Structural and functional properties of BaTX,a new Lys49 phospholipase A2 homologue isolated from the venom of the snake Bothrops alternatus

BaTX PLA₂, a K49 phospholipase A₂ homologue was purified from Bothrops alternatus venom after two chromatographic steps, molecular exclusion on Superdex 75 and reverse phase HPLC on μ-Bondapack C-18. A molecular mass of 13898.71 Da was determined by MALDI-TOF mass spectrometry. The amino acid composition showed that BaTX has a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA₂. The complete amino acid sequence of BaTX PLA₂ contains 121 residues, resulting in a calculated pI value of 8.63. This sequence shows high identity values when compared to other K49 PLA₂s isolated from the venoms of viperid snakes. Lower identity is observed in comparison to D49 PLA₂s. The sequence was SLFELGKMIL QETGKNPAKS YGAYYCYCGW GGQGQPKDAT DRCCYVHKCC YKKLTGCNPK KDRYSYSWKD KTIVCGENNS CLKELCECDK AVAICLRENL NTYNKKYRYY LKPLCKKADA C. In mice, BaTX induced myonecrosis and edema, upon intramuscular or subcutaneous injections, respectively. The LD₅₀ of BaTX was 7 μg/g body weight, by intravenous route. In vitro, the toxin caused a potent blockade of neuromuscular transmission in young chicken biventer cervicis preparations. The blockage 50% was achieved at a concentration of 0.03 μM: 40 ± 0.4 min and 0.07 μM: 35 ± 0.3 min. Moreover, this protein induced a rapid cytolytic effect upon mouse skeletal muscle myoblasts in culture. Thus, the combined structural and functional information obtained identify BaTX as a new member of the K49 PLA₂ family, which presents the typical bioactivities described for such proteins.     

Structural and Functional Properties of Cr 5, a New Lys49 Phospholipase A2 Homologue Isolated from the Venom of the Snake Calloselasma rhodostoma

Cr 5 PLA₂ homologous (K49) was isolated from Calloselasma rhodostoma venom in only one chromatographic step in reverse phase HPLC (RP-HPLC) (on μ-Bondapack C-18). A molecular mass of 13.965 Da was determined by MALDI-TOF mass spectrometry. The amino acid composition showed that Cr 5 had a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical residues of a basic PLA₂. The complete amino acid sequence of Cr 5 PLA₂ contains 120 residues, resulting in a calculated pI value of 5.55. This sequence shows high identity values when compared to other K49 PLA₂s isolated from the venoms of viperid snakes. Lower identity is observed in comparison to D49 PLA₂s. The sequence found was SLVELGKMIL QETGKNPAKS YGAYGCNCGV LGRHKPKDAT DRCCFVHKCC YKKLTGCDPK KDRYSYSWKD KTIVCGENNP CLKEMCECDK AVAICLRENL DTYNKKYRYL KPFCKKADDC. In mice, Cr 5 induced myonecrosis and edema upon intramuscular and intravenous injections, respectively. The LD₅₀ of Cr 5 was 0.070 mg/kg of the animal weight, by intracerebroventricular (i.c.v.) route. In vitro, the toxin caused rapid cytolytic effect upon mouse skeletal muscle myoblasts in culture. The isolation of this PLA₂ and the combined structural and functional information obtained classify Cr 5 as a new member of the K49 PLA₂ family, since it presents typical features from such proteins.     

Inhibition of the calcium-activated chloride current in cardiac ventricular myocytes by N-(p-amylcinnamoyl)anthranilic acid (ACA)

N-(p-amylcinnamoyl)anthranilic acid (ACA), a phospholipase A₂ (PLA₂) inhibitor, is structurally-related to non-steroidal anti-inflammatory drugs (NSAIDs) of the fenamate group and may also modulate various ion channels. We used the whole-cell, patch-clamp technique at room temperature to investigate the effects of ACA on the Ca²⁺-activated chloride current (I_Cl(Ca)) and other chloride currents in isolated pig cardiac ventricular myocytes. ACA reversibly inhibited I_Cl(Ca) in a concentration-dependent manner (IC₅₀ = 4.2 μM, n_Hill = 1.1), without affecting the L-type Ca²⁺ current. Unlike ACA, the non-selective PLA₂ inhibitor bromophenacyl bromide (BPB; 50 μM) had no effect on I_Cl(Ca). In addition, the analgesic NSAID structurally-related to ACA, diclofenac (50 μM) also had no effect on I_Cl(Ca), whereas the current in the same cells could be suppressed by chloride channel blockers flufenamic acid (FFA; 100 μM) or 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS;100 μM). Besides I_Cl(Ca), ACA (50 μM) also suppressed the cAMP-activated chloride current, but to a lesser extent. It is proposed that the inhibitory effects of ACA on I_Cl(Ca) are PLA₂-independent and that the drug may serve as a useful tool in understanding the nature and function of cardiac anion channels.     

