Shao-Wen Zhu

Characterization of a fibrinogen-clotting enzyme from Trimeresurus stejnegeri venom, and comparative study with other venom proteases

Trimeresurus stejnegeri venom which contains TSV-PA (a specific plasminogen activator sharing 60-70% sequence homology with venom fibrinogen-clotting enzymes), also possesses fibrinogen-clotting activity in vitro. A fibrinogen-clotting enzyme (stejnobin) has been purified to homogeneity by gel filtration and ion-exchange chromatography on a Mono-Q column. It is a single-chain glycoprotein with a mol. wt of 44,000. The NH2-terminal amino acid sequence of stejnobin shows great homology with venom fibrinogen-clotting enzymes and TSV-PA. Like TSV-PA, stejnobin was able to hydrolyse several chromogenic substrates. Comparative study of substrate specificities of stejnobin and other venom proteases purified in our laboratory was carried out on five chromogenic substrates. Stejnobin clotted human fibrinogen with a specific activity of 122 NIH thrombin-equivalent units/mg protein. However, stejnobin did not act on other blood coagulation factors, such as factor X, prothrombin and plasminogen. Diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride inhibited its activity, whereas ethylenediamine tetracetic acid had no effect on it, indicating that it is a serine protease. Although stejnobin showed strong immunological cross-reaction with polyclonal antibodies raised against TSV-PA, it was interesting to observe that, unlike the case of TSV-PA, these antibodies did not inhibit the amidolytic and fibrinogen-clotting activities of stejnobin.

Characterization and cloning of a novel phospholipase A2 from the venom of Trimeresurus jerdonii snake

A phospholipase A(2) (PLA(2)), called jerdoxin, was isolated from Trimeresurus jerdonni snake venom and partially characterized. The protein was purified by three chromatographic steps. SDS-polyacrylamide gel electrophoresis in the presence or absence of dithiothreitol showed that it had a molecular mass of 15 kDa. Jerdoxin had an enzymatic activity of 39.4 micro mol/min/mg towards egg yolk phosphatidyl choline (PC). It induced edema in the footpads of mice. In addition, jerdoxin exhibited indirect hemolytic activity. About 97% hemolysis was observed when 2 micro g/ml enzyme was incubated for 90 min in the presence of PC and Ca(2+). No detectable hemolysis was noticed when PC was not added. Ca(2+) was necessary for jerdoxin to exert its hemolytic activity, since only 52% hemolysis was seen when Ca(2+) was absent in the reaction mixture. Furthermore, jerdoxin inhibited ADP induced rabbit platelet aggregation and the inhibition was dose dependent with an IC(50) of 1.0 micro M. The complete amino acid sequence of jerdoxin deduced from cDNA sequence shared high homology with other snake venom PLA(2)s, especially the D 49 PLA(2)s. Also, the residues concerned to Ca(2+) binding were conserved. This is the first report of cDNA sequence of T. jerdonii venom PLA(2).

A novel short neurotoxin, cobrotoxin c, from monocellate cobra (Naja kaouthia) venom: isolation and purification, primary and secondary structure determination, and tertiary structure modeling.

A novel short neurotoxin, cobrotoxin c (CBT C) was isolated from the venom of monocellate cobra (Naja kaouthia) using a combination of ion-exchange chromatography and FPLC. Its primary structure was determined by Edman degradation. CBT C is composed of 61 amino acid residues. It differs from cobrotoxin b (CBT B) by only two amino acid substitutions, Thr/Ala11 and Arg/Thr56, which are not located on the functionally important regions by sequence similarity. However, the LD50 is 0.08 mg/g to mice, i.e. approximately five-fold higher than for CBT B. Strikingly, a structure-function relationship analysis suggests the existence of a functionally important domain on the outside of Loop III of CBT C. The functionally important basic residues on the outside of Loop III might have a pairwise interaction with alpha subunit, instead of gamma or delta subunits of the nicotinic acetylcholine receptor (nAChR).

