Search Results (40)

Scorpion venom toxins are important peptides being studied for their clinical significance. These peptides act by binding to ion channels in the membrane of nerve cells, causing the symptoms associated with scorpion stings (scorpionism). They principally affect the function of voltage-gated sodium channels (Nav) and are valuable for studying ion channels. Scorpions from the Buthidae family contain toxins that affect sodium channels and have a high affinity for mammalian channels. In this study, two sodium toxins isolated from the venom of the scorpion Centruroides hirsutipalpus, a member of the Buthidae family, were identified as belonging to the beta-type subfamily. These toxins were purified from whole venom using molecular exclusion, cationic-exchange, and reverse-phase chromatography techniques. Their molecular masses were determined using mass spectrometry, while their amino acid sequences were obtained by Edman degradation. A comparative analysis revealed that the sequences are identical to ChiNaBet60 and ChiNaBet50 toxins (now named Chirp7 and Chirp9, respectively) previously identified in the venom gland transcriptomics from C. hirsutipalpus. Furthermore, toxicity studies showed that these toxins were lethal to mammals. Electrophysiological analysis revealed that these peptides act as sodium channel–modulating toxins. In addition, interaction assays with antibodies were performed to analyze the structural determinants governing the binding mechanism. Full article

The discovery of novel cytotoxic drugs is of paramount importance in contemporary medical research, particularly in the search for treatments with fewer side effects and higher specificity. Antimicrobial peptides are an interesting class of molecules for this endeavor. In this context, the LyeTx III, a new peptide extracted from the venom of the Lycosa erythrognatha spider, stands out. The peptide exhibits typical antimicrobial traits: a positive net charge and amphipathic α -helix structure in lipid-like environments. Its unique sequence (GKAMKAIAKFLGR-NH₂), identified via mass spectrometry and Edman degradation, shows limited similarity to existing peptides. Significantly, when liposome-encapsulated, LyeTx III demonstrates selective activity against tumor cells in culture. Our MTT results showed that the cytotoxicity of the peptide increased against HN13 cells when administered as liposomes, with their viability in HN13 cells alone being 98%, compared to 38% in liposome-encapsulated form. This finding underscores that the LyeTx III peptide may be a good candidate for the development of new drugs against cancer. Its activity when encapsulated is promising, as it can increase its half-life in the body and can also be targeted to specific tumors. Full article

This study explores the potential of natural bioactive peptides from animal venoms as targeted anti-cancer agents with reduced toxicity. Initially, we screened a broad collection of animal venoms for their antiproliferative activity against cancer cell lines. From this collection, we selected venoms from Naja anchietae and Naja senegalensis due to their promising activity. Utilizing reverse- phase high-performance liquid chromatography (RP HPLC), mass spectrometry (MALDI-TOF MS and MALDI-TOF TOF MSMS), and Edman degradation sequencing, we isolated and characterized three peptides named CTNanc1, CTNanc2, and CTNanc3 from Naja anchietae, and three others named CTNsen1, CTNsen2, and CTNsen3 from Naja senegalensis, each with a molecular weight of around 7 kDa. These purified peptides demonstrated inhibition of U87 glioblastoma cell proliferation, but not of U251 and T98G cells, in cell viability assays. To assess the impact of these treatments on cell viability, apoptosis, and necrosis, flow cytometry assays were conducted on U87 cells at 72 h. The results showed a decrease in cell viability and an increase in dead cells, suggesting that the treatments not only promote apoptosis, but may also lead to increased necrosis or late-stage apoptosis as the exposure time increases. These findings suggest that these peptides could be developed as leads for cancer therapy. Full article

Body temperature is primarily regulated by the hypothalamus, ensuring proper metabolic function. Envenomation by Phoneutria nigriventer can cause symptoms such as hypothermia, hyperthermia, sweating, and shivering, all related to thermoregulation. This study aims to analyze and identify components of the venom that affect thermoregulation and to evaluate possible mechanisms. Rats were used for thermoregulation analysis, venom fractionation by gel filtration and reverse-phase chromatography (C18), and sequencing by Edman degradation. The venom exhibited hypothermic effects in rats, while its fractions demonstrated both hypothermic (pool II) and hyperthermic (pool III) effects. Further separations of the pools with C18 identified specific peaks responsible for these effects. However, as the peaks were further purified, their effects became less significant. Tests on U87 human glioblastoma cells showed no toxicity. Sequencing of the most active peaks revealed masses similar to those of the Tachykinin and Ctenotoxin families, both known to act on the nervous system. The study concludes that molecules derived from venom can act synergistically or antagonistically. Additionally, toxins that affect thermoregulation are poorly studied and require further characterization. These toxins could potentially serve as sources for the development of new thermoregulatory drugs. Full article

