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PubMed1996

Peptide libraries define the fine specificity of anti-polysaccharide antibodies to Cryptococcus neoformans.

Cryptococcus neoformans is an encapsulated fungus that causes a life-threatening meningoencephalitis in patients with AIDS. Monoclonal antibodies to the capsular glucuronoxylomannan can modulate the infection in mice, but the epitopes on this complex polysaccharide recognized by protective and non-protective antibodies have not been defined. We have used 2H1, one of our most protective antibodies, to screen phage display peptide libraries for peptide mimotopes that would allow us to explore the fine specificity of anti-cryptococcal polysaccharide antibodies. Hexa- and decapeptides have been identified with sequence homologies that define four motifs: 1, (E)TPXWM/LM/L; 2, W/YXWM/ LYE; 3, DWXDW; and 4, (Ar)WDGQ(Ar). Peptides representing these motifs compete with each other for a shared binding site that overlaps the polysaccharide binding site. Motifs 1 and 2 confer high affinity binding, and PA1, which displays a motif 1 peptide with the sequence LQYTPSWMLV, binds to 2H1 with a Kd of 295 nM. Analysis of the interaction between the 2H1 binding peptides and 24 structurally related anti-polysaccharide antibodies reveals a complex pattern of reactivity that strongly suggests binding to or close to the complementary determining regions. Furthermore, those antibodies that have been shown to have different specificity, and in some cases different protective potential, do not bind any of the peptides selected by the protective 2H1 antibody. This study shows that peptide mimotopes for a complex microbial polysaccharide can be identified by screening phage peptide libraries and demonstrates the usefulness of such peptides in analyzing closely related interactive sites of proteins in general and of antibodies in particular.

Amino Acid Sequence↗
PubMedDate not supplied

Use of synthetic peptide libraries for the H-2Kd binding motif identification.

To identify Kd-binding peptides, an approach based on small peptide libraries has been developed. These peptide libraries correspond to all possible single-amino acid variants of a particular Kd-binding peptide, SYIPSAEYI, an analog of the Plasmodium berghei 252-260 antigenic peptide SYIPSAEKI. In the parent sequence, each position is replaced by all the genetically encoded amino acids (except cysteine). The multiple analog syntheses are performed either by the Divide Couple and Recombine method or by the Single Resin method and generate mixtures containing 19 peptides. The present report deals with the synthesis, the purification, the chemical characterization by amino acid analysis and electrospray mass spectrometry (ES-MS), and the application of such mixtures in binding tests with a soluble, functionally empty, single-chain H-2Kd molecule denoted SC-Kd. For each mixture, bound peptides were eluted and analyzed by sequencing. Since the binding tests were realized in noncompetitive conditions, our results show that a much broader set of peptides bind to Kd than expected from previous studies. This may be of practical importance when looking for low affinity peptides such as tumor peptides capable of eliciting protective immune response.

Amino Acid Sequence↗
PubMed2005

Were arachnids the first to use combinatorial peptide libraries?

Spiders, scorpions, and cone snails are remarkable for the extent and diversity of gene-encoded peptide neurotoxins that are expressed in their venom glands. These toxins are produced in the form of structurally constrained combinatorial peptide libraries in which there is hypermutation of essentially all residues in the mature-toxin sequence with the exception of a handful of strictly conserved cysteines that direct the three-dimensional fold of the toxin. This gene-based combinatorial peptide library strategy appears to have been first implemented by arachnids almost 400 million years ago, long before cone snails evolved a similar mechanism for generating peptide diversity.

Animals↗
PubMed2006

GD3-replica peptides selected from a phage peptide library induce a GD3 ganglioside antibody response.

GD3-replica peptides were obtained from a phage peptide library and an anti-GD3 monoclonal antibody (Mab) (4F6), and anti-GD3 Mabs were generated by immunizing a peptide GD3P4. A Mab, 3D2 was found to recognize GD3 by immunohistochemical approaches. Amino acid analysis of heavy and light chain variable regions of 4F6 and 3D2 showed that the respective chains had the same length, and only a few different amino acid substitutions were found. The present data indicate that the immunogenic GD3P4 is processed in a certain size and exposed on the antigen-presenting cells with a molecular shape quite similar to that of the GD3 epitope in 4F6.

Amino Acids↗
PubMed1997

The potential of combinatorial peptide libraries for the identification of inhibitors of TNF-alpha mediated cytotoxicity in vitro.

