Control of function of a small peptide by a protein

A peptide that functions only in the presence of a protein has been developed using reaction-based selection from peptide phage libraries. The peptide was not functional in the absence of the protein, but formed enaminones with 1,3-diketone derivatives when bound to the protein.     

Identification of Mimotope Peptides Which Bind to the Mycotoxin Deoxynivalenol-Specific Monoclonal Antibody

Monoclonal antibody 6F5 (mAb 6F5), which recognizes the mycotoxin deoxynivalenol (DON) (vomitoxin), was used to select for peptides that mimic the mycotoxin by employing a library of filamentous phages that have random 7-mer peptides on their surfaces. Two phage clones selected from the random peptide phage-displayed library coded for the amino acid sequences SWGPFPF and SWGPLPF. These clones were designated DONPEP.2 and DONPEP.12, respectively. The results of a competitive enzyme-linked immunosorbent assay (ELISA) suggested that the two phage displayed peptides bound to mAb 6F5 specifically at the DON binding site. The amino acid sequence of DONPEP.2 plus a structurally flexible linker at the C terminus (SWGPFPFGGGSC) was synthesized and tested to determine its ability to bind to mAb 6F5. This synthetic peptide (designated peptide C430) and DON competed with each other for mAb 6F5 binding. When translationally fused with bacterial alkaline phosphatase, DONPEP.2 bound specifically to mAb 6F5, while the fusion protein retained alkaline phosphatase activity. The potential of using DONPEP.2 as an immunochemical reagent in a DON immunoassay was evaluated with a DON-spiked wheat extract. When peptide C430 was conjugated to bovine serum albumin, it elicited antibody specific to peptide C430 but not to DON in both mice and rabbits. In an in vitro translation system containing rabbit reticulocyte lysate, synthetic peptide C430 did not inhibit protein synthesis but did show antagonism toward DON-induced protein synthesis inhibition. These data suggest that the peptides selected in this study bind to mAb 6F5 and that peptide C430 binds to ribosomes at the same sites as DON.     

A peptide mimetic of Gal-alpha 1,3-Gal is able to block human natural antibodies

The carbohydrate of Gal-alpha1,3-Gal is thought to be the major antigenic epitope present on pig vascular endothelium. The peptides that mimic the binding of antigenic epitope (Gal-alpha1,3-Gal) to lectin BS-I-B4 were identified from screening a filamentous phage-displayed random library. A phage bearing the peptide NCVSPYWCEPLAPSARA has been identified to bind the lectin strongly. Melibiose was able to inhibit the binding of the human natural anti-alpha Gal antibody to the peptide competitively. Our experiments show that the peptide mimetic of Gal-alpha1,3-Gal is able to inhibit the agglutination of pig RBCs by human natural antibody or lectin BS-I-B4. The peptide inhibitor of human natural antibodies may prove useful in pig-to-human xenotransplantation.     

Identification of mimotope peptides which bind to the mycotoxin deoxynivalenol-specific monoclonal antibody.

Monoclonal antibody 6F5 (mAb 6F5), which recognizes the mycotoxin deoxynivalenol (DON) (vomitoxin), was used to select for peptides that mimic the mycotoxin by employing a library of filamentous phages that have random 7-mer peptides on their surfaces. Two phage clones selected from the random peptide phage-displayed library coded for the amino acid sequences SWGPFPF and SWGPLPF. These clones were designated DONPEP.2 and DONPEP.12, respectively. The results of a competitive enzyme-linked immunosorbent assay (ELISA) suggested that the two phage displayed peptides bound to mAb 6F5 specifically at the DON binding site. The amino acid sequence of DONPEP.2 plus a structurally flexible linker at the C terminus (SWGPFPFGGGSC) was synthesized and tested to determine its ability to bind to mAb 6F5. This synthetic peptide (designated peptide C430) and DON competed with each other for mAb 6F5 binding. When translationally fused with bacterial alkaline phosphatase, DONPEP.2 bound specifically to mAb 6F5, while the fusion protein retained alkaline phosphatase activity. The potential of using DONPEP.2 as an immunochemical reagent in a DON immunoassay was evaluated with a DON-spiked wheat extract. When peptide C430 was conjugated to bovine serum albumin, it elicited antibody specific to peptide C430 but not to DON in both mice and rabbits. In an in vitro translation system containing rabbit reticulocyte lysate, synthetic peptide C430 did not inhibit protein synthesis but did show antagonism toward DON-induced protein synthesis inhibition. These data suggest that the peptides selected in this study bind to mAb 6F5 and that peptide C430 binds to ribosomes at the same sites as DON.     

