Improved substrate specificity of water-soluble pyrroloquinoline quinone glucose dehydrogenase by a peptide ligand

A new approach in altering the substrate specificity of enzyme is proposed using glucose dehydrogenase, with pyrroroquinoine quinone (PQQGDH) as co-factor, as the model. This approach is based on the selection of random peptide phage displayed library. Using an M13 phage-display random peptide library, we have selected peptide ligands. Among the peptide ligands, a 7-mer peptide, composed of Thr-Thr-Ala-Thr-Glu-Tyr-Ser, caused PQQGDH substrate specificity to decrease significantly toward disaccharides, such as maltose and lactose, while a smaller effect was observed toward glucose. Consequently, this peptide narrowed the substrate specificity of PQQGDH, without a significant loss of the enzyme activity.     

Mast cell degranulating (MCD) peptide: a prototypic peptide in allergy and inflammation.

The solid phase synthesis of mast degranulating peptide (MCD peptide) raised the possibility of preparing analogs and examining the pharmacology and the proposed role of this peptide as a potential agent in allergy and inflammation. MCD peptide, a cationic 22-amino acid residue peptide with two disulfide bridges, causes mast cell degranulation and histamine release at low concentrations and has anti-inflammatory activity at higher concentrations. Because of these unique immunologic properties, MCD peptide may serve as a useful tool for studying secretory mechanisms of inflammatory cells such as mast cells, basophils, and leukocytes, leading to the design of compounds with therapeutic potential.     

Isolation and sequence determination of frog C-type natriuretic peptide

A new bioactive peptide was isolated from frog brain using a bioassay for chick rectum relaxant activity. Amino acid sequence of this peptide was determined to be Gly-Tyr-Ser-Arg-Gly-Cys-Phe-Gly-Val-Lys-Leu-Asp-Arg-Ile-Gly-Ala-Phe-Ser- Gly- Leu-Gly-Cys, in which two cysteines were linked by a disulfide bond. The peptide was found to belong structurally to the natriuretic peptide family and to exert diuretic-natriuretic activity as well as hypotensive activity when injected into rats. The peptide showed a high homology to recently identified porcine C-type natriuretic peptide (CNP) and a pharmacological spectrum highly similar to porcine CNP. Thus, the peptide was designated frog C-type natriuretic peptide (frog CNP). Frog CNP may participate in the central control of body fluid homeostasis, since its tissue concentration is high in brain.     

A novel peptide, vasoactive intestinal contractor, of a new (endothelin) peptide family. Molecular cloning, expression, and biological activity

A new peptide family (endothelin (ET] consisting of three members in mammals appears to be present in mice according to genomic Southern blot analysis. Two ET-related genes were identified by cloning and sequence analysis of a mouse genome. One encoded a peptide identical to porcine and human vasoconstrictor peptide ET, and the other encoded a novel peptide differing from ET in 3 amino acid residues, with 4 cysteines in the same positions as in ET. This novel peptide was synthesized and confirmed to have in vivo pressor activity similar to that of ET. Northern blot analysis, however, indicated the gene of this novel peptide to be expressed only in the intestine, and not in other tissues or cell lines, or endothelial cells. Furthermore, the peptide evoked a strong contractile response in the guinea pig ileum. This peptide may thus be reasonably classified as a gastrointestinal peptide, vasoactive intestinal contractor.     