Determination of the Amino Acid Sequence of a New Phospholipase A<Subscript>2</Subscript> (MIDCA1) Isolated from <Emphasis Type="Italic">Micrurus dumerilii carinicauda</Emphasis> Venom

A new Phospholipase A₂ (PLA₂) from Micrurus dumerilii carinicauda venom was isolated and its primary structure determined. This new PLA₂ showed a low enzymatic activity when compared with other PLA₂s and it is moderately basic with an isoelectric point of 8.0. Its amino acid sequence showed the presence of 120 amino acid residues and its sequence was: NLIQFLNMIQCTTPGREPLVAFANYGCYCGRGGSGTPVDELDRCCQVHDNCYDTAKKVFGCSPYFTMYSYDCSEGKLTCKDNNTKCKAAVCNCDRTAALCFAKAPYNDKNYKIDLTKRCQ. The structural model of MIDCA1, when compared with other strong neurotoxic PLA₂s, such as Naja naja, showed significant differences in the β-wing and neurotoxic sites, despite the high level of amino acid sequence similarity. These observations indicate a dissociation between the biological and catalytic activity of this new PLA₂, supporting the view that other regions of the protein are involved in the biological effects.     

Isolation of an acidic phospholipase A2 from the venom of the snake Bothrops asper of Costa Rica: Biochemical and toxicological characterization

Phospholipases A₂ (PLA₂) are major components of snake venoms, exerting a variety of relevant toxic actions such as neurotoxicity and myotoxicity, among others. Since the majority of toxic PLA₂s are basic proteins, acidic isoforms and their possible roles in venoms are less understood. In this study, an acidic enzyme (BaspPLA₂-II) was isolated from the venom of Bothrops asper (Pacific region of Costa Rica) and characterized. BaspPLA₂-II is monomeric, with a mass of 14,212 ± 6 Da and a pI of 4.9. Its complete sequence of 124 amino acids was deduced through cDNA and protein sequencing, showing that it belongs to the Asp49 group of catalytically active enzymes. In vivo and in vitro assays demonstrated that BaspPLA₂-II, in contrast to the basic Asp49 counterparts present in the same venom, lacks myotoxic, cytotoxic, and anticoagulant activities. BaspPLA₂-II also differed from other acidic PLA₂s described in Bothrops spp. venoms, as it did not show hypotensive and anti-platelet aggregation activities. Furthermore, this enzyme was not lethal to mice at intravenous doses up to 100 μg (5.9 μg/g), indicating its lack of neurotoxic activity. The only toxic effect recorded in vivo was a moderate induction of local edema. Therefore, the toxicological characteristics of BaspPLA₂-II suggest that it does not play a key role in the pathophysiology of envenomings by B. asper, and that its purpose might be restricted to digestive functions. Immunochemical analyses using antibodies raised against BaspPLA₂-II revealed that acidic and basic PLA₂s form two different antigenic groups in B. asper venom.     

Plant low-molecular-weight phospholipase A2s (PLA2s) are structurally related to the animal secretory PLA2s and are present as a family of isoforms in rice (Oryza sativa)

