Leptoglycin: A new Glycine/Leucine-rich antimicrobial peptide isolated from the skin secretion of the South American frog Leptodactylus pentadactylus (Leptodactylidae)

Antimicrobial peptides are components of innate immunity that is the first-line defense against invading pathogens for a wide range of organisms. Here, we describe the isolation, biological characterization and amino acid sequencing of a novel neutral Glycine/Leucine-rich antimicrobial peptide from skin secretion of Leptodactylus pentadactylus named leptoglycin. The amino acid sequence of the peptide purified by RP-HPLC (C₁₈ column) was deduced by mass spectrometric de novo sequencing and confirmed by Edman degradation: GLLGGLLGPLLGGGGGGGGGLL. Leptoglycin was able to inhibit the growth of Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli and Citrobacter freundii with minimal inhibitory concentrations (MICs) of 8 μM, 50 μM, and 75 μM respectively, but it did not show antimicrobial activity against Gram-positive bacteria (Staphylococcus aureus, Micrococcus luteus and Enterococcus faecalis), yeasts (Candida albicans and Candida tropicalis) and dermatophytes fungi (Microsporum canis and Trichophyton rubrum). No hemolytic activity was observed at the 2-200 μM range concentration. The amino acid sequence of leptoglycin with high level of glycine (59.1%) and leucine (36.4%) containing an unusual central proline suggests the existence of a new class of Gly/Leu-rich antimicrobial peptides. Taken together, these results suggest that this natural antimicrobial peptide could be a tool to develop new antibiotics.     

Structural identification by mass spectrometry of a novel antimicrobial peptide from the venom of the solitary bee Osmia rufa (Hymenoptera: Megachilidae)

The venom from the solitary bee Osmia rufa (Hymenoptera: Megachilidae) was analyzed using mass spectrometry (MS)-based techniques. Sensitive proteomic methods such as on-line LC-ESI-MS and nanoESI-MS analyses revealed more than 50 different compounds with molecular masses ranging from 400 to 4000 Da. The major component has a monoisotopic molecular mass of 1924.20 Da and its amino acid sequence was elucidated by de novo sequencing using tandem mass spectrometry and Edman degradation. This 17-residue cysteine-free peptide, named osmin, shows some similarities with the mast cell degranulation (MCD) peptide family. Free acid and C-terminally amidated osmins were chemically synthesized and tested for antimicrobial and haemolytic activities. The synthetic C-amidated peptide (native osmin) was found to be about three times more haemolytic than its free acid counterpart, but both peptides are much less lytic than melittin from social bee venom. Preliminary antimicrobial and antifungal tests indicate that both peptides are able to inhibit bacterial and fungal growth at micromolar concentrations.     

Molecular cloning and characterization of alboaggregin D, a novel platelet activating protein, from Green pit viper (Cryptelytrops albolabris) venom

Viper venoms are abundant sources of proteins affecting hemostasis. This study aimed to clone and purify a high-molecular-weight C-type lectin-like protein (snaclec) from Green pit viper (Cryptelytrops albolabris) venom, as well as to characterize its effects on human platelets.Based on the partial sequences from the C. albolabris venom gland library, we cloned full-length cDNAs encoding the snaclec subunits using 5′RACE and 3′RACE methods. The cDNA sequence of the α subunit contained 477 base pairs (bp) that were translated into 23 amino acid residue signal peptide and a 135-residue mature protein. The cDNA sequence of the β subunit contained 447 bp that were translated into 23-residue signal peptide and a 125-residue mature protein. Compared with known sequences of dimeric snaclecs, these peptides contained extra cysteines that probably formed a high-order multimer. In parallel, a snaclec was isolated from C. albolabris crude venom using gel filtration followed by ion-exchange chromatography. The purified C. albolabris snaclec on SDS-PAGE showed the apparent molecular mass of 120 kDa under native condition and 2 bands of 14 and 17 kD under reduced condition suggesting a tetramer of heterodimers (αβ)₄. Liquid chromatography-tandem mass spectrometry analysis of the peptides found perfect matches with the conceptually translated sequences from the cDNA library. This protein was unique from any other snaclecs previously purified from C. albolabris and named alboaggregin D. It induced human platelet aggregation in the absence of any cofactor with the EC₅₀ of 0.25 nM and caused tyrosine phosphorylation in human platelets. Antibodies against either platelet glycoprotein (GP) Ib or GPVI could inhibit alboaggregin D-induced platelet aggregation. This snaclec may be useful for dissecting the mechanisms of platelet activation.     

