Phosphoinositide-Containing Polymerized Liposomes: Stable

Bioconjugate Chem. 2005, 16, 1475−1483

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Phosphoinositide-Containing Polymerized Liposomes: Stable Membrane-Mimetic Vesicles for Protein-Lipid Binding Analysis Colin G. Ferguson,*,† Robyn D. James,† Cleve S. Bigman,† Donnie A. Shepard,† Yasmina Abdiche,‡ Phinikoula S. Katsamba,‡ David G. Myszka,‡ and Glenn D. Prestwich†,§ Echelon Biosciences Inc., 675 Arapeen Drive, Suite 302, Salt Lake City, Utah 84108, Center for Biomolecular Interaction Analysis, University of Utah, 50 North Medical Drive, Salt Lake City, Utah 84132, and Department of Medicinal Chemistry, University of Utah, 419 Wakara Way, Suite 205, Salt Lake City, Utah 84108. Received July 6, 2005; Revised Manuscript Received September 2, 2005

Stable phosphoinositide (PIPn)-containing liposomes were prepared using polydiacetylene photochemistry. Tethered pentacosadiynyl inositol polyphosphate (InsPn) analogues of Ins(1,3,4)P3, Ins(1,4,5)P3, and Ins(1,3,4,5)P4 were synthesized, incorporated into vesicles made up of diyne-phosphatidylcholine and -phosphatidylethanolamine, and polymerized by UV irradiation. The polymerized liposome nanoparticles showed markedly increased stability over conventional PIPn-containing vesicles as a result of the covalent conjugated ene-yne network in the acyl chains. The polymerized liposomes were specifically recognized by PIPn binding PH domains in liposome overlay assays and amplified luminescent proximity homogeneous assays. Moreover, the biotin moiety allowed attachment of the nanoparticles to a streptavidin-coated sensor chips in surface plasmon resonance (SPR) sensor. The PIPn headgroups displayed on SPR sensors showed higher affinities for PH domains and PIPn monoclonal antibodies than did monomeric PIPn-analogues with biotinylated acyl chains.

INTRODUCTION

Phosphatidylinositol polyphosphates (PIPns) are important second messengers for a diverse array of cellular functions (1, 2). These charged lipids are minor components of cellular membranes and are biosynthesized by the interplay of kinases (3), lipases, and phosphatases (4). PIPns are essential elements in tyrosine kinase growth factor receptor and G-protein receptor signaling pathways (5). Furthermore, they have important roles in membrane traffic (6), including endocytosis, exocytosis, Golgi vesicle movement and protein trafficking (7), in cell adhesion and migration, in remodeling of the actin cytoskeleton, and in mitogenesis and oncogenesis (8, 9). Activation of cellular signaling pathways often results from specific PIPn production in response to stimuli. The common mechanism employed by PIPns appears to be localized signal generation and the spatially localized recruitment of effector proteins (1, 10). Recruitment generally occurs through a specific PIPn binding domain on the protein. For example, pleckstrin homology (PH) domains bind to PIPns with varying affinities and specificities and are present in over 110 proteins (11, 12). Other domains include PX and FYVE, both of which predominantly bind PI(3)P (13, 14), and the COOHterminal domain of the tubby protein which binds to PI(4,5)P2 (15). Finally, the first known PIPn-binding nuclear receptor ING2, a putative tumor suppressor protein that promotes p53 acetylation, predominantly binds to the rare species, PI(5)P by a plant homeodomain (PHD) zinc * To whom correspondence should be addressed. Tel: (801) 588-0455. Fax: (801) 588-0497. E-mail: [email protected]. † Echelon Biosciences Inc. ‡ Center for Biomolecular Interaction Analysis, University of Utah. § Department of Medicinal Chemistry, University of Utah.

finger (16). The association of ING2 with chromatin is modulated by phosphoinositides and it was shown that ING2-dependent p53 acetylation and transactivation requires an intact PIPn-binding domain. An example of the importance of PIPn binding domains is that of the PH domains of PDK1 and Akt (protein kinase B) and their involvement in the PI-3 kinase pathway and cancer. Both PDK1 and Akt are sequestered to the membrane by binding to PI(3,4,5)P3 and Akt possibly undergoes a conformational change thought to make it susceptible to activation through phosphorylation on Ser473 by PDK1 (17). Activated Akt then inhibits apoptosis by phosphorylating a critical serine residue (Ser136) which suppresses pro-apoptic activity of BAD leading to subsequent cell death (18). Rationally designed PI analogues have been prepared that act as specific Akt PH domain inhibitors with low µM activities and increasing apoptosis 20-30 times in cancer cells with high Akt activity and 4-5 times in those with low activity (19, 20). Results such as these demonstrate the importance of PIPn-protein interactions in disease and suggest that small molecule modulators of these processes are an attractive new avenue for therapeutics (21, 22). Presentation of the PIPn ligand in liposomes has been a common technique employed to mimic the cell membrane for binding studies. For example, binding between the effector domain of the myristoylated alanine-rich C kinase substrate (MARCKS) and PI(4,5)P2 has been measured using sucrose laden vesicles (23) and vesicles containing spin labeled (24) and fluorescently labeled PI(4,5)P2 analogues (25). PIPn-containing liposomes have found extensive use in surface plasmon resonance (SPR) measurements of the PHD finger of ING2 (16), mammalian PH domains (26), and a recent genome-wide analysis of yeast PH domains (27). However, liposomes have some drawbacks, in particular, their instability and short lifetime. After 24-48 h at 4 °C, the carefully

