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.     

从噬菌体表面展示肽库中筛选葡萄球菌B型肠毒素抑制剂

通过生物淘选,从噬菌体表面展示12肽肽库中筛选能与葡萄球菌B型肠毒素(staphylococcalenterotoxinB ,SEB)结合且能抑制其肠毒活性的特异性短肽.采用Phage ELISA和MTT鉴定所得目的肽的亲和性;根据优势噬菌体阳性克隆序列合成相应多肽.利用竞争ELISA研究合成肽与SEB单克隆抗体竞争结合SEB的情况;通过动物实验考察其抑制SEB的超抗原特性和肠毒活性情况.筛选所得短肽在一定浓度范围内可以抑制SEB对鼠脾淋巴细胞的激活;合成肽与SEB质量比为16 0∶1时,合成肽可较好地抑制SEB对乳猫的肠毒活性,并对SEB引起的小鼠致死具有明显保护作用.结果表明,初步得到了能与SEB特异结合并能抑制SEB超抗原特性和肠毒活性的短肽,为进一步研制SEB高效抑制剂奠定了基础.     

Antagonists of Hsp16.3, a low-molecular-weight mycobacterial chaperone and virulence factor, derived from phage-displayed peptide libraries

The persistence of Mycobacterium tuberculosis is a major cause of concern in tuberculosis (TB) therapy. In the persistent mode the pathogen can resist drug therapy, allowing the possibility of reactivation of the disease. Several protein factors have been identified that contribute to persistence, one of them being the 16-kDa low-molecular-weight mycobacterial heat shock protein Hsp16.3, a homologue of the mammalian eye lens protein alpha-crystallin. It is believed that Hsp16.3 plays a key role in the persistence phase by protecting essential proteins from being irreversibly denatured. Because of the close association of Hsp16.3 with persistence, an attempt has been made to develop inhibitors against it. Random peptide libraries displayed on bacteriophage M13 were screened for Hsp16.3 binding. Two phage clones were identified that bind to the Hsp16.3 protein. The corresponding synthetic peptides, an 11-mer and a 16-mer, were able to bind Hsp16.3 and inhibit its chaperone activity in vitro in a dose-dependent manner. Little or no effect of these peptides was observed on alphaB-crystallin, a homologous protein that is a key component of human eye lens, indicating that there is an element of specificity in the observed inhibition. Two histidine residues appear to be common to the selected peptides. Nuclear magnetic resonance studies performed with the 11-mer peptide indicate that in this case these two histidines may be the crucial binding determinants. The peptide inhibitors of Hsp16.3 thus obtained could serve as the basis for developing potent drugs against persistent TB.     

Antagonists of Hsp16.3, a Low-Molecular-Weight Mycobacterial Chaperone and Virulence Factor, Derived from Phage-Displayed Peptide Libraries

The persistence of Mycobacterium tuberculosis is a major cause of concern in tuberculosis (TB) therapy. In the persistent mode the pathogen can resist drug therapy, allowing the possibility of reactivation of the disease. Several protein factors have been identified that contribute to persistence, one of them being the 16-kDa low-molecular-weight mycobacterial heat shock protein Hsp16.3, a homologue of the mammalian eye lens protein alpha-crystallin. It is believed that Hsp16.3 plays a key role in the persistence phase by protecting essential proteins from being irreversibly denatured. Because of the close association of Hsp16.3 with persistence, an attempt has been made to develop inhibitors against it. Random peptide libraries displayed on bacteriophage M13 were screened for Hsp16.3 binding. Two phage clones were identified that bind to the Hsp16.3 protein. The corresponding synthetic peptides, an 11-mer and a 16-mer, were able to bind Hsp16.3 and inhibit its chaperone activity in vitro in a dose-dependent manner. Little or no effect of these peptides was observed on alphaB-crystallin, a homologous protein that is a key component of human eye lens, indicating that there is an element of specificity in the observed inhibition. Two histidine residues appear to be common to the selected peptides. Nuclear magnetic resonance studies performed with the 11-mer peptide indicate that in this case these two histidines may be the crucial binding determinants. The peptide inhibitors of Hsp16.3 thus obtained could serve as the basis for developing potent drugs against persistent TB.     

