Accelerated evolution of small serum proteins (SSPs)-The PSP94 family proteins in a Japanese viper

Five small serum proteins (SSPs) with molecular masses of 6.5-10 kDa were detected in Habu (Trimeresurus flavoviridis) serum; this included two novel proteins SSP-4 and SSP-5. The amino acid sequences of these proteins and of SSP-1, SSP-2, and SSP-3, which were reported previously, were determined on the basis of the nucleotide sequences of their cDNAs. Although these proteins exhibited only limited sequence identity to mammalian prostatic secretory protein of 94 amino acids (PSP94), the topological pattern of disulfide bonds in SSPs was identical to that of the mammalian proteins. SSP-3 and SSP-4 lacked approximately 30 residues at the C-terminal. Each of the full-length cDNAs encoded a mature protein of 62-90 residues and a highly conserved signal peptide. The evolutionary distances between SSPs estimated on the basis of the amino acid changes were significantly greater than those of the synonymous nucleotide substitutions; these finding, together with results from analyses of nonsynonymous to synonymous rates of change (dN/dS) suggest that snake SSPs have endured substantial accelerated adaptive protein evolution. Such accelerated positive selection in SSPs parallels other findings of similar molecular evolution in snake venom proteins and suggests that diversifying selection on both systems may be linked, and that snake SSP genes may have evolved by gene duplication and rapid diversification to facilitate the acquisition of various functions to block venom activity within venomous snakes.     

Loss of Introns Along the Evolutionary Diversification Pathway of Snake Venom Disintegrins Evidenced by Sequence Analysis of Genomic DNA from <Emphasis Type="Italic">Macrovipera lebetina transmediterranea</Emphasis> and <Emphasis Type="Italic">Echis ocellatus</Emphasis>

Analysis of cDNAs from Macrovipera lebetina transmediterranea (Mlt) and Echis ocellatus (Eo) venom gland libraries encoding disintegrins argued strongly for a common ancestry of the messengers of short disintegrins and those for precursors of dimeric disintegrin chains. We now report the sequence analysis of disintegrin-coding genes from these two vipers. Genomic DNAs for dimeric disintegrin subunits Ml_G1 and Ml_G2 (Mlt) and Eo_D3 (Eo) contain single 1-kb introns exhibiting the 5′-GTAAG (donor)/3′-AG (acceptor) consensus intron splicing signature. On the other hand, the short RTS-disintegrins Ml_G3 (Mlt) and Eo_RTS (Eo) and the short RGD-disintegrin ocellatusin (Eo) are transcribed from intronless genomic DNA sequences, indicating that the evolutionary pathway leading to the emergence of short disintegrins involved the removal of all intronic sequences. The insertion position of the intron within Ml_G1, Ml_G2, and Eo_D3 is conserved in the genes for vertebrate ADAM (A disintegrin and metalloproteinase) protein disintegrin-like domains and within the gene for the medium-size snake disintegrins halystatins 2 and 3. However, a comparative analysis of currently available disintegrin(-like) genes outlines the view that a minimization of both the gene organization and the protein structure underlies the evolution of the snake venom disintegrin family. [Reviewing Editor: Dr. Bryan Fry] Amine Bazaa and Paula Juárez contributed equally to this work and may both be considered first authors.     

Rapid Evolution by Positive Selection and Gene Gain and Loss: PLA<Subscript>2</Subscript> Venom Genes in Closely Related <Emphasis Type="Italic">Sistrurus</Emphasis> Rattlesnakes with Divergent Diets

