Inn-Ho Tsai

Serine protease isoforms of Deinagkistrodon acutus venom: cloning, sequencing and phylogenetic analysis.

The major coagulating fibrinogenase of Deinagkistrdon acutus venom, designated acutobin, was purified by anion-exchange chromatography, gel filtration and reverse-phase HPLC. Approximately 80% of its protein sequence was determined by sequencing the various fragments derived from CNBr cleavage and digestion with endoprotease. Extensive screening of the venom gland cDNA species after amplification by PCR resulted in the isolation of four distinct cDNA clones encoding acutobin and three other serine proteases, designated Dav-PA, Dav-KN and Dav-X. The complete amino acid sequences of these enzymes were deduced from the cDNA sequences. The amino-acid sequence of acutobin contains a single chain of 236 residues including four potential N-glycosylation sites. The purified acutobin (40 kDa) contains approx. 30% carbohydrate by weight, which could be partly removed by N-glycanase. The phylogenetic tree of the complete amino acid sequences of 40 serine proteases from 18 species of Crotalinae shows functional clusters reflecting parallel evolution of the three major venom enzyme subtypes: coagulating enzymes, kininogenases and plasminogen activators. The possible structural elements responsible for the functional specificity of each subtype are discussed.

Molecular evolution and structure–function relationships of crotoxin-like and asparagine-6-containing phospholipases A2 in pit viper venoms

Some myotoxic or neurotoxic PLA2s (phospholipases A2) from pit viper venoms contain characteristic N6 substitutions. Our survey of the venoms of more than ten pit viper genera revealed that N6-PLA2s exist only in limited Asian pit vipers of two genera, Protobothrops and Gloydius, and exist as either monomers or the basic subunits of heterodimers in some New World pit vipers. For the newly identified N6-PLA2s, the neuromuscular blocking activities were assayed with the chick biventer cervicis neuromuscular tissue, whereas the increased serum creatine kinase level assessed their myotoxicities. The purified N6-PLA2s from Protobothrops mangshanensis and Gloydius intermedius saxatilis were found to be presynaptic neurotoxins. In contrast, all N6-PLA2s from the venoms of Sistrurus miliarius strackeri, S. m. barbouri, Crotalus viridis viridis, C. lepidus lepidus, Cerrophidion godmani and Bothreichis schlegelii were myotoxins without neurotoxicity even in the presence of crotoxin A. Crotoxin-like complexes were for the first time purified from the venoms of Sitrurus catenatus tergeminus, C. mitchelli mitchelli, C. horridus atricaudatus, C. basiliscus and C. durissus cumanensis. The cDNAs encoding six novel N6-PLA2s and subunits of the crotoxin-like complex from S. c. tergeminus were cloned and fully sequenced. Phylogeny analysis showed that two structural subtypes of N6-PLA2s with either F24 or S24 substitution have been evolved in parallel, possibly descended respectively from species related to present-day Protobothrops and Gloydius. Calmodulin binds all the N6-PLA2s but crotoxin A may inhibit its binding to crotoxin B and to other neurotoxic N6-PLA2s. Structure-activity relationships at various regions of the PLA2 molecules were extensively discussed.

Geographic variations, cloning, and functional analyses of the venom acidic phospholipases A2 of Crotalus viridis viridis.

Geographic venom samples of Crotalus viridis viridis were obtained from South Dakota, Wyoming, Colorado, Oklahoma, Texas, New Mexico, and Arizona. From these samples, the phospholipases A(2) (PLA(2)s) were purified and their N-terminal sequences, precise masses, and in vitro enzymatic activities were determined. We purified two to four distinct acidic PLA(2)s from each sample; some of them displayed different inhibition specificities toward mammalian platelets. One of the acidic PLA(2)s induced edema, but had no anti-platelet activity. There was also a common basic PLA(2) myotoxin in all the samples. We have cloned five acidic PLA(2)s and several hybrid-like nonexpressing PLA(2)s. Molecular masses and N-terminal sequences of the purified PLA(2)s were matched with those deduced from the cDNA sequences, and the complete amino acid sequences of five novel acidic PLA(2)s were thus solved. They share 78% or greater sequence identity, and a cladogram based on the sequences of many venom acidic PLA(2)s of New World pit vipers revealed at least two subtypes. The results contribute to a better understanding of the ecogenetic adaptation of rattlesnakes and the structure-activity relationships and evolution of the acidic PLA(2)s in pit viper venom.

