Cloning and functional expression of venom prothrombin activator protease from Pseudonaja textilis with whole blood procoagulant activity

The snake venom group C prothrombin activators contain a number of components that enhance the rate of prothrombin activation. The cloning and expression of full-length cDNA for one of these components, an activated factor X (factor Xa)-like protease from Pseudonaja textilis as well as the generation of functional chimeric constructs with procoagulant activity were described. The complete cDNA codes for a propeptide, light chain, activation peptide (AP) and heavy chain related in sequence to mammalian factor X. Efficient expression of the protease was achieved with constructs where the AP was deleted and the cleavage sites between the heavy and light chains modified, or where the AP was replaced with a peptide involved in insulin receptor processing. In human kidney cells (H293F) transfected with these constructs, up to 80% of the pro-form was processed to heavy and light chains. Binding of the protease to barium citrate and use of specific antibodies demonstrated that gamma-carboxylation of glutamic acid residues had occurred on the light chain in both cases, as observed in human factor Xa and the native P. textilis protease. The recombinant protease caused efficient coagulation of whole citrated blood and citrated plasma that was enhanced by the presence of Ca2+. This study identified the complete cDNA sequence of a factor Xa-like protease from P. textilis and demonstrated for the first time the expression of a recombinant form of P. textilis protease capable of blood coagulation.

Comparative analysis of prothrombin activators from the venom of Australian elapids

A key component of the venom of many Australian snakes belonging to the elapid family is a toxin that is structurally and functionally similar to that of the mammalian prothrombinase complex. In mammals, this complex is responsible for the cleavage of prothrombin to thrombin and is composed of factor Xa in association with its cofactors calcium, phospholipids, and factor Va. The snake prothrombin activators have been classified on the basis of their requirement for cofactors for activity. The two major subgroups described in Australian elapid snakes, groups C and D, are differentiated by their requirement for mammalian coagulation factor Va. In this study, we describe the cloning, characterization, and comparative analysis of the factor X- and factor V-like components of the prothrombin activators from the venom glands of snakes possessing either group C or D prothrombin activators. The overall domain arrangement in these proteins was highly conserved between all elapids and with the corresponding mammalian clotting factors. The deduced protein sequence for the factor X-like protease precursor, identified in elapids containing either group C or D prothrombin activators, demonstrated a remarkable degree of relatedness to each other (80%-97%). The factor V-like component of the prothrombin activator, present only in snakes containing group C complexes, also showed a very high degree of homology (96%-98%). Expression of both the factor X- and factor V-like proteins determined by immunoblotting provided an additional means of separating these two groups at the molecular level. The molecular phylogenetic analysis described here represents a new approach for distinguishing group C and D snake prothrombin activators and correlates well with previous classifications.

Comparison of active venom components between Eastern brown snakes collected from South Australia and Queensland

The abundance and activity of the prothrombin activator (pseutarin C) within the venom of the Eastern brown snake (Pseudonaja textilis textilis) is the primary determinant of its coagulation potency. Textilinin-1, also in this venom, is a plasmin inhibitor which is thought to exert its toxic effects through the slowing of fibrinolysis. The aim of this report is to determine if there are differences in the potency of the venom from Eastern brown snakes collected from South Australia (SA) compared to those from Queensland (QLD). A concentration of 0.4 mu g/ml venom protein from six QLD specimens clotted citrated plasma in an average time of 21.4 +/- 3.3 s compared to 68.7 +/- 2.4 s for the same amount of SA venom (averaged for six individuals). The more potent procoagulant activity of the QLD venom was measured between 0.4 and 94 mu g/ml venom protein in plasma. The anti-plasmin activity of textilinin was also greater in the venom of the snakes collected from QLD, causing full inhibition of plasmin at approximately 1.88 mu g/ml of venom protein compared to approximately 7.5 mu g/ml for the SA venoms. It is concluded that geographic differentiation of the Eastern brown snakes results in significant differences venom potency.

