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

Brain-derived neurotrophic factor (BDNF) shows potential in the treatment of neurodegenerative diseases, but the therapeutic application of BDNF has been greatly limited because it is too large in molecular size to permeate blood-brain barrier. To develop low-molecular-weight BDNF-like peptides, we selected a phage-displayed random peptide library using trkB expressed on NIH 3T3 cells as target in the study. With the strategy of peptide library incubation with NIH 3T3 cells and competitive elution with 1 μg/mL of BDNF in the last round of selection, the specific phages able to bind to the natural conformation of trkB and antagonize BDNF binding to trkB were enriched effectively. Five trkB-binding peptides were obtained, in which a core sequence of CRA/TXΦXXΦXXC (X represents the random amino acids, Φ represents T, L or I) was identified. The BDNF-like activity of these five peptides displayed on phages was not observed, though all of them antagonized the activity of BDNF in a dose-dependent manner. Similar results were obtained with the synthetic peptide of C1 clone, indicating that the 5 phage-derived peptides were trkB antagonists. These low-molecular-weight antagonists of trkB may be of potential application in the treatment of neuroblastoma and chronic pain. Meanwhile, the obtained core sequence also could be used as the base to construct the secondary phage-displayed peptide library for further development of small peptides mimicking BDNF activity.     

Structure-Activity Relationships of Conformationally Constrained Peptide Analogues of Loop 2 of Brain-Derived Neurotrophic Factor

Abstract: Brain-derived neurotrophic factor (BDNF) promotes the survival of various neuronal populations and thus shows potential in the treatment of neurodegenerative disease. However, BDNF is not pharmacokinetically optimal for use as a therapeutic agent. As a step toward the development of low-molecular-weight BDNF-like drugs, we have designed a series of small, conformationally constrained peptides of various sizes using the three-dimensional structure of BDNF derived by homology modeling as a template. When tested in cultures of embryonic chick sensory neurons the peptides produced concentration-dependent inhibition of BDNF-mediated neuronal survival and caused both a rightward shift and depression of the maximum of the BDNF concentration-response curve. The compounds had no effect on the survival response to nerve growth factor and were without intrinsic trophic or toxic effects when added to cultures alone. With the aid of pharmacodynamic simulations we demonstrated that the inhibitory activity of the active peptides is consistent with them acting as competitive antagonists of BDNF for its high-affinity receptor, trkB. An alanine scan of the largest peptide identified several residues important in mediating the inhibitory action of the peptides. We intend to use the data from these studies to develop small peptidic BDNF-like agonists.     

脑源性神经营养因子受体trkB在NIH 3T3细胞上的表达

构建了克隆有大鼠脑源性神经营养因子（BDNF）受体trkB全长基因的真核表达载体pcDNA3.1( )-rat trkB.用脂质体介导法将重组载体转入小鼠NIH3T3细胞,在mRNA和蛋白质水平检测到了trkB基因在用G418筛选到的抗性NIH 3T3细胞中的表达,表达的trkB蛋白定位于细胞膜上。BDNF能够剂量依赖性地促进NIH 3T3－trkB细胞的增殖,说明表达的trkB是有功能的。该表达trkB和NIH3T3细胞为研究BDNF的生理功能、活性测定和从噬菌体展示肽库中筛选BDNF肽提供了一个简便的细胞模型。     

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。     

Peptides selected from phage display library may change the conformation of S protein of rice stripe virus

Summary Phages with high affinity to the S protein obtained from rice stripe virus (RSV) were enriched from phage-displayed random 12-mer peptide library after three rounds of biopanning. 9 different peptides from the enriched library were selected by ELISA. Circular dichroism (CD) spectra of the GST-S fusion protein with binding phages and non-binding phages showed that structure of the S protein was changed after it bound to each of these 9 selected 12-mer peptides, which suggested that these peptides might disrupt the function of S protein. Thus, those peptides might be used to develop plant resistance and disrupt virus transmission. 3 of the 12-mer peptide genes were fused with the GST gene in pGEX 3X. The fusion proteins were also obtained usingE. coli expression system and purified.     

