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1.
Detection of antibodies in serum has many important applications. Our goal was to develop a facile general experimental approach for identifying antibody-specific peptide ligands that could be used as the reagents for antibody detection. Our emphasis was on an approach that would allow identification of peptide ligands for antibodies in serum without the need to isolate the target antibody or to know the identity of its antigen. We combined ribosome display (RD) with the analysis of peptide libraries by next generation sequencing (NGS) of their coding RNA to facilitate identification of antibody-specific peptide ligands from random sequence peptide library. We first demonstrated, using purified antibodies, that with our approach-specific peptide ligands for antibodies with simple linear epitopes, as well as peptide mimotopes for antibodies recognizing complex epitopes, were readily identified. Inclusion of NGS analysis reduced the number of RD selection rounds that were required to identify specific ligands and facilitated discrimination between specific and spurious nonspecific sequences. We then used a model of human serum spiked with a known target antibody to develop NGS-based analysis that allowed identification of specific ligands for a target antibody in the context of an overwhelming amount of unrelated immunoglobins present in serum. 相似文献
2.
K R Prilliman K W Jackson M Lindsey J Wang D Crawford W H Hildebrand 《Journal of immunology (Baltimore, Md. : 1950)》1999,162(12):7277-7284
Therapies to elicit protective CTL require the selection of pathogen- and tumor-derived peptide ligands for presentation by MHC class I molecules. Edman sequencing of class I peptide pools generates "motifs" that indicate that nonameric ligands bearing conserved position 2 (P2) and P9 anchors provide the optimal search parameters for selecting immunogenic epitopes. To determine how well a motif represents its individual constituents, we used a hollow-fiber peptide production scheme followed by the mapping of endogenously processed class I peptide ligands through reverse-phase HPLC and mass spectrometry. Systematically mapping and characterizing ligands from B*1508, B*1501, B*1503, and B*1510 demonstrate that the peptides bound by these B15 allotypes i) vary in length from 7 to 12 residues, and ii) are more conserved at their C termini than their N-proximal P2 anchors. Comparative peptide mapping of these B15 allotypes further pinpoints endogenously processed ligands that bind to the allotypes B*1508, B*1501, and B*1503, but not B*1510. Overlapping peptide ligands are successful in binding to B*1501, B*1503, and B*1508 because these B15 allotypes share identical C-terminal anchoring pockets whereas B*1510 is divergent in the C-terminal pocket. Therefore, endogenous peptide loading into the B15 allotypes requires that a conserved C terminus be anchored in the appropriate specificity pocket while N-proximal anchors are more flexible in their location and sequence. Queries for overlapping and allele-specific peptide ligands may thus be contingent on a conserved C-terminal anchor. 相似文献
3.
This paper describes direct binding of a small vancomycin to peptide ligands immobilized on a sensor chip using quartz crystal microbalance. In this study, the binding ligands were composed of three components: a molecular recognition element (peptide), a conformationally flexible and hydrophilic linker, and a long-chain alkanethiol. These peptide ligands were used to establish the well-packed, self-assembled monolayers on quartz chips and could be readily synthesized using conventional organic chemistry protocols. Results of quartz crystal microbalance measurements showed that vancomycin specifically associated with the d-Ala-d-Ala-containing peptide with an affinity of 3.2+/-0.3 microM and was, as expected, completely inactive to the self-assembled monolayer presenting l-Ala-l-Ala peptide. The dissociation constant obtained correlated well with values reported in literature and was further confirmed by surface plasmon resonance measurement (2.7+/-0.7 microM). The technique used in this study should be applicable to both peptidyl and nonpeptidyl ligands of greater complexity than that used here. This method is practical, it provides quantitative binding information, and complicated analysis is avoided. 相似文献
4.
One main issue with peptide-based molecular imaging probes is their relatively low tumor affinity and short retention time.
To improve peptide binding affinity, multivalency approach has been introduced. Traditionally, this approach involves the
use of peptide homodimers or homomultimers in which peptide ligands of the same type are constructed with suitable linkers.
