Conservative and variable regions of homologous snake phospholipases A2 sequences: Prediction of the taxonspecific peptides structure

Homologous amino acid sequences of phospholipases A₂ (PLA₂) of snakes belonging to the families Elapidae, Viperidae, and Colubridae were considered in order to study the conservative and variable regions location. The PLA₂ sequences were divided into two groups (taxons) according to the phylogenetic tree reconstructed from the pair similarity matrix. Results of the intergroup comparison were plotted to facilitate the identification of significant conservative and variable regions. It was shown that the results of the comparison between two phylogenetic groups of snake PLA₂ did not much depend on the number of each group representatives and did not markedly change if one of the groups was represented by the single sequence. The knowledge of the number and location of conservative and variable regions and their dependence on the phylogenetic relations between compared taxa may be used to predict a synthetic peptide structure to obtain specific antibodies against PLA₂ of one of these taxons. Such prediction is possible if there is a specific region conservative for one taxon but variable for two of them.     

Conserved and variable segments of the snake phospholipase A2 amino acid domain]

Homologous amino acid sequences of phospholipases A2 of snakes belonging to families Elapidae, Viperidae and Colubridae were considered in order to study the location of conservative and variable regions. To identify significant conservative and variable regions a comparison between two groups of aligned sequences of snake phospholipases A2 was successfully applied. The phospholipases A2 sequences were divided into two groups (taxons) according to the phylogenetic tree reconstructed from the pair distance matrix. Results of the comparison were plotted to facilitate the identification of significant conservative and variable regions. It was shown, that the results of the comparison between two phylogenetic groups of snake phospholipases A2 didn't depend much on the number of each group representatives, and the location of conservative and variable regions didn't significantly change if one of the groups was represented by the single sequence. It should be mentioned, that the more the phylogenetic difference between groups of phospholipases A2 the more was the number of significant conservative and variable regions. The knowledge of the number and location of conservative and variable regions and their dependence on phylogenetic relations between the compared taxons can be used to predict the synthetic peptide structure to obtain antibodies of various specificity. These antibodies may have either a wide range of cross-reactivity against all of phospholipases A2 or a limited range of cross-reactivity against phospholipases A2 of one taxon.     

Molecular evolution of group II phospholipases A2

The nucleotide sequences of 13 cDNAs encoding group II phospholipases A₂ (PLA₂ ^S), which are from viperidae snake venoms and from mammalian sources, were aligned and analyzed by phylogenetic trees constructed using various components of the sequences. The evolutionary trees derived from the combined sequences of the untranslated (5 and 3) region and the signal peptide region of cDNAs were in accord with the consequences from taxonomy. In contrast, the evolutionary trees from the mature protein-coding region sequences of cDNAs and from the amino acid sequences showed random patterns. These observations indicated that the mature protein-coding region has evolved through a process differently from the untranslated and signal peptide regions. The trees built from the nucleotide differences at each of three positions of codons in the mature protein-coding region suggested that snakevenom-gland PLA₂ genes have evolved via a process different from mammalian PLA₂ genes. The occurrence of accelerated evolution has been recently discovered in Trimeresurus flavoviridis venom-gland group II PLA₂ isozyme genes (Nakashima et al. 1993, Proc Natl Acad Sci USA 90:5964–5968), so the present phylogenetic analysis together with the estimation of nucleotide divergence of cDNAs provides further evidence that snakevenom-group II PLA₂ isozyme genes have evolved by accelerated evolution to gain diverse physiological activities. Correspondence to: M. Ohno     

Interisland Mutation of a Novel Phospholipase A<Subscript>2</Subscript> from <Emphasis Type="Italic">Trimeresurus flavoviridis</Emphasis> Venom and Evolution of Crotalinae Group II Phospholipases A<Subscript>2</Subscript>

