Insect immunity. Isolation from a coleopteran insect of a novel inducible antibacterial peptide and of new members of the insect defensin family.

Injection of heat-killed bacteria into larvae of the large tenebrionid beetle Zophobas atratus (Insecta, Endopterygota, Coleoptera) results in the appearance in the hemolymph of a potent antibacterial activity as evidenced by a plate growth inhibition assay. We have isolated three peptides (A-C) from this immune hemolymph which probably account for most of this activity. Their primary structures were established by a combination of peptide sequencing and molecular mass determination by mass spectrometry. Peptide A, which is bactericidal against Gram-negative cells, is a 74-residue glycine-rich molecule with no sequence homology to known peptides. We propose the name coleoptericin for this novel inducible antibacterial peptide. Peptides B and C are isoforms of a 43-residue peptide which contains 6 cysteines and shows significant sequence homology to insect defensins, initially reported from dipteran insects. This peptide is active against Gram-positive bacteria. The results are discussed in connection with recent studies on inducible antibacterial peptides present in the three other major orders of the endopterygote clade of insects: the Lepidoptera, Diptera, and Hymenoptera.     

A new antifungal peptide from the seeds of Phytolacca americana: characterization, amino acid sequence and cDNA cloning

An antifungal peptide from seeds of Phytolacca americana, designated PAFP-s, has been isolated. The peptide is highly basic and consists of 38 residues with three disulfide bridges. Its molecular mass of 3929.0 was determined by mass spectrometry. The complete amino acid sequence was obtained from automated Edman degradation, and cDNA cloning was successfully performed by 3'-RACE. The deduced amino acid sequence of a partial cDNA corresponded to the amino acid sequence from chemical sequencing. PAFP-s exhibited a broad spectrum of antifungal activity, and its activities differed among various fungi. PAFP-s displayed no inhibitory activity towards Escherichia coli. PAFP-s shows significant sequence similarities and the same cysteine motif with Mj-AMPs, antimicrobial peptides from seeds of Mirabilis jalapa belonging to the knottin-type antimicrobial peptide.     

Carbohydrate composition and presence of a fucose-protein linkage in recombinant human pro-urokinase

A new post-translational modification site in the growth factor domain of urinary type plasminogen activator has been identified. A glycopeptide containing the monosaccharide, fucose, covalently linked directly to the peptide backbone has been isolated from the tryptic digest of pro-urokinase expressed in a mouse hybridoma cell line Sp 2/0 Ag 14. The glycopeptide was isolated by semi-preparative reversed phase high performance liquid chromatography. The identity of a fucose containing peptide was confirmed by carbohydrate analysis, amino acid analysis and plasma desorption mass spectrometry (PDMS). A combination of these methodologies showed an equimolar ratio of peptide and fucose in the glycopeptide. This modification is not detected without mass spectrometry because the fucose residue is hydrolyzed under standard acidic conditions of amino acid composition and N-terminal sequence analysis. The site of attachment of fucose to the peptide has been localized towards the N-terminus (within first 23 amino acids) of the protein. Also, the carbohydrate composition of recombinant pro-urokinase is reported.     

A bradykinin-potentiating peptide (peptide K12) isolated from the venom of Egyptian scorpion Buthus occitanus

A nontoxic peptide with bradykinin-potentiating activity was isolated from the dialyzed venom of the scorpion Buthus occitanus by reverse-phase high performance liquid chromatography (RP-HPLC). The pharmacological activity of the peptide was bioassayed by its ability to potentiate added bradykinin (BK) on the isolated guinea pig ileum as well as the isolated rat uterus for contraction. Moreover, the peptide potentiates in vivo the depressor effect of BK on arterial blood pressure in the normotensive anesthetized rat. Chemical characterization of the peptide was also performed. The amino acid composition of the peptide showed 21 amino acid residues per molecule including three proline residues. The amino acid sequence of the purified peptide was confirmed by mass spectrometry. Either N- or C-terminal ends were free. The sequence does not show a homology with bradykinin-potentiating peptides isolated from either scorpion or snake venoms. Furthermore, we did not find a significant sequence homology between the sequence of the isolated peptide and any of proteins or peptides in GenPro or NBRF data banks. The peptide also inhibited angiotensin-converting enzyme (ACE), and could not serve as substrate for the enzyme. It could be concluded that the mechanism of bradykinin-potentiating peptide (BPP) activity may be due to ACE inhibition.     