Rapid evolution of snake venom genes by positive selection has been reported previously but key features of this process such as the targets of selection, rates of gene turnover, and functional diversity of toxins generated remain unclear. This is especially true for closely related species with divergent diets. We describe the evolution of PLA₂ gene sequences isolated from genomic DNA from four taxa of Sistrurus rattlesnakes which feed on different prey. We identified four to seven distinct PLA₂ sequences in each taxon and phylogenetic analyses suggest that these sequences represent a rapidly evolving gene family consisting of both paralogous and homologous loci with high rates of gene gain and loss. Strong positive selection was implicated as a driving force in the evolution of these protein coding sequences. Exons coding for amino acids that make up mature proteins have levels of variation two to three times greater than those of the surrounding noncoding intronic sequences. Maximum likelihood models of coding sequence evolution reveal that a high proportion (∼30%) of all codons in the mature protein fall into a class of codons with an estimated d _N /d _S (ω) ratio of at least 2.8. An analysis of selection on individual codons identified nine residues as being under strong (p < 0.01) positive selection, with a disproportionately high proportion of these residues found in two functional regions of the PLA₂ protein (surface residues and putative anticoagulant region). This is direct evidence that diversifying selection has led to high levels of functional diversity due to structural differences in proteins among these snakes. Overall, our results demonstrate that both gene gain and loss and protein sequence evolution via positive selection are important evolutionary forces driving adaptive divergence in venom proteins in closely related species of venomous snakes.     

Snake Population Venomics: Proteomics-Based Analyses of Individual Variation Reveals Significant Gene Regulation Effects on Venom Protein Expression in <Emphasis Type="Italic">Sistrurus</Emphasis> Rattlesnakes

Studies of the molecular basis of adaptations seek to understand the relative importance of structural changes in proteins versus gene regulation effects as determinants of phenotype. Amino acid substitutions in gene coding sequences are well documented as causes of variation in snake venom proteins, whereas the importance of gene regulation effects on venom protein abundance and composition is less well known. Here, we use a proteomics-based approach to infer the effects of gene regulation on protein expression by comparing the relative abundance of specific, known venom proteins among different individuals in each of two species of Sistrurus rattlesnakes. Variation in the presence or absence, and in the relative amounts, of proteins was high in both species across all major protein families. Based on our empirical criteria for inferring regulatory effects (presence-absence of specific proteins and/or more than threefold variation in abundance) between 51% and 83% of S. catenatus individuals and between 40% and 63% of S. miliarius individuals showed evidence for gene regulation across the four most abundant proteins (disintegrins, phospholipase A₂’s, serine proteinases, and snake venom metalloproteases). Thus, the effects of gene regulation should be considered an important cause of variation in the composition of whole venoms at the intraspecific level. They also suggest the need for testing the adaptive hypothesis for venom plasticity in relation to prey consumed by adult snakes. Finally, the venom variability reported may have an impact in the treatment of bite victims, highlighting the necessity of using pooled venoms as a substrate for antivenom production.     

Snake venom alpha-neurotoxins and other 'three-finger' proteins.

The review is mainly devoted to snake venom alpha-neurotoxins which target different muscle-type and neuronal nicotinic acetylcholine receptors. The primary and spatial structures of other snake venom proteins as well as mammalian proteins of the Ly-6 family, which structurally resemble the 'three-finger' snake proteins, are also briefly discussed. The main emphasis is placed on recent data characterizing the alpha-neurotoxin interactions with nicotinic acetylcholine receptors.     

The molecular basis of venom resistance in a rattlesnake‐squirrel predator‐prey system

Understanding how interspecific interactions mould the molecular basis of adaptations in coevolving species is a long‐sought goal of evolutionary biology. Venom in predators and venom resistance proteins in prey are coevolving molecular phenotypes, and while venoms are highly complex mixtures it is unclear if prey respond with equally complex resistance traits. Here, we use a novel molecular methodology based on protein affinity columns to capture and identify candidate blood serum resistance proteins (“venom interactive proteins” [VIPs]) in California Ground Squirrels (Otospermophilus beecheyi) that interact with venom proteins from their main predator, Northern Pacific Rattlesnakes (Crotalus o. oreganus). This assay showed that serum‐based resistance is both population‐ and species‐specific, with serum proteins from ground squirrels showing higher binding affinities for venom proteins of local snakes compared to allopatric individuals. Venom protein specificity assays identified numerous and diverse candidate prey resistance VIPs but also potential targets of venom in prey tissues. Many specific VIPs bind to multiple snake venom proteins and, conversely, single venom proteins bind multiple VIPs, demonstrating that a portion of the squirrel blood serum “resistome” involves broad‐based inhibition of nonself proteins and suggests that resistance involves a toxin scavenging mechanism. Analyses of rates of evolution of VIP protein homologues in related mammals show that most of these proteins evolve under purifying selection possibly due to molecular constraints that limit the evolutionary responses of prey to rapidly evolving snake venom proteins. Our method represents a general approach to identify specific proteins involved in co‐evolutionary interactions between species at the molecular level.     