Trimeresurus flavoviridis (Crotalinae) snakes inhabit the southwestern islands of Japan: Amami-Oshima, Tokunoshima, and Okinawa. Affinity and conventional chromatographies of Amami-Oshima T. flavoviridis venom led to isolation of a novel phospholipase A₂ (PLA₂). This protein was highly homologous (91%) in sequence to trimucrotoxin, a neurotoxic PLA₂, which had been isolated from T. mucrosquamatus (Taiwan) venom, and exhibited weak neurotoxicity. This protein was named PLA-N. Its LD₅₀ for mice was 1.34 µg/g, which is comparable to that of trimucrotoxin. The cDNA encoding PLA-N was isolated from both the Amami-Oshima and the Tokunoshima T. flavoviridis venom-gland cDNA libraries. Screening of the Okinawa T. flavoviridis venom-gland cDNA library with PLA-N cDNA led to isolation of the cDNA encoding one amino acid-substituted PLA-N homologue, named PLA-N(O), suggesting that interisland mutation occurred and that Okinawa island was separated from a former island prior to dissociation of Amami-Oshima and Tokunoshima islands. Construction of a phylogenetic tree of Crotalinae venom group II PLA₂s based on the amino acid sequences revealed that neurotoxic PLA₂s including PLA-N and PLA-N(O) form an independent cluster which is distant from other PLA₂ groups such as PLA₂ type, basic [Asp⁴⁹]PLA₂ type, and [Lys⁴⁹]PLA₂ type. Comparison of the nucleotide sequence of PLA-N cDNA with those of the cDNAs encoding other T. flavoviridis venom PLA₂s showed that they have evolved in an accelerated manner. However, when comparison was made within the cDNAs encoding Crotalinae venom neurotoxic PLA₂s, their evolutionary rates appear to be reduced to a level between accelerated evolution and neutral evolution. It is likely that ancestral genes of neurotoxic PLA₂s evolved in an accelerated manner until they had acquired neurotoxic function and since then they have evolved with less frequent mutation, possibly for functional conservation. The nucleotide sequences reported in this paper are available from the GenBank/EMBL/DDBJ databases under accession numbers AB102728 and AB102729.     

The molecular cloning of a phospholipase A2 from Bothrops jararacussu snake venom: Evolution of venom group II phospholipase A2's may imply gene duplications

The sequence coding for a snake venom phospholipase A₂ (PLA₂), BJUPLA₂, has been cloned from a Bothrops jararacussu venom gland cDNA library. The cDNA sequence predicts a precursor containing a 16-residue signal peptide followed by a molecule of 122 amino acid residues with a strong sequence similarity to group II snake venom PLA₂'s. A striking feature of the cDNA is the high sequence conservation of the 5 and 3 untranslated regions in cDNAs coding for PLA₂'s from a number of viper species. The greatest sequence variation was observed between the regions coding for the mature proteins, with most substitutions occurring in nonsynonymous sites. The phylogenetic tree constructed by alignment of the amino acid sequence of BJUPLA₂ with group II PLA₂'s in general groups them according to current taxonomical divisions and/or functional activity. It also suggests that gene duplications may have occurred at a number of different points during the evolution of snake venom group II PLA₂'s.The nucleotide sequence reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number X76289.Correspondence to: A.M. Moura-da-Silva     

cDNA cloning and sequencing of phospholipase A2 from the pyloric ceca of the starfish Asterina pectinifera

Three cDNA from the pyloric ceca of the starfish Asterina pectinifera, (namely, cDNA 1, 2, and 3), encoding phospholipase A₂ (PLA₂), were isolated and sequenced. These cDNAs were composed of 415 bp with an open reading frame of 414 bp at nucleotide positions 1–414, which encodes 138 amino acids including N-terminal Met derived from the PCR primer. The amino acid sequence deduced from the cDNA 1 was completely consistent with the sequence determined with the starfish PLA₂ protein, while those deduced from cDNA 2 and cDNA 3 differed at one and twelve amino acid residual positions, respectively, from the sequence of the PLA₂ protein, suggesting the presence of multiple forms in the starfish PLA₂. All of the sequences deduced from cDNA 1, 2, and 3 required two amino acid deletions in pancreatic loop region, and sixteen insertions and three deletions in β-wing region when aligned with the sequence of mammalian pancreatic PLA₂. In phylogenetic tree, the starfish PLA₂ should be classified into an independent group, but hardly to the established groups IA and IB. The characteristic structure in the pancreatic loop and β-wing regions may account for the specific properties of the starfish PLA₂, e.g. the higher activity and characteristic substrate specificity compared with commercially available PLA₂ from porcine pancreas.     

