Identification and structure-activity relationship of an antimicrobial peptide of the palustrin-2 family isolated from the skin of the endangered frog Odorrana ishikawae

Recently, we identified nine novel antimicrobial peptides from the skin of the endangered anuran species, Odorrana ishikawae, to assess its innate immune system. In this study an additional antimicrobial peptide was initially isolated based on antimicrobial activity against Escherichia coli. The new antimicrobial peptide belonging to the palustrin-2 family was named palustrin-2ISb. It consists of 36 amino acid residues including 7 amino acids C-terminal to the cyclic heptapeptide Rana box domain. The peptide's primary structure suggests a close relationship with the Chinese odorous frog, Odorrana grahami. The cloned cDNA encoding the precursor protein contained a signal peptide, an N-terminal acidic spacer domain, a Lys-Arg processing site and the C-terminal precursor antimicrobial peptide. It also contained 3 amino acid residues at the C-terminus not found in the mature peptide. Finally, the antimicrobial activities against four microorganisms (E. coli, Staphylococcus aureus, methicillin-resistant S. aureus and Candida albicans) were investigated using several synthetic peptides. A 29 amino acid truncated form of the peptide, lacking the 7 amino acids C-terminal to the Rana box, possessed greater antimicrobial activities than the native structure.     

Somatocrinin,growth hormone releasing factor,stimulates secretion of growth hormone in anesthetized rats

The synthetic replicate of a 44 amino acid peptide isolated from a human pancreatic tumor which had caused acromegaly possesses high specific activity to release growth hormone (GH) in anesthetized male rats. The GH secretion induced by this peptide is dose-dependent from 50 ng to 1 μg, with plasma GH concentrations increasing more than 10-fold within 5 min of iv administration at the higher doses. Two enzymatic degradation products of the 44 residue peptide were also isolated and consist of the first 37 and 40 amino acids. All three peptides appear to possess similar potency, on a molar basis, $\text{in}\text{vivo}$, contrary to $\text{in}\text{vitro}$ results. The specificity of these peptides on GH release was shown by their failure to alter plasma concentrations of prolactin (PRL), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and corticosterone. Based on these $\text{in}\text{vivo}$ results, the three peptides with serve as powerful tools with which to investigate the mechanisms of GH secretion.     

Equilibrium of the Cis-Trans Isomerisation of the Peptide Bond with N-alkyl Amino Acids Measured by 2D NMR

Summary The conformationalcis-trans equilibrium around the peptide bond in model tripeptides has been determined by 2D NMR methods (HOHAHA, ROESY). The study was limited to three different N-substituted amino acids in position 2, namely Pro (proline), Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), and N-MePhe (N-methylphenylalanine). In all cases the amino acid in position 1 was tyrosine and in position 3, phenylalanine. The results of our studies show that thecis-trans ratio depends mostly on the configuration of the amino acids forming the peptide bond undergoing thecis-trans isomerisation. The amino acid following the sequence (in position 3) does not have much influence on thecis-trans isomerisation, indicating that there is no interaction of the side chains between these amino acids. The model peptides with the L-Tyr-L-AA-(L-or D-)Phe (where AA is N-substituted amino acid) chiralities give 80–100% more of thecis form in comparison to the corresponding peptides with the D-Tyr-L-AA-(L-or D-)Phe chiralities. These results indicate that the incorporation of N-substituted amino acids in small peptides with the same chirality as the precedent amino acid involved in the peptide bound undergoing thecis/trans isomerisation moves the equilibrium to a significant amount of thecis form.     

Dye-sensitized Photooxidation of Neutral Protease from Bacillus subtilis var. amylosacchariticus: Assignment of Histidine Residue Oxidized

The neutral pro tease of Bacillus subtilis var. amylosacchariticus was photooxidized in the presence of methylene blue, by which treatment the enzyme was rapidly inactivated. The inactive enzyme was digested with endoproteinase Asp-N, the resultant peptides were separated by HPLC, and their amino acid sequences were compared with those obtained from the unmodified enzyme. Of four peptides that contained histidine residues, only the recovery of one peptide was found to be decreased by the photooxidation with the appearance of a new peptide. Comparisons of amino acid compositions and sequences between these two peptides showed that the latter peptide lacked His²²⁸ of the former one, indicating that His²²⁸was photooxidized. This result suggests that His²²⁸ is involved in the catalytic reaction of the neutral protease or interaction with substrates.     

