Isolation and characterization of the thrombin-like enzyme from Cryptelytrops albolabris (white-lipped tree viper) venom

A thrombin-like enzyme (termed albolabrase) was isolated in purified form from the venom of Cryptelytrops albolabris (white-lipped tree viper) using high performance anion ion exchange and gel filtration chromatography. The molecular mass of albolabrase was 33.7 kDa as determined by SDS-PAGE and 35.8 kDa as determined by Superose gel filtration chromatography. The N-terminal sequence was determined to be VVGGDECNINE which is homologous to many snake venom thrombin-like enzymes. Albolabrase exhibits both arginine ester hydrolase and arginine amidase activities and the enzyme is fastidious towards tripeptide chromogenic anilide substrates. The fibrinogen clotting activity was optimum at 3 mg/mL bovine fibrinogen, and showed distinct species differences in the following decreasing order: bovine fibrinogen > dog fibrinogen ≈ human fibrinogen > goat fibrinogen. The enzyme failed to clot both rabbit and cat fibrinogens. Reversed-phase HPLC analysis on the breakdown products of fibrinogenolytic action of albolabrase indicated that the enzyme belongs to the AB class of snake venom thrombin-like enzyme. In the indirect ELISA, IgG anti-albolabrase reacted extensively with most crotalid venoms, except with Tropidolaemus wagleri and Calloselasma rhodostoma venoms. The double sandwich ELISA, however, showed that anti-albolabrase reacted strongly only with venoms from the Trimeresurus complex, and that the results support the proposed new taxonomy changes concerning the Trimeresurus complex.     

Alsophinase, a new P-III metalloproteinase with α-fibrinogenolytic and hemorrhagic activity from the venom of the rear-fanged Puerto Rican Racer Alsophis portoricensis (Serpentes: Dipsadidae)

Metalloproteinases from snake venoms are often multi-domain enzymes involved in degradation of a variety of structural proteins. Hemorrhage and tissue necrosis are common manifestations of viperid envenomations in humans, largely due to the actions of prominent metalloproteinases, and envenomation by rear-fanged snakes may also cause hemorrhage. We purified the major metalloproteinase in Alsophis portoricensis (Puerto Rican Racer) venom through HPLC size exclusion and ion exchange chromatography. Named alsophinase, it is the first protein purified and characterized from the venom of Alsophis. Alsophinase is a single polypeptide chain protein, and based on mass, activity and complete inhibition by 1,10-phenanthroline, it is a class P-III snake venom member of the M12 ADAM family of metalloproteinases. Alsophinase has a molecular mass of 56.003 kDa and an N-terminal sequence of QDTYLNAKKYIEFYLVVDNGMFxKYSxxFTV, with 67% sequence identity to a metalloproteinase isolated from venom of Philodryas olfersii (another rear-fanged species). Alsophinase rapidly catalyzed cleavage of only the Ala14–Leu15 bond of oxidized insulin B chain, had potent hemorrhagic activity in mice, and degraded only the α-subunit of human fibrinogen in vitro. Alsophinase is responsible for hemorrhagic and fibrinogenolytic activity of crude venom, and it may contribute to localized edema and ecchymosis associated with human envenomations by A. portoricensis. It may be more specific in peptide bond recognition than many well-characterized viperid P-III metalloproteinases, and it could have utility as a new protein fragmentation enzyme for mass spectrometry studies.     

Identification and characterization of a taxon-specific three-finger toxin from the venom of the Green Vinesnake (Oxybelis fulgidus; family Colubridae)

Snake venoms contain a variety of protein and peptide toxins, and the three-finger toxins (3FTxs) are among the best characterized family of venom proteins. The compact nature and highly conserved molecular fold of 3FTxs, together with their abundance in many venoms, has contributed to their utility in structure-function studies. Although many target the nicotinic acetylcholine receptor of vertebrate skeletal muscle, often binding with nanomolar K_ds, several non-conventional 3FTxs show pronounced taxon-specific neurotoxic effects. Here we describe the purification and characterization of fulgimotoxin, a monomeric 3FTx from the venom of Oxybelis fulgidus, a neotropical rear-fanged snake. Fulgimotoxin retains the canonical 5 disulfides of the non-conventional 3FTxs and is highly neurotoxic to lizards; however, mice are unaffected, demonstrating that this toxin is taxon-specific in its effects. Analysis of structural features of fulgimotoxin and other colubrid venom 3FTxs indicate the presence of a “colubrid toxin motif” (CYTLY) and a second conserved segment (WAVK) found in Boiga and Oxybelis taxon-specific 3FTxs, both in loop II. Because specific residues in loop II conventional α-neurotoxic 3FTxs are intimately associated with receptor binding, we hypothesize that this loop, with its highly conserved substitutions, confers taxon-specific neurotoxicity. These findings underscore the importance of rear-fanged snake venoms for understanding the evolution of toxin molecules and demonstrate that even among well-characterized toxin families, novel structural and functional motifs may be found.     

