Jüri Siigur

Biochemical Characterization of Lebetase, a Direct-Acting Fibrinolytic Enzyme from Vipera Lebetina Snake Venom

Lebetase, the fibrinolytic enzyme isolated from Vipera lebetina (Levantine viper) snake venom is a metalloenzyme that contains one mole of Zn2+ and one mole of Ca2+ per mole of protein. Lebetase is inhibited by dithiothreitol, suggesting that disulfide bonds are necessary for holding the structure. Vipera lebetina venom contains several isoforms of lebetase in the interval of pI 4.6-5.4. Two lebetase fractions I (pI of the main component 5.0) and II (pI of the main component 5.3) degrade fibrin and fibrinogen by hydrolysis of the alpha and beta chains. The molecular weights of the cleavage products produced by the two different lebetase fractions are identical. The metal ions, Cd2+, Cu2+, Co2+, inhibit fibrinolytic and caseinolytic activity of lebetase I and II. Using mass spectrometry we characterized differences in molecular masses of lebetase I and II (22719 Da and 22912 Da). Vipera lebetina venom from a single snake contains mainly one form of lebetase. Lebetase I is more stable at low pH than lebetase II. The lebetases I and II inhibit platelet aggregation induced by ADP in a dose-dependent manner.

Factor X activator from Vipera lebetina snake venom, molecular characterization and substrate specificity

Our studies of the venom from the Levantine viper Vipera lebetina have demonstrated the existence of both coagulants and anticoagulants of the hemostatic system in the same venom. We showed that V. lebetina venom contains factor X activator (VLFXA) and factor V activator, fibrinolytic enzymes. VLFXA was separated by gel filtration on Sephadex G-100 superfine and ion exchange chromatography on CM-cellulose and on TSK-DEAE (for HPLC) columns. VLFXA is a glycoprotein composed of a heavy chain (57.5 kDa) and two light chains (17.4 kDa and 14.5 kDa) linked by disulfide bonds. VLFXA has multiple molecular forms distinguished by their isoelectric points. The differences in their pI values may be caused by dissimilarities in the respective charged carbohydrate content or in the primary sequence of amino acids. We synthesized 6-9 amino acid residues containing peptides according to physiological cleavage regions of human factor X and human factor IX. The peptides (Asn-Asn-Leu-Thr-Arg-Ile-Val-Gly-Gly - factor X fragment, and Asn-Asp-Phe-Thr-Arg-Val-Val-Gly-Gly - factor IX fragment) were used as substrates for direct assay of VLFXA. Cleavage products of peptide hydrolysis and the molecular masses of cleavage products of human factor X were determined by MALDI-TOF MS. The MALDI-TOF MS was highly efficient for the recovery and identification of peptides released by VLFXA hydrolysis. We can conclude that 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. VLFXA could not activate prothrombin nor had any effect on fibrinogen, and it had no arginine esterase activity toward benzoylarginine ethyl ester.

L-Amino acid oxidase from Naja naja oxiana venom

A new l-amino acid oxidase (LAAO) was isolated from the Central Asian cobra Naja naja oxiana venom by size exclusion, ion exchange and hydrophobic chromatography. The N-terminal sequence and the internal peptide sequences share high similarity with other snake venom l-amino acid oxidases, especially with those isolated from elapid venoms. The enzyme is stable at low temperatures (-20 degrees C, -70 degrees C) and loses its activity by heating at 70 degrees C. Specific substrates for the isolated protein are l-phenylalanine, l-tryptophan, l-methionine and l-leucine. The enzyme has antibacterial activity inhibiting the growth of Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria. N. naja oxiana LAAO dose-dependently inhibited ADP- or collagen-induced platelet aggregation with IC(50) of 0.094 microM and 0.036 microM, respectively. The antibacterial and anti-aggregating activity was abolished by catalase.

MALDI-TOF mass spectrometry analysis of substrate specificity of lebetase, a direct-acting fibrinolytic metalloproteinase from Vipera lebetina snake venom.

Lebetase is a direct-acting fibrinolytic zinc metalloendopeptidase related in amino acid sequence to reprolysins which include both hemorrhagic and non-hemorrhagic proteinases. Despite apparent structural similarities, fibrinolytic and hemorrhagic proteinases differ significantly in substrate specificity. In this study, we have examined the activity of lebetase I against biologically active peptides (bradykinin, kallidin, substance P) and 6-10 amino acid residues containing peptides synthesized according to cleavage regions of alpha(2)-macroglobulin, pregnancy zone protein (PZP) and fibrinogen. Lebetase was found to have no activity against studied hexapeptides. Surprisingly, the best substrates for lebetase were substance P, and peptide fragment of PZP, both were cleaved at position Pro-Gln. Identification of the hydrolysis products of 15 peptides by MALDI-TOF mass spectrometry analysis indicates that lebetase possesses broad substrate specificity. The MALDI-TOF MS technique was proven to be highly efficient for the recovery and identification of the peptides released by lebetase hydrolysis.