Recently, a new approach using peptide heterodimers has emerged as a promising method for targeting multi-receptor over-expressed
tumor cells. Significant affinity enhancements have been observed with peptide heterodimers compared with their parent peptide
monomers. In a peptide heterodimer, two different peptide ligands capable of targeting two different receptors are covalently
linked. The binding modes of peptide heterodimers can be monovalent or bivalent depending on whether simultaneous binding
of two ligands can be achieved. Increased local ligand concentration and improved binding kinetics contribute to enhanced
binding in both monovalent- and bivalent binding modes, while multivalency effect also plays an important role in bivalent
binding mode. As many tumors overexpress multiple receptors, more peptide heterodimer-based molecular imaging probes are expected
to be developed in future. This review article will discuss the peptide homodimers and heterodimers for molecular imaging
with special emphasis on peptide heterodimers. 相似文献
5.
A technical challenge in the development of biosensor devices for cancer detection and diagnosis is the identification of ligands that recognize cancer cells with high affinity and specificity. Furthermore, it is unlikely that one cell-binding ligand will provide sufficient biological information, thus, multiple ligands for a given cancer type will be needed for confident clinical diagnosis. Biopanning of phage displayed peptide libraries is a route to isolation of specific cell-binding reagents. A potential approach towards isolation of multiple ligands for a single cell type is to pan against the same cell type using different peptide libraries. Here we report the synthesis of a new 20-mer peptide-phage library and its use to select a peptide that binds to the large cell lung carcinoma cell line, H1299. The isolated phage clone binds H1299 cells 80 times better than a control phage and can distinguish between H1299 and normal control cells. The phage clone also binds to the lung pleura epidermoid cell line, Calu-1 but not to all lung carcinoma cell lines. The peptide is functional outside the context of the phage and tetramerization of the peptide on a trilysine core improves the affinity of the peptide. The tetrameric peptide can be used to deliver a fluorescent quantum dot to H1299 cells. Unexpectedly, the peptide shares sequence similarity to a previously isolated H1299-binding peptide isolated from a different 20-mer peptide library. Data suggests that the two peptides target the same cellular receptor. Our results imply that cell-based biopanning can isolate cell-binding ligands that may be of utility for cancer diagnosis, and isolation of cell-targeting peptides from different peptide libraries can expand the repertoire of cell-binding reagents. 相似文献
6.
Yumiko Mizukoshi Michiko Nagasu Ichio Shimada Hideo Takahashi 《Journal of biomolecular NMR》2010,46(4):299-305
Structural determination of target-bound conformations of peptides is of primary importance for the optimization of peptide
ligands and peptide–mimetic design. In the structural determination of weakly binding ligands, transferred nuclear Overhauser
effect (TrNOE) methods have been widely used. However, not many distance constraints can be obtained from small peptide ligands
by TrNOE, especially for peptides bound to a target molecule in an extended conformation. Therefore, for precise structural
determination of weakly binding peptides, additional structural constraints are required. Here, we present a strategy to systematically
introduce dihedral angle constraints obtained from multiple transferred cross-correlated relaxation experiments and demonstrate
precise structures of weakly binding peptides. As a result, we could determine the bioactive conformations of phage-derived
peptide ligands and define their core binding motifs. 相似文献
7.
Wilson DB Pinilla C Wilson DH Schroder K Boggiano C Judkowski V Kaye J Hemmer B Martin R Houghten RA 《Journal of immunology (Baltimore, Md. : 1950)》1999,163(12):6424-6434
Recent studies have demonstrated the utility of synthetic combinatorial libraries for the rapid identification of peptide ligands that stimulate clonotypic populations of T cells. Here we screen a decapeptide combinatorial library arranged in a positional scanning format with two different clonotypic populations of CD4+ T cells to identify peptide epitopes that stimulate proliferative responses by these T cells in vitro. An extensive collection of mimic peptide sequences was synthesized and used to explore the fine specificity of TCR/peptide/MHC interactions. We also demonstrate that many of these deduced ligands are not only effective immunogens in vivo, but are capable of inducing T cell responses to the original native ligands used to generate the clones. These results have significant implications for considerations of T cell specificity and the design of peptide vaccines for infectious disease and cancer using clinically relevant T cell clones of unknown specificity. 相似文献
8.