Recently, we purified to homogeneity and characterized a low-molecular-weight calcium-dependent phospholipase A₂ (PLA₂) from developing elm seed endosperm. This represented the first purified and characterized PLA₂ from a plant tissue. The full sequences of two distinct but homologous rice (Oryza sativa) cDNAs are given here. These encode mature proteins of 119 amino acids (PLA₂-I, preceded by a 19 amino acid signal peptide) and 128 amino acids (PLA₂-II, preceded by a 25 amino acid signal peptide), and were derived from four expressed sequence tag (EST) clones. Both proteins were homologous to the N-terminal amino acid sequence of the elm PLA₂. They contained twelve conserved cysteine residues and sequences that are likely to represent the Ca²⁺-binding loop and active-site motif, which are characteristic of animal secretory PLA₂s. A soluble PLA₂ activity was purified 145 000-fold from green rice shoots. This had the same biochemical characteristics as the elm and animal secretory PLA₂s. The purified rice PLA₂ consisted of two proteins, with a molecular weight of 12 440 and 12 920, that had identical N-terminal amino acid sequences. This sequence was different from but homologous to the PLA₂-I and PLA₂-II sequences. Taken together, the results suggest that at least three different low-molecular-weight PLA₂s are expressed in green rice shoots. Southern blot analysis suggested that multiple copies of such genes are likely to occur in the rice and in other plant genomes.     

Biological and Structural Characterization of a New PLA<Subscript>2</Subscript> from the Crotalus durissus collilineatus Venom

In the present article we report on the biological characterization and amino acid sequence of a new basic Phospholipases A₂ (PLA₂) isolated from the Crotalus durissus collilineatus venom (Cdcolli F6), which showed the presence of 122 amino acid residues with a pI value of 8.3, molecular mass of 14 kDa and revealed an amino acid sequence identity of 80 with crotalic PLA₂s such as Mojave B, Cdt F15, and CROATOX. This homology, however, dropped to 50 if compared to other sources of PLA₂s such as from the Bothrops snake venom. Also, this PLA₂ induced myonecrosis, although this effect was lower than that of BthTx-I or whole crotoxin and it was able to induce a strong blockage effect on the chick biventer neuromuscular preparation, independently of the presence of the acid subunid (crotapotin). The neurotoxic effect was strongly reduced by pre-incubation with heparin or with anhydrous acetic acid and q-BPB showed a similar reduction. The q-BPB did not reduce significantly the myotoxic activity induced by the PLA₂, but the anhydrous acetic acid treatment and the pre-incu-bation of PLA₂ with heparin reduced significantly its effects. This protein showed a strong antimicrobial activity against Xanthomonas axonopodis passiflorae (Gram-negative), which was drastically reduced by incubation of this PLA₂ with q-BPB, but this effect was marginally reduced after treatment with anhydrous acetic acid. Our findings here allow to speculate that basic amino acid residues on the C-terminal and molecular regions near catalytic site regions such as Calcium binding loop or b-wing region may be involved in the binding of this PLA₂ to the molecular receptor to induce the neurotoxic effect. The bactericidal effect, however, was completely dependent on the enzymatic activity of this protein.     

Molecular cloning and characterization of a venom phospholipase A2 from the bumblebee Bombus ignitus

Phospholipase A₂ (PLA₂) is one of the main components of bee venom. Here, we identify a venom PLA₂ from the bumblebee, Bombus ignitus. Bumblebee venom PLA₂ (Bi-PLA₂) cDNA, which was identified by searching B. ignitus venom gland expressed sequence tags, encodes a 180 amino acid protein. Comparison of the genomic sequence with the cDNA sequence revealed the presence of four exons and three introns in the Bi-PLA₂ gene. Bi-PLA₂ is an 18-kDa glycoprotein. It is expressed in the venom gland, cleaved between the residues Arg44 and Ile45, and then stored in the venom sac. Comparative analysis revealed that the mature Bi-PLA₂ (136 amino acids) possesses features consistent with other bee PLA₂s, including ten conserved cysteine residues, as well as a highly conserved Ca²⁺-binding site and active site. Phylogenetic analysis of bee PLA₂s separated the bumblebee and honeybee PLA₂ proteins into two groups. The mature Bi-PLA₂ purified from the venom of B. ignitus worker bees hydrolyzed DBPC, a known substrate of PLA₂. Immunofluorescence staining of Bi-PLA₂-treated insect Sf9 cells revealed that Bi-PLA₂ binds at the cell membrane and induces apoptotic cell death.     

