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Characterization, primary structure and molecular evolution of anticoagulant protein from Agkistrodon actus venom
Abstract
An anticoagulant protein named AaACP was isolated from Agkistrodon actus (hundred-pace snake of Taiwan, Viperidae) venom. AaACP inhibited the factor Xa-induced plasma coagulation in a concentration-dependent manner. Thus, AaACP seems to bind to factor Xa in prothrombinase complex. AaACP was composed of A and B chains linked by disulphide bond(s). The amino acid sequences of A and B chains of AaACP were analysed with a few residues unidentified which were complemented from the nucleotide sequences of their cDNAs. The A chain consisted of 129 amino acid residues and the B chain 123 amino acid residues. Their amino acid sequences were highly similar to those of A and B chains of a series of anticoagulant proteins which had been purified from the venoms of some Viperidae snakes. The A and B chains structurally belong to C-type lectin-like protein family of snake venom origin. Construction of phylogenetic tree of C-type lectins and C-type lectin-like proteins based on their amino acid sequences indicated that their A and B chains diverged before speciation of snake species. The comparison of the nucleotide sequences of the cDNAs encoding A and B chains of AaACP and of Trimeresurus flavoviridis (Viperidae) venom-gland factors IX/X-binding protein and factor IX-binding protein showed that the mature protein-coding region is much more variable than the signal peptide-coding domain and the 5'- and 3'-untranslated regions, being in contrast to the case of the ordinary isoprotein genes. The ratios of the numbers of nucleotide substitutions per nonsynonymous site (K(A)) and per synonymous site (K(S)) in the mature protein-coding region in the cDNA pairs were about three times greater than those for the ordinary isoprotein genes, suggesting that these genes have been evolving in an accelerated manner. Taking account of the functional diversities of venom-gland C-type lectins and C-type lectin-like proteins including factors IX and/or X-binding proteins, it can be said that their functional diversities have been acquired by accelerated evolution.
Supplementary resources (2)
Nucleotide Sequence
January 2000
Nucleotide Sequence
January 2000
... It has been reported that the signal peptide-coding sequence conservation in CTLs/CTLDs proteins does not directly correspond to a high homology of the total deduced amino acid sequence of these proteins [24,25]. For 5 0 -UTRs of rBJcuL, a higher level of sequence identities with CTLDs signal peptide sequence than the deduced amino acid (data not shown) was observed. ...
... According to Fig. 2, the comparison of the deduced BJcuL sequence showed that the mature 135-amino acid residues present about 90% of identity with TSL [15], around 60% with BFL-1 and BFL-2, Bungarus fasciatus lectins [17], and 40% with CTLDs proteins, such as Convulxin, from Crotalus durissus terriWcus venom [26], anticoagulant IX/XBP from Trimeresurus Xavoviridis [14], and AaACP an anti-coagulant from Agkistrodon actus venom [24]. ...
... Alignments of sequence were made according to the half-cystine residues. The sequence is: TSL, the c-type lectin from T. stejnegeri [15]; BFL 1 and BFL 2, B. fasciatus lectins [17]; Convulxin from C. d. terriWcus [24]; anticoagulant IX/XBP from T. Xavoviridis [14]; AaACP, an anti-coagulant from A. actus venom [25]; and rat mannose-binding protein (RMBP, residues 115-238) [27]. The asterisk indicates identical residues with rBJcuL. ...
The lactose-binding lectin from Bothrops jararacussu venom (BJcuL) is a homodimer belonging to group VII of the c-type animal lectins. BJcuL has also been shown to serve as an interesting tool for combating tumor progression by inhibiting cancer and endothelial cell growth. However, detailed structural studies of BJcuL and its biological mechanisms of cytotoxicity are yet to be reported, perhaps because of the non-availability of recombinant proteins in necessary quantities. Intending to increase the present information about structural and consequently the understating of biological studies, the cDNA coding for BJcuL from a venom gland has been cloned and sequenced. The mature protein-coding region was amplified by PCR with specific oligonucleotides, and subcloned into the pET-15b vector to express the recombinant BJcuL in Escherichia coli BL21 (DE3). The deduced amino acid sequence exhibits a high degree of sequence identity with c-type lectins (CTLs) and c-type lectin-like domains (CTLDs). An insoluble and inactive 18.5-kDa protein was overexpressed after 1.0mM IPTG induction. The recombinant BJcuL was recovered and denatured in a buffer with 6M urea and purified on a nickel-affinity column. Protein refolding was carried out on this column, during procedure purification, followed by dialysis against CTBS and then by gel filtration for separation of the active dimmer. The refolding process of rBJcuL and the analysis of its structure were confirmed by biological assay, circular dichroism, and MALDI-TOF.
... Although highly homologous to C-type lectins, these proteins differ from C-type lectins in their physiological activities. For example, the anticoagulant proteins, factor IX/X-binding protein (Atoda et al., 1991) and factor IX-binding protein, have been isolated from Trimeresurus flavoviridis venom (Atoda et al., 1995), factor X-binding protein was also isolated from the venom of Agkistrodon actus (hundred-pace snake) (Atoda et al., 1998;Tani et al., 2002), and botrocetin, which potentiates the binding of von Willebrand factor (VWF) to glycoprotein Ib and blocks platelet aggregation (Read et al., 1989;Andrew et al., 1989), and bothrojaracin, a hirudin-like a-thrombin inhibitor, were isolated from Bothrops jararaca venom (Zingali et al., 1993). ...
... Many anticoagulant C-type lectin-like proteins have been isolated from various snake species and characterized (Table 1). Based on their ligand recognition specificity, they can be classified into three types: (1) coagulation factor IX/X-binding proteins, such as habu IX/X-binding protein from T. flavoviridis (Atoda and Morita, 1989;Atoda et al., 1991), jararaca IX/X-binding protein from B. jararaca (Sekiya et al., 1993), ECLV IX/ X-binding protein from Echis carinatus leucogaster (Chen and Tsai, 1996), and halyxin from A. halys brevicaudus (Koo et al., 2002); (2) coagulation factor IX-binding proteins, such as habu IX-binding protein from T. flavoviridis (Atoda et al., 1995), TSV FIX-binding protein from T. stejnegri (Xu et al., 1999), and AHP-IXbinding protein from A. halys pallas (Zang et al., 2003); and (3) coagulation factor X-binding proteins, such as acutus X-binding protein from Deinagkistrodon acutus (Atoda et al., 1998) and AaACP from A. actus (Tani et al., 2002). In the presence of Ca 2C , these proteins bind to the g-carboxyglutamic acid (Gla) domain of coagulation factors IX and X (Atoda et al., 1994(Atoda et al., , 1995(Atoda et al., , 1998. ...
... It is evident that the A and B chains diverged from an ancestral C-type lectin before speciation of snake species. A similar branching pattern is observed in the phylogenetic tree constructed using the amino acid sequences (Tani et al., 2002). Furthermore, the A and B chains can be divided into three groups on the basis of their target proteins and functions. ...
A number of C-type lectin-like proteins that affect thrombosis and hemostasis by inhibiting or activating specific platelet membrane receptors or blood coagulation factors have been isolated from the venom of various snake species and characterized and more than 80 have been sequenced. Recent data on the primary sequences and 3D structures of C-type lectins and C-type lectin-like proteins from snake venoms have enabled us to analyze their molecular evolution. Statistical analysis of their cDNA sequences shows that C-type lectin-like proteins, with some exceptions, have evolved in an accelerated manner to acquire their diverse functions. Phylogenetic analysis shows that the A and B chains of C-type lectin-like proteins are clearly separated from C-type lectins and that the A and B chains are further divided into a group of platelet receptor-binding proteins and a group of coagulation factor-binding proteins. Elucidation of the tertiary structures of several C-type lectin-like proteins led to the discovery of a unique domain-swapping interaction between heterodimeric subunits, which creates a concave surface for ligand binding.
... The phylogenic tree of Cc-Lec and blast results showed that the purified lectin presented similarities with snake venom CLPs and was clustered with the  chain of a CLP derived from Gloydius intrmidius. Indeed, previous studies revealed the similarity of ␣ and  chains of a CLP (Aa ACP) derived from Agkistrodon actus with Crotalos atrox and Trimeresurus steigeni galactose-binding lectins [89]. Moreover, phylogenic exploration of SVLs showed that ␣ and  chains of snake CLPs diverged from an ancestral C-type lectin before speciation of snake species and the functional diversities among CTLs and CLPs have been acquired by accelerated evolution [4,89]. ...
... Indeed, previous studies revealed the similarity of ␣ and  chains of a CLP (Aa ACP) derived from Agkistrodon actus with Crotalos atrox and Trimeresurus steigeni galactose-binding lectins [89]. Moreover, phylogenic exploration of SVLs showed that ␣ and  chains of snake CLPs diverged from an ancestral C-type lectin before speciation of snake species and the functional diversities among CTLs and CLPs have been acquired by accelerated evolution [4,89]. Cc-Lec 3D structure was modeled by homology to the B chain of the template 1J35.pdb, a CLP purified by Protobothrops flavoridis with 48% of homology. ...
In this study, we reported for the first time the biochemical and structural characterization of Cc-Lec, a C-type lectin purified from Cerastes cerastes venom by affinity chromatography. This lectin was homogeneous by SDS-PAGE, and was shown to be a 34 271.59 Da polypeptide by Electrospray mass spectrometry MS-ES-TOF. Its identified sequence of 160 amino acids corresponding to one subunit, revealed a high identity with other related proteins. Cc-Lec modeled 3D structure appeared as homodimer cross-linked by one disulfide bridge. Cc-Lec exhibited a calcium dependent hemagglutinating activity against human group O erythrocytes. Cc-Lec inhibited platelet aggregation induced by ADP, arachidonic acid or fibrinogen suggesting its interaction with their specific receptors namely P2Y1 and/or P2Y12, GPIIb/IIIa and TPα respectively. Cc-Lec was not lethal for mice until 10 mg/kg administered by i.p. route. The lectin displayed a lasting anticoagulation on mice plasma even two days post-injection. This anticoagulation seems to be related to its interaction with coagulation factors Xa and IXa. Therefore, Cc-Lec prevented FXa amidolytic activity with Km = 4.33 10⁻⁴ μg/mL and ki = 14.4 μg/mL. It seems to interact with these targets through CRD domain which could make it a good target as a pharmacological promising molecule in thrombosis diagnosis and therapy.
... Moreover, it seems reasonable to expect that snakes of the same genus contain similar subtypes or paralogs of the venom proteins. Previous findings on various venom proteins have revealed that gene duplication followed by accelerated evolution of their functional domains have made a significant contribution to the functional diversities of viperid venom proteins (Deshimaru et al., 1996; Nakashima et al., 1995; Tani et al., 2002). For adaptation and endurance of the snakes, less frequent mutations in the txr.cls ...
... 1993) and C-lectin like integrin-agonist on the platelet (Huang et al., 2004); it also causes myonecrosis due to the effects of high content of N6-PLAs (Chen et al. 1994; Tsai et al., 1995 ) and K49- PLAs (Liu et al., 1991). Envenoming by D. acutus causes severe hemorrhage and coagulopathy due to the synergism of both the Zn +2 -metalloproteases () and the thrombin like fibrinogenase (Wang et al., 2001 ), and rich contents of Clectin like antiplatelet proteins (Chen et al., 2000; Tani et al., 2002). The envenoming by T. stejnegeri and O. gracilis causes local edema, apparently due to some basic PLAs (Tsai et al., 2004) and kininogenases. ...