Two novel antimicrobial peptides from centipede venoms

Centipede venoms are complex mixtures of biochemically and pharmacologically active components such as peptides and proteins. Very few are known about their pharmacological actions. The present work reports the structural and functional characterization of two antimicrobial peptides (scolopin 1 and -2) identified from centipede venoms of Scolopendra subspinipes mutilans by Sephadex gel filtration and reverse-phase high-performance liquid chromatography (RP-HPLC). The amino acid sequences of scolopin 1 and -2 were FLPKMSTKLRVPYRRGTKDYH and GILKKFMLHRGTKVYKMRTLSKRSH determined by Edman degradation and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Both scolopin 1 and -2 showed strong antimicrobial activities against tested microorganisms including Gram-positive/negative bacteria and fungi. They also showed moderate hemolytic activity against both human and rabbit red cells. This is the first report of antimicrobial peptides from centipedes.     

ImKTx88, a novel selective Kv1.3 channel blocker derived from the scorpion Isometrus maculates

Scorpion toxins are useful in the structure-function research of ion channels and valuable resources for drug design. The Kv1.3 channel is an important pharmacological target for the therapy of T cell-mediated autoimmune diseases, and many toxin peptides targeting Kv1.3 have been identified as good drug candidates in recent years. In this study, a novel toxin gene ImKTx88 was isolated from the venom of the scorpion Isometrus maculates through the construction of the cDNA library method, and the recombinant toxin peptide was purified and characterized physiologically. The mature peptide of ImKTx88 contained 39 amino acid residues including six cysteines and was predicted to be a new member of α-KTx scorpion family by sequence analysis. The electrophysiological experiments further indicated that the rImKTx88 peptide had a novel pharmacological profile: it inhibited Kv1.3 channel current with an IC₅₀ of 91 ± 42 pM, and exhibited very good selectivity for Kv1.3 over Kv1.1 (4200-fold) and Kv1.2 (93000-fold) channels, respectively. All these results suggested that, as a new selective Kv1.3 channel blocker, the ImKTx88 peptide may serve as a potential drug candidate in the therapy of autoimmune diseases.     

A rapidly diverging superfamily of peptide toxins in venomous Gemmula species

The gem turrids (genus Gemmula Weinkauff, 1875) are venomous snails in the family Turridae. A gene superfamily of disulfide-rich peptides expressed in Gemmula venom ducts was characterized. Gemmula speciosa (Reeve, 1843) venom duct cDNA clones revealed two different conotoxin-like prepropeptide precursors, with identical signal sequences, a largely conserved pro region, and a cysteine-rich C-terminal mature peptide region. The conserved signal sequence was used to successfully amplify homologous genes from three other Gemmula species; all had the same pattern of Cys residues in the predicted mature venom peptide. Although the signal sequence and propeptide regions were highly conserved, the mature toxin regions diverged greatly in sequence, except that the Cys residues were conserved. We designate this as the Pg-gene superfamily (Pg-superfamily) of Gemmula venom peptides. Purification of two members of the family directly from G. speciosa venom was achieved; amino acid sequence analysis revealed that these peptides are highly posttranslationally modified. With at least 10-fold as many species of turrids as cone snails, identification of rapidly diversifying gene superfamilies such as the Pg-superfamily of Gemmula is essential before the facile and systematic discovery and characterization of peptide toxins from turrid venoms can be achieved.     