Identification and characterization of TCRγ and TCRδ chains in channel catfish,Ictalurus punctatus

Channel catfish, Ictalurus punctatus, T cell receptors (TCR) γ and δ were identified by mining of expressed sequence tag databases, and full-length sequences were obtained by 5′-RACE and RT-PCR protocols. cDNAs for each of these TCR chains encode typical variable (V), diversity (D), joining (J), and constant (C) regions. Three TCRγ V families, seven TCRγ J sequences, and three TCRγ C sequences were identified from sequencing of cDNA. Primer walking on bacterial artificial chromosomes (BACs) confirmed that the TRG locus contained seven TRGJ segments and indicated that the locus consists of (Vγ3-Jγ6-Cγ2)–(Vγ1_n-Jγ7-Cγ3)–(Vγ2-Jγ5-Jγ4-Jγ3-Jγ2-Jγ1-Cγ1). In comparison for TCRδ, two V families, four TCRδ D sequences, one TCRδ J sequence, and one TCRδ C sequence were identified by cDNA sequencing. Importantly, the finding that some catfish TCRδ cDNAs contain TCR Vα-D-Jδ rearrangements and some TCRα cDNAs contain Vδ-Jα rearrangements strongly implies that the catfish TRA and TRD loci are linked. Finally, primer walking on BACs and Southern blotting suggest that catfish have four TRDD gene segments and a single TRDJ and TRDC gene. As in most vertebrates, all three reading frames of each of the catfish TRDD segments can be used in functional rearrangements, and more than one TRDD segment can be used in a single rearrangement. As expected, catfish TCRδ CDR3 regions are longer and more diverse than TCRγ CDR3 regions, and as a group they utilize more nucleotide additions and contain more nucleotide deletions than catfish TCRγ rearrangements.     

Alcohol,Phospholipase A2-associated Neuroinflammation,and ω3 Docosahexaenoic Acid Protection

Chronic alcohol (ethanol) abuse causes neuroinflammation and brain damage that can give rise to alcoholic dementia. Insightfully, Dr. Albert Sun was an early proponent of oxidative stress as a key factor in alcoholism-related brain deterioration. In fact, oxidative stress has proven to be critical to the hippocampal and temporal cortical neurodamage resulting from repetitive “binge” alcohol exposure in adult rat models. Although the underlying mechanisms are uncertain, our immunoelectrophoretic and related assays in binge alcohol experiments in vivo (adult male rats) and in vitro (rat organotypic hippocampal-entorhinal cortical slice cultures) have implicated phospholipase A₂ (PLA₂)-activated neuroinflammatory pathways, release of pro-oxidative arachidonic acid (20:4 ω6), and elevated oxidative stress adducts (i.e., 4-hydroxynonenal-protein adducts). Also, significantly increased by the binge alcohol treatments was aquaporin-4 (AQP4), a water channel enriched in astrocytes that, when augmented, may trigger brain (esp. cellular) edema and neuroinflammation; of relevance, glial swelling is known to provoke increased PLA₂ activities or levels. Concomitant with PLA₂ activation, the results have further implicated binge alcohol-elevated poly (ADP-ribose) polymerase-1 (PARP-1), an oxidative stress-responsive DNA repair enzyme linked to parthanatos, a necrotic-like neuronal death process. Importantly, supplementation of the brain slice cultures with docosahexaenoic acid (22:6 ω3) exerted potent suppression of the induced changes in PLA₂ isoforms, AQP4, PARP-1 and oxidative stress footprints, and prevention of the binge alcohol neurotoxicity, by as yet unknown mechanisms. These neuroinflammatory findings from our binge alcohol studies and supportive rat binge studies in the literature are reviewed.     

