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• W1994398203 abstract "It is well established that the human immunodeficiency virus-1 envelope glycoprotein surface unit, gp120, binds to cell-associated heparan sulfate (HS). Virus infectivity is increased by such interaction, and a variety of soluble polyanions efficiently neutralize immunodeficiency virus-1 in vitro. This interaction has been mainly attributed to the gp120 V3 loop. However, although evidence suggested that this particular domain does not fully recapitulate the binding activity of the protein, the ability of HS to bind to other regions of gp120 has not been completely addressed, and the exact localizations of the polysaccharide binding sites are not known. To investigate in more detail the structural basis of the HS-gp120 interaction, we used a mapping strategy and compared the heparin binding activity of wild type and mutant gp120 using surface plasmon resonance-based binding assays. Four heparin binding domains (1–4) were identified in the V2 and V3 loops, in the C-terminal domain, and within the CD4-induced bridging sheet. Interestingly, three of them were found in domains of the protein that undergo structural changes upon binding to CD4 and are involved in co-receptor recognition. In particular, Arg419, Lys421, and Lys432, which directly interact with the co-receptor, are targeted by heparin. This study provides a complete account of the gp120 residues involved in heparin binding and identified several binding surfaces that constitute potential target for viral entry inhibition. It is well established that the human immunodeficiency virus-1 envelope glycoprotein surface unit, gp120, binds to cell-associated heparan sulfate (HS). Virus infectivity is increased by such interaction, and a variety of soluble polyanions efficiently neutralize immunodeficiency virus-1 in vitro. This interaction has been mainly attributed to the gp120 V3 loop. However, although evidence suggested that this particular domain does not fully recapitulate the binding activity of the protein, the ability of HS to bind to other regions of gp120 has not been completely addressed, and the exact localizations of the polysaccharide binding sites are not known. To investigate in more detail the structural basis of the HS-gp120 interaction, we used a mapping strategy and compared the heparin binding activity of wild type and mutant gp120 using surface plasmon resonance-based binding assays. Four heparin binding domains (1–4) were identified in the V2 and V3 loops, in the C-terminal domain, and within the CD4-induced bridging sheet. Interestingly, three of them were found in domains of the protein that undergo structural changes upon binding to CD4 and are involved in co-receptor recognition. In particular, Arg419, Lys421, and Lys432, which directly interact with the co-receptor, are targeted by heparin. This study provides a complete account of the gp120 residues involved in heparin binding and identified several binding surfaces that constitute potential target for viral entry inhibition. Human immunodeficiency virus (HIV) 4The abbreviations used are: HIV, human immunodeficiency virus; PBS, phosphate-buffered saline; HS, heparan sulfate; HBD, HS binding domain; mAb, monoclonal antibody; sCD4, soluble CD4; CD4i, CD4-induced; RU, response units. 4The abbreviations used are: HIV, human immunodeficiency virus; PBS, phosphate-buffered saline; HS, heparan sulfate; HBD, HS binding domain; mAb, monoclonal antibody; sCD4, soluble CD4; CD4i, CD4-induced; RU, response units. gains entry into permissive CD4+ cells by sequentially interacting with CD4, the primary receptor, and a co-receptor, usually either CCR5 or CXCR4 (1Berger E.A. Murphy P.M. Farber J.M. Annu. Rev. Immunol. 