AaeAP1 and AaeAP2: Novel Antimicrobial Peptides from the Venom of the Scorpion,Androctonus aeneas: Structural Characterisation,Molecular Cloning of Biosynthetic Precursor-Encoding cDNAs and Engineering of Analogues with Enhanced Antimicrobial and Anticancer Activities

The main functions of the abundant polypeptide toxins present in scorpion venoms are the debilitation of arthropod prey or defence against predators. These effects are achieved mainly through the blocking of an array of ion channel types within the membranes of excitable cells. However, while these ion channel-blocking toxins are tightly-folded by multiple disulphide bridges between cysteine residues, there are additional groups of peptides in the venoms that are devoid of cysteine residues. These non-disulphide bridged peptides are the subject of much research interest, and among these are peptides that exhibit antimicrobial activity. Here, we describe two novel non-disulphide-bridged antimicrobial peptides that are present in the venom of the North African scorpion, Androctonus aeneas. The cDNAs encoding the biosynthetic precursors of both peptides were cloned from a venom-derived cDNA library using 3''- and 5''-RACE strategies. Both translated precursors contained open-reading frames of 74 amino acid residues, each encoding one copy of a putative novel nonadecapeptide, whose primary structures were FLFSLIPSVIAGLVSAIRN and FLFSLIPSAIAGLVSAIRN, respectively. Both peptides were C-terminally amidated. Synthetic versions of each natural peptide displayed broad-spectrum antimicrobial activities, but were devoid of antiproliferative activity against human cancer cell lines. However, synthetic analogues of each peptide, engineered for enhanced cationicity and amphipathicity, exhibited increases in antimicrobial potency and acquired antiproliferative activity against a range of human cancer cell lines. These data clearly illustrate the potential that natural peptide templates provide towards the design of synthetic analogues for therapeutic exploitation.     

Purification, characterization, and cDNA cloning of acidic platelet aggregation inhibiting phospholipases A2 from the snake venom of Vipera lebetina (Levantine viper)

Two novel acidic phospholipase A₂s (PLA₂) were isolated by size exclusion chromatography and reversed-phase chromatography from the crude Vipera lebetina venom. The molecular masses of VLPLA₂-1 (13,704 Da) and VLPLA₂-2 (13,683 Da) and their internal tryptic peptides were determined by MALDI-TOF mass-spectrometry. When tested in human platelet-rich plasma, both enzymes showed a potent inhibitory effect on aggregation induced by ADP and collagen. Chemical modification with p-bromophenacylbromide abolished the enzymatic activity of PLA₂; its anti-platelet activity was fully inhibited in case of collagen as inducer and partially inhibited in case of ADP as inducer. The complete cDNAs encoding PLA₂ were cloned from a single venom gland cDNA library. Complete amino acid sequences of the VLPLA₂ were deduced from the cDNA sequences. The full-length cDNA sequences of the VLPLA₂ possess 615 bp and encode an open reading frame of 138 amino acids that include signal peptide (16 amino acids) and mature enzyme (122 amino acids). The VLPLA₂s have significant sequence similarity to many other phospholipase A₂s from snake venoms. The phylogenetic analysis on the basis of the amino acid sequence homology demonstrates that VLPLA₂s grouped with other Asp49 PLA₂s and they appear to share a close evolutionary relationship with the European vipers.     

Cloning and sequence analysis of an Ophiophagus hannah cDNA encoding a precursor of two natriuretic pepide domains

The king cobra (Ophiophagus hannah) is the largest venomous snake. Despite the components are mainly neurotoxins, the venom contains several proteins affecting blood system. Natriuretic peptide (NP), one of the important components of snake venoms, could cause local vasodilatation and a promoted capillary permeability facilitating a rapid diffusion of other toxins into the prey tissues. Due to the low abundance, it is hard to purify the snake venom NPs. The cDNA cloning of the NPs become a useful approach. In this study, a 957 bp natriuretic peptide-encoding cDNA clone was isolated from an O. hannah venom gland cDNA library. The open-reading frame of the cDNA encodes a 210-amino acid residues precursor protein named Oh-NP. Oh-NP has a typical signal peptide sequence of 26 amino acid residues. Surprisingly, Oh-NP has two typical NP domains which consist of the typical sequence of 17-residue loop of CFGXXDRIGC, so it is an unusual NP precursor. These two NP domains share high amino acid sequence identity. In addition, there are two homologous peptides of unknown function within the Oh-NP precursor. To our knowledge, Oh-NP is the first protein precursor containing two NP domains. It might belong to another subclass of snake venom NPs.     