While typical intracellular protein modules have only one ligand-binding site, there are rare examples of single modules that bind two different ligands at distinct binding sites. Here we present a detailed mutational and energetic analysis of one such domain, the phosphotyrosine binding (PTB) domain of Disabled-1 (Dab1), which binds to both peptide and phosphoinositide (PI) ligands simultaneously at structurally distinct binding sites. Through the techniques of isothermal titration calorimetry (ITC), analysis of Dab1 PTB domain mutants, and nuclear magnetic resonance (NMR), we have evaluated the characteristics of binding of the Dab1 PTB domain to various peptide and PI ligands. These studies reveal that the presence of saturating concentrations of one ligand has little effect on the binding constant for a second ligand at the other site. In addition, proteins with single-point mutations in the peptide-binding site retain native affinity for PI ligands, while proteins with mutations that prevent PI binding retain native affinity for peptide. NMR titrations show that the final structure of the ternary complex is the same independent of the order of addition of the two ligands. Together, these studies show that binding of peptide and PI ligands is energetically independent and noncooperative. 相似文献
9.
The Ag receptor of cytotoxic CD8+ T lymphocytes recognizes peptides of 8-10 aa bound to MHC class I molecules. This Ag recognition event leads to the activation of the CD8+ lymphocyte and subsequent lysis of the target cell. Altered peptide ligands are analogues derived from the original antigenic peptide that commonly carry amino acid substitutions at TCR contact residues. TCR engagement by these altered peptide ligands usually impairs normal T cell function. Some of these altered peptide ligands (antagonists) are able to specifically antagonize and inhibit T cell activation induced by the wild-type antigenic peptide. Despite significant advances made in understanding TCR antagonism, the molecular interactions between the TCR and the MHC/peptide complex responsible for the inhibitory activity of antagonist peptides remain elusive. To approach this question, we have identified altered peptide ligands derived from the vesicular stomatitis virus peptide (RGYVYQGL) that specifically antagonize an H-2Kb/vesicular stomatitis virus-specific TCR. Furthermore, by site-directed mutagenesis, we altered single amino acid residues of the complementarity-determining region 3 of the beta-chain of this TCR and tested the effect of these point mutations on Ag recognition and TCR antagonism. Here we show that a single amino acid change on the TCR CDR3 beta loop can modulate the TCR-antagonistic properties of an altered peptide ligand. Our results highlight the role of the TCR complementarity-determining region 3 loops for controlling the nature of the T cell response to TCR/altered peptide ligand interactions, including those leading to TCR antagonism. 相似文献
10.
Improved substrate specificity of water-soluble pyrroloquinoline quinone glucose dehydrogenase by a peptide ligand 总被引:1,自引:0,他引:1
A new approach in altering the substrate specificity of enzyme is proposed using glucose dehydrogenase, with pyrroroquinoine quinone (PQQGDH) as co-factor, as the model. This approach is based on the selection of random peptide phage displayed library. Using an M13 phage-display random peptide library, we have selected peptide ligands. Among the peptide ligands, a 7-mer peptide, composed of Thr-Thr-Ala-Thr-Glu-Tyr-Ser, caused PQQGDH substrate specificity to decrease significantly toward disaccharides, such as maltose and lactose, while a smaller effect was observed toward glucose. Consequently, this peptide narrowed the substrate specificity of PQQGDH, without a significant loss of the enzyme activity. 相似文献
11.
Kanako Arai Hiroshi Tsutsumi Hisakazu Mihara 《Bioorganic & medicinal chemistry letters》2013,23(17):4940-4943
A monosaccharide-modified β-loop peptide library displayed on phage has been constructed and used for the screening of glycopeptide ligands against a carbohydrate-binding protein. The β-loop peptide library was designed and modified with a mannose derivative on phage. The glycopeptide ligands to concanavalin A (ConA), a mannose-binding protein, were obtained from the mannose-modified peptide phage library. The amino acids neighboring the mannose unit of glycopeptides not only reinforced the binding affinity but also gave diverse binding characteristics. 相似文献
12.