PHOSPHOLIPASES A2 OF ASIAN SNAKE VENOMS

AbstractThis review up-dated the structural and functional information of various phospholipase A2 (PLA2) isoforms purified from Asian snake venoms. A phylogenic tree of group I PLA2s was constructed herein based on many recently resolved amino acid sequences of the venom enzymes. It was found that PLA2s of Asian elapid venoms are structurally different from those of sea-snake/Australian elapid venoms, and are usually associated with cardiovascular effect, although exceptions such as β-bungarotoxins do exist. Two types of venom PLA2s appear to be present in the venom of Asiatic viperinae such as Daboia and Echis, one has a N-terminal residue Asn and the other has the residue Ser. In the venom of Asiatic crotalinae, up to four subgroups of PLA2 isoforms are present and each of them is characterized by a distinct substitution at residue 6 (Glu, Asn or Arg) or residue 49 (Asp or Lys) in their sequences. The venom PLA2s in each of the subgroup show more or less functional similarity specific for the subgroup:...

New insights into the functions and N‐glycan structures of factor X activator from Russell’s viper venom

The coagulation factor X activator from Russell’s viper venom (RVV‐X) is a heterotrimeric glycoprotein. In this study, its three subunits were cloned and sequenced from the venom gland cDNAs of Daboia siamensis. The deduced heavy chain sequence contained a C‐terminal extension with four additional residues to that published previously. Both light chains showed 77–81% identity to those of a homologous factor X activator from Vipera lebetina venom. Far‐western analyses revealed that RVV‐X could strongly bind protein S, in addition to factors X and IX. This might inactivate protein S and potentiate the disseminated intravascular coagulation syndrome elicited by Russell’s viper envenomation. The N‐glycans released from each subunit were profiled and sequenced by MALDI‐MS and MS/MS analyses of the permethyl derivatives. All the glycans, one on each light chain and four on the heavy chain, showed a heterogeneous pattern, with a combination of variable terminal fucosylation and sialylation on multiantennary complex‐type sugars. Amongst the notable features were the presence of terminal Lewis and sialyl‐Lewis epitopes, as confirmed by western blotting analyses. As these glyco‐epitopes have specific receptors in the vascular system, they possibly contribute to the rapid homing of RVV‐X to the vascular system, as supported by the observation that slower and fewer fibrinogen degradation products are released by desialylated RVV‐X than by native RVV‐X.

Functional proteomic approach to discover geographic variations of king cobra venoms from Southeast Asia and China

UNLABELLED This study deciphers the geographic variations of king cobra (Ophiophagus hannah) venom using functional proteomics. Pooled samples of king cobra venom (abbreviated as Ohv) were obtained from Indonesia, Malaysia, Thailand, and two provinces of China, namely Guangxi and Hainan. Using two animal models to test and compare the lethal effects, we found that the Chinese Ohvs were more fatal to mice, while the Southeast Asian Ohvs were more fatal to lizards (Eutropis multifasciata). Various phospholipases A2 (PLA2s), three-finger toxins (3FTxs) and Kunitz-type inhibitors were purified from these Ohvs and compared. Besides the two Chinese Ohv PLA2s with known sequences, eight novel PLA2s were identified from the five Ohv samples and their antiplatelet activities were compared. While two 3FTxs (namely oh-55 and oh-27) were common in all the Ohvs, different sets of 3FTx markers were present in the Chinese and Southeast Asian Ohvs. All the Ohvs contain the Kunitz inhibitor, OH-TCI, while only the Chinese Ohvs contain the inhibitor variant, Oh11-1. Relative to the Chinese Ohvs which contained more phospholipases, the Southeast Asian Ohvs had higher metalloproteinase, acetylcholine esterase, and alkaline phosphatase activities. BIOLOGICAL SIGNIFICANCE Remarkable variations in five king cobra geographic samples reveal fast evolution and dynamic translational regulation of the venom which probably adapted to different prey ecology as testified by the lethal tests on mice and lizards. Our results predict possible variations of the king cobra envenoming to human and the importance of using local antivenin for snakebite treatment.

