Rana Box Research Articles

Modification and Synergistic Studies of a Novel Frog Antimicrobial Peptide against Pseudomonas aeruginosa Biofilms.

The overuse of traditional antibiotics has resulted in bacterial resistance and seriously compromised the therapeutic efficacy of traditional antibiotics, making the exploration of new antimicrobials particularly important. Several studies have shown that bioactive peptides have become an important source of new antimicrobial drugs due to their broad-spectrum antibacterial action and lack of susceptibility to resistance. In this study, a novel bioactive peptide Nigrosin-6VL was characterised from the skin secretion of the golden cross band frog, Odorrana andersonii, by using the 'shotgun' cloning strategy. Modifications on the Rana Box of Nigrosin-6VL revealed its critical role in antimicrobial functions. The peptide analogue, 2170-2R, designed to preserve the Rana Box structure while enhancing cationicity, exhibited improved therapeutic efficacy, particularly against Gram-negative bacteria, with a therapeutic value of 45.27. Synergistic studies demonstrated that 2170-2R inherits the synergistic antimicrobial activities of the parent peptides and effectively enhances the antimicrobial capacity of cefepime and gentamicin against both planktonic cells and biofilms. Specifically, 2170-2R can synergise effectively with cefepime and gentamicin against different strains of P. aeruginosa biofilms. Consequently, 2170-2R holds promise as a potent antimicrobial agent developed to combat infections induced by Pseudomonas aeruginosa.

Progressive Design of a Ranatuerin-2 Peptide from Amolops wuyiensis: Enhancement of Bioactivity and In Vivo Efficacy.

Antimicrobial peptides (AMPs) that exert multiple functions are considered promising candidates to combat the bacterial drug resistance crisis. Nowadays, targeted peptide modification has been widely recognised to improve biological activity and make up for deficiencies in clinical applications such as toxicity. In this study, a helix-loop peptide was isolated and identified from the skin secretion of the Wuyi torrent frog Amolops wuyiensis, namely, ranatuerin-2-AW (R2AW) (GFMDTAKNVAKNVAATLLDKLKCKITGGC). Target modifications were made to R2AW to study the structure-activity relationships and to optimise its bioactivities. Five analogues were progressively designed via residue substitution and truncation and the antibacterial and anticancer activities were evaluated. We found that the serine-substitution and cyclic-domain-deletion products showed similar antibacterial activity to the natural peptide R2AW, implying that the disulphide bridge and Rana box were dispensable for the antibacterial activity of ranatuerin-2 peptides. Notably, the cationicity- and hydrophobicity-enhanced variant, [Lys4,19, Leu20]R2AW(1-22)-NH2, exhibited significantly optimised antibacterial and anticancer activities. Additionally, it killed bacteria by membrane disruption at a highly efficient rate. Moreover, [Lys4,19, Leu20]R2AW(1-22)-NH2 exerted potential in vivo efficacy in a methicillin-resistant Staphylococcus aureus (MRSA)-infected waxworm model. Overall, this study demonstrated some rational design ideas for optimising the dual antibacterial and anticancer activities of ranatuerin-2 peptides and it proposes [Lys4,19, Leu20]R2AW(1-22)-NH2 as an appealing candidate for therapeutic development.

EThcD Benefits for the Sequencing Inside Intramolecular Disulfide Cycles of Amphibian Intact Peptides.

Disulfide bonds formed by a pair of cysteine residues in the peptides' backbone represent a certain problem for their sequencing by means of mass spectrometry. As a rule, in proteomics, disulfide bonds should be cleaved before the analysis followed by some sort of chemical derivatization. That step is time-consuming and may lead to losses of minor peptides of the analyzed mixtures due to incomplete reaction, adsorption on the walls of the vials, etc. Certain problems in the de novo top-down sequencing of amphibian skin peptides are caused by the C-terminal disulfide loop, called the Rana box. Its reduction with or without subsequent derivatization was considered to be an unavoidable step before mass spectrometry. In the present study, EThcD demonstrated its efficiency in sequencing intact disulfide-containing peptides without any preliminary derivatization. Applied to the secretion of three frog species, EThcD provided the full sequence inside the intramolecular disulfide cycle for all S-S-containing peptides found in the samples, with the only exception being diarginine species. Proteolytic fragments, which are shorter than the original peptides, were helpful in some cases. HCD should be mentioned as a complementary tool to the EThcD tool, being useful as a confirmation method for some sequence details.