Katz BA 《Biomolecular engineering》1999,16(1-4):57-65
Structural and mechanistic determinants of affinity of streptavidin-binding peptide ligands discovered by phage display are reviewed along with the use of streptavidin as a paradigm for structure-based design. A novel way of producing protein-dimerizing ligands in the streptavidin model system is discussed, in which crystal packing topochemically mediates or even catalyzes dimerization of adjacent bound ligands whose reactive ligating groups are presented toward one another in productive orientations in the crystal lattice. Finally, through crystallography on a set of streptavidin complexes with small molecule and peptide ligands at multiple pHs in two space groups, the mechanism by which ligands enhance intersubunit stabilization of the streptavidin tetramer is probed. 相似文献
13.
Toebes M Coccoris M Bins A Rodenko B Gomez R Nieuwkoop NJ van de Kasteele W Rimmelzwaan GF Haanen JB Ovaa H Schumacher TN 《Nature medicine》2006,12(2):246-251
Major histocompatibility complex (MHC) class I molecules associate with a variety of peptide ligands during biosynthesis and present these ligands on the cell surface for recognition by cytotoxic T cells. We have designed conditional MHC ligands that form stable complexes with MHC molecules but degrade on command, by exposure to a defined photostimulus. 'Empty MHC molecules' generated in this manner can be loaded with arrays of peptide ligands to determine MHC binding properties and to monitor antigen-specific T-cell responses in a high-throughput manner. We document the value of this approach by identifying cytotoxic T-cell epitopes within the H5N1 influenza A/Vietnam/1194/04 genome. 相似文献
14.
Discovery of high-affinity peptide ligands for vancomycin 总被引:1,自引:0,他引:1
Vancomycin, an important antibiotic against medically relevant gram-positive bacteria such as methicillin-resistant Staphylococcus aureus, exerts its antibacterial effects by binding with moderate affinity to the C-terminal Lys-D-Ala-D-Ala motif (Kaa) of the bacterial cell wall peptide precursor. Essential for Kaa binding to vancomcyin is the free-carboxyl group on the terminal D-Ala in Kaa. In efforts to identify other Kaa-based peptides which bind vancomycin with higher affinity, we utilized our one-bead-one-compound (OBOC) combinatorial library approach, a method which has been widely used to discover highly specific ligands against various receptors. In standard OBOC peptide libraries, the C-terminal end of the synthesized peptide is tethered to a solid-support/resin, however, this study reports development of a synthetic strategy for generating OBOC peptide libraries with a free D-Ala-D-Ala carboxyl end. We screened these "OBOC inverted" peptide libraries against vancomycin, and discovered a series of peptide ligands with strong consensus, which bind vancomycin. To further optimize these ligands, two highly focused Kaa-containing OBOC combinatorial peptidomimetic libraries were designed, synthesized, and screened against vancomycin under more stringent conditions. Peptidomimetic ligands which bind vancomycin with higher affinity than Kaa were identified. The dissociation constant of one of these ligands, Lys(Ac)-HOCit-Glu-Cha-Lys(3,5-dihydroxybenzoyl)-D-Ala-D-Ala (9), as determined by surface plasmon resonance, was 1.03 microM, roughly a 50-fold improvement in affinity compared to Kaa (K(D) = 50 microM). 相似文献
15.
DNA display for in vitro selection of diverse peptide libraries 总被引:2,自引:0,他引:2
16.
The G-protein coupled seven transmembrane domain receptors bind a wide variety of ligands of different molecular size ranging from small monoamines to large neuropeptides and peptide hormones. This review summarises data from studies on the localisation of the binding site for a few neuropeptides in their receptors and compares this to the binding pockets for non peptide ligands. The main conclusion is that neuropeptide binding involves residues on the top of several transmembrane domains and in extracellular loops of the receptors while the non peptide type ligands to the same receptors tend to bind deeper in the plane of the membrane, between several transmembrane domains—similarly to monoamines. Thus the antagonism exerted by most of the non peptide type ligands is an allosteric phenomenon whereby binding of these to another site than the peptide binding site stabilises a non agonist binding, and for signalling inactive, conformation of the 7 TM receptor. 相似文献
17.