Identification of Brucella cell wall antigenic determinants by immunoscreening of a random peptide library expressed at the surface of filamentous phage

Summary Antibodies against the 89 kDa Brucella abortus outer membrane protein (OMP) are detected in 68% of B. abortus infected cows. A monoclonal antibody, specifically directed against Brucella OMP89, has been used to screen a phage-displayed peptide library. We describe here results obtained from affinity selection of phages with mAb A7617E3C3 in three rounds of biopanning using a cysteine-constrained peptide library expressed on the surface of filamentous bacteriophages pVIII major coat protein. Deduced amino acid sequences of the peptide region of clones positively detected by colony immunoblotting experiments indicate the presence of a consensus sequence. Peptides corresponding to the most frequently represented sequence have been synthesized. Monoclonal antibody binding to selected phages is inhibited by corresponding cyclic peptides, but not by linear peptides. This confirms the specificity of the peptide sequence for its paratope but also the importance of a certain conformation for binding.     

The trkB tyrosine protein kinase is a receptor for brain-derived neurotrophic factor and neurotrophin-3.

trkB is a tyrosine protein kinase gene highly related to trk, a proto-oncogene that encodes a receptor for nerve growth factor (NGF) and neurotrophin-3 (NT-3). trkB expression is confined to structures of the central and peripheral nervous systems, suggesting it also encodes a receptor for neurotrophic factors. Here we show that brain-derived neurotrophic factor (BDNF) and NT-3, but not NGF, can induce rapid phosphorylation on tyrosine of gp145trkB, one of the receptors encoded by trkB. BDNF and NT-3 can induce DNA synthesis in quiescent NIH 3T3 cells that express gp145trkB. Cotransfection of plasmids encoding gp145trkB and BDNF or NT-3 leads to transformation of recipient NIH 3T3 cells. In these assays, BDNF elicits a response at least two orders of magnitude higher than NT-3. Finally, 125I-NT-3 binds to NIH 3T3 cells expressing gp145trkB; binding can be competed by NT-3 and BDNF but not by NGF. These findings indicate that gp145trkB may function as a neurotrophic receptor for BDNF and NT-3.     

Novel monocyclic and bicyclic loop mimetics of brain-derived neurotrophic factor.

Brain-derived neurotrophic factor (BDNF) is a protein that promotes the survival of neurons. It is widely thought to possess clinical potential for the treatment of neurodegenerative diseases, and in recent years, has been found to play a role in the pathogenesis of some tumours. BDNF is thought to bind to its cellular receptors trkB and p75(NTR) primarily by way of solvent-exposed loops on the BDNF dimer. In this paper, we describe our recent progress towards the development of small peptides as mimetics and inhibitors of BDNF. Two classes of peptides were prepared: disulphide-constrained monomeric monocyclic peptides designed to mimic a single solvent-exposed loop; and homo- and heterodimeric bicyclic peptides designed to mimic pairs of loops. Each peptide was examined in cultures of embryonic chick dorsal root ganglion sensory neurons, both alone, and in competition with BDNF. All peptides were found to inhibit BDNF-mediated neuronal survival, while one--a dimeric peptide based on the two loop 4 regions of BDNF--behaved as a partial BDNF-like agonist. The work described in this paper supports the proposed receptor-binding role of loops 1, 2, and 4 of BDNF, and provides valuable steps towards our long-term goal of developing BDNF mimetics and inhibitors for clinical use.     

Selection of silk-binding peptides by phage display

Peptides that bind to silkworm-derived silk fibroin fiber were selected from a phage-displayed random peptide library. The selected silk-binding peptides contained a consensus sequence QSWS which is important for silk-binding as confirmed by binding assays using phage and synthetic peptides. With further optimization, we anticipate that the silk-binding peptides will be useful for functionalization of silk for biomaterial applications.     

