A novel blood coagulation factor IX/factor X-binding protein with anticoagulant activity from the venom of Trimeresurus flavoviridis (Habu snake): isolation and characterization

Using affinity chromatography on a column of factor X-Cellulofine, we have isolated a novel blood coagulation factor X-binding protein with anticoagulant activity from the venom of Trimeresurus flavoviridis (Habu snake). This anticoagulant protein was also purified by chromatography on Sephadex G-75 and S-Sepharose Fast Flow. The yield of the purified protein was approximately 16 mg from 400 mg of crude venom. The purified protein gave a single band on both analytical alkaline disc-gel electrophoresis and SDS-PAGE. This protein had a relative molecular weight (Mr) after SDS-PAGE of 27,000 before reduction of disulfide bonds and 14,000 after reduction of disulfide bonds. The protein prolonged the clotting time induced by kaolin or factor Xa. In the presence of Ca2+, it formed a complex with factor X, the molar ratio being 1 to 1. Similar complex formation was observed with factor Xa and factor IX/factor IXa, but not with other vitamin K-dependent coagulation factors, i.e., prothrombin, factor VII, protein C, protein S, and protein Z. The interaction of this anticoagulant protein with factor IX/factor X was dependent on gamma-carboxyglutamic acid (Gla) domains, since Gla-domainless derivatives of factor X and factor IXa beta' did not interact with this anticoagulant protein.     

Crystallization and preliminary X-ray studies of human vascular anticoagulant protein

The human vascular anticoagulant protein, a 36 kDa member of the annexin/lipocortin family, has been crystallized using polyethylene glycol 20,000, by the vapour diffusion method. The crystals are monoclinic, space group P2(1), cell dimensions a = 83.9 A, b = 80.9 A, c = 71.4 A, beta = 108.7 degrees and diffract to at least 2.2 A resolution.     

Isolation of a venom factor devoid of proteolytic activity from Taiwan Habu (Trimeresurus mucrosquamatus): N-Terminal sequence homology and no functional similarity to factors IX/X-binding proteins and botrocetin

One novel venom factor was isolated and purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus) using two consecutive anion-exchange and gel-filtration chromatographies followed by cation-exchange HPLC. Further characterization of the purified protein indicated that it lacks the proteolytic activity toward fibrinogen molecules, suggesting that this protein factor does not belong to the familes of metalloproteinases and thrombin-like serine proteases commonly found in the crude venoms of various crotalid snakes. The purified protein exists as a native dimeric protein of 26 kDa, consisting of two closely similar subunits of 16 and 13 kDa, held together by disulfide linkage. N-Terminal sequence analysis revealed that both chains are homologous to each other at the N-terminal fragment and also similar to the factors IX/X-binding protein isolated fromTrimeresurus flavoviridis and botrocetin fromBothrops jararaca. This study points to the existence of one new two-chain venom factor without fibrinogenase activity from Taiwan habu, which, in contrast to botrocetin, promotes platelet agglutination even in the absence of von Willebrand factor. Unlike factors IX/X-binding proteins, it did not show affinity to coagulation factors IX and X in the presence of Ca²⁺ ion. It also shows no inhibition on thrombin, in contrast with bothrojaracin, a thrombin inhibitor isolated fromBothrops jararaca venom. We have therefore named this novel venom factortrimecetin to distinguish it from some structurally related venom factors present in various crotalid and viperid snakes.     

Isolation of a venom factor devoid of proteolytic activity from Taiwan Habu (Trimeresurus mucrosquamatus): N-Terminal sequence homology and no functional similarity to factors IX/X-binding proteins and botrocetin

One novel venom factor was isolated and purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus) using two consecutive anion-exchange and gel-filtration chromatographies followed by cation-exchange HPLC. Further characterization of the purified protein indicated that it lacks the proteolytic activity toward fibrinogen molecules, suggesting that this protein factor does not belong to the familes of metalloproteinases and thrombin-like serine proteases commonly found in the crude venoms of various crotalid snakes. The purified protein exists as a native dimeric protein of 26 kDa, consisting of two closely similar subunits of 16 and 13 kDa, held together by disulfide linkage. N-Terminal sequence analysis revealed that both chains are homologous to each other at the N-terminal fragment and also similar to the factors IX/X-binding protein isolated fromTrimeresurus flavoviridis and botrocetin fromBothrops jararaca. This study points to the existence of one new two-chain venom factor without fibrinogenase activity from Taiwan habu, which, in contrast to botrocetin, promotes platelet agglutination even in the absence of von Willebrand factor. Unlike factors IX/X-binding proteins, it did not show affinity to coagulation factors IX and X in the presence of Ca²⁺ ion. It also shows no inhibition on thrombin, in contrast with bothrojaracin, a thrombin inhibitor isolated fromBothrops jararaca venom. We have therefore named this novel venom factortrimecetin to distinguish it from some structurally related venom factors present in various crotalid and viperid snakes.     