ABRF ESRG 2006 Study: Edman Sequencing as a Method for Polypeptide Quantitation

The Edman Sequencing Research Group (ESRG) designs studies on the use of Edman degradation for protein and peptide analysis. These studies provide a means for participating laboratories to compare their analyses against a benchmark of those from other laboratories that provide this valuable service. The main purpose of the 2006 study was to determine how accurate Edman sequencing is for quantitative analysis of polypeptides. Secondarily, participants were asked to identify a modified amino acid residue, N-epsilon-acetyl lysine [Lys(Ac)], present within one of the peptides. The ESRG 2006 peptide mixture consisted of three synthetic peptides. The Peptide Standards Research Group (PSRG) provided two peptides, with the following sequences: KAQYARSVLLEKDAEPDILELATGYR (peptide B), and RQAKVLLYSGR (peptide C). The third peptide, peptide C*, synthesized and characterized by ESRG, was identical to peptide C but with acetyl lysine in position 4. The mixture consisted of 20% peptide B and 40% each of peptide C and its acetylated form, peptide C*. Participating laboratories were provided with two tubes, each containing 100 picomoles of the peptide mixture (as determined by quantitative amino acid analysis) and were asked to provide amino acid assignments, peak areas, retention times at each cycle, as well as initial and repetitive yield estimates for each peptide in the mixture. Details about instruments and parameters used in the analysis were also collected. Participants in the study with access to a mass spectrometer (MALDI-TOF or ESI) were asked to provide information about the relative peak areas of the peptides in the mixture as a comparison with the peptide quantitation results from Edman sequencing. Positive amino acid assignments were 88% correct for peptide C and 93% correct for peptide B. The absolute initial sequencing yields were an average of 67% for peptide (C+C*) and 65.6 % for peptide B. The relative molar ratios determined by Edman sequencing were an average of 4.27 (expected ratio of 4) for peptides (C+C*)/B, and 1.49 for peptide C*/C (expected ratio of 1); the seemingly high 49% error in quantification of Lys(Ac) in peptide C* can be attributed to commercial unavailability of its PTH standard. These values compare very favorably with the values obtained by mass spectrometry.     

An opioid peptide from synganglia of the tick, Amblyomma testindinarium

An opioid peptide, which shares similarity with mammalian hemorphins, has been identified from the synganglia (central nervous system) of the hard tick, Amblyomma testindiarium. Its primary sequence was established as LVVYPWTKM that contains a tetrapeptide sequence Tyr-Pro-Trp-Thr of hemorphin-like opioid peptides. By hot-plate bioassay, the purified peptide and synthetic peptide displayed dose-related antinociceptive effect in mice, as observed for other hemorphin-like opioid peptides. This is the first opioid peptide identified from ticks. Ticks may utilize the opioid peptide in their strategy to escape host immuno-surveillance as well as in inhibiting responses directed against themselves.     

Conformational behavior of the HAV-VP3(110–121) peptidic sequence and synthetic analogs in membrane environments studied by CD and computational methods

The present study was undertaken to examine the structural features that may be important to explain the immunogenicity of the (110–121) peptide sequence (FWRGDLVFDFQV) of VP3 capsid protein of hepatitis A virus. A conformational analysis of the preferred conformations by CD and molecular mechanics was carried out. Present results suggest that the interaction with liposomes as biomembrane model induces and stabilizes the amphipathic β-structure of the peptide. To study the contribution of amino acid replacements at the RGD tripeptide as well as the influence of the peptide chain length on peptide conformation, solid-phase peptide synthesis of several peptide analogs was carried out and the peptide conformation was studied using CD spectroscopy. The results show that the RGD sequence is necessary to induce the β-structure in the presence of liposomes. © 1998 John Wiley & Sons, Inc. Biopoly 45: 479–492, 1998     

Combinatorial solid phase peptide synthesis and bioassays

Solid phase peptide synthesis method, which was introduced by Merrifield in 1963, has spawned the concept of combinatorial chemistry. In this review, we summarize the present technologies of solid phase peptide synthesis (SPPS) that are related to combinatorial chemistry. The conventional methods of peptide library synthesis on polymer support are parallel synthesis, split and mix synthesis and reagent mixture synthesis. Combining surface chemistry with the recent technology of microelectronic semiconductor fabrication system, the peptide microarray synthesis methods on a planar solid support are developed, which leads to spatially addressable peptide library. There are two kinds of peptide microarray synthesis methodologies: pre-synthesized peptide immobilization onto a glass or membrane substrate and in situ peptide synthesis by a photolithography or the SPOT method. This review also discusses the application of peptide libraries for high-throughput bioassays, for example, peptide ligand screening for antibody or cell signaling, enzyme substrate and inhibitor screening as well as other applications.     