Phylogeny-Based Comparative Analysis of Venom Proteome Variation in a Clade of Rattlesnakes (Sistrurus sp.)

A long-standing question in evolutionary studies of snake venoms is the extent to which phylogenetic divergence and diet can account for between-species differences in venom composition. Here we apply phylogeny-based comparative methods to address this question. We use data on venom variation generated using proteomic techniques for all members of a small clade of rattlesnakes (Sistrurus sp.) and two outgroups for which phylogenetic and diet information is available. We first complete the characterization of venom variation for all members of this clade with a “venomic” analysis of pooled venoms from two members of this genus, S. milarius streckeri and S. m. milarius. These venoms exhibit the same general classes of proteins as those found in other Sistrurus species but differ in their relative abundances of specific protein families. We then test whether there is significant phylogenetic signal in the relative abundances of major venom proteins across species and if diet (measured as percent mammals and lizards among all prey consumed) covaries with venom composition after phylogenetic divergence is accounted for. We found no evidence for significant phylogenetic signal in venom variation: K values for seven snake venom proteins and two composite venom variables [PC 1 and 2]) were all nonsignificant and lower (mean = 0.11+0.06 sd) than mean K values (>0.35) previously reported for a wide range of morphological, life history, physiological and behavioral traits from other species. Finally, analyses based on Phylogenetic Generalized Least Squares (PGLS) methods reveal that variation in abundance of some venom proteins, most strongly CRISP is significantly related to snake diet. Our results demonstrate that venom variation in these snakes is evolutionarily a highly labile trait even among very closely-related taxa and that natural selection acting through diet variation may play a role in molding the relative abundance of specific venom proteins.     

Natural inhibitors of toxic phospholipases A(2)

Endogenous proteins isolated from the serum of snakes have been found to be natural inhibitors displaying anti-hemorrhagic, anti-neurotoxic or anti-myotoxic activity. Some of these proteins inhibit phospholipase A(2) (PLA(2)) activity. We review in brief here the properties, structure and classification of these PLA(2) inhibitors (PLIs), focusing in particular on the mechanism of neutralization of the toxic PLA(2)s by anti-neurotoxic PLIs. We also discuss: 1) the protection provided by these molecules against endogenous snake venom PLA(2)s; 2) their specificity for neurotoxic snake venom PLA(2)s (beta-neurotoxins) and non-toxic mammalian secreted sPLA(2)s; and 3) the domains of the inhibitor and PLA(2) potentially involved in the binding of these two molecules. Purified and characterized natural inhibitors of PLA(2)s may be used to develop more effective therapeutic strategies for dealing with snake envenomation. Moreover, the structural and, in some cases, functional similarity of natural inhibitors to various mammalian proteins suggests that these mammalian proteins may themselves behave as PLA(2) inhibitors. Thus, these proteins may have important physiological functions in regulating the activities of neurotoxic PLA(2) and non-toxic sPLA(2).     

蛇毒C型凝集素类蛋白cDNA与基因组DNA序列分析显示特别的转录后加工(英文)

为研究蛇毒C型凝集素类蛋白的快速进化机制和结构功能关系 ,使用PCR技术扩增了若干编码C型凝集素类蛋白 β链的cDNA分子以及agkisasinβ的基因组DNA ,并将这些扩增产物进行克隆和测序 .对测序结果与试验过程中的具体条件进行了因果关系分析 ,并且进行点阵图比较和多序列比对 .结果表明 ,可能存在“转录后同源重组”等转录后的事件 ,在蛇毒C型凝集素类蛋白的多样性上起着重要的作用 .对于解释基因数目与蛋白质数目的差异这一后基因组时代的重要问题 ,具有一定的参考价值 .首次报告蛇毒C型凝集素类蛋白的基因组DNA序列 ,其中未发现有内含子     

