the positional distribution of the fatty acids on the glycerol moiety, the presence of ether or ester linkages to the glycerol backbone, and the chain-length and degree and position of saturation of the fatty acyl chains. This informational content leads to selectivity in the functional relationship with cell receptors. Most mammalian cells express receptors for lysophosphatidic acid, and lysophosphatidic acid 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. At least five G protein-coupled receptors that are specific for lysophosphatidic acid have now been identified. They are only present in vertebrates. 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. In some instances, molecular species with specific fatty acid components may be involved.

Scottish thistle Lysophosphatidic acid signalling has regulatory functions in the mammalian reproductive system, both male and female, facilitating oocyte maturation and spermatogenesis, for example. There is also evidence that the lipid is involved in brain development, through its activity in neural progenitor cells, neurons and glia, and in vascular remodelling.

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. The expression of many of the surface receptors for lysophosphatidic acid in cancer cells is aberrant. Therefore, lysophosphatidic acid metabolism 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. As oxidized low-density lipoproteins promote the production of lysophosphatidic acid, its content in atherosclerotic plaques is high suggesting that it might serve as a biomarker for cardiovascular disease. Indeed, lysophosphatidic acid promotes pro-inflammatory events that lead to the development of atheroma as well encouraging progression of the disease. By mediating platelet aggregation, it could lead to arterial thrombus formation.

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 oesophagus. 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 de-phosphorylate sphingosine-1-phosphate, phosphatidic acid and ceramide 1-phosphate in a non-specific manner. It can be converted back to phosphatidic acid by a membrane-bound O-acyltransferase (MBOAT2) specific for lysophosphatidic acid (and lysophosphatidylethanolamine) with a preference for oleoyl-CoA as substrate.

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

5. Cyclic Phosphatidic Acid

Structural formula of cyclic phosphatidic acid Cyclic phosphatidic acid (sometimes termed ‘cyclic lysophosphatidic acid’) was isolated originally from a slime mould, but has now been detecte of cyclic phosphatidic acidd in a wide range of organisms including humans, especially in the brain but also bound to albumin in serum (at a concentration of 10^-7M, or a tenth that of lysophosphatidic acid). 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 biological activity. It is most abundant in tissues subject to injury. In human serum, the main molecular species contains palmitic acid.

While cyclic phosphatidic acid may have some similar signalling functions to lysophosphatidic acid per se in that it binds to some of the same receptors, it also has some quite distinct activities in animal tissues. 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. In addition, it inhibits the platelet aggregation induced by lysophosphatidic acid.

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.

6. Pyrophosphatidic Acid

Structural formula of 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, which is present in all plant tissues but especially the plasma membrane.

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 D signalling cascade in plants, perhaps by attenuating the effects of phosphatidic acid. 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.

7. 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.

The AOCS Lipid Library