Bactericidal activity of Lys49 and Asp49 myotoxic phospholipases A2 from Bothrops asper snake venom--synthetic Lys49 myotoxin II-(115-129)-peptide identifies its bactericidal region

Mammalian group-II phospholipases A2 (PLA2) of inflammatory fluids display bactericidal properties, which are dependent on their enzymatic activity. This study shows that myotoxins II (Lys49) and III (Asp49), two group-II PLA2 isoforms from the venom of Bothrops asper, are lethal to a broad spectrum of bacteria. Since the catalytically inactive Lys49 myotoxin II isoform has similar bactericidal effects to its catalytically active Asp49 counterpart, a bactericidal mechanism that is independent of an intrinsic PLA2 activity is demonstrated. Moreover, a synthetic 13-residue peptide of myotoxin II, comprising residues 115-129 (common numbering system) near the C-terminal loop, reproduced the bactericidal effect of the intact protein. Following exposure to the peptide or the protein, accelerated uptake of the hydrophobic probe N-phenyl-N-naphthylamine was observed in susceptible but not in resistant bacteria, indicating that the lethal effect was initiated on the bacterial membrane. The outer membrane, isolated lipopolysaccharide (LPS), and lipid A of susceptible bacteria showed higher binding to the myotoxin II-(115-129)-peptide than the corresponding moieties of resistant strains. Bacterial LPS chimeras indicated that LPS is a relevant target for myotoxin II-(115-129)-peptide. When heterologous LPS of the resistant strain was present in the context of susceptible bacteria, the chimera became resistant, and vice versa. Myotoxin II represents a group-II PLA2 with a direct bactericidal effect that is independent of an intrinsic enzymatic activity, but adscribed to the presence of a short cluster of basic/hydrophobic amino acids near its C-terminal loop.

Functional and Structural Diversification of the Anguimorpha Lizard Venom System

Venom has only been recently discovered to be a basal trait of the Anguimorpha lizards. Consequently, very little is known about the timings of toxin recruitment events, venom protein molecular evolution, or even the relative physical diversifications of the venom system itself. A multidisciplinary approach was used to examine the evolution across the full taxonomical range of this similar to 130 million-year-old clade. Analysis of cDNA libraries revealed complex venom transcriptomes. Most notably, three new cardioactive peptide toxin types were discovered (celestoxin, cholecystokinin, and YY peptides). The latter two represent additional examples of convergent use of genes in toxic arsenals, both having previously been documented as components of frog skin defensive chemical secretions. Two other novel venom gland-overexpressed modified versions of other protein frameworks were also recovered from the libraries (epididymal secretory protein and ribonuclease). Lectin, hyaluronidase, and veficolin toxin types were sequenced for the first time from lizard venoms and shown to be homologous to the snake venom forms. In contrast, phylogenetic analyses demonstrated that the lizard natriuretic peptide toxins were recruited independently of the form in snake venoms. The de novo evolution of helokinestatin peptide toxin encoding do-mains within the lizard venom natriuretic gene was revealed to be exclusive to the helodermatid/anguid subclade. New isoforms were sequenced for cysteine-rich secretory protein, kallikrein, and phospholipase A 2 toxins. Venom gland morphological analysis revealed extensive evolutionary tinkering. Anguid glands are characterized by thin capsules and mixed glands, serous at the bottom of the lobule and mucous toward the apex. Twice, independently this arrangement was segregated into specialized serous protein-secreting glands with thick capsules with the mucous lobules now distinct (Heloderma and the Lanthanotus/Varanus clade). The results obtained highlight the importance of utilizing evolution-based search strategies for biodiscovery and emphasize the largely untapped drug design and development potential of lizard venoms. Molecular & Cellular Proteomics 9:2369-2390, 2010.

Cloning of cDNAs and molecular characterisation of C-type lectin-like proteins from snake venoms

C-type lectin-like proteins (CTLPs) isolated from snake venoms are the largest and most complex non-mammalian vertebrate C-type lectin-like domain family. In the present study, we simultaneously amplified four cDNAs encoding different types of CTLP subunits from the venoms of two different species of snakes by RT-PCR with a single sense primer and a nested universal primer - two CTLP subunit-encoding cDNAs were cloned from Deinagkistrodon acutus venom and two from Agkistrodon halys Pallas venom. All four cloned CTLP subunits exhibited typical motifs in their corresponding domain regions but with relatively-low sequence similarities to each other. Compared with previously-published CTLPs, the four cloned CTLPs subunits showed slight variations in the calcium-binding sites and the disulphide bonding patterns. To our knowledge, these data constitute the first example of co-expression of CTLP platelet glycoprotein Ib-binding subunits and coagulation factors in Agkistrodon halys Pallas venom.