Agonists and antagonists of protease activated receptors (PARs)

Protease activated receptors (PARs) are a category of G-protein coupled receptors (GPCRs) implicated in the progression of a wide range of diseases, including thrombosis, inflammatory disorders, and proliferative diseases. Signal transduction via PARs proceeds via an unusual activation mechanism. Instead of being activated through direct interaction with an extracellular signal like most GPCRs. they are self-activated following cleavage of their extracellular N-terminus by serine proteases to generate a new receptor N-terminus that acts as an intramolecular ligand by folding back onto itself and triggering receptor activation. Short synthetic peptides corresponding to this newly exposed N-terminal tethered ligand can activate three of the four known PARs in the absence of proteases. and such PAR activating peptides (PAR-APs) have served as templates for agonist/antagonist development. In fact much of the evidence for involvement of PARs in diseases has relied upon use of PAR-APs. often of low potency and uncertain selectivity. This review summarizes current structures of PAR agonists and antagonists, the need for more selective and more potent PAR ligands that activate or antagonize this intriguing class of receptors, and outlines the background relevant to PAR activation, assay methods, and physiological properties anticipated for PAR ligands.

Novel gene containing multiple epidermal growth factor-like motifs transiently expressed in the papillae of the ascidian tadpole larvae

We have investigated molecular mechanisms of the embryonic development of an ascidian, a primitive chordate which shares features of both invertebrates and vertebrates, with a view to identifying genes involved in development and metamorphosis, We isolated 12 partial cDNA sequences which were expressed in a stage-specific manner using differential display, We report here the isolation of a full-length cDNA sequence for one of these genes which was specifically expressed during the tailbud and larval stages of ascidian development, This cDNA, 1213 bp in length, is predicted to encode a protein of 337 amino acids containing four epidermal growth factor (EGF)-like repeats and three novel cysteine-rich repeats, Characterization of its spatial expression pattern by in situ hybridisation in late tailbud and larval embryos demonstrated strong expression localised throughout the papillae and anteriormost trunk and weaker expression in the epidermis of the remainder of the embryo, As recent evidence indicates that the signal for metamorphosis originates in the anterior trunk region, these results suggest that this gene may have a role in signalling the initiation of metamorphosis. (C) 1997 Wiley-Liss, Inc.

Cleavage of hemoglobin by hookworm cathespin D aspartic proteases and its potential contribution to host specificity

Hookworms routinely reach the gut of nonpermissive hosts but fail to successfully feed, develop, and reproduce. To investigate the effects of host-parasite coevolution on the ability of hookworms to feed in nonpermissive hosts, we cloned and expressed aspartic proteases from canine and human hookworms. We show here that a cathepsin D-like protease from the canine hookworm Ancylosotoma caninum (Ac-APR-1) and the orthologous protease from the human hookworm Necator americanus (Na-APR-1) are expressed in the gut and probably exert their proteolytic activity extracellularly. Both proteases were detected immunologically and enzymatically in somatic extracts of adult worms. The two proteases were expressed in baculovirus, and both cleaved human and dog hemoglobin (Hb) in vitro. Each protease digested Hb from its permissive host between twofold (whole molecule) and sixfold (synthetic peptides) more efficiently than Hb from the nonpermissive host, despite the two proteases' having identical residues lining their active site clefts. Furthermore, both proteases cleaved Hb at numerous distinct sites and showed different substrate preferences. The findings suggest that the paradigm of matching the molecular structure of the food source within a host to the molecular structure of the catabolic proteases of the parasite is an important contributing factor for host-parasite compatibility and host species range.

Gene expression during early ascidian metamorphosis requires signaling by Hemps, an EGF-like protein