The major viperid snakes present in Taiwan today are: Daboia russelli siamensis, Deinagkistrodon acutus, Protobothrops mucrosquamatus, Trimeresurus stejnegeri, Ovophis monticola and Ovophis gracilis. As suggested by the species trees deduced from mtDNA sequences of the family Viperidae, these species apparently belong to five different genera. Molecular cloning, N-terminal sequencing, and mass spectrometry have facilitated the sequence-determination of venom proteins. Previous examples of using venom proteins as characteristics for the phylogenetic and evolutionary studies are reviewed herein. Two new phylogeny trees using protein sequence datasets for the crotalid venom acidic phospholipases and the disintegrins are shown. Both trees grossly reveal phylogeographic relationships between the species, although some inconsistencies exist. Moreover, distinct protein markers in each of the Taiwanese viperid venoms and their relations to the snakebite symptoms are discussed in terms of the known snake systematics.
... Recently, the cDNAs encoding the subunits of ACFI and ACFII were cloned from the cDNA library of A. acutus venom gland (Yu et al., 2003). The deduced amino acid sequences of ACFI showed high similarity to factor X-binding proteins (X-bps) from Deinagkistrodon acutus venom (Atoda et al., 1998), while ACFII was highly homologous to AaACP from A. acutus venom (Tani et al., 2002). Therefore, recombinant expression of ACFI may be helpful to investigate its structure-function relationships and clinical applications. ...
... As ACFI is highly homologous to X-bp, it should be speculated that both subunits of ACFI interact with factor X in a cooperative manner similar to that of X-bp (Fig. 7). Actually, all the coagulation factor-binding CLPs are strictly conserved in those residues involved in the direct interactions, while the two regions involved in the hydrogen bond interactions in their a subunits exhibit more variations and they were presumed to at least partially discriminate the recognizing specificity for factor IX and/or X (Atoda et al., 1995(Atoda et al., , 1998Chen et al., 1996;Lee et al., 2003;Tani et al., 2002). Our results confirmed that both subunits of ACFI are quite important and irreplaceable for its binding to coagulation factors. ...
ACFI is an anticoagulant C-type lectin-like protein (CLP) isolated from Agkistrodon acutus venom. To investigate the function of ACFI and its subunits, the cDNAs of two subunits were transformed and expressed in Pichia pastoris separately or together by a novel strategy using two vectors with different selectable markers. The results showed that recombinant homodimers were secreted when the subunits were expressed alone, while heterodimers (rACFI) were secreted when two subunits were co-expressed. The secreted proteins were purified from culture supernatants in one step by metal-chelating affinity chromatography with the yields of 1-4 mg/L. PAGE and ELISA showed that rACFI competed the binding of native ACFI for human factor X and IX with affinities of 1.6 and 30 nM, respectively. In addition, rACFI prolonged the activated partial thromboplastin time (APTT) in a concentration dependent manner as same as native ACFI. However, recombinant alpha or beta homodimers completely lost these activities, indicating the heterodimerization of two subunits is required for its function. It also suggests that P. pastoris is a promising system for structure-function studies of snake CLPs.
... On the other hand, the acidic PLA 2 isoforms dominant in B. hyoprora or B. taeniatus venom predict anticoagulant activity in these venoms. In addition, the high proportions of CTL in the venoms of B. b. smaragdinus, B. atrox, and B. taeniatus collected in Acre indicates hemostatic disturbances involving platelet activation [62] that may result in their depletion aggravated by anticoagulation as CTLs may be potent inhibitors of thrombin [63][64][65]. ...
Snake venoms have evolved in several families of Caenophidae, and their toxins have been assumed to be biochemical weapons with a role as a trophic adaptation. However, it remains unclear how venom contributes to the success of venomous species for adaptation to different environments. Here we compared the venoms from Bothrocophias hyoprora, Bothrops taeniatus, Bothrops bilineatus smaragdinus, Bothrops brazili, and Bothrops atrox collected in the Amazon Rainforest, aiming to understand the ecological and toxinological consequences of venom composition. Transcriptomic and proteomic analyses indicated that the venoms presented the same toxin groups characteristic from bothropoids, but with distinct isoforms with variable qualitative and quantitative abundances, contributing to distinct enzymatic and toxic effects. Despite the particularities of each venom, commercial Bothrops antivenom recognized the venom components and neutralized the lethality of all species. No clear features could be observed between venoms from arboreal and terrestrial habitats, nor in the dispersion of the species throughout the Amazon habitats, supporting the notion that venom composition may not shape the ecological or toxinological characteristics of these snake species and that other factors influence their foraging or dispersal in different ecological niches.
... Most of them act as antagonists to platelet aggregation but with different functional pathways. Agkicetin binds to the platelet glycoprotein (GP) Ib-IX-V receptor complex and inhibits the ability of von Willebrand factor (VWF) and thrombin to interact with GPIb-IX-V complex (Chen and Tsai, 1995;Xu et al., 2005); snaclec anticoagulant protein attaches to coagulation factor Xa, inhibiting conversion of prothrombin to thrombin (Atoda et al., 1998;Tani et al., 2002); agkisacutacin not only binds to GPIb, serving as a blocker of VWF, but also interacts with the key coagulation factors IX and X to block the coagulation cascade (Li et al., 2005). All of these activities are associated with the clinical symptoms of D. acutus envenomation. ...
DEINAGKISTRODON ACUTUS: also known as the hundred-pace viper or Chinese moccasin, is a clinically significant venomous snake in Taiwan. To address the lack of knowledge on the venom proteome of D. acutus, the venom composition was studied by a bottom-up proteomic approach combining reverse phase high-performance liquid chromatography, SDS-PAGE, and LC-MS/MS analysis. The immunoreactivity and cross-reactivity of Taiwanese freeze-dried D. acutus antivenom (DA-AV) and hemorrhagic antivenom (FH-AV) were investigated, as well. The proteomic analysis revealed the presence of 29 distinct proteins from D. acutus venom belonging to 8 snake venom protein families. Snake venom metalloproteinase (SVMP, 46.86%), C-type lectin (CLEC, 37.59%), phospholipase A2 (PLA2, 7.33%) and snake venom serine protease (SVSP, 6.62%) were the most abundant proteins. In addition to DA-AV, FH-AV also showed a profile of broad immunorecognition toward the venom of D. acutus. Remarkably, both antivenoms specifically reacted with the HPLC fractions containing SVMPs, and the titer was 5-10 times higher than fractions of other components. This information helps us to deeply understand the pathophysiology of D. acutus envenomation and guide us to development of more effective antivenom for clinical treatment.
... La comparaison des nucléotides de l'ADN codant pour chaque chaîne d'AaACP et de la protéine liant les facteurs IX/X de Trimeresurus flavoviridis (Viperidé) a prouvé que la région codant pour la protéine mature est beaucoup plus variable que le domaine du peptide signal (contrairement à se qui est trouvé d'ordinaire). Tenant compte des diversitésfonctionnelles des protéines ophidiennes on peut supposer qu'elles ont été acquises par une évolution accélérée(Tani et al., 2002). ...
Les venins des Vipéridés, Crotalidés, certaines espèces d' Elapidés australiens et quelques Colubridés sont riches en enzymes qui ont une action multifactorielle sur l'hémostase. Elles sont capables d' agir aussi bien sur l' agrégation plaquettaire que sur la cascade de la coagulation ou sur la fibrinolyse, avec un effet activateur ou inhibiteur. De plus, une même molécule peut posséder des activités différentes.Les recherches effectuées sur de nombreuses protéines ophidiennes ont permis dedécouvrir leurs structures ainsi que leurs modes d' action. Elles sont aujourd' hui utilisées dans des tests d'hémostase, en recherche fondamentale et comme outils thérapeutiques. Elles ont également des utilisations pot entielles en tant qu ' agents antithrombotiques, antiinfectieux ou anti-cancéreux.
... Finally, the amino acid sequences of the tryptic peptides resultant from P8-IIa band (Fig. 4) showed high similarity/identity with invariant or conservatively substituted amino acid residues corresponding to b-subunit snake C-type lectins (Snaclec) from Trimeresurus albolabris (alboaggregin-A; Kowalska et al., 1998), from P. flavoviridis (flavocetin-A; Shin et al., 2000), from Deinagkistrodon acutus (agkicetin-C; Xu et al., 2005), from G. blomhoffii (mamushigin; Sakurai et al., 1998). On the other hand, the amino acid sequence of the tryptic peptides resultant from the P8-IIb band showed high similarity/identity with invariant and/or conservatively substituted amino acid residues corresponding to Snaclec coagulation factor IX/factor X-binding proteins subunit A from T. stejnegeri, P. flavoviridis and G. brevicaudus (Koo et al., 2002;Tani et al., 2002), and a-subunits of snake C-type lectins (Snaclec) from Trimeresurus albolabris (alboaggregin-D, Mekchay andRojnuckarin, 2012) andB. jararaca (botrocetin, Sen et al., 2001). ...
There is a rising interest in snake venoms proteins (SVPs) because these macromolecules are related to pharmacological properties that manifest themselves during poisoning and can lead to secondary microbial infections. Interestingly, researchers have somehow neglected the antimicrobial activity of SVPs. The aims of this study were: (i) to verify whether the venom of the Peruvian snake Bothriopsis oligolepis displays such activity; (ii) to isolate and identify some its antimicrobial constituents. Liquid growth inhibition assays revealed that the crude venom inhibited the growth of Gram-positive and Gram-negative bacteria, but not of Candida species. Fractionation of the venom by anion-exchange chromatography provided fractions P2, P4 and P8 active against S. aureus. Fractionation of P2 or P8 by gel-filtration chromatography and of P4 by RP-HPLC furnished the sub-fractions P2-I, P8-II and P4-II, respectively, being both active against S. aureus. Analyses of these sub-fractions by SDS-PAGE under denaturing/reducing conditions evidenced SVPs with 59–73, 27 and 14–28 kDa, respectively. Their in-gel tryptic digestion gave peptide fragments, whose sequencing by MALDI-TOF/MS followed by protein BLAST analysis allowed identifying PIII metalloprotease(s) [SVMP(s)], in P2-I, serine protease(s), [SVMP(s)], in P4-II and lectin(s) in P8-II. Detection of gelatinolytic activity in P2-I and P4-II reinforced the existence of PIII-SVMP(s) and SVSP(s), respectively. Activation of the coagulation cascade intrinsic pathway by P8-II (probably by interaction with factors IX and/or X as some snake C-type lectins do) supported the presence of C-type lectin(s). Altogether, these new findings reveal that the venom of the Peruvian snake Bothriopsis oligolepis displays antibacterial activity and that the isolated SVMP(s), SVSP(s) and C-type lectin(s) are associated to its ability to inhibit the growth of S. aureus.
... With the limited information available on viper venom cDNAs encoding hyaluronidase and hyaluronidase-like molecules, it is too early to comment on the presumed evolutionary history of the enzyme. However, gene duplication is an important evolutionary mechanism that results in the formation of functionally distinct isomers of snake venom metalloproteases (Moura da Silva et al., 1996), PLA 2 s (Ohno et al., 2003), C-type lectins (Tani et al., 2002), Kunitz/bovine pancreatic trypsin inhibitors (Zupunski et al., 2003), and serine proteases (Deshimaru et al., 1996), and it likely plays a role in the evolution of hyaluronidase isomers as well. ...
... The K N /K S < 1 indicated that the mature protein coding region of the RVV-V cDNA evolved more rapidly than the conserved UTR. This observation was also found in serine proteases and phospholipase A2s from T. flavoviridis and Trimeresurus gramineus, and serine proteases from Deinagkistrodon actus (Deshimaru et al., 1996; Nakashima et al., 1995; Nikandrov et al., 2005; Tani et al., 2002). ...