Biological and biochemical characterization of two new PLA2 isoforms Cdc-9 and Cdc-10 from Crotalus durissus cumanensis snake venom

This work reports the purification, biological characterization and amino acid sequence of two new basic PLA₂ isoforms, Cdc-9 and Cdc-10, purified from the Crotalus durissus cumanensis venom by one step analytical chromatography reverse phase HPLC. The molecular masses of the PLA₂ were 14,175 ± 2.7 Da for Cdc-9 and 14,228 ± 3.5 Da for Cdc-10 both deduced by primary structure and confirmed by MALDI-TOF. The isoforms presented an amino acid sequence of 122 amino acid residues, being Cdc-9: SLVQFNKMIK FETRKSGLPF YAAYGCYCGW GGQRPKDATD RCCFVHDCCY GKVAKCNTKW DIYSYSLKSG YITCGKGTWC KEQICECDRV AAECLRRSLS TYKNEYMFYP DSRCREPPEY TC with pI value of 8.25 and Cdc-10: SLLQFNKMIK FETRKSGVPF YAAYGCYCGW GGRRPKDPTD RCCFVHDCCY GKLTKCNTKW DIYSYSLKSG YITCGKGTWC KEQICECDRV AAECLRRSLN TYKNEYMFYP DSRCRGPPEY TC with a pI value of 8.46, showing highly conserved Ca²⁺-binding and catalytic sites. The PLA₂ activity decreased when the isoforms Cdc-9 and Cdc-10 were incubated with 4-bromophenacyl bromide (p-BPB), anhydrous acetic acid and p-nitrobenzene sulfonyl fluoride (NBSF) when compared with the activity of both native isoforms. In mice, the PLA₂ isoforms Cdc-9 and Cdc-10 induced myonecrosis and edema. Myotoxic and edema activities were reduced after treatment of the isoforms with p-BPB; acetylation of the lysine residues and the treatment of PLA₂ with NBSF have also induced edema reduction. However, p-BPB strongly diminishes the local and systemic myotoxic effects.     

In silico and in vitro characterization of phospholipase A2 isoforms from soybean (Glycine max)

At the present, no secreted phospholipase A₂ (sPLA₂) from soybean (Glycine max) was investigated in detail. In this work we identified five sequences of putative secreted sPLA₂ from soybean after a BLAST search in G. max database. Sequence analysis showed a conserved PA2c domain bearing the Ca²⁺ binding loop and the active site motif. All the five mature proteins contain 12 cysteine residues, which are commonly conserved in plant sPLA₂s. We propose a phylogenetic tree based on sequence alignment of reported plant sPLA₂s including the novel enzymes from G. max. According to PLA₂ superfamily, two of G. max sPLA₂s are grouped as XIA and the rest of sequences as XIB, on the basis of differences found in their molecular weights and deviating sequences especially in the N- and C-terminal regions of the isoenzymes. Furthermore, we report the cloning, expression and purification of one of the putative isoenzyme denoted as GmsPLA₂-XIA-1. We demonstrate that this mature sPLA₂ of 114 residues had PLA₂ activity on Triton:phospholipid mixed micelles and determine the kinetic parameters for this system. We generate a model based on the known crystal structure of sPLA₂ from rice (isoform II), giving first insights into the three-dimensional structure of folded GmsPLA₂-XIA-1. Besides describing the spatial arrangement of highly conserved pair HIS-49/ASP-50 and the Ca⁺² loop domains, we propose the putative amino acids involved in the interfacial recognition surface. Additionally, molecular dynamics simulations indicate that calcium ion, besides its key function in the catalytic cycle, plays an important role in the overall stability of GmsPLA₂-XIA-1 structure.     

Isolation,enzymatic characterization and antiedematogenic activity of the first reported rattlesnake hyaluronidase from Crotalus durissus terrificus venom

