Characterization of a new bradykinin-potentiating peptide (TmF) from Trimeresurus mucrosquamatus

A novel bradykinin-potentiating peptide (BPP), designated as TmF, has been purified to homogeneity from the venom of Trimeresurus mucrosquamatus by 70% cold methanol extraction, Sephadex G-15 gel filtration and reverse-phase high performance liquid chromatography (RP-HPLC). The amino acid sequence of TmF was determined to be pGlu-Gly-Arg-Pro-Leu-Gly-Pro-Pro-Ile-Pro-Pro (pGlu denotes pyroglutamic acid), which shared high homology with other BPPs. The molecular mass of TmF was 1.1107 kD as determinated by electrospray ionization-mass spectrometry (ESI-MS), which was in accordance with the calculated value of 1.1106 kD. The potentiating "unit" of TmF to bradykinin-induced (BK-induced) contraction on the guinea-pig ileum in vitro was (1.13 +/- 0.3) unit (mg/L), and TmF (5.0 x 10(-4) mg/kg) increased the pressure-lowering-effect of bradykinin (5.0 x 10(-5) mg/kg) with approximate descent value of (14 +/- 2) mmHg. In addition, TmF inhibited the conversion of angiotensin I to angiotensin 11, 2 x 10(-3) mg of TmF caused 50% inhibition (IC50) of angiotensin-converting enzyme (ACE) hydrolyzing activity to bradykinin.

A novel bradykinin potentiating peptide isolated from Bothrops jararacussu venom using catallytically inactive oligopeptidase EP24.15

Characterization of the peptide content of venoms has a number of potential benefits for basic research, clinical diagnosis, development of new therapeutic agents, and production of antiserum. Here, we use a substrate-capture assay that employs a catalytically inactive mutant of thimet oligopeptidase (EC 3.4.24.15; EP24.15) to identify novel bioactive peptides in Bothrops jararacussu venom. Of the peptides captured with inactive EP24.15 and identified by mass spectrometry, three were previously identified bradykinin-potentiating peptides (BPP), < ENWPHPQIPP (Xc), < EGGWPRPGPEIPP (XIIIa) and < EARPPHPPIPP (XIe) (where < E is a pyroglutamyl residue). In addition, we identified a novel BPP peptide containing additional AP amino acids in the C-terminus (< EARPPHPPIPPAP); this novel peptide was named BPP-AP. Next, dermal and muscle microcirculations were visualized using intravital microscopy to establish the roles of peptides BPP-XIe and BPP-AP in this process. After local administration of peptide BPP-XIe (0.5 mu g.mu L-1), leukocyte rolling flux and adhesion were increased by fivefold in post-capillary venules, without any increments in vasodilatation of arterioles compared to control experiments. In contrast, local administration of BPP-AP (0.5 mu g.mu L-1) potently induced vasodilatation of arterioles (nearly 100% increase compared with the vehicle saline control), with only a small increase in leukocyte rolling flux. Therefore, the novel BPP-AP described herein has pharmacological advantages compared to the BPP-XIe. The present study further suggests that inactive oligopeptidase EP24.15 is a useful tool for the isolation of bioactive peptides from crude biological samples.

A bradykinin-potentiating peptide (BPP-10c) from Bothrops jararaca induces changes in seminiferous tubules

