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21.3: Biosynthesis of Membrane Complex Glycerolipids

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    15175
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    by William (Bill) W. Christie and Henry Jakubowski.

    This section is an abbreviated and modified version of material from the Lipid Web, an introduction to the chemistry and biochemistry of individual lipid classes, written by William Christie.

    Introduction

    In this section will be explore the synthesis of the membrane glycerophospholipids and their metabolic derivatives. We will start with the simplest one, phosphatidic acids, and end with phosphatidylinositols.

    Phosphatidic Acids and its derivatives

    Phosphatidic acid or 1,2-diacyl-sn-glycero-3-phosphate is a key intermediate in the biosynthesis both of other glycerophospholipids and of triacylglycerols. It is structurally one of the simplest of the phospholipids and was long thought to be important only as a precursor of other lipids, where it is indeed a key molecule, but it is now known to have many other functions in animals, plants, and other organisms by its influence on membrane structure and dynamics, and by its interactions with various proteins. As a lipid mediator, it modulates various signaling and cellular processes, such as membrane tethering, conformational changes and enzymatic activities of specific proteins, and vesicular trafficking. Moreover, its metabolite lysophosphatidic acid is recognized as a key signaling molecule with a myriad of biological effects mediated through specific receptors.

    Phosphatidic Acid – Occurrence and Biosynthesis

    Phosphatidic acid is not an abundant lipid constituent of any living organism, seldom greater than picomolar concentrations in cells, but it is extremely important both as an intermediate in the biosynthesis of other glycerophospholipids and triacylglycerols and as a signaling molecule or a precursor of signaling molecules. 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. The structure of phosphatidic acid is shown in Figure \(\PageIndex{1}\) below.

    phosphatidicacid.svg
    Figure \(\PageIndex{1}\): Phosphatidic acid

    There are at least four important biosynthetic pathways for phosphatidic acid biosynthesis in different organelles under various stimuli, and possibly resulting in the formation of different molecular species. The main pathway involves sequential acylation of sn-glycerol-3-phosphate, derived from catabolism of glucose, by acyl-coA derivatives of fatty acids. First, one acyltransferases catalyses the acylation of position sn-1 to form lysophosphatidic acid (1‑acyl-sn-glycerol-3-phosphate), and then a second specific acyltransferase catalyses the acylation of position sn-2 to yield phosphatidic acid. The synthesis of phosphatidic acid from glycerol-3-phosphate is shown in Figure \(\PageIndex{2}\) below.

    SynPhosphatidicAcdvisGlycerol3PV2.svg
    Figure \(\PageIndex{2}\): Synthesis of phosphatidic acid from glycerol-3-phosphate

    In mammals, the glycerol-3-phosphate acyltransferase that catalyses the first step exists in four isoforms, two in the mitochondrial outer membrane (designated GPAT1 and 2) and two in the endoplasmic reticulum (GPAT3 and 4); all are membrane-bound enzymes, which are believed to span the membranes. GPAT1 is highly expressed in the liver and adipose tissue, where it is responsive to changes in feeding status via the sterol regulatory element binding protein-1 (SREBP-1), a master transcriptional regulator of lipogenic enzymes. It is essential in directing fatty acyl-CoA esters towards glycerolipid synthesis as opposed to β-oxidation. GPAT3 is especially important for triacylglycerol storage in adipocytes, while GPAT4 is the main contributor to lysophosphatidic acid synthesis in liver and brown adipose tissue.

    For the second step in phosphatidic acid biosynthesis, five mammalian acyl-CoA:lysophosphatidic acid acyltransferases are known of which three are in the endoplasmic reticulum (LPAAT or LPAT or AGPAT1, 2 and 3), with a further two (LPAT4 and 5) on the outer mitochondrial membrane. While LPAT1 and 2 have strict specificity for lysophosphatidic acid as acyl acceptor, other isoforms can esterify other lysophospholipids. Human LPAT1 showed higher activity with 14:0-, 16:0- and 18:2‑CoAs, while LPAT2 prefers 20:4-CoA and LPAT3 produces phosphatidic acid containing docosahexaenoic acid (22:6(n-3)); the last is especially important in retina and testes. LPAT4 and 5 have a preference for oleoyl-CoA and polyunsaturated acyl-CoAs as the acyl donor, suggesting a dual role in glycerolipid synthesis and remodeling. The activity in the endoplasmic reticulum predominates in adipose tissue, but the mitochondrial forms are believed to be responsible for half the activity in liver. However, as there is traffic of phosphatidic acid between the mitochondria and endoplasmic reticulum for remodeling or for synthesis of other lipids, the relative contributions of the two can be difficult to assess.

    In plants, the sn-glycerol-3-phosphate pathway exists both in plastids and at the endoplasmic reticulum with multiple isoforms of the two acyltransferases as well as differences in the acyl substrates. In brief most plant lipid biosynthesis begins with fatty acid biosynthesis in the chloroplasts. In plastids, the acyltransferase ATS1 transfers 18:1 acyl groups from acyl-acyl carrier protein (acyl-ACP) to position sn-1 of glycerol 3-phosphate, before ATS2 transfers a palmitoyl group from ACP to position sn-2, producing phosphatidic acid at the inner leaflet of the chloroplast inner envelope membrane (IEM). Fatty acids intended for the endoplasmic reticulum are released from ACP in the chloroplast stroma by IEM-associated thioesterases, exported and then activated by acyl-CoA synthetases of the outer envelope membrane to produce species with C18 fatty acids in both positions. Thus, acyl-CoAs are used for phosphatidic acid biosynthesis in the endoplasmic reticulum with marked differences in the specificity of the acyl substrates. Subsequently, phosphatidic acid in the plastids is utilized for biosynthesis of galactosyldiacylglycerols, while that in the endoplasmic reticulum is used for synthesis of triacylglycerols and phospholipids.

    In bacteria, two families of enzymes are responsible for acylation of position sn-1 of glycerol-3-phosphate. One present in Escherichia coli, for example, utilizes the acyl-acyl carrier protein (acyl-ACP) products of fatty acid synthesis as acyl donors as well as acyl-CoA derived from exogenous fatty acids. In a second wider group of bacteria, including cyanobacteria, there are enzymes (PlsX and PlsY) that make use of the unique acyl donors, acyl-phosphates derived in part from acyl-ACP, to acylate position sn-1. Acylation of position sn-2 in this instance is performed by a further family of enzymes (PlsC) that uses acyl-ACP as the acyl donor, although some bacterial species may use acyl-CoA also.

    In animals, a second biosynthetic pathway utilizes dihydroxyacetone phosphate (DHAP) as the primary precursor for the peroxisomal enzyme, DHAP acyltransferase, which produces acyl-DHAP. This intermediate is converted to lysophosphatidic acid in a NADPH-dependent reaction catalysed by acyl-DHAP reductase, and this is in turn acylated to form phosphatidic acid by the same LPAT as in the previous mechanism. This pathway is of particular importance in the biosynthesis of ether lipids. The synthesis of phosphatidic acid from dihydroacetone phosphate is shown in Figure \(\PageIndex{3}\) below.

    SynPhosphatidicAcdviaDHAP2.svg
    Figure \(\PageIndex{3}\):Synthesis of phosphatidic acid from dihydroacetone phosphate

    A third important route to phosphatidic acid is via hydrolysis of other phospholipids, but especially phosphatidylcholine, by the enzyme phospholipase D (or by a family or related enzymes of this kind). The enzyme is readily available for study in plants, where the special functions of phosphatidic acid have long been known (see below), but it is now recognized that phospholipase D is present in bacteria, yeasts and most animal cells. In the last, it exists in two main isoforms with differing specificities and cellular locations; PLD1 is found mainly in the Golgi-lysosome continuum, while PLD2 is present mainly in the plasma membrane. They are phosphoproteins, the activity of which is regulated by kinases and phosphatases and by binding to phosphatidylinositol-4,5-bisphosphate. In mitochondria, a distinctive enzyme of this type utilizes cardiolipin as substrate. The mechanism involves the use of water as the nucleophile to catalyse the hydrolysis of phosphodiester bonds in phospholipids. Phospholipase D activity is dependent on and regulated by neurotransmitters, hormones, small monomeric GTPases and lipids. The hydrolysis of phosphatidylcholine to phosphatidic acids by phospholipase D is shown in Figure \(\PageIndex{4}\) below.

    PChydrolysisPLD.svg
    Figure \(\PageIndex{4}\): hydrolysis of phosphatidylcholine to phosphatidic acids by phospholipase D

    In addition to its function in generating phosphatidic acid mainly for signaling purposes but also for the maintenance of membrane composition, phospholipase D is involved in intracellular protein trafficking, cytoskeletal dynamics, cell migration and cell proliferation, partly through protein-protein interactions; it is considered to be important in inflammation and in cancer growth and metastasis as a downstream transcriptional target of proteins involved in the pathophysiology of these diseases. It also has an unusual activity as a guanine nucleotide exchange factor. By a transphosphatidylation reaction with ethanol, it generates phosphatidylethanol, a useful biomarker for ethanol consumption in humans.

    Under some conditions, phosphatidic acid can be generated from 1,2-diacyl-sn-glycerols by the action of diacylglycerol kinases, for example those produced from other phospholipids by the action of phospholipase C. Such enzymes appear to be ubiquitous in nature, although those in bacteria and yeast are structurally different from the mammalian enzymes. Diacylglycerol kinases, of which at least ten isoforms (DGKα to DGKκ) exist with different sub-cellular locations and functions in animals, use ATP as the phosphate donor. While the epsilon isoform (DGKε) utilizes the 1-stearoyl-2-arachidonoyl species of diacyl-sn-glycerols preferentially to produce phosphatidic acid for the biosynthesis of phosphatidylinositol, other isoenzymes phosphorylate diverse diacylglycerol species. Aside from producing phosphatidic acid for phospholipid production or signaling , these enzymes may attenuate the signaling effects of diacylglycerols. For example, diacylglycerol kinases can contribute to cellular asymmetry and control the polarity of cells by regulating the gradients in diacylglycerol and phosphatidic acid concentrations. Figure \(\PageIndex{5}\) below showns the synthesis of phosphatidic acid via diacylglycerols and the reverse reaction.

    PA_synthesisviaDAGs2.svg
    Figure \(\PageIndex{5}\): Synthesis of phosphatidic acid via diacylglycerols and the reverse reaction

    The reverse reaction, i.e., hydrolysis by lipins (phosphatidic acid phosphohydrolases), is discussed in our web page on triacylglycerols and briefly below. These enzymes are of importance in regulating the local concentrations of phosphatidic acid and thence its biological activity.

    A further possible route to phosphatidic acid production for signaling specifically is via acylation of lysophosphatidic acid, which can be produced independently for signaling purposes as discussed below. This pathway may be especially relevant in membranes, where the protein endophilin has LPAT activity and is believed to generate phosphatidic acid from lysophosphatidic acid in order to alter the curvature of the membrane bilayer.

    Phosphatidic Acid - Role as a Lipid Precursor

    In summary, phosphatidic acid generated via 1-acyl-sn-glycerol-3-phosphate is the primary precursor of other glycerolipids, although other pathways may be more important for generating the lipid for signaling functions. Whether separate pools of this lipid for specific purposes really exist is not certain since dynamic changes of intracellular distribution occur under various cellular conditions. These are attributed to inter-organelle transfer via vesicular transport or at membrane contact sites by lipid transfer proteins. Control of its concentration in membranes, especially in the endoplasmic reticulum, is therefore of great importance, and a transcriptional repressor 'Opi1', which binds specifically to phosphatidic acid in membranes, is a key regulatory factor. However, many other phosphatidic acid-binding proteins have been identified that influence how phosphatidic acid is used either as a biosynthetic precursor or for signaling purposes. The mechanisms for phosphatidic acid homeostasis differ among animals, plants, yeasts, and bacteria in response to the differing functional requirements in these organisms. Figure \(\PageIndex{6}\) below shows the pathways for biosynthesis of complex glycerolipids.

    Phosphatidic acid metabolism
    Figure \(\PageIndex{6}\): Biosynthesis of complex glycerolipids

    In addition to dietary, hormonal and tissue-specific factors in animals, the extent to which fatty acids are channelling either into triacylglycerol synthesis for storage in lipid droplets and secretion in lipoproteins or into glycerophospholipids for membrane formation depends to a large extent upon the enzymes of glycerol-3-phosphate pathway, their isoform expression, activities and locations. On the other hand, phosphatidic acid is not only a biosynthetic precursor of other lipids but also a regulatory molecule in the transcriptional control of the genes for glycerolipid synthesis, and regulation of its concentration in cells for this purpose is similarly essential. For example, the local concentration of phosphatidic acid in the endoplasmic reticulum is an important factor in the biogenesis of lipid droplets.

    The subsequent steps in the utilization of phosphatidic acid in the biosynthesis of triacylglycerols and of the various glycerophospholipids are described in separate documents of this website. Thus, hydrolysis of phosphatidic acid by phosphatidate phosphatase enzymes (including lipins 1, 2 and 3) is the source of most other glycerolipids, e.g. sn‑1,2‑diacylglycerols (DG), 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 (CL), and of phosphatidylinositol (PI), and in prokaryotes and yeast but not animals phosphatidylserine (PS). Depending on the organism and other factors, phosphatidylserine can be a precursor for phosphatidylethanolamine, while the latter can give rise to phosphatidylcholine by way of mono- and dimethyl-phosphatidylethanolamine intermediates. The cytidine diphosphate diacylglycerol synthase is another enzyme that consumes phosphatidic acid and is important for modulating the concentration of phosphatidic acid in cells and for regulating processes mediated by this lipid.

    While the fatty acid composition of phosphatidic acid can resemble that of the eventual products, the latter are generally much altered by re-modeling after synthesis via deacylation-reacylation reactions.

    Phosphatidic Acid - Biological Functions in Animals

    In addition to its role as an intermediate in lipid biosynthesis, phosphatidic acid and especially that generated by the action of phospholipase D and by diacylglycerol kinases may have signaling 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.

    Some of the observed 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 phosphatidic acid, which promotes negative membrane curvature and fusion of membranes. 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. In model systems, phosphatidic acid can effect membrane fusion, probably because of its ability to form non-bilayer phases. For example, the phosphatidic acid biosynthesis is believed to favor intraluminal budding of endosomal membranes with the formation of exosomes, and in many cell types, vesicle trafficking, secretion and endocytosis may require phosphatidic acid derived by the action of phospholipase D.

    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. In contrast to phosphoinositide-interacting proteins, which have defined structural folds, the binding motifs of effector proteins with phosphatidic acid are not highly conserved. 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 amino acid residues leading to de-protonation of the head group, increasing its negative charge from -1 to -2 and thus enabling stronger interactions with basic residues and tight docking with the membrane interacting protein. In this way, phosphatidic acid can tether certain proteins to membranes, and it can simultaneously induce conformational changes, hinder ligand binding and/or oligomerize proteins to alter their catalytic activity, stability and interactions with other molecules. It functions as a cellular pH sensor in effect in that binding to proteins is dependent on intracellular pH and the protonation state of its phosphate headgroup.

    One key target of the lipid is mTOR, a serine/threonine protein kinase with a signaling cascade that regulates cell growth, proliferation, motility and survival, together with protein synthesis and transcription, by integrating both nutrient and growth factor signals. This forms two distinct complexes of accessory proteins that regulate downstream targets. Of these, mTORC1 interacts directly with phosphatidic acid and this interaction allosterically activates the enzyme complex to regulate protein synthesis, mitochondrial metabolism and the transcription of enzymes involved in lipid synthesis. In contrast, phosphatidic acid appears to inhibit mTORC2 activity, for example in relation to insulin signaling .

    Phosphatidic acid is believed to regulate membrane trafficking events, and it is involved in activation of the enzyme NADPH oxidase, which operates as part of the defence mechanism against infection and tissue damage during inflammation. By binding to targeted proteins, including protein kinases, protein phosphatases and G-proteins, it may increase or inhibit their activities. Effects on gene transcription have been observed that are linked to inhibition of peroxisome proliferator-activated receptor (PPAR) activity. In yeast, phosphatidic acid in 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 repress target genes. The overall effect is a mechanism to control phospholipid synthesis.

    In addition, phosphatidic acid regulates many aspects of phosphoinositide function. For example, the murine phosphatidylinositol 4-phosphate 5-kinase, the main enzyme generating the lipid second messenger phosphatidylinositol-4,5-bisphosphate, does not appear to function unless phosphatidic acid is bound to it; this lipid, generated by the action of phospholipase D, recruits the enzyme to the membrane and induces a conformational change that regulates its activity. It may have a role in promoting phospholipase A2 activity, a key enzyme in eicosanoid production from phosphoinositide precursors.

    In relation to signaling 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 signaling functions in their own right. Conversely, both of these compounds can be in effect be de-activated by conversion back to phosphatidic acid.

    Phospholipase D isoforms and phosphatidic acid have been implicated in a variety of pathologies including neurodegenerative diseases, blood disorders, late-onset Alzheimer's disease and cancer, leading to attempts to develop specific inhibitors of the enzyme for therapeutic purposes. Similarly, the expression of LPAT isoforms can enhance the proliferation and chemoresistance of some cancer cells. Diacylglycerol kinase alpha (DGKα) is highly expressed in several refractory cancer cells, where it attenuates apoptosis, and promotes proliferation. In addition, DGKα is highly abundant in T cells and induces a nonresponsive state, which enables advanced cancers to escape immune action. Inhibition of this enzyme also is seen as a promising treatment strategy.

    Phosphatidic Acid - Biological Functions in Plants

    Phosphatidic acid is present at higher levels in roots of plants in comparison to leaves and is believed to have a function in root architecture. Similarly, its concentration is elevated in flowers and reproductive tissues, but the significance of this is not known. In addition to its role as one of the central molecules in lipid biosynthesis, it facilitates the transport of lipids across plant membranes, and it is also the key plant lipid second messenger, which is rapidly and transiently generated in response to many different biotic and abiotic stresses. In contrast to animal metabolism, the diacylglycerol signaling pathway is believed to be relatively insignificant in plants.

    The main source of phosphatidic acid for these purposes is the action of phospholipase D (PLD) on membrane phospholipids, such as phosphatidylcholine and phosphatidylethanolamine. Plants contain numerous related enzymes of this type, 12 in Arabidopsis and 17 in rice, in comparison with two in humans and one in yeast, and individual iso-enzymes may elicit specific responses. In the former, the isoforms are grouped into six classes, based on the genic architecture, sequence similarities, domain structures and biochemical properties. These depend mainly on their lipid-binding domains, with some homologous to the human and yeast enzymes and with most containing a characteristic ‘C2’ (calcium- and lipid-binding) domain. The most widespread of these is PLDα, which does not require binding to phosphatidylinositol 4,5-bisphosphate, in contrast to other PLD isoforms and the mammalian enzyme, but millimolar levels of Ca2+ are necessary. Studies with fluorescent biosensors suggest that phosphatidic acid accumulates in the subapical region of the cytosolic leaflet of the plasma membrane.

    Phosphatidic acid can also be produced by the sequential action of phospholipase C and diacylglycerol kinase on membrane inositol phospholipids, with diacylglycerols as an intermediate (there are 7 isoenzymes in A. thaliana). One difference from animal metabolism is that diacylglycerol pyrophosphate can be synthesized from phosphatidic acid in plants (see below).

    Phosphatidic acid is required to bind and allosteric activate the monogalactosyldiacylglycerol synthase (MGDG1), located in the inner envelope membrane of the chloroplast, and it may be a regulator of the biosynthesis of thylakoid membranes. Phospholipase D activity and the phosphatidic acid produced have long been recognized as of importance during germination and senescence, and they have an essential role in the response to stress damage and pathogen attack, both in higher plants and in green algae. A high content of phosphatidic acid induced by phospholipase D action during wounding or senescence brings about a loss of the membrane bilayer phase, because of the conical shape of this negatively charged phospholipid in comparison to the cylindrical shape of structural phospholipids. This change in ionization properties has crucial effects upon lipid-protein interactions, "the electrostatic-hydrogen bond switch model" described above. By promoting negative curvature at the plasma membrane and binding to clathrin proteins, it is believed to facilitate the process of endocytosis. Similar phenomena may explain why 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, it is believed to promote the response to the plant hormone abscisic acid. In addition, phosphatidic acid may interact with salicylic acid to mediate defence responses.

    In plants, phosphatidic acid is involved in many different cell responses induced by hormones, stress and developmental processes. In relation to cellular signaling, it often acts in concert with phosphatidylinositol 4,5-bisphosphate by binding to specific proteins rather than acting via a receptor. As in mammalian cells, targets for such signaling include protein kinases and phosphatases in addition to proteins involved in membrane trafficking and the organization of the cytoskeleton. It can both activate or inhibit enzymes. If the target protein is soluble, binding to phosphatidic acid can cause the protein to be sequestered into a membrane with effects upon downstream targets. For example, it is involved in promoting the growth of pollen-tubes and root hairs, decreasing peroxide-induced cell death, and mediating the signaling processes that lead to responses to ethylene and again to the 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 signaling pathway that regulates water loss from plants.

    It is noteworthy that phosphatidic acid production can be initiated by opposing stress factors, such as cold and heat, as well as by hormones that are considered to be antagonistic, such as abscisic acid and salicylic acid. It is possible that phosphatidic acid molecules synthesized by the two main pathways differ in composition and cellular distributions and so may produce different responses, but this is an open question. Certainly, during low temperature stress, phosphatidic acid is generated by the action of diacylglycerol kinase. It also seems likely that these differing activities are controlled by the cellular environment where the lipid is produced and by the availability of target proteins or other molecules with which it can act synergistically. Genes encoding enzymes involved in phosphatidic acid metabolism have been manipulated to explore their potential application for crop improvements, based on effects on plant growth, development, and stress responses.

    As in animals, phosphatidic acid is catabolized and its signaling functions are terminated by lipid phosphate phosphatases and phosphatidic acid hydrolases, and by acyl-hydrolases and lipoxygenases with the production of fatty acids and other small molecules, which are subsequently absorbed and recycled.

