Isolation of a proteinase with plasminogen-activating activity from Lachesis muta muta (bushmaster) snake venom

A plasminogen activator enzyme (LV-PA) from Lachesis muta muta venom was purified to homogeneity using gel filtration and anion exchange chromatography. SDS-PAGE under reducing conditions showed a single protein band with an Mr of 33,000 Da. It is an acidic glycoprotein which activates plasminogen to plasmin indirectly, functioning via prior formation of a molecular complex, known as plasminogen activator. The purified preparation catalyzes the hydrolysis of several p-nitroanilide peptide substrates containing Lys at the scissile bond. In contrast, no hydrolysis was detected on the synthetic substrates TAME and BAPNA, which contain arginine. By the use of the plasmin-specific chromogenic substrate Tos-Gly-Pro-Lys-pNA, the preparation had a plasmin-like activity of 0.68 U/mg, which was 35.8-fold higher than that of the crude venom from which it was prepared. In vitro, fibrin hydrolysis using LV-PA as plasminogen activator displayed more similarity with the effect produced by streptokinase (SK). SDS-PAGE (10%) analysis showed a 115-kDa complex formation after incubation of plasminogen with either LV-PA or SK. At a molar ratio of 50:1 (fibrinogen:enzyme), the preparation exhibited weakly fibrinogenolytic activity. However, LV-PA is distinguished from thrombin in that it does not clot fibrinogen. After incubation of LV-PA with platelet-rich plasma, the enzyme (2 microM) showed no effect on platelet aggregation induced by ADP, epinephrine, or collagen. Comparison of the N-terminal sequence of LV-PA with other snake venom plasminogen activators revealed that LV-PA exhibits a high degree of sequence identity with the TsVPA from Trimeresurus stejnegeri (90%) and with the Haly-PA from Agkistrodon halys (85%). LV-PA also has homology with other snake venom serine proteinases such as the thrombin-like/gyroxin analogue (38%) from bushmaster venom and with other coagulation serine proteases. The proteinase was readily inhibited by treatment with p-nitrophenyl p-guanidinebenzoate, p-aminobenzamidine, and phenylmethanesulfonyl fluoride but was not affected by metal chelators.     

Kallikrein-like proteinase from bushmaster snake venom

A kallikrein-like proteinase of Lachesis muta muta (bushmaster) venom, designated LV-Ka, was purified by gel filtration and anion exchange chromatographies. Physicochemical studies indicated that the purified enzyme is a 33 kDa monomeric glycoprotein, the Mr of which fell to 28 kDa after deglycosylation with PNGase F. Approximately 77% of the protein sequence was determined by sequencing the various fragments derived from digestions with endoproteases. The partial sequence obtained suggests that LV-Ka is of a similar size to other serine proteinases (i.e., approximately 234 amino acid residues). Sequence studies on the NH2-terminal region of the protein indicate that LV-Ka shares a high degree of sequence homology with the kallikrein-like enzymes EI and EII from Crotalus atrox, with crotalase from Crotalus adamanteus and significant homology with other serine proteinases from snake venoms and vertebrate serum enzymes. LV-Ka showed kallikrein-like activity, releasing bradikinin from kininogen as evidenced by guinea pig bioassay. In addition, intravenous injection of the proteinase (0.8 microg/g) was shown to lower blood pressure in experimental rats. In vitro, the isolated proteinase was shown to have neither fibrin(ogeno)lytic activity nor coagulant effect. LV-Ka was active upon the kallikrein substrates S-2266 and S-2302 (specific activity=13.0 and 31.5 U/mg, respectively; crude venom=0.25 and 6.0 U/mg) but had no proteolytic effect on dimethylcasein and insulin B chain. Its enzymatic activity was inhibited by NPGB and PMSF, indicating that the enzyme is a serine proteinase. Interestingly, one of the other reactions catalyzed by plasma kallikrein, the activation of plasminogen was one of the activities exhibited by LV-Ka.     

