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At least 1,333 records · Page 74Linked to original sources
PubMed2003

Use of cyclic peptide phage display library for the identification of a CD45RC epitope expressed on murine B cells and their precursors.

The alternative splicing and variable expression of the exons near to the N-terminus of the leukocyte common antigen (L-CA, CD45) result in distinct extracellular isoforms expressed by cells with different functional and developmental properties. Here we report the tissue reactivity pattern and epitope specificity of a novel rat monoclonal antibody (IBL-8) against a restricted epitope of mouse CD45. We found that this mAb reacts with an epitope displayed by B cells and their precursors (both in newborn spleen and adult bone marrow). Moreover, peripheral CD8-positive T cells were also recognised at an intermediate intensity, whereas the CD4 T cell subset was weakly reactive. The epitope of this mAb was determined with M13 filamentous phages that display cysteine constrained nonapeptides on their coat proteins. The isolated bacteriophages expressing the putative epitope showed an isoform-specific inhibition of the binding of exon-specific mAbs. Deduced amino acid sequence data of these phages indicate that the epitope recognised by the IBL-8 mAb lies at the 136-144 region of the mouse CD45 molecule within its C exon, with a TAFP consensus sequence at its centre.

Amino Acid Sequence↗
PubMed1995

Mapping sites of interaction of p47-phox and flavocytochrome b with random-sequence peptide phage display libraries.

During assembly of the phagocyte NADPH oxidase, cytosolic p47-phox translocates to the plasma membrane and binds to flavocytochrome b, and binding domains for p47-phox have been identified on the C-terminal tails of both flavocytochrome b subunits. In the present report, we further examine the interaction of these two oxidase components by using random-sequence peptide phage display library analysis. Screening p47-phox with the peptide libraries identified five potential sites of interaction with flavocytochrome b, including three previously reported regions of interaction and two additional regions of interaction of p47-phox with gp91-phox and p22-phox. The additional sites were mapped to a domain on the first predicted cytosolic loop of gp91-phox encompassing residues S86TRVRRQL93 and to a domain near the cytosolic C-terminal tail of gp91-phox encompassing residues F450EWFADLL457. The mapping also confirmed a previously reported binding domain on gp91-phox (E554SGPRGVHFIF564) and putative Src homology 3 domain binding sites on p22-phox (P156PRPP160 and G177GPPGGP183). To demonstrate that the additional regions identified were biologically significant, peptides mimicking the gp91-phox sequences F77LRGSSACCSTRVRRQL93 and E451WFADLLQLLESQ463 were synthesized and assayed for their ability to inhibit NADPH oxidase activity. These peptides had EC50 values of 1 microM and 230 microM, respectively, and inhibited activation when added prior to assembly but did not affect activity of the preassembled oxidase. Our data demonstrate the usefulness of phage display library analysis for the identification of biologically relevant sites of protein-protein interaction and show that the binding of p47-phox to flavocytochrome b involves multiple binding sites along the C-terminal tails of both gp91- and p22-phox and other regions of gp91-phox nearer to the N terminus.

Binding Sites↗
PubMed2006

Ribosomal synthesis of dehydroalanine-containing peptides.

Dehydroalanine is a nonproteinogenic amino acid, but it is a component of a wide variety of natural products with therapeutic activities. Indeed, this alpha,beta-unsaturated residue is a highly versatile building block due to its rigidifying effect on peptide backbones and its electrophilicity which allows site-specific thiol ligations of peptides with small molecules or proteins. To harness such versatility in genetically encoded, combinatorial peptide libraries, we report a simple and robust method for the ribosomal synthesis of dehydroalanine-containing peptides. Selenalysine, a selenium-containing lysine analogue, was recruited as a masked dehydroalanine equivalent. This residue is efficiently incorporated by a reconstituted Escherichia coli translation system at high fidelity and efficiency despite the presence of low levels of lysine. Mild oxidative conditions were used to convert selenalysine into dehydroalanine post-translationally. Using this method, we demonstrate the preparation of polyunsaturated and highly decorated peptides. This report is an important step toward the preparation and selection of large libraries of protein-reactive compounds with potential use as novel drugs or as analytical tools.

Alanine↗
PubMed1996

Substrate specificity and inhibitor profile of human recombinant p56lck from a baculovirus expression vector.

p56lck, a member of the src family of non-receptor protein receptor kinases, is required for normal signal transduction through the T cell receptor. Inappropriate T cell activation and proliferation has been identified as an early event in auto-immune disease-agents which control T cell activation through modulation of p56lck kinase activity could therefore be potential therapeutic agents for a range of pathological conditions. To identify p56lck inhibitors, we have established an assay system suitable for the high throughput screening of compound libraries. The assay uses enzyme purified from baculovirus infected SF9 cells, and a novel peptidic substrate identified by L. Cantley from a degenerate combinatorial peptide library. We have used this assay system to screen a number of different compounds as potential inhibitors of p56lck. In addition, peptides based on the substrate sequence were also tested to identify a sequence that could be used in the rational design of peptide inhibitors of p56lck.