    Lysophosphatidic Acid

    Figure \(\PageIndex{7}\) below shows the structure of a lysophosphatidic acid (note the absence of an acyl group at C2).

    lysophosphatidicacid.svg
    Figure \(\PageIndex{7}\): lysophosphatidic acid

    Lysophosphatidic acid (LPA) or 1-acyl-sn-glycerol-3-phosphate differs structurally from phosphatidic acid in having only one mole of fatty acid per mole of lipid. As such, it is one of the simplest possible glycerophospholipids. It exists in the form of many different molecular species, i.e., esterified to 16:0 to 22:6 fatty acids, and there is preliminary evidence that saturated and polyunsaturated species may differ in their biological properties in some circumstances. As the sn-1-acylated form is more stable thermodynamically, facile isomerization ensures that this tends to predominate. As it lacks one fatty acid in comparison to phosphatidic acid, it is a much more hydrophilic molecule, while the additional hydroxyl group strengthens hydrogen bonding within membranes, properties that may be important for its function in cells.

    Although lysophosphatidic acid is present at very low levels only in animal tissues, it is extremely important biologically, influencing many biochemical processes. It is a biosynthetic precursor of phosphatidic acid, but there is particular interest in its role as a lipid mediator with growth factor-like activities. For example, it is rapidly produced and released from activated platelets to influence target cells.

    Biosynthesis: In the circulation, the most important source of lysophosphatidic acid is the activity of an enzyme with lysophospholipase D-like activity and known as ‘autotaxin’ on lysophosphatidylcholine (200 μM in plasma) to yield LPA in an albumin-bound form mainly, although it is relatively soluble in aqueous media because of its polarity and small size. This lipid is more abundant in serum (1 to 5 μM) than in plasma (100 nM), because of the release of its main precursor, lysophosphatidylcholine, from activated platelets during coagulation. Autotaxin is a member of the nucleotide pyrophosphatase-phosphodiesterase family and is also present in cerebrospinal and seminal fluids and many other tissues including cancer cell lines from which it was first isolated and characterized. Indeed, the name derives from the finding that it promoted chemotaxis on melanoma cells in an autocrine fashion. It binds to target cells via integrin and heparan sulfate proteoglycans and this may assist the delivery of lysophosphatidic acid to its receptors. Genetic deletion of the enzyme in mice results in aberrant vascular and neuronal development and soon leads to death of the embryos. However, overexpression of autotaxin causes physical defects also and is eventually lethal to embryos.

    Figure \(\PageIndex{8}\) shows the pathways for synthesis of lysophosphatidic acid.

    biosyn_lysophosphatidicacid.svg
    Figure \(\PageIndex{8}\): Pathways for synthesis of lysophosphatidic acid.

    While autotaxin is the primary source of extracellular lysophosphatidic acid, it is now established that it is produced intracellularly by a wide variety of cell types by various mechanisms often with phosphatidic acid, derived from other phospholipids by the action of phospholipase D, as the primary precursor. For example, hydrolysis of phosphatidic acid by a phospholipase A2 (PLA2) is the main mechanism in platelets, but other cellular enzymes involved include a phosphatidic acid-selective phospholipase A1 (PLA1) producing sn-2-acyl-lysophosphatidic acid, a monoacylglycerol kinase (utilizing monoacylglycerols produced by the action of lipid phosphate phosphatases) and glycerol-3-phosphate acyltransferase (the first step in phosphatidic acid biosynthesis). In particular, secretory PLA2-IIA (sPLA2-IIA) is able to induce the release of LPA from phosphatidic acid exposed on the surface of extracellular vesicles derived from platelets and Ca2+-loaded erythrocytes upon stimulation by pro-inflammatory cytokines.

    General function: 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. Lysophosphatidic acid acts upon nearly all cell types, often as a proliferative and pro-survival signal, inducing cellular invasion, migration and differentiation, while stimulating smooth muscle and fibroblast contraction, cytoskeletal rearrangement, secretion of cytokines/chemokines and numerous other effects. Many of these activities are displayed also by the 1-O-alkyl- and alkenyl-ether forms, which can be derived from platelet activating factor. On the other hand, it is possible that much of the lysophosphatidic acid produced intracellularly is used for synthesis of other phospholipids rather than for signaling purposes.

    Receptors: The informational content of the lysophosphatidic acid molecule leads to selectivity in the functional relationship with cell receptors. As most mammalian cells express receptors for lysophosphatidic acid, this lipid may initiate signaling in the cells in which it is produced, as well as affecting neighbouring cells. Characterization of cloned lysophosphatidic acid receptors in combination with strategies of molecular genetics has allowed determination of both signaling and biological effects that are dependent on receptor mechanisms. At least six G protein-coupled receptors that are specific for lysophosphatidic acid have now been identified in vertebrates, each found in particular organs and coupled to at least one or more of the four heterotrimeric Gα proteins and designated LPAR1 to LPAR6, of which LPAR1 is virtually ubiquitous in tissues. These vary appreciably amino acid sequences but are classified into two subgroups, the EDG (LPAR1-3) and P2Y (LPAR4-6) families, with differing tissue distributions. Most cell types express these receptors in different combinations. There is also some interaction with transient receptor potential cation channel V1 (TRPV1), peroxisome proliferator-activated receptor gamma (PPARγ) and other proteins. Plasma lysophosphatidic acid binds to its receptors while it is bound to albumin.

    Experimental activation of the LPAR receptors has shown that a range of downstream signaling cascades are mediated by lysophosphatidic acid signaling via these various receptors. These include activation of adenylyl cyclase, cAMP production, intracellular Ca2+ and K+ production (by activating ion channels), protein kinases, phospholipase C, phosphatidylinositol 3-kinase, small GTPases (Ras, Rho, Rac), release of arachidonic acid, and much more. In this way, lysophosphatidic acid regulates cell survival, proliferation, cytoskeleton re-arrangement, motility, cytokine secretion, cell differentiation and many other vital cellular processes. Sometimes, lysophosphatidic acid appears to function in contradictory ways, and there is evidence that it is involved in cell survival in some circumstances and in programmed cell death in others, for example.

    Signaling by lysophosphatidic acid has regulatory functions in the mammalian reproductive system, both male and female, facilitating oocyte maturation and spermatogenesis through the action of the receptors LPAR1 to LPAR3. During early gestation, it regulates vascular remodeling at the maternal-fetal interface. 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 remodeling. In the central nervous system, these receptors are thought to play a central role in both triggering and maintaining neuropathic pain by mechanisms that may involve demyelination of damaged nerves.

    Lysophosphatidic acid has 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. When applied topically to skin wounds, it has similar effects probably by stimulating proliferation of new cells to seal the wound. Receptor LPAR6 together with the phospholipase A1 is required for the development of hair follicles, and this receptor is also involved in the regulation of endothelial blood-brain barrier function. The proliferation and survival of stem cells and their progenitors is regulated by lysophosphatidic acid signaling, while in bone cells, acting via LPAR1, lysophosphatidic acid is important for bone mineralization and repair.

    Disease: There is particular interest in the activity of lysophosphatidic acid in various disease states and cancer especially, as increased expression of autotaxin and the subsequent increased levels of lysophosphatidic acid have been reported in several primary tumors. For example, a finding that lysophosphatidic acid is markedly elevated in the plasma and peritoneal fluid (ascites) of ovarian cancer patients compared to healthy controls may be especially significant. Also, 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. It is believed that the secretory form of phospholipase A2 acts preferentially on lipids from damaged membranes or microvesicles, such as those produced by malignant cells, and this eventually results in increased levels of this lipid. Lysophosphatidic acid has been shown to stimulate the expression of genes for many different enzymes that lead to the proliferation of ovarian and other cancer cells and may induce cell migration via receptors LPAR1 to LPAR3 and possibly LPAR6, while LPAR4 and LPAR5 have opposing effects. Autotaxin and LPARs have been implicated in resistance to chemotherapy and radiation treatment in cancer therapy.

    As lysophosphatidic acid has growth-factor-like activities for many cell types that induce cell proliferation and migration, changes in cellular shape and increasing of endothelial permeability, it is perhaps not surprising that it is relevant to tumor biology. Treatment of various cancer cell types with lysophosphatidic acid promotes the expression and release of interleukin 8 (IL-8), which is a potent angiogenic factor, and thus it has a critical role in the growth and spread of cancers by enhancing the availability of nutrients and oxygen. There is evidence that signaling by lysophosphatidic acid is causally linked to hyperactive lipogenesis in cancer. For example, it activates the sterol regulatory element-binding protein (SREBP) together with the fatty acid synthase and AMP-activated protein kinase–ACC lipogenic cascades leading to elevated synthesis of lipids de novo. Increased autotaxin expression has been demonstrated in many different cancer cell lines, and the expression of many of the surface receptors for lysophosphatidic acid in cancer cells is aberrant. Cancer cells must evade the immune system during metastasis, and lysophosphatidic acid facilitates this process by inhibiting the activation of T cells. Therefore, lysophosphatidic acid metabolism is a target of the pharmaceutical industry in the search for new drugs for cancer therapy, aided by a knowledge of the crystal structures of three of the receptors.

    Signaling by lysophosphatidic acid has been implicated in many aspects of chronic inflammation, which it promotes by affecting the endothelium in several ways, for example by stimulating endothelial cell migration, the secretion of chemokines-cytokines and regulating the integrity of the endothelial barrier. Problems with lysophosphatidic acid signaling together with changes in autotaxin expression are believed to be factors in such metabolic and inflammatory disorders as obesity, insulin resistance, non-alcoholic fatty liver disease, rheumatoid arthritis, multiple sclerosis and cardiovascular disease. Further, there is evidence it contributes to neurological disorders, such as Alzheimer's disease and neuropathic pain, and to asthma, fibrosis and bone malfunction. Drugs that interact with the lysophosphatidic acid receptors are reported to be effective in attenuating symptoms of several diseases in animal models, and three have passed phase I and II clinical trials for idiopathic pulmonary fibrosis and systemic sclerosis in human patients. Drugs that target autotaxin production and catabolism of lysophosphatidic acid are also in development, and the steroidal anti-inflammatory agent, dexamethasone, appears to be especially useful.

    Under certain conditions, lysophosphatidic acid 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.

    Related lipids: The sphingolipid analogue, sphingosine-1-phosphate, shows a similar range of activities to lysophosphatidic acid and the two lipids are often discussed together in the same contexts, although they may sometimes have opposing effects. Acute leukemia cells produce methyl-lysophosphatidic acids (the polar head-group is methylated). As these act as antigens to which a specific group of human T cells react strongly, it is possible that they might be a target for the immunotherapy of hematological malignancies. Other lysophospholipids are known to have distinctive biological functions (see separate web pages).

    Catabolism: Deactivation of lysophosphatidic acid is accomplished by dephosphorylation to produce monoacylglycerols by a family of three lipid phosphate phosphatases (LPP1, 2 and 3), which also de-phosphorylate sphingosine-1-phosphate, phosphatidic acid and ceramide 1-phosphate in a non-specific manner. These are integral membrane proteins with the active site in the plasma membrane facing the extracellular environment, enabling them to access and hydrolyse extracellular lysophosphatidic acid and other phospholipids. Mice with a constitutive LPP3 deficiency are not viable, but this is not true for LPP1 and LPP2 knockout mice. Lysophosphatidic acid 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.

    Phosphatidylcholine and Related Lipids

    Phosphatidylcholine - Structure and Occurrence

    Phosphatidylcholine or 1,2-diacyl-sn-glycero-3-phosphocholine (once given the trivial name 'lecithin') is a neutral or zwitterionic phospholipid over a pH range from strongly acid to strongly alkaline. It is usually the most abundant phospholipid in animals and plants, often amounting to almost 50% of the total complex lipids, and as such it is obviously a key building block of membrane bilayers. In particular, it makes up a very high proportion of lipids of the outer leaflet of the plasma membrane in animals. Virtually all the phosphatidylcholine in human erythrocyte membranes is present in the outer leaflet, for example, while in the plasma membranes of nucleated cells, 80 to 90% of this lipid is located on the outer leaflet. Phosphatidylcholine is also the principal phospholipid circulating in plasma, where it is an integral component of the lipoproteins, especially the HDL. On the other hand, it is less often found in bacterial membranes, perhaps ~10% of species, but there is none in the 'model' organisms Escherichia coli and Bacillus subtilis. In animal tissues, some of its membrane functions appear to be shared with the structurally related sphingolipid, sphingomyelin, although the latter has many unique properties of its own.

    Figure \(\PageIndex{9}\) below shows the structure of phosphatidylcholine

    PC.svg
    Figure \(\PageIndex{9}\): Phosphatidylcholine

    In animal tissues, phosphatidylcholine tends to exist in mainly in the diacyl form, but small proportions (in comparison to phosphatidylethanolamine and phosphatidylserine) of alkyl,acyl and alkenylacyl forms may also be present. As a generalization, animal phosphatidylcholine tends to contain lower proportions of arachidonic and docosahexaenoic acids and more of the C18 unsaturated fatty acids than the other zwitterionic phospholipid, phosphatidylethanolamine. Saturated fatty acids are most abundant in position sn-1, while polyunsaturated components are concentrated in position sn-2. Indeed, C20 and C22 polyenoic acids are exclusively in position sn-2, yet in brain and retina the unusual very-long-chain polyunsaturated fatty acids (C30 to C38) of the n-6 and n-3 families occur in position sn-1. Dietary factors obviously influence fatty acid compositions, but in comparing animal species, it would be expected that the structure of the phosphatidylcholine in the same metabolically active tissue would be somewhat similar in terms of the relative distributions of fatty acids between the two positions. Table \(\PageIndex{1}\) lists some representative data.

    Table \(\PageIndex{1}\). Positional distribution of fatty acids in the phosphatidylcholine of some animal tissues.
    Position Fatty acid
    16:0 16:1 18:0 18:1 18:2 20:4 22:6
     
    Rat liver [1]
    sn-1 23 1 65 7 1 trace
    sn-2 6 1 4 13 23 39 7
     
    Rat heart [2]
    sn-1 30 2 47 9 11 - -
    sn-2 10 1 3 17 20 33 9
     
    Rat lung [3]
    sn-1 72 4 15 7 3 - -
    sn-2 54 7 2 12 11 10 1
     
    Human plasma [4]
    sn-1 59 2 24 7 4 trace -
    sn-2 3 1 1 26 32 18 5
     
    Human erythrocytes [4]
    sn-1 66 1 22 7 2 - -
    sn-2 5 1 1 35 30 16 4
     
    Bovine brain (gray matter) [5]
    sn-1 38 5 32 21 1 - -
    sn-2 33 4 trace 48 1 9 4
     
    Chicken egg [6]
    sn-1 61 1 27 9 1 - -
    sn-2 2 1 trace 52 33 7 4
    1, Wood, R. and Harlow, R.D. Arch. Biochem. Biophys., 131, 495-501 (1969); DOI.
    2, Kuksis, A. et al. J. Lipid Res., 10, 25-32 (1969); DOI.
    3, Kuksis, A. et al. Can. J. Physiol. Pharm., 46, 511-524 (1968); DOI.
    4, Marai, L. and Kuksis, A. J. Lipid Res., 10, 141-152 (1969); DOI.
    5, Yabuuchi, H. and O'Brien, J.S. J. Lipid Res., 9, 65-67 (1968); DOI.
    6, Kuksis, A. and Marai, L. Lipids, 2, 217-224 (1967); DOI.

    There are some exceptions to the rule as the phosphatidylcholine in some tissues or organelles contains relatively high proportions of disaturated molecular species. For example, it is well known that lung phosphatidylcholine in most if not all animal species studied to date contains a high proportion (50% or more) of dipalmitoylphosphatidylcholine.

    The positional distributions of fatty acids in phosphatidylcholine in representative plants and yeast are listed in Table \(\PageIndex{2}\). In the leaves of the model plant Arabidopsis thaliana, saturated fatty acids are concentrated in position sn-1, but monoenoic fatty acids are distributed approximately equally between the two positions, and there is a preponderance of di- and triunsaturated fatty acids in position sn-2; the same is true for soybean ‘lecithin’. In the yeast Lipomyces lipoferus, the pattern is somewhat similar except that much of the 16:1 is in position sn-1.

    Table \(\PageIndex{2}\)​​​​​​​: Composition of fatty acids (mol %) in positions sn-1 and sn-2 in the phosphatidylcholine from plants and yeast.
    Position Fatty acid
    16:0 16:1 18:0 18:1 18:2 18:3
     
    Arabidopsis thaliana (leaves) [1]
    sn-1 42 4 5 23 26
    sn-2 1 trace 5 47 47
     
    Soybean 'lecithin' [2]
    sn-1 24 9 14 47 4
    sn-2 5 1 13 75 6
     
    Lipomyces lipoferus [3]
    sn-1 24 18 trace 37 16 4
    sn-2 4 5 trace 39 31 19
    1, Browse, J., Warwick, N., Somerville, C.R. and Slack, C.R. Biochem. J., 235, 25-31 (1986); DOI.
    2, Blank, M.L., Nutter, L.J. and Privett, O.S. Lipids, 1, 132-135 (1966); DOI.
    3, Haley, J.E. and Jack, R.C. Lipids, 9, 679-681 (1974); DOI.

    Phosphatidylcholine – Biosynthesis

    There are several mechanisms for the biosynthesis of phosphatidylcholine in animals, plants and micro-organisms. Choline itself is not synthesized as such by animal cells and is an essential nutrient, not only for phospholipid synthesis but also for cholinergic neurotransmission (acetylcholine synthesis) and as a source for methyl groups for numerous other metabolites. It must be obtained from dietary sources or by degradation of existing choline-containing lipids, for example those produced by the second pathway described below. Once taken across membranes and into cells by specific transporters, choline is immediately phosphorylated by a choline kinase (1) in the cytoplasm of the cell to produce phosphocholine, which is reacted with cytidine triphosphate (CTP) by the enzyme CTP:phosphocholine cytidylyltransferase (CCT) (2) to form cytidine diphosphocholine (CDP-choline).

    CTP + PC → CDP-choline + Pi

    The latter enzyme exists in two isoforms of which CCTα is the more important and is a soluble protein found first in the nucleoplasm, but then in the nucleoplasmic reticulum. This is considered to be the rate-limiting step in phosphatidylcholine biosynthesis, and the activity of the enzyme is regulated by signals from a sensor in the membrane that reports on the relative abundance of the final product. However, choline kinase (ChoKα) also has regulatory functions.

    Figure \(\PageIndex{10}\) below shows an interactive iCn3D model of the mammalian (rat) CTP: Phosphocholine cytidylyltransferase catalytic domain (3HL4).

    Mammalian (rat) CTP-Phosphocholine cytidylyltransferase catalytic domain (3HL4).png

    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{10}\): Mammalian (rat) CTP-Phosphocholine cytidylyltransferase catalytic domain (3HL4). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...tBYRYk7vPKD7f6

    The biologically active homodimer is shown. The B chains is colored by secondary structure and the A chain is shown in gray. Key active site residues (only shown in the A chain) are in CPK-colored sticks and labeled. A bound CDP-choline analog ((2-cytidylate-O'-phosphonyloxy)-ethyl-trimethylammonium) is shown in spacefill CPK colors.

    This enzyme (CCT) catalyzes the key regulatory and rate-limiting step in PC synthesis. The C-terminal domain binds membrane lipids and regulates the enzyme. The iCn3D model above is for the catalytic domain with the regulatory domain deleted. Two nonconserved active site side chains, His 168 and Tyr 173 interact with and position the phosphocholine. Other active site residues include Arg-196 in L6, Lys-122 in L2, and Asp-94. Figure \(\PageIndex{11}\) below shows a simplified mechanism for the reaction

    MechCTP_PCcytidylyltransferase.svg
    Figure \(\PageIndex{11}\): Simplified mechanism for CTP-Phosphocholine cytidylyltransferase

    In plants, nematodes and certain parasites, most phosphocholine is synthesized by sequential methylation of phosphoethanolamine by phospho-base N‑methyltransferases, but phosphatidylethanolamine is only methylated in this way in a few plant species. This is also the main route to free choline and betaine in plants.

    The CDP-choline produced is acted upon by the membrane-bound enzyme CDP-choline:1,2-diacylglycerol choline/ethanolamine-phosphotransferase in the endoplasmic reticulum (CEPT1), and a related choline phosphotransferase 1 (CPT1) in the trans-Golgi, which catalyse the reaction with sn-1,2-diacylglycerols to form phosphatidylcholine. The first of these is responsible for most phosphatidylcholine biosynthesis but with a somewhat different molecular species composition from the second, which has a preference for 1-alkyl precursors. This is the main pathway for the synthesis of phosphatidylcholine in animals and plants, and it is analogous to that for a major route to phosphatidylethanolamine; it is also found in a few bacterial species (e.g. Sinorhizobium meliloti). Phosphatidylcholine in mitochondria is obtained by transfer from the endoplasmic reticulum.

    Figure \(\PageIndex{12}\) below shows the main pathways for PC synthesis in plants and animals.

    PCSynthesis.svg
    Figure \(\PageIndex{172}\): Main pathways for PC synthesis in plants and animals.

    The discovery of the importance of this pathway depended a little on serendipity in that in experiments in the laboratory of Professor Eugene Kennedy, samples of adenosine triphosphate (ATP) contained some cytidine triphosphate (CTP) as an impurity. However, luck is of little value without receptive minds, and Kennedy and co-workers demonstrated that the impurity was an important metabolite that was essential for the formation of phosphatidylcholine.

    The above reaction, together with the biosynthetic mechanism for phosphatidylethanolamine, is significantly different from that for phosphatidylglycerol, phosphatidylinositol and cardiolipin. Both make use of nucleotides, but with the latter, the nucleotide is covalently linked directly to the lipid intermediate, i.e., cytidine diphosphate diacylglycerol. However, a comparable pathway to the latter for biosynthesis of phosphatidylcholine occurs in bacteria (see below).

