Cloning of cDNAs encoding C-type lectins from Elapidae snakes Bungarus fasciatus and Bungarus multicinctus

A number of C-type lectins with various biological activities have been purified and characterized from Viperidae snake venoms. In contrast, only a few reports could be found in literature concerning the C-type lectins in Elapidae snake venoms. Based on the published cDNA sequences of C-type lectins from Viperidae snake venoms, oligonucleotide primers were designed and used to screen the cDNA libraries made from the venom glands of Bungarus fasciatus and Bungarus multicinctus. This allowed the cloning of three full length cDNAs encoding C-type lectins. The encoded proteins, named BFL-1, BFL-2 and BML, exhibit high degrees of sequence identities with Viperidae snake venom saccharide-binding lectins (around 60% with Trimeresurus stejnegeri venom lectin, Crotalus atrox venom lectin and Agkistrodon piscivorus venom lectin). They show much less identities with other venom C-type lectin-like proteins (around 30% with the platelet glycoprotein Ib-binding protein from Agkistrodon blomhoffi venom and the factor IX/X-binding protein from Trimeresurus flavoviridis venom). The cDNAs revealed that the precursors contain potential signal peptides characterized by a hydrophobic core. To our knowledge, these are the first cDNA cloning of group VII C-type lectins (Drickamer K. 1993. Prog. Nucleic Acid Res. Mol. Biol. 45, 207–232) from Elapidae snake venom glands.     

Molecular cloning of serine proteases from elapid snake venoms

Serine proteases are widely distributed in viperid snake venoms, but rare in elapid snake venoms. Previously, we have identified a fibrinogenolytic enzyme termed OhS1 from the venom of Ophiophagus hannah. The results indicated that OhS1 might be a serine protease, but there was no structural evidence previously. In the present study, the primary structure of OhS1 was determined by protein sequencing, in combination with RT-PCR and 5′-RACE methods. OhS1 precursor is composed of an 18-amino acid signal peptide, a 6-amino acid putative activation peptide and 236-amino acid mature protein. OhS1 homologues from Naja atra and Bungarus multicinctus were also cloned and reported. These elapid venom serine proteases exhibited 60% sequence identity with serine proteases from the snake venoms of the Viperidae and Colubridae family. Phylogenetic analysis indicated that snake venom serine protease might have a common ancestor.     

Characterization of the coagulant activity of some snake venoms

The venoms of Agkistrodon contortrix mokeson, Crotalus durissus, C. adamanteus, Trimeresurus flavoviridis and Agkistrodon bilineatus were shown to have a thrombin-like activity. Echis carinatus has a Factor X activator activity and C. viridis helleri has both thrombin-like and Factor X activator components. None of the venoms are able to convert prothrombin to thrombin directly.     

Antihemorrhagin in the blood serum of king cobra (Ophiophagus hannah): purification and characterization

King cobra (Ophiophagus hannah) serum was found to possess antihemorrhagic activity against king cobra hemorrhagin. The activity was stronger than that in commercial king cobra antivenom. An antihemorrhagin has been purified by ion exchange chromatography, affinity chromatography and gel filtration with a 22-fold purification and an overall yield of 12% of the total antihemorrhagic activity contained in crude serum. The purified antihemorrhagin was homogeneous in disc-PAGE and SDS–PAGE. Its apparent molecular weight determined by SDS–PAGE was 120 kDa. The antihemorrhagin was also active against other hemorrhagic snake venoms obtained in Thailand and Japan such as Calloselasma rhodostoma, Trimeresurus albolabris, Trimeresurus macrops and Trimeresurus flavoviridis (Japanese Habu). It inhibited the proteolytic activity of king cobra venom. It is an acid- and thermolabile protein and does not form precipitin lines against king cobra venom.     

Molecular cloning of albolatin, a novel snake venom metalloprotease from green pit viper (Trimeresurus albolabris), and expression of its disintegrin domain