Determination of the amino acid sequence of an intramolecular disulfide linkage-containing sperm-activating peptide by tandem mass spectrometry.

A sperm-activating peptide (SAP) was isolated from the egg jelly of the sea urchin Stomopneustes variolaris. The presence of an intramolecular disulfide linkage in the peptide was demonstrated by fast atom bombardment (FAB) mass spectrometry with the intact and reduced peptides. The amino acid sequence of the reduced peptide was determined to be Lys-Phe-Cys-Pro-Glu-Gly-Lys-Cys-Val by tandem mass spectrometry from the spectrum produced by a collision-induced decomposition method. Furthermore, it was also demonstrated that SAPs obtained from sea urchins Arbacia punctulata and Glyptocidaris crenularis are cyclic peptides containing one cystine residue by FAB mass spectrometry.     

Extensive post-translational modification, including serine to D-alanine conversion, in the two-component lantibiotic, lacticin 3147

Lacticin 3147 is a two-component bacteriocin produced by Lactococcus lactis subspecies lactis DPC3147. In order to further characterize the biochemical nature of the bacteriocin, both peptides were isolated which together are responsible for the antimicrobial activity. The first, LtnA1, is a 3,322 Da 30-amino acid peptide and the second component, LtnA2, is a 29-amino acid peptide with a mass of 2,847 Da. Conventional amino acid analysis revealed that both peptides contain the thioether amino acid, lanthionine, as well as an excess of alanine to that predicted from the genetic sequence of the peptides. Chiral phase gas chromatography coupled with mass spectrometry of amino acid composition indicated that both LtnA1 and LtnA2 contain D-alanine residues and amino acid sequence analysis of LtnA1 confirmed that the D-alanine results from post-translational modification of a serine residue in the primary translation product. Taken together, these results demonstrate that lacticin 3147 is a novel, two-component, D-alanine containing lantibiotic that undergoes extensive post-translational modification which may account for its potent antimicrobial activity against a wide range of Gram-positive bacteria.     

Wheat proteomics: relationship between fine chromosome deletion and protein expression

To explore the relationship between fine chromosome deletion and protein expression in common wheat, the changes in protein composition of wheat seed proteome were investigated by using chromosome 1B. A momosomic alien chromosome addition line of common wheat was used to produce the fine deletion lines. Endosperm and embryo proteins were separated by two-dimensional gel electrophoresis (2-DE) and visualized by staining with Commassie Brilliant Blue, and gel images were analyzed with a computer assisted image analyzer. For the first time, fine gene locations of a few endosperm and embryo proteins were identified on the chromosome 1B. These proteins with their specific gene location on the chromosome can be used as protein markers in breeding programs for quality of wheat proteins. To identify wheat seed proteins and to understand their expression in relation to chromosome deletion, the feasibility of a new analytical approach based on isotope coded affinity tag labeling (ICAT) of peptides in tryptic digests followed by electrospray ionization mass spectrometry has been described. Simplification of the complex tryptic digest prior to mass spectral analysis was performed by treating the samples with light and heavy ICAT labeling reagents. A clear separation of peptide fragment containing the light and heavy reagents was achieved in mass spectral analysis. Out of the 14 peptides detected by mass fragment analysis of the euploid, four were down-regulated, nine up-regulated and one did not show any change due to the terminal deletion of chromosome 1B. Selected peptide fragments were subjected to tandem mass spectrometry analysis for sequence information and the resulting sequence information was submitted to databases for protein identification. Of the five proteins submitted, four were identified as alpha-amylase inhibitor, alpha-amylase/subtilisin inhibitor precursor, proteasome subunit alpha-type 7 and 1,4 alpha-glucan-D-maltohydrolase. With this approach it is possible to identify wheat seed proteins and to understand their expression, which have been reported to be difficult by 2-DE due to cosynthesis of proteins by genes from three genomes, A, B and D.     