Molecular Characterization of Three Novel Phospholipase A2 Proteins from the Venom of Atheris chlorechis, Atheris nitschei and Atheris squamigera

Secretory phospholipase A2 (sPLA2) is known as a major component of snake venoms and displays higher-order catalytic hydrolysis functions as well as a wide range of pathological effects. Atheris is not a notoriously dangerous genus of snakes although there are some reports of fatal cases after envenomation due to the effects of coagulation disturbances and hemorrhaging. Molecular characterization of Atheris venom enzymes is incomplete and there are only a few reports in the literature. Here, we report, for the first time, the cloning and characterization of three novel cDNAs encoding phospholipase A2 precursors (one each) from the venoms of the Western bush viper (Atheris chlorechis), the Great Lakes bush viper (Atheris nitschei) and the Variable bush viper (Atheris squamigera), using a “shotgun cloning” strategy. Open-reading frames of respective cloned cDNAs contained putative 16 residue signal peptides and mature proteins composed of 121 to 123 amino acid residues. Alignment of mature protein sequences revealed high degrees of structural conservation and identity with Group II venom PLA2 proteins from other taxa within the Viperidae. Reverse-phase High Performance Liquid Chromatography (HPLC) profiles of these three snake venoms were obtained separately and chromatographic fractions were assessed for phospholipase activity using an egg yolk suspension assay. The molecular masses of mature proteins were all identified as approximately 14 kDa. Mass spectrometric analyses of the fractionated oligopeptides arising from tryptic digestion of intact venom proteins, was performed for further structural characterization.

Identification and functional analysis of a novel bradykinin inhibitory peptide in the venoms of New World Crotalinae pit vipers.

A novel undecapeptide has been isolated and structurally characterized from the venoms of three species of New World pit vipers from the subfamily, Crotalinae. These include the Mexican moccasin (Agkistrodon bilineatus), the prairie rattlesnake (Crotalus viridis viridis), and the South American bushmaster (Lachesis muta). The peptide was purified from all three venoms using a combination of gel permeation chromatography and reverse-phase HPLC. Automated Edman degradation sequencing and MALDI-TOF mass spectrometry established its peptide primary structure as: Thr-Pro-Pro-Ala-Gly-Pro-Asp-Val-Gly-Pro-Arg-OH, with a non-protonated molecular mass of 1063.18 Da. A synthetic replicate of the peptide was found to be an antagonist of bradykinin action at the rat vascular B2 receptor. This is the first bradykinin inhibitory peptide isolated from snake venom. Database searching revealed the peptide to be highly structurally related (10/11 residues) with a domain residing between the bradykinin-potentiating peptide and C-type natriuretic peptide domains of a recently cloned precursor from tropical rattlesnake (Crotalus durissus terrificus) venom gland. BIP thus represents a novel biological entity from snake venom.

Cloning and characterisation of three novel disintegrin precursors from the venoms of three Atheris species: Atheris chlorechis, Atheris nitschei and Atheris squamigera

Snake venom constitutes one of the most complex mixtures of naturally-occurring toxic proteins/polypeptides and a large number of these possess very profound biological activities. Disintegrins, that are commonly found in viper venoms, are low molecular weight proteins that usually contain an -Arg-Gly-Asp- (-RGD-) motif that is known to be involved in cell adhesion ligand recognition, binding specifically to cell surface integrin receptors and also exhibiting platelet anti-aggregation activity.

Here, we report for the first time, the successful cloning of three cDNAs encoding disintegrin precursors from lyophilised venom-derived libraries of Atheris chlorechis, Atheris nitschei and Atheris squamigera, respectively. All of these disintegrins belong to the short-coding class and all exhibit high degrees of structural identity, both in their amino acid sequences and in the arrangement of their functional domains. Mass spectrometric analyses of the HPLC-separated/in-gel digested venom proteins was performed to characterise the mature disintegrins as expressed in the venom proteome. Studies on both the structures and conserved sites within these disintegrins are of considerable theoretical interest in the field of biological evolution and in the development of new research tools or novel templates for drug design.

Unmasking venom gland transcriptomes in reptile venoms

While structural studies of reptile venom toxins can be achieved using lyophilized venom samples, until now the cloning of precursor cDNAs required sacrifice of the specimen for dissection of the venom glands. Here we describe a simple and rapid technique that unmasks venom protein mRNAs present in lyophilized venom samples. To illustrate the technique we have RT-PCR-amplified a range of venom protein transcripts from cDNA libraries derived from the venoms of a hemotoxic snake, the Chinese copperhead (Deinagkistrodon acutus), a neurotoxic snake, the black mamba (Dendroaspis polylepis), and a venomous lizard, the Gila monster (Heloderma suspectum). These include a metalloproteinase and phospholipase A2 from D. acutus, a potassium channel blocker, dendrotoxin K, from D. polylepis, and exendin-4 from H. suspectum. These findings imply that the apparent absence and/or lability of mRNA in complex biological matrices is not always real and paves the way for accelerated acquisition of molecular genetic data on venom toxins for scientific and potential therapeutic purposes without sacrifice of endangered herpetofauna.