Disintegrins are snake venom-derived, RGD- or KGD-containing peptides that can inhibit integrin-mediated platelet aggregation and cell–matix interactions. The aim of this study is to analyze the full-length cDNA sequence of a snake venom metalloprotease (SVMP) from green pit viper (Trimeresurus albolabris) venom and characterize functions of its disintegrin domain on human platelets. From the primary cDNA library of venom glands, a partial sequence of a novel SVMP (Albolatin) was obtained. Using the 5′-RACE, the 2040 bp full-length sequence of albolatin mRNA was derived. The deduced amino acid sequence revealed a type P-II SVMP of 484 amino acid residues comprising a signal region, pro-peptide, inactive metalloprotease domain and a disintegrin domain. It showed 85% amino acid identical to Trimeresurus jerdonii jerdonitin and 81% to Gloydius halys agkistin. Sequence alignment revealed that all cysteines were conserved except for an extra cysteine in the protease domain of albolatin. The disintegrin domain of albolatin, which comprised 76 amino acids with a KGDW sequence, was expressed in Pichia pastoris with the yield of 3.3 mg/L of culture medium. The molecular weights were 11 kDa in reduced and 22 kDa in non-reduced states indicating a homodimer. It can inhibit collagen-induced platelet aggregation with IC₅₀ of 976 nM and, therefore, should be investigated for a potential to be a novel therapeutic agent.     

Immunological studies on Egyptian polyvalent antivenins

Polyvalent antivenin was prepared against the Egyptian snake venoms Naja haje, N. nigricollis, Cerastes cerastes and C. vipera, and tested by neutralization and immunodiffusion tests. The antivenin was of high potency against the Egyptian snake venoms, especially C. cerastes and C. vipera, followed by Echis carinatus, E. coloratus, N. haje, N. nigricollis and Walterinnesia aegyptia. The titre against the African and Indian cobra venoms N. nivea, N. haje (Ethiopian) and N. naja was lower, while poor or no effect was shown against Bitis arietans, B. gabonica and Trimeresurus flavoviridis venoms. The polyvalent serum prepared in horses not previously immunized, was comparable to sera obtained by using horses, which were previously immunized against a single venom. However, the latter sera, still maintained their higher titre against the original sensitizing venom. Comparison of polyvalent and monovalent N. nigricollis antivenins, revealed that while the polyvalent serum was of better effectiveness against the Egyptian viper venoms especially cerastes venoms, more protection was provided by the monovalent serum against the naja venoms.     

Characterization and cDNA cloning of aminopeptidase A from the venom of Gloydius blomhoffi brevicaudus

The aminopeptidase activities of snake venoms from Gloydius blomhoffi brevicaudus, Gloydius halys blomhoffii, Trimeresurus flavoviridis, Bothrops jararaca and Crotalus atrox were investigated. Aminopeptidase A (APA), aminopeptidase B and aminopeptidase N activities were present in all snake venoms. The strongest APA activity was found in venom from G. blomhoffi brevicaudus. The susceptibility to metallopeptidase inhibitors and the pH optimum of the partially purified enzyme from G. blomhoffi brevicaudus venom were similar to those of known APAs from mammals. A G. blomhoffi brevicaudus venom gland cDNA library was screened to isolate cDNA clones using probes based on highly conserved amino acid sequences in known APAs. Molecular cloning of APA from G. blomhoffi brevicaudus venom predicted that it was a type II integral membrane protein containing 958 amino acid residues with 17 potential N-linked glycosylation sites. It possessed a His-Glu-Xaa-Xaa-His-(Xaa)₁₈-Glu zinc binding motif that allowed the classification of this protein as a member of the M1 family of zinc-metallopeptidases, or gluzincins. The deduced amino acid sequence shows approximately 60% sequence identity to mammalian APA sequences. This is the first study to report the primary structure of APA from a reptile.     

Molecular diversity and accelerated evolution of C-type lectin-like proteins from snake venom.

A number of C-type lectin-like proteins that affect thrombosis and hemostasis by inhibiting or activating specific platelet membrane receptors or blood coagulation factors have been isolated from the venom of various snake species and characterized and more than 80 have been sequenced. Recent data on the primary sequences and 3D structures of C-type lectins and C-type lectin-like proteins from snake venoms have enabled us to analyze their molecular evolution. Statistical analysis of their cDNA sequences shows that C-type lectin-like proteins, with some exceptions, have evolved in an accelerated manner to acquire their diverse functions. Phylogenetic analysis shows that the A and B chains of C-type lectin-like proteins are clearly separated from C-type lectins and that the A and B chains are further divided into a group of platelet receptor-binding proteins and a group of coagulation factor-binding proteins. Elucidation of the tertiary structures of several C-type lectin-like proteins led to the discovery of a unique domain-swapping interaction between heterodimeric subunits, which creates a concave surface for ligand binding.     

Characterization, primary structure and molecular evolution of anticoagulant protein from Agkistrodon actus venom.

