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W2073983206 abstract "The conserved 664DKWASLWNWFNITNWLWYIK683 (preTM) sequence preceding the transmembrane anchor of human immunodeficiency virus (HIV-1) gp41 glycoprotein subunit is accessible to the broadly neutralizing 4E10 antibody and, therefore, constitutes a potential target for vaccine design. Recently reported structural data are compatible with preTM insertion into the viral external membrane monolayer in the gp41 pre-fusion state (Zhu, P., Liu, J., Bess, J., Chertova, E., Lifson, J. D., Grisé, H., Ofek, G. A., Taylor, K. A., and Roux, K. H. (2006) Nature 441, 847-852). Here we demonstrate that the broadly neutralizing 4E10 antibody is able to specifically block the membrane-restructuring activity of a peptide mimic inserted into membranes. Recognition and restructuring blocking occurred in the presence of cholesterol, whereas transmembrane versions as those promoted in 1-palmitoyl-2-oleoylphosphatidylcholine:sphingomyelin mixtures could not be effectively arrested. Spectrofluorimetric assays using rhodamine-labeled peptides revealed that recognition correlated better with pore-formation blocking than with membrane-fusion inhibition. The capacity of the antibody to recognize preTM peptides in a raft-like environment was further corroborated employing planar-supported lipid layers and fluorescence microscopy. These data support that membrane-bound epitope recognition by 4E10 results in clustering reorganization of preTM at the membrane interface. We propose that this process might interfere with the formation of fusion-competent complexes at the low spike densities existing in the HIV-1 membrane. This work comprises the first experimental report on a lipid-modulated antibody capacity to bind a membrane-bound epitope sequence and arrest its restructuring activity. The conserved 664DKWASLWNWFNITNWLWYIK683 (preTM) sequence preceding the transmembrane anchor of human immunodeficiency virus (HIV-1) gp41 glycoprotein subunit is accessible to the broadly neutralizing 4E10 antibody and, therefore, constitutes a potential target for vaccine design. Recently reported structural data are compatible with preTM insertion into the viral external membrane monolayer in the gp41 pre-fusion state (Zhu, P., Liu, J., Bess, J., Chertova, E., Lifson, J. D., Grisé, H., Ofek, G. A., Taylor, K. A., and Roux, K. H. (2006) Nature 441, 847-852). Here we demonstrate that the broadly neutralizing 4E10 antibody is able to specifically block the membrane-restructuring activity of a peptide mimic inserted into membranes. Recognition and restructuring blocking occurred in the presence of cholesterol, whereas transmembrane versions as those promoted in 1-palmitoyl-2-oleoylphosphatidylcholine:sphingomyelin mixtures could not be effectively arrested. Spectrofluorimetric assays using rhodamine-labeled peptides revealed that recognition correlated better with pore-formation blocking than with membrane-fusion inhibition. The capacity of the antibody to recognize preTM peptides in a raft-like environment was further corroborated employing planar-supported lipid layers and fluorescence microscopy. These data support that membrane-bound epitope recognition by 4E10 results in clustering reorganization of preTM at the membrane interface. We propose that this process might interfere with the formation of fusion-competent complexes at the low spike densities existing in the HIV-1 membrane. This work comprises the first experimental report on a lipid-modulated antibody capacity to bind a membrane-bound epitope sequence and arrest its restructuring activity. Only three broadly neutralizing anti-HIV-1 4The abbreviations used are: HIV-1, human immunodeficiency virus type-1; Chol, cholesterol; mAb, monoclonal antibody; MBCD, methyl-β-cyclodextrin; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; preTM, pre-transmembrane; SPM, sphingomyelin; LUV, large unilamellar vesicles; NBD, 7-nitro-benz-2-oxa-1,3-diazol-4-yl; ANTS, 8-aminonaphtalene-1,3,6-trisulfonic acid sodium salt; DPX, p-xylenebis(pyridinium). 