Structure-function Study Of Several Toxins From Spider Venoms

Chilobrachys jingzhao was identified recently as a new spider species of the family of Theraphosidae in the Hainan province of China. By means of ion-exchange and reverse phase HPLC, we have isolated two new components, named jingzhaotoxin-V (JZTX-V) and jingzhaotin-I (JZTIN-I), from its venom. The molecular weights of the two peptides were determined to be 3605.73 and 3631.42 Da by MALDI-TOF mass spectrometry, respectively. JZTX-V is composed of 29 residues while JZTIN-I has 34 residues. By using 3'-RACE and 5'-RACE, the full-length cDNAs were obtained and deduced to encode a 83-residue precursor of JZTX-V and a 66-residue precursor of JZTTN-I. To better understand their structure-function relationships, we constructed two models of JZTX-V and JZTIN-I based on the NMR structures of their homologous toxins respectively. JZTIN-I is a new disintegrin containing the KGD sequence, which is the first disintegrin found in spider venoms. It inhibits platelet aggregation induced by adenosine 5-diphosphate (ADP) in human platelet-rich plasma and possesses anticoagulant activity. JZTIN-I at low concentration facilitated the platelet aggregation, but JZTIN-I restrained the platelet aggregation at high concentration. It is further assumed that the binding site of JZTIN-I on platelets is the platelet glycoprotein IIb/IIIa. JZTX-V inhibits both tetrodotoxin-resistant and tetrodotoxin-sensitive sodium currents in adult rat dorsal root ganglion neurons with IC₅₀ values of 19.8 and 35.4 nM, respectively. Moreover, the toxin exhibits high affinity to both the resting closed and inactivated states of the channels. JZTX-V can inhibits Kv4.2potassium channels expressed in Xenpus Laevis oocytes (IC50 = 604.2 nM), but has no effects on Kvl.2, Kvl.3 and Kvl.4 expressed in Xenopus laevis oocytes. JZTX-V alters the gating properties of both tetrodotoxin-resistant and tetrodotoxin-sensitive sodium channels by causing a depolarization shift of about 10 mV in the threshold of the initial activated voltage or the active voltage of peak inward currents and a hyperpolarizing shift of about 7 mV in the voltage midpoint of steady-state sodium channel inactivation. Small unilamellar vesicles binding assays show that the partitioning of JZTX-V into lipid bilayer requires negatively charged phospholipids. Tryptophan fluorescence and acrylamide quenching experiments hint that tryptophan residues on the hydrophobic surface of JZTX-V have a reduced exposure to water when the toxin interacts with membranes. Furthermore, JZTX-V exhibits a red edge excitation shift of 5.7 nm when bound to acidic phospholipid membranes. The present studies hinted that when bound to the phospholipid bilayer JZTX-V may be located at the interface between polar headgroups and the hydrophobic phase of the membrane. Mutants R26A-JZTX-V, K27A-JZTX-V, K22A-JZTX-V and R20V-JZTX-V were synthesized by solid-phase Fmoc chemistry, followed by oxidative refolding of purified peptides under the optimal conditions. Human tetrodotoxin-resistant sodium channel was reported to be a potential target for treatment of pain, highlighting the importance of JZTX-V as potential lead for drug development.Using two-dimensional lH NMR techniques, the complete sequence-specific assignments of proton resonance in the !H-NMR spectra of JZTX-I were obtained by analyzing a series oftwo-dimensional spectra, including DQF-COSY, TOCSY and NOESY spectra in H2O and D2O. All the backbone protons except Gln4 and more than 95% of the side-chain protons were identified by daN, d^, dpN and dun connectivities in NOESY spectrum. Furthermore, the secondary structure of JZTX-I was identified from NMR data. It contained mainly a short triple-stranded antiparallel p-sheet consisted of residue Trp7-Cys9, Phe20-Lys23, and Leu28-Trp31. Small unilamellar vesicles binding assays showed that the partitioning of JZTX-I into lipid bilayer does not require negatively charged phospholipids. Tryptophan fluorescence and acrylamide quenching experiments hint that tryptophan residues on the hydrophobic surface of JZTX-I have a reduced exposure to water when the toxin interacts with membranes. Furthermore, JZTX-I exhibits a red edge excitation shift of 8.0 and 7.4 nm respectively when bound to acidic or neutral phospholipid membranes. By contrast, interaction between BMK Ml (a sodium channel site 3 toxin) and lipid vesicles could not observed under the same condition. It is further assumed that the mechanism of JZTX-I may be different from these site 3 toxins that have been reported up to the present.Five neurotoxic peptides, named Raventoxin-I (RVTX-I) Raventoxin-HA (RVTX-HA), Raventoxin-HB (RVTX-HB), Raventoxin-IIC (RVTX-HC) and Raventoxin-m (RVTX-III) were isolated from the venom of the spider Macrothele raveni. The molecular weights of these peptides were determined to be 4840.11, 3133.48, 3020.35, 3115.63 and 3286.58 Da by MALDI-TOF mass spectrometry, respectively. These toxins are composed of 43, 34, 33, 34 and 29 residues respectively. Both RVTX-I and RVTX-III are lethal to mice and blockneuromuscular transmission in an isolated mouse phrenic nerve diaphragm preparation. The LD50 of Raventoxin-I in mice is 0.772mg/kg. Moreover, Raventoxin-I could exert an effect of exciting firstly and followed with inhibition on the contraction of mouse diaphragm muscle caused by electrically stimulating phrenic nerve, but Raventoxin-EQ could not. Electrophysiological experiments showed that RVTX-I not only could delay TTX-S sodium channels fast inactivation, but also inhibited the peak inward currents of sodium channels on rat DRG neurons, and that RVTX-III could inhibit high-voltage-activated calcium channels on rat DRG neurons. RVTX-HA, RVTX-IIB and RVTX-IIC are a group of natural mutants. RVTX-IIA contains 6 cysteines stabilized by three intracellular disulfide bridges (I-IV, II-V and ffl-VI), assigned by partial reduction and sequencing. Raventoxin-IIA and Raventoxin-IIB are lethal to tettigoniidaes (Holochlora nawae Mats et Shiraki,) and their LD50 value in tettigoniidaes are 17.99 nmol/g and 54.95nmol/g, respectively. While Raventoxin-HC completely lacks anti-tettigoniidae activity. The structure-activity relationship indicates that the residue Asp5 may be a key amino acid residue for the anti-insect activity of Raventoxin-IIA, while the residue Leu1 is not.