Spider venoms and toxins are valuable sources of lead compounds for drug development due to their essential role in cellular and physiological processes targeting various receptors. Here, we present the protein profile of the venom of Phlogiellus bundokalbo, an endemic Philippine tarantula, to screen and characterize its cytotoxicity against MCF-7 cells, secretory phospholipase a2 (sPLA2), and neurotoxicity to evaluate its potential anticancer properties. Spider venom was extracted via electrical stimulation. Venom components were fractionated by reversed-phase high-performance liquid chromatography and characterized through liquid chromatography-mass spectrometry (LC-MS) and SDS-PAGE analysis before assay. The resulting five venom fractions were amphiphilic peptides showing cytotoxicity against MCF-7 cells in a concentrationdependent manner (IC50 ranging from 52.25μg/ml to 110.20μg/ml) after 24-hour incubation. Cells appeared detached, rounded, and shrunk with cytoplasmic condensation upon overnight incubation with venom fractions. The sPLA2 was observed in all the venom fractions tested for cytotoxicity. Venom fractions revealed a predominant mass of ~3-5 kDa with LC-MS analysis. Results showed distinct similar mass as μ- theraphotoxin-Phlo1a, an Australian tarantula, Phlogiellus sp. toxin with inhibitor cystine knot motif. The venom fractions exhibit excitatory neurotoxins that might activate presynaptic voltage-gated ion channels, such as an agonist or gating modifier toxins that slow down the channel inactivation similar to spider toxins. In conclusion, the spider venom of P. bundokalbo exhibits cytotoxic, phospholipase A2, and neuroactive properties suggesting that its venom components, upon further purification and structure-function analysis, can be potential tools in the development of targeted breast chemotherapeutics.
Spider Venoms ; Phospholipases

A total of 631 species of spiders have been reported in Korea. However, there are no spiders with noxious venom such as Latrodectus sp. Because of this, to date, no serious medical problems due to spider bites have been reported in Korea, and only two cases of spider bite were officially reported. However, as the number of earning spiders from other countries is increased, the number of cases of spider bite has also shown a recent increase. A 17-year-old man presented with numbness of both extremities after being bitten by a spider which he had as a pet. The spider is called an Indian ornamental tree spider (Poecilotheria regalis sp.), one kind of tarantula species. Herein, we report on a case of a spider bite by a tarantula.
Adolescent ; Extremities ; Humans ; Hypesthesia ; Korea ; Spider Bites* ; Spider Venoms ; Spiders ; Venoms

One of the distinct characters of Latrodectus tredecimguttatus is that its toxic components exist not only in the venomous glands, but also in the tissues outside the venomous glands and even in the eggs. Investigation on the toxins outside the venomous glands can deepen our understanding of spider toxins and discover new lead molecules with important application prospects. In order to explore the low-abundance proteinaceous toxins in the L. tredecimguttatus eggs, we used bioinformatic strategies to mine a gene sequence encoding a peptide toxin from the transcriptome of L. tredecimguttatus eggs, and then heterologously expressed the gene successfully with a 3'-RACE combined with nest PCR strategy. Biological activity analyses indicated that the expressed peptide toxin, named latroeggtoxin-Ⅵ (LETX-Ⅵ), could inhibit Na⁺ channel currents in ND7/23 cells and promote dopamine release from PC12 cells, without obvious toxicity against Periplaneta americana and bacteria as well as fungi including Staphylococcus aureus and Candida albicans, demonstrating that LETX-Ⅵ is a mammal-specific neurotoxin with a potential application prospect in development of the tool reagents for neurobiological study and the drugs for treating related diseases.
Animals ; Arthropod Proteins/genetics* ; Black Widow Spider/genetics* ; Cloning, Molecular ; Rats ; Spider Venoms/genetics* ; Transcriptome

Latrodectus tredecimguttatus (commonly known as black widow spiders) have toxins not only in their venom glands, but also in other parts of their body, in their eggs and even in the newborn spiderlings. The study on the toxins in venom and materials outside the venom glands of the spiders to elucidate their differences and similarities, evolutional relationship and biological functions is of important theoretical and applicable significance. The development of modern protein chemistry and proteomics techniques has provided efficient means for the study of protein and peptide toxins of L. tredecimguttatus. By using such techniques, the molecular base and action mechanism of the toxins can be revealed at the levels of both single purified proteins and omics. Up to now, although protein chemistry and proteomics study on L. tredecimguttatus toxins have achieved a certain progress, the relevant work particularly that on the toxins in the materials outside the venom glands has to be further deepened.
Animals ; Arthropod Proteins ; chemistry ; Black Widow Spider ; chemistry ; Proteomics ; Venoms ; chemistry