The use of random peptide libraries has recently received considerable attention as a new tool for identifying novel therapeutic molecules. The libraries represent an enormous number of peptide sequence displayed either on the virion surface of filamentous phage clones or on a solid phase synthetic support. In this minireview we summarize our recent findings on the use of combinatorial peptide libraries to identify antagonists of the biological activities of TNF-alpha, a cytokine which plays a key role in immune and inflammatory responses.

Animals↗
PubMed1998

Affinity selection from peptide libraries to determine substrate specificity of protein tyrosine phosphatases.

Affinity selection from peptide libraries is a powerful tool that has been used for determining the sequence specificities of a number of enzymes and protein binding domains, including protein kinases, src homology 2 domains, and PDZ domains. We have extended this approach to protein tyrosine phosphatases using peptide libraries containing a nonhydrolyzable phosphotyrosine analog, difluorophosphonomethylphenylalanine. A size-exclusion method is used to separate enzyme-peptide complexes from free peptide, providing several advantages over the traditional immobilized protein affinity column approach. In addition, the feasibility of using mass spectrometric detection to quantitate peptides rapidly and reproducibly is demonstrated as an alternative to quantitation by peptide sequencing. The validity of this analysis is demonstrated by synthesizing individual peptides and comparing their affinity for enzyme with the predictions from the affinity selection process. As a model for these studies the protein tyrosine phosphatase PTP1B is used, providing additional insights into the sequence specificity of this enzyme. In particular, a selection for aromatic amino acids at the pY - 1 position (immediately N-terminal to the phosphotyrosine), as well as a broad pY + 1 selectivity, is observed in addition to the general preference for acidic residues N-terminal to the phosphotyrosine. The approach described here should prove applicable to protein tyrosine phosphatases in general as well as for the study of nonpeptidyl combinatorial libraries.

Amino Acid Sequence↗
PubMed2000

Identification of chymotrypsin inhibitors from a second-generation template assisted combinatorial peptide library.

In an earlier study (McBride JD, Freeman N, Domingo GJ, Leatherbarrow RJ. Selection of chymotrypsin inhibitors from a conformationally-constrained combinatorial peptide library. J. Mol. Biol. 1996; 259: 819-827) we described a resin-bound cyclic peptide library, constructed based on the sequence of the anti-tryptic reactive site loop of Bowman Birk Inhibitor (BBI), a proteinase inhibitor protein. This library was used to identify re-directed chymotrypsin inhibitors with Ki values as low as 17 nM. We have now extended this work by constructing an enhanced library in which a further position, at the P4 site of the inhibitor, has been randomized. This new library has variation at three target locations (P4, P1 and P2) within the inhibitory loop region, producing 8,000 variants. Screening this library allowed selection of new inhibitor sequences with Ki values as low as 3.4 nM. The success of this approach is reflected by the fact that the inhibition constant given by the selected peptide sequence is slightly lower than that reported against chymotrypsin for the most studied full length BBI protein, Soybean BBI 2-IV.

Amino Acid Sequence↗
PubMed2001

Identification of enzyme inhibitors from phage-displayed combinatorial peptide libraries.

In recent years, there have been a growing number of examples of the successful isolation of peptide ligands for enzymes from phage-displayed combinatorial peptide libraries. These peptides typically bind at or near the active site of the enzymes and can inhibit their activity. We review the literature on peptide ligands that have been isolated for enzymes other than proteases as well as present data on peptide ligands we have identified for E. coli dihydrofolate reductase (DHFR) which bind at, or near, the same site as the known inhibitors methotrexate or trimethoprim. Thus, while the peptide ligand isolated from phage-displayed libraries may not resemble the chemical structure of the normal substrate of the enzyme, the peptide can be used as an inhibitor to evaluate the function of the enzyme or for drug discovery efforts (i.e., as a lead compound for peptidomimetic design or as displaceable probe in high-throughput screens of libraries of small molecules).

Amino Acid Sequence↗
PubMed2003

Peptabodies as tools to test ligands isolated from phage-displayed peptide libraries.

