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PubMed2007

The mechanism by which molecules containing the HIV gp41 core-binding motif HXXNPF inhibit HIV-1 envelope glycoprotein-mediated syncytium formation.

The HIV-1 gp41 (glycoprotein 41) core plays a critical role in fusion between the viral and target cell membranes. We previously identified a gp41 core-binding motif, HXXNPF, by screening the phage display peptide libraries. In the present study, we elucidated the mechanism of action of HXXNPF motif-containing molecules of different sizes, including the phage clone L7.8 (a selected positive phage clone), L7.8-g3p* (a 10-kDa fragment of the gene 3 protein) and JCH-4 (a peptide containing 13 residues of L7.8-g3p*), regarding their respective binding abilities to the six-helix bundle and inhibition on syncytium formation at different temperatures. We found that all of the HXXNPF motif-containing molecules could bind to the gp41 core, and that their binding sites may be located in the N-helix domain. L7.8-g3p* and JCH-4 effectively inhibited HIV-1 Env (envelope glycoprotein)-mediated syncytium formation at 37 degrees C, while the phage clone L7.8 showed no inhibition under the same conditions. However, at suboptimal temperature (31.5 degrees C), all of these HXXNPF motif-containing molecules were capable of inhibiting syncytium formation. These results suggest that these HXXNPF motif-containing molecules mainly bind to the gp41 core and stop the fusion process mediated by the fusion-active core, resulting in inhibition of HIV-1 fusion and entry. The HXXNPF motif-containing molecules may be used as probes for studying the role of the HIV-1 gp41 core in the late stage of the membrane-fusion process.

3T3 Cells↗

PubMed2006

Decreased specific CD8+ T cell cross-reactivity of antigen recognition following vaccination with Melan-A peptide.

The aim of T cell vaccines is the expansion of antigen-specific T cells able to confer immune protection against pathogens or tumors. Although increase in absolute cell numbers, effector functions and TCR repertoire of vaccine-induced T cells are often evaluated, their reactivity for the cognate antigen versus their cross-reactive potential is rarely considered. In fact, little information is available regarding the influence of vaccines on T cell fine specificity of antigen recognition despite the impact that this feature may have in protective immunity. To shed light on the cross-reactive potential of vaccine-induced cells, we analyzed the reactivity of CD8(+) T cells following vaccination of HLA-A2(+) melanoma patients with Melan-A peptide, incomplete Freund's adjuvant and CpG-oligodeoxynucleotide adjuvant, which was shown to induce strong expansion of Melan-A-reactive CD8(+) T cells in vivo. A collection of predicted Melan-A cross-reactive peptides, identified from a combinatorial peptide library, was used to probe functional antigen recognition of PBMC ex vivo and Melan-A-reactive CD8(+) T cell clones. While Melan-A-reactive CD8(+) T cells prior to vaccination are usually constituted of widely cross-reactive naive cells, we show that peptide vaccination resulted in expansion of memory T cells displaying a reactivity predominantly restricted to the antigen of interest. Importantly, these cells are tumor-reactive.

Antigens, Neoplasm↗

PubMed2002

Affinity purification of polyclonal antibodies using a new all-D synthetic peptide ligand: comparison with protein A and protein G.

This study investigates the applicability of the protein A Mimetic (PAM) affinity ligand, obtained from the screening of a multimeric combinatorial peptide library, in immunoglobulin isolation from serum. To avoid protease degradation, the ligand has been substituted by its inverso form, named D-PAM, synthesized by replacing all amino acids with the corresponding D derivatives. D-PAM affinity columns, prepared by immobilizing the all-D peptide on the commercially available support Emphaze, were able to capture IgG directly from the serum in a single chromatographic step, with a recovery yield ranging from 60% to 90%, a purity degree higher than 90%, and with a full recovery of antibody activity. Column capacity, determined by applying a large excess of purified IgG to 1 ml bed volume column, was close to 52 mg/ml for bovine IgG, 58 mg/ml for goat IgG, 66 mg/ml for horse IgG, 50 mg/ml for human IgG, 52 mg/ml for mouse IgG, 36 mg/ml for rabbit IgG and 48 mg/ml for sheep IgG. D-PAM peptide was found to be very stable to protease activity, and after prolonged incubation with mouse serum. Similarly, the corresponding derivatized matrix tested before and after various treatments, including sanitization and autoclaving procedures maintained its IgG binding properties, thus indicating a very high stability in terms of ligand leakage and degradation.

Adaptor Proteins, Signal Transducing↗

PubMed2007

Specific binding of GM1-binding peptides to high-density GM1 in lipid membranes.