Evolutionary relationships and implications for the regulation of phospholipase A2 from snake venom to human secreted forms

Summary The amino acid sequences of 40 secreted phospholipase A₂'s (PLA₂) were aligned and a phylogenetic tree derived that has three main branches corresponding to elapid (group I), viperid (group II), and insect venom types of PLA₂. The human pancreatic and recently determined nonpancreatic sequences in the comparison align with the elapid and viperid categories, repectively, indicating that at least two PLA₂ genes existed in the vertebrate line before the divergence of reptiles and mammals about 200–300 million years ago. This allows resolution for the first time of major genetic events in the evolution of current PLA₂'s and the relationship of human PLA₂'s to those of snake venom, many of which are potent toxins. Implications for possible mechanisms of regulation of mammalian intra- and extracellular PLA₂'s are discussed, as well as issues relating to the search for the controlling enzymes in arachidonic acid release, prostaglandin generation, and signal transduction.     

Studies on the status of tyrosyl residues in phospholipases A2 fromNaja naja atra andNaja nigricollis snake venoms

Two phospholipases A₂ (PLA₂) fromNaja naja atra andNaja nigricollis snake venoms were subjected to tyrosine modification withp-nitrobenzenesulfonyl fluoride (NBSF) atpH 8.0. Three major NBS derivatives from each PLA₂ were separated by high-performance liquid chromatography. The results of amino acid analysis showed that only two Tyr residues out of nine were modified, and the modified residues were identified to be Tyr-3 and Tyr-63 (or Tyr-62) in the sequence. Spectrophotometric titration indicated that the phenolic group of Tyr-3 and Tyr-63 (or Tyr-62) had apK of 10.1 and 11.0, respectively. The reactivity of Tyr-3 toward NBSF was not affected in the presence or absence of Ca ²⁺; however, the reactivity of Tyr-63 (or Tyr-62) toward NBSF was greatly enhanced by Ca²⁺. Modification of Tyr-63 (or Tyr-62) resulted in a marked decrease in both lethality and enzymatic activity. Conversely, modification of Tyr-3 inN. naja atra PLA₂ could cause more than a sixfold increase in lethal potency, in sharp contrast to the loss of enzymatic activity. Tyrosine-63-modifiedN. naja atra PLA₂ exhibited the same Ca²⁺-induced difference spectra as that of native PLA₂, indicating that the Ca²⁺-binding ability of Tyr-63-modifiedN. naja atra PLA₂ was not impaired. However, Tyr-3-modified PLA₂ and all Tyr-modifiedN. nigricollis CMS-9 were not perturbed by Ca²⁺, revealing that the Ca²⁺-binding ability have been lost after tyrosine modification. These results suggest that Tyr-62 inN. nigricollis CMS-9 and Tyr-3 in both enzymes are involved in Ca²⁺ binding. AtpH 8.0, both native PLA₂ enzymes enhance the emission intensity of 8-anilinonaphthalene sulfonate (ANS) dramatically, while all of the Tyr-modified derivatives did not enhance the emission intensity at all either in the presence or absence of Ca²⁺, suggesting that the hydrophobic pocket that interacts with ANS might be the substrate binding site, in which Tyr-3 and Tyr-63 (or Tyr-62) are involved.     

Phylogenetic and genetic characterization of Acidithiobacillus strains isolated from different environments

To study the phylogenetic relationships and genetic heterogeneity of 21 Acidithiobacillus strains isolated from different environments, we amplified and sequenced the 16S–23S rRNA gene intergenic spacers (ITS) of all these strains. These sequence data, combined with related sequences available from GenBank, were divided into six phylogenetic groups by 16S rRNA gene and by 16S–23S rRNA gene sequence analysis. The results of phylogenetic analysis were consistent with those obtained by repetitive element PCR and arbitrarily primed PCR. In this research, the Acidithiobacillus ferrooxidans (A. ferrooxidans) strains were always separated into two groups in phylogenetic and cluster analyses. Genotypic analyses of the genes rusA, rusB, hip and iro suggest that these two groups may have different biochemical mechanisms for oxidizing ferrous iron. Strains in one A. ferrooxidans group were detected with rusA gene that encodes rusticyanin A which plays a very important role in the iron respiratory chain. The second A. ferrooxidans group was found to contain rusB gene which encode a homologous protein (RusB). The data suggested that ITS-based phylogeny is an effective tool to elucidate the relationships of Acidithiobacillus and that a different iron oxidation pathway may exist in different A. ferrooxidans groups.     