We have previously isolated filamentous bacteriophages, expressing linear hexa-peptides and homing to bone marrow endothelium (BME), by panning in vivo of a phage-displayed peptide library in mice. Here, we used peptabody fusion proteins to test the binding capacity of the hexa-peptide SSLTTG to BME cells in vitro. To display this motif in a multimeric form, as originally presented on the bacteriophage, we expressed it N-terminally as a fusion with the peptabody cartilage oligomeric matrix assembly protein (COMP) pentamerization domain, either alone or followed by the N1 domain of the pIII phage coat protein. Binding of the peptabody constructs to the mouse BME cell line STR-10 was investigated by immunofluorescence using anti-COMP antibodies. Only peptabody fusion proteins co-expressing pIII-N1 exhibited binding to STR-10, regardless of the presence or absence of SSLTTG. These results indicate that the phage coat protein pIII-N1 domain is the principle determinant responsible for the binding of filamentous bacteriophages to cells of the reticulo-endothelial system (RES). Peptabodies expressing pIII-N1 did not bind to the osteoblast-like cell line MC3T3-E1, indicating that binding is mediated by receptors specifically expressed by BME cells in vivo. Polyinosinic acid (poly-I) was able to inhibit binding of bacteriophages and pIII-N1 expressing peptabodies to STR-10, confirming our previous studies showing that bacteriophages bind to scavenger receptors (SR) expressed by BME cells. In summary, the present study shows the usefulness of peptabodies as a general tool to test the binding capacity of peptide ligands identified by phage display.

Amino Acid Sequence↗
PubMed1994

Random peptide libraries.

Over the past year, great strides have been made in the design of peptide libraries, and new approaches have been developed for identifying peptide ligands. The libraries comprise large collections of peptides, ranging from 1 million to 1 billion different sequences, which can be screened using monoclonal and polyclonal antibodies, receptors, enzymes or other target molecules. The power of this technology stems from the chemical diversity of the amino acids coupled with the large number of sequences in a library. As such, peptide libraries may be useful for finding ligands that can serve as leads for pharmaceutical development and other purposes.

Amino Acid Sequence↗
PubMed1998

Phage-displayed peptide libraries.

Over the past year, significant advances have been achieved through the use of phage-displayed peptide libraries. A wide variety of bioactive molecules, including antibodies, receptors and enzymes, have selected high-affinity and/or highly-specific peptide ligands from a number of different types of peptide library. The demonstrated therapeutic potential of some of these peptides, as well as new insights into protein structure and function that peptide ligands have provided, highlight the progress made within this rapidly-expanding field.

Antibodies↗
PubMed2006

Conformationally constrained CCK8 analogues obtained from a rationally designed peptide library as ligands for cholecystokinin type B receptor.

A library of 14 cyclic peptide analogues derived from the octapeptide C-terminal sequence of the human cholecystokinin hormone (CCK(26-33), or CCK8) was designed, synthesized, and characterized. The 14 peptide analogues were rationally designed to specifically interact with the CCK type B receptor (CCK(B)-R) on the basis of the structure of the bimolecular complex between CCK8 and the third extracellular loop of CCK(B)-R, namely CCK(B)-R(352-379). The rational design of new ligands for CCK(B)-R has relied on stabilization by cyclic constraints of the structural motifs that bring the key residues of the ligand (especially Trp 30, Met 31, and Phe 33) in the proper spatial orientation for optimal interaction with the receptor. The binding affinity of the new ligands for CCK(B)-R was assessed by displacement experiments of (111)In-radiolabeled CCK8 in cells that overexpress the CCK(B) receptor. The new ligands generally showed binding affinities lower than that of parent CCK8, with the best compounds having IC50 values around 10 microM. Structure-activity relationship data show that preservation of the Trp 30-Met 31 motif is essential and that the Phe 33 side chain must be present. NMR conformational studies of the compound with maximal binding affinity (cyclo-B11, IC50=11 microM) in DPC micelles shows that this compound presents a turn-like conformation centered at the Trp 30-Met 31 segment, as planned by rational design. Such a conformation is stabilized by its interaction with the micelle rather than by the cyclic constraint.

Cholecystokinin↗
PubMed1997

Mapping epitopes of neutralizing monoclonal antibodies using phage random peptide libraries.

Identification of protective determinants from microbial proteins is a necessary step in the rational design of subunit vaccines. We have previously used a synthetic peptide scan (Pepscan) assay to map a panel of eight neutralizing monoclonal antibodies (mAb; designated as C1.1 to C1.8) to a common motif sequence from Chlamydia trachomatis. In the present study, five of the eight mAbs were used to screen phage random peptide libraries. mAbs C1.1 and C1.3 selected a motif sequence of G-L-X-N-D from a pIII-based phage random peptide library and a motif sequence of G-X-X-N-D from a pVIII-based random peptide library while mAbs C1.6 to C1.8 failed to select recognizable motifs from either of the phage libraries. However, C1.6 to C1.8 bound to the same motif sequence displayed on phage when the appropriate conformational constraints were imposed onto the motif sequence. Thus the specificity of the mAbs identified on Pepscan assays correlates with the mAbs' dependence on local epitope constraints displayed on the phage surface.

