Analysis of intraspecific variation in venoms of Acanthophis antarcticus death adders from South Australia.

Intraspecific variation in venom composition and activity has been reported from a wide range of snakes. Geographical origin can be one cause for this variation and has recently been documented from Acanthophis antarcticus death adders sampled across four different Australian states. The present study examined whether a narrower sampling range of A. antarcticus from four collection sites within one Australian state (i.e., South Australia) would also exhibit variation in venom composition and/or activity. The present LC-MS results reveal marked differences in the venom composition from different collection sites. The most striking difference was the reduced venom complexity found in the only venom originating from a mallee scrub habitat in comparison to the venoms from coastal heath scrub habitats. Interestingly, the pharmacological activity of all venoms was found to be the same, independent of the collection site.

The x-ray crystal structure of mannose-binding lectin-associated serine proteinase-3 reveals the structural basis for enzyme inactivity associated with the Carnevale, Mingarelli, Malpuech, and Michels (3MC) syndrome.

The mannose-binding lectin associated-protease-3 (MASP-3) is a member of the lectin pathway of the complement system, a key component of human innate and active immunity. Mutations in MASP-3 have recently been found to be associated with Carnevale, Mingarelli, Malpuech, and Michels (3MC) syndrome, a severe developmental disorder manifested by cleft palate, intellectual disability, and skeletal abnormalities. However, the molecular basis for MASP-3 function remains to be understood. Here we characterize the substrate specificity of MASP-3 by screening against a combinatorial peptide substrate library. Through this approach, we successfully identified a peptide substrate that was 20-fold more efficiently cleaved than any other identified to date. Furthermore, we demonstrated that mutant forms of the enzyme associated with 3MC syndrome were completely inactive against this substrate. To address the structural basis for this defect, we determined the 2.6-Å structure of the zymogen form of the G666E mutant of MASP-3. These data reveal that the mutation disrupts the active site and perturbs the position of the catalytic serine residue. Together, these insights into the function of MASP-3 reveal how a mutation in this enzyme causes it to be inactive and thus contribute to the 3MC syndrome.

S1 pocket of a bacterially derived subtilisin-like protease underpins effective tissue destruction.

The ovine footrot pathogen, Dichelobacter nodosus, secretes three subtilisin-like proteases that play an important role in the pathogenesis of footrot through their ability to mediate tissue destruction. Virulent and benign strains of D. nodosus secrete the basic proteases BprV and BprB, respectively, with the catalytic domain of these enzymes having 96% sequence identity. At present, it is not known how sequence variation between these two putative virulence factors influences their respective biological activity. We have determined the high resolution crystal structures of BprV and BprB. These data reveal that that the S1 pocket of BprV is more hydrophobic but smaller than that of BprB. We show that BprV is more effective than BprB in degrading extracellular matrix components of the host tissue. Mutation of two residues around the S1 pocket of BprB to the equivalent residues in BprV dramatically enhanced its proteolytic activity against elastin substrates. Application of a novel approach for profiling substrate specificity, the Rapid Endopeptidase Profiling Library (REPLi) method, revealed that both enzymes prefer cleaving after hydrophobic residues (and in particular P1 leucine) but that BprV has more restricted primary substrate specificity than BprB. Furthermore, for P1 Leu-containing substrates we found that BprV is a significantly more efficient enzyme than BprB. Collectively, these data illuminate how subtle changes in D. nodosus proteases may significantly influence tissue destruction as part of the ovine footrot pathogenesis process.

The subtilisin-like protease AprV2 is required for virulence and uses a novel disulphide-tethered exosite to bind substrates.