Purification and characterization of a potent hemolytic toxin with phospholipase A2 activity from the venom of Indian Russell's viper

A potent toxin with phospholipase A₂ (PLA₂) and hemolytic activity in vitro was purified from the Russell's viper venom of eastern India (RVV-EI). The purified protein (RVV-PFIIc) of 15.3 kDa molecular weight, and a lethal toxicity dose (LD₅₀ i.p.) of 0.1 mg/kg body weight, was the most toxic PLA₂ so far reported from the Indian subcontinent. The material also possessed anticoagulant activity as it enhanced the prothrombin induced plasma clotting time in vitro. The PLA₂ toxin (RVV-PFIIc) was shown to be different from other PLA₂s of RVV in respect to one or more of these parameters e.g. molecular weight, isoelectric pH, in vivo toxicity, specific activity of the enzyme and certain other biological activities. The first 19 amino terminal sequence (NLFQFAEMIVKMTGKEAVH) of RVV-PFIIc showed variable degree of homology (42.1–94.7%) with those of other RVV-PLA₂s described in the literature. Antisera raised against RVV-EI or RVV-PFIIc, though completely neutralized the in vivo lethal toxicity of RVV-EI or RVV-PFIIc, failed to inhibit their PLA₂ activity in vitro thereby suggesting that in vivo toxicity and in vitro activity of the enzyme may not be directly related. Apart from RVV-PFIIc, at least two other PLA₂ isozymes were found to be present in RVV-EI that were distinct from RVV-PFIIc in respect to their molecular, biological as well as serological properties. The significance of these and related data in antivenom therapy is discussed.     

Identification of hypervariable regions within the 16S–23S rRNA intergenic spacer region of Flavobacterium columnare and its application in assigning genomovar group to an individual strain

Flavobacterium columnare is an important bacterial pathogen of fish with a wide genetic variability within the species. This intra-species diversity has been termed as genomovars and genomovar groups on the basis of Restriction Fragment Length Polymorphisms of 16S rDNA and 16S–23S rDNA intergenic spacer region (ISR), respectively. In this study, we demonstrate the source of genetic heterogeneity in the F. columnare by sequence analysis of ISR. The length of ISR sequences of different genomovars varied from 553 to 592 nucleotides, while the similarity among sequences ranged from 76.1 to 92.6%. A common ISR structure with tRNA^Ala and tRNA^Ile embedded within the sequence was identified in all the genomovars of F. columnare. The results show that strains of F. columnare can be categorized into five genomovar groups based on the heterogeneity of the ISR sequences. Of these, strains belonging to Genomovar I and II can be sub-divided into two groups each; while strains of Genomovar III belong to one group. Sequence similarity between genomovar groups was lower for ISR (76.1–92.6%) as compared to 16S rDNA (96.1–99.4%) indicating its ability to resolve closely related groups within the genomovars of F. columnare. The main source of variation between the genomovar groups is the presence of three hyper variable regions (V1, V2, and V3) in the ISR. Of the three, V3 was found to be the most heterogeneous region and was found to be useful in assigning a genomovar group to an individual strain of F. columnare.     

Interactions of selected indole derivatives with phospholipase A<Subscript>2</Subscript>: <Emphasis Type="Italic">in silico and in vitro</Emphasis> analysis

Phospholipase A2 (PLA₂) is one of the key enzymes involved in the formation of inflammatory mediators. Inhibition of PLA₂ is considered to be one of the efficient methods to control inflammation. In silico docking studies of 160 selected indole derivatives performed against porcine pancreatic PLA₂ (ppsPLA₂) suggested that, CID2324681, CID8617 (indolebutyric acid or IBA), CID22097771 and CID802 (indoleacetic acid or IAA) exhibited highest binding energies. In silico analysis was carried out to predict some of the ADME properties. The binding potential of these compounds with human non pancreatic secretory PLA₂ (hnpsPLA₂) was determined using molecular docking studies. In order to corroborate the in silico results, enzyme kinetics and isothermal titration calorimetric analysis of the two selected compounds, IAA and IBA were performed against ppsPLA₂. From the analysis, it was concluded that IAA and IBA can act as competitive inhibitors to the enzyme and may be used as anti inflammatory agents.

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Figure Inhibitory activity of IAA and IBA against PLA₂