Suppression of Helicobacter pylori-induced gastritis by green tea extract in Mongolian gerbils

Since urease of Helicobacter pylori is essential for its colonization, we focused attention on foodstuffs which inhibit the activity of this enzyme. Among plant-derived 77 foodstuff samples tested, some tea and rosemary extracts were found to clearly inhibit H. pylori urease in vitro. In particular, green tea extract (GTE) showed the strongest inhibition of H. pylori urease, with an IC(50) value of 13 microg/ml. Active principles were identified to be catechins, the hydroxyl group of 5(')-position appearing important for urease inhibition. Furthermore, when H. pylori-inoculated Mongolian gerbils were given GTE in drinking water at the concentrations of 500, 1000, and 2000 ppm for 6 weeks, gastritis and the prevalence of H. pylori-infected animals were suppressed in a dose-dependent manner. Since the acquisition by H. pylori of resistance to antibiotics has become a serious problem, tea and tea catechins may be very safe resources to control H. pylori-associated gastroduodenal diseases.     

基质金属蛋白酶2抑制剂的筛选及鉴定

以原核表达的具有明胶水解活性的人基质金属蛋白酶 2的催化区 (MCD)为靶标 ,筛选噬菌体随机环七肽库和十二肽库 .找到 6种与MCD特异结合的小肽 ,将 6种小肽基因分别与GST表达质粒重组 ,进行GST融合表达 ,制备融合蛋白 .采用Glutathione Sepharose 4B亲和层析法纯化融合蛋白 ,通过酶抑制实验、体外侵袭实验检测融合蛋白的活性 .结果表明 ,GST C71能够抑制MCD水解 β酪蛋白的活性 ,并且对人纤维肉瘤细胞HT10 80的体外侵袭有明显的抑制作用     

Dependence of Helicobacter pylori urease activity on the nickel-sequestering ability of the UreE accessory protein

The Helicobacter pylori ureE gene product was previously shown to be required for urease expression, but its characteristics and role have not been determined. The UreE protein has now been overexpressed in Escherichia coli, purified, and characterized, and three altered versions were expressed to address a nickel-sequestering role of UreE. Purified UreE formed a dimer in solution and was capable of binding one nickel ion per dimer. Introduction of an extra copy of ureE into the chromosome of mutants carrying mutations in the Ni maturation proteins HypA and HypB resulted in partial restoration of urease activity (up to 24% of the wild-type levels). Fusion proteins of UreE with increased ability to bind nickel were constructed by adding histidine-rich sequences (His-6 or His-10 to the C terminus and His-10 as a sandwich fusion) to the UreE protein. Each fusion protein was overexpressed in E. coli and purified, and its nickel-binding capacity and affinity were determined. Each construct was also expressed in wild-type H. pylori and in hypA and hypB mutant strains for determining in vivo urease activities. The urease activity was increased by introduction of all the engineered versions, with the greatest Ni-sequestering version (the His-6 version) also conferring the greatest urease activity on both the hypA and hypB mutants. The differences in urease activities were not due to differences in the amounts of urease peptides. Addition of His-6 to another expressed protein (triose phosphate isomerase) did not result in stimulation of urease, so urease activation is not related to the level of nonspecific protein-bound nickel. The results indicate a correlation between H. pylori urease activity and the nickel-sequestering ability of the UreE accessory protein.     