1999; 17: 657-700Crossref PubMed Scopus (1876) Google Scholar). Both receptor and co-receptors are recognized by gp120, the glycoprotein that constitutes the surface unit of HIV envelope spikes. This protein consists of five relatively conserved regions (C1 to C5) that fold into a “core” comprising two distinct domains termed “inner” and “outer” and five variable regions (V1 to V5). This protein is prone to structural changes and is believed to sample a number of different conformations. A wealth of evidence showed that upon binding to CD4, roughly half of the gp120 core structure undergoes structural rearrangements, in particular within the inner domain. In the CD4-bound form, the base of the V1/V2 region of the inner domain (β2 and β3 strands) is brought to close proximity to a β-hairpin of the outer domain (β20 and β21 strands) and forms a four-stranded β-sheet located within the bridging sheet that connects the inner and the outer domain of the glycoprotein. Importantly, this highly conserved structure forms, in conjunction with the V3 loop, the binding site for CCR5 or CXCR4 (2Chen B. Vogan E.M. Gong H. Skehel J.J. Wiley D.C. Harrison S.C. Nature. 2005; 433: 834-841Crossref PubMed Scopus (464) Google Scholar, 3Kwong P.D. Wyatt R. Robinson J. Sweet R.W. Sodroski J. Hendrickson W.A. Nature. 1998; 393: 648-659Crossref PubMed Scopus (2502) Google Scholar, 4Poignard P. Saphire E.O. Parren P.W. Burton D.R. Annu. Rev. Immunol. 2001; 19: 253-274Crossref PubMed Scopus (226) Google Scholar, 5Rizzuto C.D. Wyatt R. Hernandez-Ramos N. Sun Y. Kwong P.D. Hendrickson W.A. Sodroski J. Science. 1998; 280: 1949-1953Crossref PubMed Scopus (752) Google Scholar, 6Wu L. Gerard N.P. Wyatt R. Choe H. Parolin C. Ruffing N. Borsetti A. Cardoso A.A. Desjardin E. Newman W. Gerard C. Sodroski J. Nature. 1996; 384: 179-183Crossref PubMed Scopus (1081) Google Scholar, 7Wyatt R. Sodroski J. Science. 1998; 280: 1884-1888Crossref PubMed Scopus (1299) Google Scholar).It has been well known that HIV also binds to CD4- cells in a gp120-dependent manner through interactions with cell surface molecules including heparan sulfates (HSs) (8Mondor I. Ugolini S. Sattentau Q.J. J. Virol. 1998; 72: 3623-3634Crossref PubMed Google Scholar). HSs and the closely related heparin belong to a large family of anionic polysaccharides, collectively known as glycosaminoglycans. They occur covalently bound to core proteins and are ubiquitously expressed at most cell surfaces (9Bernfield M. Gotte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2293) Google Scholar). These molecules are structurally complex, characterized by very large interactive properties, and recognize many unrelated protein via clusters of basic amino acids exposed on the surface of their targets (10Whitelock J.M. Iozzo R.V. Chem. Rev. 2005; 105: 2745-2764Crossref PubMed Scopus (341) Google Scholar). As such, they are exploited as a prevalent source of docking sites by a large array of pathogens (11Liu J. Thorp S.C. Med. Res. Rev. 2002; 22: 1-25Crossref PubMed Scopus (244) Google Scholar, 12Spillmann D. Biochimie (Paris). 2001; 83: 811-817Crossref PubMed Scopus (147) Google Scholar, 13Vives R.R. Lortat-Jacob H. Fender P. Curr. Gene Ther. 2006; 6: 35-44Crossref PubMed Scopus (30) Google Scholar) that interact with highly sulfated heparin-like regions of these polysaccharides (14Harrop H.A. Rider C.C. Glycobiology. 1998; 8: 131-137Crossref PubMed Scopus (58) Google Scholar). Regarding HIV, it has earlier been described that removal of HSs from the cell membrane inhibited infection by reducing viral particle concentration at the cell surface (15Roderiquez G. Oravecz T. Yanagishita M. Bou-Habib D.C. Mostowski H. Norcross M.A. J. Virol. 