Venom Serine Proteases Research Articles

Membrane independent activation of fibroblast proMMP-2 by snake venom: novel roles for venom proteinases

ProMMP-2 activation by Bothrops asper venom was investigated in mouse gastrocnemius muscle, mammalian cell culture and a cell-free system. Zymography revealed an increment of latent and activated forms of MMP-2 in muscle homogenates 1–3 days after venom injection. To clarify if venom can induce expression and activation of MMP-2, independently of the inflammatory response, venom was added to cultured human fibroblasts, endothelial and skeletal muscle cells, which expressed proMMP-2 constitutively. Venom activated proMMP-2 without promoting its expression. Venom also activated and degraded proMMP-2 in supernatants collected from fibroblast cultures, indicating that cells are not required for this activation. Pretreatment with EDTA increased MMP-2 activation and reduced degradation. Venom serine proteinases activated proMMP-2, whereas BaP1, a P-I metalloproteinase, predominantly degraded the latent and active forms of MMP-2. Moreover, pretreatment of conditioned medium with serine proteinase inhibitors greatly reduced the venom-induced activation, suggesting that venom proteinases activate MMP-2 via a serine proteinase secreted by fibroblasts. Venom also directly activated and degraded purified proMMP-2, albeit requiring a high concentration. Thus, B. asper venom proteinases activate and degrade proMMP-2 without inducing its synthesis. Serine proteinases play a dominant role in the activation, whereas metalloproteinases predominantly degrade MMP-2. Activation of proMMP-2 by snake venom proteinases, independently of the MT1-MMP/TIMP-2 pathway, extracellular matrix degradation or apoptosis, represents a novel mechanism in human fibroblasts.

Hymenoptera venom protease allergens

Recent studies have shown the presence of additional allergenic proteins in honeybee and paper wasp venoms. Both venoms contain serine protease enzymes. We isolated and obtained complete sequences of honeybee and Mediterranean paper wasp venom proteases, both of which have significant IgE binding activity. The structures are compared with bumblebee venom protease. Venom proteases were chromatographically isolated from venoms and partial amino acid sequences determined. RT-PCR and rapid amplification of cDNA ends methods were used to clone cDNA, and complete sequences were determined for honeybee and a paper wasp venom protease. The venom proteases are all serine proteases of the trypsin type. The honeybee protease contains a complement, embryonic sea urchin protein, bone morphogenetic protein interaction domain as well as a linker and propeptide sequence, and a unique methionine residue near the active site. It has IgE binding activity. The paper wasp protease is a single trypsin domain and is an important allergen. The framework residues are poorly conserved among honeybee, bumblebee, and paper wasp enzymes. The 3 venom serine proteases have significant IgE binding activities. The structures are poorly conserved even among the Apidae , suggesting little cross-reactivity among the protein portions. The paper wasp venom proteases are important allergens.

On the modeling of snake venom serine proteinase interactions with benzamidine-based thrombin inhibitors.

Pit viper venoms contain a number of serine proteinases that exhibit one or more thrombin-like activities on fibrinogen and platelets, this being the case for the kinin-releasing and fibrinogen-clotting KN-BJ from the venom of Bothrops jararaca. A three-dimensional structural model of the KN-BJ2 serine proteinase was built by homology modeling using the snake venom plasminogen activator TSV-PA as a major template and porcine kallikrein as additional structural support. A set of intrinsic buried waters was included in the model and its behavior under dynamic conditions was molecular dynamics simulated, revealing a most interesting similarity pattern to kallikrein. The benzamidine-based thrombin inhibitors alpha-NAPAP, 3-TAPAP, and 4-TAPAP were docked into the refined model, allowing for a more insightful functional characterization of the enzyme and a better understanding of the reported comparatively low affinity of KN-BJ2 toward those inhibitors.