A switchable stapled peptide

The O‐N acyl transfer reaction has gained significant popularity in peptide and medicinal chemistry. This reaction has been successfully applied to the synthesis of difficult sequence‐containing peptides, cyclic peptides, epimerization‐free fragment coupling and more recently, to switchable peptide polymers. Herein, we describe a related strategy to facilitate the synthesis and purification of a hydrophobic stapled peptide. The staple consists of a serine linked through an amide bond formed from its carboxylic acid function and the side chain amino group of diaminopropionic acid and through an ester bond formed from its amino group and the side chain carboxylic acid function of aspartic acid. The α‐amino group of serine was protonated during purification. Interestingly, when the peptide was placed at physiological pH, the free amino group initiated the O‐N shift reducing the staple length by one atom, leading to a more hydrophobic stapled peptide. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of stable C‐linked ferrocenyl amino acids and their use in solution‐phase peptide synthesis

Incorporation of ferrocenyl group to peptides is an efficient method to alter their hydrophobicity. Ferrocenyl group can also act as an electrochemical probe when incorporated onto functional peptides. Most often, ferrocene is incorporated onto peptides post‐synthesis via amide, ester or triazole linkages. Stable amino acids containing ferrocene as a C‐linked side chain are potentially useful building units for the synthesis of ferrocene‐containing peptides. We report here an efficient route to synthesize ferrocene‐containing amino acids that are stable and can be used in peptide synthesis. Coupling of 2‐ferrocenyl‐1,3‐dithiane and iodides derived from aspartic acid or glutamic acid using n‐butyllithium leads to the incorporation of a ferrocenyl unit to the δ‐position or ε‐position of an α‐amino acid. The reduction or hydrolysis of the dithiane group yields an alkyl or an oxo derivative. The usability of the synthesized amino acids is demonstrated by incorporating one of the amino acids in both C‐terminus and N‐terminus of tripeptides in solution phase. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Amino acid replacements in proteins S5 and S12 of two Escherichia coli revertants from streptomycin dependence to independence

Summary Ribosomes were isolated from two E. coli revertants from streptomycin dependence to independence, N660 and d1023. After separation of subunits, proteins were extracted from ribosomal 30S subunits and separated by CM-cellulose column chromatography and gel filtration. Pure S5 and S12 proteins of the two mutants were digested with trypsin and all resulting peptides were isolated by column and paper chromatography. The amino acid compositions of the peptides from the four mutant proteins were compared with the corresponding peptides of the wild type strain A19. The amino acid sequences of non-identical peptides were determined.The following amino acid replacements were found: Glycine by arginine in peptide T2 of protein S5 from mutant N660 and glycine by aspartic acid in peptide T15 of protein S12 from the same mutant. In the other mutant, d1023, arginine in peptide T2 of protein S5 was replaced by leucine and furthermore arginine by serine in peptide T10 of protein S12. Besides the single amino acid replacements mentioned above which are compatible with alterations of single nucleotides, a rather drastic difference between peptides T15 of proteins S12 isolated from strain A19 and mutant d1023 has been detected.The results presented in this paper are compared with amino acid replacements in proteins S5 and S12 from other ribosomal mutants of E. coli.Paper No. 62 on Ribosomal Proteins. Preceding paper is by Wittmann et al., Molec. gen. Genet., in press.     

The amino acid sequence of the peptide moiety of the pseudomurein from Methanobacterium thermoautotrophicum

The amino acid sequence of the peptide subunits of the peptide moiety of the sacculus polymer (pseudomurein) of Methanobacterium thermoautotrophicum was elucidated by analysing overlapping peptides obtained from partial acid hydrolysates of isolated sacculi. It is suggested that the peptide subunits are attached to glycan strands via one of their glutamyl residues. Another glutamyl residue may crosslink two adjacent peptide subunits to form a dimer. The calculated molar ratios of the amino acids and the percentages of the N-or C-terminal amino acid residues of the supposed dimers are compatible with those actually found in the sacculus polymer.     