A frog-derived antimicrobial peptide as a potential anti-biofilm agent in combating Staphylococcus aureus skin infection.

Staphylococcus aureus (S. aureus), one of the most prevalent bacteria found in atopic dermatitis lesions, can induce ongoing infections and inflammation by downregulating the expression of host defence peptides in the skin. In addition, the emergence of the 'superbug' Methicillin-resistant S. aureus (MRSA) has made the treatment of these infections more challenging. Antimicrobial peptides (AMPs), due to their potent antimicrobial activity, limited evidence of resistance development, and potential immunomodulatory effects, have gained increasing attention as potential therapeutic agents for atopic dermatitis. In this study, we report a novel AMP, brevinin-1E-OG9, isolated from the skin secretions of Odorrana grahami, which shows potent antibacterial activity, especially against S. aureus. Based on the characteristics of the 'Rana Box', we designed a set of brevinin-1E-OG9 analogues to explore its structure-activity relationship. Brevinin-1E-OG9c-De-NH2 exhibited the most potent antimicrobial efficacy in both in vitro and ex vivo studies and attenuated inflammatory responses induced by lipoteichoic acid and heat-killed microbes. As a result, brevinin-1E-OG9c-De-NH2 might represent a promising candidate for the treatment of S. aureus skin infections.

Targeted Modification and Structure-Activity Study of GL-29, an Analogue of the Antimicrobial Peptide Palustrin-2ISb.

Antimicrobial peptides (AMPs) are considered as promising antimicrobial agents due to their potent bioactivity. Palustrin-2 peptides were previously found to exhibit broad-spectrum antimicrobial activity with low haemolytic activity. Therefore, GL-29 was used as a template for further modification and study. Firstly, the truncated analogue, GL-22, was designed to examine the function of the ‘Rana box’, which was confirmed to have no impact on antimicrobial activity. The results of antimicrobial activity assessment against seven microorganisms demonstrated GL-22 to have a broad-spectrum antimicrobial activity, but weak potency against Candida albicans (C. albicans). These data were similar to those of GL-29, but GL-22 showed much lower haemolysis and lower cytotoxicity against HaCaT cells. Moreover, GL-22 exhibited potent in vivo activity at 4 × MIC against Staphylococcus aureus (S. aureus)-infected larvae. Several short analogues, from the C-terminus and N-terminus of GL-22, were modified to identify the shortest functional motif. However, the results demonstrated that the shorter peptides did not exhibit potent antimicrobial activity, and the factors that affect the bioactive potency of these short analogues need to be further studied.

Truncated analog Brevinin2-CE-N26V5K: Revelation the Augmentation of Antimicrobial Activity.

Brevinin2-CE (B2CE), a natural peptide containing 37 amino acids, was first isolated from the skin secretions of the Chinese forest frog Rana chensinensis. B2CE shows good antibacterial activity. In this study, a series of B2CE analogs with differences in cationicity, α-helicity, hydrophobicity and amphipathic properties were designed through chain-length deletion and amino acid substitution. The most potent, nontoxic analog, B2CE-N26V5K, was identified by examination of its antibacterial activity, hemolytic activity, and stability under physiological conditions. The increased cationicity, hydrophobicity and more obvious hydrophilic and hydrophobic surface of B2CE-N26-N16WA18KG23K did not improve the antibacterial activity but increased the hemolytic activity of this modified peptide. The helicity might promote antibacterial activity for brevinin-2 peptides, as the 15-aa analogs with lower helicity show decreased potency against different test bacteria (approximately 2- to 72-fold) compared to B2CE-N26V5K. Additionally, the results indicated that the "Rana box" does not affect the antimicrobial activity of brevinin-2 peptides, as B2CE, B2CE-nonDS and B2CE-C31-37S have similar strong inhibitory effects on both gram-positive and gram-negative bacteria. However, the "Rana box" does affect the hemolytic activity, as the HC50 values of the 3 peptides range from 25 ~ 130 µM. Furthermore, B2CE-N26V5K caused obvious morphological alterations of the bacterial surfaces, as shown by atomic force microscopy. Additionally, B2CE-N26V5K exhibited strong membrane-disrupting activity when examined using the LIVE/DEAD Bac Light Bacterial Viability Kit. Thus, the antibacterial effect of B2CE-N26V5K on gram-negative and gram-positive bacteria may be caused by cell membrane attack. In conclusion, the excellent candidate B2CE-N26V5K was obtained and has application prospects as a novel anti-infective agent.