Sequence and structural diversity in endotoxin-binding dodecapeptides

For the study of sequence or structure requirement of short peptides for endotoxin binding, and to search for potential endotoxin antagonists, biopanning was carried out on a phage-displayed random dodecapeptide library against immobilized lipopolysaccharide (LPS) or lipid A (LA), the core toxic portion of LPS. Specific binding of selected phage-displayed peptides to LPS/LA was confirmed by surface plasmon resonance (SPR) analysis. These peptides are rich in basic and hydrophobic amino acids, especially histidine, proline and tryptophan, highlighting apparent amphiphilicity and bacterial membrane activity. These dodecapeptide sequences have no predictable secondary structure in solution, indicating the importance of a random structure before their interaction with LPS/LA. Sequence alignment reveals various potential secondary structures with these selected peptides, which contain specific signature motifs such as b(p)hb(p)hb(p), bbbb, hhhh (b-basic, p-polar, h-hydrophobic residue), capable of binding LPS/LA. However, none of these peptides exhibit a significant calculated structural amphiphilicity while assuming a secondary structure. This study suggests that for these short dodecapeptides to bind LPS/LA, the potential for their structural adaptation is more important than an amphipathic structure.     

The neurotrophic factors brain-derived neurotrophic factor and neurotrophin-3 are ligands for the trkB tyrosine kinase receptor.

Neurotrophic factors are essential for neuronal survival and function. Recent data have demonstrated that the product of the tyrosine kinase trk proto-oncogene binds NGF and is a component of the high affinity NGF receptor. Analysis of the trkB gene product, gp145trkB, in NIH 3T3 cells indicates that this tyrosine kinase receptor is rapidly phosphorylated on tyrosine residues upon exposure to the NGF-related neurotrophic factors BDNF and NT-3. Furthermore, gp145trkB specifically binds BDNF and NT-3 in NIH 3T3 cells and in hippocampal cells, but does not bind NGF. Thus, the trk family of receptors are likely to be important signal transducers of NGF-related trophic signals in the formation and maintenance of neuronal circuits.     

Peptides selected from phage display library may change the conformation of S protein of rice stripe virus

Phages with high affinity to the S protein obtained fromrice stripe virus (RSV) were enriched fromphage-displayed random 12-mer peptide library after threerounds of biopanning. 9 different peptides from theenriched library were selected by ELISA. Circulardichroism (CD) spectra of the GST-S fusion protein withbinding phages and non-binding phages showed thatstructure of the S protein was changed after it bound toeach of these 9 selected 12-mer peptides, which suggestedthat these peptides might disrupt the function of Sprotein. Thus, those peptides might be used to developplant resistance and disrupt virus transmission. 3 of the12-mer peptide genes were fused with the GST gene in pGEX3X. The fusion proteins were also obtained using E.coli expression system and purified.     

Analysis of the humoral immune response to immunoselected phage-displayed peptides by a microarray-based method

We describe a novel approach for high-throughput analysis of the immune response in cancer patients using phage-based microarray technology. The recombinant phages used for fabricating phage arrays were initially selected via the use of random peptide phage libraries and breast cancer patient serum antibodies. The peptides displayed by the phages retained their ability to be recognized by serum antibodies after immobilization. The recombinant phage microarrays were screened against either breast cancer or healthy donor serum antibodies. A model-based statistical method is proposed to estimate significant differences in serum antibody reactivity between patients and normals. A significant tumor effect was found with most of the selected phage-displayed peptides, suggesting that recombinant phage microarrays can serve as a tool in monitoring humoral responses towards phage-displayed peptides.     

Dense surface functionalization using peptides that recognize differences in organized structures of self-assembling nanomaterials