Alternative recombinant sources of antivenoms have been successfully generated. The application of such strategies requires the characterization of the venoms for the development of specific neutralizing molecules against the toxic components. Five toxic peptides to mammals from the Mexican scorpion Centruroides villegasi were isolated by chromatographic procedures by means of gel filtration on Sephadex G-50, followed by ion-exchange columns on carboxy-methyl-cellulose (CMC) resins and finally purified by high-performance chromatography (HPLC) columns. Their primary structures were determined by Edman degradation. They contain 66 amino acids and are maintained well packed by four disulfide bridges, with molecular mass from 7511.3 to 7750.1 Da. They are all relatively toxic and deadly to mice and show high sequence identity with known peptides that are specific modifiers of the gating mechanisms of Na⁺ ion channels of type beta-toxin (β-ScTx). They were named Cv1 to Cv5 and used to test their recognition by single-chain variable fragments (scFv) of antibodies, using surface plasmon resonance. Three different scFvs generated in our laboratory (10FG2, HV, LR) were tested for recognizing the various new peptides described here, paving the way for the development of a novel type of scorpion antivenom. Full article

The deformed wing virus (DWV) belongs to the genus Iflavirus and the family Iflaviridae within the order Picornavirales. It is an important pathogen of the Western honey bee, Apis mellifera, causing major losses among honey bee colonies in association with the ectoparasitic mite Varroa destructor. Although DWV is one of the best-studied insect viruses, the mechanisms of viral replication and polyprotein processing have been poorly studied in the past. We investigated the processing of the protease-polymerase region at the C-terminus of the polyprotein in more detail using recombinant expression, novel serological reagents, and virus clone mutagenesis. Edman degradation of purified maturated polypeptides uncovered the C- and N-termini of the mature 3C-like (3CL) protease and RNA-dependent RNA polymerase (3DL, RdRp), respectively. Autocatalytic processing of the recombinant DWV 3CL protease occurred at P1 Q₂₁₁₈ and P1′ G₂₁₁₉ (KPQ/GST) as well as P1 Q₂₃₉₃ and P1′ S₂₃₉₄ (HAQ/SPS) cleavage sites. New monoclonal antibodies (Mab) detected the mature 3CL protease with an apparent molecular mass of 32 kDa, mature 3DL with an apparent molecular mass of 55 kDa as well as a dominant 3CDL precursor of 90 kDa in DWV infected honey bee pupae. The observed pattern corresponds well to data obtained via recombinant expression and N-terminal sequencing. Finally, we were able to show that 3CL protease activity and availability of the specific protease cleavage sites are essential for viral replication, protein synthesis, and establishment of infection using our molecular clone of DWV-A. Full article

Envenomation by the Trinidad thick-tailed scorpion Tityus trinitatis may result in fatal myocarditis and there is a high incidence of acute pancreatitis among survivors. Peptidomic analysis (reversed-phase HPLC followed by MALDI-TOF mass spectrometry and automated Edman degradation) of T. trinitatis venom led to the isolation and characterization of three peptides with antimicrobial activity. Their primary structures were established asTtAP-1 (FLGSLFSIGSKLLPGVFKLFSRKKQ.NH2), TtAP-2 (IFGMIPGLIGGLISAFK.NH2) and TtAP-3 (FFSLIPSLIGGLVSAIK.NH2). In addition, potassium channel and sodium channel toxins, present in the venom in high abundance, were identified by CID-MS/MS sequence analysis. TtAP-1 was the most potent against a range of clinically relevant Gram-positive and Gram-negative aerobes and against the anaerobe Clostridioides difficile (MIC = 3.1–12.5 µg/mL). At a concentration of 1× MIC, TtAP-1 produced rapid cell death (<15 min against Acinetobacter baumannii and Staphylococcus aureus). The therapeutic potential of TtAP-1 as an anti-infective agent is limited by its high hemolytic activity (LC₅₀ = 18 µg/mL against mouse erythrocytes) but the peptide constitutes a template for the design of analogs that maintain the high bactericidal activity against ESKAPE pathogens but are less toxic to human cells. It is suggested that the antimicrobial peptides in the scorpion venom facilitate the action of the neurotoxins by increasing the membrane permeability of cells from either prey or predator. Full article