Synthesis of peptide gels for the investigation of oligopeptide-oligonucleotide interactions

Peptide gels usable as protein model systems have been synthesized by a cross-linking copolymerization of acryloyl substituted peptides with 1,4-tetramethylene dimethacrylate. A specially adapted approach to peptide synthesis allows the removal of the amino terminal Cbo group at the end of the peptide synthesis, followed by the introduction of an acryloyl group. The polymerizable peptide monomers obtained can be transferred into insoluble peptide gels by radical copoylmerization with cross-linking agents. After cleavage of the protecting groups of the side chains, these peptide gels can be used both as protein model systems for investigating peptide–oligonucleotide interaction and as sorbents for affinity chromatography. The preparation and characterization of the peptide gels Ala-Lys-Glu-Lys-Ala-OMe (I), Ala-Arg-Glu-Arg-Ala-OMe (II), Ala-Arg-Glu-Lys-Ala-OMe (III), and Ala-Arg-Ala-Lys-Ala-OMe (IV) as well as the conditions for the removal of the protecting groups is presented. Gel III contains the natural peptide sequence Arg-Glu-Lys while the other gels are analogs of this sequence.     

Determination of the minimal fusion peptide of bovine leukemia virus gp30

In this study, we determined the minimal N-terminal fusion peptide of the gp30 of the bovine leukemia virus on the basis of the tilted peptide theory. We first used molecular modelling to predict that the gp30 minimal fusion peptide corresponds to the 15 first residues. Liposome lipid-mixing and leakage assays confirmed that the 15-residue long peptide induces fusion in vitro and that it is the shortest peptide inducing optimal fusion since longer peptides destabilize liposomes to the same extent but not shorter ones. The 15-residue long peptide can thus be considered as the minimal fusion peptide. The effect of mutations reported in the literature was also investigated. Interestingly, mutations related to glycoproteins unable to induce syncytia in cell-cell fusion assays correspond to peptides predicted as non-tilted. The relationship between obliquity and fusogenicity was also confirmed in vitro for one tilted and one non-tilted mutant peptide.     

Requirement of the proteasome for the trimming of signal peptide-derived epitopes presented by the nonclassical major histocompatibility complex class I molecule HLA-E

The nonclassical major histocompatibility complex class I molecule HLA-E acts as a ligand for CD94/NKG2 receptors on the surface of natural killer cells and a subset of T cells. HLA-E presents closely related nonameric peptide epitopes derived from the highly conserved signal sequences of classical major histocompatibility complex class I molecules as well as HLA-G. Their generation requires cleavage of the signal sequence by signal peptidase followed by the intramembrane-cleaving aspartic protease, signal peptide peptidase. In this study, we have assessed the subsequent proteolytic requirements leading to generation of the nonameric HLA-E peptide epitopes. We show that proteasome activity is required for further processing of the peptide generated by signal peptide peptidase. This constitutes the first example of capture of a naturally derived short peptide by the proteasome, producing a class I peptide ligand.     

Isolation of the membrane-binding peptide of NADPH-cytochrome P-450 reductase. Characterization of the peptide and its role in the interaction of reductase with cytochrome P-450

A peptide identified as the membrane-associated segment of NADPH-cytochrome P-450 reductase has been generated by steapsin protease treatment of vesicle-incorporated reductase and isolated by preparative gel electrophoresis. This peptide remains associated with vesicles when steapsin protease digests of vesicle-incorporated reductase were fractionated by Sepharose 4B chromatography, confirming its identity as the membrane-binding peptide. The molecular weight of the membrane-binding peptide was 6400 as determined by gel filtration in 8 M guanidine hydrochloride, and its amino acid content was not especially hydrophobic. The activity of reconstituted hydroxylation systems consisting of reductase, cytochrome P-446, and dilauroyl phosphatidylcholine was not inhibited by large molar excesses of purified membrane-binding peptide. Moreover, when purified reductase and cytochrome P-446 were added to liposomes which contained the membrane-binding peptide, it was determined that mixed function oxidase activity was reconstituted as effectively as when vesicles without the membrane-binding peptides were used. Similar results were obtained with reductase, cytochrome P-450, and detergent-solubilized liposomes (with or without the membrane-binding peptide). Thus, the membrane-binding peptide does not appear to interact with either of these two forms of the hemoprotein in a site-specific manner to prevent reconstitution of hydroxylation activity.     