Structure-function studies on neurohormone D: activity of naturally-occurring hormone analogues

Summary The relative potencies of 11 naturally-occurring peptides of the adipokinetic hormone/red pigmentconcentrating hormone family (AKH/RPCH-family) have been assessed with respect to increase in heart rate in adult, female American cockroaches,Periplaneta americana, in in vitro and in vivo bioassays. In addition, analogues that lacked the N-terminal pyroglutamate residue or had a free threonine acid at the C-terminus were also investigated. In both bioassays the N- or C-terminal-modified analogues give no or little response suggesting that blocked termini are essential for receptor-binding. In both bioassays the naturally-occurring peptide from the cockroach corpus cardiacum Pea-CAH-I (neurohormone D) is more potent than the second endogenous peptide, Pea-CAH-II. On the basis of this result and previous data it is proposed that neurohormone D is the only physiologically important true cardioactive peptide. The dose-response curves of the other peptides indicate that in octapeptides, amino acid residues at positions 2, 6, and 7 are important for receptor-recognition, and that decapeptides are not as effective as octapeptides (exception: the peptide Rom-CC-I isolated from the grasshopperRomalea microptera).     

Isolation and characterization of red-pigment-concentrating hormone (RPCH) from six crustacean species

Summary Red-pigment-concentrating hormone (RPCH) has been isolated from nerve tissue of six decapod crustaccan species. The primary structure of three of the six hormones, i.e., those ofCancer magister, Carcinus maenas andOrconectes limosus, was determined by manual microsequencing as: pELNFSPGW-NH₂. This sequence is identical to that of RPCH fromPandalus borealis, the only previously known sequence of a crustacean RPCH. The other three hormones fromLiocarcinus puber, Nephrops norvegicus, andPacifastacus leniusculus could not be characterized completely. However, amino acid compositions, the presence of N-terminal pGlu, and the blocked N-terminal ends are in accordance with the primary structure established for the other three RPCHs. We suggest that all six peptides have the same amino acid sequence. These results indicate that RPCH, which is likely to be related to the peptides of the AKH family in insects, is highly conserved among crustacean species. This is in remarkable contrast to the high degree of molecular evolution exemplified by the many different AKH-like peptides among insect species.Abbreviations AKH adipokinetic hormone - Aufs absorption units full scale - BSA bovine serum albumin - ECH erythrophore concentrating hormone (=RPCH) - EDTA ethylenediamine-tetra-acetic acid - HOAc acetic acid - HPLC high pressure liquid chromatography - O.D. optical density - PDH pigment dispersing hormone - PGAP pyroglutamate aminopeptidase - RPCH red-pigment-concentrating hormone (=ECH) - SOG suboesophageal ganglion - TFA trifluoroacetic acid     

Acid Soluble Peptides in Rat Skeletal Muscle

The acid soluble peptide fraction was prepared from rat skeletal muscle, and the amino acid composition of the fraction was analyzed. The peptide fraction was rich in glutamic acid (or glutamine) and glycine and was poor in branched chain or aromatic amino acids. Since the peptide fraction contained N^τ-methylhistidine, the fraction or at least a part of it was presumed to be composed of intermediate peptides of protein degradation in skeletal muscle. At least 31 spots were detected in the fraction by one dimensional paper electrophoresis.     