Characterization, crystallization and preliminary X—ray diffraction analysis of acutohaemolysin, a haemolytic toxin from Agkistrodon acutus venom

Acutohaemolysin, a phospholipase A2 (PLA2) from the venom of the snake Agkistrodon acutus, has been isolated and purified to homogeneity by anion-exchange chromatography on a DEAE-Sepharose column followed by cation-exchange chromatography on a CM-Sepharose column. It is an alkaline protein with an isoelectric point of 10.5 and is comprised of a single polypeptide chain of 13 938 Da. Its N-terminal amino-acid sequence shows very high similarity to Lys49-type PLA2 proteins from other snake venoms. Although its PLA2 enzymatic activity is very low, acutohaemolysin has a strong indirect haemolytic activity and anticoagulant activity. Acutohaemolysin crystals with a diffraction limit of 1.60 Å were obtained by the hanging-drop vapour-diffusion method. The crystals belong to the space group C2, with unit-cell parameters a = 45.30, b = 59.55, c = 46.13 Å, [beta] = 117.69°. The asymmetric unit contains one molecule

Children with stable asthma have reduced airway matrix metalloproteinase-9 and matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ratio

Background Childhood asthma is characterized by inflammation of the airways. Structural changes of the airway wall may also be seen in some children early in the course of the disease. Matrix metalloproteinases (MMPs) are key mediators in the metabolism of the extracellular matrix (ECM). Objective To investigate the balance of MMP-8, MMP-9 and tissue inhibitor of metalloproteinases (TIMP)-1 in the airways of children with asthma. Methods One hundred and twenty-four children undergoing elective surgical procedures also underwent non-bronchoscopic bronchoalveolar lavage (BAL). MMP-8, MMP-9 and TIMP-1 were measured by ELISA. Results There was a significant reduction in MMP-9 in atopic asthmatic children (n=31) compared with normal children (n=30) [median difference: 0.57 ng/mL (95% confidence interval: 0.18–1.1 ng/mL)]. The ratio of MMP-9 to TIMP-1 was also reduced in asthmatic children. Levels of all three proteins were significantly correlated to each other and to the relative proportions of particular inflammatory cells in BAL fluid (BALF). Both MMP-8 and MMP-9 were moderately strongly correlated to the percentage neutrophil count (r=0.40 and 0.47, respectively, P

Alterations in vascular matrix metalloproteinase due to ageing and chronic hypertension: effects of endothelin receptor blockade.

OBJECTIVE: To determine the effects of age and dual endothelin (ET)A/ETB receptor antagonism (bosentan) on aortic matrix metalloproteinase (MMP) abundance and tissue inhibitor of metalloproteinase (TIMP) expression in normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). METHODS: Male SHR and control WKY rats were randomly assigned to receive placebo or bosentan (100 mg/kg per day) for 3 months. Animals were killed under terminal anaesthesia at either 20 weeks (adult) or 17-20 months (senescent). Aortic gelatinase activity was determined by zymography, whereas MT-1 MMP and TIMP-1 expression were assessed by immunoblotting. RESULTS: In WKY rats, aortic MMP-2 but not proMMP-2 activity was 3.6-fold higher (P <0.02) in the senescent compared with the adult group. TIMP-1 (twofold) and MT-1 MMP (3.8-fold) expression increased (P <0.05) with age in the WKY groups. Short-term hypertension (adult SHR versus adult WKY) increased MMP-2 to 74.7 +/- 14.1 from 18.9 +/- 3.5 arbitrary units (AU) (P = 0.0012), but did not alter proMMP-2 activity. This increased further on progression to chronic hypertension (117.4 +/- 12.2 versus 74.7 +/- 14.1 AU; P <0.02). Bosentan decreased MMP-2 (78.9 +/- 3.8 versus 117.4 +/- 12.2 AU; P = 0.014) and proMMP-2 activity (P <0.006) in the senescent SHR group. CONCLUSION: Ageing and the development/progression of hypertension are associated with increased MMP-2 activity in the aorta, which is consistent with ongoing remodelling of the vasculature. However, the underlying mechanisms regulating MMP-2 abundance in ageing and hypertension appear to be divergent, as MT-1 MMP expression is differentially altered. Dual ETA/ETB receptor antagonism did not alter the age-dependent increase in aortic MMP activity in normotensive rats. However, bosentan decreased pro and active MMP-2 activity in senescent SHR rats, indicating that ET modulates late events in vascular remodelling in hypertension.