Isolation and identification of a new diuretic peptide from the tobacco hornworm, Manduca sexta.

A 30-amino acid diuretic peptide was isolated from the corpora cardiaca-corpora allata complexes and, separately, from medial neurosecretory cells of the Sphingid moth, Manduca sexta. The peptide was found to have the following sequence, determined by automated Edman degradation and mass spectrometry: SFSVNPAVDILQHRYMEKV AQNNRNFLNRV-NH2. We have named the peptide Mas-DP II. The peptide was synthesized and shown to possess diuretic activity in decapitated moths. Mas-DP II is related by sequence homology to a 41-amino acid diuretic peptide identified previously from M. sexta, and it belongs to the family of corticotropin releasing factor-like peptides.     

Identification and characterization of a novel antimicrobial peptide from the venom of the ant Tetramorium bicarinatum

A novel antimicrobial peptide, named Bicarinalin, has been isolated from the venom of the ant Tetramorium bicarinatum. Its amino acid sequence has been determined by de novo sequencing using mass spectrometry and by Edman degradation. Bicarinalin contained 20 amino acid residues and was C-terminally amidated as the majority of antimicrobial peptides isolated to date from insect venoms. Interestingly, this peptide had a linear structure and exhibited no meaningful similarity with any known peptides. Antibacterial activities against Staphylococcus aureus and S. xylosus strains were evaluated using a synthetic replicate. Bicarinalin had a potent and broad antibacterial activity of the same magnitude as Melittin and other hymenopteran antimicrobial peptides such as Pilosulin or Defensin. Moreover, this antimicrobial peptide has a weak hemolytic activity compared to Melittin on erythrocytes, suggesting potential for development into an anti-infective agent for use against emerging antibiotic-resistant pathogens.     

Primary structure of CHH/MIH/GIH-like peptides in sinus gland extracts from Penaeus vannamei

Peptides belonging to the CHH/MIH/GIH-family of crustacean hormones were isolated from acetic acid extracts of sinus glands isolated from eyestalks of the shrimp, Penaeus vannamei. The peptides were isolated by chromatography and molecular weights determined by MALDI mass spectrometry. Peptides in the range of 7-9 kDa and containing three disulfide bridges were selected for amino acid sequence analysis. Three peptides with the requisite properties were present in sufficient amounts for sequence analysis. Two peptides had unique sequences similar to CHH/MIH/GIH peptides from other crustaceans. A third peptide seemed to be a truncated form of one of the previous sequences.     

Anglerfish preprosomatostatin II is processed to somatostatin-28 and contains hydroxylysine at residue 23

A peptide fraction containing two 28-residue somatostatins, both products of the anglerfish somatostatin II gene, has been isolated, characterized, and subjected to amino acid sequence analysis. The structural data indicate that one of the two forms of the 28-residue peptide contains 5-hydroxylysine. Hydroxylysine was identified in an acid hydrolysate of somatostatin-28 by gas chromatography/mass spectrometry. Fast-atom bombardment mass spectrometry indicated that the two forms of somatostatin-28 have molecular weights of 3220 and 3204, representing the hydroxylated and nonhydroxylated peptides, respectively. The location of the hydroxylated lysine was deduced by analysis of proteolytic fragments to be position 23. This represents the first observation of a hydroxylated peptide hormone and one of the few reported occurrences of hydroxylysine in non-collagen proteins.     

Biochemical and mass spectrometry recognition of phospholipid–peptide complexes in wheat sprouts extract

Total hydroalcoholic extract of wheat sprouts was treated with 90% cold acetone as a preliminary step directed to separate antioxidant peptides from antioxidant polyphenols. Surprisingly, the addition of acetone causes the formation of a yellow buoyant gelatinous drop that prevailingly contains peptides and phospholipids. In this context, evidences have been presented that support the hypothesis that peptides (and perhaps other active molecules) are complexed with phospholipids. In fact, the MS/MS analysis of some main ions, present in RP HPLC fractions of wheat sprout extract, generates several ions that correspond to molecular weight of phospholipids or phospholipid fragments. Moreover, several ions were detected that correspond to lysophosphatidylcholine or phosphatidylcholine–peptide complexes. The possibility that phospholipids can be complexed with peptides has been discussed in the light of potential involvement in the peptide bioavailability. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Isolation and structural characterization of novel Rugosin A-like insulinotropic peptide from the skin secretions of Rana saharica frog