4The abbreviations used are: HIV-1, human immunodeficiency virus type-1; Chol, cholesterol; mAb, monoclonal antibody; MBCD, methyl-β-cyclodextrin; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; preTM, pre-transmembrane; SPM, sphingomyelin; LUV, large unilamellar vesicles; NBD, 7-nitro-benz-2-oxa-1,3-diazol-4-yl; ANTS, 8-aminonaphtalene-1,3,6-trisulfonic acid sodium salt; DPX, p-xylenebis(pyridinium). monoclonal antibodies (mAbs), 2F5, 4E10, and Z13, have been identified to react with the membrane-integral gp41 Env subunit that promotes viral-cell fusion (for a recent review, see Ref. 1Zwick M.B. AIDS. 2005; 19: 1725-1737Crossref PubMed Scopus (82) Google Scholar). These three antibodies recognize epitopes within the conserved aromatic-rich domain that precede the gp41 transmembrane anchor (2Suárez T. Gallaher W.R. Agirre A. Goñ F.M. Nieva J.L. J. Virol. 2000; 74: 8038-8047Crossref PubMed Scopus (153) Google Scholar, 3Sáez-Cirión A. Nir S. Lorizate M. Agirre A. Cruz A. Pérez-Gil J. Nieva J.L. J. Biol. Chem. 2002; 277: 21776-21785Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 4Sáez-Cirión A. Arrondo J.L.R. Gómara M.J. Lorizate M. Lloro I. Melikyan G. Nieva J.L Biophys. J. 2003; 85: 3769-3780Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 5Salzwedel K. West J. Hunter E. J. Virol. 1999; 73: 2469-2480Crossref PubMed Google Scholar, 6Muñoz-Barroso I. Salzwedel K. Hunter E. Blumenthal R. J. Virol. 1999; 73: 6089-6092Crossref PubMed Google Scholar, 7Zwick M.B. Labrijn A.F. Wang M. Spenlehauer C. Ollmann E. Binley J.M. Moore J.P. Stiegler C. Katinger H. Burton D.R. Parren P.W.H.I. J. Virol. 2001; 75: 10892-10905Crossref PubMed Scopus (698) Google Scholar). mAb4E10 bears the broadest activity, showing the capacity of neutralizing all tested M group clades and newly transmitted virus isolated from infected patients (8Stiegler G. Kunert R. Purtscher M. Wolbank S. Voglauer R. Steindl F. Katinger H. AIDS Res. Hum. Retroviruses. 2001; 17: 1757-1765Crossref PubMed Scopus (443) Google Scholar, 9Kunert R. Wolbank S. Stiegler G. Weik R. Katinger H. AIDS Res. Hum. Retroviruses. 2004; 20: 755-762Crossref PubMed Scopus (60) Google Scholar, 10Binley J.M. Wrin T. Korber B. Zwick M.B. Wang M. Chappey C. Stiegler G. Kunert R. Zolla-Pazner S. Katinger H. Petropoulos C.J. Burton D.R. J. Virol. 2004; 78: 13232-13252Crossref PubMed Scopus (634) Google Scholar, 11Mehandru S. Wrin T. Galovich J. Stiegler G. Vcelar B. Hurley A. Hogan C. Vasan S. Katinger H. Petropoulos C.J. Markowitz M. J. Virol. 2004; 78: 14039-14042Crossref PubMed Scopus (71) Google Scholar). Thus, there exists a considerable interest in unraveling the mechanistic basis of the broad neutralizing activity of mAb4E10. The epitope core 671NWF(D/N)IT676 recognized by mAb4E10 locates at the center of the gp41 664DKWASLWNWFNITNWLWYIK683 sequence. This sequence has been defined as a distinct domain according to its interfacial hydrophobicity (designated as pre-transmembrane (preTM)) (2Suárez T. Gallaher W.R. Agirre A. Goñ F.M. Nieva J.L. J. Virol. 2000; 74: 8038-8047Crossref PubMed Scopus (153) Google Scholar, 3Sáez-Cirión A. Nir S. Lorizate M. Agirre A. Cruz A. Pérez-Gil J. Nieva J.L. J. Biol. Chem. 2002; 277: 21776-21785Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 4Sáez-Cirión A. Arrondo J.L.R. Gómara M.J. Lorizate M. Lloro I. Melikyan G. Nieva J.L Biophys. J. 2003; 85: 3769-3780Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) and also attending to its implication in fusion and neutralization processes (designated as a membrane-proximal external region) (1Zwick M.B. AIDS. 2005; 19: 1725-1737Crossref PubMed Scopus (82) Google Scholar, 5Salzwedel K. West J. Hunter E. J. Virol. 