The ganglioside Galbeta1-3GalNAcbeta1-4(Neu5Acalpha2-3)Galbeta1-4Glcbeta1-1'Cer (GM1) is an important receptor. We have previously identified GM1-binding peptides based on affinity selection from a random peptide library. In the present study, we determined the amino acids essential for binding GM1 and investigated the specific interaction with GM1 in the lipid membrane. Arginines and aromatic amino acids in the consensus sequence (W/F)RxL(xP/Px)xFxx(Rx/xR)xP contributed to the ability of the peptides to bind GM1. The peptide p3, VWRLLAPPFSNRLLP, having the consensus sequence, showed high affinity for GM1 with a dissociation constant of 1.2 microM. Furthermore, the density-dependent binding of p3 was investigated using mixed monolayers of GM1 and Glcbeta1-1'Cer (GlcCer). p3 binds preferentially to high-density GM1, and its interaction with GM1 was found to be cooperative based on a Hill plot. These results indicated that a lateral assembly of GM1 molecules was required for the recognition of carbohydrates by p3. The GM1-binding peptide played a role as a unique anti-GM1 probe differing from the cholera toxin B subunit or antibodies.

Amino Acid Sequence↗

PubMed1998

A basis for alloreactivity: MHC helical residues broaden peptide recognition by the TCR.

The high frequency of alloreactive T cells is a major hindrance for transplantation; however, the molecular basis for alloreactivity remains elusive. We examined the I-Ep alloreactivity of a well-characterized Hb(64-76)/I-Ek-specific murine T cell. Using a combinatorial peptide library approach, we identified a highly stimulatory alloepitope mimic and observed that the recognition of the central TCR contact residues (P3 and P5) was much more flexible than that seen with Hb(64-76)/I-Ek, but still specific. Therefore, alloreactive T cells can recognize a self-peptide/MHC surface; however, the allogeneic MHC molecule changes the recognition requirements for the central region of the peptide, allowing a more diverse repertoire of ligands to be recognized.

Animals↗

PubMed2002

The thioesterase domain from a nonribosomal peptide synthetase as a cyclization catalyst for integrin binding peptides.

Nonribosomal peptide synthetases responsible for the production of macrocyclic compounds often use their C-terminal thioesterase (TE) domain for enzymatic cyclization of a linear precursor. The excised TE domain from the nonribosomal peptide synthetase responsible for the production of the cyclic decapeptide tyrocidine A, TycC TE, retains autonomous ability to catalyze head-to-tail macrocyclization of a linear peptide thioester with the native sequence of tyrocidine A and can additionally cyclize peptide analogs that incorporate limited alterations in the peptide sequence. Here we show that TycC TE can catalyze macrocyclization of peptide substrates that are dramatically different from the native tyrocidine linear precursor. Several peptide thioesters that retain a limited number of elements of the native peptide sequence are shown to be substrates for TycC TE. These peptides were designed to integrate an Arg-Gly-Asp sequence that confers potential activity in the inhibition of ligand binding by integrin receptors. Although enzymatic hydrolysis of the peptide thioester substrates is preferred over cyclization, TycC TE can be used on a preparative scale to generate both linear and cyclic peptide products for functional characterization. The products are shown to be inhibitors of ligand binding by integrin receptors, with cyclization and N(alpha)-methylation being important contributors to the nanomolar potency of the best inhibitors of fibrinogen binding to alpha IIb beta 3 integrin. This study provides evidence for TycC TE as a versatile macrocyclization catalyst and raises the prospect of using TE catalysis for the generation of diverse macrocyclic peptide libraries that can be probed for novel biological function.

Amino Acid Sequence↗

PubMedDate not supplied

Specificity of human cathepsin S determined by processing of peptide substrates and MHC class II-associated invariant chain.

Cathepsin S (CatS) is a lysosomal cysteine protease of the papain family, the members of which possess relatively broad substrate specificities. It has distinct roles in major histocompatibility complex (MHC) class II-associated peptide loading and in antigen processing in both the MHC class I and class II pathways. It may therefore represent a target for interference with antigen presentation, which could be of value in the therapy of (auto)immune diseases. To obtain more detailed information on the specificity of CatS, we mapped its cleavage site preferences at subsites S3-S1' by in vitro processing of a peptide library. Only five amino acid residues at the substrate's P2 position allowed for cleavage by CatS under time-limited conditions. Preferences for groups of amino acid residues were also observed at positions P3, P1 and P1'. Based on these results, we developed highly CatS-sensitive peptides. After processing of MHC class II-associated invariant chain (Ii), a natural protein substrate of CatS, we identified CatS cleavage sites in Ii of which a majority matched the amino acid residue preference data obtained with peptides. These observed cleavage sites in Ii might be of relevance for its in vivo processing by CatS.