Skin secretions of Rana saharica were evaluated for the isolation and characterization of novel insulinotropic peptides. Crude secretions obtained from young adult frogs by mild electrical stimulation of the dorsal skin surface were purified by reversed-phase HPLC yielding 80 fractions. In acute incubations with glucose-responsive BRIN-BD11cells, fractions 36-43, 46-54 and 57-63 showed the significant 2-8-fold increase in insulin-releasing activity (P<0.001) compared with 5.6mM of glucose alone. A pool of fractions 36-43 was subsequently rechromatographed to 28 homogenous peaks out of which 7 were capable of subsequent 1.5-3-fold increase in insulin release (P<0.001). Structural analysis of the non-toxic peptides with greatest insulin-releasing activity was performed by mass spectrometry and Edman degradation. Mass spectrometry analysis of two peaks indicated the molecular masses of 1892.6 and 2930.8Da. The sequence of the 1892.6-Da peptide was determined as KGAAKGLLEVASCKLSKSC, which has 68% homology with Rugosin A originally isolated from the skin secretion of Rana rugosa. A partial N-terminal sequence was determined for the 2930.8-Da peptide as AVITGACERDVQCGGGTCCAVSLI.... These data indicate that the skin secretions of Rana saharica frogs contain novel peptides with insulin-releasing activity.     

Bacteriocins of Lactobacillus gasseri K7 – Monitoring of gassericin K7 A and B genes’ expression and isolation of an active component

The genome of Lactobacillus gasseri K7, isolated from baby's faeces, contains gene regions encoding two-component bacteriocins named gassericin K7 A (GenBank EF392861) and gassericin K7 B (GenBank AY307382). The strain has been known to exhibit bacteriocin activity in vitro, however, no data exist on the expression of particular genes of bacteriocins’ operons or on the activity of individual components of this bacteriocin complex, which has not been isolated so far. The objectives of this study were to examine bacteriocin genes’ expression during the growth of L. gasseri K7 and to isolate individual components in order to reveal the contribution of individual peptides to the overall bacteriocin activity. All eight target genes were expressed during exponential phase of growth in MRS broth. Mass spectrometry analysis revealed that the amino acid sequence of isolated peptide matched the deduced amino acid sequence of putative active peptide of gassericin K7 B (Gas K7 B_AcP) and GatX, a complementary peptide of gassericin T, previously supposed to have no antimicrobial activity. The isolated peptide showed a broad spectrum of antimicrobial activity. Furthermore, the isolation protocol developed in this study will enable to obtain a considerable amount of purified bacteriocins needed for further investigation of their functionality.     

A novel small antifungal peptide from Bacillus strain B-TL2 isolated from tobacco stems

A novel small antifungal peptide produced by a Bacillus strain B-TL2 isolated from tobacco stems was purified. The purification procedure consisted of ammonium sulfate precipitation, cation exchange chromatography on CM-Sepharose Fast Flow column and reverse-phase HPLC on SOURCE 5RPC column. After the final isolation step, one peptide with antifungal activity, designated as BTL, was obtained. The molecular mass of the purified BTL was determined as 2500 Da and 2237.7 Da by SDS-PAGE and matrix-assisted laser desorption/ionization time of flight mass spectrometry, respectively. The N-amino acid sequence of BTL was determined to be NH(2)-KQQLATEAESAGPIL, which shows relatively low identity to other antimicrobial peptides from bacteria. The peptide exhibited strong inhibitory activity against mycelial growth of Bipolaris maydis, Alternaria brassicae, Aspergillus niger, Cercospora personata. The purified BTL displayed thermostability, almost retaining 100% activity at 100 degrees C for 15 min.     