Many bacterial pathogens produce extracellular proteases that degrade the extracellular matrix of the host and therefore are involved in disease pathogenesis. Dichelobacter nodosus is the causative agent of ovine footrot, a highly contagious disease that is characterized by the separation of the hoof from the underlying tissue. D. nodosus secretes three subtilisin-like proteases whose analysis forms the basis of diagnostic tests that differentiate between virulent and benign strains and have been postulated to play a role in virulence. We have constructed protease mutants of D. nodosus; their analysis in a sheep virulence model revealed that one of these enzymes, AprV2, was required for virulence. These studies challenge the previous hypothesis that the elastase activity of AprV2 is important for disease progression, since aprV2 mutants were virulent when complemented with aprB2, which encodes a variant that has impaired elastase activity. We have determined the crystal structures of both AprV2 and AprB2 and characterized the biological activity of these enzymes. These data reveal that an unusual extended disulphide-tethered loop functions as an exosite, mediating effective enzyme-substrate interactions. The disulphide bond and Tyr92, which was located at the exposed end of the loop, were functionally important. Bioinformatic analyses suggested that other pathogenic bacteria may have proteases that utilize a similar mechanism. In conclusion, we have used an integrated multidisciplinary combination of bacterial genetics, whole animal virulence trials in the original host, biochemical studies, and comprehensive analysis of crystal structures to provide the first definitive evidence that the extracellular secreted proteases produced by D. nodosus are required for virulence and to elucidate the molecular mechanism by which these proteases bind to their natural substrates. We postulate that this exosite mechanism may be used by proteases produced by other bacterial pathogens of both humans and animals.

Variations in the pharmacological profile of post-synaptic neurotoxins isolated from the venoms of the Papuan (Oxyuranus scutellatus canni) and coastal (Oxyuranus scutellatus scutellatus) taipans.

Based on murine LD(50) values, the taipans (i.e. Oxyuranus microlepidotus, Oxyuranus scutellatus and Oxyuranus scutellatus canni) are the most venomous snake genus in the world. Despite this, little is known about the toxins contained in their venoms. The aim of the present study was to isolate and characterise post-synaptic neurotoxins from the venoms of the Papuan taipan (O. s. canni) and coastal taipan (O. scutellatus), and to compare their pharmacology. A 6770Da toxin (i.e. alpha-oxytoxin 1) and a 6781Da toxin (i.e. alpha-scutoxin 1) were isolated from the venoms of O. s. canni and O. scutellatus, respectively, using reverse-phase high performance liquid chromatography. Both alpha-oxytoxin 1 (0.3-1 microM) and alpha-scutoxin 1 (0.1-1 microM) caused concentration-dependent inhibition of indirect twitches in the chick biventer cervicis nerve-muscle preparation. Contractile responses to exogenous carbachol (CCh), but not potassium chloride (KCl), were inhibited by both toxins, suggesting a post-synaptic mode of action. The inhibitory effect of alpha-oxytoxin 1 was reversed by washing. Cumulative concentration-response curves to CCh were obtained in the presence and absence of the toxins with the subsequently determined pA(2) of alpha-scutoxin 1 being 44.7-fold higher than alpha-oxytoxin 1 (i.e. 8.38+/-0.59 versus 7.62+/-0.04). The current study shows that Papuan taipan and coastal taipan venom both contain potent post-synaptic neurotoxins which exhibit different pharmacological profiles. The effect of alpha-oxytoxin 1 is atypical of most snake venom post-synaptic neurotoxins displaying a 'competitive' mode of action, whereas alpha-scutoxin 1 possesses pseudo-irreversible or non-competitive activity.

The innate immune molecule, NOD1, regulates direct killing of Helicobacter pylori by antimicrobial peptides.

The cytosolic innate immune molecule, NOD1, recognizes peptidoglycan (PG) delivered to epithelial cells via the Helicobacter pylori cag pathogenicity island (cagPAI), and has been implicated in host defence against cagPAI(+)H. pylori bacteria. To further clarify the role of NOD1 in host defence, we investigated NOD1-dependent regulation of human beta-defensins (DEFBs) in two epithelial cell lines. Our findings identify that NOD1 activation, via either cagPAI(+) bacteria or internalized PG, was required for DEFB4 and DEFB103 expression in HEK293 cells. To investigate cell type-specific induction of DEFB4 and DEFB103, we generated stable NOD1'knockdown' (KD) and control AGS cells. Reporter gene assay and RT-PCR analyses revealed that only DEFB4 was induced in an NOD1-/cagPAI-dependent fashion in AGS cells. Moreover, culture supernatants from AGS control, but not AGS NOD1 KD cells, stimulated with cagPAI(+)H. pylori, significantly reduced H. pylori bacterial numbers. siRNA studies confirmed that human beta-defensin 2 (hBD-2), but not hBD-3, contributes to the antimicrobial activity of AGS cell supernatants against H. pylori. This study demonstrates, for the first time, the involvement of NOD1 and hBD-2 in direct killing of H. pylori bacteria by epithelial cells and confirms the importance of NOD1 in host defence mechanisms against cagPAI(+)H. pylori infection.