Hemps, a novel epidermal growth factor (EGF)-like protein, is expressed during larval development and early metamorphosis in the ascidian Herdmania curvata and plays a direct role in triggering metamorphosis. In order to identify downstream genes in the Hemps pathway we used a gene expression profiling approach, in which we compared post-larvae undergoing normal metamorphosis with larval metamorphosis blocked with an anti-Hemps antibody. Molecular profiling revealed that there are dynamic changes in gene expression within the first 30 minutes of normal metamorphosis with a significant portion of the genome (approximately 49%) being activated or repressed. A more detailed analysis of the expression of 15 of these differentially expressed genes through embryogenesis, larval development and metamorphosis revealed that while there is a diversity of temporal expression patterns, a number of genes are transiently expressed during larval development and metamorphosis. These and other differentially expressed genes were localised to a range of specific cell and tissue types in Herdmania larvae and post-larvae. The expression of approximately 24% of the genes that were differentially expressed during early metamorphosis was affected in larvae treated with the anti-Hemps antibody. Knockdown of Hemps activity affected the expression of a range of genes within 30 minutes of induction, suggesting that the Hemps pathway directly regulates early response genes at metamorphosis. In most cases, it appears that the Hemps pathway contributes to the modulation of gene expression, rather than initial gene activation or repression. A total of 151 genes that displayed the greatest alterations in expression in response to anti-Hemps antibody were sequenced. These genes were implicated in a range of developmental and physiological roles, including innate immunity, signal transduction and in the regulation of gene transcription. These results suggest that there is significant gene activity during the very early stages of H. curvata metamorphosis and that the Hemps pathway plays a key role in regulating the expression of many of these genes.

Lipopolysaccharide and lipoteichoic acid induce different innate immune responses in bovine mammary epithelial cells

The objective of the present study was to characterize the innate immune responses induced by in vitro stimulation of bovine primary mammary epithelial cells (bMEC) using gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. Quantitative real-time PCR (qRT-PCR) was employed to examine the mRNA expression of a panel of 22 cytokines, chemokines, beta-defensins and components of the Toll-Like Receptor signaling pathway. Stimulation of bMEC with LPS for 24 h elicited a marked increase in mRNA expression for IL-1 beta, IL-8, TNF alpha, CXCL6 and beta-defensin while members of the Toll-Like Receptor pathway.. although present, were largely unaffected. Surprisingly, stimulation of these cells with LTA for 24 h did not significantly alter the expression of these genes. A time course of the expression of IL-1 beta, IL-8, TNF alpha, CXCL6 and beta-defensin was subsequently performed. The mRNA levels of all genes increased rapidly after stimulation for 2-4 h with both LPS and LTA but only the former treatment resulted in sustained responses. In contrast, the increased gene expression for LTA stimulated cells returned to resting levels after 8-16 h with the exception of beta-defensin, which remained up-regulated. The limited and unsustained cytokine response to LTA may explain why mastitis caused by gram-positive bacteria has greater potential for chronic intra-mammary infection than gram-negative infection. It was concluded that bovine mammary epithelial cells have a strong but differential capacity to mount innate immune responses to bacterial cell wall components. Crown Copyright (c) 2005 Published by Elsevier Ltd. All rights reserved.

Suppressor of cytokine signaling 2 regulates neuronal differentiation by inhibiting growth hormone signaling

The intracellular mechanisms that determine the response of neural progenitor cells to growth factors and regulate their differentiation into either neurons or astrocytes remain unclear. We found that expression of SOCS2, an intracellular regulator of cytokine signaling, was restricted to mouse progenitor cells and neurons in response to leukemia inhibitory factor (LIF)-like cytokines. Progenitors lacking SOCS2 produced fewer neurons and more astrocytes in vitro, and Socs2(-/-) mice had fewer neurons and neurogenin-1 (Ngn1)-expressing cells in the developing cortex, whereas overexpression of SOCS2 increased neuronal differentiation. We also report that growth hormone inhibited Ngn1 expression and neuronal production, and this action was blocked by SOCS2 overexpression. These findings indicate that SOCS2 promotes neuronal differentiation by blocking growth hormone-mediated downregulation of Ngn1.

Streptococcus pyogenes prtFII, but not sfbI, sfbII or fbp54, is represented more frequently among invasive-disease isolates of tropical Australia

Streptococcus pyogenes (group A streptococcus) strains may express several distinct fibronectin-binding proteins (FBPs) which are considered as major streptococcal adhesins. Of the FBPs, SfbI was shown in vitro to promote internalization of the bacterium into host cells and has been implicated in persistence. In the tropical Northern Territory, where group A streptococcal infection is common, multiple genotypes of the organism were found among isolates from invasive disease cases and no dominant strains were observed. To determine whether any FBPs is associated with invasive disease propensity of S. pyogenes, we have screened streptococcal isolates from bacteraemic and necrotizing fasciitis patients and isolates from uncomplicated infections for genetic endowment of 4 FBPs. No difference was observed in the distribution of sfbII, fbp54 and sfbI between the blood isolates' and isolates from uncomplicated infection. We conclude that the presence of sfbI does not appear to promote invasive diseases, despite its association with persistence. We also show a higher proportion of group A streptococcus strains isolated from invasive disease cases possess prtFII when compared to strains isolated from non-invasive disease cases. We suggest that S. pyogenes may recruit different FBPs for different purposes.

