Identification and Functional Analysis of Novel Bradykinin-Related Peptides (BRPs) from Skin Secretions of Five Asian Frogs.

In recent decades, various types of bioactive substances have been identified from amphibian skin and its secretions. Bradykinin-related peptides (BRPs) are among these compounds that make up the host defence system of amphibians. In the present study, we identified six novel BRPs, amolopkinin-GN1, amolopkinin-RK1, amolopkinin-TR1, amolopkinin-LF1, ranakinin-MS1, and ranakinin-MS2, from five East Asian amphibians, Amolops granulosus, Amolops ricketti, Amolops torrentis, Amolops lifanensis, and Hylarana maosonensis. This is the first report on BRPs in the skin of these species. Physiological assays reveal that these peptides have a contractive effect on the smooth muscle of rat ileum.

Theoretical and experimental comparison of one dimensional versus on-line comprehensive two dimensional liquid chromatography for optimized sub-hour separations of complex peptide samples.

This study was devoted to the search for conditions leading to highly efficient sub-hour separations of complex peptide samples with the objective of coupling to mass spectrometry. In this context, conditions for one dimensional reversed phase liquid chromatography (1D-RPLC) were optimized on the basis of a kinetic approach while conditions for on-line comprehensive two-dimensional liquid chromatography using reversed phase in both dimensions (on-line RPLCxRPLC) were optimized on the basis of a Pareto-optimal approach. Maximizing the peak capacity while minimizing the dilution factor for different analysis times (down to 5min) were the two objectives under consideration. For gradient times between 5 and 60min, 15cm was found to be the best column length in RPLC with sub-2µm particles under 800bar as system pressure. In RPLCxRPLC, for less than one hour as first dimension gradient time, the sampling rate was found to be a key parameter in addition to conventional parameters including column dimension, particle size, flow-rate and gradient conditions in both dimensions. It was shown that the optimum sampling rate was as low as one fraction per peak for very short gradient times (i.e. below 10min). The quality descriptors obtained under optimized RPLCxRPLC conditions were compared to those obtained under optimized RPLC conditions. Our experimental results for peptides, obtained with state of the art instrumentation, showed that RPLCxRPLC could outperform 1D-RPLC for gradient times longer than 5min. In 60min, the same peak intensity (same dilution) was observed with both techniques but with a 3-fold lower injected amount in RPLCxRPLC. A significant increase of the signal-to-noise ratio mainly due to a strong noise reduction was observed in RPLCxRPLC-MS compared to the one in 1D-RPLC-MS making RPLCxRPLC-MS a promising technique for peptide identification in complex matrices.

Evaluation of Thr6-bradykinin purified from Polybia occidentalis wasp venom in the choline uptake of mammal cortices.

CONTEXT: Thr6-bradykinin is a peptide found in the venom of social and solitary wasps. This kinin, along with other bradykinin-like peptides, is known to cause irreversible paralysis in insects by presynaptic blockade of cholinergic transmission. However, this activity has never been tested in mammals. OBJECTIVE: As such, the objective of this study was to evaluate the effect of Thr6-bradykinin on the cholinergic system of rats. MATERIALS AND METHODS: The peptide was isolated from the venom of the Neotropical social wasp Polybia occidentalis Olivier (Vespidae). After correct identification and quantification by ESI-MS and MS/MS, the peptide was tested in [14C]-choline uptake using rat cortical synaptosomes. Each uptake assay was accompanied by lactic acid dehydrogenase (LDH) activity measurement to evaluate synaptosome integrity in the presence of six increasing concentrations of BK or Thr6-BK (0.039, 0.156, 0.625, 2.500, 10.000 and 40.000 µM). RESULTS: Data revealed that neither BK nor Thr6-BK at any of the six concentrations tested (from 0.039 to 40.000 µM) affected [14C]-choline uptake in synaptosomes. Moreover, there was no increase in LDH in the supernatants, indicating that BK and Thr6-BK did not disrupt the synaptosomes. DISCUSSION AND CONCLUSION: In contrast to previous reports for the insect central nervous system (CNS), Thr6-BK had no effect on mammalian cholinergic transmission. Nevertheless, this selectivity for the insect CNS, combined with its irreversible mode of action may be relevant to the discovery of new sources of insecticides and could contribute to understanding the role of kinins in the mammalian CNS.

