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Lipid modulation of membrane-bound epitope recognition and blocking by HIV-1 neutralizing antibodies
FEBS Letters 582 (2008) 3798–3804
Lipid modulation of membrane-bound epitope recognition and blocking by HIV-1 neutralizing antibodies Nerea Huartea, Maier Lorizatea,1, Renate Kunertb, Jose´ L. Nievaa,* a
Biophysics Unit (CSIC-UPV/EHU) and Biochemistry and Molecular Biology Department, University of the Basque Country, P.O. Box 644, 48080 Bilbao, Spain b Institute of Applied Microbiology, University of Agriculture, A-1190 Vienna, Austria Received 21 August 2008; revised 29 September 2008; accepted 5 October 2008 Available online 16 October 2008 Edited by Jacomine Krijnse-Locker
Abstract The conserved, aromatic-rich membrane-proximal external region (MPER) of gp41 is functional in human immunodeficiency virus (HIV)-cell fusion by perturbing membrane integrity. Broadly-neutralizing 2F5 and 4E10 monoclonal antibodies (MAb-s) recognize amino- and carboxy-terminal epitope sequences within this domain, respectively. An MPER peptide overlapping 2F5 and 4E10 epitope sequences was capable of breaching the permeability barrier of lipid vesicles. Cholesterol and sphingomyelin raft-lipids, present at high quantities in the HIV-1 envelope, promoted exposure or occlusion of 4E10 epitope, respectively. Conversely, 2F5 epitope accessibility was affected to a lesser extent by these envelope lipids. These observations support the idea that MPER epitopes on membranes are segmented in terms of how they are affected by envelope lipids, which may have implications for MPER-based vaccine development. Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: HIV-1; HIV-1 gp41; HIV-1 neutralization; MAb2F5; MAb4E10; MPER; Cholesterol; Sphingomyelin
1. Introduction The potential to elicit broadly neutralizing antibodies (NAbs) is a long-pursued goal for an human immunodeficiency virus type-1 (HIV-1) vaccine [1]. Monoclonal antibodies (MAb-s) 2F5 and 4E10 are one of the few examples of identified NAb-s showing capacity to neutralize a broad spectrum of HIV-1 clades and primary isolates [2–7]. These NAb-s have evolved to recognize epitopes within the membrane-proximal * Corresponding author. Address: Biofisika Unitatea (CSIC-UPV/ EHU) and Biokimika Saila, Euskal Herriko Unibertsitatea, Posta Kutxa Box 644, 48080 Bilbao, Spain. Fax: +34 94 6013360. E-mail address: [email protected] (J.L. Nieva). 1
Present Address: Abteilung Virologie, Universita¨tsklinikum Heidelberg, im Neuenheimer Feld 324, D-69120 Heidelberg, Germany.
Abbreviations: Chol, cholesterol; CRAC, Cholesterol Recognition/ interaction Amino acid Consensus motif; DPC, dodecylphosphocholine; HIV-1, human immunodeficiency virus type-1; MAb, monoclonal antibody; MPER, membrane-proximal external region; NAb-s, neutralizing antibodies; POPC, phosphatidylcholine; PL, phospholipid; PreTM, pretransmembrane; SPM, sphingomyelin; WW, Wimley– White
external region (MPER), preceding the envelope gp41 subunitÕs transmembrane domain. Thus, MPER constitutes a candidate target vaccine for the development of antibodies that can neutralize the wide array of circulating HIV-1 isolates [8,9]. Based on a site-directed mutagenesis study, Salzwedel et al. [10] defined MPER as the aromatic-rich, conserved gp41sequence 665KWASLWNWFNITNWLWYIK683. This domain was primarily implicated in gp41-induced membrane fusion and envelope glycoprotein incorporation into virions [10,11]. The same gp41 sequence was designated as the pretransmembrane (PreTM) domain according to its tendency to stably associate with the membrane interface as an a-helix and promote lipid vesicle destabilization [12–14]. The interfacial location of the helical MPER/PreTM sequence was subsequently supported by structural studies [15–17]. Additionally, viral envelope raft-lipids such as cholesterol (Chol) and sphingomyelin (SPM) were shown to modulate MPER capacity to destabilize membranes [14,18,19]. Experimental evidence and modeling studies further supported MPER–Chol interactions through the carboxy-terminal ‘‘Cholesterol Recognition/interaction Amino acid Consensus’’ (CRAC) LWYIK motif [20,21]. Just recently, Vishwanathan and Hunter [22] replaced MPER with indolicidin-based sequences. Indolicidin is a Trprich, antimicrobial peptide that forms pores in membranes. Some of the produced gp41 chimeras retained fusion activity, suggesting that membrane-perturbing properties are key to MPER membrane fusion function [22]. Thus, the experimental evidence produced in cells and model systems supports MPER function in fusion (recently reviewed in [23]). Attending to this evidence it has been hypothesized that NMAb-s 2F5 and 4E10 might neutralize the virus by blocking membrane-perturbing MPER activity [17,24]. In support of this hypothesis MAb-s 2F5 and 4E10 are adapted for recognition of membrane embedded MPER sequences [17,24–29]. Recent experiments described by Sun et al. [17] demonstrate that MAb4E10 binds to membrane-embedded MPER and induces its partial extraction. These authors postulate that the solved structure of MPER inserted into dodecylphosphocholine (DPC) micelles represents a minimal native-like gp41 structure with the potential of eliciting NAb-s. Accordingly, it has been suggested that MPER-derived peptides in a lipid environment might constitute useful platforms for purposes of vaccine design [17,24,29]. Here, in search for a minimal lipid composition allowing simultaneous and effective presentation of 2F5 and 4E10 epitopes at the membrane interface, we have comparatively
0014-5793/$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.10.012
N. Huarte et al. / FEBS Letters 582 (2008) 3798–3804
analyzed the effects of SPM and Chol, the main raft-lipids existing in the viral envelope [19]. Effective binding has been derived from the antibody-induced inhibition of the MPER membrane-perturbing activity measured in solution-diffusing lipid vesicles [24]. Since the MPER native-like structure relevant for antibody recognition has been solved in contact with a phosphocholine-based lipid [17], the vesicles used in this study as a reference were made of pure phosphatidylcholine (POPC). Our results suggest that the lipid environment affects predominantly 4E10 epitope accessibility. This observation might guide the development of immunogens combining MPER with a minimal amount of chemically defined lipids.