Abstract: Background: The testis-specific isoform of angiotensin-converting enzyme (tACE) is exclusively expressed in germ cells during spermatogenesis. Although the exact role of tACE in male fertility is unknown, it clearly plays a critical function in spermatogenesis. The dipeptidase domain of tACE is identical to the C-terminal catalytic domain of somatic ACE (sACE). Bradykinin potentiating peptides (BPPs) from snake venoms are the first natural sACE inhibitors described and their structure–activity relationship studies were the basis for the development of antihypertensive drugs such as captopril. In recent years, it has been showed that a number of BPPs – including BPP-10c – are able to distinguish between the N- and C-active sites of sACE, what is not applicable to captopril. Considering the similarity between tACE and sACE (and since BPPs are able to distinguish between the two active sites of sACE), the effects of the BPP-10c and captopril on the structure and function of the seminiferous epithelium were characterized in the present study. BPP-10c and captopril were administered in male Swiss mice by intraperitoneal injection (4.7 μmol/kg for 15 days) and histological sections of testes were analyzed. Classification of seminiferous tubules and stage analysis were carried out for quantitative evaluation of germ cells of the seminiferous epithelium. The blood-testis barrier (BTB) permeability and distribution of claudin-1 in the seminiferous epithelium were analyzed by hypertonic fixative method and immunohistochemical analyses of testes, respectively. Results: The morphology of seminiferous tubules from animals treated with BPP-10c showed an intense disruption of the epithelium, presence of atypical multinucleated cells in the lumen and degenerated germ cells in the adluminal compartment. BPP-10c led to an increase in the number of round spermatids and total support capacity of Sertoli cell in stages I, V, VII/VIII of the seminiferous epithelium cycle, without affecting BTB permeability and the distribution of claudin-1 in the seminiferous epithelium. Interestingly, no morphological or morphometric alterations were observed in animals treated with captopril. Conclusions: The major finding of the present study was that BPP-10c, and not captopril, modifies spermatogenesis by causing hyperplasia of round spermatids in stages I, V, and VII/VIII of the spermatogenic cycle.

Identification and functional analysis of a novel bradykinin inhibitory peptide in the venoms of New World Crotalinae pit vipers.

A novel undecapeptide has been isolated and structurally characterized from the venoms of three species of New World pit vipers from the subfamily, Crotalinae. These include the Mexican moccasin (Agkistrodon bilineatus), the prairie rattlesnake (Crotalus viridis viridis), and the South American bushmaster (Lachesis muta). The peptide was purified from all three venoms using a combination of gel permeation chromatography and reverse-phase HPLC. Automated Edman degradation sequencing and MALDI-TOF mass spectrometry established its peptide primary structure as: Thr-Pro-Pro-Ala-Gly-Pro-Asp-Val-Gly-Pro-Arg-OH, with a non-protonated molecular mass of 1063.18 Da. A synthetic replicate of the peptide was found to be an antagonist of bradykinin action at the rat vascular B2 receptor. This is the first bradykinin inhibitory peptide isolated from snake venom. Database searching revealed the peptide to be highly structurally related (10/11 residues) with a domain residing between the bradykinin-potentiating peptide and C-type natriuretic peptide domains of a recently cloned precursor from tropical rattlesnake (Crotalus durissus terrificus) venom gland. BIP thus represents a novel biological entity from snake venom.

A novel physiological property of snake bradykinin-potentiating peptides-Reversion of MK-801 inhibition of nicotinic acetylcholine receptors

The first naturally occurring angiotensin-converting enzyme (ACE) inhibitors described are pyroglutamyl proline-rich oligopeptides, found in the venom of the viper Bothrops jararaca, and named as bradykinin-potentiating peptides (BPPs). Biochemical and pharmacological properties of these peptides were essential for the development of Captopril, the first active site-directed inhibitor of ACE, currently used for the treatment of human hypertension. However, a number of data have suggested that the pharmacological activity of BPPs could not only be explained by their inhibitory action on enzymatic activity of somatic ACE. In fact, we showed recently that the strong and long-lasting anti-hypertensive effect of BPP-10c [peptide, however, alleviated MK-801-induced inhibition of nicotinic acetylcholine receptor activity. Although more data are needed for understanding the mechanism of the BPP-10c effect on nicotinic receptor activity and its relationship with the anti-hypertensive activity, this work reveals possible therapeutic applications for BPP-10c in establishing normal acetylcholine receptor activity. (C) 2008 Elsevier Inc. All rights reserved.