Aberrant protein expression in plasma and kidney tissue during experimental obstructive nephropathy.

Kidney failure is a major health problem worldwide. Patients with end-stage renal disease require intensive medical support by dialysis or kidney transplantation. Current methods for diagnosis of kidney disease are either invasive or insensitive, and renal function may decline by as much as 50% before it can be detected using current techniques. The goal of this study was, therefore, to identify biomarkers of kidney disease (associated with renal fibrosis) that can be used for the development of a non-invasive clinical test for early disease detection. We utilized two protein-profiling technologies (SELDI-TOF MS and 2-D) to screen the plasma and kidney proteome for aberrantly expressed proteins in an experimental mouse model of unilateral uretric obstruction, which mimics the pathology of human renal disease. Several differentially regulated proteins were detected at the plasma level of day-3-obstructed animals, which included serum amyloid A1, fibrinogen α, haptoglobin precursor protein, haptoglobin and major urinary proteins 11 and 8. Differentially expressed proteins detected at the tissue level included ras-like activator protein 2, haptoglobin precursor protein, malate dehydrogenase, α enolase and murine urinary protein (all p<0.05 versus controls). Immunohistochemistry was used to confirm the up-regulation of fibrinogen. Interestingly, these proteins are largely separated into four major classes: (i) acute-phase reactants (ii) cell-signaling molecules (iii) molecules involved in cell growth and metabolism and (iv) urinary proteins. These results provide new insights into the pathology of obstructive nephropathy and may facilitate the development of specific assay(s) to detect and monitor renal fibrosis.

Characterisation of endothelin converting enzyme-1 shedding from endothelial cells.

The aim of this study was to determine if endothelin converting enzyme-1 (ECE-1) like other members of this metalloprotease family undergoes ectodomain shedding. The release/shedding of catalytically active ECE-1 was measured by monitoring the fluorescence resulting from the cleavage of a specific quenched fluorescent substrate. Catalytically active ECE-1 was detected in the media of human umbilical vein endothelial cells, and was confirmed by mass spectrometry based assays. Specificity of cleavage was confirmed by using both narrow and broad specificity inhibitors. In conclusion we demonstrate and characterize for the first time, ECE-1 shedding from the surface of endothelial cells.

Oxylepitoxin-1, a reversible neurotoxin from the venom of the inland taipan (Oxyuranus microlepidotus).

This study describes the characterization of oxylepitoxin-1 (MW 6789), the first postsynaptic neurotoxin isolated from the venom of the Inland taipan (Oxyuranus microlepidotus), which is the most venomous snake in the world. Oxylepitoxin-1, purified using successive steps of size-exclusion and reverse phase-high performance liquid chromatography, produced concentration-dependent (0.3-1.0 microM) inhibition of nerve-mediated (0.1 Hz, 0.2 ms, supramaximal V) twitches of the chick biventer cervicis nerve-muscle preparation. Taipan antivenom (5units/ml) prevented the neurotoxic activity of whole venom (10 microg/ml), but had no significant effect on oxylepitoxin-1 (1 microM). The toxin-induced inhibition of nerve-mediated twitches was significantly reversed upon washing the tissue at 5 min intervals. Oxylepitoxin-1 (30-300 nM) displayed competitive antagonism at the skeletal muscle nicotinic receptor with a pA(2) value of 7.16+/-0.28 (i.e. approximately 10-fold more potent than tubocurarine). The venom had a high level of PLA(2) activity (765+/-73 micromol/min/mg) while oxylepitoxin-1 displayed no PLA(2) activity. Partial N-terminal sequencing of oxylepitoxin-1 shows high sequence identity (i.e. 93%) to postsynaptic toxins isolated from the venom of the closely related coastal taipan (Oxyuranus scutellatus scutellatus).

Isolation and pharmacological characterisation of hostoxin-1, a postsynaptic neurotoxin from the venom of the Stephen's banded snake (Hoplocephalus stephensi).