Anxiolytic activity and evaluation of potentially adverse effects of a bradykinin-related peptide isolated from a social wasp venom.

Anxiety disorders are major health problems in terms of costs stemming from sick leave, disabilities, healthcare and premature mortality. Despite the availability of classic anxiolytics, some anxiety disorders are still resistant to treatment, with higher rates of adverse effects. In this respect, several toxins isolated from arthropod venoms are useful in identifying new compounds to treat neurological disorders, particularly pathological anxiety. Thus, the aims of this study were to identify and characterize an anxiolytic peptide isolated from the venom of the social wasp Polybia paulista. The peptide was identified as Polisteskinin R, with nominal molecular mass [M+H](+)=1301Da and primary structure consisting of Ala-Arg-Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg-OH. The anxiolytic effect was tested using the elevated plus maze test. Moreover, adverse effects on the spontaneous behavior and motor coordination of animals were assessed using the open field and rotarod tests. Polisteskinin R induced a dose-dependent anxiolytic effect. Animals treated with the peptide and diazepam spent significantly more time into the open arms when compared to the groups treated with the vehicle and pentylenetetrazole. No significant differences in spontaneous behavior or motor coordination were observed between the groups, showing that the peptide was well tolerated. The interaction by agonists in both known BK receptors induces a variability of physiological effects; Polisteskinin R can act on these receptors, inducing modulatory activity and thus, attenuating anxiety behaviors. The results of this study demonstrated that the compound Polisteskinin R exerted potent anxiolytic effects and its analogues are promising candidates for experimental pharmacology.

Angioedema hereditario / Hereditary angioedema

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A review on bradykinin-related peptides isolated from amphibian skin secretion.

Amphibian skin secretion has great potential for drug discovery and contributes hundreds of bioactive peptides including bradykinin-related peptides (BRPs). More than 50 BRPs have been reported in the last two decades arising from the skin secretion of amphibian species. They belong to the families Ascaphidae (1 species), Bombinatoridae (3 species), Hylidae (9 speices) and Ranidae (25 species). This paper presents the diversity of structural characteristics of BRPs with N-terminal, C-terminal extension and amino acid substitution. The further comparison of cDNA-encoded prepropeptides between the different species and families demonstrated that there are various forms of kininogen precursors to release BRPs and they constitute important evidence in amphibian evolution. The pharmacological activities of isolated BRPs exhibited unclear structure-function relationships, and therefore the scope for drug discovery and development is limited. However, their diversity shows new insights into biotechnological applications and, as a result, comprehensive and systematic studies of the physiological and pharmacological activities of BRPs from amphibian skin secretion are needed in the future.

C-terminal protein characterization by mass spectrometry using combined micro scale liquid and solid-phase derivatization.

A sample preparation method for protein C-terminal peptide isolation has been developed. In this strategy, protein carboxylate glycinamidation was preceded by carboxyamidomethylation and optional α- and ϵ-amine acetylation in a one-pot reaction, followed by tryptic digestion of the modified protein. The digest was adsorbed on ZipTip(C18) pipette tips for sequential peptide α- and ϵ-amine acetylation and 1-ethyl-(3-dimethylaminopropyl) carbodiimide-mediated carboxylate condensation with ethylenediamine. Amino group-functionalized peptides were scavenged on N-hydroxysuccinimide-activated agarose, leaving the C-terminal peptide in the flow-through fraction. The use of reversed-phase supports as a venue for peptide derivatization enabled facile optimization of the individual reaction steps for throughput and completeness of reaction. Reagents were exchanged directly on the support, eliminating sample transfer between the reaction steps. By this sequence of solid-phase reactions, the C-terminal peptide could be uniquely recognized in mass spectra of unfractionated digests of moderate complexity. The use of the sample preparation method was demonstrated with low-level amounts of a model protein. The C-terminal peptides were selectively retrieved from the affinity support and proved highly suitable for structural characterization by collisionally induced dissociation. The sample preparation method provides for robustness and simplicity of operation using standard equipment readily available in most biological laboratories and is expected to be readily expanded to gel-separated proteins.