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     

Purification and Functional Characterisation of Rhiminopeptidase A,a Novel Aminopeptidase from the Venom of Bitis gabonica rhinoceros

Background

Snake bite is a major neglected public health issue within poor communities living in the rural areas of several countries throughout the world. An estimated 2.5 million people are bitten by snakes each year and the cost and lack of efficacy of current anti-venom therapy, together with the lack of detailed knowledge about toxic components of venom and their modes of action, and the unavailability of treatments in rural areas mean that annually there are around 125,000 deaths worldwide. In order to develop cheaper and more effective therapeutics, the toxic components of snake venom and their modes of action need to be clearly understood. One particularly poorly understood component of snake venom is aminopeptidases. These are exo-metalloproteases, which, in mammals, are involved in important physiological functions such as the maintenance of blood pressure and brain function. Although aminopeptidase activities have been reported in some snake venoms, no detailed analysis of any individual snake venom aminopeptidases has been performed so far. As is the case for mammals, snake venom aminopeptidases may also play important roles in altering the physiological functions of victims during envenomation. In order to further understand this important group of snake venom enzymes we have isolated, functionally characterised and analysed the sequence-structure relationships of an aminopeptidase from the venom of the large, highly venomous West African gaboon viper, Bitis gabonica rhinoceros.

Methodology and Principal Findings

The venom of B. g. rhinoceros was fractionated by size exclusion chromatography and fractions with aminopeptidase activities were isolated. Fractions with aminopeptidase activities showed a pure protein with a molecular weight of 150 kDa on SDS-PAGE. In the absence of calcium, this purified protein had broad aminopeptidase activities against acidic, basic and neutral amino acids but in the presence of calcium, it had only acidic aminopeptidase activity (APA). Together with the functional data, mass spectrometry analysis of the purified protein confirmed this as an aminopeptidase A and thus this has been named as rhiminopeptidase A. The complete gene sequence of rhiminopeptidase A was obtained by sequencing the PCR amplified aminopeptidase A gene from the venom gland cDNA of B. g. rhinoceros. The gene codes for a predicted protein of 955 amino acids (110 kDa), which contains the key amino acids necessary for functioning as an aminopeptidase A. A structural model of rhiminopeptidase A shows the structure to consist of 4 domains: an N-terminal saddle-shaped β domain, a mixed α and β catalytic domain, a β-sandwich domain and a C-terminal α helical domain.

Conclusions

This study describes the discovery and characterisation of a novel aminopeptidase A from the venom of B. g. rhinoceros and highlights its potential biological importance. Similar to mammalian aminopeptidases, rhiminopeptidase A might be capable of playing roles in altering the blood pressure and brain function of victims. Furthermore, it could have additional effects on the biological functions of other host proteins by cleaving their N-terminal amino acids. This study points towards the importance of complete analysis of individual components of snake venom in order to develop effective therapies for snake bites.     

Eggs-Only Diet: Its Implications for the Toxin Profile Changes and Ecology of the Marbled Sea Snake (Aipysurus eydouxii)

Studies so far have correlated the variation in the composition of snake venoms with the target prey population and snakes diet. Here we present the first example of an alternative evolutionary link between venom composition and dietary adaptation of snakes. We describe a dinucleotide deletion in the only three finger toxin gene expressed in the sea snake Aipysurus eydouxii (Marbled Sea Snake) venom and how it may have been the result of a significant change in dietary habits. The deletion leads to a frame shift and truncation with an accompanying loss of neurotoxicity. Due to the remarkable streamlining of sea snake venoms, a mutation of a single toxin can have dramatic effects on the whole venom, in this case likely explaining the 50- to 100-fold decrease in venom toxicity in comparison to that of other species in the same genus. This is a secondary result of the adaptation of A. eydouxii to a new dietary habit — feeding exclusively on fish eggs and, thus, the snake no longer using its venom for prey capture. This was parallel to greatly atrophied venom glands and loss of effective fangs. It is interesting to note that a potent venom was not maintained for use in defense, thus reinforcing that the primary use of snake venom is for prey capture.Nucleotide sequence data reported here have been deposited in the GenBank database under accession number AY559317.Reviewing Editor: Dr. Martin Kreitman     