Nerve growth factor from Vipera lebetina venom

Nerve growth factor was isolated from the Vipera lebetina venom by a four-step procedure including gel filtration, ion exchange, heparin and hydrophobic chromatography. The purified protein is a glycosylated non-covalently bound homodimer with monomeric molecular mass of 14,380 Da. The cDNA encoding NGF is cloned and sequenced. The amino acid sequence translated from the cDNA comprises 117 or 119 amino acids depending on the N-terminus (truncated or not). The recombinant NGF (expressed in Escherichia coli) was used to prepare the anti-NGF antiserum. The antiserum interacted with the wild-type NGF and enabled to localize NGF during the purification procedure in parallel with MALDI-TOF analysis of tryptic peptides. The isolated NGF caused neurite outgrowth from PC12 cells in concentrations beginning from 2.5 ng/ml.     

Turkish scorpion Buthacus macrocentrus: General characterization of the venom and description of Bu1, a potent mammalian Na+-channel α-toxin

The venom of the scorpion Buthacus macrocentrus of Turkey was fractionated by high performance liquid chromatography (HPLC) and its mass finger print analysis was obtained by spectrometry. More than 70 different fractions were obtained, allowing the determination of the molecular masses of at least 60 peptides ranging between 648 and 44,336 Da. The venom is enriched with peptides containing molecular masses between 3200–4500 Da, and 6000–7500 Da. They very likely correspond to K⁺-channel and Na⁺-channel specific peptides, respectively, as expected from venoms of scorpions of the family Buthidae, already determined for other species. The major component obtained from HPLC was shown to be lethal to mice and was further purified and characterized. It contains 65 amino acid residues maintained closely packed by 4 disulfide bridges, and shows a molecular weight of 7263 Da. Additionally, a cDNA from the venomous glands of this scorpion was used in conjunction with sequence data from Edman degradation and mass spectrometry for cloning the gene that codes for Bu1 as we named this toxin. This gene codes for a 67 amino acid residues peptide, where the two last are eliminated post-translationally for production of an amidated C-terminal arginine. Its sequence is closely related to toxins from the species Leiurus quinquestriatus, as revealed by a phylogenetic tree analysis. Electrophysiological results conducted with Bu1 using patch-clamp techniques indicate that it modifies the Na⁺ currents, in a similar way as other well known α-scorpion toxins. These results support the conclusion that this species of scorpions is dangerous to humans, having an epidemiological interest for the country.     

A biochemical characterization of the major peptides from the Venom of the giant Neotropical hunting ant Dinoponera australis

Venom from the “false tocandira” Dinoponera australis, a giant Neotropical hunting ant, paralyzes small invertebrate prey and induces a myriad of systemic effects in large vertebrates. HPLC/DAD/MS analyses revealed that the venom has over 75 unique proteinaceous components with a large diversity of properties ranging in size, hydrophobicity, and overall abundance. The six most abundant peptides, demonstrative of this diversity and hereafter referred to as Dinoponeratoxins, were de novo sequenced by exact mass precursor ion selection and Edman degradation. The smallest peptide characterized, Da-1039, is hydrophilic and has similarities to vasoactive peptides like kinin and bombesin. The two largest and most abundant peptides, Da-3105 and Da-3177, have a 92.9% identity in a 28 residue overlap and share ∼50 of their sequence with ponericin G2 (an antimicrobial from another ponerine ant Pachycondyla goeldii). One peptide, Da-1585, is a hydrophilic cleavage product of an amphipathic peptide, Da-2501. The most hydrophobic peptide, Da-1837, is amidated (a PTM observed in one half of the major peptides) and shares homology with poneratoxin, a sodium channel modifier found in the bullet ant Paraponera clavata. This study is the first examination of potential pharmacophores from venom of the genus Dinoponera (Order: Hymenoptera).     

