Antigenic Properties of Peptide Mimotopes of HIV-1-associated Carbohydrate Antigens
2005; Elsevier BV; Volume: 280; Issue: 32 Linguagem: Inglês
10.1074/jbc.m502964200
ISSN1083-351X
AutoresAnastas Pashov, Gabriela Canziani, Behjatolah Monzavi‐Karbassi, Srini V. Kaveri, Stewart MacLeod, Rinku Saha, Marty Perry, Thomas C. VanCott, Thomas Kieber‐Emmons,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoThe glycan shield of the human immunodeficiency virus (HIV) envelope protein presents many potential epitopes for vaccine development. To augment immune responses to HIV, type 1 (HIV-1), envelope-associated carbohydrate antigens, we are defining peptide mimics of HIV-associated carbohydrate antigens that function as antigen mimotopes that upon immunization will induce antibodies cross-reactive with carbohydrate antigens. We have previously defined peptides with a putative sequence tract RYRY that mimic concanavalin A-binding glycans. To imitate the multivalent binding of carbohydrates, we compared the avidity of a linear (911) and cyclic peptide (D002) reactive with concanavalin A presented in a multiple antigen peptide (MAP) format. The affinity of the MAP-D002 peptide was higher than that of the peptide MAP-911, whereas the avidity of D002 peptide was lower than that of 911. Serum from mice immunized with MAP-911 had lower titer for oligomannose-9 than those elicited by MAP-D002 under the same conditions, but both immunogens elicited antibodies that can block the binding of GP120 to dendritic cells. Antibodies that bind to the studied MAPs were found in a preparation of normal human immunoglobulin for intravenous use. Those that were purified on 911 bound back to 911 and D002, whereas anti-D002 antibodies were specific only for D002. Human antibodies reactive with both mimotopes and with a mannosyl preparation were observed to bind to envelope protein. These results suggested the potential to fine-tune the antibody response to carbohydrate antigens by modifying structural features of peptide mimotope-based immunogens. The glycan shield of the human immunodeficiency virus (HIV) envelope protein presents many potential epitopes for vaccine development. To augment immune responses to HIV, type 1 (HIV-1), envelope-associated carbohydrate antigens, we are defining peptide mimics of HIV-associated carbohydrate antigens that function as antigen mimotopes that upon immunization will induce antibodies cross-reactive with carbohydrate antigens. We have previously defined peptides with a putative sequence tract RYRY that mimic concanavalin A-binding glycans. To imitate the multivalent binding of carbohydrates, we compared the avidity of a linear (911) and cyclic peptide (D002) reactive with concanavalin A presented in a multiple antigen peptide (MAP) format. The affinity of the MAP-D002 peptide was higher than that of the peptide MAP-911, whereas the avidity of D002 peptide was lower than that of 911. Serum from mice immunized with MAP-911 had lower titer for oligomannose-9 than those elicited by MAP-D002 under the same conditions, but both immunogens elicited antibodies that can block the binding of GP120 to dendritic cells. Antibodies that bind to the studied MAPs were found in a preparation of normal human immunoglobulin for intravenous use. Those that were purified on 911 bound back to 911 and D002, whereas anti-D002 antibodies were specific only for D002. Human antibodies reactive with both mimotopes and with a mannosyl preparation were observed to bind to envelope protein. These results suggested the potential to fine-tune the antibody response to carbohydrate antigens by modifying structural features of peptide mimotope-based immunogens. Immune targeting of carbohydrate antigens on the Env 1The abbreviations used are: Env, envelope; P-20, polyoxyethylene (20Haury M. Grandien A. Sundblad A. Coutinho A. Nobrega A. Scand. J. Immunol. 