1995; 69: 2233-2239Crossref PubMed Google Scholar). It is now, however, appreciated that HIV binding to HSs can occur at many places and serves many functions. At the mucosal surface, the main point of entry for the virus into the host, HSs sequester viral particles and are involved in their translocation across epithelial barriers (16Bomsel M. Alfsen A. Nat. Rev. Mol. Cell Biol. 2003; 4: 57-68Crossref PubMed Scopus (145) Google Scholar, 17Saidi H. Magri G. Nasreddine N. Requena M. Belec L. Virology. 2007; 358: 55-68Crossref PubMed Scopus (60) Google Scholar, 18Wu Z. Chen Z. Phillips D.M. J. Infect. Dis. 2003; 188: 1473-1482Crossref PubMed Scopus (90) Google Scholar). Similarly, HSs expressed by microvasculature endothelial cells of the blood brain barrier capture HIV and contribute to HIV brain invasion (19Argyris E.G. Acheampong E. Nunnari G. Mukhtar M. Williams K.J. Pomerantz R.J. J. Virol. 2003; 77: 12140-12151Crossref PubMed Scopus (87) Google Scholar, 20Banks W.A. Robinson S.M. Wolf K.M. Bess Jr., J.W. Arthur L.O. Neuroscience. 2004; 128: 143-153Crossref PubMed Scopus (30) Google Scholar, 21Bobardt M.D. Salmon P. Wang L. Esko J.D. Gabuzda D. Fiala M. Trono D. Van der Schueren B. David G. Gallay P.A. J. Virol. 2004; 78: 6567-6584Crossref PubMed Scopus (94) Google Scholar). Several studies also showed that HSs play an active role in sequestering, protecting, and transferring viruses to CD4+ susceptible cells, with conditions that boost their replication (in trans mechanism) (22Bobardt M.D. Saphire A.C. Hung H.C. Yu X. Van der Schueren B. Zhang Z. David G. Gallay P.A. Immunity. 2003; 18: 27-39Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 23Olinger G.G. Saifuddin M. Spear G.T. J. Virol. 2000; 74: 8550-8557Crossref PubMed Scopus (51) Google Scholar). On permissive cells, HIV binding to HSs is thought to increase infectivity by favoring viral particle concentration at the cell surface (in cis process). For some cells, such as macrophages, they may compensate for low CD4 expression (24Saphire A.C. Bobardt M.D. Zhang Z. David G. Gallay P.A. J. Virol. 2001; 75: 9187-9200Crossref PubMed Scopus (205) Google Scholar). It has also been suggested that HSs could be directly involved in the infection of CD4- cells, including endothelial cells and possibly neurons (19Argyris E.G. Acheampong E. Nunnari G. Mukhtar M. Williams K.J. Pomerantz R.J. J. Virol. 2003; 77: 12140-12151Crossref PubMed Scopus (87) Google Scholar, 25Alvarez Losada S. Canto-Nogues C. Munoz-Fernandez M.A. Neurobiol. Dis. 2002; 11: 469-478Crossref PubMed Scopus (33) Google Scholar). Finally, HSs and heparin have been found to inhibit the protein disulfide isomerase-mediated reduction of gp120, although the exact role of this effect has not been elucidated yet (26Barbouche R. Lortat-Jacob H. Jones I.M. Fenouillet E. Mol. Pharmacol. 2005; 67: 1111-1118Crossref PubMed Scopus (29) Google Scholar). In addition to mediating viral attachment and entry, gp120 (either free or virion associated) targets a number of uninfected or non-permissive cells. As such, it is involved in some aspects of the AIDS-associated pathologies, including apoptosis and oxidative stress, and this has motivated the development of heparin-mimetics that inhibit gp120-HS interactions (27Bugatti A. Urbinati C. Ravelli C. De Clercq E. Liekens S. Rusnati M. Antimicrob. Agents Chemother. 2007; 51: 2337-2345Crossref PubMed Scopus (41) Google Scholar).A large body of work, aiming at characterizing the gp120/HS complex, showed that heparin, HSs, or other polyanions including dextran sulfate bind to gp120 V3 loop-derived peptides or compete with V3 loop-specific monoclonal antibodies (mAbs) for binding to gp120 (28Batinic D. Robey F.A. J. Biol. Chem. 1992; 267: 6664-6671Abstract Full Text PDF PubMed Google Scholar, 29Callahan L.N. Phelan M. Mallinson M. Norcross M.A. J. Virol. 