    The source of the sn-1,2-diacylglycerol precursor, which is also a key intermediate in the formation of phosphatidylethanolamine and phosphatidylserine, and of triacylglycerols, is phosphatidic acid. In this instance, the important enzyme is phosphatidic acid phosphatase (also known as lipin or phosphatidate phosphatase’ or ‘lipid phosphate phosphatase’ or ‘phosphatidate phosphohydrolase’).

    Figure \(\PageIndex{13}\) below shows the biosynthesis of the diacyl precursor of PC

    PA_toPC.svg
    Figure \(\PageIndex{13}\): Synthesis of the diacyl precursor of PC

    This enzyme is also important for the production of diacylglycerols as essential intermediates in the biosynthesis of triacylglycerols and of phosphatidylethanolamine. Yeasts contain two such enzymes, one of which is Mg2+-dependent (PAP1) and the other Mg2+-independent (PAP2). In mammals, much of the phosphatidic acid phosphatase activity resides in three related cytoplasmic proteins, termed lipins-1, -2, and -3. Lipin-1 is found mainly in adipose tissue, while lipin-2 is present mainly in liver. They are unique among biosynthetic enzymes for glycerolipids in that they can transit among cellular membranes rather than remain tethered to membranes. Of these lipin-1 is most important and exists in three isoforms, lipin-1α, lipin-1β and lipin-1γ with lipin-1α located mainly in the nucleus and lipin-1β in the cytoplasm. Lipin-1γ is present primarily in brain.

    The second pathway for biosynthesis of phosphatidylcholine involves sequential methylation of phosphatidylethanolamine, with S-adenosylmethionine (SAM) as the source of methyl groups, with mono- and dimethylphosphatidylethanolamine as intermediates and catalysed by the enzyme phosphatidylethanolamine N‑methyltransferase. A single enzyme (~20 KDa) in two isoforms catalyses all three reactions in hepatocytes; the main form is located in the endoplasmic reticulum (ER) where it spans the membrane, while the second is found in the mitochondria-associated ER membrane. At least two N-methyltransferases are present in yeasts. This is a major pathway in the liver, generating one third of the phosphatidylcholine in this organ, but not in other animal tissues or in general in higher organisms. It may be the main route to phosphatidylcholine in those bacterial species that produce this lipid and in yeasts, but it appears to operate in only a few species of higher plants. When choline is deficient in the diet, this liver pathway is especially important.

    Figure \(\PageIndex{14}\) shows the synthesis of PC via methylation of PE.

    PCSynthesisfromSAM.svg
    Figure \(\PageIndex{14}\): synthesis of PC via methylation of PE

    A by-product of the biosynthesis of phosphatidylcholine from phosphatidylethanolamine is the conversion of S‑adenosylmethionine to S‑adenosylhomocysteine, which is hydrolyzed in the liver to adenosine and homocysteine. An elevated level of the latter in plasma is a risk factor for cardiovascular disease and myocardial infarction.

    Phosphatidylcholine biosynthesis by both pathways in the liver is necessary for normal secretion of the plasma lipoproteins (VLDL and HDL), and it is relevant to a number of human physiological conditions. It should be noted that all of these pathways for the biosynthesis of diacylphosphatidylcholine are very different and are separated spatially from that producing alkyl, acyl- and alkenylacyl-phosphatidylcholines de novo. Also, synthesis of phosphatidylcholine does not occur uniformly throughout the endoplasmic reticulum but is located at membrane interfaces or where it meets other organelles, and especially where the membrane is expanding dynamically.

    The enzymes in the endoplasmic reticulum responsible for the synthesis of all phospholipids are orientated in such a manner that their active sites are exclusively facing the cytosol. Problems would arise if there were a rapid expansion of the cytosolic leaflet while the luminal leaflet did not change, but a phospholipid transporter known as a scramblase enables a rapid bidirectional flip-flop of phospholipids between leaflets of the bilayer in an energy-independent manner. Compositional asymmetry in first seen in the trans-Golgi and is completed before the plasma membrane is formed with phosphatidylcholine and sphingolipids present mainly in the exofacial (outer) leaflet while phosphatidylethanolamine and phosphatidylserine are enriched in the cytosolic leaflet.

    Dietary phosphatidylcholine is rapidly hydrolysed in the proximal small intestine by pancreatic enzymes with formation of lysophosphatidylcholine (and free fatty acids). Further hydrolysis can occur in the jejuno-ileal brush-border by the action of the membrane phospholipases, with the release of glycerophosphocholine, but much of the lysophosphatidylcholine is reacylated by the lyso-PC-acyl-CoA-acyltransferase 3 for export in chylomicrons.

    In plant cells, phosphatidylcholine biosynthesis occurs mainly in the endoplasmic reticulum, and it is a major components of most membranes other than the internal membranes of plastids; it is absent from the thylakoids and the inner envelope membrane, but is the main glycerolipid of the outer monolayer of the outer envelope membrane. Further complications arise in plants in that turnover or partial synthesis via lysophosphatidylcholine occurs in different organelles from different fatty acid pools or with enzymes with differing specificities, and in addition fatty acids esterified to phosphatidylcholine serve as substrates for desaturases. The result is that an appreciable pool of the diacylglycerols for the biosynthesis of triacylglycerols, galactosyldiacylglycerols and other glycerolipids pass through phosphatidylcholine as an intermediate, so that the fatty acid compositions in different membranes change after the initial synthetic process. This mechanism has obvious differences from the remodeling of molecular species in animal tissues discussed next, although a comparable exchange of acyl groups does occur in part catalysed by acyl transferases (see next section). Some transfer of phosphatidylcholine per se from the endoplasmic reticulum to plastids may occur via contact points between the two membranes or may be facilitated by specific transport proteins.

    While phosphatidylcholine is a major lipid in yeasts, recent work suggests that it is not essential if suitable alternative growth substrates are available, unlike higher organisms where perturbation of phosphatidylcholine synthesis can lead to inhibition of growth or even cell death.

    Remodeling of Phosphatidylcholine - the Lands' cycle

    Whatever the mechanism of biosynthesis of phosphatidylcholine in animal tissues, it is apparent that the fatty acid compositions and positional distributions on the glycerol moiety are determined post synthesis by extensive re-modeling involving orchestrated reactions of hydrolysis (phospholipase A2 mainly) to lysophosphatidylcholine, acyl-CoA synthesis and re-acylation by lysophospholipid acyltransferases or transacylases, a series of reactions that is sometimes termed the 'Lands' Cycle' after its discoverer W.E.M. (Bill) Lands. Similar processes occur with all glycerophospholipid classes.

    The final composition of the lipid is achieved by a mixture of synthesis de novo and the remodeling pathway. There are at least fifteen different groups of enzymes in the phospholipase A2 super-family, which differ in calcium dependence, cellular location and structure (discussed on another web page in greater detail in relation to eicosanoid production). All hydrolyse the sn-2 ester bond of phospholipids specifically, generating a fatty acid and lysophospholipid, both of which have important functions in their own right in addition to their role in the Lands cycle. There is also a phospholipase A1 family of enzymes, which are esterases that are able to cleave the sn-1 ester bond but are less important in this context.

    Figure \(\PageIndex{15}\) below shows Land's cycle

    PC_to_lysoPC_LandsCyclev2.svg
    Figure \(\PageIndex{15}\): Land's cycle - 1

    The re-acylation step is catalysed by membrane-bound coenzyme A-dependent lysophosphatidylcholine acyltransferases such as LPCAT3 (also designated ‘MBOAT5’), which is located chiefly within the endoplasmic reticulum, though also in mitochondria and the plasma membrane in organs such as the liver, adipose tissue and pancreas. It maintains systemic lipid homeostasis by regulating lipid absorption and composition in the intestines, the secretion of lipoproteins, and lipogenesis de novo in liver, and is notable in that it incorporates linoleoyl and arachidonoyl chains specifically into lysophosphatidylcholine (as does a related enzyme LPCAT2). There is also a CoA-independent acyltransferase in inflammatory cells that transfers arachidonic acid from phosphatidylcholine to ethanolamine-containing phospholipids. While LPCAT3 prefers 1-acyl lysophosphatidylcholine as an acyl acceptor, LPCAT2 utilizes both 1-acyl and 1-alkyl precursors. LPCAT2 is highly expressed in inflammatory cells such as macrophages and neutrophils, which contain ether-phospholipids, where it contributes to the production of eicosanoid lipid mediators. The highly saturated molecular species of phosphatidylcholine found in lung surfactant are formed from species with a more conventional composition by remodeling by an acyltransferase with a high specificity for palmitoyl-CoA acid (LPCAT1). In other tissues, those species containing high proportions of polyunsaturated fatty acids depend more on synthesis de novo. These and further related enzymes are involved in remodeling of all other phospholipids. Over-expression of the genes for these enzymes is associated with the progression of many different cancers and may be involved in other pathological conditions.

    Phosphatidylcholine has a central role in glycerolipid metabolism in plants and remodeling occurs for reasons and by mechanisms that are rather different from those in animal cells as described briefly above. For example, there is extensive remodeling as a site of fatty acid desaturation (see our web page on polyunsaturated fatty acids) and as the main entry point for acyl groups exported from the plastid into the endoplasmic reticulum. In addition, remodeling of phosphatidylcholine provides fatty acids for triacylglycerol synthesis in developing seeds and diacylglycerols for the synthesis of thylakoid lipids such as galactosyldiacylglycerols. In Arabidopsis, two lysophosphatidylcholine acyltransferases, LPCAT1 and LPCAT2, are involved in remodeling in developing seeds and leaves, with some preference for position sn-2 using fatty acids exported from the plastid. In some plant species, there is a strong preference for C18‑unsaturated acyl chains over 16:0. However, the lipases that generate lysophosphatidylcholine from phosphatidylcholine for this purpose are not yet known. Some remodeling in plant membranes occurs in response to stress.

    The yeast Saccharomyces cerevisiae is able to reacylate glycerophosphocholine, generated endogenously by the action of phospholipase B (an enzyme with both phospholipase A1 and A2 activities) on phosphatidylcholine, with acyl-CoA in the microsomal membranes by means of a glycerophosphocholine acyltransferase (Gpc1) to produces lysophosphatidylcholine, which can be converted back to phosphatidylcholine by the lysophospholipid acyltransferase (Ale1) with appreciable changes in the molecular species composition. The process is regulated in coordination with the other main lipid pathways and affects yeast growth. The enzyme Gpc1 does not affect other phospholipids in yeasts. A similar mechanism appears to operate in some plant species. Figure \(\PageIndex{16}\) below shows variants of the Land's cycle.

    Remodelling of phosphatidylcholine species in yeasts
    Figure \(\PageIndex{16}\): Variants of the Land's cycle - 2

    Catabolism

    Phosphatidylcholine (and most other glycerophospholipids) in membranes can be metabolized by lipolytic enzymes, especially phospholipases, some isoforms of which are specific for particular lipid classes in humans. For example, in addition to the action of phospholipase A (discussed above), phospholipase C (six families in mammals differing in expression and subcellular distribution) yields diacylglycerols together with phosphocholine by hydrolysing glycerophospholipids at the phosphodiester bond, a process that is especially important in relation to phosphoinositide metabolism. The sphingomyelin synthases also have phospholipase C activity (in the absence of ceramide). Phospholipase D generates phosphatidic acid and choline, while phospholipase B removes both fatty acids to yield glycerophosphocholine.

    Figure \(\PageIndex{17}\) below shows the activities of phospholipases on phosphatidyl choline.

    HydrolysisPC.svg
    Figure \(\PageIndex{17}\): Activities of phospholipases on phosphatidyl choline

    On catabolism in this way, the lipid components are re-cycled or they may have signaling functions, while much of the choline is re-used for phosphatidylcholine biosynthesis, often after being returned to the liver (the CDP-choline cycle). Some choline is oxidized in the kidney and liver to betaine, which serves as a donor of methyl groups for S-adenosylmethionine production, and some is lost through excretion of phosphatidylcholine in bile. A proportion is used in nervous tissues for production of acetylcholine, a neurotransmitter of importance to learning, memory and sleep. Phosphatidylcholine in the high-density lipoproteins of plasma is taken up by the liver, and perhaps surprisingly a high proportion of this is eventually converted to triacylglycerols via diacylglycerol intermediates.

    Phosphatidylcholine – Biological Functions

    Because of the generally cylindrical shape of the molecule, phosphatidylcholine organizes spontaneously into bilayers, so it is ideally suited to serve as the bulk structural element of biological membranes, and as outlined above it is makes up a high proportion of the lipids in the outer leaflet of the plasma membrane. The unsaturated acyl chains are kinked and confer fluidity on the membrane. Such properties are essential to act as a balance to those lipids that do not form bilayers or that form specific micro-domains such as rafts. While phosphatidylcholine does not induce curvature of membranes, as may be required for membrane transport and fusion processes, it can be metabolized to form lipids that do.

    In contrast, dipalmitoyl phosphatidylcholine is the main surface-active component of human lung surfactant, although in other animals the lung surfactant can be enriched in some combination of short-chain disaturated and monounsaturated species, mainly palmitoylmyristoyl- and palmitoylpalmitoleoyl- in addition to the dipalmitoyl-lipid. This is believed to provide alveolar stability by decreasing the surface tension at the alveolar surface to a very low level during inspiration while preventing alveolar collapse at the end of expiration. Also, the internal lipids of the animal cell nucleus (after the external membrane has been removed) contain a high proportion of disaturated phosphatidylcholine. This is synthesized entirely within the nucleus, unlike phosphatidylinositol for example, and in contrast to other cellular lipids its composition cannot be changed by extreme dietary manipulation; it has been suggested that it may have a role in stabilizing or regulating the structure of the chromatin, as well as being a source of diacylglycerols with a signaling function. A further unique molecular species, 1-oleoyl-2-palmitoyl-phosphatidylcholine, is located specifically at the protrusion tips of neuronal cells and appears to be essential for their function, while 1-palmitoyl-2-arachidonoyl-phosphatidylcholine is important in the regulation of the progression of the cell cycle and cell proliferation, and this is independent of eicosanoid production.

    Phosphatidylcholine is present bound non-covalently in the crystal structures of a number of membrane proteins, including cytochrome c oxidase and yeast cytochrome bc1. The ADP/ATP carrier protein has two binding sites for phosphatidylcholine, one on each side. In addition, it is known that the enzyme 3‑hydroxybutyrate dehydrogenase must be bound to phosphatidylcholine before it can function optimally. Both the head group and the acyl chains may be involved in the interactions depending on the protein.

    As noted above, phosphatidylcholine is by far the most abundant phospholipid component in plasma and in all plasma lipoprotein classes. Although it is especially abundant in high density lipoproteins (HDL), it influences strongly the levels of all circulating lipoproteins and especially of the very-low-density lipoproteins (VLDL), which are surrounded by a phospholipid monolayer. Indeed, phosphatidylcholine with polyunsaturated fatty acids in position sn-2 is essential for the assembly and secretion of VLDLs and chylomicrons in liver and the intestines, and it must be synthesized de novo in the latter. Similarly, phosphatidylcholine synthesis is required to stabilize the surface of lipid droplets in tissues where triacylglycerols are stored.

    Some of the phosphatidylcholine synthesized in the liver is secreted into bile by a specific flippase together with bile acids where it assists in the emulsification of dietary triacylglycerols in the intestinal lumen to facilitate their hydrolysis and uptake. Eventually, it is absorbed across the intestinal brush border membrane after hydrolysis to lysophosphatidylcholine, which may then be involved in the initiation of chylomicron formation in the endoplasmic reticulum of enterocytes by activation of a protein kinase. In addition, phosphatidylcholine produced in enterocytes is secreted into the intestinal lumen and forms part of the hydrophobic mucus layer that protects the intestinal surface.

    Phosphatidic acid generated from phosphatidylcholine by the action of phospholipase D in plants has key signaling functions. Similarly, phosphatidic acid generated in this way from phosphatidylcholine in animals is involved in the metabolism and signaling function of phosphoinositides by activating phosphatidylinositol 4-phosphate 5-kinase, the main enzyme generating the lipid second messenger phosphatidylinositol-4,5-bisphosphate. The plasmalogen form of phosphatidylcholine may also have a signaling function, as thrombin treatment of endothelial cells activates a selective hydrolysis (phospholipase A2) of molecular species containing arachidonic acid in the sn-2 position, releasing this fatty acid for eicosanoid production, while the diacyl form of phosphatidylcholine may have a related function in signal transduction in other tissues. In addition, phosphatidylcholine may have a role in signaling via the generation of diacylglycerols by phospholipase C, especially in the nucleus. Although the pool of the precursor is so great in many tissues that turnover is not easily measured, the presence of phospholipases C and D that are specific for phosphatidylcholine and are activated by a number of agonists suggests such a function especially in the cell nucleus. Diacylglycerols formed in this way would be much more saturated than those derived from phosphatidylinositol, and would not be expected to be as active in some functions.

    Phosphatidylcholine is the biosynthetic precursor of sphingomyelin and as such must have some influence on the many metabolic pathways that constitute the sphingomyelin cycle. It is also a precursor for phosphatidic acid, lysophosphatidylcholine and platelet-activating factor, each with important signaling functions, and of phosphatidylserine.

    Because of the increased demand for membrane constituents, there is enhanced synthesis of phosphatidylcholine in cancer cells and solid tumours; the various biosynthetic and catabolic enzymes are seen as potential targets for the development of new therapeutic agents. Impaired phosphatidylcholine biosynthesis has been observed in a number of pathological conditions in the liver in humans, including the development of non-alcoholic fatty liver disease, liver failure and impaired liver regeneration. Similarly, a deficiency in phosphatidylcholine or an imbalance in the ratio of phosphatidylcholine to phosphatidylethanolamine has negative effects upon insulin sensitivity and glucose homeostasis in skeletal muscle.

    Plants and bacteria: In addition to its structural role in plant membranes, phosphatidylcholine levels at the shoot apex correlate with flowering time, and this lipid is believed to bind to the Flowering Locus T, a master regulator of flowering. Molecular species containing relatively low levels of α-linolenic acid are involved. Diacylglycerols formed by the action of a family of enzymes of the phospholipase C type on phosphatidylcholine, as opposed to phosphatidylinositol, may be more important in plants and especially during phosphate deprivation for the generation of precursors for galactolipid biosynthesis and perhaps for lipid re-modelling more generally. In prokaryotes, phosphatidylcholine is essential for certain symbiotic and pathogenic microbe-host interactions. For example, in human pathogens such as Brucella abortus and Legionella pneumophila, this lipid is necessary for full virulence, and the same is true for plant pathogens, such as Agrobacterium tumefaciens. Bacteria symbiotic with plants, e.g. the rhizobial bacterium Bradyrhizobium japonicum, require it to establish efficient symbiosis and root nodule formation.

    Lysophosphatidylcholine

    Figure \(\PageIndex{18}\) shows the structure of lysophosphatidylcholine

    lysoPC.svg
    Figure \(\PageIndex{18}\): Lysophosphatidylcholine

    Lysophosphatidylcholine (LPC), with one mole of fatty acid per mole of lipid in position sn-1, is found in trace amounts in most animal tissues, although there are relatively high concentrations in plasma (150–500µM). It is produced by hydrolysis of dietary and biliary phosphatidylcholine and is absorbed as such in the intestines, but it is re-esterified before being exported in the lymph. In addition, it is formed in most tissues by hydrolysis of phosphatidylcholine by means of the superfamily of phospholipase A2 enzymes as part of the de-acylation/re-acylation cycle that controls the overall molecular species composition of the latter, as discussed above. Much of the LPC in the plasma of animal species is secreted by hepatocytes into plasma in a complex with albumin, but an appreciable amount is formed in plasma by the action of the enzyme lecithin:cholesterol acyltransferase (LCAT), which is secreted from the liver. This catalyses the transfer of fatty acids from position sn-2 of phosphatidylcholine to free cholesterol in plasma, with formation of cholesterol esters and of course of lysophosphatidylcholine, which consists of a mixture of molecular species with predominately saturated and mono- and dienoic fatty acid constituents. Some LPC is formed by the action of an endothelial lipase on phosphatidylcholine in HDL.

    At high concentrations, lysophosphatidylcholine can disrupt membranes, while some biological effects at low concentrations may be simply due to its ability to diffuse readily into membranes, altering their curvature and indirectly affecting the properties of membrane proteins. In plasma, it is bound to albumin and lipoproteins so that its effective concentration is reduced to a relatively safe level.

    Lysophosphatidylcholine is considered to be an important factor in cardiovascular and neurodegenerative diseases. It is usually considered to have pro-inflammatory properties and it is known to be a pathological component of oxidized lipoproteins (LDL) in plasma and of atherosclerotic lesions, when it is generated by overexpression or enhanced activity of phospholipase A2. Its concentration is elevated in joint fluids from patients with rheumatoid arthritis. In addition, it is a major component of platelet-derived microvesicles and activates a specific receptor in platelets that ultimately leads to vascular inflammation, increasing the instability of atherosclerotic plaques. The intracellular acyltransferase LPCAT cannot remove lysophosphatidylcholine directly from plasma or lipoproteins, nor do there appear to be any enzymes with lysophospholipase A1 activity in the circulation. Lysophosphatidylcholine blocks the formation of early hemifusion intermediates required for cell-cell fusions. Lysophosphatidylcholine in insect bites attracts inflammatory cells to the site, enhances parasite invasion, and inhibits the production of nitric oxide, for example in Chagas disease. Elevated levels of 26:0‑lysophosphatidylcholine in blood are reported to be characteristic of Zellweger spectrum disorders (the result of a defect in peroxisome biogenesis).

    Elevated levels of lysophosphatidylcholine have been identified in cervical cancer and may be diagnostic for the disease. On the other hand, reduced concentrations of lysophosphatidylcholine are observed in some malignant cancers, and it has protective effects in patients undergoing chemotherapy. Stearoyl-lysophosphatidylcholine has an anti-inflammatory role in that it is protective against lethal sepsis in experimental animals by various mechanisms, including stimulation of neutrophils to eliminate invading pathogens through a peroxide-dependent reaction. Similarly, there are reports that lysophosphatidylcholine may have beneficial effects in rheumatoid arthritis and a number of other diseases. However, there are suggestions that some experimental studies in vitro of the activity of lysophosphatidylcholines may be flawed because insufficient levels of carrier proteins were used. A further point for consideration is that lysophosphatidylcholine is the precursor of the key lipid mediator lysophosphatidic acid via the action of the enzyme autotaxin in plasma, and this may be the true source of some of the effects described for the former, especially on cell migration and survival.