10.1021/bc050197q CCC: $30.25 © 2005 American Chemical Society Published on Web 10/22/2005

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prepared unilamellar liposomes begin to fuse and form an irregular mixture of sizes. Thus, new batches need to be frequently prepared leading to potential inconsistencies and difficulties in experimental reproducibility. To make more robust liposomes capable of providing in highly reproducible data we report herein the preparation of stable, polymerized, PIPn-containing liposomes. These new nanoparticles show properties similar to those of conventional liposomes and monomeric PIPns in three binding assays: liposome overlays, the amplified luminescence proximity homogeneous assay (ALPHA), and SPR. EXPERIMENTAL PROCEDURES

General. Thin-layer chromatography was performed on 0.25 mm precoated glass plates (Merck silica gel 60F254) with detection by phosphomolybdic acid (5% in EtOH). Flash column chromatography (FCC) used Merck silica gel 60 (230-400 mesh). 1H, 13C, and 31P NMR spectra were recorded on a Varian INOVA 400 instrument. Coupling constants are recorded in Hz and chemical shifts in ppm. Mass spectra were measured at the University of Utah Medicinal Chemistry Department using matrix-assisted laser desorption ionization (MALDI). Bis-10,12-tricosadiynoyl-phosphatidylcholine and -phosphatidyletheanolamine (diyne-PC and diyne-PE) were purchased from Avanti Polar Lipids (Alabaster, AB). Biotin-X, succinimide ester was purchased from Invitrogen (Eugene, OR). All other reagents were purchased from Aldrich or Acros and used without further purification. N-Succinimidyl-10,12-pentacosadiynoate was prepared from 10,12-pentacosadiynoic acid (GFS Chemicals Inc) as previously described (28). Aminopropyl-Ins(1,3,4)P3, -Ins(1,4,5)P3, and -Ins(1,3,4,5)P4 were synthesized according to established protocols (29-31). DiC8-PIPns, acyl-biotinylated PIPns, and anti-PIPn mAbs were from Echelon. PH domain GST fusions were prepared at Echelon except for Akt-PH (Upstate Biotech). 1-O-(10,12-Pentacosadiynylaminopropyl-1-phospho)-myo-inositol-4,5-diphosphate (triethylammonium salt) (1a). A solution of N-succinimidyl-10,12pentacosadiynoate (7.0 mg, 14.8 mmol) in DMF (0.6 mL) was added to a solution of (3-aminopropyl-1-phospho)myo-inositol-4,5-diphosphate, sodium salt (5.8 mg, 9.8 mmol) in TEAB (0.5 M, 0.6 mL, pH 7.8). THF (∼10 drops) was added to the suspension to dissolve solids, and the reaction was stirred in the dark for 16 h. The reaction mixture was dried in vacuo, and the solid residue was washed with acetone (5 × 1.5 mL). The product was dissolved in water, passed through a short column (4 × 40 mm) of DOWEX 50 × 8-100 (triethylammonium form) to convert the product from the sodium salt, then lyophilized. Yield: 10.6 mg (77%). 1H NMR (400 mHz, D2O, free acid) δ 0.79 (m, 3H), 1.20 (m, 26H), 1.43 (m, 4H), 1.72 (m, 2H), 1.88 (quin, J ) 6.0 Hz, 2H), 2.10 (m, 6H), 3.04 (t, J ) 7.2 Hz, 2H), 3.58 (dd, J ) 9.6, 2.8 Hz, 1H), 3.77-3.98 (m, 5H), 4.10-4.17 (m, 2H). 31P NMR (D2O, 162 MHz) δ 0.61, 3.20, 3.66 (1:1:1). MALDI-TOF MS: 832.0 (M - H)- (free acid). 1-O-(10,12-Pentacosadiynylaminopropyl-1-phospho)-myo-inositol-3,4-diphosphate (triethylammonium salt) (1b). The same procedure was followed as for 1a. 1H NMR (400 MHz, CD3OD, 3-TEAH+ salt) δ 0.89 (t, J ) 6.4 Hz, 3H), 1.28 (m, 24H), 1.30 (t, J ) 7.6 Hz, 27H), 1.50 (quin, J ) 6.8 Hz, 4H), 1.59 (m, 2H), 1.79 (quin, J ) 5.6 Hz, 2H), 2.15-2.21 (m, 2H), 2.23 (t, J ) 8.0 Hz, 4H), 3.19 (quart, J ) 7.6 Hz, 18H), 3.47 (t, J ) 8.4 Hz, 1H), 3.86 (t, J ) 9.2 Hz, 1H), 3.95 (m, 3H), 1.38