Effective separation of peptides using highly dense thermo-responsive polymer brush-grafted porous polystyrene beads.

For the development of well-defined highly dense thermo-responsive polymer grafted surface as an improved stationary phase for thermo-responsive chromatography, poly(N-isopropylacrylamide) (PIPAAm) brush-grafted porous polystyrene beads were prepared by surface-initiated atom transfer radical polymerization (ATRP). The PIPAAm grafted region of polystyrene beads was adjusted by the addition of isooctane as a poor solvent for polystyrene upon the reaction of ATRP initiator immobilization. Using a thermo-responsive HPLC column containing the prepared beads with PIPAAm brush grafted on the inside pores nearby the outer surfaces, angiotensin subtypes were effectively separated with aqueous mobile phase, because the densely grafted PIPAAm on nearby the outer surface effectively interacted with the peptides hydrophobically. Retention of basic peptide was achieved by the beads with basic mobile phase. These results indicated that the prepared beads with grafted PIPAAm nearby the outer surface became an effective chromatographic stationary phase for retaining basic peptides using wide pH range of mobile phase.

High-resolution differential ion mobility separations using helium-rich gases.

Analyses of complex mixtures and characterization of ions increasingly involve gas-phase separations by ion mobility spectrometry (IMS) and particularly differential or field asymmetric waveform IMS (FAIMS) based on the difference of ion mobility in strong and weak electric fields. The key advantage of FAIMS is substantial orthogonality to mass spectrometry (MS), which makes FAIMS/MS hybrid a powerful analytical platform of broad utility. However, the potential of FAIMS has been constrained by limited resolution. Here, we report that the use of gas mixtures comprising up to 75% He dramatically increases the FAIMS separation capability, with the resolving power for peptides and peak capacity for protein digests reaching and exceeding 100. The resolution gains extend to small molecules, where previously unresolved isomers can now be separated. These performance levels open major new applications of FAIMS in proteomic and other biomolecular analyses.

Bradykinin-related peptides in the venom of the solitary wasp Cyphononyx fulvognathus.

Bradykinin (BK) and its related peptides are widely distributed in venomous animals, including wasps. In fact, we have previously purified a novel BK-related peptide (BRP) named Cd-146 and the threonine(6)-bradykinin (Thr(6)-BK) from the venom of the solitary wasp Cyphononyx fulvognathus. Further survey of this same wasp venom extract allowed the structural characterization of two other novel BRPs, named here as fulvonin and cyphokinin. Biochemical characterization performed here showed that although the high primary structure similarity observed with BK, these wasp peptides are not good substrates for angiotensin I-converting enzyme (ACE) acting more likely as inhibitors of this enzyme. In pharmacological assays, only those more structurally similar to BK, namely cyphokinin and Thr(6)-BK, were able to promote the contraction of guinea-pig ileum smooth muscle preparations, which was completely blocked by the B(2) receptors antagonist HOE-140 in the same way as observed for BK. Only fulvonin was shown to potentiate BK-elicited smooth muscle contraction. Moreover, the 2 new wasp BRPs, namely fulvonin and cyphokinin, as well as Cd-146 and Thr(6)-BK, showed hyperalgesic effect in the rat paw pressure test after intraplantar injection. This effect was shown here to be due to the action of these peptides on BK receptors, since the hyperalgesia induced by both Cd-146 and fulvonin was blocked by B(1) receptor antagonist, while the effect of both cyphokinin and Thr(6)-BK was reversed by B(2) antagonist. This data give support to a better understanding of the function and targets of the kinin-related peptides widely found in several insect venoms.

A novel bradykinin-like peptide from skin secretions of the frog, Rana nigrovittata.