Tentacle type peptides as artificial lectins against sulfated Lewis X and A

An effort has generated peptides from a phage-displayed library that selectively bind to the sulfated carbohydrates HSO3-LeA and HSO3-LeX. Even though more than six phaged peptides were identified by using the biopanning procedure, only one synthesized peptide displayed a consistently high binding affinity and specificity against the cognate HSO3-LeA. This dimeric, tentacle type peptide has a low micromolar affinity against the cognate sugar, which is even more specific than an antibody (Table 2(b)). Thus, it suggests that tentacle type peptides can be used as alternatives to antibodies to bind to aberrant cell-surface carbohydrates that are either the causes or results of carbohydrate-indicating disease states.     

A new approach for drug discovery from glycobiology and phage-displayed peptide library technology

Peptides which mimic functional activities of glycosphingolipids were prepared by a technology of phage-displayed peptide library using monoclonal antibodies against glycosphingolipids. These peptides were named glyco-replica peptides. Peptides prepared with anti-GD1alpha antibody by this technology were found to contain WHW as common motif, and they showed suppressive activity not only on adhesion between hepatic sinusoidal endothelial cells and lymphosarcoma RAW117-H10 cells, but also on metastasis of the tumor cell to the liver and lung. The WHW motif seems to be important to mimic the functional activity of the ganglioside GD1alpha. Next, we prepared GD3-replica peptides using a monoclonal antibody against GD3 (4F6). A peptide, GD3-P4 with highest affinity to 4F6 was used to immunize mice to examine if the mice show their immune response to raise antibodies against GD3. We confirmed the immune response and succeeded in the production of a monoclonal antibody (3D2) against GD3. The monoclonal antibody 3D2 showed specific binding to GD3 on a thin-layer chromatography plate and also melanoma tissues. Interestingly, the amino acid sequence of the CDR regions of light and heavy chains showed high similarity with those of the original GD3 monoclonal antibody (4F6) used for the preparation of GD3-replica peptide. The technology of the phage-displayed peptide library was applied to in vivo bio-panning study using an angiogenesis experimental model. The obtained peptides were found to show strong binding property to the neo-vasculature system and to be quite useful to carry an anti-tumor drug to the tumor tissue. Based on these experimental results, we discuss about some applications of this method to drug discovery.     

The antibody binding site. Labelling of a specific antibody against the photo-precursor of an aryl nitrene

The isolation of specific rabbit antibodies for the haptenic group 4-azido-2-nitrophenyl, is described. These antibodies bind 1.8-2.0mol of hapten [in-(4-azido-2-nitrophenyl)-l-lysine]/mol with an association constant of nearly 10(7)m(-1) at 4 degrees C. On photolysis of the antibody-hapten complex, resulting in the formation of an aryl nitrene at the binding site, hapten was covalently bound to the antibody, and the antibody binding site was blocked. The ratio of labelling of heavy- and light-chains was 2.5:1. Two small peptides were isolated from digests of labelled heavy-chain, indicating that some 13% of the label in the antibody was attached to cysteine-92 and to alanine-93. These residues are adjacent to the major hypervariable region in rabbit heavy-chain (residues 95-105).     

Identification of peptide mimotopes for the fluorescein hapten binding of monoclonal antibody B13‐DE1

Using 6mer and 12mer phage peptide libraries three unique phage clones were identified which specifically bind to a monoclonal anti‐FITC antibody, B13‐DE1. The two 6mer and one 12mer peptide insert sequences are clearly related to each other and contain a high proportion of hydrophobic amino acids. The peptides are bound by the antibody combining site of B13‐DE1 probably in a similar manner to FITC and represent therefore true peptidic mimics of the fluorescein hapten. No reactivity of the peptides could be demonstrated with another monoclonal anti‐fluorescein antibody or with polyclonal anti‐fluorescein antibodies. Immunization of mice with the peptides resulted in the production of antibodies cross‐reacting with all peptides but not with fluorescein. The results show that phage peptide libraries can be used to isolate mimotope peptides which can mimic low molecular weight structures seen by a specific antibody and probably other recognition molecules. Copyright © 1999 John Wiley & Sons, Ltd.     