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(m, 4H), 4.10 (t, J ) 9.2 Hz, 1H), 4.41 (quart, J ) 8.8 Hz, 1H).31P NMR (162 MHz, CD3OD) δ 1.50, 1.79, 2.87 (1:1: 1). MALDI-TOF MS: 832.1 (M - H)- (free acid). 1-O-(10,12-Pentacosadiynylaminopropyl-1-phospho)-myo-inositol-3,4,5-trisphosphate (triethylammonium salt) (1c). The same procedure was followed as for 1a. 1H NMR (400 MHz, CD3OD) δ 0.89 (J ) 6.8 Hz, t, 3H), 1.28 (m, 26 H), 1.30 (J ) 6.8 Hz, t, 45H), 1.49 (J ) 7.2 Hz, quin, 4H), 1.79 (m, 2H), 2.19 (J ) 8.0 Hz, t, 2H), 1.58 (m, 2H), 2.23 (J ) 6.8 Hz, t, 4H), 3.19 (J ) 7.2 Hz, quart, 30H), 3.91-4.19 (m, 5H), 4.41 (m, 2H), 4.56 (m, 1H). 31P NMR (162 MHz, CD3OD) δ 1.33, 1.87, 2.20 (2:1:1). MALDI-TOF MS: 912.2 (M - H)- (free acid). Biotin-diyne-PE. NEt3 (10 µL, 72 µmol) was added to a solution of Biotin-X, succinimde ester (11.6 mg, 25 µmol) and diyne-PE (17.7 mg, 20 µmol) in chloroform (2 mL), and the reaction was stirred at room temperature for 2 h. The solution was evaporated to half the volume and purified by flash column chromatography (70:20:4 CHCl3:MeOH:concentrated NH4OH). Yield: 22.8 mg (95%). 1H NMR (400 MHz, CDCl3) δ 0.87 (t, J ) 6.8 Hz, 6H), 1.22-1.76 (m, 70H), 2.20 (m, 4H), 2.23 (t, J ) 7.2 Hz, 8H), 2.28 (q, J ) 7.2 Hz, 4H), 2.75 (d, J ) 12.8 Hz, 1H), 2.89 (dd, J ) 12.8, 4.8 Hz, 1H), 3.01-3.12 (m, 3H), 3.14 (q, J ) 4.4 Hz, 1H), 3.21 (q, J ) 6 Hz, 2H), 3.46 (d, J ) 4.0 Hz, 2H), 3.92-4.06 (m, 5H), 4.26-4.39 (m, 2H), 4.51 (dd, J ) 4.8 Hz, 1H), 5.21 (m, 1H), 6.73 (bs, 1H), 6.94 (bs, 1H), 7.39 (bs, 1H). 13C NMR (101 MHz CDCl3) δ 8.79, 14.32, 19.42, 22.89, 25.03, 25.07, 25.37, 25.74, 26.52, 27.96, 28.06, 28.55, 28.57, 29.02, 29.08, 29.16, 29.31, 29.34, 29.51, 29.69, 29.77, 32.10, 32.68, 34.25, 34.43, 35.71, 36.34, 39.41, 40.85, 45.90, 55.97, 60.49, 62.10, 64.21, 65.18, 65.44, 65.54, 173.62, 173.63, 173.72, 173.97. 31P NMR (162 MHz, CDCl3) δ 0.88. MALDI-TOF MS: 1209.3 (M - H)- (free acid). Polymerized Liposome Preparation The following details a typical preparation of polymerized liposomes. Lipid constituents 1,2-bis(10,12-tricosadiynoyl-sn-glycero3-phosphatidylcholine (diyne-PC), 1,2-bis(10,12-tricosadiynoyl-sn-glycero-3-phosphatidylethanolamine (diynePE) (Avanti Polar Lipids,), InsPn-diyne, and biotin-diynePE) were dissolved in CHCl3/MeOH (9:1) and dried to a film on a rotary evaporator then dried further under high vacuum for 2 h. The proportions were typically 65:24: 10:1 or 65:29:5:1 diyne-PC:diyne-PE:InsPn-diyne:biotindiyne-PE [mol %]. Deionized water was added to give a 1 mM (total lipid) suspension. The mixture was heated to 70 °C and probe sonicated (Fisher Scientific 60 Sonic Dismembrator, setting 8) for 30 min, forming a clear solution. The solution was cooled to 4 °C for 2 h and then polymerized by UV irradiation (254 nm) at the same temperature using a handheld lamp. The faintly red solutions were filtered through 0.45 µm cellulose acetate syringe filters to remove any insoluble aggregates. Liposome solutions were stored at 4 °C in amber vials with 0.02% sodium azide to prevent bacterial growth. Liposome Overlay Assays. Aliquots of the desired protein solutions were spotted on a piece of nitrocellulose membrane (Hybond-C, Amersham Life Sciences) by hand and the membrane was allowed to dry for 5 min. The membrane was blocked for 2 h in blocking buffer (PBS (10 mM sodium phosphate, 138 mM NaCl, 2.7 mM KCl, pH 7.4), 3% BSA) at room temperature. The membrane was incubated in the polymerized liposome solution (5 µg/mL in PBS, 3% BSA) for 1 h at room temperature. The membrane was washed (4 × 10 min) in blocking buffer, then incubated in streptavidin-horseradish peroxidase (1:50 000) (Biosource International) in blocking buffer for 30-60 min at room temperature. Finally, the