1994; 39: 79-87Crossref PubMed Scopus (110) Google Scholar) sorbitan mono-oleate; SPR, surface plasmon resonance; ConA, concanavalin A; MAP, multiple antigen peptide; ELISA, enzyme-linked immunosorbent assay; HIV, human immunodeficiency virus; HIV-1, HIV, type 1; Man-9, oligomannose-9; PBS, phosphate-buffered saline; GP, glycoprotein; DC, dendritic cells; RU, response units; SA, streptavidin. 1The abbreviations used are: Env, envelope; P-20, polyoxyethylene (20Haury M. Grandien A. Sundblad A. Coutinho A. Nobrega A. Scand. J. Immunol. 1994; 39: 79-87Crossref PubMed Scopus (110) Google Scholar) sorbitan mono-oleate; SPR, surface plasmon resonance; ConA, concanavalin A; MAP, multiple antigen peptide; ELISA, enzyme-linked immunosorbent assay; HIV, human immunodeficiency virus; HIV-1, HIV, type 1; Man-9, oligomannose-9; PBS, phosphate-buffered saline; GP, glycoprotein; DC, dendritic cells; RU, response units; SA, streptavidin. protein of HIV-1 has gained interest with the realization that the human antibody 2G12, which neutralizes a broad range of HIV-1 isolates and shows protective activity against viral challenge in animal models (1Ferrantelli F. Hofmann-Lehmann R. Rasmussen R.A. Wang T. Xu W. Li P.L. Montefiori D.C. Cavacini L.A. Katinger H. Stiegler G. Anderson D.C. McClure H.M. Ruprecht R.M. AIDS. 2003; 17: 301-309Crossref PubMed Scopus (104) Google Scholar), binds a dense cluster of carbohydrate moieties on the “silent” face of the GP120 Env glycoprotein (2Scanlan C.N. Pantophlet R. Wormald M.R. Saphire E.O. Calarese D. Stanfield R. Wilson I.A. Katinger H. Dwek R.A. Burton D.R. Rudd P.M. Adv. Exp. Med. Biol. 2003; 535: 205-218Crossref PubMed Google Scholar). Defining “protective” epitopes on HIV-associated carbohydrate antigens is a critical first step in carbohydrate-based vaccine design strategies (3Lee R.T. Lee Y.C. Glycoconj. J. 2000; 17: 543-551Crossref PubMed Scopus (410) Google Scholar, 4Li H. Wang L.X. Org. Biomol. Chem. 2004; 2: 483-488Crossref PubMed Scopus (67) Google Scholar, 5Dudkin V.Y. Orlova M. Geng X. Mandal M. Olson W.C. Danishefsky S.J. J. Am. Chem. Soc. 2004; 126: 9560-9562Crossref PubMed Scopus (87) Google Scholar). Crystallographic studies of 2G12 coupled with direct binding assays suggest a high binding interaction of 2G12 with mannosyl glycans at nanomolar affinity. This interaction makes use of an unusual but distinct interface between the heavy chains of 2G12 (2Scanlan C.N. Pantophlet R. Wormald M.R. Saphire E.O. Calarese D. Stanfield R. Wilson I.A. Katinger H. Dwek R.A. Burton D.R. Rudd P.M. Adv. Exp. Med. Biol. 2003; 535: 205-218Crossref PubMed Google Scholar, 6Wang L.X. Ni J. Singh S. Li H. Chem. Biol. 2004; 11: 127-134Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar, 7Calarese D.A. Scanlan C.N. Zwick M.B. Deechongkit S. Mimura Y. Kunert R. Zhu P. Wormald M.R. Stanfield R.L. Roux K.H. Kelly J.W. Rudd P.M. Dwek R.A. Katinger H. Burton D.R. Wilson I.A. Science. 2003; 300: 2065-2071Crossref PubMed Scopus (673) Google Scholar, 8Lee H.K. Scanlan C.N. Huang C.Y. Chang A.Y. Calarese D.A. Dwek R.A. Rudd P.M. Burton D.R. Wilson I.A. Wong C.H. Angew. Chem. Int. Ed. Engl. 2004; 43: 1000-1003Crossref PubMed Scopus (122) Google Scholar, 9Sanders R.W. Venturi M. Schiffner L. Kalyanaraman R. Katinger H. Lloyd K.O. Kwong P.D. Moore J.P. J. Virol. 