Nucleotide and protein sequences of a proteinase inhibitor from the vitelline envelope of dace (Tribolodon hakonensis) eggs

Our experimental purpose is to probe the structure(s) of the chorionic proteinase inhibitor and its cDNA sequence(s) and to develop the application of safe medicines for protection of human and other animal bodies from pathogenic microbe attacks. In this study, chorionic proteinase inhibitor protein was isolated, sequenced and used to base the design of PCR primers, which were then used to amplify DNA using RT-PCR. A cDNA clone of the protein which inhibited the activities of serine proteinases and thermolysin was obtained on the basis of mRNA extracted from ovarian tissue of dace, Tribolodon hakonensis, and the deduced amino acid sequence was determined. Chorionic proteinase inhibitor (TribSPI) peptides of about 9.0 kDa (TribSPI) and 14 kDa (TribSPI-S) were purified from vitelline envelope extracts by thermolysin-immobilized affinity-chromatography. The cloned TribSPI cDNA was 1806 bp in length, and the open reading flame (ORF) was 915 bp encoding a protein of 305 amino acid residues. The inhibitor protein had a molecular mass of 33,550 daltons and was composed of five similar domains. Each domain contained eight cysteine residues, and it's deduced amino acid sequence was only 33 approximately 34% identical to those of human and porcine antileukoproteinases (hALP and pALP, respectively). A possible binding-site for serine proteinases, Arg-Ile, was contained in three domains.     

The contribution of residues 192 and 193 to the specificity of snake venom serine proteinases

Snake venom serine proteinases, which belong to the subfamily of trypsin-like serine proteinases, exhibit a high degree of sequence identity (60-66%). Their stringent macromolecular substrate specificity contrasts with that of the less specific enzyme trypsin. One of them, the plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA), which shares 63% sequence identity with batroxobin, a fibrinogen clotting enzyme from Bothrops atrox venom, specifically activates plasminogen to plasmin like tissue-type plasminogen activator (t-PA), even though it exhibits only 23% sequence identity with t-PA. This study shows that TSV-PA, t-PA, and batroxobin are quite different in their specificity toward small chromogenic substrates, TSV-PA being less selective than t-PA, and batroxobin not being efficient at all. The specificity of TSV-PA, with respect to t-PA and batroxobin, was investigated further by site-directed mutagenesis in the 189-195 segment, which forms the basement of the S(1) pocket of TSV-PA and presents a His at position 192 and a unique Phe at position 193. This study demonstrates that Phe(193) plays a more significant role than His(192) in determining substrate specificity and inhibition resistance. Interestingly, the TSV-PA variant F193G possesses a 8-9-fold increased activity for plasminogen and becomes sensitive to bovine pancreatic trypsin inhibitor.     

Purification, Properties, and N-Terminal Amino Acid Sequence of a Kallikrein-like Enzyme from the Venom of Lachesis muta rhombeata (Bushmaster)

Pit viper venoms contain multiple proteinases which cause considerable damage in tissues and systemic effects after envenomation. A proteinase, kallikrein-like enzyme, belonging to the serine group must play a very important role on systemic effects. The corresponding enzyme from Lachesis muta rhombeata venom was purified to homogeneity by a combination of isoelectrofocusing fractionation followed by one step of gel filtration HPLC. The enzyme focused with pI 5.0–6.5, it had a molecular mass of 32 kDa by gel filtration HPLC, had edematogenic activity, and induced a hypotensic effect in anesthetized rats. It exhibited strong N-α-tosyl-L-Arg methyl esterase (955.38 units/mg) and N-BZ-DL-Arg-pNA amidolytic (233.02 units/mg) activities, hydrolyzed tripeptide nitroanilide derivatives weakly or not at all, and cleaved selectively the A-α and B-β chains of fibrinogen, apparently leaving the Y-chain unaffected. The 30 N-terminal amino acid sequence of the L. m. rhombeata protein showed greatest identity (74% in 26 amino acids) with Crotalus viridis kallikrein-like protein, but significant similarities in sequence were observed with enzymes from other snake venoms and pig pancreatic kallikrein.     