Amino Acid Sequence↗
PubMed1999

The C-terminal region of the P3 structural protein of rice dwarf phytoreovirus is important for P3-P3 interaction.

Using a random peptide library designed for the yeast two-hybrid system, we identified a peptide that binds strongly to the P3 structural protein of rice dwarf phytoreovirus (RDV). The amino acid sequence of the peptide showed a high homology to the C-terminal region of P3. C-terminally truncated P3 lost its ability to interact with authentic P3. Our observations suggest that the C-terminal region of P3 is important for the P3-P3 interaction, which forms the core shell structure of RDV.

Amino Acid Sequence↗
PubMed2005

Expression mapping using a retroviral vector for CD8+ T cell epitopes: definition of a Mycobacterium tuberculosis peptide presented by H2-Dd.

Identification of CD8+ T cell epitopes is important because detection of specific CD8+ T cells after infection or immunization requires prior knowledge of epitope specificity. Furthermore, identification of CD8+ T cell epitopes permits the development of specific preventive and therapeutic approaches to both infections and tumors. Thus far, CD8+ T cell epitopes have been identified either using an overlapping peptide library covering an entire protein, or using algorithms designed to identify likely peptides that bind to major histocompatibility complex (MHC) class I molecules. The synthesis of overlapping peptides can be prohibitively expensive, and the algorithm programs used to predict CD8+ T cell epitopes are not always accurate. Here we describe a retroviral expression system that specifically allows longer polypeptides and shorter peptides to be expressed in the cytoplasm, and thereby to be processed onto class I MHC molecules. T cells from mice that were immunized with a DNA vaccine encoding MPT-51 were probed against MHC-compatible cell lines retrovirally transduced with overlapping gene fragments encoding 120-140 amino acids of the MPT-51 molecule. After further testing of shorter peptide sequences, we identified a CD8+ T cell epitope using cell lines expressing a relatively small number of algorithm-predicted candidate epitopes. We found that one of the requirements for cell surface display of the 20-mer peptide was the need for cotranslational ubiquitination. The restriction molecule was identified as Dd following transduction with MHC class I genes followed by transduction with the oligonucleotide encoding the epitope. The retroviral expression system described here is cost-effective, particularly if the target molecule is large, and could be adapted to identifying T cell epitopes recognized in infectious disease and against tumor cell antigens.

Algorithms↗
PubMed2006

Identification of key peptide-specific CD4+ T cell responses to human cytomegalovirus: implications for tracking antiviral populations.

Human cytomegalovirus (CMV) infection is normally controlled effectively by the immune response, including CD4(+) T cells. Large numbers of these cells are present in healthy seropositive individuals but their loss in immunosuppression leads to reactivation and disease. Tracking such responses in vivo is hampered by poor definition of their peptide targets. In this study, we defined the key targets of the peptide-specific CD4(+) T cell responses to the CMV pp65 protein using functional assays and a peptide library. Despite a good deal of interindividual variation in the numbers of peptides recognized, responses to CMV pp65 were strikingly targeted at three key epitopes. A response to one or more of these three key peptides was seen in all individuals tested (P < 0.0001) and this finding was tested and reproduced in a second independent population. The most common response identified was that to a DR53 restricted epitope, aa281-295. HLA-DR1 restricted CMV pp65-specific populations, although reproducibly detected, were of low frequency ex vivo. However, it was possible to detect and phenotype these cells using an enrichment protocol and this revealed them to have 'effector memory' status although, in contrast to CD8(+) T cell responses, these were CD45RA(-). These data suggest that CD4(+) T cell responses to CMV can be identified reliably using a pool of just three peptides. This simple approach will provide a robust and reliable as well as economic method for tracking peptide specific populations in health and disease.

Adult↗
PubMed1994

Rules for peptide binding to MHC class II molecules.