2002; 76: 7293-7305Crossref PubMed Scopus (505) Google Scholar). Specificity analysis of 2G12 indicates preferred interactions with Env-associated Man9GlcNAc antigen, which is also recognized by cyanovirin-N (10Botos I. Wlodawer A. Cell. Mol. Life Sci. 2003; 60: 277-287Crossref PubMed Scopus (51) Google Scholar). There is strong evidence that lectins bind specifically to HIV-1-associated sugar epitopes (11Bewley C.A. Cai M. Ray S. Ghirlando R. Yamaguchi M. Muramoto K. J. Mol. Biol. 2004; 339: 901-914Crossref PubMed Scopus (52) Google Scholar), permitting a better understanding of carbohydrate profiles displayed on the Env protein that may be targeted in HIV-directed drug or vaccine design applications. The lectin concanavalin A (ConA) recognizes mannosyl antigens on the Env protein (12Robinson Jr., W.E. Montefiori D.C. Mitchell W.M. AIDS Res. Hum. Retroviruses. 1987; 3: 265-282Crossref PubMed Scopus (64) Google Scholar) but might do so differently than 2G12 because ConA can tolerate a larger number of sugar residues in its carbohydrate-binding site compared with that suggested for 2G12. ConA recognizes carbohydrate structures similar to the C-type lectin DC-specific inter-cellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN), preferring interactions with core oligosaccharide with specificity for α1–3- and α1–6-linked residues, whereas cyanovirin-N and 2G12 interact with terminal carbohydrates displaying specificity for α1–2-linked Man residues. ConA can also recognize α1–2-linked Man residues; therefore, ConA can recognize a larger set of mannosyl residues on HIV-1 Env than 2G12 or cyanovirin-N. These fine recognition features may affect affinity/avidity of binding of these proteins as several sugar residues of an oligosaccharide can interact with an extended binding site as observed for ConA. Alternatively, higher affinity may also be affected by multiplicity of binding sites. In this latter case, the multiplicity can compensate for weak affinity of individual sites. Therefore, as antigens display many epitopes, the interaction of ConA with mannosyl glycan expressed on the Env protein represents a model of ligand presentation. N-Linked glycosylation patterns in HIV Env are complicated by the fact that different cell types can affect glycosylation. DC-SIGN preferentially binds to the Env protein enriched for high mannose oligosaccharides, which are typically produced by peripheral blood mononuclear cells and T cells, compared with macrophage-produced GP120, which contains more complex carbohydrates, including modification by lactosaminoglycans (13Lin G. Simmons G. Pohlmann S. Baribaud F. Ni H. Leslie G.J. Haggarty B.S. Bates P. Weissman D. Hoxie J.A. Doms R.W. J. Virol. 2003; 77: 1337-1346Crossref PubMed Scopus (223) Google Scholar). DC-SIGN, however, appears to have evolved its binding specificity in that it is found to bind to mannan, fucose, and all fucosylated lactosamines excluding sLex and sLea (14Appelmelk B.J. van Die I. van Vliet S.J. Vandenbroucke-Grauls C.M.J.E. Geijtenbeek T.B.H. van Kooyk Y. J. Immunol. 2003; 170: 1635-1639Crossref PubMed Scopus (379) Google Scholar). We have used ConA previously as a template to define a peptide mimotope of Env-associated mannosyl residues (15Cunto-Amesty G. Dam T.K. Luo P. Monzavi-Karbassi B. Brewer C.F. Van Cott T.C. Kieber-Emmons T. J. Biol. Chem. 2001; 276: 30490-30498Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). We have shown that peptide mimotopes of the neolactoseries (16Agadjanyan M. Luo P. Westerink M.A. Carey L.A. Hutchins W. Steplewski Z. Weiner D.B. Kieber-Emmons T. Nat. Biotechnol. 1997; 15: 547-551Crossref PubMed Scopus (81) Google Scholar, 17Monzavi-Karbassi B. Cunto-Amesty G. Luo P. Lees A. Kieber-Emmons T. Biologicals. 2001; 29: 249-257Crossref PubMed Scopus (17) Google Scholar, 18Monzavi-Karbassi B. Shamloo S. Kieber-Emmons M. Jousheghany F. Luo P. Lin K.Y. Cunto-Amesty G. Weiner D.B. Kieber-Emmons T. Vaccine. 