Temperature dependence of the thrombin-catalyzed proteolysis of prothrombin

Measurement of the temperature-dependence of thrombin-catalyzed cleavage of the Arg(155)-Ser(156) and Arg(284)-Thr(285) peptide bonds in prothrombin and prothrombin-derived substrates has yielded Arrhenius parameters that are far too large for classical mechanistic interpretation in terms of a simple hydrolytic reaction. Such a difference from the kinetic behavior exhibited in trypsin- and chymotrypsin-catalyzed proteolysis of peptide bonds is attributed to contributions by enzyme exosite interactions as well as enzyme conformational equilibria to the magnitudes of the experimentally determined Arrhenius parameters. Although the pre-exponential factor and the energy of activation deduced from the temperature-dependence of rate constants for proteolysis by thrombin cannot be accorded the usual mechanistic significance, their evaluation serves a valuable role by highlighting the existence of contributions other than those emanating from simple peptide hydrolysis to the kinetics of proteolysis by thrombin and presumably other enzymes of the blood coagulation system. (C) 2004 Elsevier B.V. All rights reserved.

LPS regulates a set of genes in primary murine macrophages by antagonising CSF-1 action

We previously reported that bacterial products such as LPS and CpG DNA down-modulated cell surface levels of the Colony Stimulating Factor (CSF)-1 receptor (CSF-1R) on primary murine macrophages in an all-or-nothing manner. Here we show that the ability of bacterial products to down-modulate the CSF-IR rendered bone marrow-derived macrophages (BMM) unresponsive to CSF-1 as assessed by Akt and ERK 1/2 phosphorylation. Using toll-like receptor (th-)9 as a model CSF-1-repressed gene, we show that LPS induced tlr9 expression in BMM only when CSF-1 was present, suggesting that LPS relieves CSF-1-mediated inhibition to induce gene expression. Using cDNA microarrays, we identified a cluster of similarly CSF-1 repressed genes in BMM. By real time PCR we confirmed that the expression of a selection of these genes, including integral membrane protein 2B (itm2b), receptor activity-modifying protein 2 (ramp2) and macrophage-specific gene 1 (mpg-1), were repressed by CSF-1 and were induced by LPS only in the presence of CSF-1. This pattern of gene regulation was also apparent in thioglycollate-elicited peritoneal macrophages (TEPM). LPS also counteracted CSF-1 action to induce mRNA expression of a number of transcription factors including interferon consensus sequence binding protein 1 (Icsbp1), suggesting that this mechanism leads to transcriptional reprogramming in macrophages. Since the majority of in vitro studies on macrophage biology do not include CSF-1, these genes represent a set of previously uncharacterised LPS-inducible genes. This study identifies a new mechanism of macrophage activation, in which LPS (and other toll-like receptor agonists) regulate gene expression by switching off the CSF-1R signal. This finding also provides a biological relevance to the well-documented ability of macrophage activators to down-modulate surface expression of the CSF-1R. (C) 2005 Elsevier GmbH. All rights reserved.

Proteins in the saliva of the Ixodida (ticks): Pharmacological features and biological significance

The saliva of ticks (Suborder Ixodida) is critical to their survival as parasites. A tick bite should result in strong responses from the host defence systems (haemostatic, immune and inflammatory) but tick saliva appears to have evolved to counter these responses. We review current knowledge of tick saliva components, with emphasis on those molecules confirmed to be present in the secreted saliva but including some that have only been confirmed to be present in salivary glands. About 50 tick saliva proteins that are well described in the literature are discussed. These saliva components include enzymes, enzyme inhibitors, amine-binding proteins and cytokine homologues that act as anti-haemostatic, anti-inflammatory or immuno-modulatory agents. Sequence comparisons are illustrated. The importance of tick saliva and the significance of the findings to date are also discussed. (C) 2006 Elsevier Ltd. All rights reserved.