Proteomic And Peptidomic Characterization Of The Venom From Chinese Bird Spider Ornithoctonus Huwena And Structure-function Study Of Several Spider Peptide Toxins

The bird spider Ornithoctonus huwena is one of the most venomous spiders in china. Several compounds with different types of biological activities have been identified previously from the venom of the spider. In this study, we have performed a proteomic and peptidomic analysis of the venom. The venom was pre-separated into two parts: the venom proteins with molecular weight (MW) higher than 10 kDa and the venom peptides with MW lower than 10 kDa. Using one-dimension gel electrophoresis (1-DE), two-dimension gel electrophoresis (2-DE) and mass spectrometry, 90 proteins were identified, including some important enzymes, binding proteins, and some proteins with significant biological functions. For venom peptides, a combination of cation-exchange and reversed-phase chromatography was employed. 133 different components were detected by mass spectrometry and 47 peptides were sequenced by Edman degradation. The peptides display structural and pharmacological diversity, and share little sequence similarity with peptides from other animal venoms, which indicates the venom of O. huwena Wang is unique. The venom peptides can be classified into several superfamilies. Our results revealed that gene duplication and focal hypermutation have taken places during the evolution of the spider toxins.A novel peptide, denoted Huwentoxin-â…ª(HWTX-â…ª), which inhibits serine proteinase such as trypsin and chymotrypsin and behaves as a blocker of voltage-sensitive K⁺ channels, has been isolated from the venom of Chinese bird spider Ornithoctonus huwena Wang. The molecular weight of HWTX-â…ªwas determined to be 6166.2Da by MALDI-TOF mass spectrometry. It is composed of 55 residues and 6 cysteines stabilized by three intramolecular disulfide bridges (â… -â…¥,â…¡-â…£andâ…¢-â…¤). The dimensional structure analysis by NMR indicates that HWTX-â…ªbelongs to a Kunitz protein family. Its dissociation constants were, from trypsin, less than 1×10^-12 M and fromα-chymotrypsin, 1.56×10^-7 M as measured by a BIAcore binding assay system, and its dissociation constants was, from trypsin, 8.92×10^-12 M, by Isother Titration Calormetry. Whole-cell patch clamp recording showed that HWTX-â…ªhad no effects on voltage-gated sodium channels or calcium channels in dorsal root ganglion neurons, whereas it significantly inhibited outward delay-rectified potassium channels in a voltage-dependent manner in rat dorsal root ganglion neurons. This was confirmed by testing the HWTX-â…ªeffect on Kv1.1, Kv1.2 and Kv1.3 channels expressed in Xenopus oocytes. It blocked the Kv1.1 channel with an IC₅₀ value of 2.57μM and the Kv1.2 and Kv1.3 channels with a much lower affinity. In order to determine the key residues of HWTX-â…ªresponsible for binding to serine proteinase or inhibiting potassium channels, eighteen mutants of native HWTX-â…ªwere designed and expressed in Saccharomyces cerevisiae. Substitution of Lys14 with Ala has led to lmost totally loss of trypsin inhibitory activity, and S16R, R12P and G36R substitution increased the dissociation constant by 1～3 fold, but decreased the dissociation constant of 6 fold for R10T substitution. The results indicated that Lys14 was the key residue for proteinase inhibitory activity and the residues around are important residues. The above substitutions did not influence the activity on potassium channels. On the other hand, the substitutions of L6A and L6Y almost lost the affinity for Kv1.1 channel; substitutions of R5I and R25A decreased the Kv1.1 affinity for 14 and 4-fold, respectively, while substitutions of T3L, T3Y, G24W, and G24Q decreased the affinity 2～10 fold. The mutant D2K increased the affinity for only 2 fold, which is much less than what we expected, since the basic residue Lys3 in DTX-K is a key residue to the Kv1.1 inhibition activity of DTX-K. The results indicated that the residues in the distortedβ-turn and neighboring 3₁₀-helix of HWTX-â…ªwere critical for its interaction with K⁺ channels. The toxin would be helpful to study the structural and functional relationship of Kunitz-type inhibitors and BPTI-like toxins, and provides significant cues for studying the evolution of Kunitz-family toxins.Chilobrachys jingzhao was identified recently as a new species of spider in the Hainan province of China. By means of ion-exchange and rpHPLC, we have isolated three neurotoxic peptides, denoted jingzhaotoxin-â…ª(JZTX-â…ª), jingzhaotoxin-â…«(JZTX-â…«), and jingzhaotoxin-â…©â…¢(JZTX-â…©â…¢), from its venom. The molecular weights of the three toxins were determined to be 3726.38, 3665.4, and 4122.5Da respectively by MALDI-TOF mass spectrometry. The three toxins consist of 29-35 residues with 6 cysteines stabilized by three intramolecular disulfide bridges. We characterized the actions of the novel toxins and other three jingzhaotoxins, including jingzhaotoxin-â… (JZTX-â… ), jingzhaotoxin -â…¢(JZTX -â…¢) and jingzhaotoxin -â…¤(JZTX -â…¤) which were determined by their sodium channel activity, on potassium channels using two-electrode voltage-clamp techniques. Electrophysiological recordings carried out inâ…©. laevis oocytes showed that these toxins acted as gating modifier of voltage-dependent K⁺ channels. They displayed different affinity to Kv2.1 and Kv4.1 channels. JZTX-â…¢and JZTX-â…ªare specific for Kv2.1 channels; JZTX-â…¤and JZTX-â…«are specific for Kv4 channels; while JZTX-â… and JZTX-â…©â…¢exhibit similar affinity to Kv2.1 and Kv4 channels. Structure-function analysis indicates that electrostatic interactions can benefit for toxin affinity and the feature of electrostatic anisotropy may be correlated with the different affinity of the toxins for the Kv2.1 and Kv4.1 channels. The onset of inhibition and recovery of these toxins to Kv channels were different. JZTX-â…¤exhibited much faster kinetics than that of the other three toxins. We have given an analysis of the differences based on the structure and the phylogenetic tree. It provided some significant information on the functional mechanism of this class of toxins. These toxins have turned out to be valuable tools for the investigation of the physiological role of the different potassium channel subunits in cellular physiology. In summary: we have performed a proteomic and peptidomic analysis of the venom of the bird spider Ornithoctonus huwena Wang. Several toxins with different biological activities have been purified from two Chinese bird spiders. The structural and functional investigation proved that they belong to two supfamilies and have specific functions targeting serine proteinase and different potassium channel subtypes. This study discovered a group of spider peptide toxins which are significant in science research.