A novel peptide AnmTX Sco 9a-1 with the β-hairpin fold was isolated from the swimming sea anemone Stomphia coccinea (Actinostolidae family). The peptide consists of 28 amino acid residues, including modified hydroxyproline residue, and its measured molecular mass is 2960 Da. The peptide was not toxic on mice; however, it stimulated their exploratory motivation and active search behavior, and demonstrated an anti-anxiety effect. AnmTX Sco 9a-1 at doses of 0.1 and 1 mg/kg reduced the volume of edema during 24 h better than the nonsteroidal anti-inflammatory drug, Diclofenac, at dose of 1 mg/kg in a model of acute local λ-carrageenan-induced inflammation. ELISA analysis of the animal’s blood showed that peptide at a dose of 1 mg/kg reduced the content of tumor necrosis factor-α (TNF-α), a pro-inflammatory mediator responsible in the edema development, up to the level of TNF-α in the intact group. Besides, AnmTX Sco 9a-1 demonstrated a significant analgesic effect on acute pain sensitivity in the carrageenan-induced thermal hyperalgesia model at doses of 0.1 and 1 mg/kg. Activity of AnmTX Sco 9a-1 was shown not to be associated with modulation of nociceptive ASIC channels. Full article

Enteromorpha prolifera (E. prolifera), a tonic food in East Asian countries, is frequently studied for their pharmaceutical and healthcare applications. However, limited research has focused on antitumor peptides derived from this edible seaweed. In this study, we aimed to investigate the anticancer properties of peptides isolated from the hydrolysate of E. prolifera generated by a plethora of proteases including trypsin, papain, bromelain, and alkaline protease. The results showed that the hydrolysate produced by papain digestion exhibited remarkably stronger anticancer activity and was subjected to further purification by ultrafiltration and sequential chromatography. One heptapeptide, designated HTDT-6-2-3-2, showed significant antiproliferation activity towards several human cancer cell lines. The IC₅₀ values for NCI-H460, HepG2, and A549 were 0.3686 ± 0.0935 mg/mL, 1.2564 ± 0.0548 mg/mL, and 0.9867 ± 0.0857 mg/mL, respectively. Moreover, results from flow cytometry confirmed that cell apoptosis was induced by HTDT-6-2-3-2 in a dose-dependent manner. The amino acid sequence for this heptapeptide, GPLGAGP, was characterized by Edman degradation and further verified by Liquid Chromatography-Tandem Mass Spectrometry. In silico analysis results suggested that XIAP could be a potential target for HTDT-6-2-3-2. Molecular docking simulation showed that HTDT-6-2-3-2 could occupy a shallow pocket in the BIR3 domain of XIAP, which is involved in the inhibitory effect of caspase-9 activation. In conclusion, this E. prolifera derived peptide exhibited strong anticancer properties, which could be explored for pharmaceutical applications. Full article

A soy protein isolate was hydrolyzed with Alcalase^®, Flavourzyme^® and their combination, and the resulting hydrolysates (A, F and A + F) were ultrafiltered and analyzed through SDS-PAGE. Fractions with MW < 1 kDa were investigated for their ACE-inhibitory activity, and the most active one (A < 1 kDa) was purified by semi-preparative RP-HPLC, affording three further subfractions. NMR analysis and Edman degradation of the most active subfraction (A1) enabled the identification of four putative sequences (ALKPDNR, VVPD, NDRP and NDTP), which were prepared by solid-phase synthesis. The comparison of their ACE-inhibitory activities suggested that the novel peptide NDRP might be the main agent responsible for A1 fraction ACE inhibition (ACE inhibition = 87.75 ± 0.61%; IC₅₀ = 148.28 ± 9.83 μg mL⁻¹). NDRP acts as a non-competitive inhibitor and is stable towards gastrointestinal simulated digestion. The Multiple Reaction Monitoring (MRM) analysis confirmed the presence of NDRP in A < 1 kDa. Full article