Interaction between bradykinin potentiating nonapeptide (BPP9a) and beta-cyclodextrin: A structural and thermodynamic study

Herein, we demonstrate the physical and chemical characterizations of the supramolecular complex formed between beta-cyclodextrin (beta CD) and bradykinin potentiating nonapeptide (BPP9a), an endogenous toxin found in Bothrops jararaca. Circular dichroism results indicate a conformational change in the BPP9a secondary structure upon its complexation with beta CD. Nuclear magnetic resonance results, mainly from NOESY experiments, and theoretical calculations showed a favorable interaction between the tryptophan residue of BPP9a and the beta CD cavity. Thermodynamic inclusion parameters were investigated by isothermal titration calorimetry, demonstrating that beta CD/BPP9a complex formation is an exothermic process that results in a reduction in entropy. Additionally, in vitro degradation study of BPP9a against trypsin (37 degrees C, pH 7.2) showed higher stability of peptide in presence of beta CD. This beta CD/BPP9a complex, which presents new chemical properties arising from the peptide inclusion process, may be useful as an antihypertensive drug in oral pharmaceutical formulations. (C) 2011 Elsevier B.V. All rights reserved.

A novel bradykinin-related peptide from skin secretions of toad Bombina maxima and its precursor containing six identical copies of the final product

Amphibian skin contains rich bradykinin-related peptides, but the mode of biosynthesis of these peptides is unknown. In the present study, a novel bradykinin-related peptide, termed bombinakinin M, was purified from skin secretions of the Chinese red bell

Cloning of bradykinin precursor cDNAs from skin of Bombina maxima reveals novel bombinakinin M antagonists and a bradykinin potential peptide

Bombinakinin M (DLPKINRKGP-bradykinin) is a bradykinin-related peptide purified from skin secretions of the frog Bombina maxima. As previously reported, its biosynthesis is characterized by a tandem repeats with various copy numbers of the peptide and sometimes co-expressed with other structure-function distinguishable peptides. At present study, two novel cDNAs encoding bombinakinin M and its variants were cloned from a cDNA library from the skin of the frog. The encoded two precursor proteins are common in that each contains three repeats of a novel 16-amino acid peptide unit and one copy of kinestatin at their N- and C-terminal parts, respectively. They differ in that the first precursor contains two copies of bombinakinin M and the second one contains one copy of a novel bombinakinin M variant. Bombinakinin M was found to elicit concentration-dependent contractile effects on guinea pig ileum, with an EC50 value of 4 nM that is four times higher than that of bradykinin (1 nM). Interestingly, the synthetic peptide (DYTIRTRLH-amide), as deduced from the 16-amino acid peptide repeats in the newly cloned cDNAs, possessed weak inhibitory activity on the contractile effects of bombinakinin M, but not on that of bradykinin. Furthermore, the newly identified bombinakinin M variant (DLSKMSFLHG-Ile(1)-bradykinin), did not show contractile activity on guinea pig ileum, but showed potentiation effect on the myotropic activity of bradykinin. In a molar raito of 1:58, it augmented the activity of bradykinin up to two-fold. (C) 2004 Elsevier B.V. All rights reserved.

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. Copyright (C) 2007 European Peptide Society and John Wiley & Sons, Ltd.

Molecular cloning of the helokinestatin/CNP precursor from a venom-derived cDNA library of the Mexican beaded lizard, Heloderma horridum, and identification of a novel encoded bradykinin-antagonist peptide, helokinestatin-6

Helokinestatins 1–5 represent a novel family of bradykinin antagonist peptides originally isolated from the venom of the Gila Monster, Heloderma suspectum. We found that they were encoded in tandem along with a single copy of C-type natriuretic peptide (CNP), by two different but almost identical biosynthetic precursors that were cloned from a venom-derived cDNA library. Here we have applied the same strategy to the venom of a related species, the Mexican beaded lizard, Heloderma horridum. Lyophilised venom was used as a surrogate tissue to generate a cDNA library that was interrogated with primers from the previous study and for reverse phase HPLC fractionation. The structure of a single helokinestatin precursor was obtained following sequencing of 20 different clones. The open-reading frame contained 196 amino acid residues, somewhat greater than the 177–178 residues of the corresponding helokinestatin precursors in H. suspectum. The reason for this difference in size was the insertion of an additional domain of 18 amino acid residues encoding an additional copy of helokinestatin-3. Helokinestatin-6 (GPPFNPPPFVDYEPR) was a novel peptide from this precursor identified in venom HPLC fractions. A synthetic replicate of this peptide antagonised the relaxation effect of bradykinin on rat arterial smooth muscle. The novel peptide family, the helokinestatins, have been shown to be present in the venom of H. horridum and to be encoded by a single precursor of different structure to those from H. suspectum. Studies such as this reveal the naturally-selected structures of bioactive peptides that have been optimised for purpose and provide the scientist with a natural analogue library for pharmacological investigation.