Amino Acid Sequence↗

PubMed2000

A peptide mimic of E-selectin ligand inhibits sialyl Lewis X-dependent lung colonization of tumor cells.

Selectins bind to carbohydrate ligands in a calcium-dependent manner and play critical roles in host defense and possibly in tumor metastasis. To isolate peptides that mimic E-selectin ligands, we screened a phage peptide library using E-selectin as a target molecule. This attempt unexpectedly failed, probably because the binding affinity of E-selectin to its ligand is low. We then took an approach that is analogous to the isolation of anti-idiotype antibodies and were able to isolate peptides that bound to anticarbohydrate antibodies recognizing E-selectin ligands. These peptides, enriched for their binding to anti-Lewis A antibody, were found to bind to E-, P- and L-selectins in a calcium-dependent manner. Phage harboring the identified peptide IELLQAR and synthetic peptides having the same sequence inhibited the binding of sialyl Lewis X or sialyl Lewis A oligosaccharides to E-selectin. The adhesion of HL-60 and B16 melanoma cells expressing sialyl Lewis X to E-selectin was also inhibited by the phage-displaying IELLQAR peptide. Moreover, i.v. injected IELLQAR peptide inhibited the lung colonization of mouse B16 melanoma and human lung tumor cells expressing sialyl Lewis X. These results demonstrate that it is possible to isolate peptides mimicking carbohydrate ligands by screening the peptides for binding to anticarbohydrate antibodies and then using them to inhibit carbohydrate-dependent experimental tumor metastasis.

Animals↗

PubMed1998

Method to compare collision-induced dissociation spectra of peptides: potential for library searching and subtractive analysis.

We report the development of a method to compare collision-induced dissociation (CID) spectra of peptides. This method employs a cross-correlation analysis of a CID spectrum to a reference spectrum and normalizes the cross-correlation score to the autocorrelation of the CID spectra. The query spectrum is compared by using both mass information and fragmentation patterns. Fragmentation patterns are compared to each other using a correlation function. To evaluate the specificity of the approach, a set of 2180 tandem mass spectra obtained from both triple-quadrupole tandem mass spectrometers (TSQ) and quadrupole ion trap mass spectrometers (LCQ) was created. Comparisons are performed between tandem mass spectra obtained on the same instrument type as well as between different instrument types. Accurate and reliable comparisons are demonstrated in both types of analyses. The scores obtained in the cross-comparison of TSQ and LCQ tandem mass spectra of the same peptide are found to be slightly lower than comparisons performed with spectra obtained on the same instrument type. The method appears insensitive to variations in day-to-day performance of the instrument, minor variations in fragment ion abundance, and instrumental differences inherent in the same instrument model. The use of this method of comparison is demonstrated for library searching and subtractive analysis of tandem mass spectra obtained during LC/MS/MS experiments.

Algorithms↗

PubMed1995

P-glycoprotein epitope mapping. II. The murine monoclonal antibody MM6.15 to human multidrug-resistant cells binds with three distinct loops in the MDR1-P-glycoprotein extracellular domain.

A new murine monoclonal antibody (MAb), MM6.15, to human MDR1 P-glycoprotein was found to be reactive in ELISA with synthetic peptides selected from the predicted sequences of the first, fourth and sixth extracellular loop of MDR1-P-glycoprotein. In order to precisely define the MM6.15-binding site, a peptide library of overlapping 5- to 9-mer residues covering the entire sixth extracellular loop of both human and rodent class-1 P-glycoproteins was synthesized on polyethylene pins and tested for MAb binding. The results of this ELISA demonstrated that the MAb MM6.15 reacts only with human synthetic peptides and that the critical component of the MAb recognition is made up of the amino-acid sequence LVAHKL (residues 963-968 of the MDR1-P-glycoprotein) with histidine (H), lysine (K) and possibly leucine (L), key residues of this immunogenic domain.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2003

Beta 2-microglobulin-free HLA class I heavy chain epitope mimicry by monoclonal antibody HC-10-specific peptide.

