PHOSPHATIDYLSERINE AND RELATED LIPIDS

STRUCTURE, OCCURRENCE, BIOCHEMISTRY AND ANALYSIS

1. Phosphatidylserine - Structure and Occurrence

Although phosphatidylserine or 1,2-diacyl-sn-glycero-3-phospho-L-serine is distributed widely among animals, plants and microorganisms, it is usually less than 10% of the total phospholipids, the greatest concentration being in myelin from brain tissue. However, it may comprise 10 to 20 mol% of the total phospholipid in the plasma membrane and endoplasmic reticulum of the cell. In yeasts, such as S. cerivisiae, it is a minor component of most cellular organelles other than the plasma membrane, where it can amount to more than 30% of the total lipids. The 1-octadecanoyl-2-docosahexaenoyl molecular species, which may be especially important in brain tissue, is illustrated here.

Formula of phosphatidylserine

Formula of calcium-chelated phosphatidylserine Phosphatidylserine is an acidic (anionic) phospholipid with three ionizable groups, i.e. the phosphate moiety, the amino group and the carboxyl function. As with other acidic lipids, it exists in nature in salt form, but it has a high propensity to chelate to calcium via the charged oxygen atoms of both the carboxyl and phosphate moieties, modifying the conformation of the polar head group. This interaction may be of considerable relevance to the biological function of phosphatidylserine, especially during bone formation for example.

In animal cells, the fatty acid composition of phosphatidylserine varies from tissue to tissue, but does not appear to resemble the precursor phospholipids, either because of selective utilization of specific molecular species for biosynthesis or because or re-modelling of the lipid via deacylation-reacylation reactions. In human plasma, 1-stearoyl-2-oleoyl and 1-stearoyl-2-arachidonoyl species predominate, but in brain, retina and many other tissues 1-stearoyl-2-docosahexaenoyl species are very abundant. Indeed, the ratio of n-3 to n-6 fatty acids in brain phosphatidylserine is very much higher than in most other lipids. The positional distribution of fatty acids in phosphatidylserine from rat liver and bovine brain are listed in Table 1. As with most phospholipids, saturated fatty acids are concentrated in position sn-1 and polyunsaturated in position sn-2.

Position	Fatty acid

Table 1. Positional distribution of fatty acids in phosphatidylserine from rat liver and bovine brain
	16:0	18:0	18:1	18:2	20:4	22:6

Rat liver [1]
sn-1	5	93	1
sn-2	6	29	8	4	32	19

Bovine brain [2]
sn-1	3	81	13
sn-2	2	1	25	trace	1	60

1. Wood, R. and Harlow, R.D. Arch. Biochem. Biophys., 135, 272-281 (1969). 2. Yabuuchi, H. and O'Brien, J.S. J. Lipid Res., 9, 65-67 (1968).

In leaves of Arabidopsis thaliana, used as a 'model' plant in many studies, the fatty acid composition of phosphatidylserine resembles that of phosphatidylethanolamine.

As phosphatidylserine is located entirely on the inner monolayer surface of the plasma membrane (and of other cellular membranes) and it is the most abundant anionic phospholipid, it may make the largest contribution to interfacial effects in membranes involving non-specific electrostatic interactions. This normal distribution is disturbed during platelet activation and cellular apoptosis.

2. Biosynthesis of Phosphatidylserine

L-Serine is a non-essential amino acid that is actively synthesised by most organisms. In animals, it is produced in nearly all cell types, although in brain it is synthesised by astrocytes but not by neurons, which must be supplied with this amino acid for the biosynthesis of phosphatidylserine (and of sphingoid bases).

In bacteria and other prokaryotic organisms, phosphatidylserine is synthesised by a mechanism comparable to that of other phospholipids, i.e. by reaction of L-serine with CDP-diacylglycerol (see our web pages on phosphatidylglycerol).