Development of a small peptide tag for covalent labeling of proteins

A 21-mer peptide that can be used to covalently introduce synthetic molecules into proteins has been developed. Phage-displayed peptide libraries were subjected to reaction-based selection with 1,3-diketones. The peptide was further evolved by addition of a randomized region and reselection for improved binding. The resulting 21-mer peptide had a reactive amino group that formed an enaminone with 1,3-diketone and was used as a tag for labeling of maltose binding protein. Using this peptide tag and 1,3-diketone derivatives, a variety of molecules such as reporter probes and functionalities may be covalently introduced into proteins of interest.     

从噬菌体展示肽库中筛选脂质A的结合肽

以脂质A为靶标,筛选噬菌体展示十二肽库,三轮后随机挑取14个噬菌体克隆进行结合活性鉴定,并对5个克隆进行序列分析。结果表明,14个克隆全部是阳性克隆,测序结果显示4个阳性克隆的序列完全一样。说明筛选到了一个脂质A的结合多肽。     

Structural Mimicry of O-Antigen by a Peptide Revealed in a Complex with an Antibody Raised against Shigella flexneri Serotype 2a

The use of carbohydrate-mimicking peptides to induce immune responses against surface polysaccharides of pathogenic bacteria offers a novel approach to vaccine development. Factors governing antigenic and immunogenic mimicry, however, are complex and poorly understood. We have addressed this question using the anti-lipopolysaccharide monoclonal antibody F22-4, which was raised against Shigella flexneri serotype 2a and shown to protect against homologous infection in a mouse model. In a previous crystallographic study, we described F22-4 in complex with two synthetic fragments of the O-antigen, the serotype-specific saccharide moiety of lipopolysaccharide. Here, we present a crystallographic and NMR study of the interaction of F22-4 with a dodecapeptide selected by phage display using the monoclonal antibody. Like the synthetic decasaccharide, the peptide binds to F22-4 with micromolar affinity. Although the peptide and decasaccharide use very similar regions of the antigen-binding site, indicating good antigenic mimicry, immunogenic mimicry by the peptide was not observed. The F22-4-antigen interaction is significantly more hydrophobic with the peptide than with oligosaccharides; nonetheless, all hydrogen bonds formed between the peptide and F22-4 have equivalents in the oligosaccharide complex. Two bridging water molecules are also in common, adding to partial structural mimicry. Whereas the bound peptide is entirely helical, its structure in solution, as shown by NMR, is helical in the central region only. Moreover, docking the NMR structure into the antigen-binding site shows that steric hindrance would occur, revealing poor complementarity between the major solution conformation and the antibody that could contribute to the absence of immunogenic mimicry.     

一种特异性识别斑马鱼卵黄蛋白原的噬菌体展示单链抗体的可溶性表达方案

为了实现特异性识别斑马鱼卵黄蛋白原的噬菌体展示单链抗体的可溶性表达,将不能以可溶性蛋白形式表达的、只能以噬菌体展示形式特异性识别斑马鱼卵黄蛋白原的单链抗体F5的基因,克隆到pET 32a载体并转化入大肠杆菌ori DE3中。结果表明,通过诱导表达,可获得可溶性的并且仍特异性识别斑马鱼卵黄蛋白原的单链抗体32a-F5。噬菌体展示单链抗体不能可溶性表达是噬菌体展示技术应用中常见的问题,该方法提供了一种表达可溶性单链抗体的可行性方案。     

Evidence that a protein-protein interaction 'hot spot' on heterotrimeric G protein betagamma subunits is used for recognition of a subclass of effectors

To understand the requirements for binding to G protein betagamma subunits, phage-displayed random peptide libraries were screened using immobilized biotinylated betagamma as the target. Selected peptides were grouped into four different families based on their sequence characteristics. One group (group I) had a clear conserved motif that has significant homology to peptides derived from phospholipase C beta (PLC beta) and to a short motif in phosducin that binds to G protein beta subunits. The other groups had weaker sequence homologies or no homology to the group I sequences. A synthetic peptide from the strongest consensus group blocked activation of PLC by G protein betagamma subunits. The peptide did not block betagamma-mediated inhibition of voltage-gated calcium channels and had little effect on betagamma-mediated inhibition of Gs-stimulated type I adenylate cyclase. Competition experiments indicated that peptides from all four families bound to a single site on betagamma. These peptides may bind to a protein-protein interaction 'hot spot' on the surface of betagamma subunits that is used by a subclass of effectors.     