Biochemical Properties of a Bushmaster Snake Venom Serine Proteinase (LV-Ka), and Its Kinin Releasing Activity Evaluated in Rat Mesenteric Arterial Rings

A serine proteinase with kallikrein-like activity (LV-Ka) has been purified to homogeneity from bushmaster snake (Lachesis muta muta) venom. Physicochemical studies indicated that LV-Ka is a single chain glycoprotein with a molecular mass (Mr) of 33 kDa under reducing conditions which was reduced to 28 kDa after treatment with N-Glycosidase F (PNGase F). LV-Ka can be bounded and neutralized by serum α2-macroglobulin (α2-M), a prevalent mammalian protease inhibitor that is capable of forming a macromolecular complex with LV-Ka (Mr >180 kDa). Cleavage of α2-M by the enzyme resulted in the formation of 90-kDa fragments. The proteolytic activity of LV-Ka against dimethylcasein could be inhibited by α2-M, and the binding ratio of the inhibitor:enzyme complex was found to be 1:1. The Michaelis constant, Km, and catalytic rate constant, kcat, of LV-Ka on four selective chromogenic substrates were obtained from Lineweaver-Burk plots. LV-Ka exhibits substrate specificities not only for the glandular kallikrein H-D-Val-Leu-Arg-pNA (S-2266) but also for the plasmin substrates S-2251 and Tos-Gly-Pro-Lys-pNA. Bovine kininogen incubated with LV-Ka generated a polypeptide that dose dependently contracted mesenteric arterial rings from spontaneously hypertensive rats (SHR) in a similar way as bradykinin (BK) does. As it happens with BK, LV-Ka generated polypeptide was inhibited by HOE-140, a bradykinin B2-receptor antagonist and by indomethacin, a cyclo-oxygenase inhibitor. These results strongly suggest that the polypeptide generated by LV-Ka by cleavage of bovine kininogen is bradykinin. In addition, our studies may help to understand the mechanism of action involved in hypotension produced by envenomation of bushmaster snake.

Identification of the substrate-binding exosites of two snake venom serine proteinases: molecular basis for the partition of two essential functions of thrombin.

The venom of the South American snake Bothrops jararaca contains two serine proteinases, bothrombin and the platelet-aggregating enzyme PA-BJ, which share 66% sequence identity. Each of these proteinases possesses one of the two essential procoagulant functions of thrombin-the clotting of fibrinogen and platelet aggregation. Thus, bothrombin clots fibrinogen but has no direct effect on platelets, unless in the presence of exogenous fibrinogen. PA-BJ induces platelet aggregation by interacting with the protease-activated platelet receptor without clotting fibrinogen. On the other hand, thrombin possesses two extended surfaces. One is composed of basic and hydrophobic residues (exosite I) and the other one of basic residues only (exosite II). These exosites are involved in the recognition of physiological macromolecular substrates. In order to identify the corresponding exosites in bothrombin and PA-BJ and understand the molecular basis of the partition of the two procoagulant functions of thrombin among the two snake venom enzymes, we used molecular modeling to obtain models of their complexes with their natural substrates fibrinogen and a fragment of the protease-activated platelet receptor, respectively. In analogy to thrombin, each of the enzymes presents two exosites. Nonetheless, the exosites contain a smaller proportion of basic residues than thrombin does (45-72%), reducing thus the functional diversity of the enzymes. In addition, the composition of exosite I is different in both enzymes. We identify those residues in exosite I that could contribute to the differences in specificity. Finally, allostery does not seem to mediate macromolecular substrate recognition by these enzymes.

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.

Anticoagulant serine fibrinogenases from Vipera lebetinavenom: structure-function relationships

Amino acid sequences of two anticoagulant serine fibrinogenases - alpha- and beta-fibrinogenase (VLAF and VLBF) from Vipera lebetina venom have been deduced from the cDNA sequences encoding the enzymes. The mature protein sequences of 234 amino acids (VLAF) and 233 amino acids (VLBF) exhibit significant similarity with other snake venom serine proteinases. Both enzymes contain the catalytic triad His57, Asp102, Ser195, and twelve conserved cysteines forming six disulfide bridges. Unlike typical trypsin-like serine proteinases, they lack the third aspartate, Asp189 which is replaced by Gly189. VLBF is a typical representative of arginine esterases - beta-fibrinogenases. alpha-Fibrinogenase, VLAF, is unique among snake venom serine proteinases with homologous structure. Until now there is no evidence of the anticoagulant serine enzymes degrading fibrinogen alpha-chain only and lacking esterolytic activity.