Mass Spectrometry Differentiation between Rana arvalis Populations Based on Their Skin Peptidome Composition.

Skin secretion of amphibians often represents the only weapon of these species against pathogens and predators. Peptides constitute the major portion of active molecules of that weapon and may be treated as potential pharmaceuticals for future generations. The first step of their efficient use involves establishing of their primary structure, i.e., sequencing. De novo sequencing by means of mass spectrometry was applied to Rana arvalis species, collected in the spring 2021 in Central Slovenia (vicinity of Ljubljana). HPLC-ESI-HRMS/MS with Orbitrap instruments was used to establish the skin peptidome of these species and compare it with the earlier identified skin peptidome of the Moscow population of Rana arvalis. Application of CID, HCD, ETD, and EThcD enabled detecting and sequencing 18 peptides; five of them were novel and may be treated as possible biomarkers of the Ljubljana population of Rana arvalis. Interestingly, representatives of two peptide families (temporins and brevinins 2) were not found in the Moscow population. MS3 modes, first of all EThcD, demonstrated their great potential in the de novo sequencing, including extraction of the sequence information from the intact peptides with disulfide cycle (rana box) in their structure and differentiation of isomeric Leu/Ile residues. Thus, all six isomeric residues were reliably distinguished in the novel melittin-related peptide AK-23-1. In addition, another post-translational modification dealing with carbonylation of the N-terminal Gly of novel temporin AVa was established using the MS3 mode. The obtained results demonstrate the efficiency of the use of MS3 tools in proteomics/peptidomics.

A novel antimicrobial peptide found in Pelophylax nigromaculatus

BackgroundMany active peptides have been found in frog skin secretions. In this paper, our research focused on Pelophylax nigromaculatus and found a broad-spectrum antimicrobial peptide Nigrocin-PN based on the molecular cloning technique. Thereafter, the “Rana box” function was briefly studied by two mutated peptides (Nigrocin-M1 and Nigrocin-M2). Furthermore, in vitro and in vivo assays were used to characterize the peptide’s biofunctions, and the peptide’s function in treating multidrug-resistant pathogens was also studied. ResultsNigrocin-PN not only displayed potent antimicrobial abilities in vitro but also significantly ameliorated pulmonary inflammation induced by Klebsiella pneumoniae in vivo. By comparing, leucine-substituted analogue Nigrocin-M1 only displayed bactericidal abilities towards gram-positive bacteria, while the shorter analogue Nigrocin-M2 lost this function. More strikingly, Nigrocin-PN exhibited synergistic effects with commonly used antibiotics; in vitro evolution experiments revealed that coadministration between Nigrocin-PN and ampicillin could delay Staphylococcus aureus antibiotic resistance acquisition. Kinetics and morphology studies indicate that antibacterial mechanisms involved membrane destruction. Furthermore, toxicities and anticancer abilities of these peptides were also studied; compared to two analogues, Nigrocin-PN showed mild haemolytic activity and indistinctive cytotoxicity towards normal cell lines HMEC-1 and HaCaT. ConclusionsA broad-spectrum antimicrobial peptide Nigrocin-PN was discovered from the skin secretion of Pelophylax nigromaculatus. Structurally, “Rana box” played a crucial role in reducing toxicities without compromising antibacterial abilities, and Nigrocin-PN could be a desired therapeutic candidate. Graphical abstract▪ Highlights•For AMPs, disulphide bond can affect their biofunction and cytotoxicity.•Frog skin secretion is a reservoir to delve valuable peptides.•AMPs-antibiotics coadministration could be a strategy to delay drug resistance.