Topological studies of hSVCT1, the human sodium-dependent vitamin C transporter and the influence of N-glycosylation on its intracellular targeting

The Na⁺-dependent transporters, hSVCT1 and hSVCT2, were assessed in COS-1 cells for their membrane topology. Antibodies to N- and C-termini of hSVCT1 and C-terminus of hSVCT2 identified positive immunofluorescence only after permeabilisation, suggesting these regions are intracellular. PNGase F treatment confirmed that WT hSVCT1 (∼ 70-100 kDa) is glycosylated and site-directed mutagenesis of the three putative N-glycosylation sites, Asn138, Asn144, Asn230, demonstrated that mutants N138Q and N144Q were glycosylated (∼ 68-90 kDa) with only 31-65% of WT l-ascorbic acid (AA) uptake while the glycosylation profile of N230Q remained unaltered (∼ 98% of WT activity). However, the N138Q/N144Q double mutant displayed barely detectable membrane expression at ∼ 65 kDa, no apparent glycosylation and minimal AA uptake (< 10%) with no discernible improvement in expression or activity when cultured at 28 °C or 37 °C. Marker protein immunocytochemistry with N138Q/N144Q identified intracellular aggregates with hSVCT1 localised at the nuclear membrane but absent at the plasma membrane thus implicating its role as a possible intracellular transporter and suggesting N-glycosylation is required for hSVCT1 membrane targeting. Also, Lys242 on the same putative hydrophilic loop as Asn230 after biotinylation was inaccessible from the extracellular side when analysed by MALDI-TOF MS. A new hSVCT1 secondary structure model supporting these findings is proposed.     

Isolation and characterization of the thrombin-like enzyme from Cryptelytrops purpureomaculatus venom

A thrombin-like enzyme, purpurase, was purified from the Cryptelytrops purpureomaculatus (mangrove pit viper) venom using high performance ion-exchange and gel filtration chromatography. The purified sample (termed purpurase) yielded a homogeneous band in SDS-polyacrylamide gel electrophoresis with a molecular weight of 35,000. The N-terminal sequence of purpurase was determined to be VVGGDECNINDHRSLVRIF and is homologous to many other venom thrombin-like enzymes. Purpurase exhibits both arginine ester hydrolase and amidase activities. Kinetic studies using tripeptide chromogenic anilide substrates showed that purpurase is not fastidious towards its substrate. The clotting times of fibrinogen by purpurase were concentration dependent, with optimum clotting activity at 3 mg fibronogen/mL. The clotting activity by purpurase was in the following decreasing order: cat fibrinogen > human fibrinogen > dog fibrinogen > goat fibrinogen >> rabbit fibrinogen. Reversed-phase HPLC analysis of the products of action of purpurase on bovine fibrinogen showed that only fibrinopeptide A was released. Indirect ELISA studies showed that anti-purpurase cross-reacted strongly with venoms of most crotalid venoms, indicating the snake venom thrombin-like enzymes generally possess similar epitopes. In the more specific double-sandwich ELISA, however, anti-purpurase cross-reacted only with venoms of certain species of the Trimeresurus complex, and the results support the recent proposed taxonomy changes concerning the Trimeresurus complex.     