2. Materials and methods NEQELLELDKWASLWNWFNITNWLWYIK (AISpreTM), NEQELLELAAWASLWNWFNITNWLWYIK (AISpreTM(9,10)A) and NEQELLELDKWASLWNAANITNWLWYIK (AISpreTM(17,18)A) peptides were synthesized by solid-phase synthesis using Fmoc chemistry, as C-terminal carboxamides and purified by HPLC at the Proteomics Unit of the University Pompeu-Fabra (Barcelona, Spain). Peptide stock solutions were prepared in dimethylsulfoxide (DMSO) (spectroscopy grade) and concentrations determined by Bicinchoninic Acid microassay (Pierce, Rockford, IL, USA). NAb expressing hybridomas were originally generated by a combined PEG-electrofusion of peripheral blood mononuclear cells of HIV infected non-symptomatic patients [2]. MAb-s 2F5 and 4E10 used in this study were subsequently produced in recombinant CHO cells after subclass switch to IgG1 [30]. 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC), SPM, 1-palmitoyl2-stearoyl(6,7)dibromo-sn-glycero-3-phosphocholine (Br6-PSPC) and 1-palmitoyl-2-stearoyl(11,12)dibromo-sn-glycero-3-phosphocholine (Br11PSPC) were purchased from Avanti Polar Lipids (Birmingham, AL, USA). 8-Aminonaphtalene-1,3,6-trisulfonic acid sodium salt (ANTS) and p-xylenebis(pyridinium)bromide (DPX) were from Molecular Probes (Junction City, OR, USA). Large unilamellar vesicles (LUV) were prepared following the extrusion method of Hope et al. [31] in 5 mM Hepes, 100 mM NaCl (pH 7.4) buffer and using polycarbonate membranes (Nuclepore Inc., Pleasanton, CA, USA) with a 0.1 lm nominal pore size. Lipid concentrations of liposome suspensions were determined by phosphate analysis. Release of encapsulated vesicular contents to the medium was fluorimetrically monitored by the ANTS/DPX assay [32] as previously described [27]. Kinetics of membrane-insertion were measured by energy transfer from peptide-Trp to the surface fluorescent probe dDHPE as described [24]. Penetration level of the peptide into the bilayer core was inferred from the Trp fluorescence quenching by the hydrophobic matrix-residing brominated phospholipids (PLs), according to the method described by Bolen and Holloway [33]. These experiments were carried out using sonicated vesicles containing a POPC fraction substituted for the brominated PLs Br6-PSPC or Br11-PSPC. Circular dichroism (CD) measurements were obtained from a thermally-controlled Jasco J-810 CD spectropolarimeter calibrated routinely with (1S)-(+)-10-camphorsulfonic acid, ammonium salt. Samples consisted of co-lyophilized peptide and lipid dissolved and sonicated in 2 mM Hepes (pH, 7.4) buffer. Spectra were measured in a 1 mm path-length quartz cell initially equilibrated at 25 °C. Data were taken with a 1 nm band-width at 100 nm/min speed, and the results of 20 scans were averaged. For the enzyme-linked immunosorbent assay (ELISA), peptides were dissolved in phosphate-buffered saline (PBS) and immobilized (100 ml/well) overnight in C96 Maxisorp microplate wells (Nunc, Denmark) at a concentration of 1 lM. Prior to incubation with the MAbs, the plates were blocked for 2 h with 3% (w/v) bovine-serum albumin in PBS. The binding of the Mabs was detected with an alkaline phosphatase-conjugated goat anti-human immunoglobulin (Pierce, Rockford, IL, USA), which then catalyzed a color reaction with the p-nitrophenyl phosphate substrate (Sigma, St. Louis, MO, USA) that could be measured by absorbance at a wavelength of 405 nm in a Synergy HT microplate reader (Bio-TEK Instruments Inc., VT, USA).
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3. Results 3.1. Lipid effects on AISpreTM-induced leakage Predictive tools based on the use of Wimley–White (WW) water-to-membrane interface transfer free energies [34] and recent structural characterization suggest that membrane-bound MPER/PreTM is segmented into two subdomains [14,17]. The N-terminal domain comprises a helical, conserved amphipathic-at-interface sequence (AIS). AIS is followed by a fully hydrophobic-at-interface stretch that precedes the transmembrane anchor. The AISpreTM peptide used in this study was designed to contain both interfacial subdomains ( Fig. 1A). In addition, AISpreTM encompasses the full-length 2F5 and 4E10 epitopes [26,35]. Consistent with its predicted role in fusion, this MPER peptide was capable of breaching the membrane permeability barrier of lipid vesicles (Fig. 1B). In a previous contribution we showed that the lytic activity of HIVc, a shorter MPER peptide devoid of NEQELLEL AIS residues, was dependent on the presence of SPM [18]. Thus, low amounts of peptide were required to permeabilize POPC:SPM (1:1 mol:mol) vesicles (in the order of 1:1000– 1:10000 peptide-to-lipid ratios) and the process could be modeled according to a pore-formation phenomenon. Relatively much higher doses were required to permeabilize POPC or POPC:Chol (2:1 mol:mol) vesicles (in the order of 1:10–1:200 peptide-to-lipid ratios) and kinetic modeling ruled out poreformation as the permeabilization mechanism in those systems. AISpreTM lytic activity was also stimulated upon addition of SPM to the liposome composition (Fig. 1B). Peptide-tolipid mole ratios in the order of 1:1000 were required to totally
Fig. 1. Definition and membrane-perturbing properties of AISpreTM peptide. (A) Schematic diagram to designate gp41 regions: FP, fusion peptide; NHR and CHR, amino- and carboxy-terminal helical regions, respectively; MPER, membrane-proximal external region; TMD transmembrane domain. AISpreTM (below) spans the 656–683 region that contains 2F5 and 4E10 epitopes. Boxes designate crucial residues according to Zwick et al. [36] mutated by Ala in AISpreTM(9,10)A and AISpreTM(17,18)A peptides. (B) Percentage of AISpreTMinduced leakage after 30 min of incubation with vesicles, plotted as a function of the peptide-to-lipid mole ratio (Ri). Filled circles and continuous line: POPC:SPM (1:1 mol:mol); filled squares and dotted line: POPC; and empty circles and slashed line: POPC:Chol(2:1 mol:mol). Arrows indicate the conditions selected to measure MAbinduced blocking of leakage. Final lipid concentration was 0.1 mM in all cases.
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permeabilize POPC:SPM (1:1 mole ratio) LUV. By comparison ca. 10 times higher peptide doses were required to permeabilize pure POPC or POPC:Chol (2:1 mole ratio) LUV. This observation confirmed previous results indicating that PreTM/MPER activity was preserved in the longer AISpreTM peptide [24]. The CD results displayed in Fig. 2A revealed similar secondary structures for AISpreTM in contact with POPC, POPC:SPM (1:1) and POPC:Chol (2.1) vesicles. The CD spectra obtained in the three systems displayed major band components at 208 and 222 nm supporting that the helical conformation was the main structure adopted by the peptide. Thus, the adoption of a distinct lytic secondary structure by the peptide did not provide an explanation for the leakage enhancement observed in the presence of SPM (Fig. 1). The intrinsic fluorescence results below ( Fig. 2B) suggested that AISpreTM may insert deeper into POPC and POPC:SPM (1:1) than into POPC:Chol (2:1) bilayers. In the case of POPC:SPM and POPC vesicles AISpreTM-Trp-s were quenched to similar extents by Br6-PSPC and Br11-PSPC (left and center panels). In contrast, Br6-PSPC quenched AISpreTM-Trp emission more efficiently than Br11-PSPC (right panel). Bromine atoms quench by a short range process that requires a close approach to the fluorophore [33]. Thus, AISpreTM Trp residues located closer to the C6–C7 than to the C11–C12 position within the acyl chain region of the bilayers containing Chol. In conclusion, Chol seemed to induce a shallower peptide insertion into the lipid bilayer, but did not appreciably affect its overall secondary structure.