2003; 21: 753-760Crossref PubMed Scopus (37) Google Scholar) and mannose glycans (15Cunto-Amesty G. Dam T.K. Luo P. Monzavi-Karbassi B. Brewer C.F. Van Cott T.C. Kieber-Emmons T. J. Biol. Chem. 2001; 276: 30490-30498Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 18Monzavi-Karbassi B. Shamloo S. Kieber-Emmons M. Jousheghany F. Luo P. Lin K.Y. Cunto-Amesty G. Weiner D.B. Kieber-Emmons T. Vaccine. 2003; 21: 753-760Crossref PubMed Scopus (37) Google Scholar) can induce serum antibodies in mice that bind to the Env protein, neutralize Lab isolates, and block the adhesion of HIV-1-infected cells to human dendritic cells (DC) (19Monzavi-Karbassi B. Luo P. Cunto-Amesty G. Jousheghany F. Pashov A. Weissman D. Kieber-Emmons T. Arch. Virol. 2004; 149: 75-91Crossref PubMed Scopus (14) Google Scholar). Here we compare the avidity and affinity of a linear peptide with the sequence YRYRYGRYRSGSYRYRYGRYRSGS (referred to as peptide 911) and a cyclic form (peptide D002) of the putative RYRY sequence tract having the sequence RGGLCYCRYRYCVCVGR, which forms disulfide bridges between the 1st and 4th and between the 2nd and 3rd cysteine residues. The multivalent peptide 911 and its monomeric form YRYRYGRYRSGS display a free energy of association comparable with those reported for core trimannoside-ConA and pentasaccharide-ConA interactions (15Cunto-Amesty G. Dam T.K. Luo P. Monzavi-Karbassi B. Brewer C.F. Van Cott T.C. Kieber-Emmons T. J. Biol. Chem. 2001; 276: 30490-30498Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). These results suggest the feasibility of designing mimotopes to render effective humoral responses to HIV Env-associated mannosyl epitopes that would simplify current carbohydrate-based vaccine approaches. By clustering a mimotope motif in different conformational contexts, it is possible to create immunogens with different properties such as broad reactivity “recruiting” priming reagents (MAP-911) versus high affinity focused specificity ones (D002). We observe that the mimotopes can bind to natural antibodies of non-HIV-infected individuals, suggesting that a prime and boost strategy may capitalize on the existence of pools of B cell clones that can be further focused to generate antibodies toward carbohydrates expressed on HIV-1 Env. Reagents—Peptides were synthesized as MAPs (Research Genetics, Huntsville, AL) by Fmoc (N-(9-fluorenyl)methoxycarbonyl) synthesis on polylysine groups, resulting in the presentation of eight peptide clusters. Antibody 2G12 was kindly provided by Dr. H. Katinger. Recombinant GP120 was supplied by the AIDS Research and Reference Reagent Program (CM235, catalog number 2968; 93TH975, catalog number 3234; BAL, catalog number 4961; CN54, catalog number 7749). The primary isolate envelope glycoproteins were expressed in insect cells, whereas the laboratory isolate was expressed in human embryonic kidney cells. ConA and biotinylated ConA were purchased from Vector Laboratories, Burlingame, CA. Multivalent Man-9, attached to a polyacrylamide polymer of ∼30 kDa, was purchased from GlycoTech Corp., Rockville, MD. Mannan coupled to cross-linked agarose was purchased from Sigma. Pooled normal human immunoglobulin for intravenous use (containing 70% IgM and 30% IgG) was a kind gift from Biotest AG, Dreieich, Germany. The preparation was dialyzed against PBS before use. Immunization—BALB/c mice (n = 4 per group), 4–6 weeks of age, were immunized intraperitoneally three times, at intervals of 2 weeks, with 100 μg of MAP-D002 polyacrylamide polymer or MAP-911 polyacrylamide polymer combined with 20 μg of the adjuvant QS-21 (Antigenics, Inc., Lexington, MA). A control group was immunized with QS-21 alone. Sera were collected on the 7th and 14th days after the last immunization and were stored at –80 °C until use. Immunoaffinity Purification of Human Antibodies to MAPs—Affinity columns were prepared by coupling MAP-911 or MAP-D002 to cyanogen bromide-activated Sepharose (Amersham Biosciences). A