PHOSPHATIDYLINOSITOL AND RELATED LIPIDS

STRUCTURE, OCCURRENCE, COMPOSITION AND ANALYSIS

1. Phosphatidylinositol

Phosphatidylinositol is an important lipid, both as a key membrane constituent and as a participant in essential metabolic processes in all plants and animals, both directly and via a number of 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), 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, is illustrated.

Formula of phosphatidylinositol

Phosphatidylinositol is especially abundant in brain tissue, where it can amount to 10% of the phospholipids, but it is present in all tissues and cell types. There is usually less of it than of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. In rat liver, it amounts to 1.7 micromoles/g..

Tissue	Fatty acids
Table 1. Fatty acid composition of phosphatidylinositol (wt % of the total) in animal and plant tissues.
	16:0	18:0	18:1	18:2	18:3	20:3	20:4	22:3	22:5	22:6

Bovine heart [1]	8	40	14	1	1	1	31	1	1	2
Bovine liver [2]	5	32	12	6	1	7	23	4	3	5
Rat liver [3]	5	49	2	2		4	35			1
Arabidopsis thaliana [4]	48	3	2	24	24
[1] = Thompson, W. and MacDonald, G., Eur. J. Biochem., 65, 107-111 (1976). [2] = Thompson, W. and MacDonald, G., J. Biol. Chem., 250, 6779-6785 (1975). [3] = Wood, R. and Harlow, R.D. Arch. Biochem. Biophys., 135, 272-281 (1969). [4] = Browse, J., Warwick, N., Somerville, C.R. and Slack, C.R. Biochem. J., 235, 25-31 (1986).

The fatty acid composition of phosphatidylinositol is rather distinctive as shown in Table 1. Thus, in 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 2 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, palmitic acid is the main saturated fatty acid while linoleic and linolenic acids are the main unsaturated components. Again, much of the saturated fatty acids are in position sn-1 and the unsaturated in position sn-2.

As with phosphatidylglycerol (and thence cardiolipin), phosphatidylinositol is formed biosynthetically from the precursor cytidine diphosphate diacylglycerol by reaction with inositol, and catalysed by the enzyme CDP-diacylglycerol inositol phosphatidyltransferase; the other product of the reaction is cytidine monophosphate (CMP). The enzyme is located in the endoplasmic reticulum mainly, although it may also occur in the plasma membrane in yeasts, and almost entirely on the cytosolic side of the bilayer. Phosphatidylinositol is then delivered to other membranes either by vesicular transport or via the agency of specific transfer proteins.

Biosynthesis of phosphatidylinositol

The 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.

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.

Release of arachidonic acid from phosphatidylinositol

In addition to functioning as negatively charged building blocks of membranes, the inositol phospholipids (including the phosphatidylinositol phosphates or 'polyphosphoinositides' discussed below) appear to have crucial roles in interfacial binding of proteins and in the regulation of proteins at the cell interface. As phosphoinositides are polyanionic, they can be very effective in non-specific electrostatic interactions with proteins. However, they are especially effective in specific binding to so-called ‘PH’ domains of cellular proteins.

More importantly, phosphatidylinositol and the phosphatidylinositol phosphates are the main source of diacylglycerols that serve as signalling 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). They 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 the last twenty years. 2-Arachidonoyl-glycerol, an endogenous cannabinoid receptor ligand, may also be a product of phosphatidylinositol catabolism.

Few bacteria appear to contain phosphatidylinositol, although inositol-containing lipids are found in the actinobacteria (lipophosphoglycans – see below). However, archaeal ether lipids contain analogues of phosphatidylinositol. In contrast, this lipid is found in all eukaryotes, which are able to synthesise inositol de novo via glucose-6-phosphate.

2. Phosphatidylinositol Phosphates

Phosphatidylinositol is phosphorylated by a number of different kinases that place the phosphate moiety on positions 4 and 5 mainly of the inositol ring, although more recently it has become evident that position 3 can be phosphorylated also by a specific kinase. Seven different isomers are known, but the most important in both quantitative and biological terms are phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate.