Mouse thymus targeted peptide isolated by in vivo phage display can inhibit bioactivity of thymus output in vivo

Heterogeneity of the vasculature in different organs has been well documented by the method of in vivo phage display. Using this technology, several peptide ligands that home to tissue-specific vascular endothelial cell have been isolated. Such peptide ligands directed against specific vascular surface molecules can be used as targeted therapeutic compounds or imaging agents to the vasculature of the specific organ in vivo. In this study, the authors perform in vivo selection in mice using a phage display random peptide library and separated phage peptides homing to mouse thymus by 3 rounds of in vivo panning. Sequence analysis showed that CHAQGSAEC is the dominant peptide sequence. Immunohistochemistry confirmed that the phage peptide CHAQGSAEC can bind specifically to thymus blood vessels in mice. Furthermore, phage peptide CHAQGSAEC and free peptide CHAQGSAEC can inhibit the bioactivity of thymus output in vivo. These results indicate the feasibility of the targeted peptide for possible function as a kind of tool to inhibit thymus bioactivity or as a targeted compound for targeted medicine.     

Affinity purification of proteins using ligands derived from peptide libraries

Peptide libraries can be used to identify ligands that bind specifically to a desired protein. These peptides may have significant advantages as specific ligands for affinity chromatography separations. This article describes the use of one of such peptide, Try-Asn-Phe-Glu-Val-Leu, as a ligand for the purification of S-protein using affinity chromatography. General strategies for peptide immobilization are discussed and the conditions for peptide immobilization to Emphazetrade mark gel are optimized. The effects of peptide orientation and peptide densities on protein binding are studied. Results indicate that the peptide affinity is not affected by the orientation of the peptide during immobilization, but association constants can be reduced by one order of magnitude when compared with the values in solution.With increased peptide density, the protein binding capacity of the gel increases, but both the percentage of peptide utilization and apparent binding constant between immobilized peptide and S-protein decrease. S-protein is separated from a mixture with BSA via affinity chromatography using specific elution with the peptide in solution.Finally, direct purification of S-protein from an enzymatic digestion mixture of ribonuclease A is demonstrated.(c) 1995 John Wiley & Sons, Inc.     

Brain natriuretic peptide-32: N-terminal six amino acid extended form of brain natriuretic peptide identified in porcine brain

Brain natriuretic peptide (BNP) is a newly identified peptide of 26 residues, which has a remarkable homology to but is distinct from atrial natriuretic peptide. The peptide exerts natriuretic-diuretic activity as well as potent chick rectum relaxant activity. By using radioimmunoassay specific to BNP and immunoaffinity chromatography, we have isolated from porcine brain a novel peptide of 32 residues carrying a BNP structure at the C-terminus. The amino acid sequence of this peptide was determined to be: Ser-Pro-Lys-Thr-Met- Arg-Asp-Ser-Gly-Cys-Phe-Gly-Arg-Arg-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly- Leu- Gly-Cys-Asn-Val-Leu-Arg-Arg-Tyr. This peptide is an N-terminal six amino acid extended form of BNP and henceforth is designated BNP-32. BNP and BNP-32 are found to be major forms of BNP family in porcine brain.     

Radioimmunoassay of human prolactin based on a 13 amino acid synthetic analog of the amino terminus

A 13 amino acid analog of the human prolactin amino terminus was synthesized, substituting tyrosine for valine at residue 13. The peptide was coupled to crystalline bovine serum albumin for antisera production. The peptide was used for iodination with 125I, and displacement curves were found to be parallel when human prolactin and the synthetic peptide were compared as standards. The radioimmunoassay using the synthetic peptide has the advantages of purity in its roles as hapten in the antigen and as labelled peptide, of ease of iodination of the peptide, of its stability after iodination, and of obviating the need for native human prolactin. The radioimmunoassay is suitable for the measurement of human prolactin concentration in plasma.     