Epitope mapping and features of the epitope for monoclonal antibodies inhibiting enzymatic activity of Helicobacter pylori urease

Two characteristic monoclonal antibodies (HpU-2 and -18) out of 26 monoclonal antibodies (HpU-1 approximately 26) produced against Helicobacter pylori (H. pylori) urease showed a strong inhibitory effect against the enzymatic activity of the urease. Epitope mapping about some monoclonal antibodies of the HpU-series inhibiting enzymatic activity was performed by using a surface plasmon resonance apparatus and by digesting H. pylori urease with trypsin, followed by mass spectroscopy. The sequences of the epitopes recognized by HpU-2 and -18 were SVELIDIGGNRRIFGFNALVDR (22 mer) and IFGFNALVDR (10 mer), respectively. The former sequence is present as a part of a loop structure at a position close to the C-terminal of the alpha-subunit of H. pylori urease, although it has been suggested that the active site of the urease resides in the beta-subunit. The above peptide (22 mer) was chemically synthesized in a linear and cyclic form, and its conjugate with BSA was immunized in rabbits. The resultant serum induced by the linear form could specifically bind to H. pylori infecting human gastric mucosa. These results suggest that the above sequence (22 mer) must be an important epitope, although it locates in the alpha-subunit but not in the beta-subunit.     

Identifying specific matrix metalloproteinase-2-inhibiting peptides through phage display-based subtractive screening

Gelatinases A and B, which are members of the matrix metalloproteinase (MMP) family, play essential roles in cancer development and metastasis, as they can break down basal membranes. Therefore, the determination and inhibition of gelatinases is essential for cancer treatment. Peptides that can specifically block each gelatinase may, therefore, be useful for cancer treatment. In this study, subtractive panning was carried out using a 12-mer peptide library to identify peptides that block gelatinase A activity (MMP-2), which is a key pharmacological target. Using this method, 17 unique peptide sequences were determined. MMP-2 inhibition by these peptides was evaluated through zymogram analyses, which revealed that four peptides inhibited MMP-2 activity by at least 65%. These four peptides were synthesized and used for in vitro wound healing using human umbilical vein endothelial cells, and two peptides, AOMP12 and AOMP29, were found to inhibit wound healing by 40%. These peptides are, thus, potential candidates for MMP-2 inhibition for cancer treatment. Furthermore, our findings suggest that our substractive biopanning screening method is a suitable strategy for identifying peptides that selectively inhibit MMP-2.     

Investigation and molecular mimicry of the antigen involved in the interaction between the monoclonal antibody 5D10 and the human breast cancer cell line MCF-7

Monoclonal antibody (mAb) 5D10 is directed against the human breast cancer cell line MCF-7. Biochemical characterization of the antibody epitope was attempted and revealed a complex, most likely carbohydrate-linked nature, which prevented isolation and further studies of the interaction. A major goal of this work was to generate structural mimics of the 5D10 epitope to serve as putative substitutes in such studies. A peptide library displayed on filamentous phage was used to select for mimotope peptide sequences. All positive phage clones selected from the library displayed the amino acid sequence H(2)N-QMNPMYYR-CO(2)H. This peptide sequence, as well as a branched form of the peptide, was found to bind mAb 5D10. Moreover, both peptide sequences were able to inhibit the binding of 5D10 to the MCF-7 cells in a concentration-dependent manner, with an EC(50) value in the range of 65 microM. According to these results, random phage peptide libraries can serve to identify mimotopic peptides for unknown complex cell surface epitopes.     

Selection of peptide inhibitors against the Pseudomonas aeruginosa MurD cell wall enzyme

The purified Pseudomonas aeruginosa cell wall biosynthesis MurD amide ligase enzyme was used to screen C-7-C and 12 mers peptides from phage display libraries using competitive biopanning approaches with the specific substrates D-glutamate and ATP. From the 60 phage-encoded peptides identified, DNA was sequenced, deduced amino acid sequences aligned and two peptides were synthesized from consensus sequences identified. The UDP-N-acetylmuramyl-L-alanine MurD substrate was synthesized, purified and used to develop a spectrophotometric assay. One peptide synthesized was found to specifically inhibit ATPase activity of MurD. The IC50 value was estimated at 4 microM for the C-7-C MurDp1 peptide. The loop conformation of MurDp1 was shown to be important for the inhibition of the UDP-N-acetylmuramyl-L-alanine:D-glutamate MurD ligase. The linear 12 mers MurD2 peptide has an IC50 value of 15 mM. A conserved amino acid motif was found between MurDp2 and the bacterial glyceraldehyde 3-phosphate dehydrogenase indicating that MurDp2 binds at a protein-protein interacting site. The approach proposed and results obtained suggest that efficient peptide inhibitors as well as protein-protein interaction domains can be identified by phage display.     