A bradykinin-like peptide has been isolated from the skin secretions of the frog Rana nigrovittata. This peptide was named ranakinin-N. Its primary structure, RAEAVPPGFTPFR, was determined by Edman degradation and mass spectrometry. It is structurally related to bradykinin-like peptides identified from skin secretions of other amphibians. Ranakinin-N is composed of 13 amino acid residues and is related to the bradykinin identified from the skin secretions of Odorrana schmackeri, which is composed of 9 amino acid residues. Ranakinin-N was found to exert concentration-dependent contractile effects on isolated guinea pig ileum. cDNA sequence encoding the precursor of ranakinin-N was isolated from a skin cDNA library of R. nigrovittata. The amino acid sequences deduced from the cDNA sequences match well with the results from Edman degradation. Analysis of different amphibian bradykinin cDNA structures revealed that the deficiency of a 15-nucleotide fragment (agaatgatcagacgc in the cDNA encoding bradykinin from O. schmackeri) in the peptide-coding region resulted in the absence of a dibasic site for trypsin-like proteinases and an unusual -AEVA- insertion in the N-terminal part of ranakinin-N. The -AEAV- insertion resulted in neutral net charge at the N-terminus of ranakinin-N. Ranakinin-N is the first reported bradykinin-like peptide with a neutral net charge at the N-terminus.

Taking the sting out of pain.

While the role of the brain kallikrein-kinin system in the development of various pathological processes, such as oedema formation following brain injury or induction of central hypertonia has generated major interest, the possible role of this system in nociceptive processing has received little attention. In their present paper, Mortari et al. (2007) show that bradykinin B2 receptor activation in the brain by the bradykinin analogue, Thr(6)-bradykinin, isolated from the venom of the social wasp, Polybia occidentalis potently reduces acute, noxious heat-evoked reflex responses in naive rats. The unknown underlying mechanism of this powerful antinociceptive effect reminds us that the supraspinal antinociceptive system is still a "black box" in many aspects and awaits thorough investigation.

Inhibition of acute nociceptive responses in rats after i.c.v. injection of Thr6-bradykinin, isolated from the venom of the social wasp, Polybia occidentalis.

BACKGROUND AND PURPOSE: In this work, a neuroactive peptide from the venom of the neotropical wasp Polybia occidentalis was isolated and its anti-nociceptive effects were characterized in well-established pain induction models. EXPERIMENTAL APPROACH: Wasp venom was analysed by reverse-phase HPLC and fractions screened for anti-nociceptive activity. The structure of the most active fraction was identified by electron-spray mass spectrometry (ESI-MS/MS) and it was further assessed in two tests of anti-nociceptive activity in rats: the hot plate and tail flick tests. KEY RESULTS: The most active fraction contained a peptide whose structure was Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg-OH, which corresponds to that of Thr(6)-BK, a bradykinin analogue. This peptide was given by i.c.v. injection to rats. In the tail flick test, Thr(6)-BK induced anti-nociceptive effects, approximately twice as potent as either morphine or bradykinin also given i.c.v. The anti-nociceptive activity of Thr(6)-BK peaked at 30 min after injection and persisted for 2 h, longer than bradykinin. The primary mode of action of Thr(6)-BK involved the activation of B(2) bradykinin receptors, as anti-nociceptive effects of Thr(6)-BK were antagonized by a selective B(2) receptor antagonist. CONCLUSIONS AND IMPLICATIONS: Our data indicate that Thr(6)-BK acts through B(2) bradykinin receptors in the mammalian CNS, evoking antinociceptive behaviour. This activity is remarkably different from that of bradykinin, despite the structural similarities between both peptides. In addition, due to the increased metabolic stability of Thr(6)-BK, relative to that of bradykinin, this peptide could provide a novel tool in the investigation of kinin pathways involved with pain.

Dual-source mass spectrometer with MALDI-LIT-ESI configuration.