Serine proteases are widely found in snake venoms. They have variety of functions including contributions to hemostasis. In this study, five serine proteases were cloned and characterized from two different cDNA libraries: factor V activator (RVV-V), alpha fibrinogenase (RVAF) and beta fibrinogenase (RVBF) from Russell's viper (Daboia russelli siamensis), and plasminogen activator (APL-PA) and protein C activator (APL-C) from Agkistrodon piscivorus leucostoma. The snake venom serine proteases were clustered in phylogenetic tree according to their functions. K(A)/K(S) values suggested that accelerated evolution has occurred in the mature protein coding regions in cDNAs of snake venom serine proteases.
... Thus, the gene organization has influenced not only its own evolution, but also the toxin diversity. For instance, gene organization contributes to accelerated evolution of polypeptides, since gene hypervariation benefits evolutionary advantage (27,32,45). ...
Snake venom (sv) C-type lectins encompass a group of hemorrhagic toxins, which are able to interfere with hemostasis. They share significant similarity in their primary structures with C-type lectins of other animals, and also present a conserved carbohydrate recognition domain (CRD). A very well studied sv C-type lectin is the heterodimeric toxin, convulxin (CVX), from the venoms of South American rattlesnakes, Crotalus durissus terrificus and C. d. cascavella. It consists of two subunits, alfa (CVXalpha , 13.9 kDa) and beta (CVXbeta , 12.6 kDa), joined by inter and intra-chain disulfide bounds, and is arranged in a tetrameric alpha4beta4 conformation. Convulxin is able to activate platelet and induce their aggregation by acting via p62/GPVI collagen receptor. Several cDNA precursors, homolog of CVX subunits, were cloned by PCR homology screening. As determined by computational analysis, one of them, named crotacetin beta subunit, was predicted as a polypeptide with a tridimensional conformation very similar to other subunits of convulxin-like snake toxins. Crotacetin was purified from C. durissus venoms by gel permeation and reverse phase high performance liquid chromatography. The heterodimeric crotacetin is expressed in the venoms of several C. durissus subspecies, but it is prevalent in the venom of C. durissus cascavella. As inferred from homology modeling, crotacetin induces platelet aggregation but noticeably exhibits antimicrobial activity against Gram-positive and Gram-negative bacteria.
... Most of the venomous proteins are encoded as multigene families in the animal genomes that have been generated through gene duplications and also exhibit accelerated molecular evolution due to positive selection acting on the molecules. From snake venoms, phospholipases A 2 (Nakashima et al. 1993Nakashima et al. , 1995 Ogawa et al. 1995, Nobuhisa et al. 1996 Gubenšek 1996, 1997 ), serine proteases (Deshimaru et al. 1996 ), metalloproteases (Moura- Silva et al. 1996 ), Kunitz/BPTI (bovine pancreatic trypsin inhibitor) proteins (Z ˇ upunski et al. 2003), three-fingered toxins (Lachemanon et al. 1998; Ohno et al. 1998; Gong et al. 2000), and C-type lectins (Tani et al. 2002; Ogawa et al. 2005 ) have been identified as multiple copied and positively selected venomous proteins. Similar evolutionary patterns have been found in scorpion sodium channel toxins () and Conus conotoxins (Duda and Palumbi 1999). ...
In social aphids of the genus Tuberaphis a cysteine protease gene of the family cathepsin B exhibits soldier-specific expression and intestinal protease production. The product is orally excreted and injected by soldier nymphs into natural enemies, thereby exerting an insecticidal activity. In an attempt to gain insights into when and how the novel venomous protease for the altruistic caste has evolved, we investigated the soldier-specific type (S-type) and nonspecific type (N-type) cathepsin B genes from social and nonsocial aphids. All the social aphids examined, representing the genera Tuberaphis, Astegopteryx, and Cerataphis, possessed both the S-type and N-type genes. Phylogenetically distant nonsocial aphids also possessed cathepsin B genes allied to the S-type and the N-type, indicating the evolutionary origin of these genes in the common ancestor of extant aphids. In Tuberaphis species the S-type genes exhibited significant soldier-specific expression and accelerated molecular evolution whereas the N-type genes did not. In Astegopteryx and Cerataphis species, meanwhile, both the S-type and N-type genes exhibited neither remarkable soldier-specific expression nor accelerated molecular evolution. These results suggest that the S-type gene acquired the soldier-specific expression and the venom function after divergence of the genus Tuberaphis. On the structural model of the S-type protease of Tuberaphis styraci the accelerated molecular evolution was associated with the molecular surface rather than the catalytic cleft, suggesting that the venom activity was probably acquired by relatively minor modifications on the molecular surface rather than by generation of a novel active site. In Cerataphis jamuritsu the S-type gene was, although containing a stop codon, structurally almost intact and still transcribed, suggesting recent pseudogenization of the gene copy and possible relevance to relaxed functional constraint in the highly multiplied protease gene family. On the basis of these results we suggest that the massive amplification in aphid cathepsin B genes might have predisposed the evolution of venomous protease in the social aphid lineage and argue that gene duplication, accelerated molecular evolution, and acquisition of novel gene function must have played considerable roles in the evolution of complex biological systems including insect sociality.
... Russel's viper (Vipera russeli) (RVV-X), which contains a metalloproteinase and a C-type lectin-like domain, activates blood coagulation factor X (Takeya et al., 1992). Some proteins binds to the Gla domain of factors IX, X, or both such as-factor IX/X-binding protein, factor IXbinding protein (Atoda et al., 1991(Atoda et al., , 1995, and factor X-binding protein (Atoda et al., 1998;Tani et al., 2002). Botrocetin binds to the von Willebrand factor (vWF) and blocks platelet aggregation and agglutination (Read et al., 1989;Andrews et al., 1989). ...
Snake venom C-type lectin-like proteins (CLPs) are ubiquitously found in Viperidae snake venoms and differ from the C-type lectins as they display different biological activities but no carbohydrate-binding activity. Previous analysis of the transcriptome obtained from the Bothrops insularis venom gland showed the presence of two clusters homologous to bothrojaracin (BJC) chains alpha and beta. In an effort to identify a new BJC-like molecule, we used an approach associated with proteomic technologies to identify the presence of the expressed protein and then to purify and characterize a new thrombin inhibitor from B. insularis venom. We also constructed homology models of this protein and BJC, which were compared with other C-type lectin-like family members and revealed several conserved features of this intriguing snake venom toxin family.
C-type lectin-like proteins of snake have a variety of biological properties, acting for example as an anticoagulant, procoagulant, and agonist/antagonist of platelet activation. Dimerization or oligomer formation of carbohydrate recognition domain (CRD) in C-type lectin by 3D domain swapping generates novel proteins with new functions such as coagulant-, anticoagulant-, and platelet-modulating activities. The structural and functional studies of the first identified C-type lectin-like proteins, IX/X-bp, have been instrumental in defining how new functionally heterodimeric C-type lectin-like proteins are generated from monomeric CRD (carbohydrate recognition domain) in C-type lectin by 3D domain swapping. The crystal structure of IX/X-bp revealed that the two subunits associated by 3 D-domain swapping, and this dimerization resulted in the creation of a concave surface serving as a binding site of Gla domain, the functionally important domain of blood coagulation factors. The strong activities by snaclecs such as IX/X-bp and X-bp are caused by the binding at the Gla domain of factors IX and X. C-type lectin-like proteins of snake venom such as IX/X-bp and its structurally-related proteins recognize various ligands by the higher frequency of mutation in the open reading frames than in the non-coding regions after duplication of a gene.
Snake venoms are rich in a large variety of proteins and peptides that can interfere with the hemostatic system. This review focuses on bothrojaracin, which is snaclec molecule, either bind to thrombin, inhibiting its biological activities, such as clotting of fibrinogen, platelet activation or to prothrombin, impairing thrombin formation. Bothrojaracin interacts with both molecules, forming a stable 1:1 complex. The calculated Kd for bothrojaracin was 0.6 nM and 100 nM for thrombin and prothrombin, respectively. Bothrojaracin binds to thrombin exosite I displacing ligands such as fibrin, hirudin, thrombomodulin and factor V and do not block the catalytic site. This protein has helped in our understanding of some molecular aspects of the thrombin and prothrombin structure-function relationship. The knowledge about the mechanism of action and details of structural aspects will certainly result in new medical and pharmacological applications. Furthermore, bothrojaracin offer attractive template for the development of rationally designed therapeutic agents.
Snake venoms are rich in a large variety of proteins and peptides that can interfere with the hemostatic system. This review focuses on snake-venom proteins, with or without enzymatic activity, that bind to blood coagulation factors and exhibit anticoagulant effects. These proteins include (1) anticoagulant phospholipases A2, which bind to factor X and impair the formation of prothrombinase complex independently from their enzymatic activity; (2) factor IX- and X-binding proteins, which belong to the family of C-type lectin-like proteins and interact with the Gla domain of factor X/Xa and/or IX/IXa; (3) prothrombin- and thrombin-binding proteins, represented by bothrojaracin and salmorin, which are also C-type lectin-like molecules and either bind to prothrombin, impairing thrombin formation, or to thrombin, inhibiting its biological activities, such as clotting of fibrinogen, platelet activation, and so forth; and (4) L-amino oxidases that have recently been described to have anticoagulant activity. The general structural characteristics of these proteins and current knowledge regarding their mechanisms of action are discussed. These proteins have helped in our understanding of some molecular aspects of the hemostatic process. A more detailed analysis of the structure–function relationship of these molecules will certainly result in new medical and pharmacological applications. Furthermore, venom components offer attractive templates for the development of rationally designed therapeutic agents.
Based on sequence comparison, snake venom components affecting hemostasis have been classified into various families, including serine proteases, metalloproteinases, C-type lectins, disintegrins, and phospholipases. These proteins affect platelet function by binding or degrading von Willebrand factor (VWF) or platelet membrane glycoproteins, activating protease-activated receptors, or modulating ADP release and thromboxane A2 formation. Many snake venom C-type lectins have now been characterized, mostly heterodime ric structures with α and β subunits that are often multimerized to form large molecules. They affect platelet activation by binding to VWF or to specific collagen receptors such as GPIb, α2β1, and GPVI. While simple heterodimeric GPIb-binding molecules mostly inhibit platelet functions, multimeric ones often activate platelets. Some act by inducing VWF to bind to GPIb. Another series of snake venom C-type lectins activates platelets by binding to GPVI, while yet another series affects, platelet function via integrin α2β 1. Snake venom C-type lectins, have a typical fold structure like that in classic C-type lectins, such as the selectins and mannose-binding proteins. More and more structures of these proteins, often complexed with their ligands, have been determined, and structure–activity studies have shown that these proteins are quite a complex group, though with similar backbone folding. Recent studies have shown that snake C-type lectins often interact with more than one platelet receptor and have complex mechanisms of action. It is also noteworthy that snake C-type lectins may act differently in vivo and in vitro.
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.
Phospholipase A2s (phosphatide sn-2 acylhydrolase, EC 3.1.1.4, PLA2) hydrolyze specifically the sn-2 ester bond of phospholipids (PL). With some exceptions, they are Ca²⁺-dependent enzymes. PLA2s are present in many organs and body fluids of vertebrates, invertebrates, and insects. They play an important role in the phospholipid digestion, remodeling, and metabolism, and are involved in human diseases such as acute pancreatitis, rheumatoid arthritis, respiratory distress, and acute chest syndrome. This review summarizes recent data on the biological function, amino acid sequence, classification, structure–function relationships, and 3D structures of PLA2s. Special attention is paid to the pharmacological activities of the snake venom enzymes. These hydrolases exert their activity through specific protein–protein interactions. Two types of structurally and functionally distinct receptors, N- and M-type, were identified during the last decade and characterized. PLA2 binding to its protein receptor can lead to cell damage, tissue necrosis, and other undesirable processes. The ‘interfacial activation’ of the enzymes and their participation in the interfacial catalysis are discussed. Protection of cell membranes from PLA2s is of medical importance.