1991; 65: 1543-1550Crossref PubMed Google Scholar, 30Okada T. Patterson B.K. Gurney M.E. Biochem. Biophys. Res. Commun. 1995; 209: 850-858Crossref PubMed Scopus (8) Google Scholar, 31Rider C.C. Coombe D.R. Harrop H.A. Hounsell E.F. Bauer C. Feeney J. Mulloy B. Mahmood N. Hay A. Parish C.R. Biochemistry. 1994; 33: 6974-6980Crossref PubMed Scopus (69) Google Scholar). However, this binding, which affinity (KD = 220 nm) has been determined by a surface plasmon resonance-based binding assay (32Moulard M. Lortat-Jacob H. Mondor I. Roca G. Wyatt R. Sodroski J. Zhao L. Olson W. Kwong P.D. Sattentau Q.J. J. Virol. 2000; 74: 1948-1960Crossref PubMed Scopus (289) Google Scholar), appears to be more complex than previously thought. The binding of polyanions to gp120 interferes with several mAbs recognizing epitopes outside the V3 loop, including those that target the bridging sheet induced upon CD4 binding (15Roderiquez G. Oravecz T. Yanagishita M. Bou-Habib D.C. Mostowski H. Norcross M.A. J. Virol. 1995; 69: 2233-2239Crossref PubMed Google Scholar, 32Moulard M. Lortat-Jacob H. Mondor I. Roca G. Wyatt R. Sodroski J. Zhao L. Olson W. Kwong P.D. Sattentau Q.J. J. Virol. 2000; 74: 1948-1960Crossref PubMed Scopus (289) Google Scholar). In agreement with these observations, it has been previously reported that gp120 in its CD4-bound state had a substantially increased binding activity toward heparin, compared with free gp120, and molecular modeling further suggested that the bridging sheet includes a possible HS binding domain (HBD) (33Vives R.R. Imberty A. Sattentau Q.J. Lortat-Jacob H. J. Biol. Chem. 2005; 280: 21353-21357Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Finally, it has also been demonstrated that the development of resistance to dextran sulfate correlates with specific mutations within and outside the V3 loop (34Este J.A. Schols D. De Vreese K. Van Laethem K. Vandamme A.M. Desmyter J. De Clercq E. Mol. Pharmacol. 1997; 52: 98-104Crossref PubMed Scopus (76) Google Scholar). Through an approach designed to simultaneously map different HBDs at the protein surface and the production of gp120 mutants, the present work identified the presence of four HBDs on the glycoprotein, some of which are potential targets for therapeutic applications.EXPERIMENTAL PROCEDURESMaterial—Insect cell culture media and the Bac-to-Bac system were purchased from Invitrogen and the QuikChange™ site-directed mutagenesis kit from Stratagene (La Jolla, CA). SP-Sepharose Fast Flow resin, Lentil-lectin resin, Superdex 200 column, N-ethyl-N′-(diethylaminopropyl)-carbodiimide/N-hydroxysuccimide reagents, and all surface plasmon resonance products were supplied from GE Healthcare. DEAE-Sephacel resin, 15-kDa heparin, 6-kDa heparin, and thermolysin were from Sigma Aldrich. Heparin-derived dodecasaccharide (dp12) was prepared as described (35Vives R.R. Sadir R. Imberty A. Rencurosi A. Lortat-Jacob H. Biochemistry. 2002; 41: 14779-14789Crossref PubMed Scopus (70) Google Scholar). The ultrafiltration unit and membranes were purchased from Millipore (Billerica, MA). Recombinant soluble CD4 (sCD4) was from Progenics Pharmaceuticals (Tarrytown, NY) and obtained through the National Institutes of Health AIDS Research and Reference Reagent Program. Monoclonal antibody 17b was from the Centre for AIDS Reagent Program, National Institute for Biological Standards and Control.Recombinant Wild Type and Mutant Protein Production—The cDNA encoding gp120HXBc2 was amplified by PCR from pSVIII-env and inserted into pNT-Bac, encoding either the melittin or the baculovirus ecdysteroid UDP glucosyltransferase signal peptide using the BamHI and HindIII restriction sites. Mutations giving rise to gp120-K121S, gp120-R419S, gp120-K421S, and gp120-K432S