(1) Background: The amino acid sequence elucidation of peptides from the gas phase fragmentation mass spectra, de novo sequencing, is a valuable method for the identification of unknown proteins complementary to Edman sequencing. It is increasingly used in shot-gun mass spectrometry (MS)-based proteomics experiments. We review the current state-of-the-art and use the identification of an unknown snake venom protein targeting the human tissue factor (TF) as an example to describe the analysis process based on manual spectrum interrogation. (2) Methods: The immobilized TF was incubated with a crude B. moojeni venom solution. The potential binding partners were eluted and further purified by gel electrophoresis. Edman degradation was performed to elucidate the N-terminus of the 31 kDa protein of interest. High-resolution MS with collision-induced dissociation was employed to generate peptide fragmentation spectra. Sequence tags were deduced and used for searches in the NCBI and Uniprot databases. Protein matches from the snake species were further validated by target MS/MS. (3) Results: Sequence tag D [K/Q] D [I/L] VDD [K/Q] led to a snake venom serine protease (SVSP) from lancehead B. jararaca (P81824). With target MS/MS, 24% of the SVSP sequence were confirmed; an additional 41% were tentatively assigned by data-independent MS. Edman sequencing provided information for 10 N-terminal amino acid residues, also confirming the match to SVSP. (4) Conclusions: The identification of unknown proteins continues to be a challenge despite major advances in MS instrumentation and bioinformatic tools. The main requirement is the generation of meaningful, high-quality MS peptide fragmentation spectra. These are used to elucidate sufficiently long sequence tags, which can subsequently be submitted to searches in protein databases. This basic method does not require extensive bioinformatics because peptide MS/MS spectra, especially of doubly-charged ions, can be analysed manually. We demonstrated the procedure with the elucidation of SVSP. While de novo sequencing quickly indicates the correct protein group, the validation of the entire protein sequence of amino acid-by-amino acid will take time. Reasons are the need to properly assign isobaric amino acid residues and modifications. With the ongoing efforts in genomics and transcriptomics and the availability of ever more data in public databases, the need for de novo MS sequencing will decrease. Still, not every animal and plant species will be sequenced, so the combination of MS and Edman sequencing will continue to be of importance for the identification of unknown proteins. Full article

Nature presents a wide range of biomolecules with pharmacological potential, including venomous animal proteins. Among the protein components from snake venoms, phospholipases (PLA₂) are of great importance for the development of new anticancer compounds. Thus, we aimed to evaluate the PLA₂ anticancer properties from Bothrops moojeni venom. The crude venom was purified through three chromatographic steps, monitored by enzymatic activity and SDS-PAGE (12%). The purified PLA₂ denominated BmPLA2 had its molecular mass and N-terminal sequence identified by mass spectrometry and Edman degradation, respectively. BmPLA2 was assayed against human epithelial colorectal adenocarcinoma cells (Caco-2), human rhabdomyosarcoma cells (RD) and mucoepidermoid carcinoma of the lung (NCI-H292), using human fibroblast cells (MRC-5) and microglia cells (BV-2) as a cytotoxicity control. BmPLA2 presented 13,836 Da and a 24 amino acid-residue homologue with snake PLA₂, which showed a 90% similarity with other Bothrops moojeni PLA₂. BmPLA2 displayed an IC₅₀ of 0.6 µM against Caco-2, and demonstrated a selectivity index of 1.85 (compared to MRC-5) and 6.33 (compared to BV-2), supporting its selectivity for cancer cells. In conclusion, we describe a new acidic phospholipase, which showed antitumor activity and is a potential candidate in the development of new biotechnological tools. Full article