Phosphoinositide-Containing Polymerized Liposomes

membrane was washed with blocking buffer (5 × 10 min), and then visualized with enhanced chemiluminescence (ECL, Amersham plc). Exposure times varied from 5 s to 2 min. Far Western/Liposome Overlay. Samples of PLCδ1-, Grp1-, LL5R-, and FAPP-PH domains (5, 1, or 0.1 µg) were dissolved in 13.3 µL of Laemilli buffer (Bio Rad Laboratories Inc.) then diluted to 20 µL with water. The samples were applied to the lanes of a 12% polyacrylamide gel, electrophoresed for 1.25 h at 100 V, and then electrotransferred to nitrocellulose in tris/glycine/SDS buffer at 300 mA for 70 min. The membrane was washed with PBS (2 × 5 min) and then subsequently treated with 6, 3, 1, and 0.1 M guanidine buffers (25 mM NaCl, 10% glycerol, 1 mM DTT) for 1 h each to renature the proteins followed by incubating with the blocking buffer overnight at 4 °C. The membrane was treated with 3 µg/mL PI(4,5)P2 liposomes in blocking buffer for 1 h, and the subsequent steps were identical to those described above for the overlay assay. Alphascreen Assays. Alphascreen assays were performed on a Fusion instrument (Perkin-Elmer, Inc.) using standard settings and the recommended buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween-20, 0.1% BSA). In a 384-well microplate was added 10 µL of buffer followed by 5 µL each of the GST-PH domain ([PLCδ1PH] ) 7.5 nM, [Akt-PH] ) 92 nM, [Grp1-PH] ) 8.4 nM final concentrated) and liposomes (0.01-10 uM PIPn ligand). A solution of anti-GST acceptor beads and streptavidin donor beads (5 µL, 5 µg/mL) were added, the plate was gently shaken, stored for 2 h in the dark, and read on the Fusion instrument. For competition experiments, solutions of diC8-PIPns (Echelon Biosciences Inc.) or biotin-free liposomes at varying concentrations were incubated with PLCδ1-PH (7.52 nM final concentrated) in buffer (10 µL) for 1 h in a 384-well microplate followed by addition of liposomes (0.20 µM final concentrated) and acceptor and donor beads (5 µL, 5 µg/mL). The plate was incubated in the dark for another hour and the luminescence was measured. Data were graphed and analyzed (nonlinear regression) using GraphPad Prism version 4.00 for Windows (GraphPad Software). Surface Plasmon Resonance. Interaction analysis was conducted at 20 °C using a Biacore 2000 biosensor (Biacore AB) equipped with a CM5 sensor chip. Streptavidin was immobilized onto the sensor surface (CM5) at 23 °C using a standard three step amine-coupling approach in HBS running buffer (10 mM HEPES pH 7.4, 150 mM NaCl). First, all flow cells were activated by injecting a freshly mixed solution of 50 mM NHS in 200 mM EDC for 7 min. Second, streptavidin (ImmunoPure grade, Pierce Biotechnology, Inc.) was reconstituted in 10 mM sodium acetate at pH 5.0 to ∼1 mg/mL and injected for 7 min. Finally, excess activated esters were blocked by treating them with 1 M sodium ethanolamine at pH 8.5, using a 7-min pulse. Liposome solutions (100 nM in HBS) and biotin-PIPns (200-250 nM in HBS) were diluted 10-fold in HBS running buffer and injected across individual flow cells at a flow rates of 5 or 10 µL/min until the desired loading was obtained. Fresh ligand surfaces were post-conditioned by injecting three 30 s pulses of 5 mM NaOH at 100 µL/min. Unmodified streptavidin flow cells served as reference and control surfaces. Antibodies were diluted into HBS running buffer supplemented with 0.5 mg/mL BSA and injected at 100 µL/min. The surfaces were regenerated with a single 15 s pulse of 10 mM H3PO4. PH domain samples were diluted into modified running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 2% v/v glycerol, and 0.1 or 0.5 mg/mL BSA (for PLCδ1-

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PH and Grp1-PH, respectively)) and injected at 30 or 100 µL/min (for PLCδ1-PH and Grp1-PH respectively) across all surfaces. A single pulse of 5 mM NaOH was used to regenerate the surfaces. Binding responses were referenced by subtracting the response generated across an unmodified streptavidin flow cell, and double referenced by subtracting an average buffer response (from at least two blank injections). The kinetic parameters were obtained by fitting globally to a mass transport model ([A0] ) [A], [A] + [B] ) [AB], where A0 ) injected analyte, A ) analyte concentration at surface, B ) immobilized ligand, AB ) bound complex). RESULTS AND DISCUSSION