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3.2. Lipid effects on specific recognition and blocking of AISpreTM AISpreTM-induced vesicle permeabilization was next used to assess the influence of SPM and Chol on antibody-induced inhibition of MPER membrane activity (Fig. 3). Panel 3A illustrates the effect of MAb addition to the ongoing AISpreTMinduced leakage in vesicles of different lipid composition. In all cases, kinetic traces of d-DHPE fluorescence increase demonstrated faster peptide incorporation into vesicles (dotted traces). AISpreTM was added to a stirred solution of lipid vesicles (1), left sufficient time for incorporation into them, and MAb was subsequently injected into the mixture (2). Consistent with recognition of membrane-bound species under these conditions, addition of MAb-s 4E10 and 2F5 arrested the leakage process (top and bottom panels, respectively). However, as compared to that induced by MAb2F5, MAb4E10 addition induced a weaker inhibition of leakage from POPC:SPM vesicles (left panels). The dose dependence of leakage inhibition illustrates further this effect (panel B). The inhibition percentages were calculated from the leakage extents measured after 5 min of incubation, and using as 0% inhibition reference vesicle samples untreated with MAb. Relatively lower doses of MAb4E10 than of MAb2F5 were required to effectively arrest AISpreTM-induced leakage in POPC and POPC:Chol vesicles. Moreover, Chol promoted the inhibitory capacity of MAb4E10. In contrast, the capacity of MAb4E10 to block AISpreTM was impaired in POPC:SPM vesicles. In this system Mab2F5 actually inhibited AISpreTM activity more efficiently than
Fig. 2. Interaction of AISpreTM with POPC:SPM (1:1 mole ratio), POPC and POPC:Chol (2:1 mole ratio) lipid vesicles. (A) CD spectra of AISpreTM peptide associated to the vesicles (lipid concentration, 1 mM). The concentration of AISpreTM peptide was 30 lM. (B) Intrinsic fluorescence emission spectra of AISpreTM incubated with the same type of vesicles (traces on top labeled with symbols). Continuous and dotted traces below correspond to spectra obtained in the presence of vesicles in which a fraction of the POPC was substituted for Br6-PSPOPC or Br11PSPOPC, respectively. The final brominated lipid content in all vesicles was 25 mol%. Vesicle lipid concentration was 0.1 mM and the peptide-tolipid mole ratios were 1:500 (POPC:SPM) and 1:100 (POPC and POPC:Chol).
N. Huarte et al. / FEBS Letters 582 (2008) 3798–3804
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Figure 3. Inhibition of AISpreTM-induced vesicle contents leakage by MAb-s 2F5 and 4E10. (A) Effect of Mab addition to the ongoing leakage. Vesicle samples (100 lM lipid) were treated with 0.2 (POPC:SPM) or 1 lM (POPC and POPC:Chol) AISPreTM and, subsequently supplemented with 50 lg/ml of MAb-s 2F5 or 4E10 (addition times indicated by ‘‘1’’ and ‘‘2’’, respectively). The dotted traces follow AISpreTM incorporation into the vesicles monitored through energy transfer from peptide tryptophans to membrane-residing d-DHPE. Addition of the antibody results in a sudden decrease of the leakage rate. The traces labeled with (+) and (–) correspond to the leakage kinetics in the presence and the absence of antibody, respectively. (B) Dose dependency of AISpreTM-induced leakage inhibition by MAb4E10 or MAb2F5 in POPC:SPM (filled circles and continuous lines) POPC (squares and dotted lines) and POPC:Chol (hollow circles and slashed lines) vesicles. Inhibition percentages, calculated from the extents of leakage after 5 min, were plotted as a function of MAb paratope:peptide mole ratio.
MAb4E10. Overall, the lipid composition had a less pronounced effect on MAb2F5 inhibitory activity. The previous results suggest that Chol and SPM can modulate membrane-inserted epitope binding, the effect being more drastic for the MAb4E10 than for the MAb2F5. Specific epitope recognition involvement in the blocking of AISpreTM-induced leakage in POPC:Chol was previously demonstrated using sequences with Ala substituting for crucial residues Asp9 and Lys10, or Trp17 and Phe18 [24,36]. The resulting AISpreTM(9,10)A and AISpreTM(17,18)A peptides functioned as specificity controls for Mab2F5 and Mab4E10, respectively. The data shown in Fig. 4 support that also blocking of the AISpreTM-induced leakage in POPC:SPM vesicles evolved as a consequence of specific epitope recognition. MAb effect on the kinetics of leakage induced by AISpreTM(9,10)A and AISpreTM(17,18)A peptides in POPC:SPM vesicles is displayed in panel A. MAb4E10 blocked AISpreTM(9,10)A-induced leakage, albeit inefficiently, but had almost no effect on AISpreTM(17,18)A-induced leakage (blue traces). Conversely, MAb2F5 arrested AISpreTM(17,18)A-induced leakage efficiently but had almost no effect on the process induced by the AISpreTM(9,10)A variant (red traces).
Panel B surveys results of specificity experiments that compare binding to these peptides inserted into POPC:SPM vesicles or directly bound to ELISA plates. MAb4E10 binding (blue bars) was specific for the AISpreTM(9,10)A sequence in POPC:SPM vesicles, following a pattern similar to that observed for the peptides adsorbed to ELISA plates. Conversely, Mab2F5 bound to AISpreTM(17,18)A, but not to AISpreTM(9,10)A, both in vesicles and ELISA plates. Thus, even if mechanistically different membrane perturbations are expected to be promoted by AISpreTM in Chol or SPM-containing membranes (see Refs. [18,27]), MAb2F5 and Mab4E10 were capable to specifically recognize their epitopes and block membrane destabilization in the presence of both lipids.
4. Discussion NMAb-s are potent tools in HIV-1 vaccine design [7]. It has been argued that NMAb affinity per se is not the single determinant of neutralization [7,17]. We have recently proposed that the inhibition of membrane-perturbing MPER activity might correlate with the biological potency of anti-gp41 2F5
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Fig. 4. Dependence of MAb-induced POPC:SPM vesicle leakage blocking on specific epitope recognition. (A) Blocking by MAb2F5 (red traces) and Mab4E10 (blue traces) of leakage induced by the addition of 0.2 lM AISpreTM(9,10)A (left panel) or AISpreTM(17,18)A (right panel) to POPC:SPM vesicles. Black trace corresponds to the control without MAb. MAbs were added at the concentration of 50 lg/ml. For better appreciation of the effect, only the kinetic trace section after Mab addition is shown. (B) Left panel: inhibition of leakage induced by 0.2 lM AISpreTM(9,10)A or AISpreTM(17,18)A in POPC:SPM vesicles. Right panel: binding measured by ELISA in plates with 1 lM AISpreTM(9,10)A or AISpreTM(17,18)A. MAb2F5 (red bars) and Mab4E10 (blue bars) were added at the concentrations of 50 and 1 lg/ml in the vesicle assay and ELISA, respectively.