human immunoglobulin for intravenous use preparation was dialyzed against PBS and circulated through the peptide-coupled columns for 3 h at room temperature at 10 mg/ml. The bound antibodies were eluted by acid (glycine/HCl, pH 2.7) followed by alkaline (diethanolamine, pH 11) elution or by peptide monomer, followed by soluble mannan, and ending with acid (glycine/HCl, pH 2.7). All fractions for a given MAP column were pooled, whereas the fractions from the mannan column were analyzed individually after being neutralized and dialyzed against PBS. The flow-through fractions of both columns were also collected and used to determine the specificity of the isolated antibodies. Cross-blot—A dot-blot technique was used to analyze the binding of antibody fractions to GP120 and peptides (20Haury M. Grandien A. Sundblad A. Coutinho A. Nobrega A. Scand. J. Immunol. 1994; 39: 79-87Crossref PubMed Scopus (110) Google Scholar, 21Pashov A. Kenderov A. Kyurkchiev S. Kehayov I. Hristova S. Lacroix-Desmazes S. Giltiay N. Sooryanarayana Kazatchkine M.D. Kaveri S.V. Int. Immunol. 2002; 14: 453-461Crossref PubMed Scopus (44) Google Scholar). Polyvinylidene difluoride membrane (Millipore) was prewetted and inserted in a Miniblotter (Immunetics, Cambridge, MA). Different antigens were added to the channels and incubated overnight at 4 °C. The membrane was washed, blocked, and inserted again in the miniblotter at 90° relative to the original orientation. Different antibody fractions were added at 1.3 μm to the channels and incubated for 4 h at room temperature. Thus each antibody fraction (∼80 μl) was in contact with all areas of immobilized antigens. This technique creates spots of equal form and size and uses very small volumes of antibodies. After additional washes the entire membrane was incubated with an alkaline phosphatase conjugate, and the reactivities were developed using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate (Sigma). The resulting pattern was scanned and digitalized using ImageJ software as described previously (21Pashov A. Kenderov A. Kyurkchiev S. Kehayov I. Hristova S. Lacroix-Desmazes S. Giltiay N. Sooryanarayana Kazatchkine M.D. Kaveri S.V. Int. Immunol. 2002; 14: 453-461Crossref PubMed Scopus (44) Google Scholar). The average staining of the two areas adjacent to the analyzed spot along the axis of the channel containing the antibody was used as a background and was subtracted from the value of the specific spot. Biosensor Experiments—Attachment of MAP mimetics to the sensor surface was performed using amine-ester coupling to carboxymethylalkane thiol-derivatized dextran, as recommended for SPR sensor chip CM5 (Biacore AB, Uppsala, Sweden). Six minutes of activation of the surface carboxyl groups using a 1:1 mixture of 3-(N,N-dimethylamino)-propyl-N-ethylcarbodiimide (0.2 m) and N-hydroxysuccinimide (0.05 m) at 5 μl/min was followed by coupling of MAP-911 and MAP-D002, diluted 1:100 with 0.1 m sodium acetate, pH 5.5, at 20 μl/min to the desired surface density. Low surface density corresponded to 40–100 RU and high surface density 1000–2000 RU. Spare activated sites were blocked with l.0 m ethanolamine, pH 8.5, for 6 min. Binding of ConA was measured at 25 °C. Both MAP mimetics and ConA interacted very strongly with the dextran matrix of the CM5 sensor chip. To control these nonspecific interactions, the net negative charge of the sensor chip matrix was reduced by replacing the CM5 surface by the low carboxyl Biacore CM4 sensor chip (60% net negative surface charge reduction). Furthermore, the ConA nonspecific interaction with the matrix was diminished by using high ionic strengths (0.5 m NaC1) in 10 mm phosphate-buffered, 0.005% polysorbate-20 solution, pH 7.0. The CM4 surface abolished nonspecific attachment