    There is evidence to suggest that lysophosphatidylcholine containing docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, presumably in position sn-2, in plasma targets more of these fatty acids into the brain, via a specific receptor/transporter at the blood-brain barrier known as the sodium-dependent LPC symporter 1 (MFSD2A), than occurs from the corresponding fatty acids in unesterified form. Hepatic lipase is especially important for generation of these lipids. This finding is now being explored in relation to potential therapeutic applications for neurological diseases, cognitive decline and dementia. Similarly, at the maternal plasma/placental interface, phosphatidylcholine is taken up and hydrolysed to sn‑2‑lysophosphatidylcholine, presumably by the endothelial lipase, to facilitate transfer of polyunsaturated fatty acids across the basal membrane into the fetal circulation with the aid of the same LPC transporter.

    Lysophosphatidylcholine has been found to have some functions in cell signaling , and specific receptors (coupled to G proteins) have been identified, i.e., GPR119, GPR40 and GPR55. It activates the specific phospholipase C that releases diacylglycerols and inositol triphosphate with resultant increases in intracellular Ca2+ and activation of protein kinase C. Increased glucose-stimulated insulin secretion has been observed in different cell systems. Lysophosphatidylcholine also activates the mitogen-activated protein kinase in certain cell types, and it promotes demyelination in the nervous system. By interacting with the TRPV4 ion channels of skin keratinocytes, it causes persistent itching. Identification of a highly specific phospholipase A2γ in peroxisomes that is unique in generating sn-2-arachidonoyl lysophosphatidylcholine suggests that this may be of relevance to eicosanoid generation and signaling . For example, there is reportedly an enrichment of 2-arachidonoyl-lysophosphatidylcholine in carotid atheroma plaque from type 2 diabetic patients. In vascular endothelial cells, it induces the important pro-inflammatory mediator cyclooxygenase-2 (COX-2), a key enzyme in prostaglandin synthesis. However, it has beneficial effects on the innate immune system as it is able to activate macrophages and increase their phagocytic activity in the presence of T lymphocytes.

    As lysophospholipids in general and lysophosphatidylcholine in particular are important signaling molecules within mammalian cells, their levels are closely regulated, mainly by the action of the lysophospholipases A1 and A2 (LYPLA1 and LYPLA2), depending on the position to which the fatty acid is esterified; these are cytosolic serine hydrolases with esterase and thioesterase activity. The glycerophosphocholine produced can enter the Lands' cycle or be further degraded.

    In relation to plants, amylose-rich starch granules of cereal grains contain lysophosphatidylcholine as virtually the only lipid in the form of inclusion complexes or lining channels in the starch macromolecules.

    Phosphatidylethanolamine and Related Lipids

    Phosphatidylethanolamine – Structure and Occurrence

    Phosphatidylethanolamine or 1,2-diacyl-sn-glycero-3-phosphoethanolamine (once given the trivial name 'cephalin') is usually the second most abundant phospholipid in animal and plant lipids, after phosphatidylcholine, and it is frequently the main lipid component of microbial membranes. It can amount to 20% of liver phospholipids and as much as 45% of those of brain; higher proportions are found in mitochondria than in other organelles. As such, it is obviously a key building block of membrane bilayers, and it is present exclusively in the inner leaflet of the plasma membrane in animal cells, for example. It is a neutral or zwitterionic phospholipid (at least in the pH range 2 to 7), with the structure shown (with one specific molecular species illustrated as an example).

    Figure \(\PageIndex{19}\) below shows the structure of phosphatidylethanolamine.

    phosphatidylethanolamine.svg
    Figure \(\PageIndex{19}\): Phosphatidylethanolamine

    In animal tissues, phosphatidylethanolamine tends to exist in diacyl, alkyl,acyl and alkenyl, acyl forms, and data for the compositions of these various forms from phosphatidylcholine from bovine heart muscle as an example are listed in our web pages on ether lipids. As much as 70% of the phosphatidylethanolamine in some cell types (especially inflammatory cells, neurons and tumor cells) can have an ether linkage, but in liver, the plasmalogen form of phosphatidylethanolamine, i.e., with an O‑alk-1’-enyl linkage, accounts for only 0.8% of total phospholipids. Generally, there is a much higher proportion of phosphatidylethanolamine with ether linkages than of phosphatidylcholine. If biosynthesis of the plasmalogen form is inhibited by physiological conditions, it is replaced by the diacyl form so that the overall content of the phospholipid remains constant.

    In general, animal phosphatidylethanolamine tends to contain higher proportions of arachidonic and docosahexaenoic acids than the other zwitterionic phospholipid, phosphatidylcholine. These polyunsaturated components are concentrated in position sn-2 with saturated fatty acids most abundant in position sn-1, as illustrated for rat liver and chicken egg in Table \(\PageIndex{3}\)​​​​​​​. In most other species, it would be expected that the structure of the phosphatidylethanolamine in the same metabolically active tissues would exhibit similar features.

    Table \(\PageIndex{3}\)​​​​​​​: Positional distribution of fatty acids in phosphatidylethanolamine in animal tissues.
    Position Fatty acid
    14:0 16:0 18:0 18:1 18:2 20:4 22:6
     
    Rat liver [1]
    sn-1 25 65 8
    sn-2 2 11 8 8 10 46 13
     
    Chicken egg [2]
    sn-1 32 59 7 1
    sn-2 1 1 25 22 29 12
    1, Wood, R. and Harlow, R.D., Arch. Biochem. Biophys., 131, 495-501 (1969);
    2, Holub, B.J. and Kuksis, A. Lipids, 4, 466-472 (1969);

    The O-alkyl and O-alkenyl chains at the sn-1 position of the analogous ether lipids generally consist of 16:0, 18:0 or 18:1 chains, whereas arachidonic and docosahexaenoic acids are the most abundant components at the sn-2 position.

    The positional distributions of fatty acids in phosphatidylethanolamine from the leaves of the model plant Arabidopsis thaliana are listed in Table \(\PageIndex{4}\)​​​​​​​. Here also saturated fatty acids are concentrated in position sn-1, and there is a preponderance of di- and triunsaturated in position sn-2. The pattern is somewhat different for the yeast Lipomyces lipoferus, where the compositions of the two positions are relatively similar.

    Table \(\PageIndex{4}\)​​​​​​​: Composition of fatty acids (mol %) in positions sn-1 and sn-2 in the phosphatidylethanolamine from leaves of Arabidopsis thaliana and from Lipoferus lipoferus .
    Position Fatty acid
    16:0 16:1 18:0 18:1 18:2 18:3
     
    A. thaliana [1]  
    sn-1 58 trace 4 5 15 18
    sn-2 trace trace trace 2 60 38
     
    L. lipoferus [2]  
    sn-1 29 18 4 28 13 6
    sn-2 23 15 3 34 17 6
    1, Browse, J., Warwick, N., Somerville, C.R. and Slack, C.R. Biochem. J., 235, 25-31 (1986); DOI.
    2, Haley, J.E. and Jack, R.C. Lipids, 9, 679-681 (1974); DOI .

    Phosphatidylethanolamine – Biosynthesis

    The two main pathways employed by mammalian cells for the biosynthesis of phosphatidylethanolamine are the CDP-ethanolamine pathway, i.e., one of the general routes to phospholipid biosynthesis de novo in plants and animals, and the phosphatidylserine decarboxylase pathway, which occur in two spatially separated organelles - the endoplasmic reticulum and mitochondria, respectively. Ethanolamine is obtained by decarboxylation of serine in plants, and in animals most must come from dietary sources and requires facilitated transport into cells. A small amount of ethanolamine phosphate comes from catabolism of sphingosine-1-phosphate, and this is essential for the survival of the protozoon Trypanosoma brucei. The initial steps in phosphatidylethanolamine biosynthesis occur in the cytosol with first the phosphorylation of ethanolamine by two specific ethanolamine kinases to produce ethanolamine phosphate; the reverse reaction can occur by means of the enzyme ethanolamine phosphate phospholyase and this may have a regulatory function in some tissues. The second step is rate-limiting, i.e., reaction of the product with cytidine triphosphate (CTP) to form cytidine diphosphoethanolamine catalysed by CTP:phosphoethanolamine cytidylyltransferase.

    In the final step, a membrane-bound enzyme CDP-ethanolamine:diacylglycerol ethanolaminephosphotransferase catalyses the reaction of cytidine diphosphoethanolamine with diacylglycerol to form phosphatidylethanolamine. There are two such enzymes, ethanolamine phosphotransferase 1 (EPT1) in the Golgi and choline/EPT1 (CEPT1) in the endoplasmic reticulum, but EPT1 is more important for the biosynthesis of the plasmalogen form, 1-alkenyl-2-acyl-glycerophosphoethanolamine, and especially molecular species containing polyunsaturated fatty acids, while CEPT1 produced species with shorter-chain fatty acids. The diacylglycerol precursor is formed from phosphatidic acid via the action of the enzyme phosphatidic acid phosphohydrolase.

    Figure \(\PageIndex{20}\) below shows the synthesis of phosphatidyethanolamine in the ER/Golgi.

    phosphatidylethanolamineSynthesisV2.svg
    Figure \(\PageIndex{20}\): Synthesis of phosphatidyethanolamine in the ER/Golgi

    The second major pathway is the conversion (decarboxylation) of phosphatidylserine to form phosphatidylethanolamine in mitochondria. Conservation of the this pathway from bacteria to humans suggests that it has been preserved to optimize mitochondrial performance. Mitochondria are not connected with the rest of the cell's membrane network by classical vesicular routes, but must receive and export small molecules through nonvesicular transport at zones of close proximity with other organelles at membrane contact sites, such as a specific domain of the endoplasmic reticulum termed the mitochondria-associated membrane (MAM). Lipid transport of phosphatidylserine is then enabled by tethers that bridge two membranes, lipid transfer proteins and recruitment proteins. In this process, the lipid must traverse two aqueous compartments, the cytosol and the mitochondrial intermembrane space, to reach the inner mitochondrial membrane.

    The phosphatidylserine decarboxylase is located on the external aspect of the mitochondrial inner membrane, and most of the phosphatidylethanolamine in mitochondria is derived from this pathway. While various isoforms of the phosphatidylserine decarboxylation exist in prokaryotes, yeasts and mammals, the main forms designated 'PSD1' are found only in mitochondria and are related structurally. An isoform designated 'PSD2' is located in the endosomal membranes of yeasts, and the phosphatidylethanolamine formed is the preferred substrate for phosphatidylcholine biosynthesis. It is evident that cellular concentrations of phosphatidylethanolamine and phosphatidylserine are closely related and tightly regulated.

    Figure \(\PageIndex{21}\) below shows the synthesis of phosphatidyethanolamine in mitochondria.

    phosphatidylethanolamineSynthesisfromPSV2.svg
    Figure \(\PageIndex{21}\): Synthesis of phosphatidyethanolamine in mitochondria

    In prokaryotic cells, such as E. coli, in which phosphatidylethanolamine is the most abundant membrane phospholipid, all of it is derived from phosphatidylserine decarboxylation. In this instance, the enzyme undergoes auto-cleavage for activation and utilizes a pyruvoyl moiety to form a Schiff base intermediate with phosphatidylserine to facilitate decarboxylation. This pathway is also important in mammalian cells and yeasts, although the relative contributions of the two main pathways for phosphatidylethanolamine synthesis in mammalian cells appears to depend on the cell type. In cells in culture, more than 80% of the phosphatidylethanolamine is reported to be derived from the phosphatidylserine decarboxylase pathway, but in hamster heart and rat hepatocytes, only ~5% of phosphatidylethanolamine synthesis comes from this route and most is from the CDP-ethanolamine pathway. In yeasts, 70% of the phosphatidylethanolamine is generated by PSD1. The spatially distinct pools within the cell are functionally distinct, but both are essential to life. For example, disruption of the phosphatidylserine decarboxylase gene causes misshapen mitochondria and has lethal consequences in embryonic mice, although phosphatidylethanolamine synthesis continues for a time in other cellular regions. Similarly, elimination of the endoplasmic reticulum route is embryonically lethal.

    Three additional minor biosynthetic pathways are known. Phosphatidylethanolamine can be formed by the enzymatic exchange reaction of ethanolamine with phosphatidylserine, or by re-acylation of lysophosphatidylethanolamine. The second of these is associated with the mitochondria-associated membrane where the phosphatidylserine synthase II is located. Finally, the bacterial plant pathogen Xanthomonas campestris is able to synthesise phosphatidylethanolamine by condensation of cytidine diphosphate diacylglycerol with ethanolamine.

    Ether lipids: It should be noted that all of these pathways for the biosynthesis of diacyl-phosphatidylethanolamine are very different and are separated spatially from that producing alkyl,acyl- and alkenyl,acyl-phosphatidylethanolamines and described in a separate web page, suggesting that there may be functional differences. In the protozoon T. brucei, for example, it has been demonstrated that the diacyl and ether pools of phosphatidylethanolamine have separate functions and cannot substitute for each other.

    Lands’ cycle: The various mechanisms produce different pools of phosphatidylethanolamine species, which are often in different cellular compartments and have distinctive compositions. Studies with mammalian cell types in vitro suggest that the CDP-ethanolamine pathway produces molecular species with mono- or di-unsaturated fatty acids on the sn-2 position preferentially, while the phosphatidylserine decarboxylation reaction generates species with polyunsaturated fatty acids on the sn-2 position mainly. However, as with other phospholipids, the final fatty acid composition in animal tissues is attained by a process of remodeling known as the Lands’ cycle (see the web page on phosphatidylcholine, for example). The first step is hydrolysis by a phospholipase A2 to lysophosphatidylethanolamine, followed by reacylation by means of various acyl-CoA:lysophospholipid acyltransferases. The enzymes LPCAT1, 2 and 3, which are involved in phosphatidylcholine biosynthesis, are also active with phosphatidylethanolamine, while LPEAT1 utilizes lysophosphatidylethanolamine mainly and is specific for oleoyl-CoA. Some of these isoforms appear to be confined to particular tissues. There is also a CoA-independent acyltransferase in macrophages that transfers arachidonic acid from phosphatidylcholine to ethanolamine-containing phospholipids.

    Phosphatidylethanolamine – Biological Function

    Physical properties: Although phosphatidylethanolamine has sometimes been equated with phosphatidylcholine in biological systems, there are significant differences in the chemistry and physical properties of these lipids, and they have different functions in biochemical processes. Both are key components of membrane bilayers and the ratio of the two may be important to many cellular functions. However, phosphatidylethanolamine has a smaller head group, which gives the lipid a cone shape. On its own, it does not form bilayers but inverted hexagonal phases. With other lipids in a bilayer, it is believed to exert a lateral pressure that modulates membrane curvature and stabilizes membrane proteins in their optimum conformations. It can hydrogen bond to adjacent lipids and to proteins through its polar head group. For example, the phosphate can be stabilized at the binding site by interactions with lysine and arginine side chains, or with hydroxyls from tyrosine side chains. There can also be strong interactions with acyl chains of the phospholipid.

    In contrast to phosphatidylcholine, phosphatidylethanolamine is concentrated with phosphatidylserine in the inner leaflet of the plasma membrane. On the other hand, it is present in both the inner and outer membranes of mitochondria, but especially the former, where it is present at much higher levels than in other organelles and facilitates membrane fusion and protein movement across membranes in addition to many other essential mitochondrial functions, including oxidative phosphorylation via the electron transport chain. It is an important functional component of membrane contact sites between the endoplasmic reticulum and mitochondria. In eukaryotic cells, especially those of insects, studies suggest that it has a similar function to cholesterol in membranes in that it increases the rigidity of the bilayer to maintain membrane fluidity. In bacterial membranes, it appears that a primary role for phosphatidylethanolamine is simply to dilute the high negative charge density of the anionic phospholipids.

    Protein Interactions: Membrane proteins amount to 30% of the genome, and they carry out innumerable biochemical functions, including transport, energy production, biosynthesis, signaling and communication. Within a membrane, most integral proteins consist of hydrophobic α-helical trans-membrane domains that zigzag across it and are connected by hydrophilic loops. Of those parts of the proteins outwith the bilayer, positively charged residues are much more abundant on the cytoplasmic side of membrane proteins as compared to the trans side (the positive-inside rule). Phosphatidylethanolamine is believed to have a key function in that it inhibits location of negative amino acids on the cytoplasmic side, supporting the positive-inside rule, and it has an appropriate charge density to balance that of the membrane surface and the protein. However, it can also permit the presence of negatively charged residues on the cytosolic surface in some circumstances in support of protein function.

    Phosphatidylethanolamine is vital for mitochondrial functionality, as demonstrated by defects in the operation of oxidative phosphorylation in the absence of PSD1, and by the role of the lipid in the regulation of mitochondrial dynamics in general and the biogenesis of proteins in the outer mitochondrial membrane. Synthesis of phosphatidylethanolamine in the inner membrane of mitochondria is critical for the function of the cytochrome bc1 complex (III), where there is a conserved binding site for the lipid in a specific subunit.

    Phosphatidylethanolamine binds non-covalently to a superfamily of cytosolic proteins with multiple functions termed 'phosphatidylethanolamine-binding proteins'. While four members have been identified in mammals (PEBP1-4), more than 400 members of the family that are conserved during evolution are known from bacteria to higher eukaryotes. These have many different functions include lipid binding, neuronal development, serine protease inhibition and the regulation of several signaling pathways; in plants, they control shoot growth and flowering. PEBP4 is of particular interest as it is highly expressed in many different cancers and can increase their resistance to therapy. In animal tissues, phosphatidylethanolamine is especially important in the sarcolemmal membranes of the heart during ischemia, it is involved in secretion of the nascent very-low-density lipoproteins from liver and it has functions in membrane fusion and fission. It has a functional role in the Ca2+-ATPase in that one molecule of phosphatidylethanolamine is bound in a cavity between two transmembrane helices, acting as a wedge to keep them apart. This is displaced when Ca2+ is bound to the enzyme. Many other important proteins bind non-covalently to phosphatidylethanolamine in a similar way, including rhodopsin and aquaporins.

    The content of phosphatidylethanolamine in newly secreted VLDL particles and in apoB-containing particles isolated from the lumen of the Golgi is much higher than that in circulating VLDLs, suggesting that this lipid is involved in VLDL assembly and/or secretion. However, it is rapidly and efficiently removed from the VLDL in the circulation. With lipid droplets in cells, phosphatidylethanolamine is believed to promote coalescence of smaller droplets into larger ones.

    Although the mechanism has yet to fully elucidated, effects on protein conformation are believed to be behind a finding that phosphatidylethanolamine is the primary factor in brain required for the propagation and infectivity of mammalian prions. Host defence peptides are antimicrobial agents produced by both prokaryotic and eukaryotic organisms, and many of these have a high affinity for phosphatidylethanolamine as a lipid receptor to modulate their activities. For example, the peptide antibiotics cinnamycin and duramycins from Streptomyces have a hydrophobic pocket that fits around phosphatidylethanolamine such that the binding is stabilized by ionic interaction between the ethanolamine group of the lipid and the carboxylate moiety of the peptide; this complex exhibits activity against other Gram-positive organisms, such as Bacillus species.

    Much of the evidence for the unique properties of phosphatidylethanolamine comes from studies of the biochemistry of the bacterium E. coli, where this lipid is a major component of the membranes. Gram-negative bacteria have two membrane bilayers in the cell wall (see our web page on lipid A), and as much 90% of the phospholipid in the inner leaflet of the outer membrane is phosphatidylethanolamine, with a high proportion in the cytoplasmic leaflet of the inner membrane also. In particular, phosphatidylethanolamine has a specific involvement in supporting active transport of lactose by the lactose permease, and other transport systems may require or be stimulated by it. There is evidence that phosphatidylethanolamine acts as a 'chaperone' during the assembly of this and other membrane proteins to guide the folding path for the proteins and to aid in the transition from the cytoplasmic to the membrane environment, although in contrast it inhibits folding of some multi-helical proteins. In the absence of this lipid, the transport membranes may not have the correct tertiary structure and so will not function correctly. Whether the lipid is required once the protein is correctly assembled is not fully understood in all cases, but it may be needed to orient enzymes correctly in the inner membrane. Some studies suggest that life in this organism can be maintained without phosphatidylethanolamine, but that life processes are inhibited.

    Autophagy and ferroptosis: A covalent conjugate of phosphatidylethanolamine with a protein designated 'Atg8' is formed by the action of cysteine protease ATG4 (belonging to the caspase family) and various other proteins, and is involved in the process of autophagy (controlled degradation of cellular components) in yeast by promoting the formation of membrane vesicles containing the components to be degraded (phosphatidylinositol 3-phosphate is also essential to this process). Similarly, oxidatively modified phosphatidylethanolamine is an important factor in ferroptosis, a form of apoptosis in which disturbances to iron metabolism lead to an accumulation of hydroperoxides.

    Precursor of other lipids: Phosphatidylethanolamine is a precursor for the synthesis of N-acyl-phosphatidylethanolamine (see below) and thence of anandamide (N‑arachidonoylethanolamine), and it is the donor of ethanolamine phosphate during the synthesis of the glycosylphosphatidylinositol anchors that attach many signaling proteins to the surface of the plasma membrane. In bacteria, it functions similarly in the biosynthesis of lipid A and other lipopolysaccharides. It is also the substrate for the hepatic enzyme phosphatidylethanolamine N-methyltransferase, which provides about a third of the phosphatidylcholine in liver.