Free energies and equilibria of peptide bond hydrolysis and formation

For every n amino acids linked in a protein there are n − 1 peptide bonds. The free energy of peptide bond hydrolysis and formation in aqueous solution defines the equilibrium position between peptide and amino acid hydrolysis products. Yet few experimental values exist. With a minimum of assumptions, this paper deduces the free energies of hydrolysis of a variety of peptide bonds. Formation of a dipeptide from two amino acids is about eight times more difficult than subsequent condensations of an amino acid to a dipeptide or longer chain. Condensation of an amino acid to a peptide of any size is five times more difficult than joining two smaller peptides of at least dipeptide size. Thus in an abiogenesis scenario there is a kind of nucleation in peptide bond formation with the initial condensation of two amino acids to yield a dipeptide more difficult than subsequent condensations to a growing chain. © 1998 John Wiley & Sons, Inc. Biopoly 45: 351–353, 1998     

Studies on Mold Proteases

The substrate specificity of the crystalline acid protease obtained from Rhizopus chinensis was determined using B-chain of oxidized beef insulin and numerous synthetic peptides, comparing with that of several acid proteases from various sources. The peptide bonds susceptible to the action of Rhiz. acid protease were found to be mainly those involving the amino group of bulky amino acids. The enzyme split the B-chain of oxidized insulin at twelve sites of the peptide linkages and a certain similarity in the specificity was observed among the three acid proteases, Rhiz. protease, rennin and pepsin, all of which were known to show potent milk clotting activities.     

Oligopeptide assimilation and transport by Oenococcus oeni

Aims: Oenococcus oeni is a slow‐growing wine bacterium with a low growth yield. It thrives better on complex nitrogen sources than on free amino‐acid medium. We aimed to characterize the oligopeptide use of this micro‐organism. Methods and Results: Several peptides of two to eight amino‐acid residues were able to provide essential amino acids. The disappearance of various peptides from extracellular medium was assessed with whole cells. Initial rates of utilization varied with the peptide, and free amino acids were released into the medium. Conclusions: Oenococcus oeni was able to transport the oligopeptides with two to five amino‐acid residues tested and to hydrolyse them further. Significance and Impact of the Study: This study has clear implications for the relationship between wine nitrogen composition and the ability of O. oeni to cope with its environment.     

Human pituitary thyrotropin: isolation and chemical characterization of its subunits

The subunits of human pituitary thyrotropin have been separated and purified by countercurrent distribution and exclusion chromatography. The NH₂-terminal sequence of the β subunit is identical to that of the β subunit of bovine thyrotropin. However, amino acid composition and peptide map of tryptic and chymotryptic digests as well as compositions of tryptic and chymotryptic peptides suggest that the amino acid sequence of the α subunit is identical to that of the α subunit of human interstitial cell stimulating hormone.     

Base Specificity and Primary Structure of Poly U-preferential Ribonuclease from Chicken Liver

The primary structure and base specificity of chicken liver RNase CL₁ which has been reported by Miura et al. [Chem. Pharm. Bull., 32,4053–4060 (1984)] as poly U-preferential RNase, were extensively studied. The sequence study of this enzyme and comparison of the amino acid sequence of the enzyme with homologous RNases from oyster and Drosophila melanogaster suggested that RNase CL₁ consists of three peptides with 17, 19, and 163 amino acid residues. The amino acid sequence of these three peptides were identified. The two small peptides are joined to the large peptide by disulfide bridges. The amino acid sequence of RNase CL₁ had 62 (31.2%) and 63 residues (31.6%) identical with oyster RNase and D. melanogaster RNase, respectively, and belongs to the RNase T₂ family RNase.

Reassessment of the base specificity of RNase CL₁ found that it is guanylic acid, then uridylic acid-preferential, and not poly U preferential.     

Structure‐activity relationship studies of shortened analogues of the antimicrobial peptide EeCentrocin 1 from the sea urchin Echinus esculentus