A novel natriuretic peptide from the cobra venom

Natriuretic peptides (NPs) play crucial roles in human physiology and pathophysiology through natriuresis, dieresis and vasorelaxation. NPs are also one of the important components of snake venoms. However, the low abundance in snake venom hampered the investigation. Here, a novel natriuretic peptide named Na-NP was purified from the cobra Naja atra venom. Na-NP consists of 45 amino acid residues and its molecular weight is 4618.5 Da. A full-length cDNA encoding Na-NP was obtained from the cDNA library constructed from the venom gland. The open reading frame of cloned Na-NP was composed of 498bp and coded for a 165-amino acid residue protein precursor. The nucleotide and deduced protein sequences of Na-NP were remarkably conserved with other elapid NPs while significant different from the viperid NPs. Na-NP showed weak activity to relax the aortic rings precontracted with phenylephrine. Meanwhile, Na-NP showed cGMP-promotion activity against primary cultured rabbit endocardial endothelial cells, but had no effect on human platelet aggregation. In conclusion, this is the first report of a natriuretic peptide from the cobra N. atra venom. Na-NP might be served as a useful tool for the study of human NPs and the development of novel therapeutic drugs.

A unique group of inactive serine protease homologues from snake venom.

A number of inactive serine protease homologues (SPHs), which have poorly understood functions, have been identified in invertebrates and vertebrates. Recently, several SPH transcripts have been reported from snake venom glands, which provide potential new tools for the study of the functions of SPHs. Herein we report for the first time a snake venom serine protease homologue (svSPH) protein, designated as TjsvSPH, isolated from the venom of Trimeresurus jerdonii. Despite its high sequence similarity to snake venom serine proteases (SVSPs), TjsvSPH is devoid of arginine esterase and proteolytic activity. This is probably due to the replacement of Arg-43 by His-43 in the catalytic triad. TjsvSPH did not influence the coagulation time of human plasma, induce human platelet aggregation, inhibit adenosine diphosphate/thrombin-induced human platelet aggregation or increase capillary permeability. Phylogenetic analysis showed that svSPHs were separated from SVSPs and formed an independent group. Structural analysis revealed that the structures of svSPHs are quite different from those of SPHs previously reported. These results indicate that snake venoms contain a unique group of svSPH proteins.

A novel platelet glycoprotein Ib-binding protein with human platelet aggregation-inhibiting activity from Trimeresurus jerdonii venom.

Platelet glycoprotein Ib (GPIb) is a primary adhesion receptor and involved in platelet-related disorders. However, it is difficult to study GPIb-specific platelet stimulation using physiological ligands in vivo. GPIb-binding snake C-type lectins (snaclecs) are useful tools for exploring GPIb in vitro because they act on platelets differently. In the present study, a novel GPIb-binding snaclec, named jerdonibitin, was purified, molecular cloned and characterized from Trimeresurus jerdonii venom. On SDS-polyacrylamide gel electrophoresis, it showed a single band with an apparent molecular weight of 25 kDa under non-reducing conditions and two distinct bands with apparent molecular weights of 15 kDa (α-subunit) and 13 kDa (β-subunit) under reducing conditions. The cDNA sequences of each subunit of jerdonibitin were identified and both deduced amino acid sequences were confirmed by N-terminal protein sequencing and trypsin-digested peptide mass fingerprinting of MALDI-TOF. Sequence alignment showed that jerdonibitin is a snaclec and has sequence similarity with TSV-GPIb-BP (a GPIb-inhibitory snaclec). Jerdonibitin dose-dependently inhibited platelet aggregation induced by ristocetin or low-dose thrombin, but not by high-dose thrombin. The GPIbα was detected by affinity chromatography on jerdonibitin. In vivo, jerdonibitin also dose-dependently induced thrombocytopenia of mice and platelet counts remained at very low level after 18 h intravenous injection. In summary, a novel GPIb-inhibitory snaclec was molecular cloned and characterized, which might provide insights into investigation of how GPIb-inhibitory snaclecs work and development of new antiplatelet agents.