A novel linear ion trap (LIT) mass spectrometer with dual matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) ionization sources has been built in the MALDI-LIT-ESI configuration. The design features two independent ion source/ion optical channels connected to opposite ends of a single mass analyzer. The instrument consists of a commercial MALDI-LIT instrument modified by the addition of a home-built vacuum manifold, ion optical system, control electronics, and programming necessary to couple an atmospheric pressure interface to the commercial instrument. In addition to the added ESI functionality, the capabilities of the system also include simultaneous dual-channel ion introduction and analysis and high-duty cycle electronic switching (<1 s) between ion channels. Analytical and ion chemical applications of the dual-source system are explored. One analytical application is the enhanced protein sequence coverage achieved when using both ESI and MALDI to examine a tryptic digest of a six-protein mixture. The differences in the efficiency with which peptides in a mixture are ionized by the two methods give improved sequence coverage when both are applied. Other analytical applications include the use of the ions from one source as intensity or mass standards for the analyte ions from the other. An ion chemistry application involves the use of energy-resolved tandem mass spectrometry (MS/MS) to seek evidence for the generation of isomeric ions from a particular compound using the two ionization methods. A high level of agreement was achieved between the MS/MS spectra recorded under a variety of conditions after ESI and MALDI ionization; this provides evidence of the reproducibility and internal consistency of data from the dual source instrument. However, each of the peptides examined generated identical populations of structures in the two ionization methods under our conditions which are interpreted as involving slow cooling into the most stable minimum on the potential energy surface.

Bradykinin-related peptides, including a novel structural variant, (Val1)-bradykinin, from the skin secretion of Guenther's frog, Hylarana guentheri and their molecular precursors.

Multiple bradykinin-related peptides including a novel bradykinin structural variant, (Val(1))-bradykinin, have been identified from the defensive skin secretion of Guenther's frog, Hylarana guentheri by a tandem mass spectrometry method. Subsequently, four different preprobradykinin cDNAs, which encoded multiple bradykinin copies and its structural variants, were consistently cloned from a skin derived cDNA library. These preprobradykinin cDNAs showed little structural similarity with mammalian kininogens and the kininogens from the skin of toads, but have regions that are highly conserved in the kininogens from another ranid frog, Odorrana schmackeri. Alignment of these preprobradykinins revealed that preprobradykinin 1, 2 and 3 may derive from a single gene by alternative exon splicing.

Expression of genes encoding antimicrobial and bradykinin-related peptides in skin of the stream brown frog Rana sakuraii.

Peptidomic analysis of an extract of the skin of the stream brown frog Rana sakuraii Matsui and Matsui, 1990 led to the isolation of a C-terminally alpha-amidated peptide (VR-23; VIGSILGALASGLPTLISWIKNR x NH2) with broad-spectrum antimicrobial activity that shows structural similarity to the bee venom peptide, melittin together with two peptides belonging to the temporin family (temporin-1SKa; FLPVILPVIGKLLNGIL x NH2 and temporin-1SKb; FLPVILPVIGKLLSGIL x NH2), and peptides whose primary structures identified them as belonging to the brevinin-2 (2 peptides) and ranatuerin-2 (1 peptide) families. Using a forward primer that was designed from a conserved region of the 5'-untranslated regions of Rana temporaria preprotemporins in a 3'-RACE procedure, a cDNA clone encoding preprotemporin-1SKa was prepared from R. sakuraii skin total RNA. Further preprotemporin cDNAs encoding temporin-1SKc (AVDLAKIANIAN KVLSSL F x NH2) and temporin-1SKd (FLPMLAKLLSGFL x NH2) were obtained by RT-PCR. Unexpectedly, the 3'-RACE procedure using the same primer led to amplification of a cDNA encoding a preprobradykinin whose signal peptide region was identical to that of preprotemporin-1SKa except for the substitution Ser18-->Asn. R. sakuraii bradykinin ([Arg0,Leu1,Thr6,Trp8] BK) was 28-fold less potent than mammalian BK in effecting B2 receptor-mediated relaxation of mouse trachea and the des[Arg0] derivative was only a weak partial agonist. The evolutionary history of the Japanese brown frogs is incompletely understood but a comparison of the primary structures of the R. sakuraii dermal peptides with those of Tago's brown frog Rana tagoi provides evidence for a close phylogenetic relationship between these species.

The first report of kininogen from invertebrates.