Formulae of phosphatidylinositol phosphates

These are usually present at low levels only in tissues, typically at about 1 to 3% of the concentration of phosphatidylinositol. The positional distributions of fatty acids in the phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate of ox brain are listed in Table 2. In each the saturated fatty acids are concentrated in position sn-1 and polyunsaturated in position sn-2. There are few differences among the three lipids.

Fatty acids	PI		PI monophosphate		PI diphosphate
Table 2. Distribution of fatty acids (mol % of the total) in positions sn-1 and sn-2 in phosphatidylinositol (PI) and the di- and triphosphoinositides of ox brain.*
Fatty acids	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
* Holub, B.J., Kuksis, A. and Thompson, W. J. Lipid Res., 11, 558-564 (1970).

The various polyphosphoinositides are maintained at steady state levels in the inner leaflet of the plasma membrane by a continuous and sequential series of phosphorylation and dephosphorylation reactions by specific kinases and phosphatases, respectively, which are regulated and/or relocated through cell surface receptors for extracellular ligands. This has been termed a ‘futile cycle’, and can consume a significant proportion of cellular ATP production. Controlled synthesis of these different phosphoinositides can occur in different intracellular compartments for distinct and independently regulated functions with differing target enzymes.

Polyphosphoinositide metabolism

The various organelles in cells have membranes with distinct functions and molecular compositions. Yet, they are all formed primarily at the endoplasmic reticulum, and the different membrane lipids and proteins must be transported to each site via specific membrane trafficking processes. These processes appear to be regulated or directed by the enzymes that determine the metabolism of particular phosphoinositides, which are segregated spatially on different membranes. 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. In these various organelles, further phosphorylation via kinases or removal of phosphates via hydrolysis continues

Phosphoinositides have a central and general position in the fields of cell signalling and regulation. They are able to achieve signalling effects directly by binding to cytosolic proteins or cytosolic domains of membrane proteins via their polar head groups. In this way, they can regulate the function of proteins integral to membranes, or they can attract cytoskeletal and signalling components to the membrane. Binding usually involves electrostatic interactions with the negative charges of the phosphate groups on the inositol ring with clusters of basic amino acid residues in proteins, and it can lead to folding and thence increased activity of unstructured peptides. The distinctive phosphoinositide composition of membranes in different organelles adds strength and specificity to the interactions by cooperative binding with other membrane proteins.

Scottish thistle The phosphatidylinositol monophosphates are present in cells at low levels only, but phosphatidylinositol 3-phosphate has been implicated in membrane trafficking through its interactions with specific proteins in endosomes. Indeed, it is a major determinant of the identity of the membrane of early endosomes, and it participates in most aspects of endosomal function. 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. In particular, it is essential for the structure and function of the Golgi complex, where it is required for the recruitment of specific proteins. 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 may also have signalling functions.

Phosphatidylinositol 4,5-bisphosphate is especially important, as a precursor of further metabolites (see below) and because of signalling functions in the plasma membrane in its own right, where it complexes with and regulates many cytoplasmic and membrane proteins, especially those concerned with ion channels for potassium, calcium, sodium and other ions. In most instances, it increases channel activity, while its hydrolysis by phospholipase C reduces such activity. In addition, it is intimately involved in the development of the actin cytoskeleton thereby controlling cell shape, motility, and many other processes. It is an essential cofactor for phospholipase D and so affects the cellular production of phosphatidic acid with its specific signalling functions. By binding specifically to ceramide kinase, the enzyme the enzyme responsible for the synthesis of ceramide-1-phosphate, it has an influence on sphingolipid metabolism. The physical properties of phosphatidylinositol 4,5-bisphosphate and its diacylglycerol metabolites are important for vesicle formation in membranes.

The major functions of phosphatidylinositol 3,5-bisphosphate are in membrane and protein trafficking, especially in the endosomes, although there is recent evidence that it has a role in the responses of mammalian cells to insulin.

3. Water-Soluble Inositol Phosphates

As mentioned briefly above, hydrolysis of phosphatidylinositol phosphates by enzymes of the phospholipase C type leads to generation of sn-1,2-diacylglycerols, which act as second messengers in the cell. Also released by this reaction are water-soluble inositol phosphates. Up to 60 different compounds are possible, and at least 37 of these have been found in nature at the last count, all of which are also extremely important biologically.