Polymerized liposomes (vesicles) offer an attractive method of presenting multiple ligands in a stable membrane mimetic system. In a highly ordered state, diacetlyenes can be polymerized by UV light (254 nm) to form a conjugated enyne system (Figure 1A). Thus, when diacetylene amphiphiles are formed into vesicles and irradiated, polymerized vesicles are created (32). Poly(diacetylene) (PDA) liposomes are not disrupted by organic solvents or osmotic shock and show no evidence of fusion into larger aggregates over time (32, 33). Polymerizable inositol polyphosphate (InsPn)-diynes (1a-c) were synthesized from the aminopropyl analogues of Ins(1,4,5)P3, Ins(1,3,4)P3, and Ins(1,3,4,5)P4 via the N-hydroxysuccinimide ester of 10,12-pentacosadiynoic acid as shown in Figure 1B. InsPns rather than acyllabeled PIPns were chosen for two reasons. First, using a simple aminopropyl linker shortens the synthesis facilitates handling of the required headgroup. Since the headgroup will be anchored to the membrane through the diyne acyl group, incorporating the diacylglycerol seemed unnecessary. Second, InsPn probes have demonstrated effectiveness in mimicking a biologically relevant context (21). For instance, Ins(1,4,5)P3- and Ins(1,3,4,5)P4-tethered resins have been used in affinity purification of PI(4,5)P2 and PI(3,4,5)P3 binding proteins with equal to or greater efficacy than PI(4,5)P2 or PI(3,4,5)P3 resins (21, 34). Similar results have been observed for photolabeling proteins with PIPn vs InsPn probes (21, 35). To act as a detection tag, biotin-diyne-PE was synthesized from commercially available diyne-PE by condensation with the active ester of biotin-X (Figure 1C). Sonication of aqueous dispersions of the lipids at 70 °C gave liposomes, appearing as clear or slightly opaque solutions at concentrations of 1 mM (total lipid). The PIPn component constituted 1-10 mol % of the total lipids. Irradiation of the cooled solutions at 254 nm polymerized the acyl chains resulting in a faintly red color. Liposome suspensions up to 5 mM total lipid could be prepared but at that point, the viscosity increased dramatically when polymerized. For the remainder of this report, the polymerized liposomes will often be referred to simply as liposomes unless otherwise indicated. PI(4,5)P2 and PI(3,4,5)P3 liposomes were analyzed using a liposome overlay assay with immobilized PIPnbinding proteins. In this assay, the polymerized liposomes are incubated with proteins bound on a membrane and binding interactions are detected via the biotin tag (a scheme of the steps is shown in Figure 2A). First, solutions of GST fusions of the PH domains of PLCδ1, Grp1, LL5R, DAPP, and/or FAPP were spotted on nitrocellulose in varying amounts (0.021-21 pmol). Next, the membranes were incubated with a solution of polymerized liposomes in PBS containing 3% BSA (to prevent nonspecific binding to nitrocellulose), followed by treat-


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Figure 1. (A) Schematic representation of the polymerization of monomeric lipids in the outer layer of a liposome, (B) synthesis of InsPn-diynes, and (C) synthesis of diyne-PE-biotin.

Figure 3. Detection of electrophoresed PH domains by polymerized liposomes. PH domain proteins were electrophoresed and electrotransferred to nitrocellulose and PLCδ1-PH (5 µg) was selectively recognized by PI(4,5)P2 liposomes in a liposome overlay assay.

Figure 2. Detection of nitrocellulose immobilized PH domains by polymerized liposomes in liposome overlay assays. (A) Schematic of steps in the liposome overlay assay. (B) PLCδ1PH was selectively recognized by PI(4,5)P2 liposomes. (C) Grp1PH was selectively detected by PI(3,4,5)P3 liposomes.

ment with streptavidin-horseradish peroxidase (SAHRP) and chemiluminescent detection. As shown in Figure 2B, PLCδ1-PH was detected preferentially by the PI(4,5)P2 liposomes down to 0.21 pmol (10 ng) while FAPP-PH which binds PI(4)P was not detected. LL5RPH is a promiscuous PIPn binder and was recognized at 21 pmol, which is consistent with its weaker affinity for all PIPns. PI(3,4,5)P3 liposomes bound preferentially to Grp1-PH down to 0.21 pmol (10 ng) but did show some cross reactivity with PLCδ1-PH (Figure 2B). Importantly, binding was not observed using liposomes prepared without Ins(1,4,5)P3-diyne clearly demonstrating that the observed interactions are due to the PIPn headgroups (data not shown). Similar experiments with conventional liposomes showed essentially identical specificity but

approximately 10-fold greater sensitivity than polymerized liposomes (data not shown). Nonetheless, the anticipated benefit of the polymerized liposomes was their stability compared with conventional liposomes. Thus, no appreciable differences in specificity or detection limits were observed between freshly prepared PI(4,5)P2 polymerized liposomes and an older batch that had been stored at 4 °C for six months. The polymerized liposomes could also be used to specifically detect electrophoretically separated proteins in a fashion analogous to a Western blot. PH domains were separated on SDS-PAGE, electrotransferred onto nitrocellulose, and partially renatured in guanidinium hydrochloride, and then the membrane was treated as described above for the overlay experiments. As shown in Figure 3, PLCδ1-PH (5 µg) was selectively detected using PI(4,5)P2 liposomes compared with FAPP-PH and Grp1-PH; however, LL5R-PH was not detected likely due to lower affinity. As little as 1 µg of protein could be detected, and this sensitivity was increased when DTT was replaced with a glutathione redox pair at the expense of an increase in background. The detection limit was not as low as the overlays described above which we attribute to incomplete renaturation of the PH domains. In a related application, Elkin et al. employed the polymerized liposomes in liposome pull-down assays to confirm the PIPn binding specificity of the PHD finger containing RAG2 (36). RAG2 is essential for generating