Much of the phosphatidylserine thus formed is decarboxylated to phosphatidylethanolamine, and this may be the major route to this lipid in bacteria. As phosphatidylcholine in yeast is produced via methylation of phosphatidylethanolamine, phosphatidylserine is the primary precursor phospholipid in this organism.

In contrast in animal tissues, there are two routes to phosphatidylserine involving distinct enzymes (PS synthase I and II, and PS decarboxylase) with different substrates and cellular locations. Phosphatidylserine is synthesised in the endoplasmic reticulum of the cell, or in a sub-fraction of this termed the mitochondria-associated membrane, by an exchange reaction of L-serine with phosphatidylcholine or phosphatidylethanolamine, catalysed by PS synthase I. This reaction is strictly dependent on calcium ions and requires no further source of energy. The new lipid is then transported to the mitochondria, where it is decarboxylated to phosphatidylethanolamine, which returns to the endoplasmic reticulum and is converted back to phosphatidylserine by the action of PS synthase II.

Biosynthesis of phosphatidylserine in animal tissues

Phosphatidylserine synthase-I is expressed in all mouse tissues, but especially the kidney, liver and brain, while phosphatidylserine synthase-II is most active in the testis and much less so in other tissues. It is not known why such a complex series of coupled reactions is necessary, or why there should be two enzymes. One virtue of the second enzyme is that the free ethanolamine and choline formed are rapidly re-utilized for phospholipid synthesis. Thus, both phosphatidylserine and phosphatidylethanolamine are produced without a reduction in the amount of phosphatidylcholine. Elimination of both enzymes is embryonically lethal in knock-out mice, but each of them can be knocked out separately and the mice survive, even though they have substantially reduced levels of phosphatidylserine and phosphatidylethanolamine.

In plants, much of the phosphatidylserine appears to be produced by a calcium-dependent base-exchange reaction in which the head-group of an existing phospholipid is exchanged for L-serine (i.e. mechanistically similar to PS synthase I), but a CDP-diacylglycerol pathway exists in some species, e.g. wheat.

N-Acylphosphatidylserine has been reported as a minor component of the lipids of sheep erythrocytes, bovine brain and the central nervous system of freshwater fish, amongst others. The N-arachidonoyl form may be the precursor of the endocannabinoid N-arachidonoylserine.

3. Phosphatidylserine – Biological Function

The presence of appreciable amounts of phosphatidylserine on the cytosolic leaflet of endosomes and lysosomes enables these compartments to dock with proteins with specific phosphatidylserine-binding domains, including several important signaling and fusogenic effectors. In addition, the high concentration of this anionic lipid results in an accumulation of negative surface charge to which poly-cationic proteins can bind. The effect is believed to be that certain proteins are re-directed from one target membrane to another.

Scottish thistle In addition to its function as a component of cellular membranes and as a precursor for other phospholipids, phosphatidylserine is an essential cofactor that binds to and activates protein kinase C, which is a key enzyme in signal transduction (see our webpage on diacylglycerols that facilitate this interaction). All the isoforms of this enzyme are strictly dependent on phosphatidylserine for activity. It is also required for other enzymes, such as Na⁺/K⁺ ATPase and neutral sphingomyelinase. It is involved in the blood coagulation process in platelets, where it is transported to the plasma-oriented surface of membrane vesicles that are derived from activated platelets. Here, phosphatidylserine enhances the activation of prothrombin to thrombin (the key molecule in the blood clotting cascade) directly, or by binding to specific sites on two key regulatory factors. On the other hand, it is not believed to be involved in cell signalling through the formation of metabolites, as is the case with phosphatidylinositol. Antibodies to phosphatidylserine are formed in some disease states, including thrombosis and recurrent spontaneous pregnancy loss.