A hyaluronidase (CdtHya1) from Crotalus durissus terrificus snake venom (CdtV) was isolated and showed to exhibit a high activity on hyaluronan cleavage. However, surveys on this enzyme are still limited. This study aimed at its isolation, functional/structural characterization and the evaluation of its effect on the spreading of crotoxin and phospholipase A₂ (PLA₂). The enzyme was purified through cation exchange, gel filtration and hydrophobic chromatography. After that, it was submitted to a reverse-phase fast protein liquid chromatography (RP-FPLC) and Edman degradation sequencing, which showed the first N-terminal 44 amino acid residues whose sequence evidenced identity with other snake venom hyaluronidases. CdtHya1 is a monomeric glycoprotein of 64.5 kDa estimated by SDS-PAGE under reducing conditions. It exhibited maximum activity in the presence of 0.2 M NaCl, at 37 °C, pH 5.5 and a specificity to hyaluronan higher than that to chondroitin-4-sulphate, chondroitin-6-sulphate or dermatan. Divalent cations (Ca²⁺ and Mg²⁺) and 1 M NaCl significantly reduced the enzyme activity. The specific activity of CdtHya1 was 5066 turbidity reducing units (TRU)/mg, against 145 TRU/mg for the soluble venom, representing a 34.9-fold purification. The pure enzyme increased the diffusion of crotoxin and PLA₂ through mice tissues. CdtHya1 (32 TRU/40 μL) potentiated crotoxin action, as evidenced by mice death, and it decreased the oedema caused by subplantar injections of buffer, crotoxin or PLA₂, thus evidencing the relevance of hyaluronidase in the crotalic envenoming. This work yielded a highly active antiedematogenic hyaluronidase from CdtV, the first one isolated from rattlesnake venoms.     

N49 phospholipase A2, a unique subgroup of snake venom group II phospholipase A2

A novel phospholipase A₂ (PLA₂) with Asn at its site 49 was purified from the snake venom of Protobothrops mucrosquamatus by using SP-Sephadex C25, Superdex 75, Heparin-Sepharose (FF) and HPLC reverse-phage C₁₈ chromatography and designated as TM-N49. It showed a molecular mass of 13.875 kDa on MALDI-TOF. TM-N49 does not possess enzymatic, hemolytic and hemorrhagic activities. It fails to induce platelet aggregation by itself, and does not inhibit the platelet aggregation induced by ADP. However, it exhibits potent myotoxic activity causing inflammatory cell infiltration, severe myoedema, myonecrosis and myolysis in the gastrocnemius muscles of BALB/c mice. Phylogenetic analysis found that that TM-N49 combined with two phospholipase A₂s from Trimeresurus stejnegeri, TsR6 and CTs-R6 cluster into one group. Structural and functional analysis indicated that these phospholipase A₂s are distinct from the other subgroups (D49 PLA₂, S49 PLA₂ and K49 PLA₂) and represent a unique subgroup of snake venom group II PLA₂, named N49 PLA₂ subgroup.     

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.     

Biochemical,Pharmacological and Structural Characterization of Two PLA<Subscript>2</Subscript> Isoforms Cdr-12 and Cdr-13 from <Emphasis Type="Italic">Crotalus durissus ruruima</Emphasis> Snake Venom

Cdr-12 and Cdr-13 isoforms of PLA₂, a D49 protein, were purified from Crotalus durissus ruruima venom after one chromatographic step, reverse phase HPLC on μ-Bondapack C-18. The molecular mass by SDS-PAGE of Cdr-12 and Cdr-13 isoforms of PLA₂ was 14333.49 Da and 14296.42 Da, respectively and confirmed by MALDI-TOF mass spectrometry .The amino acid composition showed that both isoforms Cdr-12 and Cdr-13 have a high content of Lys, Tyr, Gly, Arg, and 14 half-Cys residues, typical of a basic PLA₂. The isoforms Cdr-12 and Cdr-13 had a sequence of amino acids of 122 amino acid residues, being Cdr-12: SLLQFNKMIK FETRKNAIPF YAFYGCYCGW GGQGRPKDAT DRCCIVHDCC YGKLAKCNTK WDFYRYSLRS GYFQCGKGTW CEQQICECDR VAAECLRRSL STYRYGYMIY PDSRCREPSE TC and pI value 8.37 and Cdr-13: SLVQFEKMIK EETGKNAVPF YAFYGCYCGW GGRGRPKDAT DRCCIVHDCC YEKLVKCNTK WDFYRYSLRS GYFQCGKGTW CEQQICECDR VAAECLRRSL STYRYGKMIY PDSRCREPSE TC with a pI value of 8.13 This sequence shows high identity values when compared to other D49 PLA₂s isolated from venoms of crotalics snakes. Skeletal muscle preparations from the young chicken have been previously used in order to study the effects of toxins on neuromuscular transmission, providing an important opportunity to study the differentiated behavior of a toxin before more than one model, because it shows differences in its sensibilities. In mice, the PLA₂ isoforms Cdr-12 and Cdr-13 induced myonecrosis and edema, upon intramuscular or subcutaneous injections, respectively. In vitro, Cdr-12 and Cdr-13 isoforms of PLA₂, caused a potent blockade of neuromuscular transmission in young chicken biventer cervicis preparation and produced cytotoxicity in murine C2C12 skeletal muscle myotubes and lack cytolytic activity upon myoblasts in vitro. Thus, the combined structural and functional information obtained identify Cdr-12 and Cdr-13 isoforms as members of the PLA₂ family, which presents the typical bioactivities described for such proteins.     