Many cases of accelerated evolution driven by positive Darwinian selection are identified in the genes of venomous and reproductive proteins. This evolutional phenomenon might have important consequences in their gene-products' functions, such as multiple specific toxins for quick immobilization of the prey and the establishment of barriers to fertilization that might lead to speciation, and in the molecular evolution of novel genes. Recently, we analyzed the molecular evolution of two galectins isolated from the skin mucus of conger eel (Conger myriaster), named congerins I and II, by cDNA cloning and X-ray structural analysis, and we found that they have evolved in the rapid adaptive manner to emergence of a new structure including strand-swapping and a unique new ligand-binding site. In this review article we summarize and discuss the molecular evolution, especially the rapid adaptive evolution, and the structure-function relationships of conger eel galectins.
The protein composition of the crude venom of Sistrurus barbouri was analyzed by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were separated by reversed phase high-performance liquid chromatography and characterized by N-terminal sequence analysis. The molecular mass and number of cysteine residues of the purified proteins were determined by matrix-associated laser desorption/ionization-time of flight mass spectrometry. Selected protein bands were subjected to in-gel tryptic digestion and peptide mass fingerprinting. Analysis of the tandem mass spectrometry spectra of selected doubly-charged peptide ions was done by collision-induced dissociation in a quadrupole-linear ion trap instrument. Our results show that the venom proteome of the pigmy rattlesnake S. barbouri is composed of proteins belonging to a few protein families, which can be structurally characterized by their disulfide bond contents.
Vipera lebetina venom contains specific coagulant Factor X activator (VLFXA) that cleaves the Arg52-Ile53 bond in the heavy chain of human factor X. VLFXA is a glycoprotein that is composed of a heavy chain (HC) and two light chains (LC) linked by disulfide bonds. The complete amino acid sequences of the three chains of the factor X activator from V. lebetina snake venom are deduced from the nucleotide sequences of cDNAs encoding these chains. The full-length cDNA (2347 bp) sequence of the HC encodes an open reading frame (ORF) of 612 amino acids that includes signal peptide, propeptide and mature metalloproteinase with disintegrin-like and cysteine-rich domains. The light chain LC1 contains 123 and LC2 135 amino acid residues. Both light chains belong to the class of C-type lectin-like proteins. The N-termini of VLFXA chains and inner sequences of peptide fragments detected by liquid chromatography-electrospray ionization tandem mass spectrometry (LC MS/MS) from protein sequence are 100% identical to the sequences deduced from the cDNA. The molecular masses of tryptic fragments of VLFXA chains analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) also confirm the protein sequences deduced from the cDNAs. These are the first cloned factor X activator heavy and light chains. We demonstrate that the heavy and light chains are synthesized from different genes.
Snake venom is known to contain an abundance of enzyme isoforms, and various disorders associated with envenomation have been ascribed partially to their diversified functions. Crude venom of Bothrops jararaca was subjected to conventional two-dimensional SDS-PAGE, followed by immunoblot analysis using an antiserum raised against KN-BJ 2, a serine proteinase previously isolated from this venom. A number of immunoreactive proteins with comparable molecular masses and different pIs emerged, implying the venom contains yet-unknown serine proteinases. A B. jararaca venom gland cDNA library was subsequently screened with a labeled KN-BJ 2 cDNA as a probe. Among a number of positive cDNA clones, three--HS112, HS114, and HS120--were selected and sequenced. These clones each had an open reading frame of 759-774 bp, and their deduced amino acid sequences illustrated considerable similarities to that of KN-BJ 2 as well as to those of serine proteinases of different origins. However, no apparent match to any of the deposited sequences was found in the current GenBank/EMBL databases, indicating that each of these cDNA clones encodes a serine proteinase distinct from the known enzymes. Analyses of the nucleotide and amino acid sequences of these cDNA clones support the accelerated evolution hypothesis proposed for snake venom enzymes.
C-type lectin-like proteins (CLPs) have a variety of biological activities, including anticoagulant- and platelet-modulating activities but have no lectin activity. CLPs are made up of heterodimers or oligomers of heterodimers, while C-type lectins from snake venom are composed exclusively of homodimers or homooligomers. In the last decade, numerous CLPs, such as blood coagulation factor IX/X-binding protein and botrocetin, have been isolated from various snake venoms, sequenced, and characterized. In addition, RVV-X (factor X activator) and carinactivase-1 (prothrombin activator) are metalloproteases composed of two C-type lectin-like domains that recognize the Gla domain of factor X and prothrombin, respectively. The basic structures of these CLPs include two homologous subunits: subunit alpha (A chain) of 14-15 kDa and subunit beta (B chain) of 13-14 kDa. CLPs occur in a variety of oligomeric forms, including alphabeta, (alphabeta)(2), and (alphabeta)(4). The basic homologous dimer (alphabeta) of these CLPs is formed by three-dimensional (3D) domain swapping. The CLPs constitute a new protein family and are useful tools for elucidating the mechanisms involved in clotting and platelet activation as well as the structure-function relationships of both blood clotting factors and platelet glycoproteins.
Nonrandom patterns associated with adaptively evolving genes can shed light on how selection and mutation produce rapid changes in sequences. I examine such patterns in two independent families of antimicrobial peptide genes: those in frogs, which are known to have evolved under positive selection, and those in flatfishes, which I show have also evolved under positive selection. I address two recently proposed hypotheses about the molecular evolution of antimicrobial peptide genes. The first is that the mature peptide region is replicated by an error-prone polymerase that increases the mutation rate and the transversion/transition ratio compared to the signal sequence of the same genes. The second is that mature peptides evolve in a coordinated fashion with their propieces, such that a change in net charge in one molecular region prompts an opposite change in charge in the other region. I test these hypotheses using alternative methods that minimize alignment errors, correct for phylogenetic nonindependence, reduce sequence saturation, and account for differing selection pressures on different regions of the gene. In both gene families I show that divergence at both synonymous and nonsynonymous sites within the mature peptide region is enhanced. However, in neither gene family is there evidence of an increased mutational transversion/transition ratio or coordinated evolution. My observations are consistent with either an elevated mutation rate in an adaptively evolving gene region or widespread selection on "silent" sites. These hypotheses challenge the assumption that mutations are random and can be measured by the synonymous substitution rate.
The protein composition of the crude venoms of the three most important vipers of Tunisia was analyzed by RP-HPLC, N-terminal sequence analysis, MALDI-TOF mass determination, and in-gel tryptic digestion followed by PMF and CID-MS/MS of selected peptide ions in a quadrupole-linear IT instrument. Our results show that the venom proteomes of Cerastes cerastes, Cerastes vipera, and Macrovipera lebetina are composed of proteins belonging to a few protein families. However, each venom showed distinct degree of protein composition complexity. The three venoms shared a number of protein classes though the relative occurrence of these toxins was different in each snake species. On the other hand, the venoms of the Cerastes species and Macrovipera lebetina each contained unique components. The comparative proteomic analysis of Tunisian snake venoms provides a comprehensible catalogue of secreted proteins, which may contribute to a deeper understanding of the biological effects of the venoms, and may also serve as a starting point for studying structure-function correlations of individual toxins.
Snake venom (sv) C-type lectins encompass a group of hemorrhagic toxins that are capable of interfering with blood stasis. A very well-studied svC-type lectin is the heterodimeric toxin, convulxin (CVX), from the venom of South American rattlesnake Crotalus durissus terrificus. CVX is able to activate platelets and induce their aggregation by acting via p62/GPVI collagen receptor. By using polymerase chain reaction homology screening, we have cloned several cDNA precursors of CVX subunit homologs. One of them, named crotacetin (CTC) beta-subunit, predicts a polypeptide with a topology very similar to the tridimensional conformations of other subunits of CVX-like snake toxins, as determined by computational analysis. Using gel permeation and reverse-phase high-performance liquid chromatography, CTC was purified from C. durissus venoms. CTC can be isolated from the venom of several C. durissus subspecies, but its quantitative predominance is in the venom of C. durissus cascavella. Functional analysis indicates that CTC induces platelet aggregation, and, importantly, exhibits an antimicrobial activity against Gram-positive and -negative bacteria, comparable with CVX.
We report the cloning and sequence analysis of BA-5A from a venom gland cDNA library of the puff adder, Bitis arietans, that encodes a novel ECD-disintegrin-like domain. BA-5A is a unique PII disintegrin. It contains the 16 cysteine residues that are conserved in all known disintegrin-like domains of ADAM proteins and snake venom metalloproteinases but lacks the cysteine-rich domain. These features suggest that BA-5A may represent an intermediate in the evolutionary pathway of the long disintegrin bitistatin and that removal of the cysteine-rich domain and loss of the PIII-specific disulfide bond were separate events along the structural diversification pathway of disintegrins, the former predating the latter. The protein family composition of the Bitis arietans venom, as determined by combination of reversed-phase HPLC and proteomic analysis, was as follows: Zn(2+)-metalloproteinase (38.5%), serine proteinase (19.5%), disintegrin (17.8%), C-type lectin-like (13.2%), PLA(2) (4.3%), Kunitz-type inhibitor (4.1%), cystatin (1.7%), and unknown (0.9%). BA-5A could not be detected in the venom proteome of Bitis arietans. The occurrence of this very low-abundance (< 0.05%) or nonexpressed disintegrin transcript indicates a hitherto unrecognized structural diversity of this protein family. Whether BA-5A plays a physiological role or represents an orphan protein which could eventually evolve a role in the adaptation of snakes to changing ecological niches and prey habits deserves further investigation.
We report the cloning and sequence analysis of Echis ocellatus cDNAs coding for dimeric disintegrin subunits and for the short disintegrin ocellatusin. All the dimeric disintegrin subunit messengers belong to the short-coding class, indicating that short messengers may be more widely distributed than previously thought. Mass spectrometric analysis of the HPLC-separated venom proteins was performed to characterize the dimeric disintegrins expressed in the venom proteome. In addition to previously reported EO4 and EO5 heterodimers, a novel dimeric disintegrin containing RGD- and KGD-bearing subunits was identified. However, a WGD-containing polypeptide encoded by clone Eo1-1 was not detected in the venom, suggesting the occurrence of larger genomic than proteomic diversity, which could represent part of a non-venom-secreted reservoir of disintegrin that may eventually acquire physiological relevance for the snake upon changes of ecological niches and prey habits. On the other hand, the realization of the existence of two distinct messengers coding for the short disintegrin ocellatusin reveals key events of the evolutionary emergence of the short disintegrin ocellatusin from a short-coding dimeric disintegrin precursor by two nucleotide mutations.
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.
The immobilisation of prey by snakes is most efficiently achieved by the rapid dissemination of venom from its site of injection into the blood stream. Hyaluronidase is a common component of snake venoms and has been termed the "venom spreading factor". In the absence of nucleotide or protein sequence data to confirm the functional identity of this venom component, we interrogated a venom gland EST database for the saw-scaled viper, Echis ocellatus (Nigeria), using the gene ontology (GO) term "carbohydrate metabolism". A single hyalurononglucosaminadase-activity matching sequence (EOC00242) was found and used to design PCR primers to acquire the full-length cDNA sequence. Although very different from the bee venom and mammalian hyaluronidase sequences, the E. ocellatus sequence retained all the catalytic, positional and structural residues that characterise this class of carbohydrate metabolising hydrolases. An extraordinarily high level of sequence identity (>95%) was observed in analogous venom gland cDNA sequences isolated (by PCR) from another saw-scaled viper species, E. pyramidum leakeyi (Kenya), and from the sahara horned viper, Cerastes cerastes cerastes (Egypt) and the puff adder, Bitis arietans (Nigeria). Smaller amplicons, lacking hyaluronidase catalytic residues because of 768 bp or 855 bp central deletions, appear to encode either truncated peptides without hyaluronidase activity, or are non-translated transcripts because they lack consensus translation initiating motifs.