and 4E10 antibodies [24]. In accordance with the epitopeextraction mechanism proposed by Sun et al. [17], Mab-s 2F5 and 4E10 blocked bilayer-destabilizing MPER activity in PC vesicles. The inclusion of SPM reduced significantly the apparent affinity of MAb4E10 for AISpreTM under conditions promoting pore-formation. Conversely, Chol induced shallower AISpreTM insertion into the bilayer and higher MAb4E10 affinity, thus supporting that recognition mainly evolves upon exposure of the 4E10 epitope at the level of the membrane interface. In contrast, Mab2F5 capacity for blocking MPER activity was less affected by the membrane composition, suggesting that 2F5 epitope accessibility was similar under all conditions. This differential lipid effect on membrane-bound epitope accessibility is likely to originate from MPER segmentation into two topologically distinct interfacial subdomains [14,17,23]. In one hand, 2F5 epitope 657EQELLELDKWASLW670 sequence (red cylinders in the models depicted in Fig. 5) appears to remain anchored to the water–membrane interface and more accessible for antibody binding even under conditions that promote lytic pore-formation. On the other hand, exposure of 4E10 epitope 672WFNITNWLW680 sequence (green cylinders in the models depicted in Fig. 5), predicted to insert more deeply into the membrane interface region, appears to be distinctly affected by Chol or SPM.
N. Huarte et al. / FEBS Letters 582 (2008) 3798–3804
Fig. 5. Model for MPER interaction with lipid bilayers containing Chol (a) or SPM (b). See text for details.
AISpreTM seems to insert less deeply into the lipid bilayer in the presence of Chol, which might increase 4E10 epitope accessibility for antibody binding (Fig. 5a). Supporting this explanation, the extended 4E10 epitope includes a helical turn involving L679 and W680 residues [35,37], which overlaps partially with the 679LWYIK683 CRAC motif [20,21,38]. Thus, it might well be that MPER interacting with Chol through the CRAC domain provides the optimal conditions for Mab4E10 binding. Furthermore, the MPER sequence positioned into the interface might exert its bilayer perturbing activity by disrupting interactions between lipid molecules and/or by inducing membrane deformation and thinning. Conversely, SPM has been described to promote lytic-pore formation [23]. It appears that accessibility to the MPER stretch spanned by the 4E10 epitope is restrained within the pores promoted by this lipid (Fig. 5b). These observations support the proposal that lipid-modulation of MPER interaction might partially explain the discrepancy in MAb4E10 antibody neutralization efficiency for the same virus, produced in 293T cells versus peripheral blood mononuclear cells [7,17]. Circulating blood lymphocyte lipid composition is comparable to that of the MT-4 T-cell line used by Bru¨gger et al. to analyze the HIV lipid constituents [19,39]. Virions produced in both types of cells are therefore predicted to bear a similar SPM/PC ratio. However, a significantly higher SPM/PC ratio of ca. 2 was determined for virions produced in MT-4 cells as compared to the value of 0.5 reported for HIV pseudotypes produced in 293T cells [40]. Thus a reduction in the SPM content with respect to PC could conceivably explain, at least in part, the higher neutralization efficiency of MAb4E10 for the same virus produced in 293T cells. Finally, the results described in this work might have a direct application to the development of immunogens combining MPER peptides with lipids. For economic, safety and repro-
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ducibility reasons, a useful formulation will be probably based on a minimal number of defined lipid species. Our data on MAb-induced MPER activity inhibition suggest that the efficient and simultaneous presentation of 2F5 and 4E10 epitopes in a membrane environment might be optimally attained in lipid bilayers combining PC and Chol. Acknowledgements: This study was supported by MCyT (BFU200506095/BMC) and University of the Basque Country (UPV 042.31013552/2001 and DIPE06/11).
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3803 [15] Schibli, D.J., Montelaro, R.C. and Vogel, H.J. (2001) The membrane-proximal tryptophan-rich region of the HIV glycoprotein, gp41, forms a well-defined helix in dodecylphosphocholine micelles. Biochemistry 40, 9570–9578. [16] Zhu, P. et al. (2006) Distribution and three-dimensional structure of AIDS virus envelope spikes. Nature 441, 847–852. [17] Sun, Z.Y. et al. (2008) HIV-1 broadly neutralizing antibody extracts its epitope from a kinked gp41 ectodomain region on the viral membrane. Immunity 28, 52–63. [18] Saez-Cirion, A., Nir, S., Lorizate, M., Agirre, A., Cruz, A., PerezGil, J. and Nieva, J.L. (2002) Sphingomyelin and cholesterol promote HIV-1 gp41 pretransmembrane sequence surface aggregation and membrane restructuring. J. Biol. Chem. 277, 21776– 21785. [19] Brugger, B., Glass, B., Haberkant, P., Leibrecht, I., Wieland, F.T. and Krausslich, H.G. (2006) The HIV lipidome: a raft with an unusual composition. Proc. Natl. Acad. Sci. USA 103, 2641–2646. [20] Epand, R.F., Sayer, B.G. and Epand, R.M. (2005) The tryptophan-rich region of HIV gp41 and the promotion of cholesterolrich domains. Biochemistry 44, 5525–5531. [21] Epand, R.F., Thomas, A., Brasseur, R., Vishwanathan, S.A., Hunter, E. and Epand, R.M. (2006) Juxtamembrane protein segments that contribute to recruitment of cholesterol into domains. Biochemistry 45, 6105–6114. [22] Vishwanathan, S.A. and Hunter, E. (2008) Importance of the membrane-perturbing properties of the membrane-proximal external region of human immunodeficiency virus type 1 gp41 to viral fusion. J. Virol. 82, 5118–5126. [23] Lorizate, M., Huarte, N., Saez-Cirion, A. and Nieva, J.L. (2008) Interfacial pre-transmembrane domains in viral proteins promoting membrane fusion and fission. Biochim. Biophys. Acta. 1178, 1624–1639. [24] Huarte, N., Lorizate, M., Maeso, R., Kunert, R., Arranz, R., Valpuesta, J.M. and Nieva, J.L. (2008) The broadly neutralizing anti-human immunodeficiency virus type 1 4E10 monoclonal antibody is better adapted to membrane-bound epitope recognition and blocking than 2F5. J. Virol. 82, 8986–8996. [25] Sanchez-Martinez, S., Lorizate, M., Katinger, H., Kunert, R. and Nieva, J.L. (2006) Membrane association and epitope recognition by HIV-1 neutralizing anti-gp41 2F5 and 4E10 antibodies. AIDS Res. Hum. Retroviruses 22, 998–1006. [26] Ofek, G., Tang, M., Sambor, A., Katinger, H., Mascola, J.R., Wyatt, R. and Kwong, P.D. (2004) Structure and mechanistic analysis of the anti-human immunodeficiency virus type 1 antibody 2F5 in complex with its gp41 epitope. J. Virol. 78, 10724–10737. [27] Lorizate, M., Cruz, A., Huarte, N., Kunert, R., Perez-Gil, J. and Nieva, J.L. (2006) Recognition and blocking of HIV-1 gp41 pretransmembrane sequence by monoclonal 4E10 antibody in a Raftlike membrane environment. J. Biol. Chem. 281, 39598–39606. [28] Alam, S.M. et al. (2007) The role of antibody polyspecificity and lipid reactivity in binding of broadly neutralizing anti-HIV-1 envelope human monoclonal antibodies 2F5 and 4E10 to glycoprotein 41 membrane proximal envelope epitopes. J. Immunol. 178, 4424–4435. [29] Coutant, J., Yu, H., Clement, M.J., Alfsen, A., Toma, F., Curmi, P.A. and Bomsel, M. (2008) Both lipid environment and pH are critical for determining physiological solution structure of 3-Dconserved epitopes of the HIV-1 gp41-MPER peptide P1. Faseb J., in press. [30] Kunert, R., Steinfellner, W., Purtscher, M., Assadian, A. and Katinger, H. (2000) Stable recombinant expression of the anti HIV-1 monoclonal antibody 2F5 after IgG3/IgG1 subclass switch in CHO cells. Biotechnol. Bioeng. 67, 97–103. [31] Hope, M.J., Bally, M.B., Webb, G. and Cullis, P.R. (1985) Production of large unilamellar vesicles by a rapid extrusion procedure. Characterization of size distribution, trapped volume and ability to maintain a membrane potential. Biochim. Biophys. Acta 812, 55–65. [32] Ellens, H., Bentz, J. and Szoka, F.C. (1985) H+- and Ca2+induced fusion and destabilization of liposomes. Biochemistry 24, 3099–3106. [33] Bolen, E.J. and Holloway, P.W. (1990) Quenching of tryptophan fluorescence by brominated phospholipid. Biochemistry 29, 9638– 9643.