mAb HC-10 loses its reactivity with HLA class I (HLA-I) H chain (HC) following its association with beta(2)-microglobulin (beta(2)m). Furthermore, the HC-10 defined epitope appears to be involved in the pathogenesis of spondyloarthropathies, because HC-10 reduced their incidence in HLA-B27(+)beta(2)m degrees /MHC class II knockout mice. This study has characterized the determinant recognized by HC-10. Panning of a phage display peptide library with HC-10 resulted in isolation of the motif PxxWDR, which could be aligned with P57, W60, D61, and R62 of the first domain of the HLA-I HC allospecificities reactive with HC-10. The (55)EGPEYWDR(N/E)T(64) (p-1) is the shortest motif-bearing peptide that reacts with HC-10 and inhibits its binding to soluble HLA-B7 HC, irrespective of whether N (p-1a) or E (p-1b) is present at position 63. By contrast, HC-10 did not react with six additional peptides, each bearing motif amino acid substitutions present in HC-10-not-reactive HLA-I allospecificities. The p-1-derived Qp-1, synthesized with the additional conserved Q54, which displays the highest in vitro reactivity with HC-10, was the only one to induce in mice IgG resembling HC-10 in their fine specificity. Mapping of the HC-10-defined determinant suggests that the lack of mAb reactivity with beta(2)m-associated HLA-I HC is caused by blocking by the peptide in the groove of beta(2)m-associated HLA-I HC, though a role of HC conformational changes following its association with beta(2)m cannot be excluded. This information contributes to our understanding of the molecular basis of the antigenic profiles of beta(2)m-free and beta(2)m-associated HLA-I HC and may serve to develop active specific immunotherapy of spondyloarthropathies.

Amino Acid Motifs↗

PubMed1998

Affinity purification of immunoglobulin M using a novel synthetic ligand.

While monoclonal antibodies of the G class can be conveniently purified by affinity chromatography using immobilized protein A or G, even on a large scale, scaling up IgM purification still presents several problems, since specific and cost-effective ligands for IgM are not available. A synthetic peptide (TG19318), deduced from the screening of a combinatorial peptide library, was characterized previously by our group for its binding properties for immunoglobulins of the G class and its applicability as a synthetic ligand for polyclonal and monoclonal IgG purification, from sera or cell culture supernatants. In this study, we have examined the ligand recognition properties for IgM, immobilizing the synthetic peptide on different affinity supports and examining its ability to purify IgMs from serum, ascitic fluid and cell culture supernatants. TG19318 affinity columns proved useful for a very convenient one-step purification of monoclonal IgMs directly from crude sources, loading the samples on the columns equilibrated with saline buffers at pH values ranging from 5 to 7, and eluting adsorbed IgM by a buffer change to 0.1 M acetic acid or 0.05-0.1 M sodium bicarbonate, pH 9.0. Antibody purity after affinity purification was very high, close to 85-95%, as determined by densitometric scanning of sodium dodecyl sulfate-polyacrylamide gels of purified fractions, and by gel permeation analysis. Antibody activity was fully recovered after purification, as determined by immunoassays. Column capacity was related to the type of support used for ligand immobilization, and ranged from 2 to 8 mg of IgM/ml of support.

Animals↗

PubMed1999

Anti-free prostate-specific antigen monoclonal antibody epitopes defined by mimotopes and molecular modeling.

BACKGROUND: Prostate-specific antigen (PSA) is an important marker for the diagnosis and management of prostate cancer, and the free PSA/total PSA ratio has been shown to be efficient for distinguishing prostate cancer from benign prostatic hyperplasia. We report here the characterization of seven mouse monoclonal antibodies (mAbs) and the partial localization of two conformational epitopes identified by anti-free PSA mAbs. METHODS: The mAbs were studied by competition and sandwich assays, and the epitope localization of the two anti-free PSA mAbs (6C8D8 and 5D3D11) was performed using phage displayed peptide libraries and molecular modeling. RESULTS: The seven mAbs were classified into three groups according to their recognition specificities and their ability to inhibit the enzymatic activity of PSA and the formation of PSA-alpha1-antichymotrypsin (ACT) complex. Among the anti-free PSA mAb group, 6C8D8 recognized the phage displayed peptide RKLRPHWLHFHPVAV, two parts of which presented similarities with two regions distant on the PSA sequence but joined in the tridimensional structure. mAb 5D3D11 recognized the peptide DTPYPWGWLLDEGYD, which is similar to a PSA region located on the board of the groove containing the PSA enzymatic site. Both epitopes were located in the theoretical ACT binding site described previously. Moreover, these mAbs were able to inhibit the enzymatic activity of PSA. CONCLUSIONS: These epitope localizations are in agreement with the ability of both mAbs to inhibit enzymatic activity and ACT fixation. The results presented here could bring information for the generation of clinically relevant PSA assays.

Amino Acid Sequence↗

PubMed2002

T cell response to preproinsulin I and II in the nonobese diabetic mouse.