Study on the Structure-Activity Relationship of an Antimicrobial Peptide, Brevinin-2GUb, from the Skin Secretion of Hylarana guentheri.

Antimicrobial peptides (AMPs) are considered potential alternatives to antibiotics due to their advantages in solving antibiotic resistance. Brevinin-2GUb, which was extracted from the skin secretion of Hylarana guentheri, is a peptide with modest antimicrobial activity. Several analogues were designed to explore the structure–activity relationship and enhance its activity. In general, the Rana box is not an indispensable motif for the bioactivity of Brevinin-2GUb, and the first to the 19th amino acids at the N-terminal end are active fragments, such that shortening the peptide while maintaining its bioactivity is a promising strategy for the optimisation of peptides. Keeping a complete hydrophobic face and increasing the net charges are key factors for antimicrobial activity. With the increase of cationic charges, α-helical proportion, and amphipathicity, the activity of t-Brevinin-2GUb-6K (tB2U-6K), in combatting bacteria, drastically improved, especially against Gram-negative bacteria, and the peptide attained the capacity to kill clinical isolates and fungi as well, which made it possible to address some aspects of antibiotic resistance. Thus, peptide tB2U-6K, with potent antimicrobial activity against antibiotic-resistant bacteria, the capacity to inhibit the growth of biofilm, and low toxicity against normal cells, is of value to be further developed into an antimicrobial agent.

Generation of truncated derivatives through in silico enzymatic digest of peptide GV30 target MRSA both in vitro and in vivo

Methicillin-resistant Staphylococcus aureus (MRSA) causing serious hospital-acquired infections and skin infections has become a “superbug” in clinical treatment. Although the clinical treatment of MRSA is continuously improving, due to its unceasing global spread, MRSA has produced much heated discussion and focused study, therefore suggesting an urgent task to find new antibacterial drugs to combat this issue. Antimicrobial peptides (AMPs) are used as the last-resort drugs for treating multidrug-resistant bacterial infections, but their utilisation is still limited due to their low stability and often strong toxicity.Here, we evaluated the structure and the bioactivity of an AMP, GV30, derived from the frog skin secretions of Hylarana guentheri, and designed seven truncated derivatives based on the presence of cleavage sites for trypsin using an online proteomic bioinformatic resource PeptideCutter tool. We investigated the anti-MRSA effect, toxicity and salt- and serum-resistance of these peptides. Interestingly, the structure–activity relationship revealed that removing “Rana box” loop could significantly improve the bactericidal speed on MRSA. Among these derivatives, GV21 (GVIFNALKGVAKTVAAQLLKK-NH2), because of its faster antibacterial effect, lower toxicity, and retains the good antibacterial activity and stability of the parent peptide, is considered to become a new potential antibacterial candidate against MRSA.

Modification and Targeted Design of N-Terminal Truncates Derived from Brevinin with Improved Therapeutic Efficacy.

Antimicrobial peptides (AMPs) are a class of molecules that play an essential role in innate immune regulation. The Brevinin-1 family are AMPs that show strong pharmacological and antimicrobial potential. A novel peptide, B1A, was designed based on the primary structure of brevinin-1PLb and brevinin-1PLc. Subsequently, a synthesised replicate was subjected to a series of bioassays and was found to display antimicrobial activity. However, it also displayed high levels of haemolysis in a horse red blood cell haemolytic assay, suggesting potential toxicity. Therefore, we rationally designed a number of B1A analogues with aim of retaining antimicrobial activity, lowering toxicity, and to explore the structure–activity relationship of its N-terminus. B1A and its analogues still retained the “Rana Box” and the FLP-motif, which is a feature of this subfamily. However, the introduction of Lys and Trp residues into the peptide sequences revealed that antimicrobial activity of these analogues remained unchanged once the hydrophobicity and the charge reached the threshold. Hence, the idea that the hydrophobicity saturation in different situations is related to antimicrobial activity can be understood via the structure–activity relationship. Meanwhile, it could also be the starting point for the generation of peptides with specific antimicrobial activity.