Intramolecular proteolytic nicking and binding of Bacillus thuringiensis Cry8Da toxin in BBMVs of Japanese beetle

Bacillus thuringiensis (Bt) Cry8D insecticidal proteins are unique among Cry8 family proteins in terms of its insecticidal activity against adult Scarab beetles, such as Japanese beetle (Popillia japonica Newman). From the sequence homology with other Bt Cry proteins especially those active against beetles, such as Cry3Aa whose 3D structure is available, the structure of the Cry8D protein has been predicted to be a typical three-domain Cry protein type. In addition, the activation process of Cry8D in gut juice of susceptible insects is presumed to be similar to that of Cry3A (Yamaguchi et al., 2008). In this study, the activation process of Cry8Da in insect gut juice was closely examined. Japanese beetle gut juice proteases digested the 130 kDa Cry8Da protein to produce a 64 kDa protein. This 64 kDa protein was active against both adult and larval Japanese beetle and considered to be an activated toxin. N-terminal sequencing of this 64 kDa protein revealed that the Cry8Da leader sequence consisting of 63 amino acid residues from M¹ to F⁶³ was removed. As in the case of Cry3Aa, the proteases further digested the 64 kDa protein to two 8 kDa and 54 kDa fragments. N-terminal amino acid analysis of these smaller fragments indicated that the proteases digested the loop between Alpha Helix (Alpha for short) 3 and Alpha 4. This means that the 8 kDa fragment consists of Alpha 1-3 of Domain I and that the 54 kDa fragment contains the remaining Domain I and full Domain II and Domain III. Size exclusion chromatography and anion exchange chromatography could not separate these 64, 54 and 8 kDa proteins suggesting that the 54 kDa and 8 kDa fragments are still forming the toxin complex equivalent to the 64 kDa protein by size and ionic charge. The sequencing and chromatography results suggest that the gut juice proteases merely nicked the loop between Alpha 3 and Alpha 4. This nicking process appeared to be essential for receptor binding of the Cry8Da toxin. BBMV binding assay revealed that the Cry8Da toxin bound to BBMV preparations from both adult and larval Japanese beetle only after the loop was nicked. Only the 54 kDa fragment bound to the BBMV preparations but not the 64 kDa protein. Ligand blot showed that the protease activated Cry8Da toxin, presumably the 54 kDa fragment, bound to specific BBMV proteins, one or more of those would be receptor(s). The sizes and binding affinities of these Cry8Da-bound proteins of Japanese beetle BBMV differed between larvae and adults.     

Isolation, complete amino acid sequence and characterization of a previously unreported post-synaptic neurotoxin - AlphaN3, from the venom of Bungarus candidus

The Malayan krait (Bungarus candidus) is one of the medically most important snake species in Southeast Asia. The venom from this snake has been shown to posses both presynaptic and post-synaptic neurotoxins. We have isolated a previously uncharacterized post-synaptic neurotoxin - alphaN3 from the venom of B. candidus. Isolation of the toxin was achieved in three successive chromatography steps - gel filtration on a Sephadex G75 column, followed by ion exchange chromatography (Mono-S strong cationic exchanger) and a final reverse-phase chromatography step (PRO-RPC C18 column). Purified toxin alphaN3 was shown to have an apparent molecular weight of ∼7 to 8 kDa on SDS-PAGE. The complete amino acid sequence of toxin alphaN3 was determined by Edman degradation and was found to share a high degree of homology with known post-synaptic neurotoxins (93% with alpha-bungarotoxin from Bungarus multicinctus, 50% with alpha cobratoxin from Naja kaouthia). The intravenous LD₅₀ of toxin alphaN3 was determined to be 0.16 ± 0.09 μg/g in mice which is comparable to alpha-bungarotoxin from B. multicinctus. Experiments with isolated nerve-muscle preparations suggested that toxin alphaN3 was a post-synaptic neurotoxin that produced complete blockade of neuromuscular transmission by binding to nicotinic acetylcholine receptors.     

A novel phospholipase A2 from Agkistrodon blomhoffii ussurensis venom: Purification,proteomic, functional and structural characterizations