    Miscellaneous other functions: Phosphatidylethanolamine is the precursor of an ethanolamine phosphoglycerol moiety bound to two conserved glutamate residues in eukaryotic elongation factor 1A, which is an essential component in protein synthesis. This unique modification appears to be of great importance for the resistance of plants to attack by pathogens. Francisella tularensis bacteria, the cause of tularemia, suppresses host inflammation and the immune response when infecting mouse cells. The effect is due to a distinctive phosphatidylethanolamine species containing 10:0 and 24:0 fatty acids, and the synthetic lipid produces the same effects in vitro in human cells infected with dengue fever virus. It is hoped that this lipid will prove to be a potent anti-inflammatory therapeutic agent.

    Plants: In the seeds of higher plants, a deficiency of phosphorylethanolamine cytidylyltransferase, a rate-limiting enzyme in the biosynthesis of phosphatidylethanolamine, has profound effects upon the viability and maturation of embryos.

    Lysophosphatidylethanolamine

    Figure \(\PageIndex{22}\) below shows the the structure of lysophosphatidyethanolamine

    lysophosphatidylethanolamine.svg
    Figure \(\PageIndex{22}\): Lysophosphatidyethanolamine

    Lysophosphatidylethanolamine (LPE), with one mole of fatty acid per mole of lipid, is found in trace amounts only in animal tissues, other than plasma (10 to 50µM, or ~1% of total serum phospholipids). It is formed by hydrolysis of phosphatidylethanolamine by the enzyme phospholipase A2, as part of a de-acylation/re-acylation cycle that controls its overall molecular species composition as discussed above. A membrane-bound O-acyltransferase (MBOAT2) specific for LPE (and lysophosphatidic acid) has been characterized with a preference for oleoyl-CoA as substrate. There are reports of the involvement of LPE in cellular functions, such as differentiation and migration of certain neuronal cells, but also of various cancer cells. For example, oleoyl-LPE in brain stimulates neurite outgrowth and protects against glutamate toxicity.

    In plants, lysophosphatidylethanolamine is a specific inhibitor of phospholipase D, a key enzyme in the degradation of membrane phospholipids during the early stages of plant senescence. By this action, it retards the senescence of leaves, flowers, and post-harvest fruits. Indeed, it has a number of horticultural applications when applied externally, e.g., to stimulate ripening and extend the shelf-life of fruit, delay senescence and increase the vase life of cut flowers. In bacteria, lysophosphatidylethanolamine displays chaperone-like properties, promoting the functional folding of citrate synthase and other enzymes. Some biological properties have been reported in animal tissues in vitro, but a specific receptor has yet to be identified.

    Lysophospholipids and especially lysophosphatidylethanolamines are produced in the envelope membranes of bacteria by many different endogenous and exogenous factors and must be transported back into the bacterial cell by flippases for conversion back to the diacyl forms by the action of a peripheral enzyme, acyl-ACP synthetase/LPL acyltransferase. Lysophosphatidylethanolamines produced by certain bacteria act synergistically with sulfonolipid rosette-inducing factors (RIFs) to maximize the activity of the latter to induce choanoflagellates to move from a unicellular to a multicellular state.

    N-Acyl Phosphatidylethanolamine

    In N-Acyl phosphatidylethanolamine, the free amino group of phosphatidylethanolamine is acylated by a further fatty acid. This lipid has been detected in rather small amounts in several animal tissues (~0.01%), but especially brain, nervous tissues and the epidermis, when the N-acyl chain is often palmitic or stearic acid (human plasma: N16:0-PE (40%), N18:1-PE (23.3%), N18:0-PE (19%), N18:2-PE (16.6%) and N20:4-PE (1.4%)). Under conditions of degenerative stress, it can accumulate in significant amounts, for example as the result of ischemic injury, infarction or cancer. It is present in plasma after feeding a high fat diet to rats, and then it can cross into the brain where it accumulates in the hypothalamus.

    Figure \(\PageIndex{23}\) below shows the structures of N-Acyl Phosphatidylethanolamine.

    N-acylPE.svg
    Figure \(\PageIndex{23}\): N-Acyl Phosphatidylethanolamine

    In animals, N-Acyl phosphatidylethanolamine is of particular importance as the precursor of anandamide (see our web pages on endocannabinoids for a more detailed discussion of N-acyl phosphatidylethanolamine synthesis and metabolism), and of other biologically important ethanolamides (e.g., N-oleoylethanolamide) in brain and other tissues, but especially the intestines. In brief, it is formed biosynthetically by the action of a transferase (cytosolic phospholipase A2ε) exchanging a fatty acid from the sn-1 position of a phospholipid (probably phosphatidylcholine) to the primary amine group of phosphatidylethanolamine (without a hydrolytic step). Both diacyl- and alkenylacyl-species of phosphatidylethanolamine can serve as acceptors. In addition, some transfer can also occur from phosphatidylethanolamine per se by an intramolecular reaction. However, it should be noted that some N-acyl phosphatidylethanolamine can be formed artefactually as a result of faulty extraction procedures during analysis.

    In plants, N-acyl phosphatidylethanolamine is a common constituent of cereal grains (e.g., wheat, barley and oats) and of some other seeds (1.9% of the phospholipids of cotton seeds, but 10-12% of oats). In other plant tissues, it is detected most often under conditions of physiological stress. In contrast to animals, synthesis involves direct acylation of phosphatidylethanolamine with a free fatty acid, catalyzed by a membrane-bound transferase in a reverse serine-hydrolase catalytic mechanism. Activation of N-acyl phosphatidylethanolamine metabolism in plants with release of N-acylethanolamines and phosphatidic acid formation seems to be associated with cellular stresses, but research is at an early stage. However, both N-acyl lipid classes have been implicated in such physiological processes as the elongation of main and lateral roots, regulation of seed germination, seedling growth, and defense from attacks by pathogens.

    N-Acyl phosphatidylethanolamine has been found in a number of microbial species, while N-acetyl phosphatidylethanolamine was detected in a filamentous fungus, Absidia corymbifera, where it comprised 6% of the total membrane lipids. It was accompanied by an even more unusual lipid 1,2‑diacyl-sn-glycero-3-phospho(N-ethoxycarbonyl)-ethanolamine.

    Phosphatidylserine and Related Lipids

    Phosphatidylserine or 1,2-diacyl-sn-glycero-3-phospho-L-serine is an important anionic phospholipid, which brings essential physical properties to membranes in both eukaryotes and prokaryotes. Independently of this, it has many biological functions in cells, including effects on blood coagulation and apoptosis, and it is the biosynthetic precursor for phosphatidylethanolamine in prokaryotes and in eukaryote mitochondria. Its metabolite lysophosphatidylserine has signaling functions and operates through specific receptors. Also, there is increasing interest in a structurally related lipid phosphatidylthreonine and other phospholipids linked to amino acids.

    Phosphatidylserine - Structure and Occurrence

    Although phosphatidylserine 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. For example, mouse brain and liver contain 14 and 3% phosphatidylserine, respectively. However, it may comprise 10 to 20 mol% of the total phospholipids in the plasma membrane, where under normal conditions it is concentrated in the inner leaflet, and in the endoplasmic reticulum of cells. In the yeast Saccharomyces cerevisiae, it is a minor component of most cellular organelles other than the plasma membrane, where surprisingly it can amount to more than 30% of the total lipids. In most bacteria, it is a minor membrane constituent, although it is important as an intermediate in phosphatidylethanolamine biosynthesis. The 1‑octadecanoyl-2-docosahexaenoyl molecular species, which is especially important in brain tissue, is illustrated here.

    Figure \(\PageIndex{24}\) below shows the structure of phosphatidylserine

    PS.svg
    Figure \(\PageIndex{24}\): 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 it does not appear to resemble the precursor phospholipids, either because of selective utilization of specific molecular species for biosynthesis or because of re-modelling of the lipid via deacylation-reacylation reactions with lysophosphatidylserine as an intermediate (see below). In human plasma, 1-stearoyl-2-oleoyl and 1-stearoyl-2-arachidonoyl species predominate, but in brain (especially grey matter), retina and many other tissues 1-stearoyl-2-docosahexaenoyl species are especially abundant and appear to be essential for normal functioning of the nervous system. Indeed, the ratio of n-3 to n-6 fatty acids in brain phosphatidylserine is 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 \(\PageIndex{5}\)​​​​​​​. As with most phospholipids, saturated fatty acids are concentrated in position sn-1 and polyunsaturated in position sn-2.

    Table \(\PageIndex{5}\)​​​​​​​: Positional distribution of fatty acids in phosphatidylserine from rat liver and bovine brain
    Position Fatty acid
    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); DOI.
    2. Yabuuchi, H. and O'Brien, J.S. J. Lipid Res., 9, 65-67 (1968); DOI.

    In leaves of Arabidopsis thaliana, used as a 'model' plant in many studies, the fatty acid composition of phosphatidylserine resembles that of phosphatidylethanolamine. There is an intriguing report that the chain-lengths of the acyl groups increase with age and stress in phosphatidylserine quite specifically, and 22:0 and 24:0 fatty acids have been reported to occur in this lipid in the plasma membrane of some plant species.

    In marked contrast to phosphatidylethanolamine, phosphatidylserines with ether-linked moieties (alkyl and alkenyl) are not common in animal tissues, although they are reported to be relatively abundant in human retina and macrophages (they were first found in rat lung). As a generality, the concentration of phosphatidylserine is highest in plasma membranes and endosomes, but is very low in mitochondria. As it 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.

    N-Acylphosphatidylserine is reportedly present in the frontal cortex of patients with schizophrenia, as a minor component of the lipids of sheep erythrocytes, bovine brain and the central nervous system of freshwater fish, and Bryozoans amongst others. The N-arachidonoyl form may be the precursor of the endocannabinoid N-arachidonoylserine.

    Biosynthesis of Phosphatidylserine

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

    In animal tissues, phosphatidylserine is synthesized solely by calcium-dependent base-exchange reactions in which the polar head-group of an existing phospholipid is exchanged for L-serine. There are two routes involving distinct enzymes (PS synthase I and II) with 30% homology and several membrane-spanning domains that can utilize different substrates. Phosphatidylserine is synthesized by both enzymes on the cytosolic face of the endoplasmic reticulum (ER) of the cell, but mainly in a specific domain of this termed the mitochondria-associated membrane ('MAM'), because it is tethered transiently to the mitochondrial outer membrane, presumably by a protein bridge. In yeast, a complex of integrated proteins ('ERMES') has been characterized with a similar function. The reaction involves exchange of L-serine with either phosphatidylcholine or phosphatidylethanolamine, catalysed by PS synthase I (although it was long thought that only phosphatidylcholine was a substrate for this enzyme), while PS synthase II catalyses a similar exchange with diacyl-phosphatidylethanolamine and its the plasmalogen form. Both enzymes are subject to feedback regulation by their product phosphatidylserine, thereby maintaining the correct amounts of this lipid. Figure \(\PageIndex{25}\) below shows the synthesis and metabolism of phosphatidylserine in animals.

    Biosynthesis of phosphatidylserine in animal tissues
    Figure \(\PageIndex{25}\): Synthesis and metabolism of phosphatidylserine in animals.

    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 brain and testis and much less so in other tissues. The latter enzyme has a high specificity for molecular species containing docosahexaenoic acid. It is not known why such a complex series of coupled reactions is necessary, or why there should be two enzymes, but one virtue 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.

    As with other phospholipids, the final fatty acid composition in animal tissues is attained by a process of remodeling known as the Lands’ cycle. The first step is hydrolysis by a phospholipase A2 to lysophosphatidylserine, followed by reacylation by various acyl-CoA:lysophospholipid acyltransferases. One membrane-bound O-acyltransferase (LPCAT4 or MBOAT2) with a preference for oleoyl-CoA has been characterized, while a second (LPCAT3 or MBOAT5) incorporates linoleoyl and arachidonoyl chains (and also utilizes lysophosphatidylcholine).

    Following synthesis, phosphatidylserine molecules can diffuse laterally in a concentration-dependent manner to different regions of the membrane to fulfill their physiological functions. In humans, cytosolic transport proteins transfer phosphatidylserine and other acidic phospholipids between membranes, and this can also occur by a vesicular transport mechanism.

    Some of the newly synthesized phosphatidylserine is transferred to the plasma membrane, while a proportion is transported to the mitochondria, probably again via transient membrane contact (MAM), where it is decarboxylated to produce phosphatidylethanolamine by a specific decarboxylase in the inner mitochondrial membrane. In yeast, there is a preference for molecular species containing two monoenoic fatty acids for transport and metabolism; this process occurs also at the Golgi/endosome membranes. All the phosphatidylethanolamine in mitochondria is formed in this way, but some can return to the endoplasmic reticulum where it may be converted back to phosphatidylserine by the action of the PS synthases. Mitochondrial production of phosphatidylethanolamine from phosphatidylserine is not fully complemented by the CDP-ethanolamine pathway, as mice lacking the enzyme do not survive for long. Evidently, cellular concentrations of these two lipids are intimately related and tightly regulated. Figure \(\PageIndex{26}\) below shows the mitochondrial metabolism of phosphatidylserine

    Biosynthesis of phosphatidylserine - mitochondria
    Figure \(\PageIndex{26}\): Mitochondrial metabolism of phosphatidylserine

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

    Bacteria and plants: In bacteria and other prokaryotic organisms and in yeast, phosphatidylserine is synthesized by a mechanism comparable to that of most other phospholipids, i.e., by reaction of L-serine with CDP-diacylglycerol, and depends on Mg2+ or Mn2+. Phosphatidylserine synthases belong to two different families: type I (non-integral membrane form) in the phospholipase D-like family as in E. coli, and type II (integral membrane form) in the CDP-alcohol phosphotransferase family as in Bacillus sp. and the yeast S. cerevisiae, although the latter shows no homology with the bacterial enzymes.

    In many plants, including in the model plant Arabidopsis, much of the phosphatidylserine is produced by a calcium-dependent base-exchange reaction in which the head-group of an existing phospholipid is exchanged for L-serine in the luminal leaflet of the endoplasmic reticulum (i.e., mechanistically similar to PS synthase I). It is transferred to the cytoplasmic membrane leaflet by flippases and thence to the post-Golgi compartments before eventually accumulating at the plasma membrane. However, some vesicular transport may occur or there may be direct transfer at membrane contact sites. A CDP-diacylglycerol (prokaryotic-like) biosynthetic pathway exists in some species, e.g. wheat.

    Let's explore the mechanism of the Methanocaldococcus jannaschii phosphatidylserine synthase (MjPSS). The organism is a hyperthermophilic methanogen. Figure \(\PageIndex{27}\) below shows substrate binding by MjPSS.

    phosphatidyl serine synthaseLowerEuk_bacteria-2Fig3.svg
    Figure \(\PageIndex{27}\): Substrate binding by MjPSS. Centola, M., van Pee, K., Betz, H. et al. Crystal structures of phosphatidyl serine synthase PSS reveal the catalytic mechanism of CDP-DAG alcohol O-phosphatidyl transferases. Nat Commun 12, 6982 (2021). https://doi.org/10.1038/s41467-021-27281-w. Creative Commons Attribution 4.0 International License, http://creativecommons.org/licenses/by/4.0/.

    The large binding pocket for CDP-DAG in MjPSS extends from the hydrophobic membrane core to the active site near the cytoplasmic surface in the centre of the dimer. In the closed structures (left), both CDP-DAG alkyl chains adopt similar conformations within the binding pocket, whereas the positions of helix 7 and 8 in the open structures (right) allow one alkyl chain to reach the membrane via a different path. Serine molecules are only found in the open structures (right). In one closed conformation, there is a citrate near the substrate-binding site, whereas in the other closed structure this position is empty. A chain of three chloride ions extends parallel to the dimer interface from the active site to the cytoplasmic interface with the N-terminal helix hH from the other protomer.

    Figure \(\PageIndex{28}\) below shows the reaction cycle of MjPSS during the synthesis of PS from CDP-DAG and serine in the presence of Mg2+ and Ca2+.

    phosphatidyl serine synthaseLowerEuk_bacteriaFig6Aff.svg
    Figure \(\PageIndex{28}\): Reaction mechanism for MjPss. Centola, M. et al., ibid

    The CDPDAG binding site in MjPSS of is formed and stabilized by the divalent cations Mg2+ and Ca2+ (a). In the absence of CDP-DAG, Ca2+ most likely is coordinated by water molecules, as shown in a, or by residues in nearby loops that would be flexible in the absence of CDP-DAG. The binding of CDP-DAG (b) is driven by the coordination of Ca2+ by the negatively charged phosphates. Serine binds to the binding pocket after CDP-DAG (c). For the nucleophilic attack, the serine molecule is positioned with its hydroxyl group near the β-phosphate of CDP-DAG. The serine molecule is activated by deprotonation, attacks the β-phosphate and forms the penta-coordinated transition state (d). The proton of serine is probably removed by one of the water molecules located in the interaction network of Asp66, Arg101, and the nearby chloride ions. Hydrolysis of the CDP-DAG/serine complex from the transition state leads to the complex of MjPSS with the products PS and CMP (e). The next cycle starts after release of the products and binding of CDP-DAG. Structural data are available for the state with bound CDP-DAG (b), CDP-DAG, and serine (c), and for the transition state of the CDP-DAG/serine complex (d).

    Figure \(\PageIndex{29}\) below shows an interactive iCn3D model of the Methanocaldococcus jannaschii phosphatidyl serine synthase (PSS) in the open state with bound CDP-DAG and serine (7B1L). The enzyme is also named CDP-diacylglycerol--serine O-phosphatidyltransferase.

    Bacterial phosphatidyl serine synthase (PSS)  in transition state (7POW).png

    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{29}\): Bacterial phosphatidyl serine synthase (PSS) in the open state with bound CDP-DAG and serine (7B1L) (Note the actual PDB file title names state that this is the closed state which it is not.) Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...B3Rn3gVN4c4ge6

    The A chain of the homodimer is shown in gray and the B in plum. The active sites residues in the A chain in the above mechanism are shown in stick, CPK colors, and labeled. Hover over the large ligand (58A) in the gray subunit. Two free serines are shown near it but only one is probably the substrate.

    Phosphatidylserine – Biological Function

    Membrane location: Phosphatidylserine modulates membrane charge locally, enabling the recruitment of soluble cations and proteins, and so it contributes to the organization of processes within cell membranes. Its distribution within membranes is tightly controlled as it facilitates signaling within the various cellular compartments. Thus, it undergoes a transition from the lumenal leaflet of the endoplasmic reticulum to the cytosolic leaflet in the trans Golgi network, probably by the activity of flippases and scramblases in the Golgi, and it is highly enriched on the inner, compared to outer, leaflet of the plasma membrane. Transport to the plasma membrane against a concentration gradient is aided in part by proteins designated 'ORP5' and 'ORP8' in humans (Osh 6 and Osh7 in yeast) with a 'PH' binding domain for phosphatidylinositol 4,5-bisphosphate and an 'ORD' domain for phosphatidylserine. At a membrane contact site between the endoplasmic reticulum and plasma membrane, phosphatidylserine is exchanged for phosphatidylinositol 4-phosphate. Such transfer requires an input of energy, which can be supplied in the form of ATP or by phosphoinositides Although it does not take part in membrane raft formation, phosphatidylserine is present in caveolae, where it is believed to interact with caveolin-1. It is also present in appreciable amounts in the endosomal compartment.

    The asymmetric structure of the plasma membrane with high concentrations of anionic lipids such as phosphatidylserine in the cytosolic leaflet with zwitterionic lipids in the extracellular leaflet generates two surfaces with greatly different electrostatic potentials that influence the association of proteins with the membrane surface and the activities of integral membrane proteins. This distribution is maintained and can be altered, after specific activation, by various flippases (transfer back into the cytoplasmic leaflet), floppases (transfer out of the cytoplasmic leaflet) and scramblases (bidirectional transfer), including ATP-dependent translocases selective for phosphatidylserine. Phosphatidylserine is highly enriched in the cytosolic leaflet of the membranes of recycling endosomes, which replenish the lipids and proteins of the plasma membrane, and it is essential for their function.

    Enzyme activation: 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 a large number of proteins, especially those with signaling activities. The negative charge on the lipid facilitates the binding to proteins through electrostatic interactions or Ca2+ bridges. For example, the presence of appreciable amounts of phosphatidylserine on the cytosolic leaflet of endosomes and lysosomes enables these compartments to dock with proteins that possess specific phosphatidylserine-binding domains including several important signaling and fusogenic effectors. The cytoskeletal protein spectrin binds to phosphatidylserine in this way, and it is also required by enzymes such as the neutral sphingomyelinase and the Na+/K+ ATPase, where the 18:0/18:1 molecular species is especially important. It is believed that the fatty acyl components of this species in the inner leaflet of the plasma membrane (and potentially other intracellular membranes) may interact (interdigitation or "hand-shake") with the very-long chains of sphingolipids in the outer leaflet in raft microdomains, resulting in a high local concentration of the anionic phospholipid and an accumulation of negative surface charge to which specific poly-cationic proteins in the membranes can bind. This may then enable transfer of signals across the membrane to the cytosol.

    Similarly, phosphatidylserine participates directly in key signaling pathways in brain by binding to the cytosolic proteins involved in neuronal signaling and thereby activating them. At least three major pathways are affected, including those involving phosphatidylinositol 3-kinase and protein kinase C. For example, most enzymes of the protein kinase C family contain a 'C2' calcium-dependent cysteine-rich region that recognizes phosphatidylserine, and in coordination with the 'C1' domain that binds to diacylglycerols, is essential for activating and locating them to the plasma membrane of appropriately stimulated cells. Phosphatidylserine is not involved in cell signaling through the formation of metabolites, as is the case with phosphatidylinositol.

    Blood coagulation: Phosphatidylserine is an important element of the blood coagulation process in platelets, where it is transported from the inner to the outer surface of the plasma membrane in platelets activated by exposure to fibrin-binding receptors, for example. Here, the exposed phosphatidylserine enhances the activation of prothrombin to thrombin (the key molecule in the blood clotting cascade) by triggering a cascade of reactions and providing the negatively charged platform that enables calcium ions to form bridges with γ-carboxyglutamic acid-containing domains on the coagulation factors. Membrane vesicles with phosphatidylserine exposed on the surface can also be released from platelets and promote the coagulation process. Apolipoprotein A-1 in high-density lipoproteins has a controlling function in that it neutralizes these procoagulant properties by arranging the phospholipid in surface areas that are too small to accommodate the prothrombinase complex. Blood coagulation is beneficial when it prevents the loss of blood from the circulatory system, but it is detrimental when it causes thrombosis, and the action of phosphatidylserine is essential to the regulation of the process.