Generation of water-soluble phosphates

For example, inositol 1,4,5-trisphosphate is released from phosphatidylinositol 4,5-bisphosphate, and this is an important cellular messenger, stimulating calcium release from the endoplasmic reticulum. Indeed, 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 signalling pathways, the rearrangement of the actin cytoskeleton or intracellular vesicle trafficking. They have also been implicated in gene transcription, RNA editing, nuclear export and protein phosphorylation. As these remarkable compounds can be rapidly synthesised 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.

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. Different isomers appear to have specific functions at each level of gene expression, so extracellular events must coordinate the production of these compounds in a highly synchronous manner.

The extraordinary range of activities of phosphoinositides is relevant to major human diseases, including cancer and diabetes, making them important targets for pharmacological research and intervention.

4. Glycosyl-Phosphatidylinositol Anchors for Proteins

Formula of a glycosyl-phosphatidylinositol anchored protein Phosphatidylinositol is known to be the anchor that links a variety of proteins to the external leaflet of the plasma membrane via a glycosyl bridge (glycosylphosphatidylinositol(GPI)-anchored proteins). They are ubiquitous in eukaryotes (yeast, protozoans, plants and animals) and have also been shown to be present in some of the Archaeobacteria (but not Eubacteria). The protein is usually linked to an ethanolamine residue at the free carboxyl end. A typical molecule is illustrated. These complicated glycophospholipid-protein aggregates are abundant in nature, amounting to about 1% of all proteins and up to 20% of membrane proteins. The lipid moieties in particular have been most studied in parasitic protozoa such as Trypanosoma brucei (African sleeping sickness) or Leishmania spp., where they are more readily accessible.

The aliphatic residues are embedded in the membrane, and their chemical composition is dependent on the organism and the stage in its life cycle, but commonly position sn-1 is occupied by a long chain (C₁₈ or C₂₄) ether-linked alkyl moiety and position sn-2 by a saturated fatty acid (12:0 to 26:0). However, forms with simple fatty acid compositions, such as two myristic acid residues (14:0) are also known.

The carbohydrate moiety of the glycophospholipid is also variable in that it can be substituted in a species-specific manner, but the manα1-4GlcNα1-6-myo-inositol-1-HPO₄-lipid part is highly conserved (from yeast to humans), indicating that all are part of a single family of complex molecules.

While GPI-anchored proteins have a diverse range of functions, many are hydrolytic enzymes or serve as receptors, cell surface antigens or cell adhesion molecules. As an example, the prion protein responsible for ‘mad cow’ disease has a GPI-anchor.

Yeasts are distinctive in that they contain both GPI-anchored proteins, with a characteristic C₂₆ fatty acid component, and ceramide phosphorylinositol-anchored proteins. With the latter, the ceramide moiety is incorporated by an exchange reaction that occurs after the addition of the GPI precursor to proteins.

While many aspects of the biosynthesis of GPI-protein complexes remain to be determined, it is apparent that both the biosynthesis of GPI precursors and post-translational modification of proteins with GPI take place in the endoplasmic reticulum. The process starts on the cytoplasmic side of this membrane and is completed on the lumenal side, so the intermediate glycophospholipid must be flipped across the membrane. In mammalian cells, the lipid precursor is a conventional phosphatidylinositol molecule, which is first attached to an N-acetylglucosamine residue. This is de-acetylated before a saturated fatty acid (usually palmitate) is attached to the inositol residue, and this is followed by a sequence of reactions in which further carbohydrate moieties and phosphorylethanolamine are added.

Scottish thistle The GPI proteins all contain a characteristic carboxyl-terminal signal peptide with a hydrophobic tail, which is split off before the protein with a new carboxyl-terminal is combined with the amino group of the ethanolamine residue of the GPI moiety. A GPI-transamidase complex catalyses the overall process of cleavage and GPI attachment. The palmitate attached to inositol is then removed before the GPI-anchored proteins are transported to the Golgi. Here, the unsaturated fatty acid in position sn-2 of the glycerol moiety is removed by the action of phospholipase A₂ to form a lyso-GPI-protein, and this is re-acylated with a saturated acid (26:0 in yeast and mainly 18:0 in mammalian cells). The re-modelled GPI-anchored protein containing two saturated fatty acids is finally transferred to the outer leaflet of the plasma membrane. The nature of the hydrophobic moiety, resembling that of a ceramide, ensures that it is readily incorporated into sphingolipid-enriched ‘rafts’.