3804 [34] Wimley, W.C. and White, S.H. (1996) Experimentally determined hydrophobicity scale for proteins at membrane interfaces. Nat. Struct. Biol. 3, 842–848. [35] Cardoso, R.M., Brunel, F.M., Ferguson, S., Zwick, M., Burton, D.R., Dawson, P.E. and Wilson, I.A. (2007) Structural basis of enhanced binding of extended and helically constrained peptide epitopes of the broadly neutralizing HIV-1 antibody 4E10. J. Mol. Biol. 365, 1533–1544. [36] Zwick, M.B., Jensen, R., Church, S., Wang, M., Stiegler, G., Kunert, R., Katinger, H. and Burton, D.R. (2005) Anti-human immunodeficiency virus type 1 (HIV-1) antibodies 2F5 and 4E10 require surprisingly few crucial residues in the membrane-proximal external region of glycoprotein gp41 to neutralize HIV-1. J. Virol. 79, 1252–1261. [37] Brunel, F.M., Zwick, M.B., Cardoso, R.M., Nelson, J.D., Wilson, I.A., Burton, D.R. and Dawson, P.E. (2006) Structure–
N. Huarte et al. / FEBS Letters 582 (2008) 3798–3804 function analysis of the epitope for 4E10, a broadly neutralizing human immunodeficiency virus type 1 antibody. J. Virol. 80, 1680–1687. [38] Vishwanathan, S.A., Thomas, A., Brasseur, R., Epand, R.F., Hunter, E. and Epand, R.M. (2008) Hydrophobic substitutions in the first residue of the CRAC segment of the gp41 protein of HIV. Biochemistry 47, 124–130. [39] Leidl, K., Liebisch, G., Richter, D. and Schmitz, G. (2008) Mass spectrometric analysis of lipid species of human circulating blood cells. Biochim. Biophys. Acta. 1781, 655– 664. [40] van Til, N.P., Heutinck, K.M., van der Rijt, R., Paulusma, C.C., van Wijland, M., Markusic, D.M., Elferink, R.P. and Seppen, J. (2008) Alteration of viral lipid composition by expression of the phospholipid floppase ABCB4 reduces HIV vector infectivity. Retrovirology 5, 14.
Lipid modulation of membrane-bound epitope recognition and blocking by HIV-1 neutralizing antibodies Nerea Huartea, Maier Lorizatea,1, Renate Kunertb, Jose´ L. Nievaa,* a
Biophysics Unit (CSIC-UPV/EHU) and Biochemistry and Molecular Biology Department, University of the Basque Country, P.O. Box 644, 48080 Bilbao, Spain b Institute of Applied Microbiology, University of Agriculture, A-1190 Vienna, Austria Received 21 August 2008; revised 29 September 2008; accepted 5 October 2008 Available online 16 October 2008 Edited by Jacomine Krijnse-Locker
Abstract The conserved, aromatic-rich membrane-proximal external region (MPER) of gp41 is functional in human immunodeficiency virus (HIV)-cell fusion by perturbing membrane integrity. Broadly-neutralizing 2F5 and 4E10 monoclonal antibodies (MAb-s) recognize amino- and carboxy-terminal epitope sequences within this domain, respectively. An MPER peptide overlapping 2F5 and 4E10 epitope sequences was capable of breaching the permeability barrier of lipid vesicles. Cholesterol and sphingomyelin raft-lipids, present at high quantities in the HIV-1 envelope, promoted exposure or occlusion of 4E10 epitope, respectively. Conversely, 2F5 epitope accessibility was affected to a lesser extent by these envelope lipids. These observations support the idea that MPER epitopes on membranes are segmented in terms of how they are affected by envelope lipids, which may have implications for MPER-based vaccine development. Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: HIV-1; HIV-1 gp41; HIV-1 neutralization; MAb2F5; MAb4E10; MPER; Cholesterol; Sphingomyelin
1. Introduction The potential to elicit broadly neutralizing antibodies (NAbs) is a long-pursued goal for an human immunodeficiency virus type-1 (HIV-1) vaccine [1]. Monoclonal antibodies (MAb-s) 2F5 and 4E10 are one of the few examples of identified NAb-s showing capacity to neutralize a broad spectrum of HIV-1 clades and primary isolates [2–7]. These NAb-s have evolved to recognize epitopes within the membrane-proximal * Corresponding author. Address: Biofisika Unitatea (CSIC-UPV/ EHU) and Biokimika Saila, Euskal Herriko Unibertsitatea, Posta Kutxa Box 644, 48080 Bilbao, Spain. Fax: +34 94 6013360. E-mail address: [email protected] (J.L. Nieva). 1
Present Address: Abteilung Virologie, Universita¨tsklinikum Heidelberg, im Neuenheimer Feld 324, D-69120 Heidelberg, Germany.