Figure 4. Alphascreen binding and competition assays for liposome-PH domain interactions. Dose-dependent binding was demostrated for: (A) PI(4,5)P2 liposomes and PLCδ1-PH (7.5 nM), (B) PI(3,4)P2 liposomes and Akt-PH (92 nM), and (C) PI(3,4,5)P3 liposomes and Grp1-PH (8.4 nM). X-axes represent the concentration of PIPn ligand in the assay well. (D) Competition of PIPns with PI(4,5)P2 liposomes for binding to PLCδ1-PH. DiC8-PI(4,5)P2 (9) preferentially competed compared with diC8-PI(3,4)P2 (2) and diC8-PI(3,4,5)P3 ([). RLU ) Relative light units.

effective immune responses by catalyzing V(D)J recombination, and it is suggested that mutations in the PIPnbinding PHD finger alter the recombination activity leading to immunodeficiency. Additional control experiments in the report demonstrated the proteins PLCδ1PH [PI(4,5)P2], ING2-PHD [PI(5)P], p40-PX [PI(3)P], and FAPP1-PH [PI(4)P] specifically bound their known PIPn partners [in brackets]. The second technique used to analyze the binding characteristics of polymerized liposomes was ALPHA, a bead-based platform developed by Packard Biosciences for detecting biological interactions. The AlphaScreen system uses photosensitive donor beads, which convert ambient oxygen to singlet oxygen when irradiated at 680 nm. When the donor bead comes in close proximity to an acceptor bead, through a binding event, chemiluminescent receptor and fluorescent acceptor molecules in the acceptor bead are activated by the diffusion of singlet oxygen. The resulting emission shift from 580 to 620 nm can be measured as a luminescent signal. The pairing of acyl labeled biotin-PIPns and PH domains in this format have been demonstrated and implemented in assays for PI 3-kinase and PTEN (22, 37) suggesting that this technique would be suitable for validating the utility of the polymerized liposomes. The first experiment was designed to determine if binding occurred in this format and optimize a ratio of lipid to protein. A solution of GSTPLCδ1-PH in assay buffer was incubated with increasing amounts of PI(4,5)P2-containing polymerized liposomes. Streptavidin-coated donor and anti-GST-coated acceptor beads were added and allowed to incubate for 120 min, followed by measurement of the luminescence output. Increasing luminescence was observed with increasing amount of PI(4,5)P2-containing nanoparticles indicating dose-dependent binding to PLCδ1-PH (Figure 4A). The concentration on the X-axis represents the concentration of PI(4,5)P2 ligand in the assay well. In addition, PI(3,4)P2- and PI(3,4,5)P3-containing polymerized liposomes bound to Akt-PH and Grp1-PH domains respectively in a dose-dependent manner (Figures 4B and 4C). At higher liposome concentrations, the luminescence dropped off,

likely due to saturation of the donor beads and competition by unbound vesicles. A second experiment examined the ability of small soluble PIPns to compete with liposomes for protein binding. As observed in Figure 4D, diC8-PI(4,5)P2 preferentially competed with PI(4,5)P2 liposomes compared with diC8-PI(3,4)P2 and PI(3,4,5)P3 for binding to PLCδ1PH. Similarly, PI(4,5)P2 liposomes, prepared without diyne-PE-biotin, caused a decrease in luminescence by competing with biotinylated-PI(4,5)P2 liposomes while nonbiotinylated PI(3,4)P2 liposomes could not compete at concentrations up to 500 pmol/well confirming specificity (data not shown). Both experiments show that PI(4,5)P2 liposomes are selectively recognized in solution by PLCδ1PH. The final technique employed SPR to analyze the polymerized liposomes. SPR measurements of PIPnprotein interactions have typically used two methods of immobilizing lipids to chip surfaces to mimic membranes: (i) trapping liposomes onto a hydrophobic dextran surface (e.g. L1 chip from Biacore), or (ii) collapsing liposomes to form a lipid monolayer on the chip (e.g. HPA chip from Biacore). Initial experiments with PI(4,5)P2polymerized liposomes used a hydrophobic L1 chip for direct capture of vesicles. Although the vesicles were captured and protein binding was observed, the results were not reproducible, but some clues as to optimum running and regeneration conditions were elucidated. In contrast, streptavidin-coated sensor chips gave outstanding results with the biotinylated nanoparticles. Thus, the biotinylated polymerized liposomes were compared with acyl-biotinylated PIPns for binding to PIPnspecific monoclonal antibodies (mAbs) and PH domains. Two anti-PIPn mAbs, an anti-PI(3,4)P2 IgG and anti-PI(4,5)P2 IgM, were analyzed against their respective cognate ligands. As shown in Figure 5A-D the anti-PI(3,4)P2 IgG bound to both PI(3,4)P2 surfaces as did the anti PI(4,5)P2 IgM to the PI(4,5)P2 surfaces. The responses for the liposomes are concentration-dependent and reproducible for both cases. Binding was undetectable over an unmodified streptavidin control surface


Ferguson et al.