1999; 73: 2469-2480Crossref PubMed Google Scholar, 6Muñoz-Barroso I. Salzwedel K. Hunter E. Blumenthal R. J. Virol. 1999; 73: 6089-6092Crossref PubMed Google Scholar, 7Zwick M.B. Labrijn A.F. Wang M. Spenlehauer C. Ollmann E. Binley J.M. Moore J.P. Stiegler C. Katinger H. Burton D.R. Parren P.W.H.I. J. Virol. 2001; 75: 10892-10905Crossref PubMed Scopus (698) Google Scholar). Pioneering mutational analyses by Salzwedel et al. (5Salzwedel K. West J. Hunter E. J. Virol. 1999; 73: 2469-2480Crossref PubMed Google Scholar, 6Muñoz-Barroso I. Salzwedel K. Hunter E. Blumenthal R. J. Virol. 1999; 73: 6089-6092Crossref PubMed Google Scholar) and biophysical determinations by our group (2Suárez T. Gallaher W.R. Agirre A. Goñ F.M. Nieva J.L. J. Virol. 2000; 74: 8038-8047Crossref PubMed Scopus (153) Google Scholar, 3Sáez-Cirión A. Nir S. Lorizate M. Agirre A. Cruz A. Pérez-Gil J. Nieva J.L. J. Biol. Chem. 2002; 277: 21776-21785Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 4Sáez-Cirión A. Arrondo J.L.R. Gómara M.J. Lorizate M. Lloro I. Melikyan G. Nieva J.L Biophys. J. 2003; 85: 3769-3780Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) suggested that preTM is instrumental for successful gp41-mediated fusion. Specifically, this domain would be involved in the expansion of the fusion pores (6Muñoz-Barroso I. Salzwedel K. Hunter E. Blumenthal R. J. Virol. 1999; 73: 6089-6092Crossref PubMed Google Scholar, 12Gallo S.A. Finnegan C.M. Viard M. Raviv Y. Dimitrov A. Rawat S.S. Puri A. Durell S. Blumenthal R. Biochim. Biophys. Acta. 2003; 1614: 36-50Crossref PubMed Scopus (337) Google Scholar). However, the structural grounds sustaining this activity are poorly understood due in part to the fact that a gp41 ectodomain atomic structure including the membrane-proximal region is not available at present. Our analyses predicted a membrane interfacial location of the sequence as a monomer (2Suárez T. Gallaher W.R. Agirre A. Goñ F.M. Nieva J.L. J. Virol. 2000; 74: 8038-8047Crossref PubMed Scopus (153) Google Scholar, 4Sáez-Cirión A. Arrondo J.L.R. Gómara M.J. Lorizate M. Lloro I. Melikyan G. Nieva J.L Biophys. J. 2003; 85: 3769-3780Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). NMR studies by Schibli et al. (13Schibli D.J. Montelaro R.C. Vogel H.J. Biochemistry. 2001; 40: 9570-9578Crossref PubMed Scopus (159) Google Scholar) further demonstrated that the preTM peptide adopts a well defined helical conformation in the membrane-like environment provided by dodecylphosphocholine micelles. In addition, nuclear Overhauser effects between side chains and polar head groups suggested that aromatic residues would position within the interfacial bilayer region (Fig. 1B). Infrared (IR) spectroscopy and fluorescence measurements were compatible with the existence of helical monomers or oligomers in phospholipid bilayers depending on peptide load and lipid composition (3Sáez-Cirión A. Nir S. Lorizate M. Agirre A. Cruz A. Pérez-Gil J. Nieva J.L. J. Biol. Chem. 2002; 277: 21776-21785Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 4Sáez-Cirión A. Arrondo J.L.R. Gómara M.J. Lorizate M. Lloro I. Melikyan G. Nieva J.L Biophys. J. 2003; 85: 3769-3780Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Subsequent x-ray diffraction data obtained by Cardoso et al. (14Cardoso R.M. Zwick M.B. Stanfield R.L. Kunert R. Binley J.M. Katinger H. Burton D.R. Wilson I.A. Immunity. 