An anticoagulant protein named AaACP was isolated from Agkistrodon actus (hundred-pace snake of Taiwan, Viperidae) venom. AaACP inhibited the factor Xa-induced plasma coagulation in a concentration-dependent manner. Thus, AaACP seems to bind to factor Xa in prothrombinase complex. AaACP was composed of A and B chains linked by disulphide bond(s). The amino acid sequences of A and B chains of AaACP were analysed with a few residues unidentified which were complemented from the nucleotide sequences of their cDNAs. The A chain consisted of 129 amino acid residues and the B chain 123 amino acid residues. Their amino acid sequences were highly similar to those of A and B chains of a series of anticoagulant proteins which had been purified from the venoms of some Viperidae snakes. The A and B chains structurally belong to C-type lectin-like protein family of snake venom origin. Construction of phylogenetic tree of C-type lectins and C-type lectin-like proteins based on their amino acid sequences indicated that their A and B chains diverged before speciation of snake species. The comparison of the nucleotide sequences of the cDNAs encoding A and B chains of AaACP and of Trimeresurus flavoviridis (Viperidae) venom-gland factors IX/X-binding protein and factor IX-binding protein showed that the mature protein-coding region is much more variable than the signal peptide-coding domain and the 5'- and 3'-untranslated regions, being in contrast to the case of the ordinary isoprotein genes. The ratios of the numbers of nucleotide substitutions per nonsynonymous site (K(A)) and per synonymous site (K(S)) in the mature protein-coding region in the cDNA pairs were about three times greater than those for the ordinary isoprotein genes, suggesting that these genes have been evolving in an accelerated manner. Taking account of the functional diversities of venom-gland C-type lectins and C-type lectin-like proteins including factors IX and/or X-binding proteins, it can be said that their functional diversities have been acquired by accelerated evolution.     

Mionecrosis, hemorragia y edema inducidos por el veneno de Bothrops asper en ratón blanco

Pathogenesis of the myonecrotic, hemorrhagic and edema-forming effects in white mice inoculated with Bothrops asper venom was studied at different time intervals by means of light microscopy, determination of serum levels of the enzyme creatinephosphokinase (CPK), and evaluation of edema. Although the three effects were evident in the first hour, their development was relatively independent. Myonectoric activity reached its maximum levels in the first 3 hr, as judged by serum CPK. This indicates that myonecrotic toxins have a very rapid action on muscle fibers, after which CPK levels decrease to near normal values 24 hr after venom inoculation. Histological studies showed severe myonecrosis at 3, 6, 9, 12 and 24 hr, characterized by myolytic and coagulative necrotic fibers mixed with normal fibers. Maximum hemorrhagic effect was observed between 9 and 12 hr, whereas at 6, 9, 12 and 24 hr there was an intense polymorphonuclear leucocyte infiltration. On the other hand, 1 hr after venom injection there was a local edema of 44%, reaching a maximum value of 70% within the first 24 hr, decreasing afterward. This edema is more prolonged than that induced by Agkistrodon piscivorous or Trimeresurus elegans venoms. This could have important clinical implication since local swelling contributes to tissue compression and is related to other physiological alterations. One week after venom inoculation there were fibroblasts, fragments of necrotic muscle fibers, and a mononuclear cell exudate. At this time, we did not observe hemorrhagic areas. Determination of serum levels of CPK could be a useful and powerful technique in order to quantify muscle tissue destruction due to snake bite. Since only scanty hemorrhagic and necrotic areas were observed in heart muscle, the drastic and rapid increase in CPK levels mainly reflected skeletal muscle necrosis. CPK determination is a simple and quick laboratory technique that should be used to evaluate muscle necrosis and to improve treatment in snake venom poisoning.     

Primary structure and biological activity of snake venom lectin (APL) from Agkistrodon p. piscivorus (Eastern cottonmouth).

A lectin (APL) was purified from the venom of Agkistrodon piscivorus piscivorus (Eastern cottonmouth moccasin). APL is a disulfide-linked, homodimeric protein consisting of identical monomers of molecular weight 16,200. Native rabbit and human erythrocytes were agglutinated by APL and the activity was found to be calcium-dependent. Galactose, lactose, rhamnose and EGTA strongly inhibited the hemagglutination activity of APL. The complete amino acid sequence determined by Edman sequencing of the S-pyridylethylated derivative and its peptides derived from enzymatic digestion indicate the structure of APL to be highly homologous with lectins and the platelet glycoprotein Ib (GPIb)-binding proteins isolated from other snake venoms. These results suggest that APL belongs to the C-type beta-galactoside binding lectin family which possess structural similarities with the C-terminal carbohydrate-recognition domain (CRD) of animal membrane lectins.     