To seek the reason of heterogeneity of recombinant HWTX-I (rHWTX-I) expressed in Pichia pastoris. We expressed HWTX-I gene of interest in Pichia pastoris GS115/HWTX-I. The heterogenous product expressed was separated, purified and identified by using Ion exchange HPLC, reverse HPLC, Tricine SDS-PAGE and MALDI-TOF Mass Spectrometry and then sequenced in both N-terminus and C-terminus. These results show that the heterogeneity of rHWTX-I results from the incomplete processing of signal peptide of N-terminus and the internal degradation of C-terminus. Biological activity assay shows that the activity of the heterogenous rHWTX-I only showed 30% activity compared with the native HWTX-I. The Solutions to how to avoid the heterogeneity are also discussed.
Animals ; Neurotoxins ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Reptilian Proteins ; biosynthesis ; genetics ; Spider Venoms ; biosynthesis ; genetics

This paper focuses on the effect of the venom of the spider Macrothele raveni on the proliferation of human hepatocelluar carcinoma cell line HepG2 and the related molecular mechanism. XTT test showed that the proliferation of HepG2 cells in vitro was inhibited by the spider venom (P<0.05) in a concentration-dependent manner. By using flow cytometry, it was found that the spider venom caused selective G(2)/M cell cycle arrest in HepG2 cells. RT-PCR and Western blot indicated the expressions of p21 mRNA and protein in HepG2 cells were obviously up-regulated by the spider venom. The venom of the spider Macrothele raveni inhibited the proliferation of HepG2 cells. These results suggest that the possible mechanism of the spider venom is to activate the expressions of p21 gene and protein and to cause selective cell cycle arrest at G(2)/M phase, leading to HepG2 cell apoptosis.
Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; Hep G2 Cells ; Humans ; RNA, Messenger ; genetics ; metabolism ; Spider Venoms ; pharmacology

Hainantoxin-IV (HNTX-IV) purified from the venom of the spider Selenocosmia hainana is a potent antagonist that acts on tetrodotoxin-sensitive (TrX-S) sodium channels. It is a 35-residue polypeptide and includes three disulfide bridges. In order to investigate the structure-function relationship of HNTX-IV, two mutants (S12A-HNTX-IV and R29A-HNTX-IV) of HNTX-TV in which Ser12 and Arg29 were replaced by Ala respectively, were synthesized by solid-phase Fmoc chemistry, followed by oxidative refolding of purified peptides under the optimal conditions. The synthetic mutants were analyzed by MALDI-TOF mass spectrometry, nuclear magnetic resonance spectroscopy (NMR) and electrophysiological experiments for molecular weight, conformation and physiological activity, respectively. The results show that the mutants and native HNTX-IV (nHNTX-IV) have almost identical three-dimensional structures. The bioactivity level of S12A-HNTX-IV is also about the same as that of nHNTX-IV, suggesting that Ser12 does not play any important role for the bioactivity of this toxin. The bioactivity of R29A-HNTX-IV is reduced by at last 155 times, indicating that Arg29 is a key residue relative to the bioactivity of HNTX-IV. It is presumed that the decrease in activity of R29A-HNTX-IV is due to the changes of the property in the binding site rather than the change in the basic conformation of the molecule.
Amino Acid Substitution ; Animals ; Mutation ; Sodium Channel Blockers ; Sodium Channels ; drug effects ; physiology ; Spider Venoms ; chemical synthesis ; genetics ; Structure-Activity Relationship ; Tetrodotoxin ; pharmacology

Kv4.3 channel is present in many mammalian tissues, predominantly in the heart and central nervous system. Its currents are transient, characterized by rapid activation and inactivation. In the hearts of most mammals, it is responsible for repolarization of the action potential of ventricular myocytes and is important in the regulation of the heart rate. Because of its central role in this important physiological process, Kv4.3 channel is a promising target for anti-arrhythmic drug development. Jingzhaotoxin-V (JZTX-V) is a novel peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. Whole-cell patch clamp recording showed that it partly blocked the transient outward potassium channels in dorsal root ganglion neurons of adult rats with an IC(50) value of 52.3 nmol/L. To investigate the effect of JZTX-V on Kv4.3 channel, JZTX-V was synthesized using the solid-phase chemical synthesis and separated by reverse phase high performance liquid chromatography (HPLC). The purity was tested by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MOLDI-TOF mass spectrometry). Two-electrode voltage-clamp technique was used to characterize the action of JZTX-V on Kv4.3 channels expressed in Xenopus laevis oocytes. As a result, JZTX-V displayed fast kinetics of inhibition and recovery from inactivation. Furthermore, it could inhibit Kv4.3 channel current in a time- and concentration-dependent manner with an IC(50) value of 425.1 nmol/L. The application of JZTX-V affected the activation and inactivation characteristics of Kv4.3 channel and caused a shift of the current-voltage relationship curve and the steady-state inactivation curve to depolarizing direction by approximately 29 mV and 10 mV, respectively. So we deduced that JZTX-V is a gating modifier toxin of Kv4.3 channel. Present findings should be helpful to develop JZTX-V into a molecular probe and drug candidate targeting to Kv4.3 channel in the myocardium.
Animals ; Ganglia, Spinal ; cytology ; Neurons ; drug effects ; Neurotoxins ; pharmacology ; Oocytes ; Patch-Clamp Techniques ; Peptides ; pharmacology ; Potassium Channel Blockers ; pharmacology ; Rats ; Shal Potassium Channels ; metabolism ; Spider Venoms ; pharmacology ; Xenopus laevis

