GPI-ANCHORED PROTEINS: BIOPHYSICAL BEHAVIOUR AND CLEAVAGE BY PI-SPECIFIC PHOSPHOLIPASES Frances J. Sharom Department of Molecular and Cellular Biology University of Guelph, Guelph, ON N1G 2W1 Canada
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[email protected] Running title: Biophysical behaviour and cleavage of GPI-anchored proteins
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Abstract 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/sphingolipidrich 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 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. Abbreviations: AFM, atomic force microscopy; AP, alkaline phosphatase; BAM, Brewster angle microscopy; cIP, myo-inositol-1,2-cyclic phosphate; CEA, carcinoembryonic antigen, CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate; CRD, cross-reacting determinant; DLS, dynamic light scattering; DMPA, dimyristoylphosphatidic acid; DOPC, dioleoylphosphatidylcholine; DPPC, dipalmitoylphosphatidylcholine; DSC, differential scanning calorimetry; FRAP, fluorescence recovery after photobleaching; FRET, fluorescence resonance energy transfer; FTIR, Fourier transform infra-red; FT-IRRAS, Fourier transform infrared reflection/absorption spectroscopy; GFP, green fluorescent protein; GPI, glycosylphosphatidylinositol; GPI-PLD, glycosylphosphatidylinositol-specific phospholipase D; IBS, interfacial binding surface; IPG, inositolphosphoglycan; LFA-3, lymphocyte functionassociated antigen 3; NMR, nuclear magnetic resonance; 5N-NTase, 5N-nucleotidase; PI, phosphatidylinositol, PLAP, placental alkaline phosphatase; PLC, phospholipase C; PLD, phospholipase D; POPC, palmitoyloleoylphosphatidylcholine; OG, n-octyl-β-D-glucoside; PIPLC, phosphatidylinositol-specific phospholipase C; SM, sphingomyelin; uPAR, urokinase-type plasminogen activator receptor; VSG, variant surface glycoprotein
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Reconstitution of GPI-anchored proteins into model membrane systems Glycosylphosphatidylinositol(GPI)-anchored proteins are unique among membrane proteins in possessing a covalently-linked phospholipid as part of their structure. This lipid moiety is instrumental in anchoring them in the membrane such that their behaviour is difficult to distinguish from that of integral proteins anchored by membrane-spanning polypeptide α-helices. Indeed, many GPI-anchored ectoenzymes were believed to be held in the membrane via transmembrane protein segments until their documented release by PI-specific phospholipases forced a re-thinking of this issue. One consequence of the attached GPI anchor is that it confers a unique set of membrane interactions, which may in turn modulate the behaviour and biological activity of the protein, such as its association with lipid raft microdomains. The presence of the GPI anchor, in turn, may influence the properties of the membrane into which the protein is inserted. The best way to explore the biochemical and biophysical basis of these interactions in vitro is to reconstitute purified GPI-anchored proteins into lipid bilayer or monolayer model membrane systems with defined physicochemical properties. In reconstituted systems, the experimenter has direct control over the acyl chain length and unsaturation (which control bilayer fluidity), bilayer charge, etc., and other membrane components such as cholesterol and glycosphingolipids can be readily included. The lipid:protein ratio can also be controlled over a wide range, from as low as 2.2:1 (w/w)1 to 200:1 (w/w)2. In the case of lipid bilayers, the choice of reconstitution method and lipid components may also allow some control over the final vesicle size. A wide variety of biophysical techniques may be applied to reconstituted GPIanchored proteins, including differential scanning calorimetry (DSC), atomic force microscopy (AFM), fluorescence spectroscopy, and Fourier transform infra-red (FTIR) spectroscopy. Such reconstituted bilayers and monolayers are powerful tools to mimic more complex cellular systems and shed light on important biological processes involving proteins that possess GPI anchors in their native state. They may also be useful in the study of proteins that have been genetically manipulated to contain a GPI linkage, and thus possess specific molecular properties. Symmetric and asymmetric lipid bilayers The detergent dialysis technique commonly used for transmembrane proteins3 has also been applied to the reconstitution of GPI-anchored proteins and ectoenzymes, such as Thy-1 antigen, lymphocyte function-associated antigen 3 (LFA-3), and 5N-nucleotidase (5N-NTase)(e.g.1,2,4-7). Typically, the lipid or lipid mixture of choice is completely solubilized in a detergent with a high critical micelle concentration, such as 3-[(3-cholamidopropyl) dimethylammonio]-1propanesulfonate (CHAPS) or n-octyl-β-D-glucoside (OG). This property of the detergent is essential for its later removal by dialysis. The desired amount of the purified GPI-anchored protein is then added, and after a period of equilibration, the lipid-protein mixture is subjected to dialysis to completely remove the detergent. During this process, unilamellar bilayer vesicles form spontaneously, with the GPI-anchored protein incorporated into them, typically in a roughly symmetrical fashion, with half the protein in the outer leaflet, and the other half in the inner leaflet (see Figure 1C)2. Recovery of GPI-anchored proteins in reconstituted vesicles prepared using this approach is usually >95%. Reconstituted vesicles can also be formed by rapid dilution of small samples of lipid and GPI-anchored protein solubilized in detergent solution (for example, dilution from 40 μL to 1 mL)8. Dynamic light scattering (DLS) provides a rapid and convenient method for characterization of the diameter and polydispersity of lipid bilayer vesicles prepared by either method. The distribution of the GPI-anchored protein between the outer and inner leaflets may be determined by measuring its catalytic activity under permeabilized and non-permeabilized
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conditions. When the bilayer remains intact and assay reagents cannot penetrate to the lumen, only the enzyme activity of proteins anchored in the outer leaflet is obtained. Addition of permeabilizing concentrations of detergent allows access of the assay reagents to the vesicle interior, so that the activity of the proteins in the inner leaflet is also measured. Typically, an approximate doubling of activity on permeabilization is an indicator that the GPI-anchored protein is symmetrically distributed between the outer and inner membrane leaflets (e.g.2). Alternatively, for GPI-anchored proteins with no enzymatic activity, cleavage by bacterial PIspecific phospholipase C (PI-PLC; see below) can be used to estimate the symmetry of reconstitution, since >90% of proteins anchored in the outer leaflet are typically released. However, it should be noted that in some GPI-anchored proteins, acylation of the inositol ring leads to resistance to phospholipase cleavage. In a typical large unilamellar vesicle of diameter 100 nm, there are not expected to be any steric hindrance-related issues for proteins anchored in the inner leaflet, since the typical GPI-anchored protein, alkaline phosphatase (AP), is about 5 nm in diameter, and the fully extended anchor is