Vespid chemotactic peptide precursor from the wasp, Vespa magnifica (Smith).

Despite the evolutional distance between wasp and amphibian, vespid chemotactic peptide (VCP), an important component of wasp venom, are found sharing remarkable similarities with the temporin antimicrobial peptides (AMPs) from Ranid frog, Amolops loloensis. Not only their amino acid sequences are highly similar, but they are both microbe-killing and can induce the cellular chemotactic response. However, whether the two peptides possess identical biosynthesis pathway was still not clear due to the unsolved gene sequence of VCP putative precursor. In this paper, a cDNA encoding one of VCP precursors was cloned from the venom sac cDNA library of the wasp, Vespa magnifica (Smith), and the corresponding native VCP was purified from the venoms. It was shown that the VCP precursor highly resembled temporin precursor not only in the sequence size but also in the sequences of their corresponding mature peptides. However, the enzyme-cutting sites and the possible processing enzymes for both peptides were different, which for VCP were dipeptidyl peptidase IV and trypsin-like proteases, while for temporin were only trypsin-like protease. The current results suggested that the biosynthesis mode of VCP was different from that of temporin AMP, even though the two mature peptides were similar in many ways. It is also the first report about VCP precursor from wasp venom.     

Vejovine, a new antibiotic from the scorpion venom of Vaejovis mexicanus

Multidrug resistant bacterial infections are one of the most important health problems in recent years. Resistance to conventional antibiotics limits the therapeutic options causing increase rate in morbid-mortality in hospitals. Therefore, new antibacterial agents with new bacterial targets have been searched and found in many different sources, including scorpion venom and scorpion hemolymph. Here, we report a new anti-microbial peptide named Vejovine. This peptide was isolated from the venom of the Mexican scorpion Vaejovis mexicanus by two steps of reversed phase high performance liquid chromatography (RP-HPLC). It is composed of 47 amino acid residues with no cysteine residues in its sequence, with a molecular weight of 4873 Da. The chemical synthesis of Vejovine was performed by the solid phase method of Merrifield, using fluoren-9-ylmethoxycarbonyl (Fmoc)-amino acids. Both the native and synthetic peptides were shown to have essentially the same activity. Vejovine inhibits growth of clinical isolates of Gram-negative multidrug resistant (Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae and Acinetobacter baumanii) causing nosocomial infections with a minimum inhibitory concentration (MIC) of 4.4 μM up to 50 μM. This peptide has also hemolytic activity against human erythrocytes with a HC₅₀ value of 100 μM. A cDNA library of the venomous gland of this scorpion provided material for cloning the gene encoding Vejovine. This peptide is a new type of antibiotic, showing less than 50% similarity to other known scorpion peptides. Vejovine is a candidate to be used as a leading compound for future development of an effective peptide against multidrug resistant bacteria.     

Cloning and purification of alpha-neurotoxins from king cobra (Ophiophagus hannah).

Thirteen complete and three partial cDNA sequences were cloned from the constructed king cobra (Ophiophagus hannah) venom gland cDNA library. Phylogenetic analysis of nucleotide sequences of king cobra with those from other snake venoms revealed that obtained cDNAs are highly homologous to snake venom alpha-neurotoxins. Alignment of deduced mature peptide sequences of the obtained clones with those of other reported alpha-neurotoxins from the king cobra venom indicates that our obtained 16 clones belong to long-chain neurotoxins (seven), short-chain neurotoxins (seven), weak toxin (one) and variant (one), respectively. Up to now, two out of 16 newly cloned king cobra alpha-neurotoxins have identical amino acid sequences with CM-11 and Oh-6A/6B, which have been characterized from the same venom. Furthermore, five long-chain alpha-neurotoxins and two short-chain alpha-neurotoxins were purified from crude venom and their N-terminal amino acid sequences were determined. The cDNAs encoding the putative precursors of the purified native peptide were also determined based on the N-terminal amino acid sequencing. The purified alpha-neurotoxins showed different lethal activities on mice.     