Effects of C-terminal amidation and heptapeptide ring on the biological activities and advanced structure of amurin-9KY, a novel antimicrobial peptide identified from the brown frog, Rana kunyuensis.

Rana kunyuensis is a species of brown frog that lives exclusively on Kunyu Mountain, Yantai, China. In the current study, a 279-bp cDNA sequence encoding a novel antimicrobial peptide (AMP), designated as amurin-9KY, was cloned from synthesized double-strand skin cDNA of R. kunyuensis. The amurin-9KY precursor was composed of 62 amino acid (aa) residues, whereas the mature peptide was composed of 14 aa and contained two cysteines forming a C-terminal heptapeptide ring (Rana box domain) and an amidated C-terminus. These structural characters represent a novel amphibian AMP family. Although amurin-9KY exhibited high similarity to the already identified amurin-9AM from R. amurensis, little is known about the structures and activities of amurin-9 family AMPs so far. Therefore, amurin-9KY and its three derivatives (amurin-9KY1–3) were designed and synthesized. The structures and activities were examined to evaluate the influence of C-terminal amidation and the heptapeptide ring on the activities and structure of amurin-9KY. Results indicated that C-terminal amidation was essential for antimicrobial activity, whereas both C-terminal amidation and the heptapeptide ring played roles in the low hemolytic activity. Circular dichroism (CD) spectra showed that the four peptides adopted an α-helical conformation in THF/H2O (v/v 1:1) solution, but a random coil in aqueous solution. Elimination of the C-terminal heptapeptide ring generated two free cysteine residues with unpaired thiol groups, which greatly increased the concentration-dependent anti-oxidant activity. Scanning electron microscopy (SEM) was also performed to determine the possible bactericidal mechanisms.

Modification Targeting the "Rana Box" Motif of a Novel Nigrocin Peptide From Hylarana latouchii Enhances and Broadens Its Potency Against Multiple Bacteria.

Public health is confronting the threat caused by antibiotic resistance and this means new antibacterial strategies must be developed urgently. Antimicrobial peptides (AMPs) have been considered as promising therapeutic candidates against infection in the post-antibiotic era. In this paper, we dismissed the significance of “Rana box” in the natural nigrocin-HL identified from skin secretion of Hylarana latouchii by comparing its activity with nigrocin-HLD without the motif. By substituting the “Rana box” sequence with an amidated phenylalanine residue, the natural peptide was modified into a shorter AMP nigrocin-HLM. Activities and toxicities of these two peptides in vitro and in vivo were compared. As a result, nigrocin-HLM not only displayed significantly increased potency against several representative microbes, but also high activity against the antibiotic-resistant methicillin-resistant S. aureus (MRSA, NCTC 12493 and ATCC43300 and several clinical isolates) as evidenced by markedly reduced minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), and minimum biofilm eradication concentration (MBEC). More strikingly, nigrocin-HLM exhibited prominent inhibition against MRSA infection in a pneumonia mice model. In addition, the substitution attenuated the toxicity of nigrocin-HLM as evidenced by precipitously decreased hemolytic and cytotoxic activities in vitro, and acute toxicity to mice in vivo. Taken these results into consideration, nigrocin-HLM should be a promising therapeutic candidate for anti-infection. And in addition to dismiss an indispensable role of “Rana box” in maintaining antimicrobial activity of nigrocin-HL, our data provided an inspired strategy for peptide optimization.

Structure-Activity Relationship and Mode of Action of a Frog Secreted Antibacterial Peptide B1CTcu5 Using Synthetically and Modularly Modified or Deleted (SMMD) Peptides.