Heterogeneity of the vasculature in different organs has been well documented by the method of in vivo phage display. Using this technology, several peptide ligands that home to tissue-specific vascular endothelial cell have been isolated. Such peptide ligands directed against specific vascular surface molecules can be used as targeted therapeutic compounds or imaging agents to the vasculature of the specific organ in vivo. In this study, the authors perform in vivo selection in mice using a phage display random peptide library and separated phage peptides homing to mouse thymus by 3 rounds of in vivo panning. Sequence analysis showed that CHAQGSAEC is the dominant peptide sequence. Immunohistochemistry confirmed that the phage peptide CHAQGSAEC can bind specifically to thymus blood vessels in mice. Furthermore, phage peptide CHAQGSAEC and free peptide CHAQGSAEC can inhibit the bioactivity of thymus output in vivo. These results indicate the feasibility of the targeted peptide for possible function as a kind of tool to inhibit thymus bioactivity or as a targeted compound for targeted medicine. 相似文献
18.
Ramos M Paradela A Vazquez M Marina A Vazquez J Lopez de Castro JA 《The Journal of biological chemistry》2002,277(32):28749-28756
In contrast to HLA-B*2705, B*2709 is weakly or not associated to ankylosing spondylitis. Both allotypes differ by a single D116H change. We compared the B*2705- and B*2709-bound peptide repertoires by mass spectrometry to quantify the effect of B*2709 polymorphism on peptide specificity. In addition, shared and differentially bound ligands were sequenced to define the structural features of the various peptide subsets. B*2705 shared 79% of its peptide repertoire with B*2709. Shared ligands accounted for 88% of the B*2709-bound repertoire. All B*2705 ligands not bound to B*2709 had C-terminal basic or Tyr residues. Most B*2709-bound peptides had C-terminal aliphatic and Phe residues, but two showed C-terminal Arg or Tyr. The B*2709-bound repertoire included 12% of peptides not found in B*2705. These had aliphatic C-terminal residues, which are also favored in B*2705. However, these peptides bound weakly B*2705 in vitro, indicating distinct contribution of secondary anchor residues in both subtypes. Differences in peptide binding did not affect the ratio of native to beta2-microglobulin-free HLA-B27 heavy chain at the cell surface. Our results suggest that weaker association of B*2709 with ankylosing spondylitis is based on differential binding of a limited subset of natural ligands by this allotype. 相似文献
19.
Peptide libraries can be used to identify ligands that bind specifically to a desired protein. These peptides may have significant advantages as specific ligands for affinity chromatography separations. This article describes the use of one of such peptide, Try-Asn-Phe-Glu-Val-Leu, as a ligand for the purification of S-protein using affinity chromatography. General strategies for peptide immobilization are discussed and the conditions for peptide immobilization to Emphazetrade mark gel are optimized. The effects of peptide orientation and peptide densities on protein binding are studied. Results indicate that the peptide affinity is not affected by the orientation of the peptide during immobilization, but association constants can be reduced by one order of magnitude when compared with the values in solution.With increased peptide density, the protein binding capacity of the gel increases, but both the percentage of peptide utilization and apparent binding constant between immobilized peptide and S-protein decrease. S-protein is separated from a mixture with BSA via affinity chromatography using specific elution with the peptide in solution.Finally, direct purification of S-protein from an enzymatic digestion mixture of ribonuclease A is demonstrated.(c) 1995 John Wiley & Sons, Inc. 相似文献
20.
Azabicyclo[X.Y.0]alkanone amino acids are challenging synthetic targets and useful tools for studying structure-activity relationships of native peptide ligands. They have been employed to increase potency and stability in conformationally rigid enzyme inhibitors and receptor ligands. Since last reviewed in 1997, activity in their synthesis and application has increased significantly and access is now available to a wider diversity of these peptide mimics. This review focuses on recent syntheses of these heterocyclic amino acids and their application in the investigation of biologically active peptides and peptide mimics. 相似文献