Target tissue distribution of the proenkephalin peptides F, E, and B

The adrenal medulla is a rich source of endogenous opioid peptides. These peptides exist predominantly in the form of larger (25-34 amino acid long) enkephalin containing peptides, whose biological roles have yet to be elucidated. We report here the tissue binding distribution of three iodinated enkephalin containing peptides, Peptides F, E, and B, in rats, rabbits, and guinea pigs following intravenous injection. Each [125I]peptide has a unique distribution profile but all three are found to bind to the pituitary in each species of animal examined. A number of lines of evidence point toward the enkephalin containing peptides, Peptides F, E, and B, having physiological roles distinct from the enkephalins. The distribution profile of these [125I]peptides reported here gives insight into their potential effector sites.     

Identification of a disulfide bridge connecting the alpha-subunits of the extracellular domain of the insulin receptor.

The alpha 2 beta 2 structure of the insulin receptor has previously been shown to involve one disulfide bridge between the alpha-subunits in the region containing Cys435, Cys468 and Cys524. We have digested the soluble extracellular domain of the insulin receptor with succinylated trypsin, partially separated the resulting peptides, and sequenced a number of fractions. The peptides containing Cys435 and Cys468 appeared in the same fraction, indicating that these two form a disulfide bond, and in another fraction we found the sequence of the peptide containing Cys524. Since it has been shown that the extracellular domain of the insulin receptor has no free thiols and since no other sequences containing cysteine were found in these fractions, we conclude that Cys524 forms a disulfide bond to the Cys524 in the other alpha-subunit.     

Effect of various peptides on the growth of Mycobacterium tuberculosis

Ten oligopeptides containing asparagine, glutamic acid, leucine or alanine on growth of Bacillus tuberculosis were tested. The experiments were performed on AS medium free of peptones. Bacterial suspensions were inoculated and the number of colonies and rapidity of bacilli growth under an influence of peptides tested was compared. Out of peptides studied and their different combinations the best turned to be combination of 0.01% glutathione +0.002% Gly-Asn + 0.0033% Leu-Gly. This combination allowed to appear on average 46% colonies more than on medium without peptides and first growth of tubercle bacilli was seen on average 3.2 days earlier than on medium free of peptides. Addition to the medium containing three above listed peptides of 0.1% of Bacto Tryptone (Difco) caused an increase of 127% of colony number of tubercle bacilli and their growth appeared 1.7 day earlier as compared to growth on medium containing these three peptides.     

Amino acid sequence of beta-galactosidase. VII. Isolation of the 24 cyanogen bromide peptides.

All of the 24 cyanogen bromide peptides of beta-galactosidase have been isolated in pure form. Of these 8 ranged in size from 2 to 5 residues and were purified by paper electrophoresis. The 16 large peptides, from 23 to 119 residues, were chromatographed at pH 5.0 on a carboxymethyl-cellulose column in 0.02 M ammonium acetate buffer containing 8 M urea. A number of peptides were obtained in pure from following Sephadex G-50 or G-75 gel filtration. Others were separated on sulfopropyl-Sephadex or diethyl-(2-hydroxylpropylaminoethyl)-Sephadex. There large peptides were obtained in over 50% yield and several others were obtained in more than 25% yield.     

Structural Proteins of Intracisternal A-Particles: Possible Repetitive Sequences

Peptide maps of tryptic digests of the structural proteins from inner shells of intracisternal A-particles have shown common peptides for all the proteins. The terminal amino group of the three different structural proteins was identified as arginine. The major protein revealed approximately half the number of peptides expected from the amino acid composition. Since evidence for a cross-link bond has not been found, the main structural protein may be a single polypeptide chain containing a total or partial duplication of sequence.     

Potential roles of flavin-containing monooxygenases in sulfoxidation reactions of l-methionine, N-acetyl-l-methionine and peptides containing l-methionine