were introduced using the QuikChange™ site-directed mutagenesis kit. Resulting constructs were checked by restriction analysis and DNA sequencing (Genome Express) and used to prepare recombinant bacmid by transposition in Escherichia coli DH10Bac cells according to the manufacturerʼns protocol (Invitrogen). Isolated recombinant bacmid DNA was purified, analyzed by PCR, and used to transfect Spodoptera frugiperda 21 (Sf21) insect cells using Cellfectin in Sf900 II SFM medium. Recombinant baculovirus particles were collected 4 days later, titrated by virus plaque assay, and amplified as described (36King L.A. Possee R.D. The Baculovirus Expression System. Chapman & Hall, London1992: 111-114Google Scholar). In preliminary assays small-scale time-course expression experiments were conducted by infecting Sf21 cells (1.75 × 106 cells/ml) at a multiplicity of infection of 8. Samples of culture supernatants were taken 0, 24, 48, 72, 96, and 120 h post-infection and analyzed by SDS-PAGE. Material corresponding to gp120 was revealed by Western blot analysis with a primary polyclonal goat antibody directed against gp120 (1/2000; Biodesign International) coupled to a horseradish peroxidase-conjugated anti-goat antibody (1/5000, Jackson ImmunoResearch).Protein Purification—For large-scale protein production, Sf21 cells were adapted for growth in suspension. Sf21 cells (1 liter at 0.5 × 106 cells/ml) were infected for 72 h with recombinant viruses at a multiplicity of infection of 8 in Sf900 II SFM medium. Purification of both wild type and mutant proteins was performed at 4 °C using a three-step method. The supernatant (1 liter) was injected into a SP-Sepharose Fast Flow cation-exchange column (XK16) equilibrated with 50 mm HEPES pH 8.2 (solvent A) at a flow rate of 2 ml/min. The column was washed with 5 column volumes of A and 5 column volumes of A supplemented with 0.3% surfactant P20 (GE Healthcare). Elution was performed using a 2-step NaCl gradient (from 0 to 0.5 m NaCl in 12 min and 0.5 to 1 m NaCl in 2 min) and monitored by absorbance (280 nm). Fractions containing gp120 were pooled and loaded onto a lentil lectin-Sepharose column (1.6 × 10 cm) pre-equilibrated with phosphate-buffered saline (PBS) at 1 ml/min. The column was washed with 30 ml of PBS, and bound proteins were eluted with PBS containing 1 m methyl-α-d-mannopyranoside. Fractions (0.5 ml) containing gp120 were pooled and concentrated by ultrafiltration using an Amicon 8010 Filter unit fitted with a YM30 ultrafiltration membrane (30 000 molecular weight cutoff) followed by concentration on a Microcon unit (10,000 molecular weight cutoff). The sample was then size-fractionated onto a Superdex 200 HR 10/30 column equilibrated with PBS at 0.4 ml/min. Fractions (0.5 ml) containing gp120 were concentrated and quantified by amino acid analysis. Protease inhibitors (Complete; Roche Applied Science) were added, and the sample was stored at -20 °C. Throughout the procedure fractions were monitored by absorbance (280 nm) and analyzed by SDS-PAGE stained with Coomassie Blue and Western blotting, as described above.Surface Plasmon Resonance-based Binding Assays—A surface plasmon resonance (Biacore 3000) instrument was used to investigate the binding of wild type or mutant gp120 to immobilized CD4, heparin, or mAb 17b. For that purpose flow cells of a CM4 sensor chip were first activated with 50 μl of 0.2 m N-ethyl-N′-(diethylaminopropyl)-carbodiimide and 0.05 m N-hydroxysuccimide at 5 μl/min. Then soluble CD4 (5 μg/ml in 10 mm acetate buffer, pH 4.5), mAb 17b (5 μg/ml in 10 mm acetate buffer, pH 5), or streptavidin (100 μg/ml in 10 mm acetate buffer, pH 4.2) was injected at 5 μl/min over one of the activated flow cells until levels of 650, 500, or 2,500 response