Design, synthesis, and evaluation of peptides with estrogen-like activity

Currently used antiestrogenic drugs against hormone-dependent breast cancer, and estrogenic drugs used in treatment of osteoporosis, are associated with risk factors. Therefore, there is a strong need to develop selective estrogen receptor modulators with better tissue selectivity. In a recent study (Peptides, 2002, Vol. 3, 573-580), we used a monoclonal antibody to estradiol (mAb-E2) to screen a phage-display peptide library. We identified a 15-mer peptide (peptide H5) that recognizes mAb-E2 (IC(50) 1 microM) and estrogen receptor (ER)alpha (IC(50) 500 microM) but not ERbeta, and displays estrogen-like activity in vitro and in vivo. In this study, we designed and prepared peptides based on peptide H5, which possess improved estrogenic activity, by evaluating their binding to mAb-E2 and to ERs. Initially, we determined the minimal binding sequence of peptide H5 capable of binding mAb-E2 and ER. Subsequently, systematic single-residue replacements of the minimal sequence, followed by multiple-residue replacements, yielded hexa- and heptapeptides with increased affinities to mAb-E2 and to ER. The most promising peptides, VSWFFE (EMP-1) and VSWFFED (EMP-2) (EMP: estrogen-mimetic peptide), bind mAb-E2 with high affinity (IC(50) of 6 and 30 nM, respectively), recognize ERs with increased affinity (IC(50) of 100 microM for ERalpha, and 100-250 microM for ERbeta), and possess estrogenic activity in vivo. The short peptides described in this study may be used as potential lead compounds for developing new ER ligands.     

Novel peptides with tyrosinase inhibitory activity

Tyrosinase inhibition by peptides may find its application in food, cosmetics or medicine. In order to identify novel tyrosinase inhibitory peptides, protein-based peptide libraries made by SPOT synthesis were used to screen for peptides that show direct interaction with tyrosinase. One of the peptide libraries studied consists of overlapping, octameric peptides derived from industrial proteins as beta-casein, alpha-lactalbumin, beta-lactoglobulin, ovalbumin, gliadin, glycinin, and beta-conglycinin. On-membrane activity staining resulted in a set of peptides that are not only able to bind to tyrosinase, but are able to inhibit tyrosinase as well. Peptides containing aspartic or glutamic acid residues usually do not bind very well to tyrosinase. Strong tyrosinase-binding peptides always contain one or more arginine residues, often in combination with phenylalanine, while lysine residues can be found equally among nonbinding peptides as well as moderate tyrosinase-binding peptides. The presence of the hydrophobic, aliphatic residues valine, alanine or leucine appears to be important for tyrosinase inhibition. Therefore, good tyrosinase inhibitory peptides preferably contain arginine and/or phenylalanine in combination with valine, alanine and/or leucine.     

Selection of peptides targeting the human sperm surface using random peptide phage display identify ligands homologous to ZP3

Analysis of the surface architecture of human spermatozoa is a necessary step in the development of new approaches to contraception and resolving the causes of human infertility. In this study we have utilized phage display technology to identify peptides that bind with high affinity to the surface of human spermatozoa. Fifteen- and twelve-mer random peptide phage display libraries were screened against paraformaldehyde-fixed spermatozoa and a number of sperm-binding peptides were identified. One peptide, M6, displayed a high level of affinity for the sperm surface and showed sequence homology with a dominant human ZP3 epitope (hZP 25-33). This peptide bound preferentially to the equatorial and post acrosomal domains of the sperm head and exhibited contraceptive activity by virtue of its capacity to impair the fusion of acrosome-reacted spermatozoa with the vitelline membrane of the oocyte. A similar form of contraceptive activity was also observed within an unrelated peptide, K6, derived from screening the 12-mer library. These results indicate that phage display technology is a powerful tool for developing reagents capable of targeting the human sperm surface, providing insights into the composition of this structure and the identity of targets susceptible to contraceptive attack and pathological disruption.     