After two decades of study, we draw the conclusion that venom-gland phospholipase A2 (PLA2) isozymes, including PLA2 myotoxins of Crotalinae snakes, have evolved in an accelerated manner to acquire their diverse physiological activities. In this review, we describe how accelerated evolution of venom PLA2 isozymes was discovered. This type of evolution is fundamental for other venom isozyme systems. Accelerated evolution of venom PLA2 isozyme genes is due to rapid change in exons, but not in introns and the flanking regions, being completely opposite to the case of the ordinary isozyme genes. The molecular mechanism by which proper base substitutions had occurred in the particular sites of venom isozyme genes is a puzzle to be solved in future studies. It should be noted that accelerated evolution occurred until the isozymes had acquired their particular function and, since then, they have evolved with less frequent mutation, possibly for functional conservation. We also found that interisland mutations occurred in venom PLA2 isozymes. The relationships between mutation and its driving force are speculative and the real mechanism remains a mystery.
Phospholipase A2 inhibitor (PLI), purified from the blood plasma of the Habu snake (Trimeresurus flavoviridis), was separated into two distinct subunits, PLI-A and PLI-B. These subunits were shown to be glycoproteins with molecular weights of around 21,000-22,000. When they were deglycosylated chemically with trifluoromethanesulfonic acid, the molecular weights were found to be 17,000. Their amino acid sequences were determined by alignment of peptides obtained by lysyl endopeptidase digestion and Staphylococcus aureus V8 protease digestion. PLI-A and PLI-B were each composed of 147 amino acid residues with one residue, Asn103, being for N-linked glycosylation, and the molecular weights of their protein portions were calculated to be 16,368 and 16,408, respectively. Each subunit contained four cysteine residues, all of which exist in disulfide linkages (Cys64-Cys141 and Cys119-Cys133). The sequences of PLI-A and PLI-B showed 89.9% homology to each other. When the sequences were compared with those of lipocortins, no significant homologies were detected. But the sequences were significantly homologous to those of COOH-terminal carbohydrate recognition portions of pulmonary surfactant apoprotein and animal lectins.
The nucleotide sequences of four genes encoding Trimeresurus gramineus (green habu snake, crotalinae) venom gland phospholipase A_2 (PLA2; phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4) isozymes were compared internally and externally with those of six genes encoding Trim-eresurus flavoviridis (habu snake, crotalinae) venom gland PLA2 isozymes. The numbers of nucleotide substitutions per site (K_N) for the noncoding regions including introns were one-third to one-eighth of the numbers of nucleotide substitutions per synonymous site (K_S) for the protein-coding regions of exons, indicating that the noncoding regions are much more conserved than the protein-coding regions. The K_N values for the introns were found to be nearly equivalent to those of introns of T. gramineus and T. flavoviridis TATA box-binding protein genes, which are assumed to be a general (nonvenomous) gene. Thus, it is evident that the introns of venom gland PLA2 isozyme genes have evolved at a similar rate to those of nonvenomous genes. The numbers of nucleotide substitutions per nonsynonymous site (K_A) were close to or larger than the K_S values for the protein-coding regions in venom gland PLA2 isozyme genes. All of the data combined reveal that Darwinian-type accelerated evolution has universally occurred only in the protein-coding regions of crotalinae snake venom PLA2 isozyme genes.
Six Trimeresurus flavoviridis (Habu snake) venom gland phospholipase A_2 (PLA2) isozyme genes were found to consist of four exons and three introns and to encode proteins of 138 amino acid residues, including the signal sequence of 16 amino acid residues. Comparison of the nucleotide sequences showed that the introns are much more homologous than the protein-coding regions of exons except for the signal peptide-coding region of the first exon. The numbers of nucleotide substitutions per site (K_N) for introns are approximately one-fourth of the numbers of nucleotide substitutions per synonymous site (K_S) for the protein-coding regions, indicating that the introns are unusually conserved. The absence of an apparent functional role for the introns suggests that the protein-coding regions, except for the signal peptide-coding domains, have evolved at greater substitution rates than introns. The fact that the numbers of nucleotide substitutions per nonsynonymous site (K_A) are close to or larger than K_S values for relevant pairs of genes revealed that Darwinian-type accelerated substitutions have occurred in the protein-coding regions or exons. This is compatible with the presence of PLA2 species with diverse physiological activities in the venom.
As a step toward understanding the structure and function of phospholipases A_2 (PLA2s), we isolated and sequenced several cDNAs encoding Trimeresurus flavoviridis venom PLA2 isozymes including two [Lys49]PLA2s called basic proteins I and II, [Thr37]PLA2, and PLX'-PLA2. Comparison of the nucleotide sequences of these cDNAs with the previously isolated [Asp49]PLA2 cDNA revealed some interesting findings from the viewpoint of evolution. First, the homologies of the 5' and 3' untranslated regions (98% and 89%, respectively) were much higher than that of the protein-coding regions (67%). The predicted secondary structure showed the characteristic stem-loop structures for both the untranslated regions of the mRNAs, suggesting that these regions play some functional role(s) in translation or stability of mRNAs. Second, base substitutions appeared to have occurred at similar rates for the three positions of codons among these PLA2s. The results are discussed in terms of evolution of PLA2s. Northern blot analysis showed that these PLA2s are specific to venom gland.
We determined the complete amino acid sequence of RVV-X, the blood coagulation factor X activating enzyme, isolated from Russell's viper venom and studied structure-function relationships. RVV-X (M(r) 79,000) consists of a disulfide-bonded two-chain glycoprotein with a heavy chain of M(r) 59,000 and a light chain of heterogeneous M(r) 18,000 (LC1) and 21,000 (LC2). These chains were separated after reduction and S-pyridylethylation, and the isolated major component LC1 was used for sequence analysis. The heavy chain consists of 427 residues containing four asparagine-linked oligosaccharides, and its entire sequence was similar to that of the high molecular mass hemorrhagic protein, HR1B, isolated from the venom of Trimere-surus flavoviridis. The heavy chain contains three distinct domains, metalloproteinase, disintegrin (platelet aggregation inhibitor)-like and unknown cysteine-rich domains. On the other hand, light chain LC1 consists of 123 amino acid residues containing one asparagine-linked oligosaccharide and shows sequence homology similar to that found in the so-called C-type (Ca(2+)-dependent) lectins. Therefore, RVV-X is a novel metalloproteinase containing a mosaic structure with distintegrin-like, cysteine-rich, and C-type lectin-like domains. RVV-X potently inhibits collagen- and ADP-stimulated platelet aggregations, probably via its distintegrin-like domain, although this domain does not contain the Arg-Gly-Asp sequence which is conserved in various venom distintegrins and which is thought to be one of the interaction sites for platelet integrins. Our findings also indicate that snake venom factor IX/factor X-binding protein with a C-type lectin structure (Atoda, H., Hyuga, M., and Morita, T. (1991) J. Biol. Chem. 266, 14903-14911) inhibits RVV-X-catalyzed factor X activation; hence, the light chain of RVV-X probably participates in recognizing some portion of the zymogen factor X.
An anticoagulant protein, factor IX/factor X-binding protein (IX/X-bp), isolated from the venom of Trimeresurus flavoviridis, binds with factor IX and factor X in the presence of Ca2+ with a 1 to 1 stoichiometry (Atoda, H., and Morita, T. (1989) J. Biochem. (Tokyo) 106, 808-813). Analysis of S-pyridylethylated IX/X-bp by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a 16.0-kDa band (designated the A chain) and a 15.5-kDa band (designated the B chain). These two chains were separated by reversed-phase high performance liquid chromatography, and their complete amino acid sequences were determined by sequencing of the peptides obtained after digestion with lysyl endopeptidase, chymotrypsin, and V8 protease from Staphylococcus aureus and after chemical cleavage with cyanogen bromide. The A chain had an amino-terminal sequence of Asp-Cys-Leu-Ser-Gly- and consisted of 129 residues with Mr 14,830. The B chain has an amino-terminal sequence of Asp-Cys-Pro-Ser-Asp- and consists of 123 residues of Mr 14,440. There was 47% identity between the A and the B chain. The sequence of IX/X-bp showed 25-37% identity with that of the C-type carbohydrate recognition domain-like structure of acorn barnacle lectin, human and rat asialoglycoprotein receptors, the human lymphocyte Fc epsilon receptor for immunoglobulin E, proteoglycan core protein, pancreatic stone protein, and tetranectin. The sequences of the first 18 amino acid residues of both the A and B chains were also, to a certain extent, homologous to the partial amino acid sequence of the b subunit of factor XIII, a member of the beta 2-glycoprotein I-like family. In this region, some similarity with the amino-terminal amino acid sequence of botrocetin was also observed.
The complete primary structure of a galactose-specific lectin contained in the venom of the rattlesnake, Crotalus atrox, was determined. The lectin is composed of two covalently linked, identical subunits, each consisting of 135 amino acid residues. Under physiological conditions the lectin proved to be highly aggregated. The venom lectin contained 9 half-cystines, 8 of which formed four intrasubunit disulfide bridges (Cys3-Cys14, Cys31-Cys131, Cys38-Cys133, and Cys106-Cys123), while Cys86 was involved in an intersubunit disulfide bridge. Because of the high content of disulfide bridges, the intact lectin was extremely resistant to tryptic digestion. The determined amino acid sequence was found to be homologous with those of the so-called carbohydrate recognition domains of Ca2(+)-dependent-type lectins in animal. Among them, 8 amino acid residues (Cys31, Gly69, Trp92, Pro97, Cys106, Asp120, Cys123, and Cys131) were completely conserved. Leu40, Trp67, and Trp81 were also well conserved. The rattlesnake venom lectin showed high hemagglutinating activity. These results, together with the occurrence of similar lectins in crotalid venoms, suggest that these lectins have evolved in order to make the venom a more effective weapon to capture prey animals.
We have developed three computer programs for comparisons of protein and DNA sequences. They can be used to search sequence data bases, evaluate similarity scores, and identify periodic structures based on local sequence similarity. The FASTA program is a more sensitive derivative of the FASTP program, which can be used to search protein or DNA sequence data bases and can compare a protein sequence to a DNA sequence data base by translating the DNA data base as it is searched. FASTA includes an additional step in the calculation of the initial pairwise similarity score that allows multiple regions of similarity to be joined to increase the score of related sequences. The RDF2 program can be used to evaluate the significance of similarity scores using a shuffling method that preserves local sequence composition. The LFASTA program can display all the regions of local similarity between two sequences with scores greater than a threshold, using the same scoring parameters and a similar alignment algorithm; these local similarities can be displayed as a "graphic matrix" plot or as individual alignments. In addition, these programs have been generalized to allow comparison of DNA or protein sequences based on a variety of alternative scoring matrices.