CD4+ T lymphocytes recognize peptide fragments of antigens that lie in the antigen binding pocket of class II major histocompatibility complex (MHC) molecules expressed on antigen-presenting cells. Specificity of T cells is determined by structural features of both the MHC molecule and antigenic peptide. MHC class II amino acid sequences are highly polymorphic within a population, and correlate with individual differences in response to infectious agents, vaccines, tumour antigens, and autoantigens. In the last few years several important breakthroughs and technological advances have made it possible to clarify the role of polymorphism and the molecular events in peptide interaction with major histocompatibility complex (MHC) class II proteins. The X-ray structural analysis of a MHC class II molecule together with the use of peptide libraries has permitted determination of the structural features of the complex between peptide antigen fragments and MHC class II proteins. The purpose of this article is to review our own studies and those of others on the requirements for peptide-class II molecule interaction and discuss possible implications for active immune intervention.

Amino Acid Sequence↗
PubMed1999

Yeast Two-Hybrid: State of the Art.

Genome projects are approaching completion and are saturating sequence databases. This paper discusses the role of the two-hybrid system as a generator of hypotheses. Apart from this rather exhaustive, financially and labour intensive procedure, more refined functional studies can be undertaken. Indeed, by making hybrids of two-hybrid systems, customised approaches can be developed in order to attack specific function-related problems. For example, one could set-up a "differential" screen by combining a forward and a reverse approach in a three-hybrid set-up. Another very interesting project is the use of peptide libraries in two-hybrid approaches. This could enable the identification of peptides with very high specificity comparable to "real" antibodies. With the technology available, the only limitation is imagination.

Journal Article↗
PubMed1997

Absence of functional relevance of human transporter associated with antigen processing polymorphism for peptide selection.

The polymorphic TAP (TAP1 and TAP2) genes code for a heteromeric complex that translocates cytosolic peptides across the membrane of the endoplasmic reticulum where they associate with MHC class I molecules. In the rat, extensive TAP2 polymorphism modifies the spectrum of peptides presented by MHC class I molecules on the cell surface. Although polymorphism of human TAP proteins is more limited, it has been proposed to be associated with susceptibility to autoimmune diseases. To analyze the peptide selectivity and transport efficiency of human TAP alleles, we have generated the most frequent TAP1 and TAP2 alleles using site-directed mutagenesis. Alleles were cloned in the baculovirus expression system and six TAP1/2 allelic combinations were overexpressed in Sf9 insect cells. Microsomes containing TAP1/2 dimers were isolated, and peptide binding assays were performed using random sequence peptide libraries of variable length (7-18 mer) and a 9-mer polyalanine peptide substituted with various amino acids in positions 1, 2, 3, 7, and 9. All studied allelic combinations selected peptides with identical substitutions and length. Furthermore, transport assays showed similar efficiency and kinetics of peptide translocation by the different TAP alleles. These data suggest that the major human TAP alleles transport identical peptide sets and are therefore unlikely to contribute to predisposition to autoimmune diseases.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

Strategies for the construction and use of peptide and antibody libraries displayed on phages.

Combinatorial chemistry and biology have become popular methods for the identification of bio-active molecules in drug discovery. A widely used technique in combinatorial biology is "phage display", by which peptides, antibody fragments and enzymes are displayed on the surface of bacteriophages, and can be selected by simple procedures of biopanning. The construction of phage libraries of peptides or antibody fragments provides a huge source of ligands and bio-active molecules that can be isolated from the library without laborious studies on antigen characteristics and prediction of ligand structure. This "irrational" approach for the construction of new drugs is extremely rapid and is now used by thousands of laboratories world-wide. The bottleneck in this procedure is the availability of large reliable libraries that can be used repeatedly over the years without loss of ligand expression and diversity. Construction of personalized libraries is therefore important for public and private laboratories engaged in the isolation of specific molecules for therapeutic or diagnostic use. Here we report the general strategies for constructing large phage peptide and antibody libraries, based on the experience of researchers who built the world's most widely used libraries. Particular attention is paid to advanced strategies for the construction, preservation and panning.

Antibody Affinity↗
PubMed1998

Molecular approach to find target(s) for oligoclonal bands in multiple sclerosis.