EeCentrocin 1 is a potent antimicrobial peptide isolated from the marine sea urchin Echinus esculentus. The peptide has a hetero‐dimeric structure with the antimicrobial activity confined in its largest monomer, the heavy chain (HC), encompassing 30 amino acid residues. The aim of the present study was to develop a shorter drug lead peptide using the heavy chain of EeCentrocin 1 as a starting scaffold and to perform a structure‐activity relationship study with sequence modifications to optimize antimicrobial activity. The experiments consisted of 1) truncation of the heavy chain, 2) replacement of amino acids unfavourable for in vitro antimicrobial activity, and 3) an alanine scan experiment on the truncated and modified heavy chain sequence to identify essential residues for antimicrobial activity. The heavy chain of EeCentrocin 1 was truncated to less than half its initial size, retaining most of its original antimicrobial activity. The truncated and optimized lead peptide ( P6 ) consisted of the 12 N‐terminal amino acid residues from the original EeCentrocin 1 HC sequence and was modified by two amino acid replacements and a C‐terminal amidation. Results from the alanine scan indicated that the generated lead peptide ( P6 ) contained the optimal sequence for antibacterial activity, in which none of the alanine scan peptides could surpass its antimicrobial activity. The lead peptide ( P6 ) was also superior in antifungal activity compared to the other peptides prepared and showed minimal inhibitory concentrations (MICs) in the low micromolar range. In addition, the lead peptide ( P6 ) displayed minor haemolytic and no cytotoxic activity, making it a promising lead for further antimicrobial drug development.     

Self‐assembling organo‐peptide bolaphiles with KLK tripeptide head groups display selective antibacterial activity

In keeping with recent efforts to generate compounds for antibiotic and microbicide development, we focused on the creation of non‐natural organo‐peptide hybrids of antimicrobial peptide amides (KLK(L)_nKLK‐NH₂) derived from sapecin B and a self‐assembling oligoglycine organo‐peptide bolaphile containing an ω‐amino fatty acid residue. The hybrid organo‐peptide bolaphiles with two cationic KLK tripeptide motifs linked with an ω‐amino acid residue (penta‐, octa‐ or undecamethylene chain) maintained the self‐assembling properties of the root oligoglycine bolaphile. Electron microscopy clearly revealed complex supramolecular architectures for both sapecin B‐derived peptides and the hybrid analogues. FT‐IR spectroscopy indicated that the supramolecular structures were composed primarily of β‐sheets. CD revealed that the hybrid bolaphiles did not share the same secondary structures as the sapecin B peptides in solution. However, although secondary structures of antimicrobial peptides are central in the activity, the organo‐peptide bolaphiles also retained the potent antimicrobial activity of the leader sapecin B‐derived peptide against both Gram‐positive and Gram‐negative bacteria. In general, the hybrids were more selective than the sapecin B peptides, as they displayed little or no appreciable haemolytic activity. The results obtained herald a new approach for the design of purpose‐built hybrid organo‐peptide bolaphiles. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Human MR1 expression on the cell surface is acid sensitive, proteasome independent and increases after culturing at 26°C

Protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) catalyzes the transfer of GlcNAc to O-mannose of glycoproteins. Mutations in the POMGnT1 gene cause muscle–eye–brain disease (MEB). POMGnT1 is a typical type II membrane protein, which is localized in the Golgi apparatus. However, details of the catalytic and reaction mechanism of POMGnT1 are not understood. To develop a better understanding of POMGnT1, we examined the substrate specificity of POMGnT1 using a series of synthetic O-mannosyl peptides based on the human α-dystroglycan (α-DG) sequence as substrates. O-Mannosyl peptides consisting of three to 20 amino acids are recognized as substrates. Enzyme kinetics improved with increasing peptide length up to a length of 8 amino acids but the kinetics of peptides longer than 8 amino acids were similar to those of octapeptides. Our results also show that the amino acid sequence affects POMGnT1 activity. These data suggest that both length and amino acid sequence of mannosyl peptides are determinants of POMGnT1 substrate specificity.     

An HPLC‐ESMS study on the solid‐phase assembly of C‐terminal proline peptides

DKP formation is a serious side reaction during the solid‐phase synthesis of peptide acids containing either Pro or Gly at the C‐terminus. This side reaction not only leads to a lower overall yield, but also to the presence in the reaction crude of several deletion peptides lacking the first amino acids. For the preparation of protected peptides using the Fmoc/tBu strategy, the use of a ClTrt‐Cl‐resin with a limited incorporation of the C‐terminal amino acid is the method of choice. The use of resins with higher loading levels leads to more impure peptide crudes. The use of HPLC‐ESMS is a useful method for analysing complex samples, such as those formed when C‐terminal Pro peptides are prepared by non‐optimized solid‐phase strategies. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.