of ConA in 0.5 m salt, without interfering with the specific interaction of ConA with the peptide mimetics. The flow rate was kept at 50 μl/min unless otherwise noted. Binding was measured at 25 °C. Binding interactions were measured without added bivalent cations nor EDTA nor EGTA. Every binding assay included a reference blank surface but no negative or positive control surfaces. Bound analyte was removed following each binding interaction using duplicate 5–10-μl pulses of regeneration solution at 100 μl/min. Binding as a function of concentration was measured in triplicate samples of ConA at concentrations of 14.4–460 nm per experiment in 2-fold dilution series. The experiment was repeated using two different sensor surfaces and independent MAP immobilizations. The specific binding of ConA to the d-mannosyl epitopes was detected using 3600 RU for 911 and 2600 RU for D002. The alternative experiment, in which biotinylated-ConA was captured on SA surfaces, was carried out in order to compare the influence of orientation on the binding kinetics of ConA to the monomeric and MAP forms of D002 and 911 peptides. Biotinylated ConA was immobilized to an SA-dextran sensor chip at a final density of 1300 RU, and a blank channel was used as a control for buffer bulk shift subtraction. Binding interactions were measured by the injection of 2-fold dilutions of D002 and 911 monomeric peptides in duplicate across the immobilized ConA chip surface, followed by dissociation in running buffer. Steady state affinity analysis of association and dissociation curves was conducted at peptide concentrations ranging between 312.5 nm and 10 μm. Binding of a fixed concentration of ConA (57.5 nm) was competed by increasing concentrations of methyl α-d-mannopyranoside. This concentration was well below the concentration that gives half of the maximum binding signal in the surface titration of D002-binding sites, measured at steady state. To establish the specificity of the competition, lactose was employed in an identical competition experiment as a negative control. All competition experiments were run twice in triplicate. Because both methyl α-d-mannopyranoside and lactose contribute heavily to bulk refractive index changes of the binding signal, all cycles were repeated in the absence of ConA, and the bulk signal of all concentrations of carbohydrate competitor used was subtracted from the signal generated with ConA present. Triplicate bulk data were gathered in a single experiment using the same MAP surfaces as the preceding experiment. Residual ConA binding was measured under mass transport conditions determined by 10× decrease in the flow rate of the sample solution so that the initial rate of binding is reduced as a function of the ConA concentration. The concentration of free ConA in solution at each concentration of methyl α-d-mannopyranoside was calculated by plotting a titration curve from a mass transported dose titration experiment. Modeling to analyze the data assumes all or non-inhibition of the ConA tetramer. Binding of Specific Human Antibodies to MAP-911 and MAP-D002—Specific fractions of human immunoglobulin as well as the respective flow-through fractions (all at 0.67 μm) were passed over MAP-911 and MAP-D002 immobilized on a CM5 chip (Biacore). The nonspecific binding to a channel with no ligand was subtracted from the sensograms. Generation of DCs and Inhibition of GP120 Binding—Peripheral blood monocytes were isolated by Ficoll-Paque gradient centrifugation of heparinized blood obtained from healthy donors. Cells were seeded in R10 (RPMI 1640, 10% fetal bovine serum) media in 6-well tissue culture plates at a density of 5 × 106 cells/well. After 2 h of incubation at 37 °C, nonadherent cells were removed, and the adherent blood