Flavin-containing monooxygenases (FMOs) are microsomal enzymes that catalyze the NADPH-dependent oxidation of a large number of sulfur-, selenium-, and nitrogen-containing compounds. Five active isoforms (FMO1-5) have been identified and shown to be differently expressed in various mammalian tissues. Previous work from this laboratory has shown l-methionine to be S-oxidized by rat, rabbit and human FMO1-4, with FMO3 exhibiting the highest stereoselectivity for the formation of the d-diastereomer of methionine sulfoxide. In this report, we describe new studies aimed at determining if N-acetyl-l-methionine and peptides containing l-methionine can be substrates for FMOs. Experiments were carried out using either rabbit liver microsomes or human cDNA-expressed FMOs. The results show that while N-acetyl-l-methionine and peptides with a modified methionine amino group may not function as substrates for FMOs, peptides containing a free N-terminal methionine may act as FMO substrates. With human cDNA-expressed FMO1, FMO3, and FMO5, both FMO1 and FMO3 exhibited activity with the active peptides whereas FMO5 was inactive. With FMO3, the activity measured with methionine was similar (1 mM) or higher (5 mM) than the activity measured with H-Met-Val-OH and H-Met-Phe-OH. With FMO1, H-Met-Phe-OH and methionine exhibited similar activities whereas activity with H-Met-Val-OH was much lower. Collectively, the results show that FMOs can oxidize peptides containing a free N-terminal methionine. Thus, the role of FMOs in the oxidation of methionine in larger peptides or proteins warrants further investigation.     

Dependence of formation of small disulfide loops in two-cysteine peptides on the number and types of intervening amino acids

Microscopic disulfide-exchange rate constants have been measured for the formation and opening of small disulfide loops in reactions between glutathione and peptides containing 2 cysteines. Twelve cysteine-Xm-cysteine peptides have been studied, where X is an amino acid and m is the number of amino acids between the cysteines. Homopolymers of alanine for m equaling 0-5 are evaluated, as well as X1 and X2 series employing glycine, valine, or proline. Equilibrium constants Kc for loop closing are only slightly dependent on the nature of X. Loops with even values of m generally are favored relative to loops with odd values. Kc increases in the rank order X1, X3, X0, X5, X4, and X2. Formation of a disulfide between sequentially adjacent cysteines therefore is not especially difficult. The dependence of Kc on the odd-even nature of m is compared with similar patterns observed both in statistics of disulfide formation in naturally occurring proteins and in theoretical studies of peptide cyclization. The relative equilibrium populations of intramolecular disulfides in peptides containing cysteine-cysteine-cysteine and cysteine-serine-cysteine-serine-cysteine clusters are consistent with predictions based on the values of Kc in the two-cysteine peptides.     

Domain organization of calbindin D28k as determined from the association of six synthetic EF-hand fragments.

Calbindin D28k is an intracellular Ca(2+)-binding protein containing six subdomains of EF-hand type. The number and identity of the globular domains within this protein have been elucidated using six synthetic peptide fragments, each corresponding to one EF-hand subdomain. All six peptides were mixed in equimolar amounts in the presence of 10 mM Ca2+ to allow for the reconstitution of domains. The mixture was compared to native calbindin D28k and to the sum of the properties of the individual peptides using circular dichroism (CD), fluorescence, and 1H NMR spectroscopy, as well as gel filtration and ion-exchange chromatography. It was anticipated that if the peptides associate to form native-like domains, the properties would be similar to those of the intact protein, whereas if they did not interact, they would be the same as the properties of the isolated peptides. The results show that the peptides in the mixture interact with one another. For example, the CD and fluorescence spectra for the mixture are very similar to those of the intact calbindin D28k, suggesting that the mixed EF-hand fragments associate to form a native-like structure. To determine the number of domains and the subdomain composition of each domain in calbindin D28k, a variety of peptide combinations containing two to five EF-hand fragments were studied. The spectral and chromatographic properties of all the mixtures containing less than six peptides were closer to the sum of the properties of the relevant individual peptides than to the mixture of the six peptides. The results strongly suggest that all six EF-hands are packed into one globular domain. The association of the peptide fragments is observed to drive the folding of the individual subdomains. For example, one of the fragments, EF2, which is largely unstructured in isolation even in the presence of high concentrations of Ca2+, is considerably more structured in the presence of the other peptides, as judged by CD difference spectroscopy. The CD data also suggest that the packing between the individual subdomains is specific.     