Phosphatidylserine is known to have an important role in the regulation of apoptosis (programmed cell death) in response to particular calcium-dependent stimuli. The normal distribution of this lipid on the inner leaflet of the membrane bilayer is then disrupted because of stimulation of the enzyme scramblase, which can move phosphatidylserine in both directions across the membrane, and inhibition of aminophospholipid translocases, which returns the lipid to the inner side of the membrane. After transfer to the outer leaflet of the cell, a receptor on the surface of macrophages and related scavenger cells recognizes the phosphatidylserine and facilitates the removal of the apoptotic cells and their potentially toxic or immunogenic contents in a non-inflammatory manner. Binding of phosphatidylserine to specific proteins, such as apolipoprotein H (β2-glycoprotein 1), enhances the recognition and clearance. This process is essential for the development of lung and brain.

In addition, appreciable amounts of phosphatidylserine are translocated by a similar mechanism to the surface of T lymphocytes that express low levels of the trans-membrane enzyme tyrosine phosphatase. This change in distribution acts then as a signalling mechanism to modulate the activities of several membrane proteins. The protein annexin V binds with high specificity to phosphatidylserine and is used as a probe to detect apoptotic cells.

Scottish thistle A further unusual function of phosphatidylserine is that it is a key component of the lipid-calcium-phosphate complexes that initiate mineral deposition during the formation of bone. It has been established that phosphatidylserine and inorganic phosphate must be present, before calcium ions are introduced, when the high affinity of phosphatidylserine for calcium ions becomes important.

The high concentrations of docosahexaenoic acid (DHA) in brain and retinal phosphatidylserine are certainly important for the development and function of these tissues. Accumulation of phosphatidylserine in neuronal membranes is promoted by DHA, and this is important for the maintenance of neuronal survival. Phosphatidylserine may also a reservoir of DHA for protectin formation in neuronal tissue. On the other hand, the Food and Drug Administration in the USA considers that there is little scientific evidence to support claims that dietary supplements of phosphatidylserine reduce the risk of dementia or cognitive dysfunction in the elderly. Other nutritional claims also appear doubtful.

Lysophosphatidylserine, with a fatty acid in position sn-1 only, has been detected after injury to animal tissues (tumor growth, graft rejection, burns), and it may have a similar function to lysophosphatidic acid in cell signalling. In particular, soluble lysophosphatidylserine may be generated when cells are damaged, when it can diffuse and transmit the information to other cells, especially mast cells. In Schistosome infections, lysophosphatidylserine from the parasite is believed to be a key activator molecule in the host. In addition, negatively charged lysophosphatidylserine derivatives tend to organize in non-bilayer structures and are believed to facilitate folding of certain membrane proteins in situ better than bilayer-forming lipids.

4. Phosphatidylthreonine and Other Amino Acid-Containing Phospholipids

Phosphatidyl-L-threonine, which is closely related structurally and metabolically to phosphatidylserine, was first characterized as a minor component of polyoma virus-transformed embryo fibroblasts in hamsters and more recently in cultured hippocampal neurons. Biosynthetic studies with microsomes from rat brain suggest that it is synthesised by the same base-exchange enzyme involved in phosphatidylserine synthesis but with much lower activity. In laboratory animals, it is barely detectable in normal tissues such as brain, and it is decarboxylated in mitochondria in vitro to phosphatidylisopropanolamine.

Formula of phosphatidyl-L-threonine and phosphatidyl-O-[N-(2-hydroxyethyl) glycine]

A further amino acid-linked phospholipid, phosphatidyl-O-[N-(2-hydroxyethyl) glycine] has been isolated from brown algae of the family Phaeophyceae, such as Fucus serratus, where it can amount to as much as 25% of the total lipids. The fatty acid composition is distinctive in that arachidonic acid comprises about 80% of the total.

Other amino acid-containing phospholipids (complex lipoamino acids) are more closely related to phosphatidylglycerol in structure and biosynthesis.

5. Analysis

As with other acidic lipids, the metal ions associated with phosphatidylserine hamper analysis, although the problem can be solved by an acid wash. It is easily separated from other phospholipids by two-dimensional thin-layer chromatography, but poorly shaped peaks are often seen with high-performance liquid chromatography. Mass spectrometry is being used increasingly for molecular species analysis and quantification.

The Lipid Library