Figure 5. Sensorgrams comparing anti-PIPn mAbs binding to polymerized liposome and biotin-PIPn surfaces. Binding of anti-PI(3,4)P2 IgG (0.3, 0.8, 2.4, 7.3, 22, and 66 nM) to (A) PI(3,4)P2-liposome and (B) biotin-PI(3,4)P2-coated streptavidin chips at 20 °C (100 µL/min). Anti-PI(4,5)P2 IgM (0.8, 2.5, 7.4, 22.4, 66.6, and 200 nM) binding to (C) PI(4,5)P2 liposome and (D) biotin-PI(4,5)P2 at 20 °C (100 µL/min). The insets in parts A and C show the lack of binding to control surfaces. Surfaces were regenerated with single pulses of 10 mM H3PO4 (15 s.).

(insets, Figures 5A and C), demonstrating that the binding signals detected over the biotin PIPn and liposome surfaces are specific to the PIPn surfaces (0.5 mg/ mL BSA was included in the running buffer to minimize nonspecific binding to unmodified sensor chip surfaces). Kinetically, the interactions are limited by mass transport as described by the linear slopes during the association phase. The shapes appear slightly different between the PI(3,4)P2-containing polymerized liposome vs biotinPI(3,4)P2 surfaces due to mass transport. The biotin-PIPn surface has a much higher capacity and is therefore limited more by mass transport. The bivalent nature of the mAbs contributes to avidity effects making it challenging to determine binding rate constants. No binding was detected when anti-PI(3,4,5)P3 IgM was injected over the PI(4,5)P2 surfaces at 200 nM indicating specific interactions between the anti-PI(4,5)P2 mAb and the PI(4,5)P2 liposomes (data not shown). PH domain binding was analyzed under slightly modified conditions, 2% glycerol was included in the running buffer which helped stabilize the protein in addition to BSA. High-resolution data were collected across similar capacity PI(4,5)P2-liposome and biotin-PI(4,5)P2 surfaces by randomly injecting PLCδ1-PH ranging in concentration from 200 to 3.1 nM in duplicate at 30 µL/min (Figure 6A and B). After each run, the sensor chips were regenerated with single pulse of 5 mM NaOH. Binding responses were concentration dependent and reproducible. At concentrations above 100 nM, protein appeared to saturate all binding sites on the liposome surface, but not the biotin-PI(4,5)P2 surface. The reactions were analyzed globally using a mass transport model, which provided a reasonable fit to the measured data. As shown in Table 1A, PLCδ1-PH bound with a sixteen-fold higher affinity to the liposome surface than to the biotin-PI(4,5)P2 surface. The on-rates (ka) for the two surfaces were fairly similar but the off-rate (kd) was 8.5-fold slower for

Table 1. Kinetic Studies into the Binding of (A) PLCδ1-PH and (B) Grp1-PH to Monomeric PIPns and Polymerized Liposomes by SPRa ka (M-1 s-1) PI(4,5)P2 liposomes Biotin-PI(4,5)P2 PI(3,4,5)P3 liposomes Biotin-PI(3,4,5)P3

(A) PLCδ1-PH 1.2 × 106 8.9 × 105 (B) Grp1-PH 3.9 × 105 1.7 × 105

kd (s-1)

KD(nM)

7.9 × 10-3 9.3 × 10-2

6.6 104

9.5 × 10-4 2.1 × 10-3

2.5 12

a The kinetic parameters were obtained by fitting globally to a mass transport model. This gave the association rate constant (ka) and the dissociation rate constant (kd) which yielded the equilibrium dissociation constant (KD).

the liposome surface. The slower off-rates could be due to hydrophobic interactions between proteins and the bilayer interior and/or an avidity effect of close proximity of multiple headgroups on the vesicle surface. Grp1-PH was assayed under essentially identical conditions, except that BSA was increased to 0.5 mg/mL and the flow rate was increased to 100 µL/min (Figure 6C and D). Data were fit globally using a mass transport model (Table 1B) and revealed that Grp1-PH bound biotin-PI(3,4,5)P3 probe with a 5-fold lower affinity (KD ) 12 nM) than the PI(3,4,5)P3 liposome surface (KD ) 2.5 nM). As with the PLCδ1-PH/PI(4,5)P2 interactions, the liposome surfaces interacted more strongly with the protein. However, the on- and off-rates were both approximately 2.3-fold faster and slower respectively for the liposome surface, both contributing equally to the increase in affinity. The improved binding to the liposome surfaces for both proteins could be due to hydrophobic interactions between proteins and the bilayer interior and/or an avidity effect of close proximity of multiple headgroups on the vesicle surface, both of which would be missing from the acyl-linked surfaces.