Abbreviations: Chol, cholesterol; CRAC, Cholesterol Recognition/ interaction Amino acid Consensus motif; DPC, dodecylphosphocholine; HIV-1, human immunodeficiency virus type-1; MAb, monoclonal antibody; MPER, membrane-proximal external region; NAb-s, neutralizing antibodies; POPC, phosphatidylcholine; PL, phospholipid; PreTM, pretransmembrane; SPM, sphingomyelin; WW, Wimley– White
external region (MPER), preceding the envelope gp41 subunitÕs transmembrane domain. Thus, MPER constitutes a candidate target vaccine for the development of antibodies that can neutralize the wide array of circulating HIV-1 isolates [8,9]. Based on a site-directed mutagenesis study, Salzwedel et al. [10] defined MPER as the aromatic-rich, conserved gp41sequence 665KWASLWNWFNITNWLWYIK683. This domain was primarily implicated in gp41-induced membrane fusion and envelope glycoprotein incorporation into virions [10,11]. The same gp41 sequence was designated as the pretransmembrane (PreTM) domain according to its tendency to stably associate with the membrane interface as an a-helix and promote lipid vesicle destabilization [12–14]. The interfacial location of the helical MPER/PreTM sequence was subsequently supported by structural studies [15–17]. Additionally, viral envelope raft-lipids such as cholesterol (Chol) and sphingomyelin (SPM) were shown to modulate MPER capacity to destabilize membranes [14,18,19]. Experimental evidence and modeling studies further supported MPER–Chol interactions through the carboxy-terminal ‘‘Cholesterol Recognition/interaction Amino acid Consensus’’ (CRAC) LWYIK motif [20,21]. Just recently, Vishwanathan and Hunter [22] replaced MPER with indolicidin-based sequences. Indolicidin is a Trprich, antimicrobial peptide that forms pores in membranes. Some of the produced gp41 chimeras retained fusion activity, suggesting that membrane-perturbing properties are key to MPER membrane fusion function [22]. Thus, the experimental evidence produced in cells and model systems supports MPER function in fusion (recently reviewed in [23]). Attending to this evidence it has been hypothesized that NMAb-s 2F5 and 4E10 might neutralize the virus by blocking membrane-perturbing MPER activity [17,24]. In support of this hypothesis MAb-s 2F5 and 4E10 are adapted for recognition of membrane embedded MPER sequences [17,24–29]. Recent experiments described by Sun et al. [17] demonstrate that MAb4E10 binds to membrane-embedded MPER and induces its partial extraction. These authors postulate that the solved structure of MPER inserted into dodecylphosphocholine (DPC) micelles represents a minimal native-like gp41 structure with the potential of eliciting NAb-s. Accordingly, it has been suggested that MPER-derived peptides in a lipid environment might constitute useful platforms for purposes of vaccine design [17,24,29]. Here, in search for a minimal lipid composition allowing simultaneous and effective presentation of 2F5 and 4E10 epitopes at the membrane interface, we have comparatively
0014-5793/$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.10.012
N. Huarte et al. / FEBS Letters 582 (2008) 3798–3804
analyzed the effects of SPM and Chol, the main raft-lipids existing in the viral envelope [19]. Effective binding has been derived from the antibody-induced inhibition of the MPER membrane-perturbing activity measured in solution-diffusing lipid vesicles [24]. Since the MPER native-like structure relevant for antibody recognition has been solved in contact with a phosphocholine-based lipid [17], the vesicles used in this study as a reference were made of pure phosphatidylcholine (POPC). Our results suggest that the lipid environment affects predominantly 4E10 epitope accessibility. This observation might guide the development of immunogens combining MPER with a minimal amount of chemically defined lipids.
2. Materials and methods NEQELLELDKWASLWNWFNITNWLWYIK (AISpreTM), NEQELLELAAWASLWNWFNITNWLWYIK (AISpreTM(9,10)A) and NEQELLELDKWASLWNAANITNWLWYIK (AISpreTM(17,18)A) peptides were synthesized by solid-phase synthesis using Fmoc chemistry, as C-terminal carboxamides and purified by HPLC at the Proteomics Unit of the University Pompeu-Fabra (Barcelona, Spain). Peptide stock solutions were prepared in dimethylsulfoxide (DMSO) (spectroscopy grade) and concentrations determined by Bicinchoninic Acid microassay (Pierce, Rockford, IL, USA). NAb expressing hybridomas were originally generated by a combined PEG-electrofusion of peripheral blood mononuclear cells of HIV infected non-symptomatic patients [2]. MAb-s 2F5 and 4E10 used in this study were subsequently produced in recombinant CHO cells after subclass switch to IgG1 [30]. 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC), SPM, 1-palmitoyl2-stearoyl(6,7)dibromo-sn-glycero-3-phosphocholine (Br6-PSPC) and 1-palmitoyl-2-stearoyl(11,12)dibromo-sn-glycero-3-phosphocholine (Br11PSPC) were purchased from Avanti Polar Lipids (Birmingham, AL, USA). 8-Aminonaphtalene-1,3,6-trisulfonic acid sodium salt (ANTS) and p-xylenebis(pyridinium)bromide (DPX) were from Molecular Probes (Junction City, OR, USA). Large unilamellar vesicles (LUV) were prepared following the extrusion method of Hope et al. [31] in 5 mM Hepes, 100 mM NaCl (pH 7.4) buffer and using polycarbonate membranes (Nuclepore Inc., Pleasanton, CA, USA) with a 0.1 lm nominal pore size. Lipid concentrations of liposome suspensions were determined by phosphate analysis. Release of encapsulated vesicular contents to the medium was fluorimetrically monitored by the ANTS/DPX assay [32] as previously described [27]. Kinetics of membrane-insertion were measured by energy transfer from peptide-Trp to the surface fluorescent probe dDHPE as described [24]. Penetration level of the peptide into the bilayer core was inferred from the Trp fluorescence quenching by the hydrophobic matrix-residing brominated phospholipids (PLs), according to the method described by Bolen and Holloway [33]. These experiments were carried out using sonicated vesicles containing a POPC fraction substituted for the brominated PLs Br6-PSPC or Br11-PSPC. Circular dichroism (CD) measurements were obtained from a thermally-controlled Jasco J-810 CD spectropolarimeter calibrated routinely with (1S)-(+)-10-camphorsulfonic acid, ammonium salt. Samples consisted of co-lyophilized peptide and lipid dissolved and sonicated in 2 mM Hepes (pH, 7.4) buffer. Spectra were measured in a 1 mm path-length quartz cell initially equilibrated at 25 °C. Data were taken with a 1 nm band-width at 100 nm/min speed, and the results of 20 scans were averaged. For the enzyme-linked immunosorbent assay (ELISA), peptides were dissolved in phosphate-buffered saline (PBS) and immobilized (100 ml/well) overnight in C96 Maxisorp microplate wells (Nunc, Denmark) at a concentration of 1 lM. Prior to incubation with the MAbs, the plates were blocked for 2 h with 3% (w/v) bovine-serum albumin in PBS. The binding of the Mabs was detected with an alkaline phosphatase-conjugated goat anti-human immunoglobulin (Pierce, Rockford, IL, USA), which then catalyzed a color reaction with the p-nitrophenyl phosphate substrate (Sigma, St. Louis, MO, USA) that could be measured by absorbance at a wavelength of 405 nm in a Synergy HT microplate reader (Bio-TEK Instruments Inc., VT, USA).