Comparison of the D-lactate stereospecific dehydrogenase of Limulus polyphemus with active-site regions of L-lactate dehydrogenases

Lactate dehydrogenase (D-lactate:NAD+ oxidoreductase, EC 1.1.1.28) from the horseshoe crab, Limulus polyphemus, a dimeric enzyme stereospecific for D-lactate, has been purified by affinity chromatography. Maleyl tryptic peptides containing arginine residues isolated from the Limulus enzyme have been characterized and sequenced. The small peptides obtained from similarly treated L-lactate-specific enzyme homologs define major portions of the substrate and coenzyme binding regions and are virtually identical among L-lactate-specific enzymes. Although the six small peptides and free arginine isolated from the Limulus enzyme indicate that the small number of arginine tryptic peptides are located in a few discrete consecutive clusters similarly to the L-lactate dehydrogenases, the peptides nevertheless show no obvious sequence homology to the corresponding peptides from L-lactate dehydrogenases. These results indicate that this lactate dehydrogenase of altered substrate specificity either evolved with major rearrangements of the active site if it evolved from an L-lactate dehydrogenase, or that D-lactate dehydrogenases have evolved from a different protein. The results contradict proposed models which suggest that minor changes in the spatial orientation of pyruvate resulting from minimal rearrangement of the active site could accommodate the change in substrate specificity.     

Attachment of peptide building blocks to proteins through tyrosine bioconjugation

Recent efforts have yielded a number of short peptide sequences with useful binding, sensing, and cellular uptake properties. In order to attach these sequences to tyrosine residues on intact proteins, a three-component Mannich-type strategy is reported. Two solid phase synthetic routes were developed to access peptides up to 20 residues in length with anilines at either the N- or C-termini. In the presence of 20 mM formaldehyde, these functional groups were coupled to tyrosine residues on proteins under mild reaction conditions. The identities of the resulting bioconjugates were confirmed using mass spectrometry and immunoblot analysis. Screening experiments have demonstrated that the method is compatible with substrates containing all of the amino acids, including lysine and cysteine residues. Importantly, tyrosine residues on proteins exhibit much faster reaction rates, allowing short peptides containing this residue to be coupled without cross reactions.     

Peptide investigations of pairwise interactions in the collagen triple-helix

Pairwise interactions have been studied for the major secondary structures in proteins. The present work extends the characterization of interactions between side-chains to the context of a collagen triple-helix. In this study, the most frequent Gly-X-Y tripeptide sequences in collagen are characterized in terms of interchain interactions between non-imino acid X and Y residues, through the use of host-guest peptides and statistical frequency analysis. Stabilities predicted on the basis of additivity show good agreement with experimental values for almost half of the peptides, indicating a lack of interaction. A small number of peptides have a stability lower than predicted, while a larger number are more stable than expected. Of all triplets containing residues of opposite charge, only Gly-Lys-Asp and Gly-Arg-Asp exhibit stabilizing electrostatic interactions, and these pairs are found together preferentially in collagens. Repulsion of like charges is observed in Gly-Arg-Lys, Gly-Lys-Arg, and Gly-Glu-Asp sequences, and a small degree of hydrophobic stabilization was observed for the Gly-Leu-Leu guest triplet. The data reported here help clarify basic principles of triple-helix stability. In addition, the experimentally determined stabilities of the tripeptide units found most frequently in collagens constitute a database useful for predicting triple-helix stability in peptides, collagens and other triple-helix-containing proteins.     

Improvement in HPLC fractionation of thyroxine-containing thyroglobulin tryptic peptides by prior Accell ion-exchange column chromatography

The isolation and purification of the peptides containing the hormonogenic tyrosyl residues in thyroglobulin is of great interest to the understanding of structure-function relationships in this iodoprotein. This is usually performed in reduced alkylated selectively hydrolyzed thyroglobulin by subsequent HPLC fractionation. However, the main difficulty encountered when trying to isolate these peptides is their disproportion with respect to the total number of possible peptides (14 vs a total of 508). Several HPLC runs with different mobile phases are necessary and each run is accompanied with significant losses of the peptides to be purified. In an attempt to improve the separation of the T4-containing peptides in thyroglobulin tryptic digests from the much more abundant iodotyrosine-containing ones, which are present as contaminants, we have used a very simple and fast step prior to the HPLC fractionation as it is a self-packed ion-exchange column chromatography. This preliminary step results in an improvement in the separation of these peptides and leads to a relative enrichment of the hormonogenic peptides falling in different zones of the HPLC chromatogram, which facilitates their subsequent separation and purification by HPLC.     