Envenoming by the Stephen's banded snake (Hoplocephalus stephensi) is not usually characterised by neurotoxicity. The present study describes the pharmacological characterisation of hostoxin-1 (MW 6660 Da), the first neurotoxin to be isolated from the venom of the Stephen's banded snake. Hostoxin-1 (0.3-1.0 microM) caused concentration-dependent inhibition of indirect twitches of the chick biventer cervicis nerve-muscle preparation. The neurotoxic activity of hostoxin-1 (0.3 microM) was irreversible by washing, but significantly reversed by the addition of CSL tiger snake antivenom (5 units/ml) added at t90 (i.e. time at which twitches were inhibited by 90%). In addition, hostoxin-1 (0.3 microM) inhibited responses to exogenous acetylcholine and carbachol, but not KCl, indicating a postsynaptic mode of action. Hostoxin-1 (5-30 nM) displayed pseudo-irreversible antagonism at the skeletal muscle nicotinic receptor with a pA2 value of 8.45+/-0.32 (i.e. approximately 100-fold more potent than tubocurarine). H. stephensi venom displayed a high level of PLA2 activity (specific activity 100.1+/-4.4 micromol/min/mg). However, the activity of hostoxin-1 was negligible. Partial N-terminal sequencing of hostoxin-1 indicates that it has high sequence homology with other elapid short-chain neurotoxins.

Proteomic tracking of serum protein isoforms as screening biomarkers of ovarian cancer.

Epithelial ovarian cancer is the fourth leading cause of cancer death among women. Due to the asymptomatic nature and poor survival characteristic of the disease, screening for specific biomarkers for ovarian cancer is a major health priority. Differentially expressed proteins in the serum of ovarian cancer patients have the potential to be used as cancer-specific biomarkers. In this study, proteomic methods were used to screen 24 serum samples from women with high-grade ovarian cancer and compared to a control group of 11 healthy women. Affigel-Blue treated serum samples were processed either by linear (pH 4-7) or narrow range (pH 5.5-6.7) IEF strips for the first dimension. Proteins separated in first dimension were resolved by 8-16% gradient SDS-PAGE. Protein spots were visualized by SYPRO Ruby staining, imaged by FX-imager and compared and analyzed by PDQuest software. Twenty-two protein spots were consistently differentially expressed between normal and ovarian cancer patients by resolving proteins in a linear pH strip of 4-7 for the first dimension. Six of the protein spots, significantly up-regulated in grade 3 ovarian cancer patients (p < 0.05), were identified by MALDI-TOF MS and Western blotting as the isoforms of haptoglobin precursor. When serum proteins were resolved on narrow pH range strips (5.5-6.7), 23 spots were consistently differentially expressed between normal and grade 3 ovarian cancer patients. Of these, 4 protein spots significantly down regulated in grade 3 ovarian cancer patients (p < 0.05) were identified by MALDI-TOF MS and Western blotting, as isoforms of transferrin precursor. Increased expression of serum haptoglobin and transferrin was also identified in peritoneal tumor fluid obtained from women diagnosed with grade 2/3 ovarian cancer (n = 7). Changes in the expression of haptoglobin and transferrin in the serum of women with different pathological grades of ovarian cancer was examined by one-dimensional Western blotting method. Serum samples collected from women suffering from benign, borderline, grade 1, grade 2 and grade 3 cancer (n = 4 for haptoglobin and n = 5 for transferrin in each group) were analyzed and compared to the serum of normal healthy women. The mean serum haptoglobin expression in grade 3 ovarian cancer patients was fourfold higher than in the control subjects (p < 0.05). On the other hand, transferrin expression in grade 3 ovarian cancer patients was decreased by twofold than in normal healthy women (p < 0.05). Haptoglobin expression in the serum of cancer patients (n = 7) decreased following chemotherapy (six cycles of taxol/carboplatin). Concomitant with the decrease of haptoglobin, transferrin expression remained constant in four patients, but increased in three out of seven patients included in the study. Changes in serum expression of haptoglobin correlated with the change of CA 125 levels before and after chemotherapy. In conclusion, proteomic profiling of differentially expressed proteins in the sera of normal women compared to women with ovarian cancer can greatly facilitate the discovery of a panel of biomarkers that may aid in the detection of ovarian cancer with greater specificity.

Isolation and characterisation of acanmyotoxin-2 and acanmyotoxin-3, myotoxins from the venom of the death adder Acanthophis sp. Seram.