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3. Results 3.1. Lipid effects on AISpreTM-induced leakage Predictive tools based on the use of Wimley–White (WW) water-to-membrane interface transfer free energies [34] and recent structural characterization suggest that membrane-bound MPER/PreTM is segmented into two subdomains [14,17]. The N-terminal domain comprises a helical, conserved amphipathic-at-interface sequence (AIS). AIS is followed by a fully hydrophobic-at-interface stretch that precedes the transmembrane anchor. The AISpreTM peptide used in this study was designed to contain both interfacial subdomains ( Fig. 1A). In addition, AISpreTM encompasses the full-length 2F5 and 4E10 epitopes [26,35]. Consistent with its predicted role in fusion, this MPER peptide was capable of breaching the membrane permeability barrier of lipid vesicles (Fig. 1B). In a previous contribution we showed that the lytic activity of HIVc, a shorter MPER peptide devoid of NEQELLEL AIS residues, was dependent on the presence of SPM [18]. Thus, low amounts of peptide were required to permeabilize POPC:SPM (1:1 mol:mol) vesicles (in the order of 1:1000– 1:10000 peptide-to-lipid ratios) and the process could be modeled according to a pore-formation phenomenon. Relatively much higher doses were required to permeabilize POPC or POPC:Chol (2:1 mol:mol) vesicles (in the order of 1:10–1:200 peptide-to-lipid ratios) and kinetic modeling ruled out poreformation as the permeabilization mechanism in those systems. AISpreTM lytic activity was also stimulated upon addition of SPM to the liposome composition (Fig. 1B). Peptide-tolipid mole ratios in the order of 1:1000 were required to totally
Fig. 1. Definition and membrane-perturbing properties of AISpreTM peptide. (A) Schematic diagram to designate gp41 regions: FP, fusion peptide; NHR and CHR, amino- and carboxy-terminal helical regions, respectively; MPER, membrane-proximal external region; TMD transmembrane domain. AISpreTM (below) spans the 656–683 region that contains 2F5 and 4E10 epitopes. Boxes designate crucial residues according to Zwick et al. [36] mutated by Ala in AISpreTM(9,10)A and AISpreTM(17,18)A peptides. (B) Percentage of AISpreTMinduced leakage after 30 min of incubation with vesicles, plotted as a function of the peptide-to-lipid mole ratio (Ri). Filled circles and continuous line: POPC:SPM (1:1 mol:mol); filled squares and dotted line: POPC; and empty circles and slashed line: POPC:Chol(2:1 mol:mol). Arrows indicate the conditions selected to measure MAbinduced blocking of leakage. Final lipid concentration was 0.1 mM in all cases.
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permeabilize POPC:SPM (1:1 mole ratio) LUV. By comparison ca. 10 times higher peptide doses were required to permeabilize pure POPC or POPC:Chol (2:1 mole ratio) LUV. This observation confirmed previous results indicating that PreTM/MPER activity was preserved in the longer AISpreTM peptide [24]. The CD results displayed in Fig. 2A revealed similar secondary structures for AISpreTM in contact with POPC, POPC:SPM (1:1) and POPC:Chol (2.1) vesicles. The CD spectra obtained in the three systems displayed major band components at 208 and 222 nm supporting that the helical conformation was the main structure adopted by the peptide. Thus, the adoption of a distinct lytic secondary structure by the peptide did not provide an explanation for the leakage enhancement observed in the presence of SPM (Fig. 1). The intrinsic fluorescence results below ( Fig. 2B) suggested that AISpreTM may insert deeper into POPC and POPC:SPM (1:1) than into POPC:Chol (2:1) bilayers. In the case of POPC:SPM and POPC vesicles AISpreTM-Trp-s were quenched to similar extents by Br6-PSPC and Br11-PSPC (left and center panels). In contrast, Br6-PSPC quenched AISpreTM-Trp emission more efficiently than Br11-PSPC (right panel). Bromine atoms quench by a short range process that requires a close approach to the fluorophore [33]. Thus, AISpreTM Trp residues located closer to the C6–C7 than to the C11–C12 position within the acyl chain region of the bilayers containing Chol. In conclusion, Chol seemed to induce a shallower peptide insertion into the lipid bilayer, but did not appreciably affect its overall secondary structure.
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3.2. Lipid effects on specific recognition and blocking of AISpreTM AISpreTM-induced vesicle permeabilization was next used to assess the influence of SPM and Chol on antibody-induced inhibition of MPER membrane activity (Fig. 3). Panel 3A illustrates the effect of MAb addition to the ongoing AISpreTMinduced leakage in vesicles of different lipid composition. In all cases, kinetic traces of d-DHPE fluorescence increase demonstrated faster peptide incorporation into vesicles (dotted traces). AISpreTM was added to a stirred solution of lipid vesicles (1), left sufficient time for incorporation into them, and MAb was subsequently injected into the mixture (2). Consistent with recognition of membrane-bound species under these conditions, addition of MAb-s 4E10 and 2F5 arrested the leakage process (top and bottom panels, respectively). However, as compared to that induced by MAb2F5, MAb4E10 addition induced a weaker inhibition of leakage from POPC:SPM vesicles (left panels). The dose dependence of leakage inhibition illustrates further this effect (panel B). The inhibition percentages were calculated from the leakage extents measured after 5 min of incubation, and using as 0% inhibition reference vesicle samples untreated with MAb. Relatively lower doses of MAb4E10 than of MAb2F5 were required to effectively arrest AISpreTM-induced leakage in POPC and POPC:Chol vesicles. Moreover, Chol promoted the inhibitory capacity of MAb4E10. In contrast, the capacity of MAb4E10 to block AISpreTM was impaired in POPC:SPM vesicles. In this system Mab2F5 actually inhibited AISpreTM activity more efficiently than
Fig. 2. Interaction of AISpreTM with POPC:SPM (1:1 mole ratio), POPC and POPC:Chol (2:1 mole ratio) lipid vesicles. (A) CD spectra of AISpreTM peptide associated to the vesicles (lipid concentration, 1 mM). The concentration of AISpreTM peptide was 30 lM. (B) Intrinsic fluorescence emission spectra of AISpreTM incubated with the same type of vesicles (traces on top labeled with symbols). Continuous and dotted traces below correspond to spectra obtained in the presence of vesicles in which a fraction of the POPC was substituted for Br6-PSPOPC or Br11PSPOPC, respectively. The final brominated lipid content in all vesicles was 25 mol%. Vesicle lipid concentration was 0.1 mM and the peptide-tolipid mole ratios were 1:500 (POPC:SPM) and 1:100 (POPC and POPC:Chol).
N. Huarte et al. / FEBS Letters 582 (2008) 3798–3804
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Figure 3. Inhibition of AISpreTM-induced vesicle contents leakage by MAb-s 2F5 and 4E10. (A) Effect of Mab addition to the ongoing leakage. Vesicle samples (100 lM lipid) were treated with 0.2 (POPC:SPM) or 1 lM (POPC and POPC:Chol) AISPreTM and, subsequently supplemented with 50 lg/ml of MAb-s 2F5 or 4E10 (addition times indicated by ‘‘1’’ and ‘‘2’’, respectively). The dotted traces follow AISpreTM incorporation into the vesicles monitored through energy transfer from peptide tryptophans to membrane-residing d-DHPE. Addition of the antibody results in a sudden decrease of the leakage rate. The traces labeled with (+) and (–) correspond to the leakage kinetics in the presence and the absence of antibody, respectively. (B) Dose dependency of AISpreTM-induced leakage inhibition by MAb4E10 or MAb2F5 in POPC:SPM (filled circles and continuous lines) POPC (squares and dotted lines) and POPC:Chol (hollow circles and slashed lines) vesicles. Inhibition percentages, calculated from the extents of leakage after 5 min, were plotted as a function of MAb paratope:peptide mole ratio.