C1q domain-containing (C1qDC) proteins are a group of biopolymers involved in immune response as pattern recognition receptors (PRRs) in a lectin-like manner. A new protein MkC1qDC from the hemolymph plasma of Modiolus kurilensis bivalve mollusk widespread in the Northwest Pacific was purified. The isolation procedure included ammonium sulfate precipitation followed by affinity chromatography on pectin-Sepharose. The full-length MkC1qDC sequence was assembled using de novo mass-spectrometry peptide sequencing complemented with N-terminal Edman’s degradation, and included 176 amino acid residues with molecular mass of 19 kDa displaying high homology to bivalve C1qDC proteins. MkC1qDC demonstrated antibacterial properties against Gram-negative and Gram-positive strains. MkC1qDC binds to a number of saccharides in Ca²⁺-dependent manner which characterized by structural meta-similarity in acidic group enrichment of galactose and mannose derivatives incorporated in diversified molecular species of glycans. Alginate, κ-carrageenan, fucoidan, and pectin were found to be highly effective inhibitors of MkC1qDC activity. Yeast mannan, lipopolysaccharide (LPS), peptidoglycan (PGN) and mucin showed an inhibitory effect at concentrations three orders of magnitude greater than for the most effective saccharides. MkC1qDC localized to the mussel hemal system and interstitial compartment. Intriguingly, MkC1qDC was found to suppress proliferation of human adenocarcinoma HeLa cells in a dose-dependent manner, indicating to the biomedical potential of MkC1qDC protein. Full article

Thrombosis is a disease that seriously endangers human health, with a high rate of mortality and disability. However, current treatments with thrombolytic drugs (such as recombinant tissue-plasminogen activator) and the oral anticoagulants (such as dabigatran and rivaroxaban) are reported to have a tendency of major or life-threatening bleeding, such as intracranial hemorrhage or massive gastrointestinal bleed with non-specific antidotes. In contrast, lumbrokinase is very specific to fibrin as a substrate and does not cause excessive bleeding. It can dissolve the fibrin by itself or convert plasminogen to plasmin by inducing endogenous t-PA activity to dissolve fibrin clots. Therefore, searching for potentially new therapeutic molecules from earthworms is significant. In this study, we first collected a strong fibrinolytic extract (PvQ) from the total protein of the Pheretima vulgaris with AKTA pure protein purification systems; its fibrinolytic bioactivity was verified by the fibrin plate assay and zebrafish thrombotic model of vascular damage. Furthermore, according to the cell culture model of human umbilical vein endothelial cells (HUVECs), the PvQ was proven to exhibit the ability to promote the secretion of tissue-type plasminogen activator (t-PA), which further illustrated that it has an indirect thrombolytic effect. Subsequently, extensive chromatographic techniques were applied to reveal the material basis of the extract. Fortunately, six novel earthworm fibrinolytic enzymes were obtained from the PvQ, and the primary sequences of those functional proteins were determined by LC-MS/MStranscriptome cross-identification and the Edman degradation assay. The secondary structures of these six fibrinolytic enzymes were determined by circular dichroism spectroscopy and the three-dimensional structures of these proteases were predicted by MODELLER 9.23 based on multi-template modelling. In addition, those six genes encoding blood clot-dissolving proteins were cloned from P. vulgaris by RT-PCR amplification, which further determined the accuracy of proteins primary sequences identifications and laid the foundation for subsequent heterologous expression. Full article

The causative agent of white tail disease (WTD) in the giant freshwater prawn is Macrobrachium rosenbergii nodavirus (MrNV). The recombinant capsid protein (CP) of MrNV was previously expressed in Escherichia coli, and it self-assembled into icosahedral virus-like particles (VLPs) with a diameter of approximately 30 nm. Extensive studies on the MrNV CP VLPs have attracted widespread attention in their potential applications as biological nano-containers for targeted drug delivery and antigen display scaffolds for vaccine developments. Despite their advantageous features, the recombinant MrNV CP VLPs produced in E. coli are seriously affected by protease degradations, which significantly affect the yield and stability of the VLPs. Therefore, the aim of this study is to enhance the stability of MrNV CP by modulating the protease degradation activity. Edman degradation amino acid sequencing revealed that the proteolytic cleavage occurred at arginine 26 of the MrNV CP. The potential proteases responsible for the degradation were predicted in silico using the Peptidecutter, Expasy. To circumvent proteolysis, specific protease inhibitors (PMSF, AEBSF and E-64) were tested to reduce the degradation rates. Modulation of proteolytic activity demonstrated that a cysteine protease was responsible for the MrNV CP degradation. The addition of E-64, a cysteine protease inhibitor, remarkably improved the yield of MrNV CP by 2.3-fold compared to the control. This innovative approach generates an economical method to improve the scalability of MrNV CP VLPs using individual protease inhibitors, enabling the protein to retain their structural integrity and stability for prominent downstream applications including drug delivery and vaccine development. Full article