Snake Venom Proteinases as Tools in Hemostasis Studies: Structure-Function Relationship of a Plasminogen Activator Purified from Trimeresurus stejnegeri Venom

Snake venom serine proteinases affect many steps of the blood coagulation cascade. Each of them usually acts selectively on one coagulation factor. They are therefore potentially useful components to study the mechanisms of action, the regulation and the structure-function relationships of human serine proteinase coagulation factors. This strategy is illustrated for a plasminogen activator purified from Trimeresurus stejnegeri venom.

Purification and characterization of jerdofibrase, a serine protease from the venom of Trimeresurus jerdonii snake

A fibrin(ogen)olytic serine protease from Trimeresurus jerdonii venom was identified and purified to SDS–polyacrylamide gel electrophoresis homogeneity. It is a single chain polypeptide with a molecular weight of 32 kDa under reduced condition and 28 kDa under non-reduced condition, respectively. The venom protease catalysed the hydrolysis of some chromogenic substrates such as S2238, S2160, S2302 and S2251. It degraded Bβ-chain of human fibrinogen preferentially. Also the enzyme degraded fibrin directly. Its enzymatic activity was completely inhibited by phenylmethylsulfonyl fluoride (PMSF), but not affected by EDTA. That suggested it was a serine protease. N-terminal sequence of the purified component showed high homology with other snake venom serine proteases.

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.

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 μM) 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.

Characterization of a thrombin-like enzyme from the venom of Trimeresurus jerdonii

From the venom of Trimeresurus jerdonii, a distinct thrombin-like enzyme, called jerdonobin, was purified by DEAE A-25 ion-exchange chromatography, Sephadex G-75 gel filtration, and fast protein liquid chromatography (FPLC). SDS-PAGE analysis of this enzyme shows that it consists of a single polypeptide chain with a molecular weight of 38,000. The NH 2-terminal amino acid sequence of jerdonobin has great homology with venom thrombin-like enzymes documented. Jerdonobin is able to hydrolyze several chromogenic substrates. The enzyme directly clots fibrinogen with an activity of 217 NIH units/mg. The fibrinopeptides released, identified by HPLC, consisted of fibrinopeptide A and a small amount of fibrinopepide B. The activities of the enzyme were inhibited by phenylmethylsulfonyl fluoride (PMSF) and p-nitrophenyl- p-guanidinobenzoate (NPGB). However, metal chelator (EDTA) had no effect on it, indicating it is venom serine protease.

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.

Molecular Cloning and Sequence Analysis of a cDNA for Factor V Activating Enzyme, a Coagulant Protein from Vipera lebetina Snake Venom

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.

The crystal structure of the novel snake venom plasminogen activator TSV-PA: a prototype structure for snake venom serine proteinases.

Trimeresurus stejnejeri venom plasminogen activator (TSV-PA) is a snake venom serine proteinase that specifically activates plasminogen. Snake venom serine proteinases form a subfamily of trypsin-like proteinases that are characterised by a high substrate specificity and resistance to inhibition. Many of these venom enzymes specifically interfere with haemostatic mechanisms and display a long circulating half-life. For these reasons several of them have commercial applications and are potentially attractive pharmacological tools. The crystal structure of TSV-PA has been determined to 2.5 A resolution and refined to an R factor of 17.8 (R free, 24.4). The enzyme, showing the overall polypeptide fold of trypsin-like serine proteinases, displays unique structural elements such as the presence of a phenylalanine at position 193, a C-terminal tail clamped via a disulphide bridge to the 99-loop, and a structurally conserved Asp97 residue. The presence of a cis proline at position 218 is in agreement with evolutionary relationships to glandular kallikrein. We postulate that Phe 193 accounts for the high substrate specificity of TSV-PA and renders it incapable of forming a stable complex with bovine pancreatic trypsin inhibitor and other extended substrates and inhibitors. Mutational studies previously showed that Asp97 is crucial for the plasminogenolytic activity of TSV-PA, here we identify the conservation of Asp97 in both types of mammalian plasminogen activator - tissue-type (tPA) and urokinase-type (uPA). It seems likely that Asp97 of tPA and uPA will have a similar role in plasminogen recognition. The C-terminal extension of TSV-PA is conserved among snake venom serine proteinases, although its function is unknown. The three-dimensional structure presented here is the first of a snake venom serine proteinase and provides an excellent template for modelling other homologous family members.