A phospholipase A₂ was isolated from the snake venom of Chinese Agkistrodon blomhoffii Ussurensis by column chromatography using DEAE Sephadex A-50 ion-exchange chromatography, Sephadex G-75 gel filtration chromatography and Mono Q ion-exchange chromatography, and designated as Akbu-PLA₂. It showed an average molecular mass of 13,980 ± 3 amu determined by MALDI TOF mass spectrometry. Protein identification results from HPLC-nESI-MS/MS analysis indicated that the Akbu-PLA₂ was a new snake venom acidic PLA₂. Seven peptides were sequenced from Akbu-PLA₂ by HPLC-nESI-MS/MS analysis. Sequencing alignment indicated that Akbu-PLA₂ shared homolog peptides of phospholipases A₂ from the venoms of Gloydius ussurensis, Gloydius halys, Gloydius halys (halys viper), Deinagkistrodon acutus and Agkistrodon halys Pallas. Akbu-PLA₂ has an optimum hydrolytic activity temperature of ∼45 °C. The intrinsic fluorescences of Tyr and Trp residues of Akbu-PLA₂ showed emission wavelengths red-shifted by 13.6 and 1.6 nm from those of free Tyr and Trp, respectively. Akbu-PLA₂ was shown to contain one Ca²⁺ per monomer by ICP-AES measurement. The Ca²⁺ ion was found to be critical for both the hydrolytic activity and the structure of Akbu-PLA₂. Ca²⁺ increased the emission fluorescence intensity and the hydrophobicity of the environment of Akbu-PLA₂. The hydrolytic activity of Akbu-PLA₂ was accelerated due to the addition of Ca²⁺ ion by enhancing the substrate binding. However, a protein component with the molecular weight two-fold relative to that of Akbu-PLA₂ was found to be difficult to eliminate for the purification of Akbu-PLA₂. HPLC-nESI-MS/MS detected the same peptides from it as from Abku-PLA₂, which indicated that it should be a homodimer of Akbu-PLA₂. A proteomic approach, 2D SDS-PAGE coupled to HPLC-nESI-MS/MS, supported the co-existence of the Akbu-PLA₂ monomer and dimer in the crude snake venom. Results from the combination of phosphoprotein and glycoprotein specific stains combined with the HPLC-nESI-MS/MS method indicated that both the Akbu-PLA₂ monomer and dimer were both phosphorylated and glycosylated. The addition of exogenous Ca²⁺ ion was found to be able to promote the dimer formation of Akbu-PLA₂. We conclude that a novel PLA₂ was successfully obtained. The systemically biochemical, proteomic, structural and functional characterization results from Akbu-PLA₂ reveal new threads and provide valuable inputs for the study of snake venom phospholipases A₂.     

The belonging of gpMuc, a glycoprotein from Mucuna pruriens seeds, to the Kunitz-type trypsin inhibitor family explains its direct anti-snake venom activity

In Nigeria, Mucuna pruriens seeds are locally prescribed as an oral prophylactic for snake bite and it is claimed that when two seeds are swallowed they protect the individual for a year against snake bites. In order to understand the Mucuna pruriens antisnake properties, the proteins from the acqueous extract of seeds were purified by three chromatographic steps: ConA affinity chromatography, tandem anionic-cationic exchange and gel filtration, obtaining a fraction conventionally called gpMucB. This purified fraction was analysed by SDS-PAGE obtaining 3 bands with apparent masses ranging from 20 to 24 kDa, and by MALDI-TOF which showed two main peaks of 21 and 23 kDa and another small peak of 19 kDa. On the other hand, gel filtration analysis of the native protein indicated a molecular mass of about 70 kDa suggesting that in its native form, gpMucB is most likely an oligomeric multiform protein. Infrared spectroscopy of gpMucB indicated that the protein is particularly thermostable both at neutral and acidic pHs and that it is an all beta protein.All data suggest that gpMucB belongs to the Kunitz-type trypsin inhibitor family explaining the direct anti-snake venom activity of Mucuna pruriens seeds.     

The self-assembly, elasticity, and dynamics of cardiac thin filaments

Solutions of intact cardiac thin filaments were examined with transmission electron microscopy, dynamic light scattering (DLS), and particle-tracking microrheology. The filaments self-assembled in solution with a bell-shaped distribution of contour lengths that contained a population of filaments of much greater length than the in vivo sarcomere size (∼1 μm) due to a one-dimensional annealing process. Dynamic semiflexible modes were found in DLS measurements at fast timescales (12.5 ns-0.0001 s). The bending modulus of the fibers is found to be in the range 4.5-16 × 10⁻²⁷ Jm and is weakly dependent on calcium concentration (with Ca²⁺ ≥ without Ca²⁺). Good quantitative agreement was found for the values of the fiber diameter calculated from transmission electron microscopy and from the initial decay of DLS correlation functions: 9.9 nm and 9.7 nm with and without Ca²⁺, respectively. In contrast, at slower timescales and high polymer concentrations, microrheology indicates that the cardiac filaments act as short rods in solution according to the predictions of the Doi-Edwards chopsticks model (viscosity, η ∼ c³, where c is the polymer concentration). This differs from the semiflexible behavior of long synthetic actin filaments at comparable polymer concentrations and timescales (elastic shear modulus, G′ ∼ c^1.4, tightly entangled) and is due to the relative ratio of the contour lengths (∼30). The scaling dependence of the elastic shear modulus on the frequency (ω) for cardiac thin filaments is G′ ∼ ω^{3/4 ± 0.03}, which is thought to arise from flexural modes of the filaments.     

