GPI Membrane Anchors – The Much Needed link

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Sphingolipid- and cholesterol-rich membrane microdomains, often referred to as lipid rafts, harbour various plasma membrane proteins and can putatively provide a specific signalling platform for these proteins. Rafts were originally discovered as detergent-resistant lipid domains, but widely differing methods of isolation and analysis have led to numerous classifications, and there is still no coherence which further complicates the study of these domains. Subclasses of lipid rafts have been categorized depending on the specific lipid- and protein-composition, such as caveolae, raft-domains rich in caveolin proteins with tissue-specific signalling properties. Some lipid raft proteins are post-translationally modified by the covalent attachment of a glycosylphosphatidylinositol (GPI) anchor. The complexity of the GPI anchor implies a function beyond directing proteins to the plasma membrane, such as cell-cell communication, regulation of protein structure and cleavage, signal transduction and protein targeting. The mechanisms by which GPI-anchored outer membrane proteins may initiate intracellular signalling events are largely unknown; however several protein kinases have been implicated.

The importance of lipid microdomains becomes apparent in the raft localization of numerous proteins involved in the pathogenesis of diseases. These include neurological diseases such as Alzheimer’s and prion diseases, immunological diseases such as lupus erythematosus, and cardiovascular disease. Interestingly, the lipid composition of lipid rafts can be altered nutritionally, especially through dietary long chain fatty acids, which could potentially make lipid rafts an attractive pharmacological target.

Here we will analyze the signalling properties of lipid raft proteins and discuss their importance in health and disease.

Glycosylphosphatidylinositol (GPI)-anchored proteins are comprised of a relatively large hydrophilic moiety tethered to a membrane by a relatively small lipid tail. The functional role of the anchor is not clearly established, and proposals range from cell motility to cell signaling. Together with gangliosides and cholesterol, GPI-anchored proteins form membrane microdomains called lipid rafts in the outer leaflet of the plasma membrane that are involved in the regulation and modulation of numerous cellular processes.

GPI-anchored proteins can be actively shed from the membrane of one cell and taken up by other cells by insertion of their lipid anchors into the cell membrane. There are physiological processes where trans-cellular mobility of GPI-anchored proteins occurs. This process appears to be regulated, and most probably involves catalytic activity of some proteins that still have to be identified (GPI-specific phospholipases). Tumor cells and some pathogens apparently misuse this process for their own advantage, but its real physiological functions remain to be discovered. Special attention should be given when using detergents for biochemical and immunohistochemical methods in analysis of these molecules because of probable artifacts.

Glycosylphosphatidylinositol (GPI) anchors appear to confer unique biophysical properties on the proteins to which they are covalently linked. Model membrane systems provide a powerful tool to explore the effects of bilayer properties on the behaviour of GPI-anchored proteins. Such studies have typically been carried out after reconstitution/insertion of purified GPI-anchored proteins into symmetric or asymmetric lipid bilayer vesicles, supported lipid bilayers, or lipid monolayers. Biophysical studies using atomic force microscopy and Langmuir isotherms have revealed quantitative details of the interactions between GPI-anchored proteins and model membranes. Proteins carrying GPI anchors are believed to be targeted to detergent-resistant cholesterol/ sphingolipid-rich lipid rafts in both intact cells and model membranes, and the special properties of these microenvironments may also modulate their functional activity. GPI-anchored proteins are likely closely associated with the bilayer surface, so that the biophysical properties of the membrane, including curvature and lipid fluidity, modulate their conformation and activity. The GPI anchor can be cleaved by both endogenous and exogenous phosphatidylinositol (PI)-specific phospholipases C and D, from sources such as bacteria, protozoa and mammalian tissues. The release of GPI-anchored proteins in soluble form by phospholipases may play a key role in regulating their surface expression and activity. The GPI anchor appears to impose structural restraints, and its removal may alter the conformation, antigenicity and enzymatic activity of the protein. PI-specific phospholipases must interact closely with the membrane surface to cleave GPI anchors, and their activity is also greatly influenced by membrane biophysical properties.

GPI-anchored membrane proteins (GPI-APs) are a functionally and structurally diverse protein family present in eukaryote cells. Their common feature is the anchoring mechanism via glycosylphosphatidylinositol (GPI) to a lipid located in the extracellular leaflet of plasma membrane. Following a modification specific proteomic strategy, GPI-APs can be selectively isolated by a combined treatment of membrane fractions with Triton X-114 detergent and phosphatidylinositol-specific phospholipase C and/or D. The released proteins can be further identified by liquid chromatography on-line coupled to a tandem mass spectrometer. Although the method is proven to be highly specific, some non-GPI-proteins are also known to be released. Bioinformatics has been regularly used to discriminate the GPI-APs among all the identified proteins with a high degree of accuracy. Structural characterization of the GPI-anchors has been historically a laborious analytical task. Recently, HILIC (Hydrophilic Interaction Liquid Chromatography) and titanium dioxide enrichment of GPI-modified peptides in combination with mass spectrometry have been used to isolate and analyse GPI-anchors as well as for the determination of the anchor attachment site. The amino acid where the previously synthesized GPI-anchor is attached is named the omega site (ω-site). Moreover, GPI-specific diagnostic ions detected in tandem mass spectra can potentially be used in large-scale proteomic experiments to track GPI-specific peptides in complex mixtures. All the information obtained by the mentioned strategies has been used for the development of an integrated computational and experimental proteomic approach designed for identification of GPI-anchored peptides in MS/MS spectra as well as for ω-site determination.

Glycosylphosphatidylinositol (GPI) anchors are complex glycolipids, which typically anchor extracellular proteins onto the lipid membranes of eukaryotic cells. Although providing a natural platform in which to present or transfer functional molecules onto cells and viruses, GPI anchors are difficult biologics to generate in a homogenously pure form. It is also difficult, though not impossible, to elucidate and confirm their structures unambiguously. Today, chemical synthesis offers not only the versatility to make both complex and simplified GPI mimics and tools, but also the means to directly relate an exact GPI structure to its biological function. These synthetic GPIs may be further modified to allow the chemical attachment of any functional molecule, and not solely proteins, in a biologically compatible manner. Fluorescent labels and affinity tags can be exploited to investigate a particular biological response or process. Alternatively, synthetic glycans of GPI anchors can be employed to elicit a particular immune response or to generate GPI-specific antibodies. In this chapter, we shall overview the structure and synthesis of GPI anchors, and give perspectives on the biological study and therapeutic potential of synthetically-derived GPI biologics.

This chapter covers the use of glycosylphosphatidylinositol (GPI)-anchored proteins for surface modification of diverse types of biomembrane covered entities ranging from viruses and virus-related particles (section 1), to cells (section 2) and other natural and engineered micro- and nano-scaled particles (section 3). The aim is to present and review state-of-the-art research in this area and to discuss the future direction of GPI painting technology relating to applications in research, biotechnology and biomedicine.

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