Characterization of three fibrinogenolytic enzymes from Chinese green tree viper ( Trimeresurus stejnegeri) venom

From the venom of Chinese green tree viper ( Trimeresurus stejnegeri), three distinct fibrinogenolytic enzymes: stejnefibrase-1, stejnefibrase-2 and stejnefibrase-3, were purified by gel filtration, ion-exchange chromatography and reverse-phase high-performance chromatography (HPLC). SDS-PAGE analysis of those three enzymes showed that they consisted of a single polypeptide chain with mol. wt of 50 000, 31 000 and 32 000, respectively. Like TSV-PA (a specific plasminogen activator) and stejnobin (a fibrinogen-clotting enzyme) purified from the same venom, stejnefibrase-1, -2 and -3 were able to hydrolyze several chromogenic substrate. On the other hand, different from TSV-PA and stejnobin, stejnefibrase-1, -2 and -3 did not activate plasminogen and did not possess fibrinogen-clotting activity. The three purified enzymes directly degraded fibrinogen to small fragments and rendered it unclottable by thrombin. Stejnefibrase-2 degraded preferentially B β-chain while stejnefibrase-1 and -3 cleaved concomitantly A α and B β-chains of fibrinogen. None of these proteases degraded the γ-chain of fibrinogen. When correlated with the loss of clottability of fibrinogen, the most active enzyme was stejnefibrase-1. The activities of the three enzymes were inhibited by phenylmethylsulfonyl fluoride (PMSF) and p-nitrophenyl- p-guanidinobenzoate (NPGB), indicating that like TSV-PA and stejnobin, they are venom serine proteases.

Purification and amino acid sequence of halystase from snake venom of Agkistrodon halys blomhoffii, a serine protease that cleaves specifically fibrinogen and kininogen.

We have isolated a serine protease, halystase, from Agkistrodon halys blomhoffii venom by chromatography on DEAE-Sepharose, heparin-Sepharose and Q-Sepharose columns, and have determined the complete amino acid sequence by Edman degradation and by mass spectral analysis of peptides generated by enzymatic and chemical cleavage. The 238-residue sequence of halystase, containing N-linked carbohydrates (about 13%) at two sites showed significant similarity to other thrombin-like snake venom serine proteases (66-72%), mammalian tissue kallikrein (42%) and thrombin (26%). Halystase contained the tentative catalytic triad of His43, Asp88 and Ser184 common to all serine proteases and Asp178 in the primary substrate-binding site. Although halystase contained an RGD sequence at residues 181-183, it did not inhibit platelet aggregation induced by ADP or collagen. It hydrolyzed most efficiently a tissue-kallikrein substrate, prolylphenylalanylarginyl-4-methyl-coumaryl-7-amide, and released bradykinin from bovine kininogen. Halystase did not coagulate human plasma, but it cleaved the fibrinogen B beta chain at the carboxyl side of Arg42 and cleaved slowly the fibrogen A alpha chain. Fibrinogen thus treated gradually became insensitive to thrombin. The proteolytic activity was inhibited with diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride or leupeptin. These results indicate that halystase is a serine protease structurally similar to coagulating thrombin-like snake venom proteases, but it specifically cleaves fibrinogen at sites different from thrombin without inducing fibrin clotting, and hydrolyzes kininogen to produce bradykinin, resulting in the reduction of blood pressure.

Trimeresurus stejnegeri Snake Venom Plasminogen Activator: SITE-DIRECTED MUTAGENESIS AND MOLECULAR MODELING