Determination of hyaluronan molecular mass distribution in human breast milk

Hyaluronan (HA) in human milk mediates host responses to microbial infection via TLR4- and CD44-dependent signaling. Signaling by HA is generally size specific. Because pure HA with average molecular mass (M) of 35 kDa can elicit a protective response in intestinal epithelial cells, it has been proposed that human milk HA may have a bioactive low-M component. Here we report the size distribution of HA in human milk samples from 20 unique donors. A new method for HA analysis, employing ion exchange (IEX) chromatography to fractionate HA by size and specific quantification of each size fraction by competitive enzyme-linked sorbent assay (ELSA), was developed. When separated into four fractions, milk HA with M ? 20 kDa, M ∼ 20 to 60 kDa, and M ∼ 60 to 110 kDa comprised averages of 1.5, 1.4, and 2.0% of the total HA, respectively. The remaining 95% was HA with M ? 110 kDa. Electrophoretic analysis of the higher M HA from 13 samples showed nearly identical M distributions, with an average M of approximately 440 kDa. This higher M HA component in human milk is proposed to bind to CD44 and to enhance human beta defensin 2 (HBD2) induction by the low-M HA components.     

Desiccation induced oxidative stress and its biochemical responses in intertidal red alga Gracilaria corticata (Gracilariales, Rhodophyta)

Intertidal alga Gracilaria corticata growing in natural environment experiences various abiotic stresses during the low tides. The aim of this study was to determine whether desiccation exposure would lead to oxidative stress and its effect varies with exposure periods. This study gives an account of various biochemical changes in G. corticata following the exposure to desiccation for a period of 0 (control), 1, 2, 3 and 4 h under controlled conditions. During desiccation, G. corticata thalli showed dramatic loss of water by almost 47% when desiccated for 4 h. The enhanced production of reactive oxygen species (ROS) and increased lipid peroxidation observed during the exposure of 3-4 h were chiefly contributed by higher lipoxygenase (LOX) activity with the induction of two new LOX isoforms (LOX-2, ∼85 kDa; LOX-3, ∼65 kDa). The chlorophyll, carotenoids and phycobiliproteins (phycoerythrin and phycocyanin) were increased during initial 2 h exposure compared to control and thereafter declined in the succeeding exposure. The antioxidative enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), glutathione peroxidase (GPX) and the regeneration rate of reduced ascorbate (AsA) and glutathione (GSH) increased during desiccation up to 2-3 h. Further, the isoforms of antioxidant enzymes Mn-SOD (∼150 kDa), APX-4 (∼110 kDa), APX-5 (∼45 kDa), GPX-1 (∼80 kDa) and GPX-2 (∼65 kDa) responded specifically to the desiccation exposure. Compared to control, a relative higher content of both free and bound insoluble putrescine and spermine together with enhanced n-6 PUFAs namely C20:4(n-6) and C20:3(n-6) fatty acids found during 2 h exposure reveals their involvement in defence reactions against the desiccation induced oxidative stress.     

Comparative study of Bacillus thuringiensis Cry1Ia and Cry1Aa delta-endotoxins: Activation process and toxicity against Prays oleae

Cry1Ia and Cry1Aa proteins exhibited toxicities against Prays oleae with LC₅₀ of 189 and 116 ng/cm², respectively. The ability to process Cry1Ia11 protoxin by trypsin, chymotrypsin and P. oleae larvae proteases was studied and compared to that of Cry1Aa11. After solubilization under high alkaline condition (50 mM NaOH), Cry1Aa11 was converted into a major fragment of 65 kDa, whereas Cry1Ia11 protoxin was completely degraded by P. oleae larvae proteases and trypsin and converted into a major fragment of 70 kDa by chymotrypsin. Using less proteases of P. oleae juice, the degradation of Cry1Ia11 was attenuated. When the solubilization (in 50 mM Na₂CO₃ pH 10.5 buffer) and activation were combined, Cry1Ia11 was converted into a proteolytic product of 70 kDa after 3 h of incubation with trypsin, chymotrypsin and P. oleae juice. These results suggest that the in vivo solubilization of Cry1Ia11 was assured by larval proteases after a swelling of the corresponding inclusion due to the alkalinity of the larval midgut.     