units (RU) were, respectively, achieved. Biotinylated heparin was captured on the streptavidin surface to get a level of 25 RU. The fourth flow cell (functionalized with 2500 RU of streptavidin) served as a negative control. Samples in HBS-P (10 mm HEPES, 150 mm NaCl, and 0.005% surfactant P20, pH 7.4) were injected over the different surfaces at a flow rate of 10 μl/ml, after which the formed complexes were washed with HBS-P. Surfaces were regenerated by sequential injections of 10 mm HCl (1 min) and 2 m NaCl (2.5 min). Each binding curve was corrected for non-specific binding by subtraction of the signal obtained from the negative-control flow cell.Mapping of Heparin/HS Binding Domains within gp120—Analysis of HBD was adapted from “the beads approach” previously described (37Vives R.R. Crublet E. Andrieu J.P. Gagnon J. Rousselle P. Lortat-Jacob H. J. Biol. Chem. 2004; 279: 54327-54333Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) but performed with free heparin in solution. Heparin (200 μg) was activated with 6 mm N-ethyl-N′-(diethylaminopropyl)-carbodiimide and 15 mm N-hydroxy-succimide for 12 min at room temperature. Reactants in excess were inactivated by the addition of β-mercaptoethanol (20 mm final) for 15 min. gp120 (3.5 μm) was incubated with 35 μm concentrations of the activated heparin in 100 μl of PBS for 2 h at room temperature, then the reaction was quenched by the addition of 11 μl of 1 m Tris, pH 7.5. Proteins cross-linked to heparin were digested overnight with 53 milliinternational units of thermolysin at 60 °C. Released peptides and thermolysin were removed by purification of the conjugates using weak anion exchange chromatography. Briefly, the sample was injected at 1 ml/min on to a 2 ml of DEAE-Sephacel column equilibrated with 20 mm Na2HPO4/NaH2PO4, pH 6.5 (solvent A). The column was washed with 5–10 column volumes of A and 5–10 column volumes of A supplemented with 0.3 m NaCl. Peptide-heparin conjugates were then eluted with buffer A containing 1 m NaCl. Eluted fractions (1 ml) were analyzed by monitoring the absorbance at 280 and 232 nm. In preliminary experiments biotinylated heparin was added in the mixture (as a tracer), and blotted fractions were analyzed using peroxidase-conjugated streptavidin. Peptide-heparin conjugate-containing fractions were pooled, desalted onto a PD-10 column, and identified by N-terminal sequencing as described (37Vives R.R. Crublet E. Andrieu J.P. Gagnon J. Rousselle P. Lortat-Jacob H. J. Biol. Chem. 2004; 279: 54327-54333Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar).RESULTSTime-Course Expression and Purification of Recombinant gp120HXBc2 in Sf21 Insect Cells—To produce the HIV envelope glycoprotein, recombinant baculoviruses encoding wild type or mutant gp120s in-frame with the melittin signal peptide were generated and used to infect Sf21 cells. The cell medium was harvested at different times post-infection and analyzed for gp120 expression by Western blotting. Immunoreactive material with a molecular mass of about 110–120 kDa was detected and migrated as purified gp120 used as a standard (Fig. 1A). Significant expression was observed 48 h after infection, with a maximum after 72 h. Longer infection times resulted in proteolytic degradation. Infection time chosen for subsequent experiments was 72 h. Under these conditions gp120 was secreted into the culture medium at an estimated level of 25–30 mg/liter. Similar amounts were produced using an expression vector, including a leader sequence derived from the baculovirus ecdysteroid UDP glucosyltransferase (data not shown).For large scale preparation, wild type and mutant gp120s were expressed in Sf21 cells adapted for growth in suspension. Cells were harvested 72 h after infection and