Figure 6. Sensorgrams comparing PH domain binding to polymerized liposome and biotin-PIPn surfaces. PLCδ1-PH (0, 3.1, 6.3, 12.5, 25, 50, 100, 200 nM) binding to (A) PI(4,5)P2-liposome and (B) biotin-PI(4,5)P2-coated streptavidin chips at 20 °C (30 µL/min). Grp1-PH (1.4, 2.7, 5.4, 10.9, 21.8, 43.5, 87, 173 nM) binding to (C) PI(3,4,5)P3-liposome and (D) biotin-PI(3,4,5)P3-coated streptavidin chips at 20 °C (100 µL/min). Surfaces were regenerated with pulses of 5 mM NaOH.

Figure 7. Grp1-PH binding to varying mol % PI(3,4,5)P3liposome surfaces by surface plasmon resonance. The overlay plot of 173 nM Grp1-PH injected across three polymerized liposome surfaces (1, 2, and 5% PIP3) at 20 °C (100 µL/min) shows increasing signal comparable to the increasing PI(3,4,5)P3 composition in the liposomes. Probe surfaces were regenerated with a single 30-s pulse of 25 mM NaOH.

To examine the effect of varying ligand concentrations within the vesicles, additional liposomes were prepared with 1, 2, and 5 mol % PI(3,4,5)P3 and analyzed (for the previous experiment, the PI(3,4,5)P3 proportion in the vesicles was 10 mol %). As expected, the binding capacity increased with mol % PI(3,4,5)P3 (Figure 7). Of these probes, results with the 5% preparation were superior as these polymerized liposomes afforded surfaces that were the easiest to regenerate between runs. The affinities of the two other preparations were judged to be comparable but did not regenerate as well, requiring a 5-fold increase in NaOH to 25 mM. For all of the SPR experiments conducted, the data deviated from the simulated data, most notably in the dissociation phase. This suggested that, as expected for a multivalent ligand,

the mechanism was complex and that simple bimolecular models were not applicable. Finally, to investigate the selectivity of PI(3,4,5)P3 liposomes for Grp1-PH, competition studies were undertaken using water soluble diC8-PI(3,4,5)P3 and diC8-PI(4,5)P2. The competitors at varying concentrations were preincubated with the protein and then passed over the PI(3,4,5)P3-liposome chip surface. As shown in Figure 8, increasing diC8-PI(3,4,5)P3 competed with the liposome surface to bind Grp1-PH causing a decrease in response with almost complete competition at 87 nM. DiC8-PI(4,5)P2 did not effectively compete until much higher concentrations, with essentially complete competition at 21 µM. This demonstrates the strong selective affinity of Grp1-PH for the PI(3,4,5)P3-liposome surface as it is only interrupted by the presence of an analogue of the natural ligand. In this report, we describe the preparation of stable polymerized PIPn-containing vesicles. InsPn ligands containing a pentacosadiynyl acyl chain were synthesized for Ins(1,4,5)P2, Ins(1,3,4)P2, and Ins(1,3,4,5)P4, and a diyne-PE-biotin was prepared as a detection tag. Vesicles were prepared and polymerized at 254 nm and were validated in a number of formats. Liposome overlay assays using PI(4,5)P2- and PI(3,4,5)P3-polymerized liposomes showed selectivity comparable to that of conventional liposomes when binding to nitrocellulose-immobilized PH domains. In addition, PLCδ1-PH that had been electrophoretically separated and electrotransferred was recognized specifically by PI(4,5)P2 liposomes in the presence of other PH domains. Polymerized liposomes were applicable the ALPHA assays, binding was observed between PI(4,5)P2/PLCδ1-PH, PI(3,4)P2/Akt-PH, and PI(3,4,5)P3/Grp1-PH. The interaction of PI(4,5)P2/PLCδ1PH was specifically disrupted by soluble PI(4,5)P2. Finally, the interactions of with anti-PIPn mAbs and PLCδ1-PH and Grp1-PH were validated using SPR. The polymerized liposome nanoparticles proved to be robust


Figure 8. Sensorgrams of Grp1-PH binding to PI(3,4,5)P3liposome surfaces in the presence of water soluble PIPns. Grp1PH (87 nM) was incubated with varying concentrations of competitors: (A) diC8-PI(3,4,5)P3 and (B) diC8-PI(4,5)P2 and injected over PI(3,4,5)P3-liposome-coated surfaces at 20 °C (100 µL/min). Probe surfaces were regenerated with a single 30 s pulse of 5 mM NaOH. DiC8-PI(3,4,5)P3 competed at approximately 250-fold lower concentration than diC8-PI(4,5)P2.

coatings for streptavidin-coated chips and bound the PH domain proteins more strongly than the corresponding monomeric PIPn ligands. Polymerized PIPn-containing liposomes are thus appropriate, convenient, and biologically informative tools for studying protein lipid interactions. ACKNOWLEDGMENT

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BC050197Q

Phosphoinositide-Containing Polymerized Liposomes: Stable

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