Protamine-fragment peptides fused to an SV40 nuclear localization signal deliver oligonucleotides that produce antisense effects in prostate and bladder carcinoma cells

The development of antisense technology has focused on improving methods for oligonucleotide delivery into cells. In the present work, we describe a novel strategy for oligonucleotide delivery based on a bifunctional peptide composed of a C-terminal protamine-fragment that contains a DNA-binding domain and an N-terminal nuclear localization signal sequence derived from the SV40 large-T antigen (The sequences of two of the peptides are R6WGR6-PKKKRKV [s-protamine-NLS] and R4SR6FGR6VWR4-PKKKRKV [l-protamine-NLS]). We demonstrated, by intrinsic fluorescence quenching, that peptides of this class form complexes with oligodeoxynucleotides. To evaluate delivery, we used a 20-mer phosphorothioate oligomer (Isis 3521) targeted to the 3'-untranslated region of the PKC-alpha mRNA and G3139, an 18-mer phosphorothioate targeted to the first six codons of the human bcl-2 open reading frame, and complexed them with either of two peptides (s- or l-protamine-NLS). These peptides bind to and deliver antisense oligonucleotides to the nucleus of T24 bladder and PC3 prostate cancer cells, as demonstrated by confocal microscopy. Furthermore, as shown by Western and Northern blotting, the peptide-oligonucleotide complexes produced excellent downregulation of the expression of the complementary mRNAs, which in turn resulted in downregulation of protein expression. However, under certain circumstances (predominantly in PC3 cells), incubation of the cells with chloroquine was required to produce antisense activity. Using this strategy, PKC-alpha protein and mRNA expression in T24 and PC3 cells and bcl-2 expression in PC3 cells was reduced by approximately 75 +/- 10% at a minimum concentration of oligomer of 0.25 microM, in combination with 12-15 microM peptide. On the basis of our results, we conclude that arginine-rich peptides of this class may be potentially useful delivery vehicles for the cellular delivery of antisense oligonucleotides. This new strategy may have several advantages over other methods of oligonucleotide delivery and may complement already existing lipid-based technologies.     

Selection of peptide ligands binding to fibroblast growth factor receptor 1

Inappropriate expression of fibroblast growth factors (FGFs) or activation of FGF receptors (FGFRs) could contribute to several human angiogenic pathologies. In an attempt to design antagonists of FGF, we developed a screening procedure for identifying peptide ligands binding to FGFR1. To retain the natural conformation of FGFR1 during screening, we expressed recombinant FGFR1 on the surface of Sf9 insect cells. A 6-mer phage display peptide library was then screened on the cell surface and a group of hydrophobic peptide sequences were identified. Further experiments demonstrated that the phages displaying these sequences can specifically bind to FGFR1. The docking analysis suggests that the peptide ValTyrMetSerProPhe can specifically bind to the hydrophobic surface of FGFR1. The synthetic peptide Ac-ValTyrMetSerProPhe-NH2 can inhibit mitogenic activity of aFGF and has the potential to become a therapeutic agent as an aFGF antagonist.     