It is noteworthy that free or non-protein-bound glycosyl phosphatidylinositols are present on the external surface of the plasma membrane of some cells both in animals and protozoa (but not in yeast), albeit at very low levels, and they may be able to traverse the cell and the cellular membranes in this form from the rough endoplasmic reticulum where they are synthesised. It is then possible that they have some signalling functions in their own right or that they are involved directly in cellular recognition processes.

5. Lipophosphoglycans

Lipophosphoglycans (lipoarabinomannans and arabinogalactans). In addition to the GPI-anchor molecules, carbohydrates attached to phosphatidylinositols play a role in the surface antigenicity both of protozoal parasites and of prokaryotic organisms, especially those of actinomycetes or coryneform bacteria. Such lipophosphoglycans are present on the external cell surface, where they are intimately involved in host-pathogen interactions. Key lipids are phosphatidylinositol mannosides, with the first mannose residue attached to the 2-hydroxyl group and the second to the 6-hydroxyl of myo-inositol, which are found in the cell walls of Mycobacteria and related bacterial species. These range in structure from simple monomannosides in some Streptomyces, Mycobacterium species and in propionibacteria to molecules with 40 or more hexose units. In addition, several fatty acyl groups can be linked to the inositol-mannose chain.

Formula of a phosphatidylinositol dimannoside

The phosphatidyl dimannoside from Mycobacterium tuberculosis and M. phlei illustrated has been characterized as 1-phosphatidyl-L-myo-inositol 2,6-di-O-α-D-mannopyranoside. The main fatty acid constituents are palmitic and 10-methyl-stearic (tuberculostearic) acids. This is the basic structure from which additional phosphatidylinositol mannosides are produced by the organisms with up to four further mannose units, some of which can have fatty acyl substituents in specific positions of the inositol and/or mannose units. They are important to the immuno-pathogenesis of the organisms. As an example of the more complex mannosides, the main features of the lipoarabinomannan from Mycobacterium bovis, used as a vaccine against tuberculosis, have been determined, and they show that it is a multiglycosylated molecule with a polymannosyl phosphatidylinositol group anchoring it in the membrane. Such molecules are believed to have a function similar to that of the lipoteichoic acids. An acyl-phosphatidylinositol has been characterized from the pathogen Corynebacterium amycolatum. The fatty acid, mainly 18:1, may be linked to various positions of the inositol moiety.

Analogous compounds with the lipid backbone consisting of a ceramide, i.e. ceramide phosphorylinositol, rather than a diacylglycerol, are also found in nature, especially in yeasts and fungi.

6. Lyso-Phosphoinositides

It has long been known that the water-soluble glycerolphosphoinositides, the fully deacylated forms of phosphatidylinositol and the phosphatidylinositol phosphates have key roles in cellular signalling pathways. However, it has become apparent relatively recently that like other lysophospholipids, lysophosphatidylinositol, i.e. with a single fatty acid only linked to the glycerol moiety, and the polyphospho-analogues may have messenger functions.

7. Analysis

The book by Kuksis cited below is a definitive guide to the topic. Like all acidic phospholipids, phosphatidylinositol is not particularly easy to isolate in a pure state, special care being necessary to ensure that it is fully resolved from phosphatidylserine. However, this can be accomplished by adsorption TLC or HPLC with care. The phosphatidylinositol phosphates are a different matter, however, because of their high polarity and low abundance in tissues. It is necessary to used acidified solvents to extract them efficiently from tissues and to ensure that they are in a single salt form. For isolation of individual components, TLC methods are usually favoured, although detection can be a problem - one approach being to equilibrate with radioactive phosphorus to facilitate detection and quantification by liquid scintillation counting. HPLC with mass spectrometric (electrospray) detection is showing great promise. Analysis of the lipid-glycoconjugate-protein complexes and of the lipophosphoglycans is a rather specialised task for which modern mass spectrometric and NMR facilities are essential.

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