Identification and bioactivity evaluation of a novel bradykinin inhibitory peptide from the skin secretion of Chinese large odorous frog, Odorrana livida

A novel peptide was isolated from the skin secretion of Chinese large odorous frog, Odorrana livida, and was named as Rana-BI. The cDNA sequencing was obtained by 'shotgun' cloning. The amino acid sequence of the mature peptide was identified as Gly-Leu-Leu-Ser-Gly-Lys-Ser-Val-Lys-Gly-Ser-Ile-OH by automated Edman degradation, and the molecular weight of the peptide was confirmed to be 1144.68 Da by MALDI-TOF and liquid chromatography/MS. Subsequently, the bioactivity of synthetic peptide was evaluated by smooth muscle assay using isolated rat bladder preparation. It was demonstrated that Rana-BI inhibited the contraction of rat bladder induced by bradykinin. Comparing with other peptides by searching from database, the primary structure of Rana-BI showed high similarity with that of an antimicrobial peptide of Rana family (12/12 residues). These data revealed a novel biological function of this peptide

Ex Vivo Smooth Muscle Pharmacological Effects of a Novel Bradykinin-Related Peptide, and Its Analogue, from Chinese Large Odorous Frog, Odorrana livida Skin Secretions

Bradykinin-related peptides (BRPs) are one of the most extensively studied frog secretions-derived peptide families identified from many amphibian species. The diverse primary structures of BRPs have been proven essential for providing valuable information in understanding basic mechanisms associated with drug modification. Here, we isolated, identified and characterized a dodeca-BRP (RAP-L1, T6-BK), with primary structure RAPLPPGFTPFR, from the skin secretions of Chinese large odorous frogs, Odorrana livida. This novel peptide exhibited a dose-dependent contractile property on rat bladder and rat ileum, and increased the contraction frequency on rat uterus ex vivo smooth muscle preparations; it also showed vasorelaxant activity on rat tail artery smooth muscle. In addition, the analogue RAP-L1, T6, L8-BK completely abolished these effects on selected rat smooth muscle tissues, whilst it showed inhibition effect on bradykinin-induced rat tail artery relaxation. By using canonical antagonist for bradykinin B1 or B2 type receptors, we found that RAP-L1, T6-BK -induced relaxation of the arterial smooth muscle was very likely to be modulated by B2 receptors. The analogue RAP-L1, T6, L8-BK further enhanced the bradykinin inhibitory activity only under the condition of co-administration with HOE140 on rat tail artery, suggesting a synergistic inhibition mechanism by which targeting B2 type receptors.

Peptide DV-28 amide: An inhibitor of bradykinin-induced arterial smooth muscle relaxation encoded by Bombina orientalis skin kininogen-2

Skin kininogens from bombinid toads encode an array of bradykinin-related peptides and one such kininogen from Bombina maxima also encodes the potent bradykinin B2-receptor antagonist, kinestatin. In order to determine if the skin secretion of the closely-related toad, Bombina orientalis, contained a bradykinin inhibitory peptide related to kinestatin, we screened reverse phase HPLC fractions of defensive skin secretion using a rat tail artery smooth muscle preparation. A fraction was located that inhibited bradykinin-induced relaxation of the preparation and this contained a peptide of 3198.5 Da as determined by MALDI-TOF MS. Automated Edman degradation of this peptide established the identity of a 28-mer as: DMYEIKGFKSAHGRPRVCPPGEQCPIWV, with a disulfide-bridge between Cys18 and Cys24 and an amidated C-terminal Val residue. Peptide DV-28 was found to correspond to residues 133–160 of skin pre-kininogen-2 of B. orientalis that also encodes two copies of (Thr6)-bradykinin. The C-terminal residue, Gly-161, of the precursor open-reading frame, acts as the C-terminal amide donor of mature DV-28. DV-28 amide thus represents a new class of bradykinin inhibitor peptide from amphibian skin secretion.