All life forms are equipped with rapidly acting, evolutionally conserved components of an innate immune defense system that consists of a group of unique and diverse molecules known as host defense peptides (HDPs). A Systematic and Modular Modification and Deletion (SMMD) approach was followed to analyse the structural requirement of B1CTcu5, a brevinin antibacterial peptide amide identified from the skin secretion of frog Clinotarsus curtipes, India, to show antibacterial activity and to explore the active core region. Seventeen SMMD-B1CTcu5 analogs were designed and synthesised by C and N-terminal amino acid substitution or deletion. Enhancement in cationicity by N-terminal Lys/Arg substitution or hydrophobicity by Trp substitution produced no drastic change in bactericidal nature against selected bacterial strains except S. aureus. But the sequential removal of N-terminal amino acids had a negative effect on bactericidal potency. Analog B1CTcu5-LIAG obtained by the removal of four N-terminal amino acids displayed bactericidal effect comparable to, or in excess of, the parent peptide with reduced hemolytic character. Its higher activity was well correlated with the improved inner membrane permeabilisation capacity. This region may act as the active core of B1CTcu5. Presence of C-terminal disulphide bond was not a necessary condition to display antibacterial activity but helped to promote hemolytic nature. Removal of the C-terminal rana box region drastically reduced antibacterial and hemolytic activity of the peptide, showing that this region is important for membrane targeting. The bactericidal potency of the D-peptide (DB1CTcu5) helped to rule out the stereospecific interaction with the bacterial membrane. Our data suggests that both the C and N-terminal regions are necessary for bactericidal activity, even though the active core region is located near the N-terminal of B1CTcu5. A judicious modification at the N-terminal region may produce a short SMMD analog with enhanced bactericidal activity and low toxicity against eukaryotic cells.

Novel antibacterial peptides from the skin secretion of the Indian bicoloured frog Clinotarsus curtipes

HPLC elution profile and MALDI TOF MS analysis of electro-stimulated skin secretion of the Indian Ranid frog Clinotarsus curtipes of the Western Ghats confirmed the presence of multiple peptides. Peptides eluted out of the C18 column at higher hydrophobic solvent region showed antibacterial activity against diverse bacterial strains, including the clinical isolates of V. cholerae and methicillin resistant Staphylococcus aureus (MRSA). Peptidomic analysis of the most potent chromatographic effluent fraction identified five novel peptide amides having sequence homology with brevinin family. These peptides are named as brevinin1CTcu1 (B1CTcu1) to brevinin1CTcu5 (B1CTcu5). Peptide B1CTcu1 is non-haemolytic while the others are haemolytic in nature but all elicited potential antibacterial activity. B1CTcu5 is a twenty-one residue peptide amide having proline hinge region in the middle and the typical C-terminal intramolecular disulfide-bridged hepta peptide domain (Rana box) that is present in most of the brevinin peptides. Analysis of their killing kinetics with E. coli and S. aureus and the ability to induce membrane depolarization proved that these are two independent events. These novel multifunctional peptides play an important role to protect C. curtipes from invading pathogenic microorganisms present in the environment.

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

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

Mass spectrometric study of peptides secreted by the skin glands of the brown frog Rana arvalis from the Moscow region

A high-performance liquid chromatography nano-electrospray ionization Fourier transform mass spectrometry (HPLC/nanoESI-FTMS) approach involving recording of collision-activated dissociation (CAD) and electron-capture dissociation (ECD) spectra of an intact sample and two its modifications after performic oxidation and reduction followed by carboxamidomethylation helps to establish peptide profiles in the crude secretion of frog species at mid-throughput level, including de novo sequencing. The proposed derivatization procedures allow increasing of the general sequence coverage in the backbone, providing complementary information and, what is more important, reveal the amino acid sequence in the cystine ring ('rana box'). Thus purely mass spectrometric efficient sequencing becomes possible for longer than usual proteolytic peptides. Seventeen peptides belonging to four known families were identified in the secretion of the European brown frog Rana arvalis inhabiting the Moscow region in Russia. Ranatuerins, considered previously a unique feature of the North American species, as well as a new melittin-related peptide, are worth special mention. The developed approach was previously successfully used for the identification of peptides in the skin secretion of the Caucasian green frog Rana ridibunda.

