Vipera lebetina venom contains all types of snake venom metalloproteases.

Snake venoms contain four classes of metalloproteases that all have a typical zinc-chelating sequence (HEXXHGXXH). N-terminal sequences and internal sequences of different purified metalloproteases were determined using Edman sequencing and LC MS/MS technique. Oligonucleotides were designed and used as primers for cDNA cloning from Vipera lebetina venom gland cDNA library. We found that isoforms of fibrinolytic enzyme lebetase Le-4 and Le-3 are synthesized in different way: Le-4 is synthesized as P-I type metalloprotease, Le-3 is synthesized with disintegrin-like domain as P-II type protease and processed post-translationally. An endothelial cell apoptosis-inducing heterodimeric glycosylated metalloprotease, V. lebetina apoptosis-inducing protease (VLAIP), belongs to P-III type containing metalloprotease, disintegrin-like and cysteine-rich domains. All these enzymes hydrolyze the Aalpha-chain and more slowly the Bbeta-chain of fibrinogen. Treatment of HUVEC cells with VLAIP induces changes in the attachment of cells to the substrate and causes apoptosis. V. lebetina venom contains also P-IV type-specific coagulant factor X activator (VLFXA) that cleaves the Arg52-Ile53 bond in the heavy chain of human factor X. VLFXA is a glycoprotein composed of a heavy chain and two C-type lectin-like light chains linked by disulfide bonds. The heavy and light chains of VLFXA are synthesized from different genes.

Sequence diversity of Vipera lebetina snake venom gland serine proteinase homologs--result of alternative-splicing or genome alteration.

Four clones encoding homologous protein(ase)s were isolated from the Vipera lebetina (snake) venom gland cDNA library. One of them represented DNA encoding factor V activating enzyme (Siigur et al., 1999), the other is homologous to VLFVA but has two principal discrepancies in the translated protein sequence in comparison with snake venom serine proteinase structures: in the active site triad Ser195 is replaced by Asn195 and His57 by Arg57. The third and the fourth clone represent combinations of the first two clones. The possibilities of generation of such clones via trans-splicing of the primary gene transcript, by exon shuffling or by unequal crossing-over on the genome level are discussed.

Proteases from Vipera lebetina venom affecting coagulation and fibrinolysis.

Our studies of the venom from the Levantine viper Vipera lebetina have demonstrated the existence of both coagulants and anticoagulants in the same venom. We showed that V. lebetina venom contains: (1) proteases that degrade fibrinogen, but not fibrin; (2) fibrinolytic enzyme (lebetase); (3) factor X activator (VLFXA); (4) factor V activator (VLFVA). Fibrinolytic enzyme and VLFXA are metalloproteases; the other studied enzymes are serine proteases. alpha-Fibrinogenase has no homolog among known serine proteases. Beta-fibrinogenase is a typical thermostable arginine esterase that hydrolyzes esters and amides of arginine and attacks the beta-chain of fibrinogen. Lebetase is a direct-acting fibrinolytic zinc metalloendopeptidase related in amino acid sequence to reprolysins. We used the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technique for the recovery and identification of peptides released by protease hydrolysis and for the detection of human factor X cleavage products after VLFXA hydrolysis. VLFXA cleaves the Arg(52)-Ile(53 )bond in the heavy chain of human factor X and the Arg(226)-Val(227) bond in human factor IX precursor; VLFVA cleaves Arg(1545)-Ser(1546) in factor V.

Molecular cloning and sequence analysis of a cDNA for factor V activating enzyme.

The complete amino acid sequence of a factor V activator (VLFVA) is deduced from the nucleotide sequence of a cDNA encoding the enzyme. The cDNA was isolated by PCR screening a venomous gland cDNA library of Central Asian Vipera lebetina snake. The full-length cDNA clone, derived from two overlapping fragments, comprises 1563 basepairs which encode an open reading frame of 259 amino acids. The amino acid sequence of VLFVA (235 amino acids) shows significant homology with snake venom and mammalian serine proteinases. It contains 12 half-cysteines which form, by analogy with other serine proteinases, 6 disulfide bridges. VLFVA has the catalytic triad His43-Asp88-Ser182. The amino terminal amino acid valine is preceded by 24 amino acids: a putative signal peptide of 18, mainly hydrophobic, amino acids and an activating peptide of 6, mainly hydrophilic amino acid residues. This is the first cloned factor V activating enzyme from snake venom.

cDNA cloning and deduced amino acid sequence of fibrinolytic enzyme (lebetase) from Vipera lebetina snake venom.