Molecular cloning and expression of a functional snake venom serine proteinase,with platelet aggregating activity,from the Cerastes cerastes viper

The venoms of Viperidae snakes contain numerous serine proteinases that have been recognized to possess one or more of the essential activities of thrombin on fibrinogen and platelets. Among them, a platelet proaggregant protein, cerastocytin, has been isolated from the venom of the Tunisian viper Cerastes cerastes. Using the RACE-PCR technique, we isolated and identified the complete nucleotide sequence of a cDNA serine proteinase precursor. The recombinant protein was designated rCC-PPP (for C. cerastes platelet proaggregant protein), since its deduced amino acid sequence is more than 96% identical to the partial polypeptide sequences that have been determined for natural cerastocytin. The structure of the rCC-PPP cDNA is similar to that of snake venom serine proteinases. The expression of rCC-PPP in Escherichia coli system allowed, for the first time, the preparation and purification of an active protein from snake venom with platelet proaggregant and fibrinogenolytic activities. Purified rCC-PPP efficiently activates blood platelets at nanomolar (8 nM) concentrations, as do natural cerastocytin (5 nM) and thrombin (1 nM). It is able to clot purified fibrinogen and to hydrolyze alpha-chains. Thus, rCC-PPP could be therefore considered a cerastocytin isoform. By comparison with other snake venom serine proteinases, a Gly replaces the conserved Cys(42). This implies that rCC-PPP lacks the conserved Cys(42)-Cys(58) disulfide bridge. A structural analysis performed by molecular modeling indicated that the segment of residues Tyr(67)-Arg(80) of rCC-PPP corresponds to anion-binding exosite 1 of thrombin that is involved in its capacity to induce platelet aggregation. Furthermore, the surface of the rCC-PPP molecule is characterized by a hydrophobic pocket, comprising the 90 loop (Phe(90)-Val(99)), Tyr(172), and Trp(215) residues, which might be involved in the fibrinogen clotting activity of rCC-PPP.     

Analysis of phospholipase A2, l-amino acid oxidase, and proteinase enzymatic activities of the Lachesis muta rhombeata venom

The study of venom components is an important step toward understanding the mechanism of action of such venoms and is indispensable for the development of new therapies. This work aimed to investigate the venom of Lachesis muta rhombeata and evaluate enzymes related to its toxicity. Phospholipase A2 (PLA₂), l ‐amino acid oxidase (LAAO), and proteinase activities were measured, and the molecular weights were estimated. We found the venom to contain one PLA₂ (17 kDa), one LAAO (132 kDa), and three serine proteinases (40, 31, and 20 kDa). Although only serine proteinases were observed in the zymogram, metalloproteinases were found to contribute more to the total proteolytic activity than did serine proteinases. The work confirmed the presence of highly active enzymes; and, moreover, we proposed a novel method for confirming the presence of LAAOs by zymography. We also suggested a simple step to increase the sensitivity of proteinase assays. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:308–314, 2012; View this article online at wileyonlinelibrary.com. DOI 10:1002/jbt.21422     

Analysis of venom constituents from the parasitoid wasp Pimpla hypochondriaca and cloning of a cDNA encoding a venom protein