Characterization and cDNA cloning of halyxin, a heterogeneous three-chain anticoagulant protein from the venom of Agkistrodon halys brevicaudus.

We report upon the isolation, characterization, and cDNA cloning of an anticoagulant protein, halyxin from Agkistrodon halys brevicaudus venom. The protein exists as a 29kDa protein, and is separated into three chains on SDS-PAGE under reducing conditions. However, we cloned only two cDNAs encoding halyxin from the cDNA library of the snake venom gland, on the basis of the determined amino acid sequences. The complete amino acid sequences were deduced from their nucleotide sequences and named halyxin A (129 amino acid residues) and B chain (123 amino acid residues). The deduced amino acid sequence of halyxin A chain corresponds to the two smaller chains. Thus, it is considered that halyxin A chain could be synthesized as a single-chain protein that is subsequently cleaved to yield the mature two-chain protein. The amino acid sequence of halyxin is similar to that of other snake venom proteins of the C-type lectin superfamily, and prolongs plasma-clotting time. In the presence of Ca(2+) ions, halyxin binds to coagulation factors IX, X, IXa, and Xa, but not to other vitamin K-dependent coagulation factors. It also inhibits factor Xa in a non-competitive manner but does not affect other activated coagulation factors.     

Haemostatically active proteins in snake venoms

Snake venom proteins that affect the haemostatic system can cause (a) lowering of blood coagulability, (b) damage to blood vessels, resulting in bleeding, (c) secondary effects of bleeding, e.g. hypovolaemic shock and organ damage, and (d) thrombosis. These proteins may, or may not, be enzymes. We review the data on the most relevant haemostatically active proteinases, phospholipases A₂, l-amino acid oxidases and 5′-nucleotidases from snake venoms. We also survey the non-enzymatic effectors of haemostasis from snake venoms - disintegrins, C-type lectins and three-finger toxins. Medical applications have already been found for some of these snake venom proteins. We describe those that have already been approved as drugs to treat haemostatic disorders or are being used to diagnose such health problems. No clinical applications, however, currently exist for the majority of snake venom proteins acting on haemostasis. We conclude with the most promising potential uses in this respect.     

Snake venom C-type lectins interacting with platelet receptors. Structure–function relationships and effects on haemostasis

Snake venoms contain components that affect the prey either by neurotoxic or haemorrhagic effects. The latter category affect haemostasis either by inhibiting or activating platelets or coagulation factors. They fall into several types based upon structure and mode of action. A major class is the snake C-type lectins or C-type lectin-like family which shows a typical folding like that in classic C-type lectins such as the selectins and mannose-binding proteins. Those in snake venoms are mostly based on a heterodimeric structure with two subunits and β, which are often oligomerized to form larger molecules. Simple heterodimeric members of this family have been shown to inhibit platelet functions by binding to GPIb but others activate platelets via the same receptor. Some that act via GPIb do so by inducing von Willebrand factor to bind to it. Another series of snake C-type lectins activate platelets by binding to GPVI while yet another series uses the integrin ₂β₁ to affect platelet function. The structure of more and more of these C-type lectins have now been, and are being, determined, often together with their ligands, casting light on binding sites and mechanisms. In addition, it is relatively easy to model the structure of the C-type lectins if the primary structure is known. These studies have shown that these proteins are quite a complex group, often with more than one platelet receptor as ligand and although superficially some appear to act as inhibitors, in fact most function by inducing thrombocytopenia by various routes. The relationship between structure and function in this group of venom proteins will be discussed.     