Cloning, characterization and mutagenesis of Russell’s viper venom l-amino acid oxidase: Insights into its catalytic mechanism ☆

To investigate the structure-function relationships and geographic variations of L-amino acid oxidase (LAAO) from Daboia venoms, a single LAAO (designated as DrLAO) was purified from eastern Indian Daboia russelii venom and characterized. The purified DrLAO showed subunit molecular mass of 60-64kDa; its N-terminal sequence (1-20) was identical to those of several true viper LAAOs. Its preferred substrates were hydrophobic l-amino acids and the kinetic specificities were ordered as follows: Phe, Tyr, Met, Leu, and Trp. Enzyme assay and Western blotting showed that the venom LAAO contents of D. russelii were higher than those of Daboia siamensis. DrLAO dose-dependently inhibited ADP- and collagen-induced platelet aggregation with IC(50) values of 0.27 and 0.82μM, respectively. Apparently, DrLAO may synergize with other venom components to prolong and enhance bleeding symptoms after Daboia envenoming. The full sequence of DrLAO was deduced from its cDNA sequence and then confirmed by peptide mass fingerprinting. Molecular phylogenetic analysis revealed that SV-LAAO family members could be differentiated not only by snake taxonomy but also by the variations at position 223, and they divided into H223, S223, N223, and D223 subclasses. We have further prepared recombinant DrLAO and mutants by the Pichia expression system. Mutagenic analyses of DrLAO His223 revealed that this residue bound substrates instead of serving as an essential base in the catalytic steps. Our results suggest a direct hydride transfer from substrate to FAD as the mechanism for SV-LAAOs.

Sequences, geographic variations and molecular phylogeny of venom phospholipases and threefinger toxins of eastern India Bungarus fasciatus and kinetic analyses of its Pro31 phospholipases A2.

Eight phospholipases A2 (PLAs) and four three‐finger‐toxins (3FTx) from the pooled venom of Bungarus fasciatus (Bf) were previously studied and sequenced, but their expression pattern in individual Bf venom and possible geographic variations remained to be investigated. We herein analyze the individual venom of two Bf specimens from Kolkata (designated as KBf) to address this question. Seven PLAs and five 3FTx were purified from the KBf venoms, and respective cDNAs were cloned from venom glands of one of the snakes. Comparison of their mass and N‐terminal sequence revealed that all the PLAs were conserved in both KBf venoms, but that two of their 3FTx isoforms were variable. When comparing the sequences of these KBf‐PLAs with those published, only one was found to be identical to that of Bf Vb‐2, and the other five were 94–98% identical to those of Bf II, III, Va, VI and XI‐2, respectively. Notably, the most abundant PLA isoforms of Bf and KBf venoms contain Pro31 substitution. They were found to have abnormally low kcat values but high affinity for Ca2+. Phylogenetic analysis based on the sequences of venom group IA PLAs showed a close relationship between Bungarus and Australian and marine Elapidae. As the five deduced sequences of KBf‐3FTx are only 62–82% identical to the corresponding Bf‐3FTx from the pooled venom, the 3FTx apparently have higher degree of individual and geographic variations than the PLAs. None of the KBf‐3FTx was found to be neurotoxic or very lethal; phylogenetic analyses of the 3FTx also revealed the unique evolution of Bf as compared with other kraits.

F-spondin plays a critical role in murine neuroblastoma survival by maintaining IL-6 expression.