Translocating proline-rich peptides from the antimicrobial peptide bactenecin 7

The intracellular delivery of most peptides, proteins, and nucleotides to the cytoplasm and nucleus is impeded by the cell membrane. To allow simplified, noninvasive delivery of attached cargo, cell-permeant peptides that are either highly cationic or hydrophobic have been utilized. Because cell-permeable peptides share half of the structural features of antimicrobial peptides containing clusters of charge and hydrophobic residues, we have explored antimicrobial peptides as templates for designing cell-permeant peptides. We prepared synthetic fragments of Bac 7, an antimicrobial peptide with four 14-residue repeats from the bactenecin family. The dual functions of cell permeability and antimicrobial activity of Bac 7 were colocalized at the N-terminal 24 residues of Bac 7. In general, long fragments of Bac(1-24) containing both regions were bactericidal and cell-permeable, whereas short fragments with only a cationic or hydrophobic region were cell-permeant without the attendant microbicidal activity when measured in a fluorescence quantitation assay and by confocal microscopy. In addition, the highly cationic fragments were capable of traversing the cell membrane and residing within the nucleus. A common characteristic shared by the cell-permeant Bac(1-24) fragments, irrespective of their number of charged cationic amino acids, is their high proline content. A 10-residue proline-rich peptide with two arginine residues was capable of delivering a noncovalently linked protein into cells. Thus, the proline-rich peptides represent a potentially new class of cell-permeant peptides for intracellular delivery of protein cargo. Furthermore, our results suggest that antimicrobial peptides may represent a rich source of templates for designing cell-permeant peptides.     

Single cellular origin of somatostatin and calcitonin in the rat thyroid gland

Summary Immunostaining of thin serial paraffin sections has shown that somatostatin is present in the same parafollicular cells as calcitonin in the adult rat thyroid gland. The number of cells containing both peptides is much smaller than the number containing calcitonin but not somatostatin.     

Antibacterial activities and conformations of bovine beta-defensin BNBD-12 and analogs:structural and disulfide bridge requirements for activity

Structure and biological activities of synthetic peptides corresponding to bovine neutrophil beta-defensin BNBD-12, GPLSC(1)GRNGGVC(2)IPIRC(3) PVPMRQIGTC(4) FGRPVKC(5) C(6)RSW with disulfide connectivities C(1)-C(5), C(2)-C(4) and C(3)-C(6) and its variants with one, two and three disulfide bridges have been investigated. Selective protection of cysteine thiols was necessary in the four and six cysteine containing peptides for the formation of disulfide connectivities as observed in BNBD-12. Circular dichroism (CD) spectra indicate that in aqueous medium, only a small fraction of molecules populate turn-like conformations. In the presence of micelles and lipid vesicles, the single, two and three disulfide containing peptides adopt beta-hairpin or beta-sheet structures. Antibacterial activity was observed for all the peptides, irrespective of the number of disulfide bridges or how they were connected. Our results suggest that a rigid beta-sheet structure or the presence of three disulfide bridges does not appear to be stringent requirements for antibacterial activity in beta-defensins.     

Combinatorial discovery of tumor targeting peptides using phage display

Peptides possess appropriate pharmacokinetic properties to serve as cancer imaging or therapeutic targeting agents. Currently, only a small number of rationally-derived, labeled peptide analogues that target only a limited subset of antigens are available. Thus, finding new cancer targeting peptides is a central goal in the field of molecular targeting. Novel tumor-avid peptides can be efficiently identified via affinity selections using complex random peptide libraries containing millions of peptides that are displayed on bacteriophage. In vitro and in situ affinity selections may be used to identify peptides with high affinity for the target antigen in vitro. Unfortunately, it has been found that peptides selected in vitro or in situ may not effectively target tumors in vivo due to poor peptide stability and other problems. To improve in vivo targeting, methodological combinatorial chemistry innovations allow selections to be conducted in the environment of the whole animal. Thus, new targeting peptides with optimal in vivo properties can be selected in vivo in tumor-bearing animals. In vivo selections have been proven successful in identifying peptides that target the vasculature of specific organs. In addition, in vivo selections have identified peptides that bind specifically to the surface of or are internalized into tumor cells. In the future, direct selection of peptides for cancer imaging may be expedited using genetically engineered bacteriophage libraries that encode peptides with intrinsic radiometal-chelation or fluorescent sequences.     