Enzymatic and structural characterization of a basic phospholipase A2 from the sea anemone Condylactis gigantea

This work aimed at the isolation and structural/functional characterization of a phospholipase A₂ (CgPLA₂) from the extract of the anemone Condylactis gigantea. CgPLA₂ was isolated with a high purity level through three chromatographic steps, showing pI ˜ 8.6 and molecular weights of 14,500 and 29,000 for the monomer and dimer, respectively. CgPLA₂ showed a high catalytic activity upon fluorescent phospholipids inducing no direct hemolytic activity. This enzyme, which is Ca²⁺-dependent, showed a lower stability against temperature and pH variations when compared with snake venom enzymes. The enzymatic activity was significantly reduced or completely abolished after chemical modification of CgPLA₂ with BPB. Its cDNA was then obtained, with 357 base pairs which codified for a mature protein of 119 amino acid residues. A comparative analysis of the primary structure of CgPLA₂ revealed 84%, 61%, 43% and 42% similarity to the PLA₂s from Adamsia carciniopados, Nematostella vectensis, Vipera russelli russelli and Bothrops jararacussu, respectively.     

A lichenase-like family 12 endo-(1-->4)-beta-glucanase from Aspergillus japonicus: study of the substrate specificity and mode of action on beta-glucans in comparison with other glycoside hydrolases

Using anion-exchange chromatography on Source 15Q followed by hydrophobic interaction chromatography on Source 15 Isopropyl, a lichenase-like endo-(1→4)-β-glucanase (BG, 28 kDa, pI 4.1) was isolated from a culture filtrate of Aspergillus japonicus. The enzyme was highly active against barley β-glucan and lichenan (263 and 267 U/mg protein) and had much lower activity toward carboxymethylcellulose (3.9 U/mg). The mode of action of the BG on barley β-glucan and lichenan was studied in comparison with that of Bacillus subtilis lichenase and endo-(1→4)-β-glucanases (EG I, II, and III) of Trichoderma reesei. The BG behaved very similar to the bacterial lichenase, except the tri- and tetrasaccharides formed as the end products of β-glucan hydrolysis with the BG contained the β-(1→3)-glucoside linkage at the non-reducing end, while the lichenase-derived oligosaccharides had the β-(1→3)-linkage at the reducing end. The BG was characterized by a high amino acid sequence identity to the EG of Aspergillus kawachii (UniProt entry Q12679) from a family 12 of glycoside hydrolases (96% in 162 identified aa residues out of total 223 residues) and also showed lower sequence similarity to the EglA of Aspergillus niger (O74705).     

Identification and characterization of a phospholipase A2 from the venom of the Saw-scaled viper: Novel bactericidal and membrane damaging activities