Kv2.1 channel currents in pancreatic beta-cells are thought to contribute to action potential repolarization and thereby modulate insulin secretion. Because of its central role in this important physiological process, Kv2.1 channel is a promising target for the treatment of type 2 diabetes. Jingzhaotoxin-XI (JZTX-XI) is a novel peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. Two-microelectrode voltage clamp experiments had showed that the toxin inhibited Kv2.1 potassium currents expressed in Xenopus Laevis oocytes. In order to investigate the structure-function relationship of JZTX-XI, the natural toxin and a mutant of JZTX-XI in which Arg3 was replaced by Ala, were synthesized by solid-phase chemistry method with Fmoc-protected amino acids on the PS3 automated peptide synthesizer. Reverse-phase high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) were used to monitor the oxidative refolding process of synthetic linear peptides to find the optimal renaturation conditions of these toxins. The experiments also proved that the relative molecular masses of refolded peptides were in accordance with their theoretical molecular masses. RP-HPLC chromatogram of co-injected native and refolded JZTX-XI was a single peak. Under the whole-cell patch-clamp mode, JZTX-XI could completely inhibit hKv2.1 and hNav1.5 channels currents expressed in HEK293T cells with IC50 values of 95.8 nmol/L and 437.1 nmol/L respectively. The mutant R3A-JZTX-XI could also inhibit hKv2.1 and hNav1.5 channel currents expressed in HEK293T cells with IC50 values of 1.22 micromol/L and 1.96 micromol/L respectively. However, the prohibitive levels of R3A-JZTX-XI on hKv2.1 and hNav1.5 channels were reduced by about 12.7 times and 4.5 times respectively, indicating that Arg3 was a key amino acid residue relative to the hKv2.1 channel activity of JZTX-XI, but it is also an amino acid residue correlated with the binding activity of JZTX-XI to hNav1.5 channel. Our findings should be helpful to develop JZTX-XI into a molecular probe and drug candidate targeting to Kv2.1 potassium channel in the pancreas.
Animals ; HEK293 Cells ; Humans ; Insulin-Secreting Cells ; metabolism ; Mutant Proteins ; genetics ; pharmacology ; NAV1.5 Voltage-Gated Sodium Channel ; metabolism ; Neurotoxins ; chemical synthesis ; genetics ; pharmacology ; Protein Refolding ; Shab Potassium Channels ; antagonists & inhibitors ; metabolism ; Sodium Channel Blockers ; pharmacology ; Spider Venoms ; genetics ; pharmacology ; Transfection

Huwentoxin-XI (HWTX-XI) is a protein isolated from the crude venom of spider Ornithoctonus huwena. It has 55 amino acid residues containing 6 cysteine residues forming 3 disulfide bonds. It shows potent inhibitory effect on trypsin and voltage-gated potassium channels in rat dorsal root ganglion cells. According to the structure-function relationship of HWTX-XI, we designed two mutants through mutation of potassium channel inhibition related amino acid residues (R5I, R10T,R25A and R5I,R25A) and then expressed them with high purity by using the vector pVT102U on Saccharamyces cerevisiae strain S78; The two mutants had the same trypsin inhibition activity as HWTX-XI, whereas their potassium channel inhibition activity and animal toxicity were much lower than those of HWTX-XI. This study is helpful for designing drugs of trypsin related diseases based on HWTX-XI.
Amino Acid Sequence ; Animals ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Mutant Proteins ; biosynthesis ; genetics ; pharmacology ; Potassium Channel Blockers ; pharmacology ; Rats ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Saccharomyces cerevisiae ; genetics ; metabolism ; Spider Venoms ; biosynthesis ; genetics ; pharmacology ; Spiders ; Trypsin Inhibitors ; pharmacology