Isolation, properties and N-terminal amino acid sequence of a factor V activator from Vipera lebetina (Levantine viper) snake venom

A factor V activator (VLFVA) was separated from Vipera lebetina venom by gel filtration on Sephadex G-100 superfine, followed by chromatography on CM-cellulose and on heparin-agarose. This enzyme (VLFVA) with a molecular mass of 28.4 kDa, as determined by matrix assisted laser desorption ionization time-of-flight mass spectrometry, is a single-chain glycoprotein containing seven residues of neutral sugars, seven residues of hexosamines and three residues of neuraminic acid per molecule. The treatment with N-glycosidase F lowered the molecular mass approximately 6%. The N-terminal sequencing of VLFVA up to the 30th residue evidenced a high homology with Vipera russelli factor V activator RVV-Vgamma (90% identity). Aside from factor V, no other protein substrate for VLFVA has yet been identified. VLFVA hydrolyzes several synthetic arginine ester substrates, such as benzoylarginine ethyl ester (BAEE), tosylarginine methyl ester (TAME) and amide substrates such as Pro-Phe-Arg-MCA. The arginine ester hydrolase activity of the enzyme is markedly lower than that of the crude venom. The ability of VLFVA to activate factor V and its activity to BAEE and TAME were inhibited by the serine proteinase inhibitor, diisopropylfluorophosphate. VLFVA is thermostable protein, heating for 20 min at 70 degrees C does not alter the arginine esterase activity of the enzyme.

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. α-Fibrinogenase has no homolog among known serine proteases. β-Fibrinogenase is a typical thermostable arginine esterase that hydrolyzes esters and amides of arginine and attacks the β-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 Arg52-Ile53 bond in the heavy chain of human factor X and the Arg226-Val227 bond in human factor IX precursor; VLFVA cleaves Arg1545-Ser1546 in factor V.

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.

Biochemical characterization of fibrinogenolytic serine proteinases from Vipera lebetina snake venom.

Two glycosylated serine fibrinogenases isolated from Vipera lebetina venom have homologous N-terminal sequences and antigenic determinants but can be clearly differentiated according to substrate specificity, glycosylation levels, molecular mass and fibrinogen degradation. alpha-Fibrinogenase has no homolog among known serine proteinases. It has N-terminal similarity with snake venom arginine esterases but does not hydrolyze the esters of arginine, lysine and tyrosine. The enzyme has strong proteolytic activity and degrades alpha-chain of fibrinogen altering its clottability by thrombin. beta-Fibrinogenase is a typical arginine esterase which hydrolyzes esters and amides of arginine and attacks the beta-chain of fibrinogen.

Isolation and characterization of nerve growth factor from Vipera lebetina (snake) venom

Nerve growth factor from Vipera lebetina venom was purified by gel filtration and ion exchange chromatography steps. The NGF preparation obtained is a glycoprotein with weak arginine esterase activity. It hydrolyzes benzoylarginine ethyl ester (BAEE). Vipera lebetina NGF consists of multiple forms of protein with pI in the interval 9-10.5. All isoforms have identical molecular weights of 32,500.

Lebetase, an α(β)-fibrin(ogen)olytic metalloproteinase of Vipera lebetina snake venom, is inhibited by human α-macroglobulins

Abstract The effects of the plasma proteinase inhibitors α2-macroglobulin (α2M) and the α2M-related pregnancy zone protein (PZP) were evaluated towards the metalloproteinase lebetase, isolated from Vipera lebetina venom. We demonstrate that lebetase interacts with both inhibitors. Cleavage of α2M by lebetase resulted in the formation of 90-kDa fragments, and covalent complexes of α2M with lebetase were observed. The proteolytic activity of lebetase against fibrinogen and azocasein could be inhibited by α2M. Cleavage of PZP also resulted in the formation of 90-kDa fragments, and complexes of both dimer and tetramer forms of PZP with lebetase were detected. The amino acid sequence identification of the sites of specific proteolysis of α2M and PZP demonstrate that the cleavage sites are within the bait regions of both proteins. Lebetase I cleaves between Arg696–Leu697, which is one of the most common cleavage sites in α2M by proteinases. The other two cleavage sites in α2M by lebetase are Gly679–Leu680 and His694–Ala695. The cleavage between Pro689–Gln690 is the only cleavage site identified in PZP. In that lebetase is an anticoagulation agent in vivo, we propose that the interaction of lebetase with α2M may suggest a reduced fibrin(ogen)olytic activity of lebetase in human.