Immunization against insulin, insulin B chain, or B chain peptide B(9-23) (preproinsulin peptide II(33-47)) prevents diabetes in the nonobese diabetic (NOD) mouse. Whether or not peptide II(33-47) is the only proinsulin determinant recognized by CD4 T cells remains unclear. Using two peptide libraries spanning the entire sequence of preproinsulin I and preproinsulin II, respectively, we identified T cells specific for four proinsulin epitopes within the islet cell infiltrate of prediabetic female NOD mice. These epitopes were among immunogenic epitopes to which a T cell response was detected after immunization of NOD mice with individual peptides in CFA. Immunogenic epitopes were found on both isoforms of insulin, especially proinsulin II, which is the isoform expressed in the thymus. The autoimmune response to proinsulin represented only part of the immune response to islet cells within the islet cell infiltrate in 15-wk-old NOD mice. This is the first systematic study of preproinsulin T cell epitopes in the NOD mouse model.

Amino Acid Sequence↗

PubMed2006

Selection of peptides for lipopolysaccharide binding on to epoxy beads and selective detection of Gram-negative bacteria.

Lipopolysaccharide (LPS)-binding peptides were enriched by using epoxy beads as a novel support to immobilize LPS for a phage displayed peptide library screening. The sequence of Phe-Ala-Pro-Trp (FAPW) was the most significant consensus motif of 10 selected clones, and Pro-Phe (PF) was the key dipeptide for binding at the apex of the loop to form a characteristic structure of CXXPFXXXC. Moreover, AWLPWAK, one of the highly conserved heptamer peptides, could detect specifically Gram-negative bacteria via a whole cell binding test at 10(6) cells ml(-1).

Bacterial Typing Techniques↗

PubMed2004

Target specificity analysis of the Abl kinase using peptide microarray data.

Protein kinases play an important role in cellular signalling. The reliable prediction of their substrates is of high importance for the deciphering of signalling pathways. A recently developed peptide microarray technology for the charcterisation of protein kinases delivers data on the individual phosphorylation status of each single member of a large peptide library. This data can be used to approximate the substrate specificity of the investigated kinase. We present an approach to process the collected information using a combination of a weight matrix approach and a nearest neighbor approach. Experiments with the protein-tyrosine kinase Abl are conducted to validate the results. Randomly selected peptides (1433) are used to estimate the substrate preferences of the kinase. The obtained prediction results are compared with standard methods. The new approach is tested further on bona fide Abl phosphorylation sites.

Binding Sites↗

PubMed1997

Confronting the degeneracy of convergent combinatorial immunogens, or 'mixotopes', with the specificity of recognition of the target sequences.

Immunization by convergent combinatorial peptide libraries, or 'mixotopes' represents an interesting approach for inducing broadly cross-reactive immune response to hypervariable pathogens. The authors have immunized rabbits with a series of eight HIV-1 V3-loop derived constructs of increasing complexity, and analysed the reactivity of the corresponding antisera towards a set of V3-related peptides. Results were surprisingly homogeneous. Mixotopes containing as many as several billion closely related combinatorial sequences were immunogenic, and able to induce V3-specific antibodies. These results suggest that serological cross-reactivity depends on the sequential similarity of the antigen with the parent immunogen. Such 'mixotopes' could represent a useful approach to vaccination against hypervariable pathogens.

AIDS Vaccines↗

PubMed2006

Conformational selectivity of peptides for single-walled carbon nanotubes.

Carbon nanotubes show promising prospects for applications ranging from molecular electronics to ultrasensitive biosensors. An important aspect to understanding carbon nanotube properties is their interactions with biomolecules such as peptides and proteins, as these interactions are important in our understanding of nanotube interactions with the environment, their use in cellular systems, as well as their interface with biological materials for medical and diagnostic applications. Here we report the sequence and conformational requirements of peptides for high-affinity binding to single-walled carbon nanotubes (SWNTs). A new motif, X(1)THX(2)X(3)PWTX(4), where X(1) is G or H, X(2) is H or D or null, X(3) is null or R, and X4 is null or K, was identified from two classes of phage-displayed peptide libraries. The high affinity binding of the motif to SWNTs required constrained conformations which were achieved through either the extension of the amino acid sequence (e.g., LLADTTHHRPWT) or the addition of a constrained disulfide bond (e.g., CGHPWTKC). This motif shows specific high-affinity to the currently studied SWNTs, compared to previously reported peptides. The conformations of the identified peptides in complex with SWNTs were also characterized with a variety of biophysical methodologies including CD, fluorescence, NMR spectroscopy, and molecular modeling.

Amino Acid Sequence↗