Characterization of voltage-dependent calcium channel blocking peptides from the venom of the tarantula Grammostola rosea

Voltage-dependent calcium channel blocking peptides were purified and sequenced from the venom of the tarantula, Grammostola rosea. cDNAs encoding the peptide sequences were cloned from the venom gland cDNA library. The electrophysiological effects of the peptides on several types of voltage-dependent calcium channels were evaluated using a Xenopus laevis oocyte expression system. A peptide contained in one of the HPLC peak fractions inhibited P/Q type voltage-dependent calcium channels (Ca_v2.1). The amino acid sequence of this peptide is identical to that of ω-grammotoxin SIA. A peptide from another discrete peak, which is identical to GsAFII except for one tryptophan residue in the C-terminus, inhibited L-type voltage-dependent calcium channels (Ca_v1.2). A novel peptide, named GTx1-15 (Accession number, AB201016), shows 76.5% sequence homology with the sodium channel blocker phrixotoxin 3, however, GTx1-15 preferentially inhibited T-type voltage-dependent calcium channels (Ca_v3.1). In silico secondary and tertiary structure prediction revealed that GTx1-15 and sodium channel blockers such as hainantoxin-IV, phrixotoxin 3, and ceratotoxin 2 show very similar β-strand composition, distribution of Optimal Docking Areas (continuous surface patches likely to be involved in protein-protein interactions), and surface electrostatic potential. These findings suggest that these peptide toxins evolved from common ancestors by gene duplication to maintain surface atmospheres appropriate for interaction with low-voltage-dependent ion channels.     

Isolation and characterization of two new Lys49 PLA2s with heparin neutralizing properties from Bothrops moojeni snake venom

Among the proteins and peptides already characterized in Bothrops moojeni venom, two novel phospholipases A₂ (PLA₂) have been purified and fully sequenced by ESI-MS/MS techniques. Both of them belong to the enzymatically non-active Lys49 variants of PLA₂. They consist of 122 amino acids and share a characteristic sequence in their C-terminal region composed of clusters of basic amino acids known to interact with heparin. Thus, as already established, heparin can be used as an antidote to antagonize some myotoxic PLA₂s from venoms of Bothrops genus. The two PLA₂ variants were shown to interact in vitro with unfractionated heparin (UFH) and low molecular weight heparin (LMWH), neutralizing their anticoagulant properties. Although the influences of PLA₂s from snake venoms on the blood coagulation system are known, their use to antagonize the anticoagulant effect of heparin in vitro or in vivo has never been proposed. These finding recommend diagnostic and therapeutic applications, which are currently investigated.     

Characterization of a venom peptide from a crassispirid gastropod

The crassispirids are a large branch of venomous marine gastropods whose venoms have not been investigated previously. We demonstrate that crassispirids comprise a major group of toxoglossate snails in a clade distinct from all turrids whose venoms have been analyzed. The isolation and biochemical definition of the first venom component from any crassispirid is described.Crassipeptide cce9a from Crassispira cerithina (Anton, 1838) was purified from crude venom by following biological activity elicited in young mice, lethargy and a lack of responsiveness to external stimuli. Using Edman sequencing and mass spectrometry, the purified peptide was shown to be 29 amino acid residues long, with the sequence: GSCGLPCHENRRCGWACYCDDGICKPLRV.The sequence assignment was verified through the analysis of a cDNA clone encoding the peptide. The peptide was chemically synthesized and folded; the synthetic peptide was biologically active and coelution with the native venom peptide was demonstrated. When injected into mice of various ages, the peptide elicited a striking shift in behavioral phenotype between 14 and 16 days, from lethargy to hyperactivity.