MAb4E10. Overall, the lipid composition had a less pronounced effect on MAb2F5 inhibitory activity. The previous results suggest that Chol and SPM can modulate membrane-inserted epitope binding, the effect being more drastic for the MAb4E10 than for the MAb2F5. Specific epitope recognition involvement in the blocking of AISpreTM-induced leakage in POPC:Chol was previously demonstrated using sequences with Ala substituting for crucial residues Asp9 and Lys10, or Trp17 and Phe18 [24,36]. The resulting AISpreTM(9,10)A and AISpreTM(17,18)A peptides functioned as specificity controls for Mab2F5 and Mab4E10, respectively. The data shown in Fig. 4 support that also blocking of the AISpreTM-induced leakage in POPC:SPM vesicles evolved as a consequence of specific epitope recognition. MAb effect on the kinetics of leakage induced by AISpreTM(9,10)A and AISpreTM(17,18)A peptides in POPC:SPM vesicles is displayed in panel A. MAb4E10 blocked AISpreTM(9,10)A-induced leakage, albeit inefficiently, but had almost no effect on AISpreTM(17,18)A-induced leakage (blue traces). Conversely, MAb2F5 arrested AISpreTM(17,18)A-induced leakage efficiently but had almost no effect on the process induced by the AISpreTM(9,10)A variant (red traces).
Panel B surveys results of specificity experiments that compare binding to these peptides inserted into POPC:SPM vesicles or directly bound to ELISA plates. MAb4E10 binding (blue bars) was specific for the AISpreTM(9,10)A sequence in POPC:SPM vesicles, following a pattern similar to that observed for the peptides adsorbed to ELISA plates. Conversely, Mab2F5 bound to AISpreTM(17,18)A, but not to AISpreTM(9,10)A, both in vesicles and ELISA plates. Thus, even if mechanistically different membrane perturbations are expected to be promoted by AISpreTM in Chol or SPM-containing membranes (see Refs. [18,27]), MAb2F5 and Mab4E10 were capable to specifically recognize their epitopes and block membrane destabilization in the presence of both lipids.
4. Discussion NMAb-s are potent tools in HIV-1 vaccine design [7]. It has been argued that NMAb affinity per se is not the single determinant of neutralization [7,17]. We have recently proposed that the inhibition of membrane-perturbing MPER activity might correlate with the biological potency of anti-gp41 2F5
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Fig. 4. Dependence of MAb-induced POPC:SPM vesicle leakage blocking on specific epitope recognition. (A) Blocking by MAb2F5 (red traces) and Mab4E10 (blue traces) of leakage induced by the addition of 0.2 lM AISpreTM(9,10)A (left panel) or AISpreTM(17,18)A (right panel) to POPC:SPM vesicles. Black trace corresponds to the control without MAb. MAbs were added at the concentration of 50 lg/ml. For better appreciation of the effect, only the kinetic trace section after Mab addition is shown. (B) Left panel: inhibition of leakage induced by 0.2 lM AISpreTM(9,10)A or AISpreTM(17,18)A in POPC:SPM vesicles. Right panel: binding measured by ELISA in plates with 1 lM AISpreTM(9,10)A or AISpreTM(17,18)A. MAb2F5 (red bars) and Mab4E10 (blue bars) were added at the concentrations of 50 and 1 lg/ml in the vesicle assay and ELISA, respectively.
and 4E10 antibodies [24]. In accordance with the epitopeextraction mechanism proposed by Sun et al. [17], Mab-s 2F5 and 4E10 blocked bilayer-destabilizing MPER activity in PC vesicles. The inclusion of SPM reduced significantly the apparent affinity of MAb4E10 for AISpreTM under conditions promoting pore-formation. Conversely, Chol induced shallower AISpreTM insertion into the bilayer and higher MAb4E10 affinity, thus supporting that recognition mainly evolves upon exposure of the 4E10 epitope at the level of the membrane interface. In contrast, Mab2F5 capacity for blocking MPER activity was less affected by the membrane composition, suggesting that 2F5 epitope accessibility was similar under all conditions. This differential lipid effect on membrane-bound epitope accessibility is likely to originate from MPER segmentation into two topologically distinct interfacial subdomains [14,17,23]. In one hand, 2F5 epitope 657EQELLELDKWASLW670 sequence (red cylinders in the models depicted in Fig. 5) appears to remain anchored to the water–membrane interface and more accessible for antibody binding even under conditions that promote lytic pore-formation. On the other hand, exposure of 4E10 epitope 672WFNITNWLW680 sequence (green cylinders in the models depicted in Fig. 5), predicted to insert more deeply into the membrane interface region, appears to be distinctly affected by Chol or SPM.
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Fig. 5. Model for MPER interaction with lipid bilayers containing Chol (a) or SPM (b). See text for details.
AISpreTM seems to insert less deeply into the lipid bilayer in the presence of Chol, which might increase 4E10 epitope accessibility for antibody binding (Fig. 5a). Supporting this explanation, the extended 4E10 epitope includes a helical turn involving L679 and W680 residues [35,37], which overlaps partially with the 679LWYIK683 CRAC motif [20,21,38]. Thus, it might well be that MPER interacting with Chol through the CRAC domain provides the optimal conditions for Mab4E10 binding. Furthermore, the MPER sequence positioned into the interface might exert its bilayer perturbing activity by disrupting interactions between lipid molecules and/or by inducing membrane deformation and thinning. Conversely, SPM has been described to promote lytic-pore formation [23]. It appears that accessibility to the MPER stretch spanned by the 4E10 epitope is restrained within the pores promoted by this lipid (Fig. 5b). These observations support the proposal that lipid-modulation of MPER interaction might partially explain the discrepancy in MAb4E10 antibody neutralization efficiency for the same virus, produced in 293T cells versus peripheral blood mononuclear cells [7,17]. Circulating blood lymphocyte lipid composition is comparable to that of the MT-4 T-cell line used by Bru¨gger et al. to analyze the HIV lipid constituents [19,39]. Virions produced in both types of cells are therefore predicted to bear a similar SPM/PC ratio. However, a significantly higher SPM/PC ratio of ca. 2 was determined for virions produced in MT-4 cells as compared to the value of 0.5 reported for HIV pseudotypes produced in 293T cells [40]. Thus a reduction in the SPM content with respect to PC could conceivably explain, at least in part, the higher neutralization efficiency of MAb4E10 for the same virus produced in 293T cells. Finally, the results described in this work might have a direct application to the development of immunogens combining MPER peptides with lipids. For economic, safety and repro-
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ducibility reasons, a useful formulation will be probably based on a minimal number of defined lipid species. Our data on MAb-induced MPER activity inhibition suggest that the efficient and simultaneous presentation of 2F5 and 4E10 epitopes in a membrane environment might be optimally attained in lipid bilayers combining PC and Chol. Acknowledgements: This study was supported by MCyT (BFU200506095/BMC) and University of the Basque Country (UPV 042.31013552/2001 and DIPE06/11).
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