Muscle Tissue Damage Induced by the Venom of Bothrops asper: Identification of Early and Late Pathological Events through Proteomic Analysis

The time-course of the pathological effects induced by the venom of the snake Bothrops asper in muscle tissue was investigated by a combination of histology, proteomic analysis of exudates collected in the vicinity of damaged muscle, and immunodetection of extracellular matrix proteins in exudates. Proteomic assay of exudates has become an excellent new methodological tool to detect key biomarkers of tissue alterations for a more integrative perspective of snake venom-induced pathology. The time-course analysis of the intracellular proteins showed an early presence of cytosolic and mitochondrial proteins in exudates, while cytoskeletal proteins increased later on. This underscores the rapid cytotoxic effect of venom, especially in muscle fibers, due to the action of myotoxic phospholipases A₂, followed by the action of proteinases in the cytoskeleton of damaged muscle fibers. Similarly, the early presence of basement membrane (BM) and other extracellular matrix (ECM) proteins in exudates reflects the rapid microvascular damage and hemorrhage induced by snake venom metalloproteinases. The presence of fragments of type IV collagen and perlecan one hour after envenoming suggests that hydrolysis of these mechanically/structurally-relevant BM components plays a key role in the genesis of hemorrhage. On the other hand, the increment of some ECM proteins in the exudate at later time intervals is likely a consequence of the action of endogenous matrix metalloproteinases (MMPs) or of de novo synthesis of ECM proteins during tissue remodeling as part of the inflammatory reaction. Our results offer relevant insights for a more integrative and systematic understanding of the time-course dynamics of muscle tissue damage induced by B. asper venom and possibly other viperid venoms.     

First structure of a snake venom metalloproteinase: a prototype for matrix metalloproteinases/collagenases.

Adamalysin II, a 24 kDa zinc endopeptidase from the snake venom of Crotalus adamanteus, is a member of a large family of metalloproteinases isolated as small proteinases or proteolytic domains of mosaic haemorrhagic proteins from various snake venoms. Homologous domains have recently been detected in multimodular mammalian reproductive tract proteins. The 2.0 A crystal structure of adamalysin II reveals an ellipsoidal molecule with a shallow active-site cleft separating a relatively irregularly folded subdomain from the calcium-binding main molecular body composed of a five-stranded beta-sheet and four alpha-helices. The folding of the peptide fragment containing the zinc-binding motif HExxHxxGxxH bears only a distant resemblance to thermolysin, but is identical to that found in astacin, with the three histidines and a water molecule (linked to the glutamic acid) likewise constituting the zinc ligand; adamalysin II lacks a fifth (tyrosine) zinc ligand, however, leaving its zinc ion tetrahedrally co-ordinated. Furthermore, adamalysin II and astacin share an identical active-site basement formed by a common Metturn. Due to their virtually identical active-site environment and similar folding topology, the snake venom metalloproteinases (hitherto called adamalysins) and the astacins (and presumably also the matrix metalloproteinases/mammalian collagenases and the Serratia proteinase-like large bacterial proteinases) might be grouped into a common superfamily with distinct differences from the thermolysin family.     

Molecular diversity in venom from the Australian Brown snake, Pseudonaja textilis

Venom from the Australian elapid Pseudonaja textilis (Common or Eastern Brown snake), is the second most toxic snake venom known and is the most common cause of death from snake bite in Australia. This venom is known to contain a prothrombin activator complex, serine proteinase inhibitors, various phospholipase A2s, and pre- and postsynaptic neurotoxins. In this study, we performed a proteomic identification of the venom using two-dimensional gel electrophoresis, mass spectrometry, and de novo peptide sequencing. We identified most of the venom proteins including proteins previously not known to be present in the venom. In addition, we used immunoblotting and post-translational modification-specific enzyme stains and antibodies that reveal the complexity and regional diversity of the venom. Modifications observed include phosphorylation, gamma-carboxylation, and glycosylation. Glycoproteins were further characterized by enzymatic deglycosylation and by lectin binding specificity. The venom contains an abundance of glycoproteins with N-linked sugars that include glucose/mannose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acids. Additionally there are multiple isoforms of mammalian coagulation factors that comprise a significant proportion of the venom. Indeed two of the identified proteins, a procoagulant and a plasmin inhibitor, are currently in development as human therapeutic agents.     