A novel hepatopancreatic phospholipase A2 from Hexaplex trunculus with digestive and toxic activities

A marine snail digestive phospholipase A₂ (mSDPL) was purified from delipidated hepatopancreas. Unlike known digestive phospholipases A₂, which are 14 kDa proteins, the purified mSDPL has a molecular mass of about 30 kDa. It has a specific activity of about 180 U/mg measured at 50 °C and pH 8.5 using phosphatidylcholine liposomes as a substrate in the presence of 4 mM NaTDC and 6 mM CaCl₂. The N-terminal amino-acid of the purified mSDPL does not share any homology with known phospholipases.Moreover, the mSDPL exhibits hemolytic activity in intact erythrocytes and can penetrate phospholipid monolayers at high surface pressure, comparable to snake venom PLA₂. These observations suggest that mSDPL could be toxic to mammal cells. However, mSDPL can be classified as a member of a new family of enzymes. It should be situated between the class of toxic phospholipase A₂ from venoms and another class of non toxic pancreatic phospholipase A₂ from mammals.     

An acidic phospholipase A₂ with antibacterial activity from Porthidium nasutum snake venom

Snake venoms are complex mixtures of proteins among which both basic and acidic phospholipases A₂ (PLA₂s) can be found. Basic PLA₂s are usually responsible for major toxic effects induced by snake venoms, while acidic PLA₂s tend to have a lower toxicity. A novel PLA₂, here named PnPLA₂, was purified from the venom of Porthidium nasutum by means of RP-HPLC on a C18 column. PnPLA₂ is an acidic protein with a pI of 4.6, which migrates as a single band under both non-reducing and reducing conditions in SDS-PAGE. PnPLA₂ had a molecular mass of 15,802.6 Da, determined by ESI-MS. Three tryptic peptides of this protein were characterized by HPLC-nESI-MS/MS, and N-terminal sequencing by direct Edman degradation showing homology to other acidic PLA₂s from viperid venoms. PnPLA₂ displayed indirect hemolytic activity in agarose erythrocyte-egg yolk gels and bactericidal activity against Staphylococcus aureus in a dose-dependent manner, with a MIC and MBC of 32 μg/mL. In addition, PnPLA₂ showed a potent inhibitory effect on platelet aggregation with doses up to 40 μg/mL. This acidic PLA₂, in contrast to basic enzymes isolated from other viperid snake venoms, was not cytotoxic to murine skeletal muscle myoblasts C₂C₁₂. This is the first report on a bactericidal protein of Porthidium nasutum venom.     

Ginger rhizome as a potential source of milk coagulating cysteine protease

A milk coagulating protease was purified ∼10.2-fold to apparent homogeneity from ginger rhizomes in 34.9% recovery using ammonium sulfate fractionation, together with ion exchange and size exclusion chromatographic techniques. The molecular mass of the purified protease was estimated to be ∼36 kDa by SDS-PAGE, and exhibited a pI of 4.3. It is a glycoprotein with 3% carbohydrate content. The purified enzyme showed maximum activity at pH 5.5 and at a temperature of ∼60 °C. Its protease activity was strongly inhibited by iodoacetamide, E-64, PCMB, Hg²⁺ and Cu²⁺. Inhibition studies and N-terminal sequence classified the enzyme as a member of the cysteine proteases. The cleavage capability of the isolated enzyme was higher for α_s-casein followed by β- and κ-casein. The purified enzyme differed in molecular mass, pI, carbohydrate content, and N-terminal sequence from previously reported ginger proteases. These results indicate that the purified protease may have potential application as a rennet substitute in the dairy industry.     