The hornet possesses highly toxic venom, which is rich in toxin, enzymes, and biologically active peptides. Several bradykinin-like peptides, vespakinins, have been found in wasp venoms since 1970s, but the mode of biosynthesis of these peptides is unknown. In the present study, a vespakinin M was purified from venom of Vespa magnifica. Its primary sequence was established as GRPPGFSPFRID. The cDNA encoding the vespakinin M was cloned from the cDNA library of V. magnifica venom gland. The cDNA structure of vespakinin M was found to contain a coding region of 168 nucleotides. The encoded precursor of vespakinin M is composed of a signal peptide, an acidic peptide, and a mature peptide of vespakinin M. This is the first kininogen from insects; it is also the first kininogen from invertebrates. The cDNA structure encoding vespakinin M suggests that the generation mode of bradykinin-related peptides in wasp is different from amphibian skin and mammalian blood system.

Direct coupling of polymer-based microchip electrophoresis to online MALDI-MS using a rotating ball inlet.

We report on the coupling of a polymer-based microfluidic chip to a MALDI-TOF MS using a rotating ball interface. The microfluidic chips were fabricated by micromilling a mold insert into a brass plate, which was then used for replicating polymer microparts via hot embossing. Assembly of the chip was accomplished by thermally annealing a cover slip to the embossed substrate to enclose the channels. The linear separation channel was 50 microm wide, 100 microm deep, and possessed an 8 cm effective length separation channel with a double-T injector (V(inj) = 10 nL). The exit of the separation channel was machined to allow direct contact deposition of effluent onto a specially constructed rotating ball inlet to the mass spectrometer. Matrix addition was accomplished in-line on the surface of the ball. The coupling utilized the ball as the cathode transfer electrode to transport sample into the vacuum for desorption with a 355 nm Nd:YAG laser and analyzed on a TOF mass spectrometer. The ball was cleaned online after every rotation. The ability to couple poly(methylmethacrylate) microchip electrophoresis devices for the separation of peptides and peptide fragments produced from a protein digest with subsequent online MALDI MS detection was demonstrated.

High-performance SPME/AP MALDI system for high-throughput sampling and determination of peptides.

This paper presents the performance characteristics for a new multiplexed solid-phase microextraction/atmospheric pressure matrix-assisted laser desorption/ionization (SPME/AP MALDI) source configuration for a hybrid quadrupole-linear ion trap instrument. The results demonstrate that thorough optimization of parameters such as SPME coating material, optics configurations, extraction solvents, and fiber capacity provides dramatic sensitivity improvements (>1000x) over previous reports in the literature. The multiplexed SPME plate is capable of simultaneous extraction from 16 different wells on a multiwell plate, eliminating the need for extensive sample preparation. Subfemtomole sensitivity is demonstrated for peptide standards and protein digests with run-run reproducibility ranging from approximately 13 to 31%. This high-performance SPME/AP MALDI system shows potential for high-throughput extraction from biological samples.

Determination of bradykinin and arg-bradykinin in rat muscle tissue by microdialysis and capillary column-switching liquid chromatography with mass spectrometric detection.

Quantification of bradykinin peptides in limited amounts of rat muscle tissue dialysate has been performed using a packed capillary LC-ESI-TOF-MS method. The micro dialysate samples (450 microL) with added internal standard were loaded onto a 1 mm x 5 mm loading column packed with 5 microm Kromasil C18 particles by a carrier solution of 0.1% formic acid in ACN/water (5:95, v/v) at a flow rate of 250 microL/min for online preconcentration of the analytes. Back-flushed elution onto a 150 mm x 0.5 mm Zorbax C18 column packed with 5 microm particles was conducted using a linear solvent ACN/H2O gradient containing 0.1% formic acid. (Tyr8)-bradykinin was used as an internal standard and was added to the dialysis sample prior to injection. Baseline separation of bradykinin, arg-bradykinin and (tyr8)-bradykinin was achieved within 10 min. Positive ESI was performed in the m/z range of 200-1300. The method was validated in the range 0.2-1.0 ng/mL dialysate, yielding correlation coefficients of 0.995 and 0.990 for bradykinin and arg-bradykinin, respectively. The within-assay and between-assay precisions were between 4.3-9.6% and 6.2-10.6%, respectively. Both arg-bradykinin and bradykinin were detected in dialysate from rat muscle tissue, at concentrations of 0.1 and 0.4 ng/mL for bradykinin and arg-bradykinin, respectively, confirming the presence of arg-bradykinin in rat muscles.