monocytes were cultured in R10 supplemented with the following cytokines: granulocyte-macrophage colony-stimulating factor (100 ng/ml; BIOSOURCE) and IL-4 (1000 IU/ml; PeproTech). Cells were fed every other day by removing 1 ml of the medium and adding back 1.5 ml of fresh medium with cytokines (200 ng/ml granulocyte-macrophage colony-stimulating factor and 2000 units/ml IL-4) and used in flow cytometry assay after 6 days. The adhesion assay was done using Vybrant™ cell adhesion assay kit (Molecular Probes inc., Eugene, OR), based on the manufacturer instructions, as described before (19Monzavi-Karbassi B. Luo P. Cunto-Amesty G. Jousheghany F. Pashov A. Weissman D. Kieber-Emmons T. Arch. Virol. 2004; 149: 75-91Crossref PubMed Scopus (14) Google Scholar). For inhibition of GP120 binding to DC, the cells were coincubated with 0.5 μg/ml GP120, which yielded low but detectable binding in flow cytometry, with or without tested serum in 1:100 final dilution. Binding and Competition ELISA—Immulon-2 microtiter plates were coated with 0.1 μg/ml GP120 in 0.05 m NaHCO3, pH 8.5, overnight at 4 °C. All binding and competition experiments were carried out at room temperature. The buffer used to block the nonspecific binding contained 0.5% bovine serum albumin, 0.2% Tween 20 in PBS. The inhibitors were preincubated with the ligand for 1 h before adding to the coated plate for incubation for an additional 2 h. All binding steps were carried out in the same blocking buffer. In the ConA experiment biotinylated ConA was used. The 2G12 binding was demonstrated by an alkaline phosphatase-conjugated anti-human IgG secondary antibody (Sigma), and the ConA-biotin binding was detected by alkaline phosphatase-conjugated streptavidin (Sigma), and 0.1% p-nitrophenyl phosphate in 0.1 m diethanolamine, pH 9.5, was used as a chromogenic substrate and read at 405 nm. For the Man-9 binding experiment, plates were coated overnight at 4 °C with 100 mm of sugar. After blocking the plates (PBS, 0.5% fetal calf serum, and 0.2% Tween 20), serial dilutions of sera were added and resolved with anti-mouse isotype-matched horseradish peroxidase conjugate (Sigma). All results were calculated from triplicate measurements. Statistics—Antibody reactivities and mean fluorescence intensity were compared using Mann-Whitney U test at level of significance p < 0.05. Interactions of ConA with Peptide Mimetics MAP-D002 and MAP-911—The glycan affinity of ConA greatly increases with the number of glycans bound (22Dam T.K. Roy R. Das S.K. Oscarson S. Brewer C.F. J. Biol. Chem. 2000; 275: 14223-14230Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). We have argued that mimicking epitope clustering would be an important general feature in immunogen design (15Cunto-Amesty G. Dam T.K. Luo P. Monzavi-Karbassi B. Brewer C.F. Van Cott T.C. Kieber-Emmons T. J. Biol. Chem. 2001; 276: 30490-30498Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). As multivalent binding depends on the spacing of binding sites and the recognition of ligands presented in particular orientations, we designed a peptide that contains repeats of the RYRY sequence, YRYRYGRYRSGSYRYRYGRYRSGS (referred to as peptide 911). This peptide and its monomeric form YRYRYGRYRSGS display a free energy of association comparable with those reported for core trimannoside-ConA and pentasaccharide-ConA interactions (15Cunto-Amesty G. Dam T.K. Luo P. Monzavi-Karbassi B. Brewer C.F. Van Cott T.C. Kieber-Emmons T. J. Biol. Chem. 2001; 276: 30490-30498Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). To test further the role of conformation in mimotope binding, we incorporated the putative RYRY sequence tract into a cyclic peptide form. This peptide (D002) has the sequence RGGLCYCRYRYCVCVGR, which forms disulfide bridges