OBJECTIVES: Oligoclonal bands are a characteristic finding in the CSF of patients with multiple sclerosis, yet their target antigen(s) remain unknown. The objective was to determine whether a filamentous phage peptide library could be employed to allow the oligoclonal bands to select their own target epitopes. METHODS: CSF IgG antibody from 14 patients with multiple sclerosis and 14 controls was used to select individual phage clones from a bacteriophage library containing approximately 4 x 10(7) different hexamers expressed on its surface pIII protein. The amino acid sequence selected was deduced by sequencing the DNA of the genetically engineered insert. RESULTS: In general, after three rounds of selection, CSF from both patients with multiple sclerosis and controls selected one to two consistent peptide motifs. Five out of 14 patients with multiple sclerosis, and one control, selected the amino acid sequence motif, RRPFF. Given 20 possible amino acids per position, the likelihood of five patients selecting the same linear five amino acid sequence is at most 1.6 x 10(-3), corrected for the number of clones sequenced. A GenBank computer search showed that this sequence is found in the Epstein-Barr Virus nuclear antigen (EBNA-1), and a heat shock protein alphaB crystallin. Human serum antibodies to a synthetic peptide containing RRPFF were virtually exclusively found in patients with prior infection by Epstein-Barr virus. Other studies have suggested a relation between Epstein-Barr virus infection and multiple sclerosis, including nearly 100% Epstein-Barr virus seropositivity among patients with multiple sclerosis and increased concentrations of antibody to EBNA in CSF of patients with multiple sclerosis. By antigen specific immunoblotting, antibodies to the RRPFF motif in the CSF were shown to correspond to a subset of oligoclonal bands in the CSF from the same patient. CONCLUSION: This study shows that phage epitope display libraries may be used to select amino acid motifs which are potentially relevant to the pathogenesis of multiple sclerosis.

Amino Acid Sequence↗
PubMed1993

Discovery of D-amino-acid-containing ligands with selectide technology.

When all L-amino acid (aa) random peptide libraries synthesized on solid-phase particles were screened (Selectide Technology), we identified several peptide ligands (YG_F_) that interacted specifically with an anti-beta-endorphin monoclonal antibody (clone 3E7) (single-letter aa symbols; symbols '_' indicate variable aa). Here, we report on the screening of three different D-aa-containing pentapeptide libraries (XxXxX, xXxXx and xxxxx, wherein X = L-aa, and x = D-aa) with the same antibody, in which several D-aa-containing ligands were identified. The binding affinities of many of these D-aa-containing ligands were as least two orders of magnitude lower than that of YGGFL, for which the Ki is 17.5 nM.

Amino Acid Sequence↗
PubMed1996

Importance of specific amino acids in protein binding sites for heparin and heparan sulfate.

Heparin and heparan sulfate bind a variety of proteins and peptides to regulate many biological activities. Past studies have examined a limited number of established heparin binding sites and have focused on basic amino acids when modeling binding site structural motifs. This study examines the prevalence of individual amino acids in peptides binding to heparin or heparan sulfate. A 7-mer random peptide library was synthesized using the 20 common amino acids. This 7-mer library was affinity separated using both heparin and heparan sulfate-Sepharose. Bound peptide populations were eluted with a salt step gradient (pH 7) and analysed for amino acid composition. Peptides released from heparin-Sepharose by 0.3 M NaCl were enriched in arginine, lysine, glycine and serine; and depleted in methionine and phenylalanine. In contrast, peptides released from heparan sulfate-Sepharose were enriched in arginine, glycine, serine, and proline (at 0.15 M NaCl). These peptides were depleted in histidine, isoleucine, methionine (not detectable) and phenylalanine. In the heparin binding sites of proteins, which have been published, the enriched amino acids were arginine, lysine and tyrosine. Depleted amino acids include aspartic acid, glutamic acid, glutamine, alanine, glycine, phenylalanine, serine, threonine and valine. This study demonstrates that heparin and heparan sulfate bind different populations of peptide sequences. The differences in amino acid composition indicate that the positive charge density and spacing requirements differ for peptides binding these two glycosaminoglycans.

Amino Acid Sequence↗
PubMed2003

Trigger Factor and DnaK possess overlapping substrate pools and binding specificities.

Ribosome-associated Trigger Factor (TF) and the DnaK chaperone system assist the folding of newly synthesized proteins in Escherichia coli. Here, we show that DnaK and TF share a common substrate pool in vivo. In TF-deficient cells, deltatig, depleted for DnaK and DnaJ the amount of aggregated proteins increases with increasing temperature, amounting to 10% of total soluble protein (approximately 340 protein species) at 37 degrees C. A similar population of proteins aggregated in DnaK depleted tig+ cells, albeit to a much lower extent. Ninety-four aggregated proteins isolated from DnaK- and DnaJ-depleted deltatig cells were identified by mass spectrometry and found to include essential cytosolic proteins. Four potential in vivo substrates were screened for chaperone binding sites using peptide libraries. Although TF and DnaK recognize different binding motifs, 77% of TF binding peptides also associated with DnaK. In the case of the nascent polypeptides TF and DnaK competed for binding, however, with competitive advantage for TF. In vivo, the loss of TF is compensated by the induction of the heat shock response and thus enhanced levels of DnaK. In summary, our results demonstrate that the co-operation of the two mechanistically distinct chaperones in protein folding is based on their overlap in substrate specificities.