F‐spondin is associated with the regulation of axonal growth and the development of the nervous system. Its mechanism of action, however, is not clearly understood. In this study, we found that murine neuroblastoma Neuro‐2a cells expressed a significant level of IL‐6, but only trace amounts of IL‐12, tumor necrosis factor α and nitric oxide. Knock‐down of F‐spondin mRNA in murine neuroblastoma NB41A3 and Neuro‐2a cells using small interfering RNAs led to decreased IL‐6 levels along with lower resistance to serum starvation and cytotoxic amyloid β1–42 (Aβ1–42) peptide. Restoring decline of F‐spondin or IL‐6 induced by F‐spondin knock‐down through adding exogenous F‐spondin, IL‐6 or over‐expressing F‐spondin reversed the cell death induced by Aβ1–42 peptide or serum starvation. The decrease of IL‐6 level was positively correlated with decrease of NF‐κB and inhibition of p38 mitogen‐activated protein kinase (MAPK). Over‐expressing MEKK, a kinase activator of the p38 MAPK pathway, increased IL‐6 production, restored the decrease of p38 induced by F‐spondin knock‐down, and rescued the cells from death caused by Aβ1–42 peptide. Taken together, these results suggest that F‐spondin may play a critical role in murine neuroblastoma survival under adverse conditions by maintaining IL‐6 level via a MEKK/p38 MAPK/NF‐κB‐dependent pathway.

Structures and functions of crotoxin-like heterodimers and acidic phospholipases A2 from Gloydius intermedius venom: Insights into the origin of neurotoxic-type rattlesnakes☆

UNLABELLED The cDNAs encoding four major phospholipases A2 (PLA2s) were sequenced while the expressed sequence tags of Gloydius intermedius venom glands were constructed. These PLA2s were designated as Gintexin-A precursor, Gintexin-B, Gin-E6a and Gin-E6b, respectively. The deduced amino acid sequences of the former two PLA2s are 80% and 90% identical to those of crotoxin-A-precursor and crotoxin-B1, respectively. We also purified Gintexin-A, Gintexin-B, Gin-E6a and Gin-E6b like PLA2 from the venom. The latter three PLA2s are enzymatically active but not strongly anticoagulant for human plasma. Gin-E6a and E6b-like PLA2s induced mouse platelet aggregation but inhibited rabbit platelet aggregation. The isolated Gintexin, a 1:1 complex of Gintexin-A and Gintexin-B, blocked the twitch of chick biventer cervicis tissue presynaptically. Results of N-terminal sequencing and peptide mass fingerprinting reveal that Gintexin-A undergoes proteolytic processing similar to crotoxin-A. This is the first time heterodimeric β-neurotoxins are found in Asian pitviper venom, and incompatible neurotoxic- and hemorrhagic-type venoms are found to evolve in parallel within the genus Gloydius, like in Crotalus. Thus, G. intermedius probably is the ancestor of rattlesnakes with type-II venom, and characterization of its venomics helps us to understand the evolution of heterodimeric neurotoxic PLA2s and the paedomorphic trend observed in Neotropical rattlesnake venoms. BIOLOGICAL SIGNIFICANCE For the first time, a heterodimeric neurotoxic PLA2 (designated as Gintexin) has been isolated from the venom of an Asian pitviper, which shows a characteristic venom gland transcriptome similar to those of the neurotoxic type rattlesnakes. The fact that the venom of G. intermedius is less hemorrhagic than those of other Gloydius species, reveals that incompatible neurotoxic- and hemorrhagic-type venoms have evolved in parallel within the genus Gloydius, like the genus Crotalus. Our findings suggest that G. intermedius is the most probable ancestor of some Neotropical rattlesnakes. The results may revolutionize the theory regarding the origin of type-II rattlesnakes and assist with the diagnosis and clinical management of G. intermedius bites. Furthermore, the possibility of using the currently available antivenoms of Neotropical rattlesnakes to treat G. intermedius bites seems feasible.