Crystallization and preliminary X-ray diffraction studies of cobra venom beta-nerve growth factor

beta-Nerve growth factor (beta-NGF) is a 118 amino acid residue polypeptide with an important role in the survival and development of certain neuronal populations. A beta-NGF purified from cobra venom was crystallized by the hanging-drop vapor diffusion method. The crystals belong to the tetragonal space group P4(1)2(1)2 (or its enantiomorph P4(3)2(1)2) with unit-cell parameters a=b=61.75, c=154.40A. X-ray data from these crystals were collected to 3.2A resolution. Analysis of the packing density shows that the asymmetric unit probably contains two molecules. The self-rotation calculation implied that a beta-NGF dimer might exist in the asymmetric unit.     

A C-type lectin-like protein from Agkistrodon acutus venom binds to both platelet glycoprotein Ib and coagulation factor IX/factor X

Agkisacutacin, a C-type lectin-like protein (CLP) isolated from Agkistrodon acutus venom, had been previously identified as an antagonist of platelet aggregation induced by ristocetin, as well as a certain extent fibrinogenlytic activity. In this study, agkisacutacin was further purified by three-step chromatography, and its biological functions were characterized. Agkisacutacin after further purification retained the effect on ristocetin-induced, von Willebrand factor-dependent platelet aggregation, while it lost the fibrinogenlytic activity. FACS and ELISA assays showed that agkisacutacin belongs to membrane glycoprotein Ib-binding protein (GPIb-bp) for it could block and inhibit the binding of anti-GPIb antibody to GPIb. Especially, agkisacutacin exhibits anti-coagulant activity and the function as IX/X-binding protein was confirmed by PAGE and ELISA. So, agkisacutacin is the first reported CLP that binds to both platelet membrane receptors and coagulation factors.     

A novel anticoagulant protein with high affinity to blood coagulation factor Va from Tegillarca granosa

A novel inhibitory protein against blood coagulation factor Va (FVa) was purified from muscle protein of granulated ark (Tegillarca granosa, order Arcoida, marine bivalvia) by consecutive FPLC method using anion exchange and gel permeation chromatography. In the results of ESIQTOF tandem mass analysis and database research, it was revealed that the purified T. granosa anticoagulant protein (TGAP) has 7.7 kDa of molecular mass and its partial sequence, HTHLQRAPHPNALGYHGK, has a high identity (64%) with serine/threonine kinase derived from Rhodopirellula baltica (order Planctomycetales, marine bacteria). TGAP could potently prolong thrombin time (TT), corresponding to inhibition of thrombin (FIIa) formation. Specific factor inhibitory assay showed that TGAP inhibits FVa among the major components of prothrombinase complex. In vitro assay for direct-binding affinity using surface plasmon resonance (SPR) spectrometer indicated that TGAP could be directly bound with FVa. In addition, the binding affinity of FVa to FII was decreased by addition of TGAP in dose-dependant manner (IC50 value = 77.9 nM). These results illustrated that TGAP might interact with a heavy chain of FVa (FVa(H)) bound to FII in prothrombin complex. The present study elucidated that non-cytotoxic T. granosa anticoagulant protein (TGAP) bound to FVa can prolong blood coagulation time by inhibiting conversion of FII to FIIa in blood coagulation cascade. In addition, TGAP did not significantly (P < 0.05) show fibrinolytic activity and cytotoxicity on venous endothelial cell line (ECV 304).     

Crystallization and preliminary X-ray study of a lipase from Pseudomonas glumae.

Lipase from Pseudomonas glumae has been purified and crystallized in two forms, using the hanging drop method of vapour diffusion at 4 degrees C and 15 degrees C. Both forms grew at pH 9.0 from 0.1 M-Tris buffer in the presence of 10% (v/v) acetone. Form 1 was crystallized from 27 to 29% polyethylene glycol in the presence of less than 0.5% (v/v) n-dodecyl-beta-D-glucopyranoside. Form 2 was grown from 17 to 19% ammonium sulphate in the presence of 1% n-octyl-beta-D-glucopyranoside. Form 1 is orthorhombic with space group P2(1)2(1)2(1), and cell dimensions of a = 158.1 A, b = 158.6 A, c = 63.4 A, Form 2 is tetragonal with space group P4(1)2(1)2 (or P4(3)2(1)2) and cell dimensions of a = 89.3 A, c = 180.4 A. Form 1 probably has four molecules per asymmetric unit and diffracts to at least 2.5 A. Form 2 has two molecules per asymmetric unit and diffracts to at least 3.0 A.     