Cloning and characterisation of novel cystatins from elapid snake venom glands

Snake venoms contain a complex mixture of polypeptides that modulate prey homeostatic mechanisms through highly specific and targeted interactions. In this study we have identified and characterised cystatin-like cysteine-protease inhibitors from elapid snake venoms for the first time. Novel cystatin sequences were cloned from 12 of 13 elapid snake venom glands and the protein was detected, albeit at very low levels, in a total of 22 venoms. One highly conserved isoform, which displayed close sequence identity with family 2 cystatins, was detected in each elapid snake. Crude Austrelaps superbus (Australian lowland copperhead) snake venom inhibited papain, and a recombinant form of A. superbus cystatin inhibited cathepsin L ≅ papain > cathepsin B, with no inhibition observed for calpain or legumain. While snake venom cystatins have truncated N-termini, sequence alignment and structural modelling suggested that the evolutionarily conserved Gly-11 of family 2 cystatins, essential for cysteine protease inhibition, is conserved in snake venom cystatins as Gly-3. This was confirmed by mutagenesis at the Gly-3 site, which increased the dissociation constant for papain by 10⁴-fold. These data demonstrate that elapid snake venom cystatins are novel members of the type 2 family. The widespread, low level expression of type 2 cystatins in snake venom, as well as the presence of only one highly conserved isoform in each species, imply essential housekeeping or regulatory roles for these proteins.     

Viperidae snake venoms block nicotinic acetylcholine receptors and voltage-gated Ca2+ channels in identified neurons of fresh-water snail Lymnaea stagnalis

Snake venoms contain a vast array of toxic polypeptide components interacting with a variety of cell targets. Thus, Elapidae snake venoms contain α-neurotoxins with very high affinity for nicotinic acetylcholine receptors (nAChRs) and a few toxins able to suppress the activity of Ca²⁺ and K⁺ channels. Experimental evidence for the presence of nAChR antagonists and voltage-gated ionic channel blockers in venoms of Viperidae snakes is very scarce. In this study, effects of crude venoms of seven snake species (Vipera nikolskii, Echis multisquamatus, Gloydius saxatilis, Bitis arietans, Vipera renardi, Vipera lebetina, and Naja kaouthia) on nAChRs and voltage-gated Ca²⁺ channels were studied for the first time. The experiments were carried out on isolated identified neurons of the fresh-water mollusc Lymnaea stagnalis using voltage-clamp and intracellular perfusion techniques. All Viperidae snake venoms under study blocked nAChRs and voltage-gated Ca²⁺ channels. The potency of these venoms against nAChRs was significantly lower in comparison with N. kaouthia venom which is rich of α-neurotoxins; however, the extent of Ca²⁺ channel block by venoms of Viperidae snakes and N. kaouthia was similar. The data obtained suggest that Viperidae snake venoms tested in this study contain peptides with affinity both for nAChRs and for voltage-gated Ca²⁺ channels.     

Purification and characterization of patagonfibrase,a metalloproteinase showing α-fibrinogenolytic and hemorrhagic activities,from Philodryas patagoniensis snake venom

Venoms of Colubridae snakes are a rich source of novel compounds, which may have applications in medicine and biochemistry. In the present study, we describe the purification and characterization of a metalloproteinase (patagonfibrase), the first protein to be isolated from Philodryas patagoniensis (Colubridae) snake venom. Patagonfibrase is a single-chain protein, showing a molecular mass of 53,224 Da and an acidic isoelectric point (5.8). It hydrolyzed selectively the Aα-chain of fibrinogen and when incubated with fibrinogen or plasma, the thrombin clotting time was prolonged. Prominent hemorrhage developed in mouse skin after intradermal injection of patagonfibrase. When administered into mouse gastrocnemius muscle, it induced local hemorrhage and necrosis, and systemic bleeding in lungs. Patagonfibrase showed proteolytic activity toward azocasein, which was enhanced by Ca²⁺ and inhibited by Zn²⁺, cysteine, dithiothreitol and Na₂EDTA. Patagonfibrase impaired platelet aggregation induced by collagen and ADP. Thus, patagonfibrase may play a key role in the pathogenesis of disturbances that occur in P. patagoniensis envenomation, and may be used as a biological tool to explore many facets of hemostasis.     

A broad glass transition in hydrated proteins

We performed Raman and Brillouin scattering measurements to estimate glass transition temperature, T_g, of hydrated protein. The measurements reveal very broad glass transition in hydrated lysozyme with approximate T_g ∼ 180 ± 15 K. This result agrees with a broad range of T_g ∼ 160–200 K reported in literature for hydrated globular proteins and stresses the difference between behavior of hydrated biomolecules and simple glass-forming systems. Moreover, the main structural relaxation of the hydrated protein system that freezes at T_g ∼ 180 K remains unknown. We emphasize the difference between the “dynamic transition”, known as a sharp rise in mean-squared atomic displacement <r²> at temperatures around T_D ∼ 200–230 K, and the glass transition. They have different physical origin and should not be confused.