The complete amino acid sequence and location of the disulfide bonds of two-chain botrocetin, which promotes platelet agglutination in the presence of von Willebrand factor, from venom of the snake Bothrops jararaca are presented. Sequences of the alpha and beta subunits were determined by analysis of peptides generated by digestion of the S-pyridylethylated protein with Achromobacter protease I or alpha-chymotrypsin and by chemical cleavage with cyanogen bromide or 2-(2'-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine. Two-chain botrocetin is a heterodimer composed of the alpha subunit (consisting of 133 amino acid residues) and the beta subunit (consisting of 125 amino acid residues) held together by a disulfide bond. Seven disulfide bonds link half-cystine residues 2 to 13, 30 to 128, and 103 to 120 of the alpha subunit; 2 to 13, 30 to 121, and 98 to 113 of the beta subunit; and 80 of the alpha subunit to 75 of the beta subunit. In terms of amino acid sequence and disulfide bond location, two-chain botrocetin is homologous to echinoidin (a sea urchin lectin) and other C-type (Ca(2+)-dependent) lectins.
Jararaca GPIb-BP, a snake venom protein composed of alpha and beta subunits purified from Bothrops jararaca, binds to platelet glycoprotein (GP)Ib and functions as a receptor blocker for von Willebrand factor binding to GPIb (Fujimura, Y., Ikeda, Y., Miura, S., Yoshida, E., Shima, H., Nishida, S., Suzuki, M., Titani, K., Taniuchi, Y., and Kawasaki, T. (1995) Thromb. Haemostasis 74, 743-750). We present here the entire 142- and 123-residue amino acid sequence of the respective alpha and beta subunits and also demonstrate that the platelet GPIb-binding site resides on the beta and not on the alpha subunit based on an enzyme-linked immunosorbent assay using biotin-labeled jararaca GPIb-BP and competing ligands. Sequences of the alpha and beta subunits were determined by analysis of the intact S-pyridylethylated proteins and their peptides generated by digestion with Achromobacter protease I, Staphyloccocus aureus V8 protease, pepsin, endoproteinase Asp-N, or L-1-tosylamino-2-phenylethyl chloromethyl ketone-trypsin. A 38-39% identity of amino acid sequence between the alpha and beta subunits of jararaca GPIb-BP was observed, as well as a high degree of sequence identities (38-64%) with the respective subunits of botrocetin (Usami, Y., Fujimura, Y., Suzuki, M., Ozeki, Y., Nishio, K., Fukui, H., and Titani, K (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 928-932) and the beta-chain of echicetin (Peng, M., Holt, J. C., and Niewiarowski, S. (1994) Biochem. Biophys. Res. Commun. 205, 68-72).
Echicetin, a heterodimeric protein from the venom of Echis carinatus, binds to platelet glycoprotein Ib (GPIb) and so inhibits platelet aggregation or agglutination induced by various platelet agonists acting via GPIb. The amino acid sequence of the beta subunit of echicetin has been reported and found to belong to the recently identified snake venom subclass of the C-type lectin protein family. Echicetin alpha and beta subunits were purified. N-terminal sequence analysis provided direct evidence that the protein purified was echicetin. The paper presents the complete amino acid sequence of the alpha subunit and computer models of the alpha and beta subunits. The sequence of alpha echicetin is highly similar to the alpha and beta chains of various heterodimeric and homodimeric C-type lectins. Neither of the fully reduced and alkylated alpha or beta subunits of echicetin inhibited the platelet agglutination induced by von Willebrand factor-ristocetin or alpha-thrombin. Earlier reports about the inhibitory activity of reduced and alkylated echicetin beta subunit might have been due to partial reduction of the protein.
A simple one-stage test for the determination of the Stuart-Prower factor is described. The method is a modification of the classical Factor VII complex assay, using a Russell’s viper venom (Stypven)-cephalin reagent instead of brain thromboplastin. The optimal conditions for the assay system have been systematically investigated. A high dilution of RVV (1 : 200 000) has given the best results. The system is not influenced by different concentrations of fibrinogen, prothrombin, Factor V, Factor VII and plasma thromboplastin precursors other than Stuart-Prower factor. Variations of lipoid and/or platelet contents of the plasma do not alter the results. Stuart-Prower factor is not activated by glass contact. The method has proved most valuable in the detection of heterozygous Stuart-Prower factor deficiencies. Comparison with a specific Stuart-Prower factor assay using patient’s plasma deficient in Stuart-Prower factor gave variations not greater than 15%. If determined simultaneously with the Quick one-stage ‘prothrombin time’, the test may provide a better control of patients under treatment with dicoumarol derivatives.
A new method called the neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data. The principle of this method is to find pairs of operational taxonomic units (OTUs [= neighbors]) that minimize the total branch length at each stage of clustering of OTUs starting with a starlike tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method. Using computer simulation, we studied the efficiency of this method in obtaining the correct unrooted tree in comparison with that of five other tree-making methods: the unweighted pair group method of analysis, Farris's method, Sattath and Tversky's method, Li's method, and Tateno et al.'s modified Farris method. The new, neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods.
We have developed three computer programs for comparisons of protein and DNA sequences. They can be used to search sequence data bases, evaluate similarity scores, and identify periodic structures based on local sequence similarity. The FASTA program is a more sensitive derivative of the FASTP program, which can be used to search protein or DNA sequence data bases and can compare a protein sequence to a DNA sequence data base by translating the DNA data base as it is searched. FASTA includes an additional step in the calculation of the initial pairwise similarity score that allows multiple regions of similarity to be joined to increase the score of related sequences. The RDF2 program can be used to evaluate the significance of similarity scores using a shuffling method that preserves local sequence composition. The LFASTA program can display all the regions of local similarity between two sequences with scores greater than a threshold, using the same scoring parameters and a similar alignment algorithm; these local similarities can be displayed as a "graphic matrix" plot or as individual alignments. In addition, these programs have been generalized to allow comparison of DNA or protein sequences based on a variety of alternative scoring matrices.
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.
Two types of cDNA clones encoding human protein kinase C (PKC) were isolated from a spleen cDNA library using rabbit protein kinase C βI/βII cDNA as a hybridization probe. Nucleotide sequence analyses of these cDNA inserts revealed complete primary structures of two distinct types of human protein kinase C βI and βII which differ only in their C-terminal 50 or 52 amino acid residues. It was concluded that there exist four distinct types of PKC, PKC α, βI, βII and γ, in human as well as rabbit, and that the corresponding sequences are strictly conserved among mammalian species.
The new amino acid, S-β-(4-pyridylethyl)-l-cysteine (PEC), was prepared by treating l-cysteine with 4-vinylpyridine in an aqueous medium containing triethylamine. The postulated structure for PEC was confirmed by infrared, nuclear magnetic resonance, and mass spectroscopic analyses. PEC is stable to acid under conditions used for protein hydrolysis, it elutes on a basic column as a discrete peak before arginine, and its ninhydrin color is linear with concentration.The new amino acid has been evaluated as a internal standard for amino acid analyses. Equally excellent results were obtained when PEC was added to the protein either before or after hydrolysis. Addition of PEC before hydrolysis eliminates the need for nitrogen analysis of the hydrolysate and for accurate sample application to the column.
We examined the structure of protein kinase C in an attempt to understand the molecular events connecting protein kinase C activation with the cellular response. Rabbit complementary DNA clones coding for three distinct types of protein kinase C, named alpha, beta and gamma, have been identified and sequenced. The deduced amino acid sequence for alpha, beta and gamma (673, 671 and 672 amino acids, respectively) are closely related. Kinases alpha and beta share an identical N-terminal sequence of 621 amino acid residues and their messenger RNAs arise from a single gene. The C-terminal halves of alpha, beta and gamma are protein kinase domains and are highly homologous to other protein kinases. The mRNAs for alpha, beta and gamma are expressed in various tissues with strikingly different tissue specificities. The one for gamma is found ubiquitously among various tissues, while those for alpha and beta predominate in the brain.
A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.
Intact ribonucleic acid (RNA) has been prepared from tissues rich in ribonuclease such as the rat pancreas by efficient homogenization in a 4 M solution of the potent protein denaturant guanidinium thiocyanate plus 0.1 M 2-mercaptoethanol to break protein disulfide bonds. The RNA was isolated free of protein by ethanol precipitation or by sedimentation through cesium chloride. Rat pancreas RNA obtained by these means has been used as a source for the purification of alpha-amylase messenger ribonucleic acid.
Phospholipase A2 inhibitor (PLI), purified from the blood plasma of the Habu snake (Trimeresurus flavoviridis), was separated into two distinct subunits, PLI-A and PLI-B. These subunits were shown to be glycoproteins with molecular weights of around 21,000-22,000. When they were deglycosylated chemically with trifluoromethanesulfonic acid, the molecular weights were found to be 17,000. Their amino acid sequences were determined by alignment of peptides obtained by lysyl endopeptidase digestion and Staphylococcus aureus V8 protease digestion. PLI-A and PLI-B were each composed of 147 amino acid residues with one residue, Asn103, being for N-linked glycosylation, and the molecular weights of their protein portions were calculated to be 16,368 and 16,408, respectively. Each subunit contained four cysteine residues, all of which exist in disulfide linkages (Cys64-Cys141 and Cys119-Cys133). The sequences of PLI-A and PLI-B showed 89.9% homology to each other. When the sequences were compared with those of lipocortins, no significant homologies were detected. But the sequences were significantly homologous to those of COOH-terminal carbohydrate recognition portions of pulmonary surfactant apoprotein and animal lectins.
Heterotrimeric guanine nucleotide-binding proteins (G proteins) are integral to the signal transduction pathways that mediate the cell's response to many hormones, neuromodulators, and a variety of other ligands. While many signaling processes are guanine nucleotide dependent, the precise coupling between a variety of receptors, G proteins, and effectors remains obscure. We found that the family of genes that encode the alpha subunits of heterotrimeric G proteins is much larger than had previously been supposed. These novel alpha subunits could account for some of the diverse activities attributed to G proteins. We have now obtained cDNA clones encoding two murine alpha subunits, G alpha q and G alpha 11, that are 88% identical. They lack the site that is ordinarily modified by pertussis toxin and their sequences vary from the canonical Gly-Ala-Gly-Glu-Ser (GAGES) amino acid sequence found in most other G protein alpha subunits. Multiple mRNAs as large as 7.5 kilobases hybridize to G alpha q specific probes and are expressed at various levels in many different tissues. G alpha 11 is encoded by a single 4.0-kilobase message which is expressed ubiquitously. Amino acid sequence comparisons suggest that G alpha q and G alpha 11 represent a third class of alpha subunits. A member of this class was found in Drosophila melanogaster. This alpha subunit, DG alpha q, is 76% identical to G alpha q. The presence of the Gq class in both vertebrates and invertebrates points to a role that is central to signal transduction in multicellular organisms. We suggest that these alpha subunits may be involved in pertussis toxin-insensitive pathways coupled to phospholipase C.
We have isolated cDNA clones from rat C6 glioma cells coding for several guanine nucleotide-binding regulatory protein (G protein) alpha subunits (G alpha). The cDNA clones were then used to isolate human chromosomal genes. Among human genomic clones isolated by cross-hybridization with the rat cDNA for the alpha subunit of the inhibitory G protein Gi2, termed Gi2 alpha, a clone designated lambda HGi62 was found to contain a sequence that is highly homologous but distinct from any of the known G alpha sequences, and we have tentatively designated this sequence Gx alpha. We have searched a rat brain cDNA library with the Gx alpha sequence and isolated a cDNA clone containing a rat sequence similar to human Gx alpha. The cDNA contained a single open reading frame of 1065 nucleotides coding for a protein of 355 amino acids with a calculated molecular weight of 40,879. The amino acid sequence of rat Gx alpha shows 66% and 40% similarity with rat Gi2 alpha and rat Gs alpha (the alpha subunit of the stimulatory G protein), respectively. By RNA blot hybridization analysis, mRNA of approximately 3.2 kilobases was detected mainly in brain. Interestingly, the deduced amino acid sequence of Gx alpha predicts that the Gx alpha protein may be refractory to modification by pertussis toxin since the cysteine residue in the fourth position from the C terminus of pertussis toxin-sensitive G alpha is replaced by isoleucine.