centrifuged, and the supernatant (1 liter) was run through a SP-Sepharose column. The column was first washed with 30 ml of 50 mm HEPES, pH 8.2, then with surfactant P20 to remove a baculovirus protein (egt) which otherwise co-eluted with gp120 during the next step (data not shown). Bound proteins were then eluted with a NaCl gradient, and the gp120-containing fractions were injected over a Lentil-lectin column. The flow-through fractions did not contain gp120 (Fig. 1B, lane 2), which was then eluted from the lectin column with 1 m methyl-α-d-mannopyranoside in PBS (Fig. 1B, lane 3). The material was further purified by gel filtration on a Superdex 200 HR column (Fig. 1B, lane 4). Fractions containing gp120 were pooled, concentrated, and stored at -20 °C. Affinity chromatography on the Lentil-lectin column was the most efficient purification step. Mutant proteins were purified using the same protocol, and purity was estimated to be at least 90% by Coomassie Blue staining on SDS-PAGE (Fig. 1C). Throughout the purification procedure, only the fractions corresponding to the apex of the eluted peaks were collected to ensure maximum purity. Purified proteins were finally quantified by amino acid analysis. This three step-purification protocol enabled us to get around 1 mg of purified gp120/liter of culture medium.CD4 Binds to Wild Type gp120 and Triggers mAb 17b Recognition—To check whether the purified gp120 was correctly folded, we first investigated its ability to interact with CD4. Surface plasmon resonance experiments were conducted for that purpose, in which gp120 was flowed across a CD4-functionalized surface. Analysis of the resulting binding curves (Fig. 2A) returned an affinity of 8 nm, in very good agreement with previously reported data using the same technique (38Wu H. Myszka D.G. Tendian S.W. Brouillette C.G. Sweet R.W. Chaiken I.M. Hendrickson W.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15030-15035Crossref PubMed Scopus (57) Google Scholar, 39Zhang W. Godillot A.P. Wyatt R. Sodroski J. Chaiken I. Biochemistry. 2001; 40: 1662-1670Crossref PubMed Scopus (47) Google Scholar). To check whether our protein undergoes conformational changes upon CD4 binding, we monitored the binding of gp120 to mAb 17b, both in the presence and in the absence of sCD4. mAb 17b belongs to a group of antibodies referred as CD4-induced (CD4i), which bind to gp120 only after CD4 engagement. It recognizes a surface that includes or is proximal to the bridging sheet and, thus, competes with co-receptor binding (40Thali M. Moore J.P. Furman C. Charles M. Ho D.D. Robinson J. Sodroski J. J. Virol. 1993; 67: 3978-3988Crossref PubMed Google Scholar). In the absence of sCD4, gp120 showed no or little binding to mAb 17b, indicating that in its CD4-unbound state the bridging sheet of gp120 remains unexposed or unfolded. Preincubation of gp120 with sCD4 dramatically increased binding to mAb 17b, indicating that sCD4 triggers the conformational changes that lead to the formation/exposure of the co-receptor binding site (Fig. 2B). Taken together, these results provide evidence that the protein was properly folded and functional.FIGURE 2Binding of gp120 to immobilized CD4 and mAb 17b. Soluble CD4 and mAb 17b were immobilized on a CM4 sensor chip, and gp120 was injected over the chip surface. Injections were carried out in duplicate and returned similar results. A, binding curves for injection of gp120 (at 50, 100, and 200 nm, from bottom to top) over the CD4-activated surface. B, binding curves for injection of gp120 (50 nm) either alone or preincubated with 50 nm of sCD4 over the mAb 17b-activated surface. The binding response in RU was recorded as a function of time.