Death adder (genus Acanthophis) venoms display neurotoxic activity but were thought to be devoid of myotoxic components. Studies from our laboratory have shown that some species (i.e. Acanthophis rugosus and Acanthophis sp. Seram) possess venom with myotoxic activity [Wickramaratna JC, Fry BG, Aguilar M, Kini RM, Hodgson WC. Isolation and pharmacological characterisation of a phospholipase A2 myotoxin from the venom of the Irian Jayan death adder (A. rugosus). Br J Pharmacol 2003;138:333-342; Wickramaratna JC, Fry BG, Hodgson WC. Species-dependent variations in the in vitro myotoxicity of death adder (Acanthophis) venoms. Toxicol Sci 2003;74:352-360]. The present study describes the isolation and characterisation of two myotoxins (acanmyotoxin-2 and acanmyotoxin-3) from A. sp. Seram venom. Venom was fractionated into approximately 12 major peaks using reverse phase high performance liquid chromatography. Two components caused concentration (0.1-1 microM) dependent inhibition of direct (2 ms, 0.1 Hz, supramaximal V) twitches and an increase in baseline tension in the chick biventer cervicis nerve-muscle. Histological examination of the muscle confirmed damage. PLA2 activity was detected in both acanmyotoxin-2 (390.2+/-19.7 micromol/(min mg); n=4) and acanmyotoxin-3 (14.2+/-7.7 micromol/(min mg); n=4). In comparison, A. sp. Seram whole venom had a specific activity of 461.3+/-90.4 micromol/(min mg) (n=3). Mass spectrometry analysis indicated acanmyotoxin-2 had a mass of 13,082 Da and acanmyotoxin-2 13,896 Da. Acanmyotoxin-2 and acanmyotoxin-3 accounted for approximately 7 and 4% of total venom composition, respectively. N-terminal sequencing of the first 30 amino acids of each toxin indicated they shared some sequence homology with known myotoxins. In conclusion, clinicians should be aware that symptoms of envenoming by some species of death adder may include signs of myotoxicity as well as neurotoxicity. Future studies will investigate the efficacy of the current antivenom treatment against the myotoxic components of A. sp. Seram venom.

Isolation and pharmacological characterization of cannitoxin, a presynaptic neurotoxin from the venom of the Papuan Taipan (Oxyuranus scutellatus canni).

The Papuan taipan (Oxyuranus scutellatus canni) is widely distributed throughout much of Papua New Guinea. Although neurotoxicity is a major symptom of envenomation, no neurotoxins have been isolated from this venom. Using a series of size exclusion chromatography steps, we report the isolation of cannitoxin, a presynaptic neurotoxin (44,848 Da) that represents approximately 16% of the whole venom. The toxin displayed high phospholipase A2 (PLA2 activity (330 +/- 5 micromol/min/mg) and caused concentration-dependent (11-66 nM) inhibition of indirect (0.2 ms; 0.1 Hz; supramaximal V) twitches of the chick biventer cervicis nerve-muscle preparation without effecting nicotinic receptor agonists. Prior addition of CSL Taipan antivenom (5 U/ml) or inhibition of phospholipase A2 activity by incubation with 4-bromophenacyl bromide prevented the inhibition of twitches. Cannitoxin is composed of three different subunits, alpha, beta, and gamma, with the possibility of two beta isomers. However, only the alpha subunit displayed in vitro neurotoxic activity of its own. Thus, cannitoxin is similar in structure and pharmacology to taipoxin, which has been isolated from the closely related Australian species O. scutellatus scutellatus (coastal taipan).

Isolation and pharmacological characterisation of papuantoxin-1, a postsynaptic neurotoxin from the venom of the Papuan black snake (Pseudechis papuanus).