2005; 22: 163-173Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar) have indeed revealed a helical structure for a 13-residue preTM derivative in complex with Fab4E10. Finally, preTM appears to bind directly cholesterol (Chol) through its C-terminal cholesterol recognition/interaction amino acid consensus LWYIK sequence (15Epand R.F. Sayer B.G. Epand R.M. Biochemistry. 2005; 44: 5525-5531Crossref PubMed Scopus (38) Google Scholar), a phenomenon that might condition gp41 fusogenic function (16Epand R.F. Thomas A. Brasseur R. Vishwanathan S.A. Hunter E. Epand R.M. Biochemistry. 2006; 45: 6105-6114Crossref PubMed Scopus (99) Google Scholar). The physiological relevance of the predicted preTM interfacial location has recently received support from the cryoelectron microscopy tomography study by Roux and co-workers (17Zhu P. Liu J. Bess J. Chertova E. Lifson J.D. Grisé H. Ofek G.A. Taylor K.A. Roux K.H. Nature. 2006; 441: 847-852Crossref PubMed Scopus (588) Google Scholar). This study has revealed that gp41 stalk regions project as “legs” from the trimeric Env complex in intact virions, with their “feet” just above the plane of the envelope (tripod-like structure). Docking of the Fab-bound atomic structures into the surface model suggests that Env native preTM would actually locate at the viral surface, inserted as a monomer in parallel to the external membrane monolayer. Thus, it is inferred that mAb4E10 bears the capacity to recognize native-like epitope sequences associated to the viral membrane interface. In this work we test this prediction by assessing the capacity of mAb4E10 to recognize and block preTM peptides inserted into lipid vesicles and monolayers. Specifically, we have compared the effects on recognition of sphingomyelin and cholesterol, the two raft-lipids postulated to be predominant at the virion external monolayer (18Aloia R.C. Jensen F.C. Curtain C.C. Mobley P.W. Gordon L.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 900-904Crossref PubMed Scopus (187) Google Scholar, 19Aloia R.C. Tian H.R. Jensen F.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5181-5185Crossref PubMed Scopus (371) Google Scholar, 20Brüger B. Glass B. Haberkant P. Leibrecht Wieland F.T. Kräus-slich H.-G. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2641-2646Crossref PubMed Scopus (545) Google Scholar). Our studies support the notion that 4E10 paratope is structurally designed to allow recognition of preTM species stabilized by cholesterol at the virion external membrane-interface. Moreover, we propose that recognition might render cross-linked spikes incompetent for fusion. These observations might be important for the design of immunogens aimed at recovering 4E10-like protective responses. Materials—The sequences DKWASLWNWFNITNWLWYIK (preTM), representing the pre-transmembrane stretch of HIV-1 (BH10 isolate) gp41, and its fluorescent derivative (Rho-preTM), labeled with rhodamine at its N terminus, were synthesized by solid-phase synthesis using Fmoc chemistry as its C-terminal carboxamide and purified by high performance liquid chromatography at the Proteomics Unit of the University Pompeu-Fabra (Barcelona, Spain). Peptide stock solutions were prepared in dimethyl sulfoxide (Me2SO) (spectroscopy grade), and concentrations were determined by bicinchoninic acid microassay (Pierce). Neutralizing antibody expressing hybridomas were generated by a combined polyethylene glycol electrofusion of peripheral blood mononuclear cells of HIV-infected non-symptomatic patients (21Buchacher A. Predl R. Strutzenberger K. Steinfellner W. Trkola A. Purtscher M. Gruber G. Tauer C. Steindl F. Jungbauer A. AIDS Res. Hum. Retroviruses. 