Isolation of dermenkephalin from amphibian skin, a high-affinity delta-selective opioid heptapeptide containing a D-amino acid residue

The predicted amino acid sequence of the biosynthetic precursor of dermorphin, a highly potent and nearly specific mu-opioid peptide from amphibian skin, contains four repeats of the dermorphin progenitor sequence and one single copy of a different heptapeptide sequence. We have developed a specific enzyme immunoassay and used synthetic peptides to detect and purify the new predicted heptapeptide (2.4 micrograms/g dry skin) from the skin of the Phyllomedusa sauvagei frog from which dermorphin was originally isolated. The identity of the novel pro-dermorphin related peptide, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2, was established by co-chromatography with synthetic peptides on reverse-phase HPLC, immunological analysis, gas-phase sequencing, mass spectrometry and by pharmacological assays. Opioid-binding assays in vitro demonstrated that both the natural and synthetic heptapeptides displayed exceptionally high selectivity and affinity towards the delta-opioid receptors. Because of its origin and its delta-opioid (enkephalin) activity and specificity, this novel D-amino acid containing peptide is named dermenkephalin.     

The utility of ETD mass spectrometry in proteomic analysis

Mass spectrometry has played an integral role in the identification of proteins and their post-translational modifications (PTM). However, analysis of some PTMs, such as phosphorylation, sulfonation, and glycosylation, is difficult with collision-activated dissociation (CAD) since the modification is labile and preferentially lost over peptide backbone fragmentation, resulting in little to no peptide sequence information. The presence of multiple basic residues also makes peptides exceptionally difficult to sequence by conventional CAD mass spectrometry. Here we review the utility of electron transfer dissociation (ETD) mass spectrometry for sequence analysis of post-translationally modified and/or highly basic peptides. Phosphorylated, sulfonated, glycosylated, nitrosylated, disulfide bonded, methylated, acetylated, and highly basic peptides have been analyzed by CAD and ETD mass spectrometry. CAD fragmentation typically produced spectra showing limited peptide backbone fragmentation. However, when these peptides were fragmented using ETD, peptide backbone fragmentation produced a complete or almost complete series of ions and thus extensive peptide sequence information. In addition, labile PTMs remained intact. These examples illustrate the utility of ETD as an advantageous tool in proteomic research by readily identifying peptides resistant to analysis by CAD. A further benefit is the ability to analyze larger, non-tryptic peptides, allowing for the detection of multiple PTMs within the context of one another.     

Isolation and structure elucidation of a novel 5-kDa peptide from neurohaemal lobes of the corpora cardiaca of Locusta migratoria (Insecta, Orthoptera)

Two predominant peptides have been isolated from neurohaemal lobes of corpora cardiaca of 8000 adults of Locusta migratoria. Both peptides have been unambiguously characterized by automated peptide microsequencing and liquid secondary-ion mass spectrometry as a 50-residue peptide (5K peptide) and a 48-residue isologue (5K' peptide). Computer search of sequence data banks did not reveal any significant similarity with other identified proteins. The 5K peptides are remarkably rich in alanine residues (25%) and contain a stretch of five consecutive alanines. This structure suggests that these molecules could correspond to spacer peptides. This assumption is corroborated in the accompanying paper [Lagueux et al. (1990) Eur. J. Biochem. 187, 249-254] on the molecular cloning of the precursor protein which attributes to the 5K peptides a role analogous to that of the C peptides of insulins.     

Novel short antimicrobial peptide isolated from Xenopus laevis skin

A rich source of bioactive peptides, including a large number of antimicrobial peptides, has been found in amphibian skin. In this study, a novel short antimicrobial peptide was purified from Xenopus laevis skin and characterised through reversed‐phase high‐performance liquid chromatography, Edman degradation and matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry. The peptide was composed of six amino acids with a sequence of DEDLDE and thus named X. laevis antibacterial peptide‐P2 (XLAsp‐P2). Transmission electron microscopy revealed that this peptide showed potential antimicrobial abilities against bacteria by damaging the bacterial cell membrane. XLAsp‐P2 maybe inhibit bacterial growth by binding to the microbial genomic DNA. The peptide also exhibited a weak haemolytic activity against rabbit red blood cells. Therefore, XLAsp‐P2 is a novel short anionic antibacterial peptide with broad activities. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.