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Mapping of Heparin Binding Domains within Wild Type gp120—Although the importance of the V3 loop for binding to heparin is well established, additional HBDs have been suggested but not identified yet. We, thus, performed a global analysis of the interaction between gp120 and heparin using a method adapted from the “beads sequencing approach” that has been previously reported (37Vives R.R. Crublet E. Andrieu J.P. Gagnon J. Rousselle P. Lortat-Jacob H. J. Biol. Chem. 2004; 279: 54327-54333Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). This approach was based on the capture of the protein of interest on heparin immobilized beads, the proteolytic digestion, and the N terminus sequencing of the peptides remaining attached to the heparin beads. However, because in some experiments beads were washed throughout the system despite the filter and blocked the sequencing machine, the cross-linking, the proteolysis, and the N terminus sequencing were now performed in solution. In addition, this enabled the use of defined oligosaccharides (instead of commercial heparin), and the results described below have been obtained with both full-length heparin and a heparin-derived 12-mer (dp12), used as a model of the S domains that characterize heparan sulfate (41Murphy K.J. Merry C.L. Lyon M. Thompson J.E. Roberts I.S. Gallagher J.T. J. Biol. Chem. 2004; 279: 27239-27245Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Carboxyl groups of soluble heparin (or dp12) were N-ethyl-N′-(diethylaminopropyl)-carbodiimide/N-hydroxysuccimide-activated, reacted with gp120, then formed protein/saccharide conjugates were submitted to proteolytic digestion. Peptides cross-linked to heparin were recovered by DEAE chromatography and analyzed by N-terminal sequencing. As shown in Fig. 3, results obtained with gp120 yielded three sequences, 165IRGKVQKEYAFFY177 (named HBD 1), 292VEINCTRPNNNTRKRIR308 (HBD 2), and 496VAPTKAKRR504 (HBD 3). Amino acids Lys168 within HBD 1, Lys305 within HBD 2, and Lys500 within HBD 3 were not detected, indicating that these were involved in the cross-linking with heparin, or dp12. The observation that a relatively small oligosaccharide (dp12) targets the same sequences and exactly returns the same results than full-length heparin indicates that having a large number of anionic charge does not give rise to additional (nonspecific) binding and supports the specificity of the assay (see Ref. 37Vives R.R. Crublet E. Andrieu J.P. Gagnon J. Rousselle P. Lortat-Jacob H. J. Biol. Chem. 2004; 279: 54327-54333Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar).FIGURE 3Mapping of gp120 heparin binding domain. A, amino acid sequence of gp120. Variable loops (V1/V2 and V3) are indicated as the β strands that fold together to form the CD4-induced bridging sheet. The amino acids are numbered according to the sequence of HXBc2 gp120, including the signal peptide (italics), with the mature full-length protein beginning at residue 31. B, sequence analysis of gp120 after immobilization on heparin and digestion with thermolysin. i and ii are the results from two different experiments, whereas iii is the results obtained when a 12-mers was used instead of full-length heparin. Amino acids involved in cross-linking, i.e. undetected" @default.
• W1994398203 created "2016-06-24" @default.
• W1994398203 creator A5001533524 @default.
• W1994398203 creator A5001553011 @default.
• W1994398203 creator A5037889329 @default.
• W1994398203 creator A5054992487 @default.
• W1994398203 date "2008-05-01" @default.
• W1994398203 modified "2023-10-12" @default.
• W1994398203 title "The HIV-1 Envelope Glycoprotein gp120 Features Four Heparan Sulfate Binding Domains, Including the Co-receptor Binding Site" @default.
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