The complete amino acid sequence of lebetase is deduced from the nucleotide sequence of a cDNA clone isolated by screening a venomous gland c DNA library of Central Asian Vipera lebetina snake. The cDNA sequence with 2011 basepairs encodes an open reading frame of 478 amino acids which includes an 18 amino acid signal peptide, plus an 175 amino acid segment of zymogen-like propeptide, a mature protein of 204 amino acids, a spacer of 18 amino acids and a disintegrin-like peptide of 63 amino acids. The mature protein lebetase as isolated from the crude venom has the molecular weight of approximately 23.7 kD and, thus, lebetase as well as several other snake venom metalloproteinases is translated as a precursor protein, which may be processed posttranslationally. The lebetase proprotein has a "cysteine switch" motif (PKMCGV) similar to that involved in the activation of matrix metalloproteinase zymogens. The mature protein (residues 223-427) shows the strongest similarity with fibrolase (63% identity), fibrinolytic enzyme from Agkistrodon contortrix contortrix venom. The metalloproteinase domain has a typical zinc-chelating sequence (HEXXHXXGXXH). In the disintegrin-like domain of protein, the RGD sequence is replaced by VGD.

The putative replicase of the cocksfoot mottle sobemovirus is translated as a part of the polyprotein by -1 ribosomal frameshift.

The polyprotein of cocksfoot mottle sobemovirus (CfMV) is encoded by two overlapping open reading frames (ORF). The ORF 2a codes for the putative VPg and serine protease and the ORF 2b codes for the putative replicase. The consensus signals for a -1 ribosomal frameshifting event are found at the very beginning of the overlapping region of these ORFs. The shifty heptanucleotide in CfMV is UUUAAAC, and the secondary structure after the shifty sequence is predicted to be a stem-loop. In vitro translation of the CfMV RNA in wheat germ extract produced proteins of several sizes, including one of 100 kDa. According to the nucleotide sequence data, no single ORF is capable of directing the synthesis of a 100-kDa protein. A chimeric beta-glucuronidase-CfMV cDNA containing the entire ORF 2a and 2b overlap region including frameshift signals was constructed. A trans-frame protein of 108 kDa was produced from this construct with an efficiency of 26-29% by in vitro translation in wheat germ extract. CfMV is the first sobemovirus in which the putative replicase is reported to be produced as a part of a polyprotein by a -1 frameshift event. The replicases of the sobemoviruses are related to the luteovirus subgroup II replicases, which are known to be produced by -1 ribosomal frameshift. The reported amino acid sequences of the putative replicases of sobemo- and subgroup II luteoviruses were compared to that of the putative replicase of CfMV. This comparison revealed more extensive homology between these groups than previously reported.

Impairment of intracellular antiviral defense with age: age-dependent changes in expression of interferon-induced and double-stranded RNA-activated 2-5A synthetase in rat.

The 2',5'-oligoadenylate (2-5A) system is involved in the defense of mammalian cells against virus infection. In a previous study [25], we demonstrated that the activities of the enzymes which synthesize and degrade 2-5A [2-5A synthetase (2-5OAS) and 2',3'-exoribonuclease] and of the enzyme that is activated by 2-5A (ribonuclease L) change during aging and development in different tissues of rat. The age-dependent decrease in 2-5OAS activity and increase in 2-5A nuclease activity results in a decrease in the cellular 2-5A content, suggesting that the efficiency of the antiviral 2-5A system is impaired in aged rats. Here we determined the age-dependent changes in the level of mRNA coding for the class I isoenzyme of 2-5OAS (M(r) 40-46 kDa) in rat liver and brain using a cDNA which was recently cloned from rat hippocampus. We found that the decrease in 2-5OAS activity is accompanied by a decrease in the level of 2-5OAS mRNA; in old animals (32-33 months old), the amount of 2-5OAS mRNA was reduced to 20-30% compared to young adult (2-3 months old) (100%) and middle-aged adult animals (12 months old) (110-120%). In addition, Western-blotting experiments revealed that the amount of class I 2-5OAS capable of binding to its activator, poly(I).poly(C), is also diminished in the livers and brains of old rats compared to those of young adult and middle-aged adult animals.