Amino Acid Sequence↗
PubMed1996

Peptides isolated from random peptide libraries on phage elicit a neutralizing anti-HIV-1 response: analysis of immunological mimicry.

Peptides binding to a murine, human immunodeficiency virus type 1 (HIV-1) neutralizing monoclonal antibody (F58/H3) were isolated from two random peptide libraries expressed on the surface of phage. The antibody was originally elicited by immunization with HIV-1 envelope protein gp120LAI, and has previously been shown to interact with the -I-GPGRA- motif of the V3 loop. The peptide libraries consisted of nine or 15 random amino acid residues flanked by two cysteines, and fused to the amino terminal end of the cpIII protein on the filamentous phage. Selection of specific peptides was carried out in three rounds, with decreasing antibody concentration. An expected peptide motif -GPGRA-, a similar segment, -GPAR-, and two unrelated motifs -FRLLG- and -WRM/ALG- were selected. Binding of antibody was tested both to synthetic peptides in solution, and the corresponding peptide on phage. The GPXR motifs bound in both formats, while the FRLLG bound antibody only when present on the phage The reactivity of peptides on phage was highly dependent on an intact disulphide bond between the cysteines flanking the peptide. The molecular mimicry of the found motifs was tested by immunizing mice and rabbits with conjugated synthetic peptides or peptide on phage. In mice, peptide-specific antisera were raised, but no reactivity to the whole protein (gp120) was detected. In rabbits, however, this was accomplished with the -GPGRA- containing peptide when present on phage. In addition, this antisera precipitated virus particles, and neutralized HIV-1SF2 virus in vitro.

Animals↗
PubMed2006

High-throughput sequence determination of cyclic peptide library members by partial Edman degradation/mass spectrometry.

Cyclic peptides provide attractive lead compounds for drug discovery and excellent molecular probes in biomedical research. Large combinatorial libraries of cyclic peptides can now be routinely synthesized by the split-and-pool method and screened against biological targets. However, post-screening sequence determination of hit peptides has been problematic. In this report, a high-throughput method for the sequence determination of cyclic peptide library members has been developed. TentaGel microbeads (90 mum) were spatially segregated into outer and inner layers; cyclic peptides were displayed on the bead surface, whereas the inner core of each bead contained the corresponding linear peptide as the encoding sequence. After screening of the cyclic peptide library against a macromolecular target, the identity of hit peptides was determined by sequencing the linear encoding peptides inside the bead using a partial Edman degradation/mass spectrometry method. On-bead screening of an octapeptide library (theoretical diversity of 160 000) identified cyclic peptides that bind to streptavidin. A 400-member library of tyrocidine A analogues was synthesized on TentaGel macrobeads and solution-phase screening of the library directly against bacterial cells identified a tyrocidine analogue of improved antibacterial activity. Our results demonstrate that the new method for cyclic peptide sequence determination is reliable, operationally simple, rapid, and inexpensive and should greatly expand the utility of cyclic peptides in biomedical research.

Anti-Infective Agents↗
PubMed1996

Linear epitopes of sperm whale myoglobin identified by polyclonal antibody screening of random peptide library.

Distinct enhancement of antibody-specific clones was apparent during the screening against random peptide libraries with antigen-specific polyclonal antibodies. Several sequence motifs obtained from these screenings were homologous with the primary sequence of myoglobin. Two of these motifs have been confirmed as antigenic determinants by competitive inhibition tests using eight-branched synthetic peptides. One of the peptides has a sequence that corresponds to amino acid residues 42-50, KFDRFKHLK, of the myoglobin sequence. This is a new epitope of myoglobin that is reported for the first time. The epitope is located precisely in the "turn' or "loop' region between helices C and D of the crystal structure of myoglobin. The second antibody binding site has a sequence of DIAAKYKELGYQG, and this is located between residues 141-153, which is the C-terminus of myoglobin. This epitope encompassed two linear epitopes of myoglobin, amino acid residues 145-151 and 147-153, that have been reported earlier based on immunochemical characterisation of cleavage fragments of the protein. These results clearly indicate that epitope mapping using polyclonal antibodies against random peptide libraries can identify new epitopes precisely, as well as confirm epitopes of myoglobin obtained earlier using established methodologies.

Animals↗