GPIs are an abundant and ubiquitous class of eukaryotic glycolipids. Although these structures were originally discovered in the form of GPI-anchored cell surface glycoproteins, a significant proportion of the GPI synthetic output of the cell is not directed to protein anchoring but to free GPIs . GPI anchors have been the subject of several reviews [1, 8, 18-21].
Preliminary structural analysis showed that the rat liver free glycolipid contained a phosphatidylinositol molecule glycosidically linked to a glucosamine residue, which was in turn bound to a glycan tail, whose composition and size have not yet been fully elucidated. Free-GPI phospholipids participating in signal transduction and GPI protein anchors share some structural motifs but have a different glycan composition. Free-GPI lacks the trimannose motif and the ethanolamine phosphate. A simplified model of the similarities and differences between the GPI anchors of membrane proteins and free-GPI phospholipids is shown in Fig. 6.1.
Several phospholipases are able to cleave free-GPI in vitro: bacterial phosphatidyl-inositol phospholipase C (PI-PLC), Trypanosoma brucei GPI-PLC and mammalian GPI-PLD. It was initially proposed that a PLC was the enzyme involved in GPI hydrolysis in mammalian cells as there was a rapid and transient intracellular rise in the level of diacylglycerol and on the ability of bacterial PI-PLC to partially mimic the effects of insulin, among other extracellular ligands, through the generation of IPG. However, a mammalian GPI-specific phospholipase C (PLC) has not yet been purified nor cloned. In contrast, a mammalian GPI-PLD has been cloned and it is an abundant, although inactive, serum protein . Furthermore, mammalian GPI-phospholipase D generates in vitro biologically active IPG from GPI [23, 24]. The relevance of GPI-PLD in insulin signaling in vivo has not been clearly demonstrated; however, the presence of GPI-PLD in serum and in mammalian cells together with its release from pancreatic islet cells in response to insulin secretago-gues suggest that GPI-PLD might be one of the enzymes responsible for free-GPI hydrolysis in vivo [25-29]. An appealing hypothesis is that differential GPI hydrolysis by phospholipases may give rise to specific IPG species in different cellular contexts, thus providing a further level of regulation in the cellular response. The cloning and characterization of mammalian GPI-lipases will certainly be a step forward in understanding this system. The recent release of the genome data of several species should help in the identification of candidate enzymes.
Fig. 6.1 Scheme representing the structure and hydrolysis of glycosyl-phosphatidylinositol (GPI) (adapted from Refs.  and ). Free GPI and GPI-anchored proteins are represented, showing their similarity in structure. The points for phospholipase C (PLC) and phospholipase D (PLD) hydrolysis are indicated. The glycan component of free GPI is
Fig. 6.1 Scheme representing the structure and hydrolysis of glycosyl-phosphatidylinositol (GPI) (adapted from Refs.  and ). Free GPI and GPI-anchored proteins are represented, showing their similarity in structure. The points for phospholipase C (PLC) and phospholipase D (PLD) hydrolysis are indicated. The glycan component of free GPI is drawn and consists of galactose (Gal), phosphate (PO2~), an unidentified residue of hex-ose (Hex), N-acetylglucosamine (GlcNAc) and glucosamine (GlcN). Below, free-GPI products of PLC and PLD hydrolysis: Ins, myo-inositol; IPG, inositol phosphoglycan; DAG, diacylgly-cerol; PA, phosphatidic acid.
Another open issue in GPI hydrolysis by phospholipases is how intracellular receptor activation couples to the hydrolysis of an extracellular plasma membrane lipid. There are many hypotheses that have not been fully explored, some of which have an experimental basis - for example, there are evidences of the presence of both free-GPI and GPI-PLD in intracellular vesicles [27, 30]. Although intracellular GPI-PLD has not been reported to hydrolyse GPI, an intracellular re lease of IPG would accord with different experimental data, for example with those reporting IPG actions on cell extracts. However, the hypothesis with the best experimental support is the presence of GPI-rich plasma membrane compartments, caveolae, where the activation of receptors could be coupled to PL-mediated GPI hydrolysis and to IPG downstream signaling. Data supporting caveolae as the key compartment for the activation of GPI/IPG signaling include: The presence of GPI in caveolae, the modulation of GPI-PLs activity by the lipidic environment, the activation of different insulin-regulated signaling molecules such as PI-3K and other kinases in caveolae and the existence of IPG carrier proteins in the plasma membrane [4, 16, 24, 32-34].
In summary, there has been a considerable advance in the knowledge of the nature of the specific phospholipases involved in GPI hydrolysis and the mechanism by which these enzymes are regulated and may couple to different receptors. But still many essential aspects of GPI-PLs structure, localization and function have to be elucidated.
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