    Apoptosis: In addition in response to particular calcium-dependent stimuli, phosphatidylserine is known to have an important role in the regulation of apoptosis or programmed cell death, the natural process by which aged or damaged cells are removed from tissues before they can exert harmful effects. When cells are damaged, a mechanism is initiated in which the normal distribution of this lipid on the inner leaflet of the plasma membrane bilayer is disrupted by stimulation of scramblases, which are ATP-independent and can move the lipid across the membrane to the outer leaflet. This occurs together with inhibition of aminophospholipid translocases, which return the lipid to the inner side of the membrane. In erythrocytes, phosphatidylserine is located in the inner leaflet of the membrane bilayer under low Ca2+ conditions when a phospholipid scramblase is suppressed by membrane cholesterol, but it is exposed to the outer leaflet under elevated Ca2+ concentrations which activate the scramblase. After the collapse of this asymmetry and transfer of phosphatidylserine to the outer leaflet of an effete cell, it is believed that it is recognized by a cohort of receptors, either directly or indirectly, through bridging ligands on the surface of macrophages and related scavenger cells. These activate a family of cysteine-dependent aspartate-specific proteases, the caspases, and other enzymes to facilitate the engulfment of the apoptotic cells and their potentially toxic or immunogenic contents in a non-inflammatory manner. It is noteworthy that the transition from a pro-inflammatory to an anti-inflammatory state is defined by phagocytosis of neutrophils by macrophages via this phosphatidylserine-dependent process.

    During apoptosis, generation of reactive oxygen species occurs, mainly hydrogen peroxide, which together with the enzyme cytochrome c bring about rapid oxidation of the fatty acids in phosphatidylserine before this lipid is externalized. Indeed, it is now apparent that molecular species of phosphatidylserine with an oxidatively truncated sn-2 acyl group that incorporates terminal γ-hydroxy(or oxo)-α,β-unsaturated acyl moieties are especially potent signals for scavenger receptors in macrophages as a prerequisite for engulfment of apoptotic cells. Such oxidized lipids are discussed in our web page dealing with oxidized phospholipids.

    This has been described as "a dominant and evolutionarily conserved immunosuppressive signal that promotes tolerance and prevents local and systemic immune activation" or more succinctly as an "eat-me signal" (externalized phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P) may have a similar function). 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, and it is also relevant to clinical situations where apoptosis plays an important part, such as cancer, chronic autoimmunity, and infections. For example, phosphatidylserine is a necessary component of the TAM family of receptor tyrosine kinases and the receptor-ligand complex of particular importance in cancer cells, where phosphatidylserine-TAM signaling regulates many aspects of inflammation and immune resolution and is seen as a target for therapeutic intervention. Exposure of phosphatidylserine is increased substantially on the surface of tumor cells or tumor cell-derived microvesicles, which have innate immunosuppressive properties and facilitate tumor growth and metastasis. Targeting phosphatidylserine is considered to be a promising strategy in cancer immunotherapy. In relation to atherosclerosis, phosphatidylserine is believed to have anti-inflammatory and protective effects as a component of the high-density lipoproteins, probably mediated by the apoptosis mechanism. In contrast, as this mechanism is important for the turnover of erythrocytes, it is relevant to thrombus formation and the stabilization of blood clots. The innate immunosuppressive effect of externalized phosphatidylserine has been hijacked by numerous viruses and bacteria to facilitate infection.

    A similar apoptopic mechanism operates in retinal pigment epithelial cells to remove the large amounts of photoreceptor cell debris that are generated continuously. In addition, appreciable amounts of phosphatidylserine are translocated by an analogous 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 signaling mechanism to modulate the activities of several membrane proteins. The anti-coagulant protein annexin V binds with high specificity to phosphatidylserine and is used as a probe to detect apoptotic cells. It is noteworthy that phosphatidylserine is a major component of the membranes of microvesicles in animal cells, and translocation to the outer leaflet upon cellular activation is essential for their biogenesis. In addition, exposure of phosphatidylserine on the cell surface is reported to be a factor in non-apoptotic forms of regulated inflammatory cell death, such as necroptosis.

    Role in infections: Unfortunately, viruses such as Ebola and HIV viruses can hijack this apoptosis machinery by incorporating phosphatidylserine into their viral envelopes so conning cells into engulfing them; the viral glycoprotein/cellular receptor complex may then facilitate the entry of foreign organisms into other cells. Similarly, parasites ingested in this manner, including Leishmania and Trypanosoma species, utilize host phosphatidylserine to establish infections and facilitate disease progression as they do not then elicit production of proinflammatory cytokines. This mechanism has been termed 'apoptotic mimicry' and is critical for survival of parasites within the macrophage.

    Other activities: Phosphatidylserine is required for the transmembrane movement of excess cholesterol, derived initially from the lysosomal degradation of low-density lipoproteins, from the plasma membrane to the endoplasmic reticulum thereby maintaining membrane integrity and ensuring cell survival. It is therefore an important element in cholesterol homeostasis. The mechanism is believed to involve proteins known as GRAMD1s embedded in the endoplasmic reticulum membrane at sites in contact with the plasma membrane. These have two functional domains: the StART-like domain that binds cholesterol and the GRAM domain that binds anionic lipids, such as phosphatidylserine, and so forms a link between the two membranes that enables the transfer of cholesterol.

    A further unusual function of phosphatidylserine is that it is a key component of the lipid-calcium-phosphate complexes that act as nucleation centers for hydroxyapatite formation and 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. Nucleation is facilitated by the protein annexin V. Similarly, during bone repair and maintenance, the fusion of osteoclasts requires the non-apoptotic exposure of phosphatidylserine at the surface of fusion-committed cells with the aid of a transmembrane protein (DC-STAMP) expressed in dendrocytes. This activity is relevant to cardiovascular disease and in particular to the phenomenon of "hardening of the arteries," where atherosclerotic plaques can undergo mineralization with the deposition of hydroxyapatite.

    Among many other functions of phosphatidylserine, it is believed to be an essential surface membrane component for the fusion of cell types other than osteoclasts, including during the formation of fibres in muscle cells, and fusion of macrophages into inflammatory giant cells and myoblasts into myotubes. Such cell fusions require the non-apoptotic exposure of phosphatidylserine at the surface of fusing cells, where it interacts with phosphatidylserine-recognizing proteins to regulate the time and place of cell-fusion. Phosphatidylserine provides stable membrane domains in spermatozoa that are essential for fertilization, and it is also an essential component of the plasma membrane microdomains known as caveolae, where it is required both for their formation and stability possibly through specific binding to the cavin proteins.

    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 be 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, and other nutritional claims appear to be dubious also. Antibodies to phosphatidylserine are formed in some disease states, including thrombosis and recurrent spontaneous pregnancy loss. The rare genetic disease Lenz-Majewski syndrome is caused by a mutation in the phosphatidylserine synthase I gene that greatly increases the activity of the enzyme while preventing feedback inhibition, and abnormal metabolism of phosphatidylserine has been implicated in other diseases.

    In yeasts such as Candida albicans, phosphatidylserine and the enzyme phosphatidylserine decarboxylase, which generates phosphatidylethanolamine, are both essential for the virulence of the organism towards a host species.

    Lysophosphatidylserine

    Figure \(\PageIndex{30}\) below shows the structure of lysophosphatidylserine

    lysoPS.svg
    Figure \(\PageIndex{30}\): lysophosphatidylserine

    Lysophosphatidylserine, i.e., with a fatty acid in one position only, is known to be a mediator of a number of biological processes, especially in the context of the immune system in animal tissues. It has been found in the thymus, peripheral lymphoid tissues, central nervous system and colon, but is barely detectable in plasma. Deacylation of the diacyl lipid by phospholipases is the primary source. For example, a secreted isoform that is phosphatidylserine-specific (PLA1A) removes the sn-1 acyl group of phosphatidylserine to generate sn‑2‑lysophosphatidylserine containing unsaturated fatty acids, and this is upregulated greatly by various inflammatory stimuli. This extracellular enzyme utilizes phosphatidylserine exposed on the cell membrane as a substrate, although other phospholipases may operate intracellularly and produce sn‑1‑lysophosphatidylserine. In addition, platelets in some species (not significantly in humans) secrete a phospholipase A2 group IIA (ABHD16A), which generates saturated sn‑1‑lysophosphatidylserine (and other lysophospholipids).

    Lysophosphatidylserine 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 signaling , for example in regulating calcium flux and stimulating immune cells through G protein-coupled receptors of which three (GPR34, P2Y10 and GPR174, LPS1-3) have been detected in mice and humans. For example, GPR174 mediates the suppression of T-cell proliferation induced in vitro by lysophosphatidylserine. When cells are damaged, lysophosphatidylserine can be generated by a reaction dependent on activation of the NADPH oxidase. It can diffuse and transmit the information to other cells, especially mast cells, and it is produced to enhance clearance of activated and dying neutrophils. It thus has a role in the resolution of inflammation. One specific molecular species, i.e., 1‑(11Z‑eicosenoyl)-glycero-3-phosphoserine, is reported to be a true agonist of the Toll-like receptor 2/6 heterodimer of importance to the immune response to pathogens; both its polar head group and the length of the acyl chain are required for this activity. On the other hand, sn-2-lysophosphatidylserine has proinflammatory reactions in that it augments mast cell degranulation and mast cell-dependent anaphylactic shock; most other lysophospholipids have no such activity.

    Deregulated lysophosphatidylserine metabolism has been linked to certain cancers, cardio-metabolic disorders, night blindness, and the human genetic neurological disorder PHARC. High serum levels of PLA1A are associated with such autoimmune disorders as Graves' disease and systemic lupus erythematosus, and there is increased expression of the enzyme in metastatic melanomas. It is necessary for assembly of the hepatitis C virus, and it can play a role in the antivirus innate immune response. In Schistosome infections, lysophosphatidylserine from the parasite is believed to be a key activator molecule in the host.

    Negatively charged lysophosphatidylserine species tend to organize in non-bilayer structures and are believed to facilitate folding of certain membrane proteins in situ better than bilayer-forming lipids.

    Phosphatidylinositol and Related Phosphoinositides

    Although it had long been recognized that phosphatidylinositol or 1,2-diacyl-sn-glycero-3-phospho-(1'-myo-inositol) was a key membrane constituent, it was initially something of a surprise when the manifold biological activities of this lipid, and then of the derived phosphatidylinositol phosphates and their hydrolysis products, were discovered in animals, plants and microorganisms. Many years after the initial discoveries in the 1950s, these lipids continue to be a major focus for research efforts around the world with considerable relevance to human health. Phosphatidylinositol and its various metabolites and relevant enzymes can be located and function within different membrane regions in cells, and they form part of what have been termed phosphoinositide and phosphatidylinositol cycles, their versatility stemming from the inositol head group, a six-carbon hexahydroxy-ring, which can be reversibly phosphorylated on the 3, 4 and 5 positions. In addition to their structural role in membranes, these lipids are intimately involved in innumerable aspects of membrane trafficking and signaling in eukaryotic cells, functions that are essential to cell growth and metabolism. Only a brief overview of such a highly complex topic is possible here.

    Glycosyl-phosphatidylinositol (GPI) is a related lipid that serves as an anchor for proteins; it is considered to be a sufficiently distinctive topic for its own web page (together with phosphatidylinositol mannosides).

    Phosphatidylinositol

    Structure and Occurrence: Phosphatidylinositol is an important lipid, both as a membrane constituent and as a participant in essential metabolic processes in all plants and animals, both directly and via a number of its metabolites. It is an acidic (anionic) phospholipid that in essence consists of a phosphatidic acid backbone, linked via the phosphate group to inositol (hexahydroxycyclohexane). In most organisms, the stereochemical form of the last is myo-D-inositol (with one axial hydroxyl in position 2 with the remainder equatorial, i.e. a chair-like structure), although other forms (scyllo- and chiro-) have been found on occasion in plants. The 1‑stearoyl,2-arachidonoyl molecular species, which is of considerable biological importance in animals, is illustrated.

    Figure \(\PageIndex{31}\) shows the structure of phosphatidylionositol.

    PI.svg
    Figure \(\PageIndex{31}\): Phosphatidylionositol

    Phosphatidylinositol is especially abundant in brain tissue, where it can amount to 10% of the phospholipids, but it is present in all tissues, cell types and membranes at relatively low levels in comparison to many other phospholipids. In rat liver, it amounts to 1.7 micromoles/g., i.e. less than phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. Under normal conditions, it is present entirely in the inner leaflet of the erythrocyte membrane and of the plasma membrane in nucleated cells. Phosphatidylinositol per se is rarely found in prokaryotes other than the Actinomycetales, although the thermophilic α-proteobacterium Rhodothermus marinus contains dialkylether glycerophosphoinositides.

    The fatty acid composition of phosphatidylinositol is rather distinctive as shown in Table \(\PageIndex{6}\)​​​​​​​. Thus, in almost all animal tissues, the characteristic feature is a high content of stearic and arachidonic acids. All the stearic acid is linked to position sn-1 and all the arachidonic acid to position sn-2, and as much as 78% of the total lipid may consist of the single molecular species sn-1-stearoyl-sn-2-arachidonoyl-glycerophosphorylinositol (see Table \(\PageIndex{7}\)​​​​​​​​​​​​​​ below). Although 1-alkyl- and alkenyl- forms of phosphatidylinositol are known, they tend to be much less abundant than the diacyl form. In plant phosphatidylinositol, e.g. Arabidopsis thaliana as listed, palmitic acid is the main saturated fatty acid in position sn-1, while linoleic and linolenic acids are the main unsaturated components in position sn-2. Similarly in yeast, palmitic acid is in position sn-1 with oleic and palmitoleic acids in position sn-2 predominantly; the Amoebozoa have a C16 alkyl group in position sn-1 and cis-vaccenic acid in position sn-2.

    Table \(\PageIndex{6}\): ​​​​​​​ Fatty acid composition of phosphatidylinositol (wt % of the total) in animal and plant tissues.
    Tissue Fatty acids
    16:0 18:0 18:1 18:2 18:3 20:3 20:4 22:3 22:5 22:6
     
    Bovine brain [1] 8 38 10 1 - 5 34 2 tr. 1
    Bovine liver [2] 5 32 12 6 1 7 23 4 3 5
    Rat liver [3] 5 49 2 2 4 35 1
     
    A. thaliana [4] 48 3 2 24 24
    [1] = Holub, B.J. et al.. J. Lipid Res.., 11, 558-564 (1970); DOI. [2] = Thompson, W. and MacDonald, G., J. Biol. Chem., 250, 6779-6785 (1975); DOI. [3] = Wood, R. and Harlow, R.D. Arch. Biochem. Biophys., 135, 272-281 (1969); DOI. [4] = Browse, J. et al. Biochem. J., 235, 25-31 (1986); DOI.

    Biosynthesis: The basic mechanism for biosynthesis of phosphatidylinositol and phosphatidylglycerol is sometimes termed a branch point in phospholipid synthesis, as phosphatidylcholine and phosphatidylethanolamine are produced by a somewhat different route.

    Phosphatidylinositol is found in all eukaryotes, which are in general able to synthesise inositol de novo via glucose-6-phosphate. As with phosphatidylglycerol (and thence cardiolipin), phosphatidylinositol is formed biosynthetically from phosphatidic acid via the intermediate cytidine diphosphate diacylglycerol, which is produced by the action of a CDP-diacylglycerol synthase believed to be the rate-limiting enzyme in phosphatidylinositol biosynthesis. Then, the enzyme CDP-diacylglycerol inositol phosphatidyltransferase ('phosphatidylinositol synthase' or 'PIS') catalyses a reaction with myo-inositol to produce phosphatidylinositol.

    Figure \(\PageIndex{32}\) below shows the synthesis of phosphatidylinositol in eukaryotes.

    SynPI.svg
    Figure \(\PageIndex{32}\): Synthesis of phosphatidylinositol in eukaryotes

    Only isoform of PIS exists in mammals and it is located in the endoplasmic reticulum, in part in a subcompartment of this associated with mitochondria (mitochondria-associated membranes - MAM) and in mitochondria per se. Indeed, it is reported that PIS is present in a mobile ER-derived subcompartment that makes transient contacts with other organelles, including the plasma membrane, and facilitates distribution of phosphatidylinositol to other subcellular compartments. The other product of the reaction is cytidine monophosphate (CMP). As PIS catalyses the reverse reaction also, the rate of phosphatidylinositol synthesis is determined by the relative concentrations of the precursors and product, and the latter must be transported away from the site of synthesis for the reaction to continue. Much of the phosphatidylinositol is delivered to other membranes by vesicular transport, but a family of soluble phosphatidylinositol transfer proteins (PITPα, PITPβ and PITPNC1) provides phosphatidylinositol from the ER to kinases for phosphorylation (see below).

    Molecular species specificity: The phosphatidylinositol synthase per se does not exhibit the fatty acyl specificity observed in the final product, but earlier in the biosynthetic process 1-stearoyl-2-arachidonoyl species of diacyl-sn-glycerols are converted preferentially into phosphatidic acid by the epsilon isoform of diacylglycerol kinase (DGKε), anchored to the membrane via its N-terminal hydrophobic helix segment; ATP is the phosphate donor. In addition, one of the CDP-diacylglycerol synthases (CDS2) has similar specificity in the generation of the immediate precursor CDP-diacylglycerols from phosphatidic acid, while some specificity may be introduced via lysophosphatidylinositol, formed as a by-product of eicosanoid formation (see below) or as an intermediate as part of the normal cycle of deacylation-acylation of phosphatidylinositol in tissues in which the fatty acid composition is remodelled to give the final distinctive composition. A membrane-bound O-acyltransferase (MBOAT7 or LPIAT1) specific for position sn-2 of lysophosphatidylinositol with a marked preference for arachidonoyl-CoA is ubiquitously expressed in animal tissues, and this may be one means by which free arachidonic acid and eicosanoid levels are regulated.

    In macrophages subjected to inflammatory stimuli, phosphatidylinositol containing two molecules of arachidonate is produced by remodeling reactions, and there is evidence that it is a novel bioactive phospholipid regulating innate immune responses in these cells. Further specificity may be introduced by lysocardiolipin acyltransferase (LYCAT; also known as LCLAT1 or ALCAT1), which exhibits a preference for lysophosphatidylinositol and lysophosphatidylglycerol over other phospholipids in vitro, and incorporates 18:0 rather than shorter chain fatty acids into position sn-1 of phosphatidylinositol and other phosphoinositides, especially phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3-phosphate; this enzyme may be located adjacent to the phosphatidylinositol synthase in the endoplasmic reticulum. Some of the phosphatidylinositol in membranes is derived from re-cycling of polyphosphoinositides via the phosphatidylinositol cycle, and this could influence the molecular species composition (see below).

    The highly specific distribution of fatty acids on the glycerol moiety of phosphatidylinositol breaks down in some cancer cells, especially those with a mutation on the transcription factor p53 gene, which is one of the most highly mutated genes in cancers.

    Plants and bacteria: In contrast to animals, plants have two phosphatidylinositol synthase isoforms, PIS1 and PIS2, which display specificities for particular species of the CDP-diacylglycerol substrate. PIS1 generates phosphatidylinositol with saturated or monounsaturated fatty acids preferentially, while PIS2 generates polyunsaturated species, the two forms possibly having different functions. In protozoan parasites, such as Trypanosoma brucei, the active site of phosphatidylinositol synthase may be the lumen of the endoplasmic reticulum and Golgi. There is evidence for two distinct pools of product in this organism, the bulk membrane form derived from inositol imported from the environment, and a second used for the synthesis of GPI anchors, which uses myo-inositol synthesized de novo. In yeasts, some biosynthesis may occur on the cytosolic side of the plasma membrane.

    The enzyme is a transmembrane protein.and use CDP-diacylglycerol as a donor and either inositol (eukaryotes) or inositol phosphate (prokaryotes) as the acceptor alcohol. The structure of a similar enzyme, phosphatidylinositol-phosphate synthase from Renibacterium salmoninarum, is shown in Figure \(\PageIndex{33}\) below.

    pISynthesisFig1Aff.svg

    Figure \(\PageIndex{33}\): Structure and reaction of phosphatidylinositol-phosphate synthase from Renibacterium salmoninarum. Clarke, O., Tomasek, D., Jorge, C. et al. Structural basis for phosphatidylinositol-phosphate biosynthesis. Nat Commun 6, 8505 (2015). https://doi.org/10.1038/ncomms9505. Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/

    Panel A shows the reaction for PIP synthases which involves the transfer of a diacylglycerol-substituted phosphate group (purple/red) from the CDP-DAG donor to the inositol phosphate acceptor (green), generating PIP and CMP. Panel B shows the structure of the RsPIPS-Δ6N homodimer in ribbon representation viewed from two orthogonal orientations (in the plane of the membrane on the left; towards the cytosol down the dimer axis on the right). One protomer is colored grey, and the helices of the other are depicted in spectral colouring, from blue (JM1) to red (TM6). The Af2299 extramembrane domain used to facilitate crystallization is not shown here.

    Figure \(\PageIndex{34}\) shows a large cavity which contains the active site of RsPIPS.

    pISynthesisFig2Aff.svg
    Figure \(\PageIndex{34}\): Large cavity containing the active site of RsPIPS . Clarke, O., Tomasek, D., Jorge, C. et al. ibid.