Immunome and venome of Bothrops jararacussu: A proteomic approach to study the molecular immunology of snake toxins

A combination of anti-bothropic and anti-crotalic sera has been reported to be more effective in neutralizing the effects of Bothrops jararacussu venom than anti-bothropic serum alone. The role of proteins from B. jararacussu venom in the horse immune response was evaluated via the analysis of cross-reactivity with homologous and heterologous sera. Many of the proteins in B. jararacussu venom were identified via 2D gel electrophoresis. Western blots revealed that anti-jararacussu showed higher reactivity to l-aminoxidase (LAOs) and snake venom metalloproteinase, (SVMPs) and weaker reactivity towards Snake venom serine proteases (SVSPs), PLA₂, C-type lectin and cysteine-rich proteins. Anti-jararaca preferentially recognized LAOs, SVMPs and SVSPs. Both of these sera failed to recognize low-molecular weight proteins. Anti-crotalic serum clearly recognized LAOs, C-type lectin, SVSP, cysteine-rich proteins, SVMP and Asp49-PLA₂. The cross-reactivity with anti-PLA₂ revealed the immunoreactivity of these antibodies to proteins with molecular masses in a range that is poorly recognized by other studied anti-sera. Our results suggest that the contribution of anti-crotalic serum to the neutralization of B. jararacussu by may be due to its cross-reactivity with proteins such as C-type lectins, SVSPs, Asp49-PLA₂. These results also reinforce the importance of neutralizing the highly toxic proteins inclusive those with low immunogenicity in commercial antivenom production to obtain a highly protective serum against snake venoms.     

Cloning, expression and characterization of two C-type lectins from the venom gland of Bungarus multicinctus.

C-type lectins found in many animals are non-enzymatic proteins and able to bind with mono- and oligosaccharides in a Ca(2+)-dependent fashion. Here, we report the cloning of two C-type lectins named BML-1 and BML-2 from the venom gland of Bungarus multicinctus, and expression of their mature peptides with 135 and 137 amino acids as inclusion bodies. Recombinant BML-1 and BML-2 proteins with 135 amino acids formed monomers, and those with 137 amino acids formed homodimers and monomers and both of them displayed certain hemagglutinating activity to rabbit erythrocytes. The results of Western blotting and immuno-affinity chromatography demonstrated that C-type lectins in B. multicinctus formed dimers in physiological conditions, and their molecular weight is lower than previous predictions. This is the first report of the cloning of the BML-2 gene from the venom gland of B. multicinctus, as well as an investigation of its confirmation and biological functions.     

cDNA cloning, sequence analysis, and recombinant expression of akitonin beta, a C-type lectin-like protein from Agkistrodon acutus

AIM: To clone the cDNA of a new member of snake venom C-type lectin-like proteins, to study its structure-function relationships and to achieve its recombinant production. METHODS: PCR primers were designed based on the homology and cDNA was amplified by RT-PCR using total RNA from snake venom gland as the template.The PCR products were cloned into the plasmid pGEM-T and sequenced. The deduced protein sequence was analyzed with some bioinformatic programs. A recombinant expression plasmid was constructed using pBAD-TOPO as vector and transformed into E.coli TOP10 competent cells. RESULTS: A novel cDNA sequence encoding akitonin β was found and accepted by GenBank (accession number AF387100). Akitonin β consists of a typical carbohydrate recognition domain (CRD) of C-type lectins, and it is homologous with other snake venom C-type lectin-like proteins. It was predicted to be a platelet antagonist. Upon induction with arabinose rAkitonin β expressing in E coli was achieved at a high level (superior to 150 mg/L). The recombinant fusion protein exhibited inhibitory activities on rat platelet aggregation in vitro. CONCLUSION: A new member of snake venom C-type lectin-like proteins was discovered and characterized, and an efficient recombinant expression system was estab-lished for its production.     

The co-purification of a lectin (BJcuL) with phospholipases A2 from Bothrops jararacussu snake venom by immunoaffinity chromatography with antibodies to crotoxin

Antigens of Bothrops jararacussu snake venom cross-reacting with specific antibodies against crotoxin, an Asp49 PLA₂-containing heterodimeric complex from Crotalus durissus terrificus snake venom, were purified by two steps of immunoaffinity chromatography. The resulting fraction (Bj-F) was shown to be non-toxic (to mice and rabbits) and immunogenic to rabbits. Antibodies raised against Bj-F were able to protect mice against the lethal effect of both B. jararacussu and Crotalus durissus terrificus snake venoms. Then, the procedure developed showed to be useful for the rapid preparation of an antigen able to elicit neutralizing antibodies against the lethal activities of both venoms. Further fractionation of Bj-F revealed the concomitant presence of two major components: BJcuL, a lectin present in B. jararacussu venom, and BthTX-I, a Lys49 PLA₂ homolog, besides other molecules in minor amounts. Our data are discussed and raise the point that the presence of unrelated molecules may be taken into account when immuno-based methods are considered for purification purposes.