蛇毒素蛋白毕赤酵母表达进展

蛇毒是许多具有独特生物活性的蛋白质与酶的混合物,在基础科学研究和临床上有重大应用价值,但是通过从蛇毒中分离获取活性组分具有局限性。巴斯德毕赤酵母表达系统是最为常用的真核表达系统之一,其真核加工、折叠、翻译后修饰等能力使得所表达的重组蛋白具有与天然蛋白近似的生物活性,因而该系统在富含二硫键或糖基化的蛇毒素蛋白表达中被广为采用。迄今为止,已经有12个属的25种蛇毒素蛋白(包括蛇毒丝氨酸蛋白酶、金属蛋白酶/去整合素、L-氨基酸氧化 酶、C-型凝集素和神经毒素、血管收缩因子、神经生长因子等家族)在毕赤酵母中获得成功表达,蛇毒富半胱氨酸蛋白、缓激肽增强肽(BPP)等至今尚未见酵母表达的报道。毕赤酵母表达蛇毒素蛋白失败的原因可能在于,有关密码子偏爱性、目的基因转录出的RNA二级结构特征、糖基化程度不均一及糖型差异、所表达毒素对酵母细胞的毒性等方面,并对解决的方法进行了讨论。     

MDC9, a widely expressed cellular disintegrin containing cytoplasmic SH3 ligand domains

Cellular disintegrins are a family of proteins that are related to snake venom integrin ligands and metalloproteases. We have cloned and sequenced the mouse and human homologue of a widely expressed cellular disintegrin, which we have termed MDC9 (for metalloprotease/disintegrin/cysteine-rich protein 9). The deduced mouse and human protein sequences are 82% identical. MDC9 contains several distinct protein domains: a signal sequence is followed by a prodomain and a domain with sequence similarity to snake venom metalloproteases, a disintegrin domain, a cysteine-rich region, an EGF repeat, a membrane anchor, and a cytoplasmic tail. The cytoplasmic tail of MDC9 has two proline-rich sequences which can bind the SH3 domain of Src, and may therefore function as SH3 ligand domains. Western blot analysis shows that MDC9 is an approximately 84-kD glycoprotein in all mouse tissues examined, and in NIH 3T3 fibroblast and C2C12 myoblast mouse cell lines. MDC9 can be both cell surface biotinylated and 125I-labeled in NIH 3T3 mouse fibroblasts, indicating that the protein is present on the plasma membrane. Expression of MDC9 in COS-7 cells yields an 84-kD protein, and immunofluorescence analysis of COS-7 cells expressing MDC9 shows a staining pattern that is consistent with a plasma membrane localization. The apparent molecular mass of 84 kD suggests that MDC9 contains a membrane-anchored metalloprotease and disintegrin domain. We propose that MDC9 might function as a membrane-anchored integrin ligand or metalloprotease, or that MDC9 may combine both activities in one protein.     

Geographic isolation facilitates the evolution of reproductive isolation and morphological divergence

Geographic isolation is known to contribute to divergent evolution, resulting in unique phenotypes. Oftentimes morphologically distinct populations are found to be interfertile while reproductive isolation is found to exist within nominal morphological species revealing the existence of cryptic species. These disparities can be difficult to predict or explain especially when they do not reflect an inferred history of common ancestry which suggests that environmental factors affect the nature of ecological divergence. A series of laboratory experiments and observational studies were used to address what role biogeographic factors may play in the ecological divergence of Hyalella amphipods. It was found that geographic isolation plays a key role in the evolution of reproductive isolation and divergent morphology and that divergence cannot be explained by molecular genetic variation.