Crystallization, preliminary X-ray study and crystal activity of the hydrogenase from Desulfovibrio gigas

Hydrogenase (EC 1.12) from Desulfovibrio gigas is a dimeric enzyme (26 and 62 (X 10(3) Mr) that catalyzes the reversible oxidation of molecular hydrogen. Single crystals of hydrogenase have been produced using the hanging drop method, with either PEG (polyethylene glycol) 6000 or ammonium sulfate as precipitants at pH 6.5. X-ray examination of the crystals indicates that those obtained with ammonium sulfate are suitable for structure determination to at least 3.0 A resolution when synchrotron radiation Sources are used (1 A = 0.1 nm). The crystals are monoclinic, with space group C2, and cell dimensions a = 257.0 A, b = 184.7 A, c = 148.3 A and beta = 101.3 degrees, and contain between four and ten molecules per asymmetric unit. The enzyme can be reactivated within the crystals under reducing conditions without crystal damage.     

Calcium-binding analysis and molecular modeling reveal echis coagulation factor IX/factor X-binding protein has the Ca-binding properties and Ca ion-independent folding of other C-type lectin-like proteins

Many biologically active heterodimeric proteins of snake venom consist of two C-type lectin-like subunits. One of these proteins, habu IX/X-bp, is a Gla domain-binding protein whose subunits both bind to a Ca2+ ion, with a total of two Ca2+-binding sites. The molecular modeling and Ca2+-binding analysis of echis IX/X-bp revealed that it lacks one of two Ca2+-binding sites, though the folding of this subunit is conserved. It is concluded that heterodimeric C-type lectin-like proteins function independent of Ca2+ and have essentially a similar folding to habu IX/X-bp.     

Crystallization and preliminary crystallographic study of stonustoxin, a protein lethal factor isolated from the stonefish (Synanceja horrida) venom

Crystals of stonustoxin have been obtained and diffract to 3.4 A resolution. Stonustoxin is a protein lethal factor isolated from the venom of the stonefish, Synanceja horrida. The crystals belong to the tetragonal space group P422, with unit cell constants a = b = 109.0 A, c = 245.7 A. A native stonustoxin molecule has two subunits, designated alpha and beta, respectively, and there is one stonustoxin molecule per asymmetric unit.     

Crystallization and preliminary X-ray crystallographic analysis of PAS factor from Vibrio vulnificus

Plasmid Achromobacter secretion (PAS) factor is a putative secretion factor that induces the secretion of periplasmic proteins. PAS factor from Vibrio vulnificus was crystallized at 294 K by the hanging drop vapor-diffusion method. It was isolated as a monomer during the purification procedures. The native crystal belongs to the F222 space group with unit cell parameters a=56.1, b=74.4, c=80.0 A, a=b=g=90 degrees. The crystal was soaked in cryoprotectant containing 1 M NaBr for 1 h for MAD phasing. The diffraction limit of the Br-MAD data set was 1.9 A using synchrotron X-ray irradiation at beam line BL-18B at the Photon Factory, Japan.     

Crystallization of kappa-bungarotoxin: preliminary X-ray data obtained from the venom-derived protein.

kappa-Bungarotoxin is a 66 residue polypeptide found in the venom of the Taiwanese banded krait, Bungarus multicinctus. It binds tightly to neuronal nicotinic acetylcholine receptors and inhibits nerve transmission mediated by these postsynaptic receptors. It is related, by similarity in amino acid sequence, to alpha-bungarotoxin and other alpha-neurotoxins, but differs sharply in physiologic action. The alpha-neurotoxins inhibit nerve transmission in nicotinic acetylcholine receptors associated with vertebrate skeletal muscle and fish electric organs. The kappa-neurotoxins inhibit nerve transmission in neuronal nicotinic acetylcholine receptors such as those found in chick ciliary ganglia. The kappa-neurotoxins display a low level of interaction with receptors that are strongly affected by alpha-neurotoxins, but alpha-neurotoxins are completely without effect on receptors that are affected by kappa-bungarotoxin. The structural basis for this physiologic differentiation is not known. Crystals of kappa-bungarotoxin have now been obtained that diffract to at least 2.3 A. These crystals are hexagonal, space group P6, and have dimensions of a = b = 80.2 A, c = 39.6 A, and angles of alpha = beta = 90 degrees and gamma = 120 degrees. Each unit cell contains 12 molecules of the 66 residue protein or two molecules per asymmetric unit. Comparison of the structure of kappa-bungarotoxin, which will result from further diffraction analysis of these crystals, with the structures of the alpha-neurotoxins that have been determined may provide information on the structural basis of physiologic action in these acetylcholine receptor antagonists.     