The Papuan black snake (Pseudechis papuanus) is found throughout the southern coastal regions of Papua New Guinea and is thought to occur in the adjacent region of Iriyan Jaya. Neurotoxicity is a major symptom of envenomation by this species. This study describes the isolation of the first neurotoxin papuantoxin-1 from the venom of P. papuanus. Papuantoxin-1 (6738Da), which accounts for approximately 5% of the whole venom, was purified to homogeneity using successive steps of RP-HPLC. The toxin (0.3-1.0 microM) caused concentration dependent inhibition of indirect twitches (0.1 Hz, 0.2 ms and supramaximal V) and inhibited the responses to nicotinic agonists in the chick biventer cervicis nerve-muscle preparation, indicating a postsynaptic mode of action. However, papuantoxin-1 displayed no signs of myotoxicity. Papuantoxin-1 displayed pseudo-irreversible antagonism of cumulative concentration-response curves to carbachol at the skeletal muscle nicotinic receptors with an estimated pA2 value of 6.9+/-0.3. CSL black snake antivenom, which is raised against the venom of the Australian black snake Pseudechis australis, appears to be effective in reversing the effects of papuantoxin-1. Thus, black snake antivenom should be considered for the treatment of the neurotoxic effects following envenomation by the Papaun black snake.

The high resolution crystal structure of a native thermostable serpin reveals the complex mechanism underpinning the stressed to relaxed transition.

Serpins fold into a native metastable state and utilize a complex conformational change to inhibit target proteases. An undesirable result of this conformational flexibility is that most inhibitory serpins are heat sensitive, forming inactive polymers at elevated temperatures. However, the prokaryote serpin, thermopin, from Thermobifida fusca is able to function in a heated environment. We have determined the 1.8 A x-ray crystal structure of thermopin in the native, inhibitory conformation. A structural comparison with the previously determined 1.5 A structure of cleaved thermopin provides detailed insight into the complex mechanism of conformational change in serpins. Flexibility in the shutter region and electrostatic interactions at the top of the A beta-sheet (the breach) involving the C-terminal tail, a unique structural feature of thermopin, are postulated to be important for controlling inhibitory activity and triggering conformational change, respectively, in the native state. Here we have discussed the structural basis of how this serpin reconciles the thermodynamic instability necessary for function with the stability required to withstand elevated temperatures.

Endopeptidases 3.4.24.15 and 24.16 in endothelial cells: potential role in vasoactive peptide metabolism.

The closely related metalloendopeptidases EC (EP24.15; thimet oligopeptidase) and 24.16 (EP24.16; neurolysin) cleave a number of vasoactive peptides such as bradykinin and neurotensin in vitro. We have previously shown that hypotensive responses to bradykinin are potentiated by an inhibitor of EP24.15 and EP24.16 (26), suggesting a role for one or both enzymes in bradykinin metabolism in vivo. In this study, we have used selective inhibitors that can distinguish between EP24.15 and EP24.16 to determine their activity in cultured endothelial cells (the transformed human umbilical vein endothelial hybrid cell line EA.hy926 or ovine aortic endothelial cells). Endopeptidase activity was assessed using a specific quenched fluorescent substrate [7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-d-Lys(2,4-dinitrophenyl)], as well as the peptide substrates bradykinin and neurotensin (assessed by high-performance liquid chromatography with mass spectroscopic detection). Our results indicate that both peptidases are present in endothelial cells; however, EP24.16 contributes significantly more to substrate cleavage by both cytosolic and membrane preparations, as well as intact cells, than EP24.15. These findings, when coupled with previous observations in vivo, suggest that EP24.16 activity in vascular endothelial cells may play an important role in the degradation of bradykinin and/or other peptides in the circulation.

Albumin fragments in normal rat urine are derived from rapidly degraded filtered albumin.

Filtered albumin is excreted as a heterogeneous population of albumin-derived molecules resulting from degradation during renal passage. In order to understand the dynamics of this degradation process, albumin clearance was studied over a short-term (minutes) and a long-term (7 days) by both radioactivity and radioimmunoassay. The radiolabelled material in the urine was also analysed extensively by using size exclusion chromatography, size selective filtration and high performance liquid chromatography. These studies demonstrate that during renal passage, albumin degradation to fragments in the size range of 500-10,000 occurs in a matter of minutes. The fragments are not detected by using radioimmunoassay. Steady state excretion rates or fractional clearance of radiolabelled albumin occur over a similar time period. Both rates of degradation and approach to steady-state clearance, while rapid, were considerably slower than the transit time for molecules in the Bowman's capsule and early tubular lumen. The results are consistent with an extremely rapid lysosomal uptake of filtered albumin, and degradation and regurgitation of the albumin-derived peptide fragments into the tubular lumen prior to excretion.