    Panel (a) shows the structure of RsPIPS-Δ6N is shown in ribbon representation, with one protomer colored grey and the other coloured by the Kyte–Doolitle hydrophobicity scale, from −4.5 (most polar, light blue) to 4.5 (most hydrophobic, orange). Two orthogonal representations are shown, on the left is a view in the plane of the membrane and on the right is a view from the cytosol along the dimer axis. A transparent purple surface delineates the borders of the interfacial cavity, which contains three subregions as follows: 1, the inositol phosphate acceptor-binding pocket; 2, the nucleotide-binding pocket between TM2 and TM3; and 3, a hydrophobic groove between TM2 and JM1. (b) Detail of the active site viewed in the plane of the membrane, with side chains that contact the bound Mg2+ and SO42- ions labeled and depicted in stick representation.

    A nucleotide-binding site formed from transmembrane segments 1, 2 and 3 contains 8 conserved residues (D1xxD2G1xxAR…G2xxxD3xxxD4). The first 3 aspartic acids side chains coordinate a metal ion while the 4th is likely a general base in catalysis.

    Figure \(\PageIndex{35}\) below shows an interactive iCn3D model of the phosphatidylinositolphosphate (PIP) synthase with bound CDP-DAG from Renibacterium Salmoninarum (5D92).

    PIPsynthase with bound CDP-DAG from Renibacterium Salmoninarum (5D92).png

    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{35}\): Phosphatidylinositolphosphate (PIP) synthase with bound CDP-DAG from Renibacterium Salmoninarum (5D92).. Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...i9nhSw9U32Man8

    The two identical subunits of the homodimer are shown in gray and plum. The two CDP-DAGs are shown in spacefill and CPK colors

    Function: In addition to functioning as negatively charged building blocks of membranes, the inositol phospholipids (including the phosphatidylinositol phosphates or 'polyphosphoinositides' discussed below) have crucial roles in interfacial binding of proteins and in the regulation of protein activity at the cell interface. As phosphoinositides are polyanionic, they can be very effective in non-specific electrostatic interactions with proteins. However, they are especially efficient in specific binding to so-called ‘PH’ domains of cellular proteins. At least three phosphatidylinositol molecules are present in the crystal structure of human erythrocyte glycophorin, for example, and they are believed to influence binding to other proteins via their head groups. The lipid is a structural component of yeast cytochrome bc1.

    In animal tissues, phosphatidylinositol is the primary source of the arachidonic acid required for biosynthesis of eicosanoids, including prostaglandins, via the action of the enzyme phospholipase A2, which releases the fatty acids from position sn-2. The reverse reaction also occurs.

    Figure \(\PageIndex{36}\) belows shows the generation of arachidonic acid and eicosanoids from PI by means of phospholipase A2

    ArachandEicosofromPIcdx.svg
    Figure \(\PageIndex{36}\): Generation of arachidonic acid and eicosanoids from PI by means of phospholipase A2.

    Similarly, phosphatidylinositol and the phosphatidylinositol phosphates are the main source of diacylglycerols that serve as signaling molecules in animal and plant cells, via the action of a family of highly specific enzymes collectively known as phospholipase C (see our web pages on diacylglycerols for further discussion). In brief, diacylglycerols regulate the activity of a group of at least a dozen related enzymes known as protein kinase C, which in turn control many key cellular functions, including differentiation, proliferation, metabolism and apoptosis. Indeed, the biological actions of the various components released have been the subject of intensive study over many years. 2‑Arachidonoylglycerol, an endogenous cannabinoid receptor ligand, may also be a product of phosphatidylinositol catabolism.

    Phosphatidylinositol Phosphates (Polyphosphoinositides) in Animals

    Structure and Occurrence: The pioneering work of Mable and Lowell Hokin in the 1950s lead to the discovery that phosphatidylinositol was converted to polyphosphoinositides with important signaling and other functional activities, including cell communication via signal transduction, cell survival and proliferation, membrane trafficking and modulation of gene expression. Phosphatidylinositol is now known to be phosphorylated by a number of substrate-selective kinases that place the phosphate moiety on positions 3, 4 and 5 of the inositol ring with the balance among them maintained by distinct phosphatases and phospholipases. Seven different isomers are known (mono-, bis-, and tris-phosphorylated), which are produced in tightly coordinated manner, and all of these have characteristic biological activities. They each turn over much more rapidly than the parent phosphatidylinositol molecule. In addition, there can be an array of molecular species of each of these isomers that differ in the nature of the fatty acyl groups. Although the most significant in quantitative and possibly biological terms were long thought to be phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5‑bisphosphate, it is now recognized that phosphatidylinositol 3-phosphate and its metabolites are as important biologically at least.

    Figure \(\PageIndex{37}\) below shows the structures of phosphatidylinositol phosphates

    PIDerivatives.svg
    Figure \(\PageIndex{37}\): Structures of phosphatidylinositol phosphates in animals

    These lipids are usually present at low levels only in tissues, typically at about 0.5 to 1% of the total lipids of the inner leaflet of the plasma membrane, so they are unlikely to have an appreciable structural role. On the other hand, static measurements of lipids that turn over very rapidly do not provide a meaningful assessment of their cellular functions. The positional distributions of fatty acids in the phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate of ox brain are listed in Table \(\PageIndex{7}\)​​​​​​​:. In each the saturated fatty acids are concentrated in position sn-1 and polyunsaturated, especially arachidonate, in position sn-2; there are few differences among the three lipids in this instance.

    Table \(\PageIndex{7}\)​​​​​​​: Distribution of fatty acids (mol % of the total) in positions sn‑1 and sn‑2 in phosphatidylinositol (PI) and the phosphatidylinositol mono- and diphosphates of ox brain.
    Fatty acids PI PI monophosphate PI diphosphate
    sn-1 sn-2 sn-1 sn-2 sn-1 sn-2
     
    16:0 15 9 7
    18:0 74 69 69
    18:1 10 10 20 13 21 10
    18:2 1 2 trace 1 1 1
    20:3(n-9) 5 10 10
    20:3(n-6) 5 11 12
    20:4(n-6) 67 49 52
    22:3 7 10 7
    22:6(n-3) trace trace trace
    Data from Holub, B.J. et al., J. Lipid Res., 11, 558-564 (1970); DOI.
    Molecular species data, see Traynor-Kaplan, A. et al., Biochim. Biophys. Acta, 1862, 513-522 (2017); DOI.

    Biosynthesis: Phosphatidylinositol per se is the ultimate precursor of all phosphoinositides, the head groups of which have different charges and structures that impact directly on membrane properties and via metabolic interactions can function as chemical switches. The individual phosphoinositides are maintained at steady state levels in membranes by a continuous and sequential series of phosphorylation and dephosphorylation reactions by specific kinases, phosphatases and phospholipase C enzymes, which are regulated and/or relocated through cell surface receptors for extracellular ligands, the phosphoinositide cycle. While this has been termed a ‘futile cycle’, which can consume a significant proportion of cellular ATP production, it is only part of a wider pattern of reactions - the phosphatidylinositol cycle (see below). Controlled synthesis of these different phosphoinositides occurs in different intracellular compartments for distinct and independently regulated functions with spacially distinct target enzymes or receptors. In mammals, the complexity is such that 18 phosphoinositide inter-conversion reactions have been identified to date, and these are mediated by at least 20 phosphoinositide kinases and 34 phosphoinositide phosphatases that span 8 and 10 classes, respectively; some have yet to be characterized. Most of these enzymes are conserved across all of the eukaryota, and each has distinct functions and specificities that cannot be replaced by the activity of related isoforms.

    As a generality, most mono-phosphorylations occur in endomembranes, such as the endosomes and the Golgi network, while second and third phosphorylations occur primarily at the plasma membrane, and this is reflected in the lipid composition of each membrane. While these enzymes are believed to work independently and sequentially to produce a specific product, there remains a possibility that some participate in protein complexes to coordinate their activities. Specific transporters, especially the 'Nir2' protein, facilitate the exchange of phosphoinositides between membranes. It should be noted that there are links to the metabolism of phosphatidylcholine, which can be hydrolysed by phospholipase D to phosphatidic acid, an important activator of key kinases. Figure \(\PageIndex{38}\) shows an overview of polyphosphoinositide metabolism in animal tissues.

    Polyphosphoinositide metabolism in animal tissues
    Figure \(\PageIndex{38}\): Overview of polyphosphoinositide metabolism in animal tissues

    Thus as an example, phosphatidylinositol 4-phosphate (PI(4)P) is produced by the action of a phosphatidylinositol 4-kinase (PI4K) in the Golgi, and is in turn phosphorylated by a phosphatidylinositol phosphate 5-kinase (PIPK I) to form phosphatidylinositol 4,5-bisphosphate (PI(4,5)P) at the plasma membrane, although this can also be formed by phosphorylation of phosphatidylinositol 5-phosphate by a specific 4-kinase (PIPK II). Four isoforms of PI4K in two structural families are known that each operate in different subcellular membrane compartments to produce phosphatidylinositol 4-phosphate for particular signaling functions. Some selectivity in the formation of molecular species or remodeling may occur to further enrich the arachidonic acid content.

    Subsequently, it was discovered that phosphatidylinositol is also phosphorylated by a 3-kinase (PI3K III or the VPS 34 complex) to produce phosphatidylinositol 3-phosphate (PI(3)P) in the early endosomes. In fact, three phosphatidylinositol 3-kinases families (eight isoforms) have been described, each with distinct substrate specificities. A second phosphoinositide signaling pathway involves activation of two of these 3‑kinases, stimulated by growth factors and hormones, which phosphorylate phosphatidylinositol 4,5-bisphosphate (by PI3K I - four isoforms) and phosphatidylinositol 4‑phosphate (by PI3K II - three isoforms) to produce phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P) and phosphatidylinositol 3,4‑bisphosphate (PI(3,4)P), respectively. While phosphatidylinositol 3-phosphate and other 3‑phosphorylated metabolites amount to only about 0.5% of the total phosphoinositides in resting mammalian cells, they are now recognized to be of profound importance for cellular metabolism.

    In addition to the activity of kinases, the amounts of these various metabolites are regulated by the activities of specific phosphoinositide phosphatases, which are highly conserved in eukaryotes and dephosphorylate phosphoinositides at the 3, 4 and 5 positions of the inositol ring. For example, so-called ‘SHIP’ phosphatases convert phosphatidylinositol 4,5‑bisphosphate back to phosphatidylinositol 4-phosphate by hydrolysis of the 5-phosphate group. 3‑Phosphorylated phosphoinositides are only degraded by phosphatases, especially those of the PTEN family, and not by phospholipase C (see below).

    The various organelles in cells have membranes with distinct functions and molecular compositions. Yet, all the phosphatidylinositol precursor is formed primarily at the endoplasmic reticulum, and the different membrane lipids must be transported between membrane sites via specific trafficking processes/proteins. There is selective recruitment of effector proteins to particular membranes by binding only to a single type of phosphoinositide, and this is followed by interactions between the phosphoinositide-binding proteins and various enzymes to channel phosphoinositide production to the required biological outcomes and to regulate signaling. For example, much of the phosphatidylinositol 4‑phosphate and phosphatidylinositol 4,5-bisphosphate involved in signaling is believed to be formed at contact sites between the endoplasmic reticulum and plasma membrane.

    A concept has emerged in which each phosphoinositide has its own role – the ‘lipid code’ hypothesis, in which defined lipids act as labels for each cellular membrane to organize cells into dynamic and responsive membrane-bound compartments and maintain the orderly flow required for the complexities of membrane trafficking and spatio-temporal signaling reactions. Thus, phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5‑bisphosphate, phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5‑bisphosphate are found mainly on the Golgi, plasma membrane, early endosomes and late endocytic organelles, respectively, where they are sometimes regarded as landmarks for these compartments. For example, phosphatidylinositol 4,5‑bisphosphate is present throughout the plasma membrane and is considered a general marker for this, while phosphatidylinositol 3,4,5-triphosphate, is a characteristic component of the basolateral region of this membrane in a polarized cell but is absent from the apical part. On the other hand, it should be noted that this map of phosphoinositides to specific organelles is derived from their steady state distributions, but the highly dynamic generation and consumption of different phosphoinositides in response to different stimuli in the various sub-cellular compartments in living cells by the action of kinases and phosphatases together with lipase reactions, may lead to the formation of transient pools of distinct molecular forms. There must be a continuous replenishment of the precursors by new synthesis.

    Function: The distinctive subcellular location of the different phosphoinositide species, together with the rapid and reversible nature of phosphorylation, gives them a central and general position in the fields of cell signaling cascades and intracellular membrane trafficking. The precise locations of particular phosphoinositides are factors that contribute a specific identity to each organelle and sometimes even to each face of an organelle, such as the cis and trans faces of the Golgi apparatus, and this enables directional transport of cellular constituents between organelles or membranes. Phosphoinositides are able to achieve signaling effects directly by binding to specific cytosolic domains of membrane proteins via their polar head groups, thereby triggering downstream signaling cascades, often in conjunction with an acidic phospholipid, such as phosphatidylserine or phosphatidic acid at an adjacent-binding site. The term 'lipidon' has been coined to describe the unique collection of co-located lipids that distinguish biological membrane nano-environments and which provide the context for PI recognition in vivo. In this way, they can regulate the function of innumerable proteins integral to membranes, for example by relocating a protein from one area of the cell to another, e.g., from the cytosol to the inner leaflet of the plasma membrane, or they can attract cytoskeletal and signaling components to the membrane. Amongst the proteins that bind to phosphoinositides in this way are phospholipases, protein kinases, regulators of membrane trafficking, and cytoskeletal, scaffold and ion channel proteins. Dysregulation of phosphoinositide metabolism and signaling is a factor in a number of diseases, including cancer.

    Binding usually involves electrostatic interactions with the negative charges of the phosphate groups on the inositol ring with characteristic clusters of basic amino acid residues in proteins to recruit them to intracellular membranes, while often leading to specific folding and thence increased activity of unstructured peptides. At least 70 distinct types of binding sites for phosphoinositides have been identified in proteins. In particular, a binding region termed the pleckstrin homology (PH) domain, consisting of ~100 amino acids, is the most abundant lipid-binding domain with more than 225 examples identified, and this can exhibit great specificity for particular polyphosphoinositides, often binding simultaneously with other proteins. While the interaction is driven by non-specific electrostatic interactions initially, it is followed by specific binding to increase the membrane residence time. The phox homology (PX) domain family with 49 members in humans is unique in in that it can recognize all seven phosphoinositide forms, while proteins with a FYVE domain, which is enriched in cysteine and is stabilized by two zinc atoms, binds specifically to phosphatidylinositol 3-phosphate (PI(3)P). The protein kinase C family have C1 or C2 domains which recognize phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate specifically (and sometimes other lipids). The distinctive phosphoinositide composition of membranes in different organelles adds strength and specificity to the interactions by cooperative binding with other membrane proteins.

    Phosphatidylinositol 3-phosphate and the other phosphatidylinositol monophosphates are present in cells at low levels only, although their levels do not appear to fluctuate greatly. PI(3)P has been implicated in membrane trafficking through its interactions with certain proteins in endosomes. In particular, it plays a pivotal role in the initiation of autophagy, i.e. the controlled internal degradation and turnover of cellular constituents, while PI(3,5)P2 is important in the autophagosome–lysosome fusion step and in the subsequent acidification of this organelle. After sorting of the lysosomal contents, components of the internalized cargo are recycled to the plasma membrane and PI(3)P is dephosphorylated to phosphatidylinositol by a specific phosphatase, and this is in turn phosphorylated to PI(4)P. Thus the processes of internalization, sorting, and trafficking of membrane proteins depend on the interconversion of phosphoinositide species by coordinated phosphorylation-dephosphorylation reactions.

    In general, PI(3)P controls cellular processes by recruiting effector proteins through low to moderate affinity interaction with specific PI(3)P binding domains. A protein designated Akt (protein kinase B) is recognized as a direct effector of the PI3K signaling cascade with receptor tyrosine kinases as the main upstream activators, for example, but it is now known that every phosphatidylinositol phosphate has a specific set of effector proteins that are recruited to target membranes or are allosterically regulated by the specific receptors; each function may require a different effector. A further function of PI(3)P is in the regulation of the final stage of cell division (cytokinesis), and the lipid is known to accumulate where cells divide. As the class I PI3K isoforms especially have been implicated in the aetiology and maintenance of various diseases and metabolic disorders, including cancer, inflammation and autoimmunity, drug companies are actively pursuing the development of inhibitors. In particular, they mediate insulin-independent glucose transport and many of the physiological actions of insulin. In relation to lung cancer especially, RAS proteins, which are key signaling switches essential for the control of proliferation, differentiation, and survival of eukaryotic cells, regulate the activity of type I phosphatidylinositol 3-kinase (PI3K); this is essential for tumor initiation and maintenance.

    Phosphatidylinositol 4-phosphate is the precursor for the 4,5-bisphosphate, but it binds to a protein on the cytoskeleton of the cell and has its own characteristic functions. It is the most widely distributed of the phosphoinositides, and in addition to the Golgi and the plasma membrane, it is present in late endosomes, lysosomes, secretory vesicles and autophagosomes. As a part of protein-lipid complexes, it is believed to have a role in essential nuclear processes. In yeast, it has a function in the anterograde transport from the trans-Golgi and the retrograde transport from the Golgi to the endoplasmic reticulum; it is also necessary for the formation of secretory vesicles in the Golgi that are targeted to the plasma membrane. Some of that in the plasma membrane is exchanged for phosphatidylserine by the action of specific transport proteins at junctions with the endoplasmic reticulum.

    In addition, PI(4)P is essential for the structure and function of the late endosomes, where it is required for the recruitment of specific proteins that control cargo exit (following hydrolysis of PI(3)P). Some of these participate in vesicle formation, while others like the oxysterol binding protein (OSBP) are involved in lipid transfer. After initiation of the process by PI(3)P, PI(4)P, PI(4,5)P2 and their binding proteins are modulators of autophagy at most stages of the process. PI(4)P has been called the 'fuel' that drives cholesterol transport, as its hydrolysis provides the energy that enables the establishment of active sterol concentration gradients across membrane-bound compartments with the aid of OSBP, which is a key regulator of cholesterol, oxysterol and PI(4)P concentrations in membranes, as discussed in our web page on cholesterol. In the plasma membrane, PI(4)P can support the functions of ion channels, and it contributes to the anchoring of proteins with polybasic domains, although it is not utilized for synthesis of PI(4,5)P2 in this membrane. On the other hand, PI(4)P derived from PI(4,5)P2 in the membrane of primary cilia in the retina is important for vision. PI(4)P has an important influence on the progression of many diseases, especially virus replication, cancer and various inflammatory diseases, and inhibitors of PI4-kinase are under study for their therapeutic potential.

    While the biological properties of phosphatidylinositol 5-phosphate have taken longer to unravel, because of the difficulties of separation of this isomer, it is now apparent that it is involved in osmoregulation both in plants and animals. It also has signaling functions, and although it is the least abundant phosphatidylinositol monophosphate, it is involved in signaling at the nucleus and in the cytoplasm, modulating cellular responses to various stresses, hormones and growth factors. In the endosomes, it is a regulator of protein sorting.

    Although phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is found primarily in the inner leaflet of the plasma membrane, where it may define membrane identity in eukaryotic cells, it is also present in endosomes, the endoplasmic reticulum and nucleus. It is an essential precursor of lipid second messengers such as diacylglycerols with vital signaling functions that operate through plasma membrane G-protein coupled receptors, receptor tyrosine kinases and immune receptors. Because of its large head group and multivalent negative charge, PI(4,5)P2 has been described as an "electrostatic beacon" that interacts in various ways with membrane proteins, other lipids and cellular cations. In consequence and in spite of its relatively low concentration, it is a key regulator of innumerable events at the plasma membrane, including cell adhesion and motility, vesicle endocytosis and exocytosis, and the function of ion channels, especially those for potassium, calcium and sodium. With ion channels, for example, it appears to be an obligatory factor, increasing their activity by activating key proteins, while its hydrolysis by phospholipase C reduces such activity.

    PI(4,5)P2 interacts with cationic residues of a large array of proteins in concert with cholesterol to form localized membrane domains that are distinct from the sphingolipid-enriched rafts. Indeed, it has a much higher concentration than other phosphoinositide species in cells, although most of this is in effect sequestered by binding proteins. Also, phosphatidylinositol 4,5-bisphosphate and its diacylglycerol metabolites are important for vesicle formation in membranes. For example, a major pathway in cells for internalization of cell surface proteins such as transferrin is the clathrin-coated vesicle pathway. PI(4,5)P2 is essential to this process in that it binds to the machinery involved in the membrane, increasing the number of clathrin-coated pits and permitting internalization of proteins. It has a related function in caveolae, where it is concentrated at the rim.

    Through its attachment to the apical plasma membrane, phosphatidylinositol 4,5-bisphosphate is intimately involved in the development of the actin cytoskeleton and thereby controls cell shape, motility, and many other processes. In particular, it binds with high specificity to effectors such as vinculin, a membrane-cytoskeletal protein that is involved in linkage of integrin adhesion molecules to the actin cytoskeleton. Dysregulation of this function has been implicated in the migration and metastasis of tumour cells. In yeasts, it appears that the presence of stearic acid in position sn-1 is essential for this function. In the cell nucleus, this lipid is believed to be involved in maintaining chromatin, the complex combination of DNA, RNA, and protein that makes up chromosomes in a transcriptionally active conformation, as well as being a precursor for further signaling molecules. It has a role in gene transcription, and RNA processing, especially in the modulation of RNA polymerase activity, and in other nuclear processes.

    Via its binding to specific proteins, the lipid is an essential component of the immune response of animal tissues to toxic bacterial lipopolysaccharides. It is also involved in the pathophysiology of the HIV virus via an interaction with the Tat protein secreted by infected cells.

    PI(4,5)P2 is the primary precursor of the endocannabinoid 2-arachidonoylglycerol in neurons, and it is also an essential cofactor for phospholipase D and so affects the cellular production of phosphatidic acid with its specific signaling functions. By binding specifically to ceramide kinase, the enzyme responsible for the synthesis of ceramide-1-phosphate, it has an influence on sphingolipid metabolism. Like ceramide-1-phosphate, it binds to and activates the Ca2+-dependent phospholipase A2, which generates the arachidonate for eicosanoid production. One molecule of phosphatidylinositol 4,5‑bisphosphate is bound to each subunit of the protein in the X-ray crystal structures of mammalian GIRK2 potassium channel, where it enables a conformational change that assists the transport function of the protein.