Two simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions are presented. Although they give no weights to different types of codon substitutions, these methods give essentially the same results as those obtained by Miyata and Yasunaga's and by Li et al.'s methods. Computer simulation indicates that estimates of synonymous substitutions obtained by the two methods are quite accurate unless the number of nucleotide substitutions per site is very large. It is shown that all available methods tend to give an underestimate of the number of nonsynonymous substitutions when the number is large.
A new method called the neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data. The principle of this method is to find pairs of operational taxonomic units (OTUs [= neighbors]) that minimize the total branch length at each stage of clustering of OTUs starting with a starlike tree. The branch lengths as well as the topology of a parsimonious tree can quickly be obtained by using this method. Using computer simulation, we studied the efficiency of this method in obtaining the correct unrooted tree in comparison with that of five other tree-making methods: the unweighted pair group method of analysis, Farris's method, Sattath and Tversky's method, Li's method, and Tateno et al.'s modified Farris method. The new, neighbor-joining method and Sattath and Tversky's method are shown to be generally better than the other methods.
When the coding regions of 11 genes from rodents (mouse or rat) and man are compared with those from another mammalian species (usually bovine), it is found that rodents evolve significantly faster than man. The ratio of the number of nucleotide substitutions in the rodent lineage to that in the human lineage since their divergence is 2.0 for synonymous substitutions and 1.3 for nonsynonymous substitutions. Rodents also evolve faster in the 5' and 3' untranslated regions of five different mRNAs; the ratios are 2.6 and 3.1, respectively. The numbers of nucleotide substitutions between members of the beta-globin gene family that were duplicated before the man-mouse split are also higher in mouse than in man. The difference is, again, greater for synonymous substitutions than for nonsynonymous substitutions. This tendency is more consistent with the neutralist view of molecular evolution than with the selectionist view. A simple explanation for the higher rates in rodents is that rodents have shorter generation times and, thus, higher mutation rates. The implication of our findings for the study of molecular phylogeny is discussed.
Three different methods for hydrolysis and determination of amino acid composition of peptides and proteins were compared. We found, that the method of Matsubara and Sasaki (using 6N HCl and thioglycolic acid) gives comparatively low recoveries for tryptophan, while Liu and Chang's method, using p-toluenesulfonic acid and tryptamine, is more suitable. To eliminate the difficulties of the latter method, we used mercaptoethane-sulfonic acid, which, in the concentration used, results in total hydrolysis of peptide bonds within 22 hr and gives very high tryptophan recoveries. Both sulfonic acid methods were used for hydrolysis of the pentapeptide “pentagastrine” as well as of the proteins lysozyme, cytochrome c, and chymotrypsine. Their amino acid composition was determined using an automatic amino acid analyzer. Similarly to the p-toluenesulfonic acid method, the results of our method are totally reliable only for pure peptides and proteins, though the results obtained with our method using samples containing carbohydrates are better than those of all earlier methods.
By means of DEAE-Sephadex column chromatography, Agkistrodon acutus venom was separated into 12 fractions. Fractions 6 and 7 had marked anticoagulant action in tests of whole blood clotting time, calcium clotting time and plasma prothrombin time. The activity of Fraction 6 was the highest. Fraction 6 was rechromatographed twice on Sephadex G-100, and a single peak was obtained. The patterns of microzone and disc electrophoresis also showed a single band. A single, symmetrical boundary with a value of 2.00 S was obtained by ultracentrifugation. The estimated molecular weight was 20 650. The isoelectric point was pH 4.7. Chemical analysis showed that the anticoagulant principle was a glycoprotein and that it was thermolabile. The anticoagulant activity of this purified principle was four times higher than that of the crude venom. This principle did not possess caseinolytic, tosyl-l-arginine methyl ester esterase, phospholipase A, phosphodiesterase, alkaline phosphomonoesterase or fibrinolytic activities.
The rate of amino acid substitutions in the evolution of homologous proteins is remarkably constant. Furthermore, estimated rates of amino acid substitutions based on comparisons of the alpha hemoglobin chains of various mammals with that of the carp are about the same as those based on comparisons of the carp alpha and mammalian beta or the alpha and beta chains in mammals. These uniformities are regarded as evidence for the hypothesis that a majority of amino acid substitutions that occurred in these proteins are the result of random fixation of selectively neutral or nearly neutral mutations.
Two implications of this possibility are discussed: (a) Random gene frequency drift is playing an important role in determining the genetic structure of biological populations and (b) genes in “living fossils” may be expected to have undergone as many DNA base (and therefore amino acid) substitutions as corresponding genes (proteins) in more rapidly evolving species.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
Comparison of about 50 pairs of homologous nucleotide sequences for different genes revealed that the substitutions between synonymous codons occurred at much higher rates than did amino acid substitutions. Furthermore, five pairs of mRNA sequences for different genes were compared in species that had diverged at the same time. The evolutionary rate of synonymous substitution was estimated to be 5.1 X 10(-9) per site per year on the average and is approximately constant among different genes. It also is suggested that this property would be suitable for a molecular clock to determine the evolutionary relationships and branching order of duplicated genes. Each functional block of the noncoding region evolves with a rate that is almost constant, regardless of the types of genes. The intervening sequence and the 5' portion of the 3' noncoding region show considerable divergence, the extent of which is almost comparable to that in the synonymous codon sites, whereas the other blocks consisting of the 5' noncoding region and the 3' portion of the 3' noncoding region are strongly conserved, showing approximatley half of the divergence of the synonymous sites. This strong sequence preservation might be due to the functional requirements for transcription and modification of mRNA.
IX/X-bp, an anticoagulant protein isolated from the venom of the habu snake Trimeresurus flavoviridis, has a structure homologous to the carbohydrate-recognition domains of C-type (Ca(2+)-dependent) animal lectins, and it binds to the gamma-carboxyglutamic acid (Gla) domains of coagulation factors IX and X in a Ca(2+)-dependent fashion. In the present study, we elucidated the role of Ca2+ ions in this binding. The binding of 125I-labeled IX/X-bp to both coagulation factors required about 1 mM Ca2+ ions in this at pH 7.5. A decrease in the pH to 6.5 had a striking negative effect on the binding, and the Ca(2+)-requirement curve was shifted rightward. We investigated the binding of Ca2+ ions to IX/X-bp directly by equilibrium dialysis and identified two independent binding sites with different affinities. At pH 7.5, the apparent Kd values for these sites were 25 and 200 microM, respectively. When the pH was decreased to 6.5, the affinity of the high-affinity binding site was reduced only slightly but that of the low-affinity site was reduced considerably. Moreover, it was evident from observations of Ca(2+)-induced changes in the intrinsic fluorescence that IX/X-bp underwent a conformational change upon binding of Ca2+ ions.(ABSTRACT TRUNCATED AT 250 WORDS)
The sensitivity of the commonly used progressive multiple sequence alignment method has been greatly improved for the alignment
of divergent protein sequences. Firstly, individual weights are assigned to each sequence in a partial alignment in order
to downweight near-duplicate sequences and up-weight the most divergent ones. Secondly, amino acid substitution matrices are
varied at different alignment stages according to the divergence of the sequences to be aligned. Thirdly, residue-specific
gap penalties and locally reduced gap penalties in hydrophilic regions encourage new gaps in potential loop regions rather
than regular secondary structure. Fourthly, positions in early alignments where gaps have been opened receive locally reduced
gap penalties to encourage the opening up of new gaps at these positions. These modifications are incorporated into a new
program, CLUSTAL W which is freely available.
Echicetin is a dimeric protein isolated from the venom of Echis carinatus that is a potent inhibitor of von Willebrand Factor and thrombin binding to glycoprotein Ib. Here, we report isolation and amino acid sequence of the beta subunit of echicetin that contains 123 amino acids, including 7 cysteines, and shows similarity with amino acid sequences of botrocetin and Factor IXa/Xa binding protein. We provide evidence that biological activity of echicetin which resides in this beta subunit is relatively resistant to reduction of the molecule.
A new thrombin inhibitor, bothrojaracin, has been identified and purified to homogeneity from the venom of Bothrops jararaca, the most common venomous snake of South America. Bothrojaracin has an isoelectric point of 4.2 and a molecular mass of 27 kDa and is made of two distinct polypeptide chains of 15 and 13 kDa, linked by disulfide bridges. Purified bothrojaracin is devoid of phospholipase A2, amidolytic, or fibrino (geno)lytic activity. Bothrojaracin forms a noncovalent complex with alpha-thrombin, without changing its catalytic activity on small peptide substrates. Bothrojaracin behaves as a potent and specific antagonist of thrombin-induced platelet aggregation and secretion, characterized by an IC50 ranging from 1 to 20 nM depending on the alpha-thrombin concentration. Bothrojaracin prolongs fibrinogen clotting time, and this effect is related to a competitive inhibition of the binding of alpha-thrombin to fibrin(ogen) (Ki 15 nM). Binding of alpha-thrombin to thrombomodulin is inhibited up to 87% by bothrojaracin, and the rate of protein C activation by alpha-thrombin is also decreased. Bothrojaracin antagonizes the inhibition of thrombin amidolytic activity by hirudin. These results indicate that bothrojaracin acts as a very potent ligand of the exosite of alpha-thrombin.
Blood coagulation factor IX/factor X-binding protein (IX/X-bp) is a two-chain anticoagulant protein that was isolated from the venom of Trimeresurus flavoviridis. The amino acid sequence of IX/X-bp is homologous to the sequences of C-type lectin-like proteins, such as asialoglycoprotein receptor, tetranectin, and the low-affinity Fc epsilon receptor of immunoglobulin E. The amino acid composition and amino acid sequence of cystine-containing peptides, formed as a result of enzymatic digestion of CNBr-generated fragments of IX/X-bp, were analyzed to determine the location of the seven disulfide bridges in the protein. Three disulfide bridges in the A chain link Cys2 to Cys13, Cys30 to Cys127, and Cys102 to Cys119. Three disulfide bridges in the A chain link Cys2 to Cys13, Cys30 to Cys119, and Cys96 to Cys111. An interchain disulfide bond links Cys79 of the A chain and Cys75 of the B chain. The intrachain disulfide-bonding patterns of both the A and B chains of IX/X-bp are similar to those found in other C-type lectin-like proteins. We discuss in this report the sequence homology between IX/X-bp and other two-chain, C-type lectin-like proteins that have been isolated from snake venoms and we compare the S-S bonding patterns of proteins that are homologous to IX/X-bp.
IX/X-bp is an anticoagulant protein isolated from the venom of the habu snake (Trimeresurus flavoviridis). It is a heterogeneous two-chain protein linked by an interchain S-S bond. We prepared a cDNA library from the venom gland of the habu snake in the vector pSPORT1. cDNA clones containing the coding sequences for IX/X-bp were isolated and sequenced to determine the structure of the proprotein of IX/X-bp. All cDNA clones containing coding sequences of either chain of IX/X-bp consisted of the 5'-end noncoding bases, the first ATG codon, a typical signal peptide sequence that was immediately followed by mature protein sequence that corresponded to one of the chains, a stop codon, the 3'-end noncoding bases, a polyadenylation signal, and a poly(A)+ region. These data indicate that the gene for each chain of the two-chain protein is transcribed and translated separately.