Phospholipase A₂ (PLA₂), a common toxic component of snake venom, has been implicated in various pharmacological effects. In this study, a basic myotoxic PLA₂, named EcTx-I was isolated from Echis carinatus snake venom by using gel filtration on Superdex G-75, and reverse phase HPLC on C18 and C8 Sepharose columns. PLA₂, EcTx-I was 13,861.72 molecular weight as estimated by MALDI-TOF (15 kD by SDS-PAGE), and consisted of 121 amino acid residues cross-linked by seven disulfide bonds. The N-terminal sequences revealed significant homology with basic myotoxic PLA₂s from other snake venoms. The purified PLA₂ EcTx-I was evaluated (250 μg/ml) for bactericidal activity of a wide variety of human pathogens against Burkholderia pseudomallei (KHW&TES), Enterobacter aerogenes, Escherichia coli, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa and Staphylococcus aureus. EcTx-I showed strong antibacterial activity against B. pseudomallei (KHW) and E. aerogenes among the tested bacteria. Other Gram-negative and Gram-positive bacteria showed only a moderate effect. However, the Gram-positive bacterium E. aerogenes failed to show any effect on EcTx-I protein at tested doses. The most significant bacteriostatic and bactericidal effect of EcTx-I was observed at MICs of >15 μg/ml against (B. pseudomallei, KHW) and MICs >30 μg/ml against E. aerogenes. Mechanisms of bactericidal and membrane damaging effects were proved by ultra-structural analysis. EcTx-I was able to induce cytotoxicity on THP-1 cells in vitro as well as lethality in BALB/c mice. EcTx-I also induced mild myotoxic effects on mouse skin, but was devoid of hemolytic effects on human erythrocytes up to 500 μg/ml. It is shown that the toxic effect induced by E. carinatus venom is due to the presence of myotoxic PLA₂ (EcTx-I). The result also corroborates the hypothesis of an association between toxic and enzymatic domains. In conclusion, EcTx-I displays a heparin binding C-terminal region, which is probably responsible for the cytotoxic and bactericidal effects.     

Ontogeny of gene expression of group IB phospholipase A₂ isoforms in the red sea bream, Pagrus (Chrysophrys) major

The red sea bream (Pagrus major) was previously found to express mRNAs for two group IB phospholipase A₂ (PLA₂) isoforms, DE-1 and DE-2, in the digestive organs, including the hepatopancreas, pyloric caeca, and intestine. To characterize the ontogeny of the digestive function of these PLA₂s, the present study investigated the localization and expression of DE-1 and DE-2 PLA₂ genes in red sea bream larvae/juveniles and immature adults, by in situ hybridization. In the adults, DE-1 PLA₂ mRNA was expressed in pancreatic acinar cells. By contrast, DE-2 PLA₂ mRNA was detected not only in digestive tissues, such as pancreatic acinar cells, gastric glands of the stomach, epithelial cells of the pyloric caeca, and intestinal epithelial cells, but also in non-digestive ones, including cardiac and lateral muscle fibers and the cytoplasm of the oocytes. In the larvae, both DE-1 and DE-2 PLA₂ mRNAs first appeared in pancreatic tissues at 3 days post-hatching (dph) and in intestinal tissue at 1 dph, and expression levels for both gradually increased after this point. In the juvenile stage at 32 dph, DE-1 PLA₂ mRNA was highly expressed in pancreatic tissue, and DE-2 PLA₂ mRNA was detected in almost all digestive tissues, including pancreatic tissue, gastric glands, pyloric caeca, and intestine, including the myomere of the lateral muscles. In conclusion, both DE-1 and DE-2 PLA₂ mRNAs are already expressed in the digestive organs of red sea bream larvae before first feeding, and larvae will synthesize both DE-1 and DE-2 PLA₂ proteins.     

Physalin B inhibits Rhodnius prolixus hemocyte phagocytosis and microaggregation by the activation of endogenous PAF-acetyl hydrolase activities

The effects of physalin B (a natural secosteroidal chemical from Physalis angulata, Solanaceae) on phagocytosis and microaggregation by hemocytes of 5th-instar larvae of Rhodnius prolixus were investigated. In this insect, hemocyte phagocytosis and microaggregation are known to be induced by the platelet-activating factor (PAF) or arachidonic acid (AA) and regulated by phospholipase A₂ (PLA₂) and PAF-acetyl hydrolase (PAF-AH) activities. Phagocytic activity and formation of hemocyte microaggregates by Rhodnius hemocytes were strongly blocked by oral treatment of this insect with physalin B (1 μg/mL of blood meal). The inhibition induced by physalin B was reversed for both phagocytosis and microaggregation by exogenous arachidonic acid (10 μg/insect) or PAF (1 μg/insect) applied by hemocelic injection. Following treatment with physalin B there were no significant alterations in PLA₂ activities, but a significant enhancement of PAF-AH was observed. These results show that physalin B inhibits hemocytic activity by depressing insect PAF analogous (iPAF) levels in hemolymph and confirm the role of PAF-AH in the cellular immune reactions in R. prolixus.