    Perhaps, the best characterized of the phosphoinositide signaling functions results from the hydrolysis of phosphatidylinositol phosphates by phospholipase C isoforms, in this instance to produce sn-1,2-diacylglycerols and inositol 3,4,5-trisphosphate (see below), which act as second messengers. Only those polyunsaturated diacylglycerol species derived from PI(4,5)P2 are able to bind and activate protein kinase C (α, ε, δ) isoforms both in vitro and in vivo. This lipid is doubly important as it binds strongly to these enzymes via a basic patch distal to a Ca2+ binding site, and this targets them selectively to the plasma membrane. Aberrant expression of phospholipase Cγ2 may be a factor in neurodegenerative diseases. Via the action of PI3 kinase, PI(4,5)P2 is the precursor of PI(3,4,5)P3 with its own distinctive signaling properties.

    Phosphatidylinositol 3,4-bisphosphate can be produced by two routes and regulates a variety of cellular processes with relevance to health and disease that include B cell activation and autoantibody production, insulin sensitivity, neuronal dynamics, endocytosis and cell migration. It is known to bind selectively to a number of proteins, and it acts as a secondary messenger by recruiting the protein kinases Akt (protein kinase B) and so may influence the cell cycle, cell survival, angiogenesis and glucose metabolism. During endocytosis in the endolysosomal system, it is produced from PI(4,5)P2 and controls the maturation of endocytic coated pits. Its synthesis and turnover of are spatially segregated within the endocytic pathway. In epithelial cells, it is located on the apical membrane, i.e. facing the lumen, as opposed to the basolateral membranes, and it is believed to be is a determinant of the identity and function of the apical membrane.

    Phosphatidylinositol 3,5-bisphosphate is present at low levels only in cells (0.04-0.1% of the total phosphatidylinositides), unless stimulated by growth factors, but it is important in membrane and protein trafficking, especially in the late endosomes in eukaryotes and in yeast vacuoles. For example, conversion of PI(3)P to PI(3,5)P2 promotes endosomal maturation and degradative sorting. It is involved in the mediation of signaling in response to stress and hormonal cues and in the control of ion transport in membranes, while genetic studies confirm that it is essential for healthy embryonic development, especially in the nervous system.

    Phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) is almost undetectable in quiescent cells, but its intracellular level rises very rapidly from synthesis at the plasma membrane in response to agonists such as extracellular growth factors and hormonal stimuli. By recruiting proteins with pleckstrin homology (PH) domains to the plasma membrane, it has been implicated in a variety of cellular functions that include growth, cell survival, proliferation, cytoskeletal rearrangement, intracellular vesicle trafficking, and cell metabolism. In particular, it is an important component of a signaling pathway in the cell nucleus. In epithelial cells, it is located on the basolateral membrane, i.e. facing adjacent cells, where it may be a determinant of the identity and function of this membrane. In contrast to phosphatidylinositol 3-phosphate, it opposes autophagy by binding to and activating the PH domain of Akt, so inducing cell proliferation. During feeding, various physiological responses lead to the secretion of insulin, which stimulates the phosphorylation of phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5-trisphosphate and triggers a signaling cascade that leads to the suppression of autophagy. When this pathway is impaired it has deleterious effects upon the insulin resistance associated with various metabolic diseases including obesity and diabetes. It has been implicated in tumor cell migration and metastasis. PI(3,4,5)P3 is also present in the nucleus and nucleoli of cells where it is believed to have functions in RNA processing/splicing, cytokinesis, protein folding, and DNA repair. In complete contrast, like phosphatidylserine, it is reportedly transferred to the outer leaflet of the plasma membrane in aged or damaged cells as an 'eat‑me' signal for phagocytes and apoptosis.

    The human immune system utilizes neutrophils, which are highly mobile cells, to eliminate pathogens from infected tissue. The first step is to track and then pursue molecular signals, such as cytokines, emitted by pathogens. It has been established that two phospholipids operate in sequence to point the neutrophils in the correct direction. The first of these is phosphatidylinositol 3,4,5-trisphosphate, which binds to a specific protein DOCK2 and enables it to translocate to the plasma membrane. Then phosphatidic acid, generated by the action of phospholipase D on phosphatidylcholine, takes over and directs the DOCK2 to the leading edge of the plasma membrane. This causes polymerization of actin within the cell and in effect reshapes the neutrophil and points it in the direction from which the pathogens signals are coming. On the other hand, Mycobacterium tuberculosis is able to subvert phosphoinositide signaling to arrest phagosome maturation by dephosphorylation of phosphatidylinositol 3-phosphate.

    Water-Soluble Inositol Phosphates

    As mentioned briefly above, hydrolysis of phosphatidylinositol phosphates by c alcium-dependent phospholipase C (or 'phosphoinositidase C') leads to generation of sn‑1,2‑diacylglycerols, which act as second messengers in animal cells and are of enormous metabolic importance. There are many different enzymes of this type, but the activity of the phosphoinositide-specific phospholipase C constitutes an essential step in the inositide signaling pathways. The enzyme exists in six families consisting of at least 13 isoenzymes, all of which have conserved regions such as the plekstrin homology (PH) binding domain. Each one has a distinctive role and can have a characteristic cell distribution that is linked to a specific function. Activity of these enzymes is stimulated by signaling molecules such as G-protein coupled receptors, receptor tyrosine kinases, Ras-like GTPases and calcium ions, thus linking the hydrolysis of phosphatidylinositol phosphates to a wide range of other cellular signals. As phospholipase C is a soluble protein located mainly in the cytosol, translocation to the plasma membrane is a crucial step in signal transduction. Regulation of these isoenzymes and the form PLCγ1 in particular is vital for health as they are associated with the activation or inhibition of important pathophysiological processes, especially in relation to cancer.

    Some phosphatidic acid is synthesized from the diacylglycerols produced within the plasma membrane through the activity of diacylglycerol kinases, and this is transported back to the endoplasmic reticulum and ultimately can be re-utilized for phosphatidylinositol biosynthesis.

    The other products of the phospholipase C reaction that are of special relevance because of their many essential functions are water-soluble inositol phosphates. Up to 60 different compounds of this type are possible, and at least 37 of these have been found in nature at the last count, all of which are extremely important biologically. However, polyphosphoinositides with a phosphate in position 3 are not substrates for phospholipase C.

    Figure \(\PageIndex{39}\) shows the generation of inositol phosphates by phospholipase C.

    solublePIDeriv.svg
    Figure \(\PageIndex{39}\): Generation of inositol phosphates by phospholipase C

    For example, under the action of various physiological stimuli in animals, including sphingosine-1-phosphate, and acting via various G-protein-coupled receptors, phosphatidylinositol 4,5-bisphosphate in the plasma membrane is hydrolysed to release inositol 1,4,5-trisphosphate, an important cellular messenger that diffuses into the cytosol and stimulates calcium release from an ATP-loaded store in the endoplasmic reticulum via ligand-gated calcium channels (the diacylglycerols remain in the membrane to recruit and activate members of the protein kinase C family). The increase in calcium concentration, together with the altered phosphorylation status, activates or de-activates many different protein targets, enabling cells to respond in an appropriate manner to the extracellular stimulus. To enable rapid replenishment of the phosphatidylinositol 4,5‑bisphosphate used in this way, a cycle of reactions - the phosphatidylinositol cycle - must occur (see below). On the other hand, a recent publication suggests that phosphatidylinositol 4-phosphate in the plasma membrane may be a more important source of diacylglycerols following stimulation of G protein–coupled receptors.

    All of the various inositol phosphates appear to be involved in the control of cellular events in very specific ways, but especially in the organization of key signaling pathways, the rearrangement of the actin cytoskeleton or intracellular vesicle trafficking. They have been implicated in gene transcription, RNA editing, nuclear export and protein phosphorylation. As these remarkable compounds can be rapidly synthesized and degraded in discrete membrane domains or even sub-nuclear structures, they are considered to be ideal regulators of dynamic cellular mechanisms. From structural studies of inositol polyphosphate-binding proteins, it is believed that the inositides may act in part at least by modifying protein function by acting as structural cofactors, ensuring that proteins adopt their optimum conformations. In addition, phosphoinositides and the inositol polyphosphates are key components of the nucleus of the cell, where they have many essential functions, including DNA repair, transcription regulation and RNA dynamics. It is believed that they may be activity switches for the nuclear complexes responsible for such processes, with the phosphorylation state of the inositol ring being of primary importance. As different isomers appear to have specific functions at each level of gene expression, extracellular events must coordinate the production of these compounds in a highly synchronous manner.

    In organisms from plants to mammals, an extra tier of regulatory mechanisms is produced by kinases that generate energetic diphosphate (pyrophosphate)-containing molecules from inositol phosphates. Conversely, these can by dephosphorylated by polyphosphate phosphohydrolase enzymes to regenerate the original inositol phosphates. These inositol pyrophosphates and the enzymes involved in their metabolism are also involved in the regulation of cellular processes by modulating the activity of proteins by a variety of mechanisms.

    It should be noted that the phospholipase C isoenzymes regulate the concentration of phosphatidylinositol 4,5-bisphosphate and related lipids and thence their activities in addition to the generation of new biologically active metabolites.

    Phosphatidylinositides in Plants

    In plants as in animals, phosphatidylinositol and polyphosphoinositides have essential biological functions, exerting their regulatory effects by acting as ligands that bind to protein targets via specific lipid-binding domains and so alter the location of proteins and their enzymatic activities. However, it appears that polyphosphoinositide metabolism developed in different ways after the divergence of the animal and plant kingdoms so the details of the processes in each are very different, not least because the subcellular locations of phosphoinositides differ appreciably between plants and animals. Phosphatidylinositol per se is of course the precursor of the phosphorylated forms and determines their fatty acid compositions. It also has a role in inhibiting programmed cell death by acting as the biosynthetic precursor of the sphingolipid ceramide phosphoinositol and so reducing the levels of ceramide.

    As in animals, the various phosphoinositides (five in total) are produced and inter-converted rapidly by a series of kinases and phosphatases (in many isoforms) in different cellular membranes in response to environmental or developmental cues. For example, phosphatidylinositol is generated mainly in the endoplasmic reticulum, while PI 4-kinases and their product are located in the trans-Golgi network and nucleus, and PI4P 5-kinases and product are present in the plasma membrane. During the biosynthesis of polyphosphoinositides, the first phosphorylation occurs at the hydroxyl group at positions 3 or 4 of the inositol ring, catalysed by the appropriate kinases, while the second phosphorylation then takes place at position 5; PI 5-phosphate is produced by the action of a phosphatase on PI 3,5‑bisphosphate. Most other metabolites are produced via phosphatidylinositol 3-phosphate, and reports that some phosphatidylinositol 3,4,5-trisphosphate may be produced from phosphatidylinositol 4,5‑bisphosphate require confirmation. In contrast to mammalian phosphatidylinositol 3-kinases, which accept both phosphatidylinositol and its monophosphates as substrates, the plant enzyme acts only on the former.

    Figure \(\PageIndex{40}\) below shows polyphosphoinositide metabolism in plants

    Polyphosphoinositide metabolism in plants
    Figure \(\PageIndex{40}\): Polyphosphoinositide metabolism in plants

    The reverse reaction in plants is accomplished by phosphoinositide phosphatases, which can be grouped into three main families, the phosphatase/tensin (PTEN) family, 5-phosphatases (5-PTases) and phosphatases containing Suppressor of Actin (SAC) domains, each with differing subcellular locations, substrate specificities and regulatory mechanisms.

    Although what might be considered normal levels of phosphatidylinositol 4-phosphate are present, the concentrations of phosphatidylinositol 4,5‑bisphosphate and other phosphoinositides are extremely low in plants (10 to 20-fold lower than in mammalian cells), although they still have vital functions. There are differences between cell types, but in Arabidopsis epidermal root cells, PI(4,5)P2 is present at highest concentration in the plasma membrane (apex region) and nucleus, while PI4P slowly distributes between the plasma membrane and Golgi, with the highest concentration in the former. Multivesicular bodies/late endosomes accumulate both PI3P and PI(3,5)P2, and the tonoplast and autophagosomes contain PI3P. How the various metabolites are transported between membranes has yet to be determined, but non-vesicular transport is believed to occur at membrane contact sites and vesicular transport probably occurs also.

    Highly polarized distributions of phosphoinositides are found within membranes, in general oriented toward the cytosolic leaflet, and they are believed to be organized in nanoclusters together with other lipids and proteins. For example, phosphatidylinositol-4-phosphate is an important constituent of the plasma membrane in plant cells, where it controls the electrostatic state and is involved in cell division. It may control the location and function of many membrane proteins, including those required for development, reproduction, immunity, nutrition and signaling . PI(4)P is the only phosphoinositide present at the cell plate, i.e. the membrane separating two daughter cells during cell division. In addition, PI(4)P may interact with salicylic acid in the plant immune response, and it is produced during salt stress. However, specific functions are now being discovered for each of the plant phosphoinositides, which are produced rapidly in response to osmotic and heat stress, and it has become evident that a continuous turnover is essential for cell growth and development. For example, they have marked effects on the growth of many cell types and on guard cell function. In the nucleus, proteins have been identified that bind to phosphoinositides via the acyl chains, leaving the head group exposed for enzymatic modifications and signal transduction.

    Phosphoinositides are of special importance in microdomains at the tip of growing tissues such as the shoot apical meristem, pollen tubes and root hairs where phosphatidylinositol 4,5-bisphosphate functions in stem cell maintenance and organogenesis. In the plasma membrane, it is enriched in the detergent-resistant component commonly equated with 'rafts'. Although its concentration is low, PI(4,5)P2 has been shown to have signaling functions by binding to a number of different target proteins, which have characteristic binding domains. For example, together with phosphatidic acid, PI(4,5)P2 regulates the activity of a number of actin-binding proteins, which in turn control the activity of the actin cytoskeleton. This has a key role in plant growth, the movement of subcellular organelles, cell division and differentiation, and plant defence. In addition, this lipid exerts a control over ion channels, ATPases and phospholipase C-mediated lipid degradation and the production of further second messengers. It is an important factor in both clathrin-mediated endocytosis and in exocytosis. The specificity of the interactions may be dependent on the fatty acid composition of the lipid and on the activity of phosphatidylinositol 4-phosphate 5-kinase.

    As in animals, phosphoinositides have a role in endosomal sorting but through the central vacuole, which is a plant specific organelle with both lytic and storage functions. Phosphatidylinositol 3,5-bisphosphate is the least abundant of the phosphoinositides, but it is a crucial lipid for membrane trafficking systems. The PI to PI(3)P to PI(3,5)P2 cascade, the second step requiring a kinase designated FAB1, is required for endosomal sorting events leading to membrane protein degradation or retrieval, vacuolar morphogenesis and autophagy. PI(3,5)P2 is involved in stomatal closure and the growth of root hairs, and it is also induced in salt stress.

    A number of different enzymes of the phospholipase C type that are specific for polyphosphoinositides have been isolated from higher plants; they are activated by Ca2+ and unlike their mammalian counterparts, they are not regulated by G proteins. It is not certain whether phosphatidylinositol is itself a substrate for these enzymes in vivo. Less is known of the metabolism of the water-soluble inositol phosphates produced in comparison to animals, and plants appear to lack a receptor for inositol 1,4,5-trisphosphate (IP3), although it is the most abundant metabolite of this type and is reported to induce release of calcium ions to trigger stomatal closure. However, there is increasing evidence for lipid signaling mediated by phospholipase C in abiotic stress tolerance and development in plants. There is a general if contested belief that inositol hexakisphosphate (phytic acid or IP6), produced at least in part by sequential phosphorylation of inositol 1,4,5-trisphosphate, is a more important cellular messenger in plants and mobilizes an endomembrane store of calcium ions. Inositol-1,2,4,5,6-pentakisphosphate (IP5) is a structural co-factor of the jasmonic acid receptor coronatine insensitive 1, linking phosphoinositide signaling with phytohormone-controlled pathways.

    In plants in contrast to animals, diacylglycerols, the other product of phospholipase C hydrolysis of phosphoinositides, are rapidly converted to phosphatidic acid by diacylglycerol kinases and have not been considered important in signal transduction. Plants lack protein kinase C but they do have proteins with related properties that appear to be influenced by diacylglycerols. Via the action of phospholipase D, inositol phospholipids are a source of phosphatidic acid with its well-characterized signaling functions in plants, especially in defence.

    Lyso-Phosphoinositides

    Figure \(\PageIndex{41}\) below shows the structure of lysophosphatidylinositol

    lysoPI.svg
    Figure \(\PageIndex{41}\): Lysophosphatidylinositol

    Lysophosphatidylinositols: Lysophosphatidylinositols (LPI), i.e. with a single fatty acid only linked to the glycerol moiety, are formed as intermediates in the remodeling of the fatty acid compositions of the lipids by the action of phospholipase A1 or phospholipase A2 (e.g. cPLA2α), and when arachidonic acid is released for eicosanoid biosynthesis (see above). In ovarian cancer, LPI is elevated appreciably to around 15µM in ascites, and it is also present at high levels in obese subjects.

    It has become apparent relatively recently that like other lysophospholipids, lysophosphatidylinositol and the polyphospho-analogues may have messenger functions. For example, it has long been known to stimulate the release of insulin from pancreatic cells, suggesting a role in glucose homeostasis. sn-2-Arachidonoyl-lysophosphatidylinositol, in particular, is an endogenous ligand for a G protein-coupled receptor GPR55, and thereby can induce rapid phosphorylation of certain enzymes, including a protein kinase, which promote cancer cell proliferation, migration and metastasis. Indeed, lysophosphatidylinositol is a biomarker for poor prognosis in cancer patients, and its concentration is elevated significantly in highly proliferative cancer cells in vitro. GPR55 is expressed in many regions of the brain, the intestines, endocrine pancreas and islets (where it may stimulate insulin release). It has been implicated in macrophage activation and inflammation. In addition to its role in cancer, lysophosphatidylinositol has been implicated in a number of metabolic diseases. It is reported to be a precursor of the endocannabinoid 2‑arachidonoylglycerol by the action of human glycerophosphodiesterase 3 as a lysophospholipase C. This enzyme suppresses the receptor for lysophosphatidylinositol, and so acts as a switch between GPR55 and endocannabinoid (CB2) signaling .

    Glycerophosphoinositol: Sequential removal of both fatty acids from phosphatidylinositol by a specific phospholipase A2 (PLA2IVα) with both phospholipase A2 and lysophospholipase activities releases water-soluble glycerophosphoinositol. While this can be hydrolyzed by a glycerophosphodiester phosphodiesterase to inositol 1-phosphate, glycerophosphoinositol per se has distinctive biological activities and functions, as do related compounds derived from the phosphatidylinositol phosphates. In particular, glycerophosphoinositol has anti-inflammatory activity in that it inhibits the inflammatory and thrombotic responses induced by bacterial lipopolysaccharides (endotoxins).

    Figure \(\PageIndex{42}\) below shows the structure of glycerolphosphoinositol

    glycerolphosphoinositol.svg
    Figure \(\PageIndex{42}\): Glycerolphosphoinositol

    The Phosphatidylinositol Cycle

    Phosphatidylinositol can be considered to be at the centre of a cycle of reactions and intermediates that are involved in innumerable aspect of cellular signaling in animals (a similar cycle could be described for plants). These are discussed individually at length above, but it is useful to point out how each component forms part of a larger pattern. In brief as illustrated, the various synthetic and hydrolytic reactions involved in phosphoinositide metabolism can be considered to constitute a phosphatidylinositol cycle with enzymes located both in the endoplasmic reticulum and plasma membrane, so lipids have to be transferred across the cytosol in both directions between the two to complete the cycle, probably via adjacent membrane structures and facilitated by proteins of the phosphatidylinositol transfer protein membrane-associated family (PITPNM or nir2), which may channel phosphoinositide production to specific biological outcomes. Phospholipase C and phosphatidylinositol-4-phosphate 5-kinase (PI4P 5K) are located in the plasma membrane, while the cytidine diphosphate-diacylglycerol synthase (CDS2) and phosphatidylinositol synthase are in the endoplasmic reticulum. The epsilon isoform of diacylglycerol kinase (DGKε) is located at contact sites between the endoplasmic reticulum and plasma membrane, but there are nine further isoforms with differing cellular and subcellular locations that may be involved in the cycle. Each turn of the cycle uses a great deal of energy and consumes three moles of ATP, together with cytidine triphosphate and inositol. If it is assumed that the pyrophosphate is hydrolyzed by endogenous pyrophosphatases to inorganic phosphate, the cycle can proceed in one direction only.

    Figure \(\PageIndex{43}\) below shows the phosphatdylionositol cycle.

    The phosphatidylinositol cycle
    Figure \(\PageIndex{43}\): Phosphatdylionositol cycle

    Factors such as membrane curvature must be taken into account, and the diagram is of necessity a considerable over-simplification. In addition to participating in this cycle, many of the lipid intermediates can be precursors for other lipids, and for example, diacylglycerols are potential precursors for triacylglycerols, while phosphatidic acid is a precursor for phosphatidylcholine and phosphatidylethanolamine. Each lipid intermediate is subject to remodeling of the acyl chains via the Lands cycle, and polyunsaturated fatty acids released can be utilized for eicosanoid production. A further by-product of the cycle is inositol triphosphate, which contributes to the regulation of intracellular calcium levels.

    It has been suggested that the unique molecular species composition of phosphoinositides (18:0-20:4) could influence their selective recycling back into phosphatidylinositol as many of the enzymes involved have a preference for this substrate. A further proposal is that the phosphatidylinositol cycle could act to enrich this species through multiple passages around the cycle.


    21.3: Biosynthesis of Membrane Complex Glycerolipids is shared under a not declared license and was authored, remixed, and/or curated by Henry Jakubowski.

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