Purification and characterization from human parotid secretion of a peptide which inhibits hemagglutination of Bacteroides gingivalis 381

A peptide from human parotid secretion which inhibited hemagglutination of Bacteroides gingivalis 381 was purified by ultrafiltration followed by DEAE-Sephadex A-25 column chromatography and by gel filtration on Sephadex G-25, and then by reversed-phase HPLC. The complete amino acid sequence of the peptide, determined by automated Edman degradation was as follows; Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr. The peptide contained 12 residues and the charged amino acids predominated with 4 histidine, 2 lysine, 1 arginine and 1 glutamic acid residues, thus being a histidine-rich peptide. The peptide was an active inhibitor of the hemagglutinating activity of B. gingivalis. Specific binding of tritium-labeled peptide to B. gingivalis cells was demonstrated. These results suggest that the histidine-rich peptide may function as a binding domain for the hemagglutinins of B. gingivalis during agglutination.     

Purification and sequencing of a trypsin-sensitive cholecystokinin-releasing peptide from rat pancreatic juice. Its homology with pancreatic secretory trypsin inhibitor

The trypsin-sensitive cholecystokinin-releasing peptide is a peptide purified from rat pancreatic juice on the basis of its stimulatory activity toward pancreatic enzyme secretion. We postulate that the peptide acts as a mediator of pancreatic enzyme secretion in response to dietary protein intake and that it (designated as "monitor peptide" from its role in the intestine) could be responsible for the feedback regulation of pancreatic enzyme secretion. About 20 nmol of the highly purified peptide were obtained from 800 ml of rat pancreatic juice by reverse-phase high performance liquid chromatography. It was then sequenced. The peptide comprises 61 amino acid residues (Table I). It has a sequence that closely resembles that of a highly conserved region in pancreatic secretory trypsin inhibitors (PSTIs, Kazal type inhibitor): -Ile-Tyr-Asx-Pro-Val-Cys-Gly-Thr-Asx-Gly-. However, the peptide is less related to other mammalian PSTIs than they are to each other. The additional 5 residues at the NH2 terminus make the peptide larger than the common 56-residue PSTIs. The trypsin-sensitive cholecystokinin-releasing peptide is to be classified as a Kazal-type inhibitor and may be one of the rat PSTIs or a related peptide. The present results and increasing evidence from other laboratories and ours suggest that Kazal-type inhibitors play previously unrecognized multiple physiological roles.     

Characterization of inhibitory mechanism and antifungal activity between group-1 and group-2 phytocystatins from taro (Colocasia esculenta)

Tarocystatin from Colocasia esculenta, a group-2 phytocystatin, is a defense protein against phytopathogenic nematodes and fungi. It is composed of a highly conserved N-terminal region, which is homological to group-1 cystatin, and a repetitive peptide at the C-terminus. The purified recombinant proteins of tarocystatin, such as full-length (FL), N-terminus (Nt) and C-terminus (Ct) peptides, were produced and their inhibitory activities against papain as well as their antifungal effects were investigated. Kinetic analysis revealed that FL peptide exhibited mixed type inhibition (K(ia) = 0.098 microM and K(ib) = 0.252 microM) and Nt peptide showed competitive inhibition (K(i) = 0.057 microM), whereas Ct peptide possessed weak papain activation properties. A shift in the inhibitory pattern from competitive inhibition of Nt peptide alone to mixed type inhibition of FL peptide implied that the Ct peptide has an regulatory effect on the function of FL peptide. Based on the inhibitory kinetics of FL (group-2) and Nt (group-1) peptides on papain activity, an inhibitory mechanism of group-2 phytocystatins and a regulatory mechanism of extended Ct peptide have each been proposed. By contrast, the antifungal activity of Nt peptide appeared to be greater than that of FL peptide, and the Ct peptide showed no effect on antifungal activity, indicating that the antifungal effect is not related to proteinase inhibitory activity. The results are valid for most phytocystatins with respect to the inhibitory mechanism against cysteine proteinase.     

Covalent capture: a natural complement to self-assembly

The utility of peptide self-assembly can be extended by covalent capture of these supramolecular materials. Disulfide bond formation, native chemical ligation, olefin metathesis, radical capture and oxidative lysine cross-linking have been used recently to help stabilize and characterize a variety of self-assembled peptides. These include natural peptides, proteins and protein mimics such as alpha-helical coiled coils, amyloid-like beta-sheet fibres, portions of p53, glutathione S-transferase and elastin as well as unnatural peptide constructs such as cyclic peptide nanotubes and cylindrical micelles of peptide amphiphiles.