Rapid screening and identification of ACE inhibitors in snake venoms using at-line nanofractionation LC-MS.

This study presents an analytical method for the screening of snake venoms for inhibitors of the angiotensin-converting enzyme (ACE) and a strategy for their rapid identification. The method is based on an at-line nanofractionation approach, which combines liquid chromatography (LC), mass spectrometry (MS), and pharmacology in one platform. After initial LC separation of a crude venom, a post-column flow split is introduced enabling parallel MS identification and high-resolution fractionation onto 384-well plates. The plates are subsequently freeze-dried and used in a fluorescence-based ACE activity assay to determine the ability of the nanofractions to inhibit ACE activity. Once the bioactive wells are identified, the parallel MS data reveals the masses corresponding to the activities found. Narrowing down of possible bioactive candidates is provided by comparison of bioactivity profiles after reversed-phase liquid chromatography (RPLC) and after hydrophilic interaction chromatography (HILIC) of a crude venom. Additional nanoLC-MS/MS analysis is performed on the content of the bioactive nanofractions to determine peptide sequences. The method described was optimized, evaluated, and successfully applied for screening of 30 snake venoms for the presence of ACE inhibitors. As a result, two new bioactive peptides were identified: pELWPRPHVPP in Crotalus viridis viridis venom with IC50 = 1.1 µM and pEWPPWPPRPPIPP in Cerastes cerastes cerastes venom with IC50 = 3.5 µM. The identified peptides possess a high sequence similarity to other bradykinin-potentiating peptides (BPPs), which are known ACE inhibitors found in snake venoms.

Molecular isoforms of cobra venom factor-like proteins in the venom of Austrelaps superbus.

Cobra venom factor (CVF) is characteristic of the elapid cobras and has not been reported from venoms of any other families of snakes. During our search for novel proteins, we isolated a polypeptide from the venom of the snake Austrelaps superbus (Lowland Copperhead) that showed structural similarity to C-terminal segment of the alpha-chain of CVF and hence named as AVFalphac (AVF-A. superbus venom factor). cDNA sequence of AVFalphac and its precursor indicated the presence of two isoforms of CVF-like proteins in A. superbus venom gland. This is the first report of molecular isoforms of CVF-like proteins in the venom of an Australian elapid snake. We have determined the complete cDNA sequence of both the isoforms (AVF-1 and AVF-2). They differ in their potential glycosylation sites and the characteristic thioester bond sequence. They display the overall domain structure of CVF and complement C3 proteins. By real-time quantitative analysis, we show that there is a 140-fold difference in the mRNA expression levels of the two isoforms in the venom gland of A. superbus. We also show the presence of AVF-1 and its variant (not AVF-2) in A. superbus venom by partial purification, dot blots, Western blots and peptide mapping using mass spectrometry. Partially purified proteins activate human Factor B in the presence of Factor D and Mg(2+), and deplete the complement activity in human and guinea pig serum. The bimolecular complex (AVFBb) formed activates complement C3 but not complement C5. Thus, AVF proteins may serve as potential candidates for therapeutic complement depletion without side effects. Thus, the discovery of CVF-like proteins in the venom of this Australian elapid snake provides an alternative source of research tools, and contributes to our understanding of the structure-function relationships and evolution of new members of CVF-like proteins.

Expression and characterization of haemathrins, madanin-like thrombin inhibitors, isolated from the salivary gland of tick Haemaphysalis bispinosa (Acari: Ixodidae).

Saliva of hematophagous animals, such as ticks, is an excellent source of anticoagulant proteins and polypeptides. Here we describe the identification and characterization of two thrombin inhibitors named as haemathrin 1 and 2 from the salivary gland of tick Haemaphysalis bispinosa using genomic approach. Haemathrins are cysteine-less peptide anticoagulants, which share about 65-70% identity with madanins, and belong to inhibitor I53 superfamily of inhibitors of the MEROPS database. Haemathrins were overexpressed in E. coli and characterized to understand its mechanism of anticoagulant activity. Recombinant haemathrins (rHaemathrins) delayed the thrombin time, prothrombin time, activated partial thromboplastin time and fibrinogen clotting time. Selectivity screening against serine proteases of coagulation cascade reveals that rHaemathrins 1 and 2 specifically inhibit thrombin with an IC50 of 46.13±0.04µM and 40.05±0.05µM respectively. Similar to madanin, rHaemathrin 1 and 2 were cleaved by thrombin and consequently lost their inhibitory function over time. Analyses of the cleavage products revealed that the first cleavage, which occurs at the C-terminal end of rHaemathrins, drastically reduced their inhibitory activity. The synthetic peptides corresponding to the cleaved fragments showed significant loss in their ability to prolong plasma clotting times and to inhibit the amidolytic activity of thrombin. Thus haemathrins are the first cleavable thrombin inhibitors characterized from the salivary glands of H. bispinosa.