Crystallization and preliminary X-ray study of AMP nucleosidase

Adenosine-5'-monophosphate nucleosidase from Escherichia coli has been crystallized in the presence of its strong competitive inhibitor formycin 5'-monophosphate and its allosteric activator adenosine 5'-triphosphate. Crystals are tetragonal bipyramids which grow to 1.2 mm in the longest dimension, are resistant to radiation damage, and diffract to a resolution of 3.5 A. The space group is P4(1)2(1)2 or P4(3)2(1)2, and the unit cell dimensions are a = 120.1 A and c = 243.7 A. The asymmetric unit is estimated to contain four subunits of 52,000 daltons. The crystals appear suitable for single crystal x-ray structure investigation.     

Crystallization and preliminary X-ray data of a phleomycin-binding protein from Streptoalloteichus hindustanus

Large single crystals of a glycopeptide resistance protein encoded by the SH-ble gene from Streptoalloteichus hindustanus have been grown using vapor diffusion techniques with ammonium sulfate as the precipitant. The diffraction pattern extends to 2.0 A resolution. The crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2 and have unit cell dimensions of a = 48.8 A and c = 112.5 A.     

Crystallization and preliminary X-ray analysis of fluorescent protein mBanana

mBanana is a novel monomeric red fluorescent protein mutant. It was cloned and expressed in Escherichia coli with 10 histidine residues at its N-terminal. After cleavage of the His tag by TEV protease, the mBanana was further purified and crystallized by the hanging-drop vapor-diffusion technique. The crystals can diffract to 2.0A resolution and one set of completed data was collected. It showed that the orthorhombic mBanana crystal was in space group P21 with unit cell parameters (48.629, 42.667, 61.714, 90, 111.676, 90) and contained one molecule in one asymmetric unit.     

Crystallization and preliminary X-ray investigation of a sarcoplasmic calcium-binding protein from amphioxus

Crystals of a sarcoplasmic Ca(2+)-binding protein from the protochordate amphioxus have been grown from solutions of ammonium sulfate. The crystals are orthorhombic, space group C222(1), with unit cell axes a = 59.6(1) A, b = 81.3(1) A and c = 82.4(1) A. There is one molecule in the asymmetric unit. The crystals diffract beyond 2.5 A and show less than 20% decline in diffraction intensities after a three day exposure to X-rays from a laboratory rotating anode source.     

Crystallization and preliminary X-ray crystallographic studies of Protac, a commercial protein C activator isolated from Agkistrodon contortrix contortrix venom

The protein C pathway plays an important role in the control and regulation of the blood coagulation cascade and prevents the propagation of the clotting process on the endothelium surface. In physiological systems, protein C activation is catalyzed by thrombin, which requires thrombomodulin as a cofactor. The protein C activator from Agkistrodon contortrix contortrix acts directly on the zymogen of protein C converting it into the active form, independently of thrombomodulin. Suitable crystals of the protein C activator from Agkistrodon contortrix contortrix were obtained from a solution containing 2 M ammonium sulfate as the precipitant and these crystals diffracted to 1.95 A resolution at a synchrotron beamline. The crystalline array belongs to the monoclinic space group C2 with unit cell dimensions a=80.4, b=63.3 and c=48.2 A, alpha=gamma=90.0 degrees and beta=90.8 degrees.     

Crystallization and preliminary x-ray diffraction study of neurotoxin-I from Naja naja oxiana venom

Crystals of the neurotoxin-I (NTX-I) from the venom of the middle Asian cobra Naja naja oxiana have been grown by vapour diffusion and dialysis methods. The crystals belong to space group P2(1)2(1)2 with dimension of a = 25.19 A, b = 75.59 A, c = 36.09 A and diffract to 1.9 A resolution. The asymmetric unit contains one molecule (Vm = 2.2 A/Da). Using the molecule of alpha-cobratoxin (CTX) as a starting model for NTX-I structure determination coordinates of C alpha atoms of the NTX-I molecule were obtained and the position of NTX-I in the unit cell was derived.