Chaperonin 60↗
PubMed2002

The Erbin PDZ domain binds with high affinity and specificity to the carboxyl termini of delta-catenin and ARVCF.

Erbin is a recently described member of the LAP (leucine-rich repeat and PDZ domain) protein family. We used a C-terminally displayed phage peptide library to identify optimal ligands for the Erbin PDZ domain. Phage-selected peptides were type 1 PDZ ligands that bound with high affinity and specificity to the Erbin PDZ domain in vitro. These peptides most closely resembled the C-terminal PDZ domain-binding motifs of three p120-related catenins: delta-catenin, ARVCF, and p0071 (DSWV-COOH). Analysis of the interactions of the Erbin PDZ domain with synthetic peptides matching the C termini of ARVCF or delta-catenin also demonstrated specific high affinity binding. We characterized the interactions between the Erbin PDZ domain and both ARVCF and delta-catenin in vitro and in vivo. The Erbin PDZ domain co-localized and coprecipitated with ARVCF or delta-catenin complexed with beta-catenin and E/N-cadherin. Mutagenesis and peptide competition experiments showed that the association of Erbin with the cadherin-catenin complex was mediated by the interaction of its PDZ domain with the C-terminal PDZ domain-binding motifs (DSWV-COOH) of ARVCF and delta-catenin. Finally, we showed that endogenous delta-catenin and Erbin co-localized in and co-immunoprecipitated from neurons. These results suggest that delta-catenin and ARVCF may function to mediate the association of Erbin with the junctional cadherin-catenin complex. They also demonstrate that C-terminal phage-display technology can be used to predict physiologically relevant ligands for PDZ domains.

Adaptor Proteins, Signal Transducing↗
PubMed2005

The periplasmic chaperone SurA exploits two features characteristic of integral outer membrane proteins for selective substrate recognition.

The Escherichia coli periplasmic chaperone and peptidyl-prolyl isomerase (PPIase) SurA facilitates the maturation of outer membrane porins. Although the PPIase activity exhibited by one of its two parvulin-like domains is dispensable for this function, the chaperone activity residing in the non-PPIase regions of SurA, a sizable N-terminal domain and a short C-terminal tail, is essential. Unlike most cytoplasmic chaperones SurA is selective for particular substrates and recognizes outer membrane porins synthesized in vitro much more efficiently than other proteins. Thus, SurA may be specialized for the maturation of outer membrane proteins. We have characterized the substrate specificity of SurA based on its natural, biologically relevant substrates by screening cellulose-bound peptide libraries representing outer membrane proteins. We show that two features are critical for peptide binding by SurA: specific patterns of aromatic residues and the orientation of their side chains, which are found more frequently in integral outer membrane proteins than in other proteins. For the first time this sufficiently explains the capability of SurA to discriminate between outer membrane protein and non-outer membrane protein folding intermediates. Furthermore, peptide binding by SurA requires neither an active PPIase domain nor the presence of proline, indicating that the observed substrate specificity relates to the chaperone function of SurA. Finally, we show that SurA is capable of associating with the outer membrane. Together, our data support a model in which SurA is specialized to interact with non-native periplasmic outer membrane protein folding intermediates and to assist in their maturation from early to late outer membrane-associated steps.

Amino Acid Sequence↗
PubMed2004

A 38-kilobase pathogenicity island specific for Mycobacterium avium subsp. paratuberculosis encodes cell surface proteins expressed in the host.

We have used representational difference analysis to identify a novel Mycobacterium avium subsp. paratuberculosis-specific ABC transporter operon (mpt), which comprises six open reading frames designated mptA to -F and is immediately preceded by two putative Fur boxes. Functional genomics revealed that the mpt operon is flanked on one end by a fep cluster encoding proteins involved in the uptake of Fe(3+) and on the other end by a sid cluster encoding non-ribosome-dependent heterocyclic siderophore synthases. Together these genes form a 38-kb M. avium subsp. paratuberculosis-specific locus flanked by an insertion sequence similar to IS1110. Expression studies using Western blot analyses showed that MptC is present in the envelope fraction of M. avium subsp. paratuberculosis. The MptD protein was shown to be surface exposed, using a specific phage (fMptD) isolated from a phage-peptide library, by differential screening of Mycobacterium smegmatis transformants. The phage fMptD-derived peptide could be used in a peptide-mediated capture PCR with milk from infected dairy herds, thereby showing surface-exposed expression of the MptD protein in the host. Together, these data suggest that the 38-kb locus constitutes an M. avium subsp. paratuberculosis pathogenicity island.

ATP-Binding Cassette Transporters↗