between the 1st and 4th and between the 2nd and 3rd cysteine residues. Peptide 911 was made as a MAP on an 8-lysine core, whereas the D002 peptide was made as a MAP on a 4-lysine core. To assess binding behavior of the immobilized mannose mimetics, SPR assays were conducted using ConA in solution. The two MAPs studied differed both in cluster structure and monomer properties. The interactions of ConA with the surface immobilized MAPs deviate from simple models as expected; however, the average stability of the complex formed under the same conditions may be compared. The binding experiments were run simultaneously for the MAP-911 and MAP-D002, at surface densities of 2000 and 3600 RU, respectively. The dissociation constant was calculated from the separate fit of the dissociation phase to a single exponential model. At the lowest concentration of ConA, the complexes at the MAP-911 surface were more stable (koff = 0.00179 ± 6.3 × 10–5 s–1) than the complexes formed with MAP-D002 (koff = 0.005 ± 1.4 × 10–5s–1). At the highest ConA concentration, the lectin off-rate from MAP-911 surfaces was faster (koff = 0.00594 ± 1.05 × 10–4 s–1) than at the lower ConA concentration for the same surface (koff = 0.00179 ± 6.3 × 10–5 s–1) and was very similar to that from MAP-D002 surface for the same concentration of ConA (koff = 0.00585 ± 2 × 10–4 s–1). The maximum association responses measured at near equilibrium after 30 min for multiple ConA concentrations indicated these interactions were complex. Scatchard analysis (Fig. 1) of the relationship v/[ConA] = Kd(l-v), where v is the bound fraction of the ligand, is nonlinear for MAP-911 interactions (Hill coefficient >1.3) but hardly deviates from linearity for the MAP-D002 interaction. To demonstrate the complexity of ConA/MAP-911 binding is indeed the consequence of avidity, both MAP-911 and MAP-D002 were immobilized at the same high surface density to minimize the spatial distance of D002-binding sites (23Muller K.M. Arndt K.M. Pluckthun A. Anal. Biochem. 1998; 261: 149-158Crossref PubMed Scopus (111) Google Scholar). However, only ConA-MAP-911 interactions at low and high MAP surface densities were complex and denoted the avidity of the lectin interaction. MAP-D002-ConA interactions were linear despite the possible aggregation artifacts that have been described in interaction analysis by using high surface densities of ligand (24Myszka D.G. J. Mol. Recognit. 1999; 12: 279-284Crossref PubMed Scopus (649) Google Scholar). To dissect the relative importance of the MAP structure, the binding of 911 and D002 monomers to ConA was studied next in the inverse orientation using monomeric 911 and D002. Biotinylated lectin was immobilized on SA-dextran surfaces, and binding of duplicate peptide monomer samples was monitored at increasing concentrations. Because of the high rate of interaction, kinetic parameters could not be determined reliably, but the equilibrium constant was derived instead by steady state affinity analysis at peptide concentrations ranging between 312.5 nm and 10 μm (Fig. 2). The constrained peptide (D002) showed a higher affinity (Kd value of 2.61 × 10–6 m for the D002 versus 1.27 × 10–5 m for the 911 monomer), although the linear peptide (911) presented multiple mimic motifs in its sequence. The Scatchard plot for both 911 and D002 was linear (Fig. 2B) with a Hill coefficient for 911 equal to 1.000008. Competition of ConA Binding to MAPs by α-d-Mannopyranoside—The competition of ConA binding to MAPs by its specific α-d-mannopyranoside ligand in solution was performed by SPR to prove the specificity of the MAP peptide interactions and to estimate the differences in their avidity. The competition analysis included the negati
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