Molecular Cloning of cDNAs Encoding Antimicrobial Peptide Precursors from the Skin of the Chinese Brown Frog,Rana chensinensis

Skin plays a key role in the daily survival of amphibians. In the present study, six cDNAs encoding amphibian skin antimicrobial peptide precursors from the Chinese brown frog Rana chensinensis, were cloned and identified as preprobrevinin-1CEc, preprobrevinin-1CEb, preprotemporin-1CEa, preprotemporin-1CEb, preprotemporin-1CEc, and preprochensinin-1. Preprotemporin-1CEa, CEb, and CEc are members of the temporin family, which are usually short, hydrophobic, and C-terminally alpha-amidated antimicrobial peptides. Preprobrevinin-1CEa and CEb were identified as members of the brevinin-1 family of antimicrobial peptides, because both peptides contain a "Rana box" that is responsible for forming C-terminal Cys-bridged cyclic heptapeptides. The nucleotide and deduced amino acid sequences of preprochensinin-1 were not similar to any known amphibian skin defensive peptides. Four bioactive peptides were chemically synthesized according to the deduced amino acid sequences of six prepropeptides from R. chensinensis skin, and their antimicrobial, cytotoxic, and haemolytic properties were evaluated. All of the synthesized peptides inhibited the growth of Gram-positive bacteria. Brevinin-1CEa showed a broad spectrum of antimicrobial activity. The novel amphibian skin peptide chensinin-1 was active against Bacillus cereus and Streptococcus lactis at a concentration of 11.6 microM, but did not inhibit the growth of MCF-7 and HeLa cells at 200 microM, and had no haemolytic activity at a concentration of 500 microM. Temporin-1CEa exhibited the greatest ability to inhibit the growth of MCF-7 cells. Its antimicrobial and cytotoxic activities may be due to its high degree of alpha-helical confirmation and amphipathic nature.

Electrospray Ionization Tandem Mass Spectrometry Sequencing of Novel Skin Peptides from Ranid Frogs Containing Disulfide Bridges

Tandem mass spectrometry sequencing, as well as Edman sequencing of peptides belonging to the Rana genus, represents a difficult task due to the presence of a disulfide bridge at the C-terminus and their rather high molecular masses (over 2000 Da). The present study throws light upon the sequence of three rather long peptides (more than 20 amino acid residues each) isolated from the skin secretion of Russian frogs, Rana ridibunda and Rana arvalis. This novel aspect involves the fact that the sequences (including two sequences established de novo) were determined exclusively by means of mass spectrometry. A combination of electron capture dissociation (ECD) and collision-induced dissociaiton (CID) data accompanied by exact mass measurements (LTQ Fourier transform ion cyclotron resonance mass spectrometer) facilitated reaching the goal. To overcome the difficulty dealing with disulphide bridges ("Rana box"), reduction of the S-S bond with dithiotreitol followed by derivatization of Cys residues with iodoacetamide was used. The sequence was determined using combined spectral data on y and b series of fragment ions. A multiple mass spectrometry (MS(3)) experiment was also used to elucidate the sequence inside the "Rana box" after cysteine derivatization. Exact mass measurements were used to differentiate between Lys and Gln residues, while characteristic losses of 29 and 43 Da (d and w fragment ions) in CID and ECD experiments allowed us to distinguish between Ile and Leu isomeric acids.

Solution structure and membrane interaction mode of an antimicrobial peptide gaegurin 4

We have applied NMR spectroscopy to determine the high-resolution structure of gaegurin 4, a 37-residue antimicrobial peptide from Rana rugosa, under varying hydrophobic conditions. Even in 100% H 2O, gaegurin 4 contains a nascent turn near its C-terminal Rana box. Under a more hydrophobic condition it forms two amphipathic helices, one long encompassing residues 2–23 and the other consisting of residues 25–34, similar to what has been observed in cecropin A. Functional implication of the helix-breaking kink at Gly 24 in gaegurin 4 was investigated by preparing several analogs. Based upon the current and previous results, we propose a novel seaanemone-like ion pore-forming model for gaegurin 4.