1994; 10: 359-369Crossref PubMed Scopus (476) Google Scholar). Neutralizing antibodies 2F5 and 4E10 were produced in recombinant Chinese hamster ovary cells after a subclass switch to IgG1 (9Kunert R. Wolbank S. Stiegler G. Weik R. Katinger H. AIDS Res. Hum. Retroviruses. 2004; 20: 755-762Crossref PubMed Scopus (60) Google Scholar, 22Kunert R. Steinfellner W. Purtscher M. Assadian A. Katinger H. Biotechnol. Bioeng. 2000; 67: 97-103Crossref PubMed Scopus (47) Google Scholar). 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC), Chol, sphingomyelin (SPM), and the fluorescent probes N-(7-nitro-benz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine (N-(NBD)-phosphatidylethanolamine) and N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine were purchased from Avanti Polar Lipids (Birmingham, AL). The N-(5-dimethylaminonaphtalene-1-sulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine), 8-aminonaphtalene-1,3,6-trisulfonic acid sodium salt (ANTS), and p-xylenebis(pyridinium)bromide (DPX) fluorescent probes and the goat anti-human IgG antibody labeled with Alexa-Fluor 488 were from Molecular Probes (Junction City, OR). Methyl-β-cyclodextrin (MBCD) and sphingomyelinase (from Bacillus cereus) were from Sigma. Production of Lipid Vesicles—Large unilamellar vesicles (LUVs) were prepared following the extrusion method of Hope et al. (23Hope M.J. Bally M.B. Webb G. Cullis P.R. Biochim. Biophys. Acta. 1985; 812: 55-65Crossref PubMed Scopus (2008) Google Scholar) in 5 mm Hepes, 100 mm NaCl (pH 7.4) buffer. Lipid concentrations of liposome suspensions were determined by phosphate analysis (24Böttcher C.S.F. van Gent C.M. Fries C. Anal. Chim. Acta. 1961; 24: 203-204Crossref Scopus (848) Google Scholar). Extraction of cholesterol from the vesicles was performed using MBCD as previously described (25Ohvo-Rekilä H. Akerlund B. Slotte J.P. Chem. Phys. Lipids. 2000; 105: 167-178Crossref PubMed Scopus (57) Google Scholar, 26John K. Kubelt J. Muller P. Wustner D. Herrmann A. Biophys. J. 2002; 83: 1525-1534Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Fluorimetric Assays—Release of vesicular contents to the medium was monitored by the ANTS/DPX assay (27Ellens H. Bentz J. Szoka F.C. Biochemistry. 1985; 24: 3099-3106Crossref PubMed Scopus (445) Google Scholar). LUV containing 12.5 mm ANTS, 45 mm DPX, 20 mm NaCl, and 5 mm Hepes were obtained by separating the unencapsulated material by gel filtration in a Sephadex G-75 column eluted with 5 mm Hepes, 100 mm NaCl (pH 7.4). Osmolarities were adjusted to 200 mosmol in a cryoscopic osmometer (Osmomat 030, Gonotec, Berlin, Germany). Fluorescence measurements were performed by setting ANTS emission at 520 nm and excitation at 355 nm. A cutoff filter (470 nm) was placed between the sample and the emission monochromator. The 0% leakage corresponded to the fluorescence of the vesicles at time 0; 100% leakage was the fluorescence value obtained after the addition of Triton X-100 (0.5%, v/v). Membrane lipid mixing was monitored using the resonance energy transfer assay, described by Struck et al. (28Struck D.K. Hoekstra D. Pagano R.E. Biochemistry. 1981; 20: 4093-4099Crossref PubMed Scopus (1133) Google Scholar). The assay is based on the dilution of N-(NBD)-phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine. Dilution due to membrane mixing resulted in an increased N-NBD-phosphatidylethanolamine fluorescence. Vesicles containing 0.6 mol % of each probe were mixed with unlabeled vesicles at a 1:4 ratio (final lipid concentration, 0.1 mm). The NBD emission was monitored at 530 nm with the excitation wavelength set at 465 nm. A cutoff filter at 515 nm was used between the sample and the emission monochromator to avoid scattering interferences. The fluorescence scale was calibrated such that the zero level corresponded to