Venom from Pimpla hypochondriaca, an endoparasitoid of pupae, was size-fractionated using gel filtration chromatography and analysed by SDS-PAGE in the presence and absence of reducing agent. A complex mixture of more than 20 venom constituents was identified which ranged in M(r) between approximately 5 and 100 kDa. Venom from a wide range of size fractions inhibited the motility of larval haemocytes and prevented the formation of cell aggregates when analysed in vitro, indicating that anti-haemocytic activity is mediated by multiple venom components. Sephadex A25 beads injected into the haemocoel of pupae were encapsulated within 24h. This reaction was abolished when the pupae were injected with 30 microg of venom protein, equivalent to one-fifth of a venom sac, 1h prior to implantation of the beads, confirming that venom suppresses encapsulation in pupae. Using random 5' end sequencing of a P. hypochondriaca venom gland cDNA library, we have isolated a cDNA encoding a 25.3 kDa protein containing a signal peptide and having sequence similarity to serine proteases. The N-terminal sequence of six residues from two venom proteins of 28 and 30 kDa was the same and identical to amino acids encoded by the cDNA, confirming that two mass forms of the protein are secreted into the venom sac. The N-terminal sequence of both venom proteins began nine residues towards the C terminus following the predicted signal sequence cleavage site, suggesting that the proteins are proteolytically processed before or during storage in the venom sac. The general applicability of using random 5' sequencing to identify cDNAs encoding secretory products is discussed.     

Cloning and molecular characterization of a cubilin-related serine proteinase from the hard tick Haemaphysalis longicornis

Serine proteinases are one of the largest proteolytic families of enzymes, and have diverse cellular activities in mammalian tissues. We report here the cloning and molecular characterization of a cDNA encoding the serine proteinase of the hard tick Haemaphysalis longicornis (HlSP). The HlSP cDNA is 1570 bp long and the deduced precursor protein consists of 464 amino acids with a predicted molecular mass of 50.4 kDa and a pI of 8.2. The preprotein, consisting of 443 amino acids, was predicted to include a complement C1r/C1s, Uegf, and bone morphogenic protein-1 domain, a low-density lipoprotein receptor class A domain, and a catalytic domain. HlSP sequence analysis showed high similarity to serine proteinases reported from arthropods and vertebrate animal species. Two-dimensional immunoblot analysis revealed endogenous HlSP in adult tick extracts at 50 kDa. Endogenous HlSP was also expressed in all lifecycle stages of H. longicornis. Immunohistochemical studies detected the endogenous enzyme in the midgut epithelial cells of an adult tick. The Escherichia coli-expressed recombinant HlSP was demonstrated to degrade bovine serum albumin and hydrolyze the substrate Bz-L-Arg-pNA at the rate of 30.2 micromol/min/mg protein. Further, HlSP expression was up-regulated during a blood-feeding process, indicating its involvement in the digestion of host blood components.     

Molecular cloning and analysis of cDNA sequences encoding serine proteinase and Kunitz type inhibitor in venom gland of Vipera nikolskii viper

Serine proteinases and Kunitz type inhibitors are widely represented in venoms of snakes from different genera. During the study of the venoms from snakes inhabiting Russia we have cloned cDNAs encoding new proteins belonging to these protein families. Thus, a new serine proteinase called nikobin was identified in the venom gland of Vipera nikolskii viper. By amino acid sequence deduced from the cDNA sequence, nikobin differs from serine proteinases identified in other snake species. Nikobin amino acid sequence contains 15 unique substitutions. This is the first serine proteinase of viper from Vipera genus for which a complete amino acid sequence established. The cDNA encoding Kunitz type inhibitor was also cloned. The deduced amino acid sequence of inhibitor is homologous to those of other proteins from that snakes of Vipera genus. However there are several unusual amino acid substitutions that might result in the change of biological activity of inhibitor.     

Molecular cloning and characterization of a midgut chymotrypsin-like enzyme from the lesser grain borer, Rhyzopertha dominica