Isolation of multiple cell-binding ligands from different phage displayed-peptide libraries

A technical challenge in the development of biosensor devices for cancer detection and diagnosis is the identification of ligands that recognize cancer cells with high affinity and specificity. Furthermore, it is unlikely that one cell-binding ligand will provide sufficient biological information, thus, multiple ligands for a given cancer type will be needed for confident clinical diagnosis. Biopanning of phage displayed peptide libraries is a route to isolation of specific cell-binding reagents. A potential approach towards isolation of multiple ligands for a single cell type is to pan against the same cell type using different peptide libraries. Here we report the synthesis of a new 20-mer peptide-phage library and its use to select a peptide that binds to the large cell lung carcinoma cell line, H1299. The isolated phage clone binds H1299 cells 80 times better than a control phage and can distinguish between H1299 and normal control cells. The phage clone also binds to the lung pleura epidermoid cell line, Calu-1 but not to all lung carcinoma cell lines. The peptide is functional outside the context of the phage and tetramerization of the peptide on a trilysine core improves the affinity of the peptide. The tetrameric peptide can be used to deliver a fluorescent quantum dot to H1299 cells. Unexpectedly, the peptide shares sequence similarity to a previously isolated H1299-binding peptide isolated from a different 20-mer peptide library. Data suggests that the two peptides target the same cellular receptor. Our results imply that cell-based biopanning can isolate cell-binding ligands that may be of utility for cancer diagnosis, and isolation of cell-targeting peptides from different peptide libraries can expand the repertoire of cell-binding reagents.     

Characterization of oligopeptides that cross-react with carbohydrate-specific antibodies by real time kinetics, in-solution competition enzyme-linked immunosorbent assay, and immunological analyses

Phage displaying random cyclic 7-mer, and linear 7-mer and 12-mer peptides at the N terminus of the coat protein, pIII, were panned with the murine monoclonal antibody, 9-2-L379 specific for meningococcal lipo-oligosaccharide. Five cyclic peptides with two sequence motifs, six linear 7-mers, and five linear 12-mers with different sequence motifs were identified. Only phage displaying cyclic peptides were specifically captured by and were antigenic for 9-2-L379. Monoclonal antibody 9-2-L379 exhibited "apparent" binding affinities to the cyclic peptides between 11 and 184 nm, comparable with lipo-oligosaccharide. All cyclic peptides competed with the binding of 9-2-L379 to lipo-oligosaccharide with EC(50) values in the range 10-105 microm, which correlated with their apparent binding affinities. Structural modifications of the cyclic peptides eliminated their ability to bind and compete with monoclonal antibody 9-2-L379. Mice (C3H/HeN) immunized with the cyclic peptide with optimal apparent binding affinity and EC(50) of competition elicited cross-reactive antibodies to meningococcal lipo-oligosaccharide with end point dilution serum antibody titers of 3200. Cyclic peptides were converted to T-cell-dependent immunogens without disrupting these properties by C-terminal biotinylation and complexing with NeutrAvidin. The data indicate that constrained peptides can cross-react with a carbohydrate-specific antibody with greater specificity than linear peptides, and critical to this specificity is their structural conformation.     

Screening of ACE-inhibitory peptides from a random peptide-displayed phage library using ACE-coupled liposomes

Angiotensin I converting enzyme (ACE)-inhibitory peptides were screened from a random peptide-displayed phage library using ACE-coupled liposomes. Among four kinds of inhibitory peptides selected by biopanning with two different elution strategies, a peptide (LSTLRSFCA) showed the highest inhibitory activity with an IC(50) value of 3microM. By measuring inhibitory activities of fragments of the peptide, it was found that the RSFCA region was a functional site to inhibit strongly the ACE catalytic activity, and particularly both Arg and Cys residues were essential for the strong inhibitory activity. The inhibitory activity of RRFCA was slightly increased, while that of the RSFRA, in which the Cys residue was replaced by Arg, was decreased to greater extent in comparison with the inhibitory activity of RSFCA. Taking into account the results obtained from the SPOT analysis, it was suggested that the Arg and Phe residues in RSFCA were important for a specific interaction with ACE, and the Cys residue inhibited the ACE activity. The cystein-based ACE-inhibitory peptides have not been isolated from processed food materials. These findings suggested that the biopanning method utilizing protein-coupled liposomes and random peptide libraries might have a possibility to screen new functional peptides that are not found in processed food materials.