Human immunodeficiency virus type 1-neutralizing monoclonal antibody 2F5 is multispecific for sequences flanking the DKW core epitope

Human monoclonal antibody 2F5 is one of a few human antibodies that neutralize a broad range of HIV-1 primary isolates. The 2F5 epitope on gp41 includes the sequence ELDKWA, with the core residues, DKW, being critical for antibody binding. HIV-neutralizing antibodies have never been elicited by immunization with peptides bearing ELDKWA, suggesting that important part(s) of the 2F5 paratope remain unidentified. The use of longer peptides extending beyond ELDKWA has resulted in increased epitope antigenicity, but neutralizing antibodies have not been generated. We sought to develop peptides that bind to 2F5, and that function as specific probes of the 2F5 paratope. Thus, we used 2F5 to screen a set of phage-displayed, random peptide libraries. Tight-binding clones from the random peptide libraries displayed sequence variability in the regions flanking the DKW motif. To further reveal flanking regions involved in 2F5 binding, two semi-defined libraries were constructed having 12 variegated residues either N-terminal or C-terminal to the DKW core (X(12)-AADKW and AADKW-X(12), respectively). Three clones isolated from the AADKW-X(12) library had similar high affinities, despite a lack of sequence homology among them, or with gp41. The contribution of each residue of these clones to 2F5 binding was evaluated by Ala substitution and amino acid deletion studies, and revealed that each clone bound 2F5 by a different mechanism. These results suggest that the 2F5 paratope is formed by at least two functionally distinct regions: one that displays specificity for the DKW core epitope, and another that is multispecific for sequences C-terminal to the core epitope. The implications of this second, multispecific region of the 2F5 paratope for its unique biological function are discussed.     

Identification of B cell epitopes of dengue virus 2 NS3 protein by monoclonal antibody

Dengue virus is a major international public health concern, and there is a lack of available effective vaccines. Virus-specific epitopes could help in developing epitope peptide vaccine. Previously, a neutralizing monoclonal antibody (mAb) 4F5 against nonstructural protein 3 (NS3) of dengue virus 2 (DV2) was developed in our lab. In this work, the B cell epitope recognized by mAb 4F5 was identified using the phage-displayed peptide library. The results of the binding assay and competitive inhibition assay indicated that the peptides, residues 460–469 (U460-469 RVGRNPKNEN) of DV2 NS3 protein, were the B cell epitopes recognized by mAb 4F5. Furthermore, the epitope peptides and a control peptide were synthesized and then immunized female BALB/c mice. ELISA analysis showed that immunization with synthesized epitope peptide elicited a high level of antibody in mice, and immunofluorescent staining showed that the antisera from fusion epitope-immunized mice also responded to DV2 NS3 protein, which further characterized the specific response of the present epitope peptide. Therefore, the present work revealed the specificity of the newly identified epitope (U460-469) of DV2 NS3 protein, which may shed light on dengue virus (DV) vaccine design, DV pathogenesis study, and even DV diagnostic reagent development.     

Insights into the molecular interactions between aminopeptidase and amyloid beta peptide using molecular modeling techniques

Amyloid beta (Aβ) peptides play a central role in the pathogenesis of Alzheimer’s disease. The accumulation of Aβ peptides in AD brain was caused due to overproduction or insufficient clearance and defects in the proteolytic degradation of Aβ peptides. Hence, Aβ peptide degradation could be a promising therapeutic approach in AD treatment. Recent experimental report suggests that aminopeptidase from Streptomyces griseus KK565 (SGAK) can degrade Aβ peptides but the interactive residues are yet to be known in detail at the atomic level. Hence, we developed the three-dimensional model of aminopeptidase (SGAK) using SWISS-MODEL, Geno3D and MODELLER. Model built by MODELLER was used for further studies. Molecular docking was performed between aminopeptidase (SGAK) with wild-type and mutated Aβ peptides. The docked complex of aminopeptidase (SGAK) and wild-type Aβ peptide (1IYT.pdb) shows more stability than the other complexes. Molecular docking and MD simulation results revealed that the residues His93, Asp105, Glu139, Glu140, Asp168 and His255 are involved in the hydrogen bonding with Aβ peptide and zinc ions. The interactions between carboxyl oxygen atoms of Glu139 of aminopeptidase (SGAK) with water molecule suggest that the Glu139 may be involved in the nucleophilic attack on Ala2–Glu3 peptide bond of Aβ peptide. Hence, amino acid Glu139 of aminopeptidase (SGAK) might play an important role to degrade Aβ peptides, a causative agent of Alzheimer’s disease.     