The specific plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) is a serine proteinase presenting 23% sequence identity with the proteinase domain of tissue type plasminogen activator, and 63% with batroxobin, a fibrinogen clotting enzyme from Bothrops atrox venom that does not activate plasminogen. TSV-PA contains six disulfide bonds and has been successfully overexpressed in Escherichia coli (Zhang, Y., Wisner, A., Xiong, Y. L., and Bon, C. (1995) J. Biol. Chem. 270, 10246-10255). To identify the functional domains of TSV-PA, we focused on three short peptide fragments of TSV-PA showing important sequence differences with batroxobin and other venom serine proteinases. Molecular modeling shows that these sequences are located in surface loop regions, one of which is next to the catalytic site. When these sequences were replaced in TSV-PA by the equivalent batroxobin residues none generated either fibrinogen-clotting or direct fibrinogenolytic activity. Two of the replacements had little effect in general and are not critical to the specificity of TSV-PA for plasminogen. Nevertheless, the third replacement, produced by the conversion of the sequence DDE 96a-98 to NVI, significantly increased the Km for some tripeptide chromogenic substrates and resulted in undetectable plasminogen activation, indicating the key role that the sequence plays in substrate recognition by the enzyme.

Purification, characterization, and amino acid sequence of a serine proteinase, PA-BJ, with platelet-aggregating activity from the venom of Bothrops jararaca.

A platelet-aggregating enzyme, PA-BJ, was isolated from the venom of the snake Bothrops jararaca. PA-BJ in a concentration of 3.2 x 10(-8) M promoted 95% platelet aggregation in platelet-rich plasma. SDS-polyacrylamide gel electrophoresis under reducing conditions showed a single protein band with an M(r) of 30,000. PA-BJ catalyzed the hydrolysis of several p-nitroanilide peptide substrates containing Arg or Lys at the scissile bond; among these the most sensitive were the thrombin substrates D-Phe-Pip-Arg-pNA and Tos-Gly-Pro-Arg-pNA. Both the platelet-aggregating and amidolytic activities of PA-BJ were abolished by reaction with phenylmethanesulfonyl fluoride. Several benzamidine derivatives, which are competitive inhibitors of trypsin-like serine proteinases, also inhibited the amidolytic activity of PA-BJ. Among the compounds tested, the thrombin inhibitor NAPAP [N alpha-[(2-naphthylsulfonyl)-glycyl]-4-amidinophenylalanine piperidide] showed the strongest inhibitor activity on PA-BJ. The complete amino acid sequence of PA-BJ, which, to the best of our knowledge, is the first of a platelet-aggregating enzyme from snake venom, was deduced from the N-terminal sequencing of overlapping fragments cleaved from the reduced and S-pyridylethylated protein by chemical and enzymatic methods. PA-BJ is composed of 232 amino acid residues and contains one N- and one O-glycosidically linked carbohydrate moiety at residues Asn20 and Ser23. Sequence comparison to other venom serine proteinases revealed significant homology, mainly in regions around the catalytic triad and conserved cysteine residues.

Opossum serum .alpha.1-proteinase inhibitor: Purification, linear sequence, and resistance to inactivation by rattlesnake venom metalloproteinase

Opossum (Didelphis virginiana) serum was fractionated with (NH4)2SO4 and then chromatographed on DEAE-Sepharose and phenyl-Sepharose. Affinity chromatography on a protein A-Sepharose-antibody column removed traces of opossum serum metalloproteinase inhibitors, and resulted in a homogeneous preparation of opossum alpha 1-proteinase inhibitor (alpha 1-PI). The inhibitor is a single-chain glycoprotein (17.7% carbohydrate) with an estimated M(r) = 54,000. An opossum liver cDNA library was immunoscreened, and clones containing cDNA encoding for the open reading frame for opossum alpha 1-PI were isolated. The cDNA inserts contained nucleotide sequences corresponding to the amino-terminal and an internal peptide sequence of opossum alpha 1-PI which had been separately determined by protein sequence analysis. The entire inserts coded for a protein consisting of a 21-residue signal peptide and a 389-residue mature protein. Opossum alpha 1-PI shows 51-58% identity with other mammalian alpha 1-PI amino acid sequences, and the conserved residues expected for a member for the serpin family have been retained. The carbohydrate attachment sites and the reactive site residues (M-S) of opossum alpha 1-PI are identical to those of human alpha 1-PI. Opossum alpha 1-PI formed stable enzyme/inhibitor complexes with trypsin, chymotrypsin, and human neutrophil elastase, but did not react with thrombin or with snake venom serine proteinases. Opossum alpha 1-PI was inactivated by papain or Pseudomonas aeruginosa elastase, and electrophoretic analysis of the reaction products indicated limited proteolysis in the reactive site loop of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)