A cDNA encoding a chymotrypsinogen-like protein in midguts of the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) was cloned and sequenced. The 901 bp cDNA contains an 816-nucleotide open reading frame encoding 272-amino acids. The predicted molecular mass and pI of the mature enzyme are 23.7 kDa and 4.64, respectively. The encoded protein includes amino acid sequence motifs that are conserved with 5 homologous chymotrypsinogen proteins from other insects. Features of the putative chymotrypsin-like protein from R. dominica include the serine proteinase active site (His(90), Asp(133), Ser(226)), conserved cysteine residues for disulfide bridges, the residues (Gly(220), Gly(243), Asp(252)) that determine chymotrypsin specificity, and both zymogen activation and signal peptides. A TPCK-sensitive caseinolytic protein (P6) with an estimated molecular mass of 24 kDa is present in midgut extracts of R. dominica and can be resolved by electrophoresis on 4-16% polyacrylamide gels. The molecular mass of this caseinolytic enzyme is similar to that of the chymotrypsin deduced from cDNA. Midgut extracts of R. dominica readily hydrolyzed azocasein and N-succinyl-alanine-alanine-proline-phenylalanine-p- nitroanilide (SAAPFpNA), a chymotrypsin-specific substrate. Properties of the enzymes responsible for these activities were partially characterized with respect to distribution in the gut, optimum pH, and sensitivity toward selected proteinase inhibitors. Optimal activity against both azocasein and SAAPFpNA occurs in a broad pH range from about 7 to 10. Both azocasein and SAAPFpNA activities, located primarily in the anterior midgut region, are inhibited by aprotinin, phenylmethyl sulphonylfluoride (PMSF), and soybean trypsin inhibitor (STI). TPCK (N-alpha-tosyl-L-phenylalanine chloromethyl ketone) and chymostatin inhibited more than 60% of SAAPFpNA but only about 10-20% of azocasein activity. These results provide additional evidence for the presence of serine proteinases, including chymotrypsin, in midguts of R. dominica. Arch. Insect Biochem. Physiol. 43:173-184, 2000.Published 2000 Wiley-Liss, Inc.     

S1 subsite in snake venom thrombin-like enzymes: can S1 subsite lipophilicity be used to sort binding affinities of trypsin-like enzymes to small-molecule inhibitors?

Thrombin-like enzymes isolated from snake venoms comprise a group of serine proteinases responsible for many important coagulation disorders in the envenomed victims. Besides, these proteinases have great biotechnological interest as antithrombotic agents and as diagnostic tools. However, in spite of the recent overflow of snake venom thrombin-like enzymes (SVTLEs) on protein sequence databases, there is a lack of three-dimensional (3D) structural information on this family. Without such 3D structures available many aspects of the biological function and biochemical properties of these enzymes still remain obscure. Therefore, we have gone through a series of computational techniques, which enabled us to identify the set of residues involved in molecular recognition of inhibitors bound to the S1 subsite of snake venom thrombin-like enzymes (SVTLEs) and ultimately conclude that nonpolar (van der Waals) intermolecular interactions and ligand's hydrophobicity are the most important factors affecting binding affinities to the S1 subsite of a SVTLE isolated from the venom of Lachesis muta muta (Lmm-TLE). Consequently, we have proposed that S1 subsite lipophilicity may be used to sort binding affinities of trypsin-like enzymes to small molecules by showing that the inhibitory potency of several S1-directed compounds follows subsite lipophilicity among Lmm-TLE and other three homologous proteases. Noteworthy, in the course of our analyses we determined that thrombin's S1 subsite should, in fact, be considered less lipophilic than that of trypsin if we account for the presence of the sodium-controlled water channel communicating with the S1 subsite in the coagulant enzyme.     

BJ46a, a snake venom metalloproteinase inhibitor. Isolation, characterization, cloning and insights into its mechanism of action.