Development of an enzyme-linked immunosorbent assay for thiacloprid in soil and agro-products with phage-displayed peptide

A monoclonal antibody (3A5) that can recognize thiacloprid was produced, and a linear 8-residue peptide phage library was constructed. Six phage-displayed peptides were isolated from the linear 8-residue peptide phage library and a cyclic 8-residue peptide phage library. A phage enzyme-linked immunosorbent assay (ELISA) was developed to detect thiacloprid using a phage-displayed peptide. Under the optimal conditions, the half-maximal inhibition concentration (IC₅₀) and the limit of detection (IC₁₀) of the developed phage ELISA were 8.3 and 0.7 μg/L, respectively. Compared with the conventional ELISA, the sensitivity was improved more than 3-fold. The cross-reactivity (CR) was less than 0.08% for the tested structural analogues and was regarded as negligible. The recoveries of thiacloprid ranged from 80.3% to 116.3% in environmental and agricultural samples, which conformed to the requirements for residue detection. The amount of thiacloprid detected by phage ELISA in the samples was significantly correlated with that detected by high-performance liquid chromatography. The current study indicates that isolating phage-displayed peptides from phage display libraries is an alternative method for the development of a sensitive immunoassay and that the developed assay is a potentially useful tool for detecting thiacloprid in environmental and agricultural samples.     

Potential application of antibody-mimicking peptides identified by phage display in immuno-magnetic separation of an antigen

Phage display was performed against human IgG (hIgG) through five rounds of 'biopanning'. Each round consisted of: (1) incubating a library of phage-displayed 12-mer peptides sequences on hIgG-coated magnetic beads, (2) washing the unbound phages, and (3) eluting the bound phages. The eluted phages were either amplified to enrich the pool of positive clones or subjected to the next round without amplification. Through ELISA, four clones (F9, D1, G5, and A10) showing specific binding affinity to hIgG were identified. Among these, F9 had the highest affinity (K(d)=6.2nM), only one order of magnitude lower than the native anti-hIgG antibody (0.66nM). Following the DNA sequences of the selected clones, four 12-mer peptides were chemically synthesized. Among them, D1 peptide showed the highest binding affinity to hIgG via SPR biosensor measurements. This peptide was conjugated to biofunctionalized magnetic beads, and its immuno-binding ability was compared with that of the native antibody immobilized to magnetic beads. The mol-to-mol binding efficacy of the peptide-coated magnetic beads was approximately 1000-fold lower than that of the antibody-coated magnetic beads. Our results suggest a feasibility of using antibody-mimicking peptides identified by phage display technique for immuno-magnetic separation of an antigen.     

Identification of peptides that bind to the constant region of a humanized IgG1 monoclonal antibody using phage display

The pFc' fragments of a humanized IgG1 monoclonal antibody were generated by digestion with immobilized pepsin. These pFc' fragments were separated from F(ab')2 fragments by affinity chromatography. The pFc' fragments corresponding to the constant region of the humanized IgG1 monoclonal antibody were used as targets for phage display using variable-length peptide libraries. Interacting phage-displayed peptides were selected by repetitious cycles of target screening and phage amplification. Peptide sequences, deduced by sequencing DNA from isolated phage, were aligned and analyzed for amino acid motifs against each other and protein A. These results indicated that an amino acid motif has been identified using phage display technology that is sufficient for pFc' binding. Furthermore, the peptides derived from this study may prove useful in the development of peptidomimetic alternatives to protein A for use in affinity chromatography.     

Selection of trkB-binding peptides from a phage-displayed random peptide library

Brain-derivedneurotrophicfactor(BDNF),originallypurifiedfrompigbrainbyBarderetal.[1]in1982,belongstothefamilyofneurotrophins(NTs)aswellasnervegrowthfactor(NGF),neurotrophin-3(NT-3),NT-4/5.Itisabletopromotesurvivalanddifferentiationofseveralpopu-lationsofneurons,includingmesencephalicdopaminergicneurons,motorneurons,andcholiner-gicneurons,andtoprotectthemagainstneurotoxicityandischemia.BDNFplaysanimportantroleinregulatingneuronsurvivalanddifferentiationduringdevelopmentandinmaintainingthe…