We obtained novel peptides that selectively bind to self-assembling peptide nanomaterials from a random peptide library displayed on phages. Affinity-dependent peptide screening gave phage clones displaying peptides with selective affinities against two kinds of highly networked nanofibers constructed of β-sheet peptides. Both peptide nanofibers have similar primary structures. Binding analyses of phage-displayed peptides by enzyme-linked immunosorbent assays demonstrated that the screened peptides specifically bind to the target nanofibers rather than to non-target nanofibers. Dot blot assays using chemically-synthesized peptides revealed that one screened peptide with the sequence Thr-Tyr-Leu-Pro-Trp-Pro-Ala has an affinity constant of 2.6 × 10(6) M(-1) against the target nanofibers. This affinity is 350 times greater than its affinity for non-target nanofibers and 90 times greater than its affinity for mixed nanofibers that contain 50% target nanofibers. These results suggest that this screened peptide can recognize organized fine-structures. Surface modification of the peptide nanofibers with the screened peptide demonstrated that the peptide specifically binds to discrete binding sites on the target nanofibers. Cell adhesion assays on the nanofibers by means of RGDS-conjugated screened peptides showed that the number of adhered cells is largely dependent on the amount of bound RGDS-peptide. These results suggest that screened peptides can recognize the organization of self-assembled peptide nanomaterials, and that the conjugation of functional groups to the peptides can be used to functionalize the target nanomaterials. Furthermore, simultaneous use of individual specific peptides that have specificities for nanomaterials can generate highly dense functionalized self-assembling peptide nanomaterials.     

Suppression of tumor growth by novel peptides homing to tumor-derived new blood vessels

Novel peptides homing to angiogenic vessels were recently isolated from a phage-displayed random pentadecapeptide library. One of the isolated peptides, ASSSYPLIHWRPWAR, significantly suppressed the migration of VEGF-stimulated human umbilical vein endothelial cells. Dendoric ASSSYPLIHWRPWAR-peptide suppressed the formation of new blood vessels in dorsal air sac model mice. Furthermore, ASSSYPLIHWRPWAR-peptide and the fragment peptides containing WRP, which is revealed to be an epitope sequence, significantly suppressed the tumor growth, although 15-mer shuffled peptide derived from ASSSYPLIHWRPWAR and pentapeptides with alanine substitution of each residue of WRP did not. Taken together, ASSSYPLIHWRPWAR-peptide may cause tumor dormancy through inhibition of angiogenesis, and the WRP sequence may be the minimal and essential sequence for this activity.     

Selection of ganglioside GM1-binding peptides by using a phage library.

Ganglioside Gal beta1 --> 3GalNAc beta1 --> 4(NeuAc alpha2 --> 3) Gal beta1 --> 4Glc beta1 -->1'Cer (GM1)-binding peptides were obtained from a phage-displayed pentadecapeptide library by an affinity selection. The selection processes were in situ-monitored by a quartz-crystal microbalance method, on which a ganglioside GM1 monolayer was transferred. After five rounds of biopanning, the DNA sequencing of 18 selected phages showed that only three individual clones were selected. The peptide sequences of the random region were found to be DFRRLPGAFWQLRQP, GWWYKGRARPVSAVA and VWRLLAPPFSNRLLP. Binding constants of these phage clones to the GM1 monolayer were 10(10) M(-1). Three synthetic pentadecapeptides inhibited the binding of cholera toxin B subunit to the GM1 monolayer with an IC50 of 24, 13 and 1.0 microM, respectively. These peptides will be useful for searching functional roles of ganglioside GMI.     

Selection of CC chemokine receptor 5-binding peptide from a phage display peptide library

Human CC chemokine receptor (CCR) 5 is a G protein-coupled receptor involved in a broad range of human diseases that mediates HIV-1 viral entry into cells. Certain small molecule receptor antagonists to CCR5 have been useful in therapy for these diseases. In this study, CCR5-expressing CHO cells (CHO/CCR5 cells) were used to select CCR5-binding peptides from a phage-displayed 12-mers peptide library. All of the 30 clones selected from the library showed specific binding to CHO/CCR5 cells by enzyme linked immunosorbent assay (ELISA). Seventeen out of the 30 clones shared the amino acid motif AFDWTFVPSLIL. The motif-containing phages and synthetic peptide AFDWTFVPSLIL blocked the binding of mAb 2D7 to CHO/CCR5 cells and competitively inhibited the ability of chemokine regulated on activation normal T cell expressed and secreted (RANTES) binding to CHO/CCR5 cells. Furthermore, the peptide AFDWTFVPSLIL also inhibited RANTES induced increase in the intracellular Ca2+ level in CHO/CCR5 cells. These results suggest that the peptide AFDWTFVPSLIL was specific for CCR5 and that it might become a CCR5 antagonist.     

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.     

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.