Fractionation of the serum of the venomous snake Bothrops jararaca with (NH4)2SO4, followed by phenyl-Sepharose and C4-reversed phase chromatographies, resulted in the isolation of the anti-hemorrhagic factor BJ46a. BJ46a is a potent inhibitor of the SVMPs atrolysin C (class P-I) and jararhagin (P-III) proteolytic activities and B. jararaca venom hemorrhagic activity. The single-chain, acidic (pI 4.55) glycoprotein has a molecular mass of 46 101 atomic mass units determined by MALDI-TOF MS and 79 kDa by gel filtration and dynamic laser light scattering, suggesting a homodimeric structure. mRNA was isolated from the liver of one specimen and transcribed into cDNA. The cDNA pool was amplified by PCR, cloned into a specific vector and used to transform competent cells. Clones containing the complete coding sequence for BJ46a were isolated. The deduced protein sequence was in complete agreement with peptide sequences obtained by Edman degradation. BJ46a is a 322-amino-acid protein containing four putative N-glycosylation sites. It is homologous to the proteinase inhibitor HSF (member of the fetuin family, cystatin superfamily) isolated from the serum of the snake Trimeresurus flavoviridis, having 85% sequence identity. This is the first report of a complete cDNA sequence for an endogenous inhibitor of snake venom metalloproteinases (SVMPs). The sequence reveals that the only proteolytic processing required to obtain the mature protein is the cleavage of the signal peptide. Gel filtration analyses of the inhibitory complexes indicate that inhibition occurs by formation of a noncovalent complex between BJ46a and the proteinases at their metalloproteinase domains. Furthermore, the data shows that the stoichiometry involved in this interaction is of one inhibitor monomer to two enzyme molecules, suggesting an interesting mechanism of metalloproteinase inhibition.     

Characterization of primary amino acid sequence of human complement control protein factor I from an analysis of cDNA clones.

A cDNA clone of the mRNA coding for the human complement system control protein Factor I has been isolated. The predicted amino acid sequence obtained from the DNA sequence demonstrates a protein consisting of a heavy chain (Mr 35,400) linked to a light chain (Mr 27,600), both of which contain three sites for N-linked glycosylation. The light chain has clear homology with other serine proteinases, most notably in the region of the catalytically active and structurally important amino acids and shares some of the features characteristic of the plasminogen activators. The heavy chain has a clear 'mosaic' nature typical of the plasma serine proteinases; in particular it contains class A and class B LDL (low-density lipoprotein) receptor repeats with conserved cysteine residues similar to those found in other complement proteins.     

A new tyrosine-specific chymotrypsin-like and angiotensin-degrading serine proteinase from Vipera lebetina snake venom

Vipera lebetina venom contains different metallo- and serine proteinases that affect coagulation and fibrin(ogen)olysis. A novel serine proteinase from V. Lebetina venom having ChymoTrypsin Like Proteolytic activity (VLCTLP) was purified to homogeneity from the venom using Sephadex G-100sf, DEAE-cellulose, heparin-agarose and FPLC on Superdex 75 chromatographies. VLCTLP is a glycosylated serine proteinase with a molecular mass of 41926 Da. It reacts with N-acetyl-l-tyrosine ethyl ester (ATEE) but not with Suc-Ala-Ala-Pro-Phe-pNA or Suc-Ala-Ala-Pro-Leu-pNA. The complete amino acid sequence of the VLCTLP is deduced from the nucleotide sequence of the cDNA encoding this protein. The full-length cDNA sequence of the VLCTLP encodes open reading frame of 257 amino acid residues that includes a putative signal peptide of 18 amino acids, a proposed activation peptide of six amino acid residues and serine proteinase of 233 amino acid residues. VLCTLP belongs to the S1 (chymotrypsin) subfamily of proteases. The multiple alignment of its deduced amino acid sequence showed structural similarity with other serine proteases from snake venoms. The protease weakly hydrolyses azocasein, Aα-chain and more slowly Bβ-chain of fibrinogen. VLCTLP does not cleave fibrin and has no gelatinolytic activity. Specificity studies against peptide substrates (angiotensin I and II, oxidized insulin B-chain, glucagon, fibrinogen fragments etc.) showed that VLCTLP catalysed the cleavage of peptide bonds after tyrosine residues. VLCTLP is the only purified and characterized serine proteinase from snake venoms that catalyses ATEE hydrolysis. We detected ATEE-hydrolysing activities also in 9 different Viperidae and Crotalidae venoms.     

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.