Molecular evolution caught in action: gene duplication and evolution of molecular isoforms of prothrombin activators in Pseudonaja textilis (brown snake).

BACKGROUND: The evolution of structurally and functionally similar proteins with highly diverse physiological roles within a single organism is of great interest. Australian elapid snakes offer an excellent opportunity to study the molecular evolution of prothrombin activators. Venom from Pseudonaja textilis contains pseutarin C, a group C prothrombin activator. Its enzymatic subunit is structurally and functionally similar to mammalian factor (F) Xa, whereas its non-enzymatic subunit is similar to FVa. As vertebrates, the snakes also contain a system to activate prothrombin in their own blood during injury. These hemostatic factors are produced in the liver. RESULTS: Here we describe the presence of two molecular forms of FX expressed in the liver of P. textilis. Both isoforms have molecular signatures and domain architecture of FX. However, one isoform shows approximately 94% sequence identity with the snake FX from Tropidechis carinatus, whereas the other is much closer (90% identity) to the catalytic subunit of pseutarin C (PCCS). Real-time polymerase chain reaction reveals that the latter isoform is expressed approximately 56 000 times lower in the liver of P. textilis. However, the isoforms are not expressed in the venom gland. CONCLUSION: A detailed analysis of deletions and insertions along with the sequence indicates that the second isoform is an intermediate caught in the evolution of venom prothrombin activator from the blood coagulation FX. Thus, this isoform represents a 'molecular fossil' and reveals the likely evolutionary path of recruitment of FX in the venom gland.

Snake venom glutaminyl cyclase.

Glutaminyl cyclase (QC) catalyzes N-terminal glutamine cyclization of many endocrine peptides and is typically abundant in brain tissue. As three-finger toxins in the venoms of colubrid snakes Boiga dendrophila and Boiga irregularis contain N-terminal pyroglutamate, we searched for QC in venom glands of both snakes. Here we report cDNA sequences of QC from brain and venom gland tissues of Boiga species. We propose that QC expressed in snake venom gland tissue plays a role in the N-terminal pyroglutamate formation of several snake venom toxins, indirectly contributing to venom potency.

Two parallel prothrombin activator systems in Australian rough-scaled snake, Tropidechis carinatus. Structural comparison of venom prothrombin activator with blood coagulation factor X.

It is uncommon for similar pathways/systems to be involved in highly divergent functions within single organisms. Earlier, we have shown that trocarin D, a venom prothrombin activator, from the Australian rough-scaled snake Tropidechis carinatus, is structurally and functionally similar to the blood coagulation factor Xa (FXa). The presence of a haemostatic system in these snakes implies that they have two parallel prothrombin activating systems: one in the plasma, that participates in the life saving process of blood clotting and the other in their venom, where it acts as a toxin. Here, we report the complete cDNA sequence encoding the blood coagulation factor X (FX) from the liver of T. carinatus. Deduced T. carinatus FX sequence shows approximately 80% identity with trocarin D but approximately 50% identity with the mammalian FX. Our present study confirms the presence of two separate genes--one each for FX and trocarin D, that code for similar proteins in T. carinatus snake. These two genes have different expression sites and divergent uses suggesting that snake venom prothrombin activators have probably evolved by the duplication of the liver FX gene and subsequently marked for tissue-specific expression in the venom gland.

The catalytic subunit of pseutarin C, a group C prothrombin activator from the venom of Pseudonaja textilis, is structurally similar to mammalian blood coagulation factor Xa.

Pseutarin C, a group C prothrombin activator from Pseudonaja textilis venom, is a large protein complex consisting of catalytic and nonenzymatic subunits, which are functionally similar to the mammalian FXa-FVa complex. Here, we present the complete cDNA sequence of the catalytic subunit of pseutarin C. The cDNA of the catalytic subunit encodes a protein of 449 amino acids, which includes a 22-residue signal peptide, 18-residue propeptide and a mature protein of 409 amino acids. The deduced amino acid sequence shows 74-83% identity to group D prothrombin activators from snake venom and approximately 42% identity to mammalian FX and has identical domain structure. The precursor of the catalytic subunit of pseutarin C has several unique features. The activation peptide of the catalytic subunit of pseutarin C is significantly smaller (27 as compared to 52 residues in mammalian FX) and does not contain any glycosylation sites. Unlike coagulation FXa, Ser52 and Asn45 of the light and heavy chains are O- and N-glycosylated in pseutarin C catalytic sub-unit. There is a 12-residue insertion in pseutarin C catalytic sub-unit close to the region that is implicated in binding to FVa. This is the first sequence of the catalytic subunit of a group C prothrombin activator.

In vitro neuromuscular activity of 'colubrid' venoms: clinical and evolutionary implications.