Separation and identification of mouse brain tissue microproteins using top‐down method with high resolution nanocapillary liquid chromatography mass spectrometry

Microproteins and endogenous peptides in the brain contain important substances that have critical roles in diverse biological processes, contributing to signal transduction and intercellular signaling. However, variability in their physical or chemical characteristics, such as molecule size, hydrophobicity, and charge states, complicate the simultaneous analysis of these compounds, although this would be highly beneficial for the field of neuroscience research. Here, we present a top‐down analytical method for simultaneous analysis of microproteins and endogenous peptides using high‐resolution nanocapillary LC‐MS/MS. This method is detergent‐free and digestion‐free, which allows for extracting and preserving intact microproteins and peptides for direct LC‐MS analysis. Both higher energy collision dissociation and electron‐transfer dissociation fragmentations were used in the LC‐MS analysis to increase the identification rate, and bioinformatics tools ProteinGoggle and PEAKS Studio software were utilized for database search. In total, we identified 471 microproteins containing 736 proteoforms, including brain‐derived neurotrophic factor and a number of fibroblast growth factors. In addition, we identified 599 peptides containing 151 known or potential neuropeptides such as somatostatin‐28 and neuropeptide Y. Our approach bridges the gap for the characterization of brain microproteins and peptides, which permits quantification of a diversity of signaling molecules for biomarker discovery or therapy diagnosis in the future.     

Enrichment and analysis of nonenzymatically glycated peptides: boronate affinity chromatography coupled with electron-transfer dissociation mass spectrometry

Nonenzymatic glycation of peptides and proteins by d-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low-abundance post-translational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron-transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. ETD fragmentation mode permitted identification of a significantly higher number of glycated peptides (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing enrichment on first the protein and then the peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS and using ETD as the fragmentation mode is an efficient approach for analysis of glycated proteins and may have broad application in studies of diabetes mellitus.     

Application of Dmb-Dipeptides in the Fmoc SPPS of Difficult and Aspartimide-Prone Sequences

Mutter’s pseudoproline dipeptides and Sheppard’s Hmb derivatives are powerful tools for enhancing synthetic efficiency in Fmoc SPPS. They work by exploiting the natural propensity of N-alkyl amino acids to disrupt the formation of the secondary structures during peptide assembly. Their use results in better and more predictable acylation and deprotection kinetics, enhanced reaction rates, and improved yields of crude products. However, these approaches have certain limitations: pseudoproline dipeptides can only be used for sequences containing serine or threonine, and the coupling of the amino acid following the Hmb residue can be extremely difficult. To alleviate some of these shortcomings, we have prepared a range of Fmoc-Aaa-(Dmb)Gly-OH dipeptides and tested their efficacy in the synthesis of a number of challenging hydrophobic peptides. We also compared the efficiency of N-Dmb against N-Hmb backbone protection in preventing aspartimide formation in the Fmoc SPPS of peptides containing the Asp-Gly sequence.     

T cell recognition of Neisseria meningitidis class 1 outer membrane proteins. Identification of T cell epitopes with selected synthetic peptides and determination of HLA restriction elements.

No vaccine is yet available against serogroup B meningococci, which are a common cause of bacterial meningitis. Some outer membrane proteins (OMP), LPS, and capsular polysaccharides have been identified as protective Ag. The amino acid sequence of the protective B cell epitopes present within the class 1 OMP has been described recently. Synthetic peptides containing OMP B cell epitopes as well as capsular polysaccharides or LPS protective B cell epitopes have to be presented to the immune system in association with T cell epitopes to achieve an optimal Ir. The use of homologous, i.e., meningococcal, T cell epitopes has many advantages. We therefore investigated recognition sites for human T cells within the meningococcal class 1 OMP. We have synthesized 16 class 1 OMP-derived peptides encompassing predicted T cell epitopes. Peptides corresponding to both surface loops and trans-membrane regions (some of which occur as amphipathic beta-sheets) of the class 1 OMP were found to be recognized by T cells. In addition, 10 of 11 peptides containing predicted amphipathic alpha-helices and four of five peptides containing T cell epitope motifs according to Rothbard and Taylor (Rothbard, J. B., and W. R. Taylor. 1988. EMBO J 7:93) were recognized by lymphocytes from one or more volunteers. Some of the T and B cell epitopes were shown to map to identical regions of the protein. At least six of the peptides that were found to contain T cell epitopes show homology to constant regions of the meningococcal class 3 OMP and the gonococcal porins PIA and PIB. Peptide-specific T cell lines and T cell clones were established to investigate peptide recognition in more detail. The use of a panel of HLA-typed APC revealed clear HLA-DR restriction patterns. It seems possible now to develop a (semi-) synthetic meningococcal vaccine with a limited number of constant T cell epitopes that cover all HLA-DR locus products.