the initial residual fluorescence of the labeled vesicles, and the 100% value corresponded to complete mixing of all the lipids in the system. The latter value was set by the fluorescence intensity of vesicles, labeled with 0.12 mol % of each fluorophore, at the same total lipid concentration as in the fusion assay. Kinetics of peptide partitioning into the membrane interface was evaluated measuring energy transfer from Trp residues to the surface fluorescent probe N-(5-dimethylaminonaphtalene-1-sulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine as in Ruiz-Argüello et al. (29Ruiz-Argüello M.B. Goñi F.M. Pereira F.B. Nieva J.L. J. Virol. 1998; 72: 1775-1781Crossref PubMed Google Scholar). In brief, 6 mol % of N-(5-dimethylaminonaphtalene-1-sulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine probe was included in the target vesicle composition, and its emission was collected in time at the wavelength of 510 nm. The excitation wavelength was that of the Trp residue (280 nm). Monolayer Penetration—Surface pressure was determined in a fixed-area circular trough (μTrough S system, Kibron, Helsinki). Measurements were carried out at room temperature and under constant stirring. The aqueous phase consisted of 1 ml of 5 mm Hepes, 100 mm NaCl (pH 7.4). Lipid mixture dissolved in chloroform was spread at the surface at the initial surface pressure (π0) of 25 mN/m. Peptide was subsequently injected into the subphase with a Hamilton microsyringe until the surface pressure reached a value of ∼32 mN/m. Finally, the antibody was injected into the subphase, and changes in surface-pressure were recorded in time. Planar Supported Phospholipid Layers—Phospholipid monolayers were spread from chloroform/methanol 3:1 (v/v) solutions ontoa5mm sodium phosphate (pH 7.4), 150 mm NaCl subphase in a thermostatted Langmuir-Blodgett trough (NIMA Technologies, Coventry) as previously described (3Sáez-Cirión A. Nir S. Lorizate M. Agirre A. Cruz A. Pérez-Gil J. Nieva J.L. J. Biol. Chem. 2002; 277: 21776-21785Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 30Ruano M.L. Nag K. Worthman L.A. Casals C. Perez-Gil J. Keough K.M. Biophys. J. 1998; 74: 1101-1109Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 31Cruz A. Worthman L.A. Serrano A.G. Casals C. Keough K.M. Perez-Gil J. Eur. Biophys. J. 2000; 29: 204-213Crossref PubMed Scopus (61) Google Scholar). After 10 min, to allow for solvent evaporation, monolayers were compressed at 25 cm2/min up to 32 mN/m and then transferred onto a glass coverslip at 5 mm/min. Lipid/Rho-PreTM monolayers were prepared by adding the proper volume of a Me2SO solution of the peptide to the chloroform/methanol lipid mixtures used in monolayer spreading. Epifluorescence microscopy observation of the planar supported monolayers was performed with a Zeiss Axioplan II fluorescence microscope (Carl Zeiss, Jena, D). Antibody was injected into the aqueous subphase and incubated for 30 min before transferring the monolayer to the solid support. Images from Alexa-Fluor-labeled antibody, and rhodamine-labeled peptide were recorded separately by switching fluorescence filters to select the proper emission wavelength range. Images presented in the figures were false-colored to show them as they look under the microscope. All experiments were carried out at 24 °C. Fig. 1A displays the average interfacial hydrophobicities of HIV-1 gp41 ectodomain and mAb4E10 heavy chain (top and bottom plots, respectively). The gp41 sequence displays a prominent hydrophobic region comprising the aromatic-rich pre-TM 664-683 sequence. The core 4E10 epitope sequence lies in the center of the preTM stretch. Therefore, mAb4E10 is predicted to recognize a sequence that may stably reside at membrane interfaces. The mAb4E10 plot below displays two hydrophobic peaks (colored in black) that correspond to loop sequences containing exposed aliphatic and aromatic residues in the heavy chains (14Cardoso R.M. Zwick M.B. Stanfield R.L. Kunert R. Binley J.M. Katinger H. Burton D.R. Wilson I.A. Immunity. 