In this study, venoms from species in the Colubrinae, Homalopsinae, Natricinae, Pseudoxyrhophiinae and Psammophiinae snake families were assayed for activity in the chick biventer cervicis skeletal nerve muscle preparation. Boiga dendrophila, Boiga cynodon, Boiga dendrophila gemincincta, Boiga drapiezii, Boiga irregularis, Boiga nigriceps and Telescopus dhara venoms (10 microg/ml) displayed postsynaptic neuromuscular activity as evidenced by inhibition of indirect (0.1 Hz, 0.2 ms, supramaximal V) twitches. Neostigmine (5 microM) reversed the inhibition caused by B. cynodon venom (10 microg/ml) while the inhibitory effects of Psammophis mossambicus venom (10 microg/ml) spontaneously reversed, indicating a reversible mode of action for both venoms. Trimorphodon biscutatus (10 microg/ml) displayed irreversible presynaptic neurotoxic activity. Detectable levels of phospholipase A2 activity were found only in T. biscutatus, T. dhara and P. mossambicus venoms. The results demonstrate a hitherto unsuspected diversity of pharmacological actions in all lineages which may have implications ranging from clinical management of envenomings to venom evolution.

Pharmacological characterization of mikatoxin, an alpha-neurotoxin isolated from the venom of the New-Guinean small-eyed snake Micropechis ikaheka.

Symptoms of envenomation by the New-Guinean small-eyed snake Micropechis ikaheka (Elapidae) include peripheral neurotoxicity and myotoxicity. We have now purified to homogeneity a long-chain neurotoxin, mikatoxin, from M. ikaheka venom by successive gel filtration and reverse-phase chromatography. Electrospray ionization mass spectrometry showed mikatoxin to be a homogenous peptide of MW 7775.6. Mikatoxin was devoid of any phospholipase A(2) activity associated with the crude venom and did not exhibit any intrinsic anticholinesterase activity. In the chick biventer cervicis muscle, it produced an irreversible, concentration-dependent block of responses to exogenously applied acetylcholine and carbachol as well as twitches evoked by nerve, but not by direct muscle stimulation. Moreover, mikatoxin, like alpha-bungarotoxin and erabutoxin-b, did not show significant fade response to train-of-four stimulation of the mouse phrenic nerve-hemi diaphragm muscle. It also failed to block ganglionic transmission in the guinea pig ileum and muscarinic responses in the rat anococcygeus muscle. Our study provides strong evidence for the presence of a neurotoxin (mikatoxin) in M. ikaheka venom that produces neuromuscular blockade in skeletal muscle attributable to selective and irreversible antagonism of postsynaptic nicotinic acetylcholine receptors of the neuromuscular junction and likely contributes to the peripheral neurotoxicity observed in M. ikaheka envenomation.

A novel prothrombin activator from the venom of Micropechis ikaheka: isolation and characterization.

A novel prothrombin activator, Mikarin, has been isolated from Micropechis ikaheka venom. It is a single polypeptide chain metalloproteinase with the apparent molecular weight of 47kDa. Mikarin exhibits Ca(2+)-independent prothrombin activation, but no effects on other blood coagulation factors, such as factor X and fibrinogen. Mikarin is the first member of group I prothrombin activators from elapid venom. Like other high-molecular-weight snake venom proteinases, it has three structural domains, metalloproteinase and disintegrin-like and Cys-rich domains, and belongs to the P-III class of snake venom metalloproteinases. The N-terminal of Mikarin exhibits 76% sequence identity with Cobrin, a metalloproteinase identified from Naja naja venom, but very lower identities were found when compared with those from viperid and crotalid venom. In addition, the presence of disintegrin-like and Cys-rich domains in snake venom metalloproteinases with diverse biological activities suggests that these domains may be important for their function.

Interaction of wheat high-mobility-group proteins with four-way-junction DNA and characterization of the structure and expression of HMGA gene.

Plant high-mobility-group (HMG) chromosomal proteins are the most abundant and ubiquitous nonhistone proteins found in the nuclei of higher eukaryotes. There are only two families of HMG proteins, namely, HMGA and HMGB in plants. The cDNA encoding wheat HMGa protein was isolated and characterized. Wheat HMGA cDNA encodes a protein of 189 amino acid residues. At its N terminus, there is a histone H1-like structure, which is a common feature of plant HMGA proteins, followed by four AT-hook motifs. Polymerase chain reaction results show that the gene contains a single intron of 134 bp. All four AT-hook motifs are encoded by the second exon. Northern blot results show that the expression of HMGA gene is much higher in organs undergoing active cell proliferation. Gel retardation analysis show that wheat HMGa, b, c and histone H1 bind to four-way-junction DNA with high binding affinity, but affinity is dramatically reduced with increasing Mg(2+) and Na(+) ion concentration. Competition binding studies show that proteins share overlapping binding sites on four-way-junction DNA. HMGd does not bind to four-way-junction DNA.