The complete amino acid sequences of alpha and beta subunits of alboaggregin-beta are presented. The alpha and beta subunits were separated by reversed-phase HPLC after reduction and S-pyridylethylation, and their sequences were determined by analysis of peptides generated by enzymatic or chemical digestion. The alpha and beta subunits consist of 133 and 123 amino acid residues, respectively. The sequences are highly homologous to each other (41.4% identity) and also to those of the alpha and beta subunits of botrocetin (a von Willebrand factor modulator) and the A and B chains of factor IX/X binding protein from other snake venoms. It is also homologous to C-type lectins with a homodimeric structure, but it shows no lectin-like activity.
Three Trimeresurus okinavensis (To; himehabu snake, Crotalinae) venom gland phospholipase A2 (PLA2) isozymeencoding genes, gPLA2-o1, gPLA2-o2 and gPLA2-o3, were isolated from its genomic DNA library. The nucleotide (nt) sequence analysis revealed that two of the three genes (gPLA2-o2 and gPLA2-o3) occasionally have been converted to inactivated genes by introduction of one base insertion or substitution. It was confirmed from Southern blot analysis that the To haploid genome contains only three venom gland PLA2 isozyme genes herein isolated. Comparison of these genes showed that nonsynonymous nt substitutions have occurred more frequently than synonymous nt substitutions in the protein-coding regions, except for the signal-peptide coding domain, implying that To venom gland PLA2 isozyme genes have evolved via accelerated evolution. Such an evolutionary feature of To venom gland PLA2 isozyme genes proves the general universality of accelerated evolution previously drawn for venom gland PLA2 isozyme genes of other crotalinae snakes. The variability in the mature protein-coding regions of three To venom gland PLA2 isozyme genes appears to have been brought about by natural selection for point mutations.
The coagulation factor IX/factor X-binding protein (IX/X-bp) from the venom of Trimeresurus flavoviridis is a heterogeneous two-chain protein, and the structure of each chain is similar to that of the carbohydrate-recognition domain of C-type lectins, such as asialoglycoprotein receptors, pancreatic stone protein, and the Fc(ε) receptor for immunoglobulin E. Analysis of the binding properties of IX/X-bp revealed that it binds to the γ-carboxyglutamic acid (Gla)-containing domains of factors IX and X. In the present study, we isolated another anticoagulant protein that binds to factor IX but is not to factor X. This protein, designated IX-bp, inhibited factor IXa-induced clotting but not factor Xa-induced clotting, whereas IX/X-bp inhibits both. The concentration of IX-bp for half-maximal binding to solid-phase bovine factor IX was 0.4 nM whereas IX-bp did not bind to factor X even at 40 nM. The binding of IX-bp to solid-phase factor IX was inhibited by the addition of Gla-domain peptide of factor IX, indicating that IX-bp binds to the Gla-domain region of factor IX. IX-bp had two Ca2+-binding sites with different affinities for Ca2+ ions. At pH 7.5, the apparent K(d) values for these sites were 14 and 130 μM, respectively. IX-bp was a two-chain protein (27.5-kDa band before reduction and 16.8- and 15.7-kDa bands after reduction on SDS-PAGE) and it reacted with immunoglobulin G against IX/X-bp. The complete amino acid sequence of IX-bp was determined. The 16.8-kDa chain (A chain) of IX-bp consisted of 129 residues, of which 19 were different from those in the A chain of IX/X-bp (129 residues). The sequence of the 15.7-kDa chain (B chain) was identical to that of the B chain of IX/X-bp (123 residues). We conclude that IX-bp is a protein that is structurally similar to but functionally different from IX/X-bp. The difference of binding specificity between IX-bp and IX/X-bp presumably arises from the sequence differences in the A chains.
Eight cDNAs encoding serine proteases isolated from Trimeresurus flavoviridis (habu snake) and T. gramineus (green habu snake) venom gland cDNA libraries showed that nonsynonymous nucleotide substitutions have accumulated in the mature protein-coding regions to cause amino acid changes. Southern blot analysis of T. flavoviridis genomic DNAs using two proper probes indicated that venom gland serine protease genes form a multigene family in the genome. These observations suggest that venom gland serine proteases have diversified their amino acid sequences in an accelerating manner. Since a similar feature has been previously discovered in crotalinae snake venom gland phospholipase A2 (PLA2) isozyme genes, accelerated evolution appears to be universal in plural isozyme families of crotalinae snake venom gland.
Inhibitors (PLIs) against snake venom gland phospholipases A2 (PLA2s) have been found in their sera. A cDNA encoding a PLI from Trimeresurus flavoviridis (Tf, habu snake, Crotalinae) serum, cPLI-A, was isolated from the Tf liver cDNA library and sequenced. Northern blot analysis with cPLI-A showed that PLIs are expressed only in liver. Genes for PLIs, gPLI-A and gPLI-B, were isolated from the Tf genomic DNA library and their nucleotide (nt) sequences were determined. The genes consisted of four exons and three introns, and exon 4 encoded the carbohydrate recognition domain (CRD)-like motif. Comparison of the nt sequences between gPLI-A and gPLI-B showed that these genes are highly homologous, including introns, except that exon 3 is rich in nonsynonymous nt substitutions which are almost four times as frequent as synonymous nt substitutions. This evolutionary feature of PLI genes is different from that of venom gland PLA2 isozyme genes in which nonsynonymous nt substitutions are spread over the entire mature protein-coding region.
IX-bp and IX/X-bp are heterogeneous two-chain anticoagulant proteins isolated from the venom of habu snake, Trimeresurus flavoviridis. The amino acid sequence of one (B chain) of their two chains is identical. We recently reported the cloning of cDNA encoding the B chain and that of the A chain of IX/X-bp. Here, we report the isolation and characterization of cDNA clones encoding the A chain of IX-bp. The 697-bp sequence showed a putative ORF capable of encoding a 152-amino-acid protein containing a 23-amino-acid signal peptide. The overall amino acid sequence identity between the pre-A chain of IX-bp and that of IX/X-bp is 84%, whereas the cDNA sequence identity of the two ORFs is 91% indicating that habu snake acquired the variety of venom proteins by efficient mutations of antonymous sites.
Bothrojaracin is a potent and selective thrombin inhibitor that has been isolated from the venom of Bothrops jararaca. It does not interact with the catalytic site of the enzyme but binds to both anion-binding exosites 1 and 2 resulting in a potent inhibition of thrombin activity towards fibrinogen and platelets [Zingali, R. B., Jandrot-Perrus, M., Guillin, M. C. & Bon, C. (1993) Biochemistry 32, 10794-108021. Bothrojaracin is a 27-kDa protein composed of two disulfide-linked polypeptide chains, A and B, of 15 kDa and 13 kDa, respectively. The sequences of A and B chains determined by molecular cloning exhibit a high degree of identity with other snake venom lectin-like proteins. In contrast to other ligands that interact with thrombin exosite 1, the amino acid sequence of bothrojaracin does not contain an acidic sequence similar to the C-terminal tail of hirudin. Expression of functional bothrojaracin was achieved in COS cells upon transfection with two pcDNA3 vectors containing the complete cDNAs. Recombinant bothrojaracin, which was secreted into the medium, was able to bind to and inhibit thrombin. When expressed alone, the B chain formed inactive dimers that were secreted into the culture medium. In contrast, no bothrojaracin-related protein was detected in conditioned media from cells transfected with the A chain.
Factor IX/factor X-binding protein (IX/X-bp) is an anticoagulant isolated from the venom of Trimeresurus flavoviridis (habu snake) and binds predominantly to factor IX. In this study, we isolated IX/X-bp-like proteins from the venom of Deinagkistrodon acutus (hundred pace snake) with binding characteristics different from those of IX/X-bp. The complete amino acid sequence and binding characteristics of the main anticoagulant protein, named X-bp, were investigated. The concentrations of X-bp at half-maximal binding to solid-phase factors X and IX were 0.4 and 3 nM, respectively. The binding of X-bp to solid-phase factor X was inhibited by 50% by 6- and 9-fold excess concentrations of factor X and Gla domain (GD) peptide 1-44, respectively, but was not influenced by GD peptide 1-41 and Gla domainless factor X. X-bp bound two Ca2+ ions per molecule with Kd values of 16 +/- 0.7 (mean +/- SE, n = 6) and 103 +/- 10 microM. X-bp was a heterodimer of C-type lectin-like subunits. The 16 kDa chain (A chain) consisted of 129 amino acid residues and was 68% identical to the sequence of the A chain of IX/X-bp. The 15 kDa chain (B chain) consisted of 123 amino acid residues and was 87% identical to IX/X-bp. Three-dimensional model construction from the known fold of IX/X-bp showed that amino acid residues different from those of IX/X-bp are mostly on the molecular surface. Some of these are concentrated on a part of the concave surface which is considered to be the coagulation factor-binding site, presumably acting as a discriminator for ligand binding. These results indicated that X-bp isolated from D. acutus venom was a GD-binding protein, and the C-terminal region of GD peptide was critical for folding of the peptide.
A galactose-binding lectin isolated from the venom of Trimeresurus stejnegeri is a homodimer C-type lectin. The cloned cDNA encoding the monomer of Trimeresurus stejnegeri lectin (TSL) was sequenced and found to contain a 5'-end non-coding region, a sequence which encodes 135 amino acids, including a typical 23 amino acid signal peptide followed by the mature protein sequence, a 3'-end non-coding region, a polyadenylation signal, and a poly(A) region. To completely characterize the deduced amino acid sequence, on-line HPLC-MS and tandem MS were used to analyse the intact monomer and its proteolytic peptides. A modified peptide fragment was also putatively identified by HPLC-MS analysis. The deduced amino acid sequence was found to contain a carbohydrate-recognition domain homologous with those of some known C-type animal lectins. Thus TSL belongs to group VII of the C-type animal lectins as classified by Drickamer [(1993) Prog. Nucleic Acid Res. Mol. Biol. 45, 207-232]. At present, a number of C-type lectins have been purified from snake venom, but most of them have been characterized only at the protein level. To our knowledge, this is the first known cDNA sequence of a true C-type lectin from snake venom.
Flavocetin-A is a strong platelet aggregation inhibitor isolated from the venom of Trimeresurus flavoviridis. It binds specifically to platelet glycoprotein Ib with high affinity and inhibits von Willebrand factor-dependent platelet aggregation. The apparent molecular weight of flavocetin-A is 149 kDa. It consists of two subunits, alpha (17 kDa) and beta (14 kDa). The amino acid sequences of the alpha and beta subunits were determined from cloned cDNAs. Deduced amino acid sequences showed signal peptide-sequences of 23 amino acids for both alpha and beta subunits, mature peptide sequences of 135 amino acids for the alpha subunit, and 125 amino acids for the beta subunit. The amino acid sequences of alpha and beta subunits show high degrees of homology to those of C-type lectin-like venom proteins such as habu coagulation factors IX/X-binding protein (IX/X-bp), botrocetin, and alboaggregin-B. The cysteinyl residues of flavocetin-A, IX/X-bp, and botrocetin are conserved, except that flavocetin-A contains Cys 135 in the alpha subunit and Cys 3 in the beta subunit. We assumed that the arrangements of disulfide bridges in flavocetin-A are similar to those of IX/X-bp and botrocetin, and the additional Cys 135 of the alpha subunit and Cys 3 of the beta subunit are involved in novel disulfide bridges. These findings suggested that the additional disulfide bridges formed with Cys 135 of the alpha subunit and Cys 3 of the beta subunit cause polymerization of C-type lectin-like heterodimers.
Tissue-speci®c expression of three distinct types of rabbit protein kinase C Puri®cation and properties of the anticoagulant principle of Agkistrodon acutus venom
- S Ohno
- H Kawasaki
- S Imajoh
- K Suzuki
- M Inagaki
- H Yokokura
- T Sakoh
- H Hidaka
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