2005; 22: 163-173Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). These two loops have been postulated to establish direct interactions with the membrane, a process that might help with recognition of the membrane-inserted epitope stretch (14Cardoso R.M. Zwick M.B. Stanfield R.L. Kunert R. Binley J.M. Katinger H. Burton D.R. Wilson I.A. Immunity. 2005; 22: 163-173Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 32Sánchez-Martínez S. Lorizate M. Katinger H. Kunert R. Nieva J.L. AIDS Res. Hum. Retroviruses. 2006; 22: 998-1006Crossref PubMed Scopus (59) Google Scholar). Fig. 1, panel B, compares the atomic structures of a preTM-derived sequence resolved by Cardoso et al. (14Cardoso R.M. Zwick M.B. Stanfield R.L. Kunert R. Binley J.M. Katinger H. Burton D.R. Wilson I.A. Immunity. 2005; 22: 163-173Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar) in complex with Fab4E10 (green chain) or immersed into dodecylphosphocholine micelles according to the NMR-based model proposed by Schibli et al. (13Schibli D.J. Montelaro R.C. Vogel H.J. Biochemistry. 2001; 40: 9570-9578Crossref PubMed Scopus (159) Google Scholar) (blue chain). The helical structure is the main conformation in both cases. However, the relative orientation of the aromatic residues is different. Specifically the Phe-673 (red side chain) is buried into a hydrophobic pocket of the Fab binding site, whereas the side chain of this residue projects toward the hydrocarbon core of the bilayer in the membraneinterface residing species. The different orientations and insertion levels suggest that the potential recognition of the membrane interface residing preTM by mAb4E10 should result in blocking of its membrane restructuring capacity. mAb4E10 Effects on PreTM-induced Membrane Restructuring—Results displayed in Figs. 2 and 3 confirmed that mAb4E10 was able to block PreTM-induced membrane restructuring in Chol-containing vesicles. We have previously reported that the PreTM peptide perturbs vesicles made of lipids that resemble the raft-like composition of HIV-1 external membrane monolayer (3Sáez-Cirión A. Nir S. Lorizate M. Agirre A. Cruz A. Pérez-Gil J. Nieva J.L. J. Biol. Chem. 2002; 277: 21776-21785Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 18Aloia R.C. Jensen F.C. Curtain C.C. Mobley P.W. Gordon L.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 900-904Crossref PubMed Scopus (187) Google Scholar, 19Aloia R.C. Tian H.R. Jensen F.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5181-5185Crossref PubMed Scopus (371) Google Scholar, 20Brüger B. Glass B. Haberkant P. Leibrecht Wieland F.T. Kräus-slich H.-G. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2641-2646Crossref PubMed Scopus (545) Google Scholar). In particular, PreTM peptide partitions almost quantitatively into POPC:SPM:Chol (1:1:1) vesicles and induces the release of the encapsulated solutes (3Sáez-Cirión A. Nir S. Lorizate M. Agirre A. Cruz A. Pérez-Gil J. Nieva J.L. J. Biol. Chem. 2002; 277: 21776-21785Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). As shown in Fig. 2A, the addition of mAb4E10 alone to these vesicles did not induce any appreciable ANTS release, suggesting that the antibody was not able to destabilize the lipid bilayer on its own. However, antibody addition before PreTM efficiently reduced the ANTS leakage induced by the peptide. The inhibitory effect was dose-dependent and more effective at low peptide-to-lipid