Phosphatidylinositol and related lipids: structure, composition, biochemistry, and analysis

PHOSPHATIDYLINOSITOL AND RELATED LIPIDS

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 (and in some bacteria (actinomycetes)), 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.

As with most other phospholipids, phosphatidylinositol is formed biosynthetically from the precursor CDP-diacylglycerol (see our web pages on phosphatidylglycerol) by reaction with inositol, catalysed by the enzyme CDP-diacylglycerol inositol phosphatidyltransferase. This 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 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..

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. 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.

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.

In addition to functioning as negatively charged building blocks of membranes, the inositol phospholipids (including the phosphatidylinositol phosphates sometimes termed '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.

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.

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. These are usually present at low levels only in tissues, typically at about 1 to 3% of the concentration of phosphatidylinositol. They are maintained at a steady state level 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. In their fatty acid compositions, the phosphorylated lipids resemble the parent molecule.

Phosphatidylinositol 4,5-bisphosphate in particular has signalling functions in the plasma membrane, where it complexes with and regulates many cytoplasmic and membrane proteins, especially those concerned with ion channels. In most instances, it increases channel activity, while its hydrolysis by phospholipase C reduces such activity.

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.

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, all of which are also extremely important biologically. Complex metabolic pathways result in further phosphorylation via kinases or removal of phosphates via hydrolysis. Controlled synthesis of these different phosphoinositides can occur in different intracellular compartments for distinct and independently regulated functions with differing target enzymes. 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. 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.

Phosphoinositides 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.

3.  Clycosyl-Phosphatidylinositol Anchors for Proteins and Lipophosphoglycans

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). The protein (over 100 are known) is usually linked to an ethanolamine residue at the free carboxyl end. A typical molecule is illustrated. These complicated glycophospholipid-protein aggregates appear to be wide spread in nature, and the prion protein responsible for ‘mad cow’ disease has a GPI-anchor, for example. However, the lipid moieties have been most studied in parasitic organisms such as Trypanosoma brucei 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, 14:0 or 16:0). 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 but the manα1-4GlcNα1-6-myo-inositol-1-HPO₄-lipid part is conserved, indicating that all are part of a single family of complex molecules.

The protein components are assumed to be especially important functionally, and they include hydrolytic enzymes, cell surface antigens, and proteins involved in interactions with other cells. They can be released by hydrolysis with the enzymes phospholipase C or D. On the other hand, it appears that free or non-protein-bound glycosyl phosphatidylinositols are also present on the surface of some cells at least and must 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.

Lipophosphoglycans.  In addition to the GPI-anchor molecules, carbohydrates attached to phosphatidylinositols play a role in the surface antigenicity 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.

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. 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.

Analogous compounds with a ceramide, rather than a diacylglycerol, backbone are also found in nature, especially in yeasts and fungi (i.e. ceramide phosphorylinositol).

4.  Lyso-phosphoinositides and the Glycerolphosphoinositides

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.

5.  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 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.

6.  Recommended reading

• Brennan, P.J. Mycobacterium and other actinomycetes. In: Microbial Lipids. Volume 1, pp. 203-298 (ed. C. Ratledge and S.G. Wilkinson, Academic Press, London) (1988).
• Christie, W.W. Lipid Analysis (3rd edition). (Oily Press, Bridgwater) (2003).
• Corda, D. Iurisci, C. and Berrie, C.P. Biological activities and metabolism of the lysophosphoinositides and glycerophosphoinositols. Biochim. Biophys. Acta, 1582, 52-69 (2002).
• Ferguson, M.A.J. The surface glycoconjugates of trypanosomatid parasites. Philos. Trans. R. Soc. London B, 352, 1295-1302 (1997).
• Gardocki,M.E., Jani,N. and Lopes,J.M. Phosphatidylinositol biosynthesis: biochemistry and regulation. Biochim. Biophys. Acta, 1735, 89-100 (2005).
• Kuksis, A. Inositol Phospholipid Metabolism and Phosphatidyl Inositol Kinases. Laboratory Techniques in Biochemistry and Molecular Biology, Volume 30, 970 pp. (Elsevier, Amsterdam) (2003).
• Menon, A.K. Structural analysis of glycosylphosphatidylinositol anchors. Methods in Enzymology, 230, 418-442 (1994).
• Meijer, H.J.G. and Munnik, T. Phospholipid-based signalling in plants. Annu. Rev. Plant Biol., 54, 265-306 (2003).
• Payrastre, B. Phosphoinositides. In: Bioactive Lipids. pp. 63-84. (edited by A. Nicolaou and G. Kokotos, The Oily Press, Bridgwater) (2004).
• Roth, M.G. Phosphoinositides in constitutive membrane traffic. Physiol. Rev., 84, 699-730 (2004).
• Suh, B.-C. and Hille, B. Regulation of ion channels by phosphatidylinositol 4,5-bisphosphate. Curr. Opinion Neurobiol., 15, 370-378 (2005).
• Vance, D.E. and Vance, J. (editors) Biochemistry of Lipids, Lipoproteins and Membranes (4th Edition). (Elsevier Science, Amsterdam) (2002) - several chapters.
• Vanhaesebroeck, B., Leevers, S.J., Ahmadi, K., Timms, J., Katso, R., Driscoll, P.C., Woscholski, R., Parker, P.J. and Waterfield, M.D. Synthesis and function of 3-phosphorylated inositol lipids. Annu. Rev. Biochem., 70, 535-602 (2001).