The bird spider Selenocosmia huwena Wang [=Ornithoctonus huwena (Wang)] is one of the most venomous spiders in China. The venom of this spider contains a mixture of compounds with different types of biological activity. About 400 proteins and peptides from the venom can be separated and detected by 2D electrophoresis. Of these, 14 peptide toxins have been purified and characterized from the venom of this spider, with several peptide toxins exhibiting structural similarity but high functional diversity. Most of these huwentoxins (HWTX) contain 30–40 amino acids with three disulfide bonds and adopt an‘inhibitor cystine-knot'(ICK) motif in their three dimensional structure, except for huwentoxin-II (HWTX-II) which adopts a novel scaffold different from the ICK motif. As a group, the toxins possess quite different biological activities including inhibition of voltage-gated calcium and sodium channels, insecticidal activity, lectin-like agglutination, and inhibition of trypsin. Eight cDNAs encoding seven toxins, HWTX-I, -II, -III, -IIIa, -IV -V, and, -VII and one lectin, S. huwena lectin-I (SHL-I), have been cloned and sequenced. Comparison of the cDNA sequences of the eight peptides from S. huwena indicates that they can be classified into two different superfamilies according to the‘prepro'region of their cDNA sequences.q 2004 Elsevier Ltd. All rights reserved.

We have isolated a cardiotoxin, denoted jingzhaotoxin-III (JZTX-III), from the venom of the Chinese spider Chilobrachys jingzhao. The toxin contains 36 residues stabilized by three intracellular disulfide bridges (I-IV, II-V, and III-VI), assigned by a chemical strategy of partial reduction and sequence analysis. Cloned and sequenced using 3 -rapid amplification of cDNA ends and 5 -rapid amplification of cDNA ends, the full-length cDNA encoded a 63-residue precursor of JZTX-III. Different from other spider peptides, it contains an uncommon endoproteolytic site (-X-Ser-) anterior to mature protein and the intervening regions of 5 residues, which is the smallest in spider toxin cDNAs identified to date.Under whole cell recording, JZTX-III showed no effects on voltage-gated sodium channels (VGSCs) or calcium channels in dorsal root ganglion neurons, whereas it significantly inhibited tetrodotoxin-resistant VGSCs with an IC50 value of0.38M in rat cardiac myocytes.Different from scorpion -toxins, it caused a 10-mV depolarizing shift in the channel activation threshold. The binding site for JZTX-III on VGSCs is further suggested to be site 4 with a simple competitive assay, which at 10M eliminated the slowing currents induced by Buthus martensi Karsch I (BMK-I, scorpion-like toxin) completely.JZTX-III shows higher selectivity for VGSC isoforms than other spider toxins affecting VGSCs, and the toxin hopefully represents an important ligand for discriminating cardiac VGSC subtype.

Hainantoxin-III and hainantoxin-IV, isolated from the venom of the Chinese bird spider Seleconosmia hainana, are neurotoxic peptides composed of 33–35 residues with three disulfide bonds. Using whole-cell patch-clamp technique, we investigated their action on ionic channels of adult rat dorsal root ganglion neurons. It was found that the two toxins did not affect Ca2+channels (both high voltage activated and low voltage activated types) nor tetrodotoxin-resistant voltage-gated Na+ channels (VGSCs). However, hainantoxin-III and hainantoxin-IV strongly depressed the amplitude of tetrodotoxin-sensitive Na+ currents with IC50 values of 1.1 and 44.6nM, respectively. Both hainantoxin-III (1nM) and hainantoxin-IV (50nM) caused a hyperpolarizing shift of about 10mV in the voltage midpoint of steady-state Na+ channel inactivation, but they showed difference in the reprime kinetics of VGSCs: hainantoxin-III significantly decreased the recovery rate from inactivation at a prepulse potential of 80mV while hainantoxin-IV did not do. It is interesting to note that similar to huwentoxin-IV, the two hainantoxins did not affect the activation and inactivation kinetics of Na+ currents and at a concentration of 1AM they completely inhibited the slowing inactivation currents induced by BMK-I (toxin I from the scorpion Buthus martensi Karsch), a scorpion a-like toxin.The results indicate that hainantoxin-III and hainantoxin-IV are novel spider toxins and affect the mammal neural Na+ channels through a mechanism quite different from other spider toxins targeting the neural receptor site 3, such as y-aractoxins and A-agatoxins.

Hainantoxin-IV (HNTX-IV) can specifically inhibit the neuronal tetrodotoxin-sensitive sodium channels and defines a new class of depressant spider toxin. The se-quence of native HNTX-IV is ECLGFGKGCNPSNDQCCKSSNLVCSRKHRWCKYEI-NH2. In the present study,to obtain further insight into the primary and tertiary structural requirements of neuronal sodium channel blockers, we determined the solution structure of HNTX-IV as a typical inhibitor cystine knot motif and synthesized four mutants designed based on the predicted sites followed by structural elucidation of two inactive mutants. Pharmacological studies indicated that the S12A and R26A mutants had activities near that of native HNTX-IV, while K27A and R29A demonstrated activities reduced by 2 orders of magnitude. 1H MR analysis showed the similar molecular conformations for native HNTX-IV and four synthetic mutants. Furthermore,in the determined structures of K27A and R29A,the side chains of residues 27 and 29 were located in the identical spatial position to those of native HNTX-IV.These results suggested that residues Ser12, Arg26, Lys27,and Arg29 were not responsible for stabilizing the distinct conformation of HNTX-IV, but Lys27 and Arg29 were critical for the bioactivities. The potency reductions produced by Ala substitutions were primarily due to the direct interaction of the essential residues Lys27 and Arg29 with sodium channels rather than to a conformational change. After comparison of these structures and activities with correlated toxins, we hypothesized that residues Lys27, Arg29, His28, Lys32, Phe5, and Trp30 clustered on one face of HNTX-IV were responsible for ligand binding.

Two-dimensional polyacrylamide gel electrophoresis (2-DE) profiles of human lung squamous carcinoma tissue and paired surrounding normal bronchial epithelial tissue were compared. Selected differential protein-spots were identified with peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching. Well-resolved and reproducible 2-DE patterns of both the tumor and the normal tissues were acquired. The average deviations of spot position were 0.873 0.125mm in IEF direction and 1.025 0.213mm in SDS–PAGE direction, respectively. For the tumor tissues, a total of 1349 67 spots were detected and 1235 48 spots were matched with an average matching rate of 91.5%. For the corresponding normal tissues, a total of 1297 73 spots were detected and 1183 56 spots were matched with an average matching rate of 91.2%. A total of 1069 45 spots were matched between the tumor and the normal tissues. Forty differential proteins between tumor and normal tissues were characterized. Some proteins were the products of oncogenes and others were involved in the regulation of cell cycle and signal transduction. These data are valuable for mass identification of differentially expressed proteins involved in lung carcinogenesis, establishing human lung cancer proteome databaseand screening molecular marker to further study human lung squamous carcinoma.2003 Published by Elsevier Inc.