ratios, suggesting that the process was directly dependent on antibody recognition (Fig. 2B).FIGURE 3Distinct effects of SPM and Chol on the membrane-restructuring blocking capacity of mAb4E10. A, inhibition of ongoing ANTS leakage with mAb4E10. POPC:Chol (2:1) (left-hand panel) or POPC: SPM (1:1) (right-hand panel) LUV suspensions (100 μm lipid) were treated with PreTM (1 μm) and, at the time indicated by the arrow, supplemented with 50 μg/ml of mAb4E10 (a traces). b traces denote the controls in absence of antibody. c traces correspond to dansyl fluorescence increase (arbitrary units) upon PreTM addition. B, specific effects of Chol and SPM on the process. Left-hand panel, control for the inhibition of ongoing ANTS leakage with mAb4E10 (25 μg/ml) as detected in POPC:SPM:Chol (1:1:1) LUV. Middle panel, vesicles were treated with MBCD (15 mm) for 15 min before preTM addition. Right-hand panel, vesicles were treated for 15 min with sphingomyelinase (SPMase; 0.16 units/ml) before preTM addition. Conditions were as in panel A. CTL, control. C, inhibition of intervesicular mixing of lipids with mAb4E10. POPC:Chol (2:1) vesicles undergoing PreTM-induced fusion were treated with 50 μg/ml mAb4E10 (arrow in the a trace). The b trace corresponds to the kinetics of the process in absence of antibody. Conditions were as in panel A.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The preTM-induced ANTS leakage process in POPC:SPM: Chol (1:1:1) vesicles satisfies the prerequisites for permeabilization occurring via lytic pore formation (3Sáez-Cirión A. Nir S. Lorizate M. Agirre A. Cruz A. Pérez-Gil J. Nieva J.L. J. Biol. Chem. 2002; 277: 21776-21785Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Accordingly, ANTS leakage kinetics reproduced the time-course required for selfaggregation and pore assembly at the membrane surface. Thus, this process may comprise a potential means for testing antibody recognition of membrane-bound species. mAb4E10 addition to vesicles undergoing permeabilization also arrested the pore formation (Fig. 2C). The PreTM peptide lacks the complete ELDKWA epitope sequence required for efficient mAb2F5 recognition, and this antibody could not arrest the leakage process (b trace). This observation reinforces the idea that blocking of membrane-bound species competent for membrane destabilization depended on specific 4E10 epitope recognition. Membrane restructuring could not be completely arrested in the previous samples, suggesting the existence of a peptide fraction not accessible to the antibody. In our previous contribution we showed that raft lipids SPM and Chol promoted different PreTM species in membranes (3Sáez-Cirión A. Nir S. Lorizate M. Agirre A. Cruz A. Pérez-Gil J. Nieva J.L. J. Biol. Chem. 2002; 277: 21776-21785Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). When mixed with POPC, SPM sustained pore-formation, whereas Chol also promoted fusion activity, a phenomenon that we interpreted as indicative of transmembrane and interfacial topologies. Therefore, the role of each raft lipid in the blocking-at-membrane processes was next analyzed separately using POPC:Chol (2:1) and POPC: SPM (1:1) vesicles (Fig. 3A). The time-course of dansylphosphatidylethanolamine fluorescence increase confirmed the immediate incorporation of peptide upon the addition to b" @default.
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W2073983206 title "Recognition and Blocking of HIV-1 gp41 Pre-transmembrane Sequence by Monoclonal 4E10 Antibody in a Raft-like Membrane Environment" @default.
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W2073983206 doi "https://doi.org/10.1074/jbc.m605998200" @default.
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