Rapid reduction of mRNA coding for 2'-5'-oligoadenylate synthetase in rat pheochromocytoma PC12 cells during apoptosis.

Apoptosis is a form of physiological cell death, characterized by DNA fragmentation, which often depends on RNA and protein synthesis. Because cellular RNA is also degraded during apoptosis we studied the role of the 2'-5'-oligoadenylate (2-5A) synthetase in this process. The product of the synthetase, 2-5A, stimulates endoribonuclease-L-mediated controlled RNA degradation. Here we show that apoptosis is induced in rat phenochromocytoma PC12 cells by tributyltin (TBT) at low concentrations (1 nM); already 5-10 min. after addition of this compound DNA fragmentation resulting in a stepladder-like gel pattern was observed. The level of mRNA coding for 2-5A synthetase was determined using a cloned cDNA from rats. Sequence analyses of the rat 2-5A synthetase (M(r) 40-46,000) revealed high homology to other members of class I synthetase cloned from mouse and human. Applying the rat cDNA as a probe we found that parallel with degradation of DNA the level of mRNA coding for 2-5A synthetase decreased already 7.5 min. after induction of apoptosis by TBT the amount of 2-5A synthetase mRNA was reduced by 60%. This finding indicates that this enzyme is among those mRNAs which are degraded during apoptosis and it suggests that 2-5A synthetase, which is involved in the antiviral response of cells and most likely in the control of cell growth and differentiation, does not play an active role during this process.

Principles and background for the construction of transgenic plants displaying multiple virus resistance.

We investigated the possibility of reconstructing the 2'-5' oligoadenylate (2-5A) pathway into the plant kingdom to achieve multiple virus resistance. Differently phosphorylated 2-5A trimers and tetramers inhibited TMV RNA translation in cell-free systems. In wheat germ extracts the most potent inhibitors were nonphosphorylated forms of 2-5A. Triphosphorylated forms of 2-5A were deposphorylated and hydrolysed in plant extracts. Since we could not detect homologous DNA to mammalian 2-5A synthetase cDNA in tobacco or potato, we cloned rat 2-5A synthetase cDNA and transformed it by the Agrobacterium-mediated mechanism into tobacco and potato. Transformed tobacco plants were resistant to PVS infection and propagation of PVX was reduced. In transgenic potatoes tolerance to PVX and, in one transgenic clone, also to PVY was observed.

Homologies between different forms of 2-5A synthetases.

Sequence analyses of 2-5A synthetases of class I (M(r) 40,000-46,000) revealed high homology among them. The cDNA coding for the M(r) 69,000 2-5A synthetase of class II displayed in the second half a likewise high homology to the complete sequences of class I enzymes. This high degree of conservation of the 2-5A synthetases supports the assumption that these enzymes play important roles during virus infection (Williams et al. 1979; Coccia et al. 1990) and in the control of growth and differentiation of mammalian cells (Williams and Silverman 1985).

Transgenic potato plants expressing mammalian 2'-5' oligoadenylate synthetase are protected from potato virus X infection under field conditions.

We cloned and sequenced a rat cDNA encoding the 2'-5' oligoadenylate synthetase, a component of the mammalian interferon-induced antiviral response, and used Agrobacterium-mediated transformation to generate transgenic potato clones expressing this mammalian enzyme. In transgenic plants infected with potato virus X and followed under field conditions, virus concentrations in leaves and in tubers were significantly lower than in nontransgenic controls. Additionally, virus concentration in the leaves of five transgenic clones and in tubers of one clone was also lower than in transgenic potatoes expressing potato virus X coat protein.