Cloning of two novel P-III class metalloproteinases from Trimeresurus stejnegeri venom gland☆
Introduction
Envenomation of a Crotalidae or Viperidae snakes results in characteristic local and systemic hemorrhage. The profuse hemorrhage observed is caused mainly by the action of snake venom metalloproteinase (SVMP) (Bjarnason and Fox, 1995). SVMPs are members of the reprolysin subfamily of the M12 family of metalloproteinases. According to the protein structure, SVMPs can be classified into four classes (Bjarnason and Fox, 1995). A large number of metalloproteinases with structural information have been isolated and characterized, mainly from crotalid and viperid snake venom. Commonly, there are different classes of metalloproteinase with various functions detected in a single species of some members of Crotalidae and Viperidae. For example, at least three P-III class (HR1a, HR1b and HV1) and three P-I class (HR2a, H2-proteinase and flavoridin precursor metalloproteinase) metalloproteinases were identified from the venom of Trimeresurus flavoviridis, a member of Crotalidae family (Masuda et al., 2001, Miyata et al., 1989, Takeya et al., 1989, Kishimoto and Takahashi, 2002a, Kishimoto and Takahashi, 2002b). In recent years, some metalloproteinases with novel structural features have been identified from snake venoms, such as jerdonitin and contortrostatin-C (Chen et al., 2003, Zhou et al., 2000), suggesting that the structures of SVMPs are more diverse than previous classification. Recently, a modified classification scheme has been presented by Fox and Serrano (2005) and subclasses of P-II and P-III were introduced. The sub-classification reflects more details of the relationship between structures and functions.
Trimeresurus stejnegeri snake venom contains various bioactive components, including C-type lectin-like proteins (Lee et al., 2003a, Lee et al., 2003b, Lee and Zhang, 2003), l-amino acid oxidase (Zhang et al., 2003) and phospholipase A2 (Tsai et al., 2004). However, there is no report on isolation or cDNA cloning of metalloproteinase from this snake venom. In this paper, we reported two cDNA sequences that both encoding a novel P-III precursor, respectively. Sequence analysis suggested that these two novel metalloproteinases, together with HR1b form T. flavoviridis, might belong to another subclass of P-III SVMPs, termed subclass P-IIIc.
Section snippets
Materials
T. stejnegeri snake was collected in the area of Guangxi province of China. The following reagents were obtained from the indicated sources: standard molecular biology reagents and plasmid cloning vector from Promega (Madison, USA) and Amersham, JM109 competent cells from Takara (Dalian, China). All other reagents were of the highest purity from Sigma (St Louis, USA).
Construction of cDNA library
Venom gland mRNA extraction and cDNA library construction were performed as described previously (Zhang et al., 1995).
Synthesis of oligonucleotide primers and PCR amplification
Based on
Results and discussion
Two cDNAs coding for two novel metalloproteinases, designed as stejnihagin-A and stejnihagin-B, were obtained (Fig. 1). Both cDNAs have an open reading frame (ORF) of 1803 bp, comprising a 54 bp signal sequence, a 516 bp proprotein region, a 597 bp metalloproteinase region, a 54 bp spacer region, a 234 bp disintegrin-like region and a 345 bp cysteine-rich region. The ORFs of stejnihagin-A and stejnihagin-B encode similar polypeptides of 600 amino acid residues, comprising a signal sequence of 18
Acknowledgements
This work was supported by the grants of ‘Western Light’ Projects from The Chinese Academy of Sciences to Dr Zhang and Dr Lee; and grants from National Natural Science Foundation (30470380, 30570359) and Yunnan Science and Technology Commission (2003C0066M).
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2013, ToxiconCitation Excerpt :The high-conserved cysteinyl residues mainly focus on the C-terminus of BumaMPs1 (Fig. 2). This phenomenon mainly occurred in the P-III SVMPs (Wan et al., 2006; Pinyachat et al., 2011), therefore we postulated BumaMPs1 is likely one member of P-III although the exact classical standard of metalloproteinase in invertebrate is not available (Sun and Bao, 2010; Tang et al., 2012). It worth noting that BumaMPs1 and metalloproteinases in other invertebrate share many conserved domains in the N-terminus (Fig. 2), assuming the conserved regions should be functioned as a clue to further explore the roles of BumaMPs1 (Macours and Hens, 2004).
Snake venomics and antivenomics of Protobothrops mucrosquamatus and Viridovipera stejnegeri from Taiwan: Keys to understand the variable immune response in horses
2012, Journal of ProteomicsCitation Excerpt :These differences in the severity of envenomings by these two species might be associated to the higher amount of venom delivered by P. mucrosquamatus [7]. Studies on the biochemical characteristics of the venoms of P. mucrosquamatus and V. stejnegeri have resulted in the isolation and characterization of P-I, P-II and P-III metalloproteinases (SVMPs) [16–21], serine proteinases [22–29], phospholipases A2 (PLA2s) and PLA2 homologues [10,30–34], l-amino acid oxidases [35,36], C-type lectin-like proteins [37–41], disintegrins [42–44], a phosphodiesterase [45], and several platelet aggregating proteins [46,47]. Despite such abundant information in the biochemical composition of these venoms, a holistic proteomic analysis, aimed at assessing the relative contribution of each type of venom protein in the overall venom composition, is pending.
Stejnihagin, a novel snake metalloproteinase from Trimeresurus stejnegeri venom, inhibited L-type Ca<sup>2+</sup> channels
2009, ToxiconCitation Excerpt :Well-established polypeptide toxins blocking L-type Ca2+ channels, for example, ω-agatoxins (Aga) purified from venom of the funnel web spider Agelenopsis aperta (Olivera et al., 1994), ω-conotoxins from the marine snail genus Conus (Cassola et al., 1998) and Calciseptine purified from the venom of the black mamba Dendroaspis polylepis polylepis (de Weille et al., 1991), have become essential research tools. Previously we have purified a mixture of Stejnihagin-A and -B (designated as Stejnihagin) from Trimeresurus stejnegeri venom, with 86% sequences identity, both as members of snake venom P-III type metalloproteinase (SVMPs) family (Wan et al., 2006). SVMPs mainly distribute in Crotalid and Viperid snake venom and are classified into the Reprolysin subfamily of the M12 family of the metzincin family among a variety of zinc-containing metalloproteinases (Fox and Serrano, 2005).
P-III hemorrhagic metalloproteinases from Russell's viper venom: Cloning, characterization, phylogenetic and functional site analyses
2008, BiochimieCitation Excerpt :In addition to hemorrhage, other versatile functions have also been reported for P-III SVMPs, e.g. activating prothrombin [5,6], inducing endothelial cell apoptosis [7], cleaving integrins [3], and inhibiting platelet functions [8], which implies that more structural diversities exist between P-III SVMPs. Based on the position of the seventh cysteinyl residue in the metalloproteinase domain, three new P-III subclasses were revealed [2,9]: P-IIIa, which undergoes autoproteolysis to release a ∼30-kDa fragment with disintegrin- and Cys-rich domains; P-IIIb, which forms a dimeric structure; and P-IIIc, which contains the seventh cysteinyl residue at position 100 in its metalloproteinase domain. However, the structural elements attributed to their functional variations and hemorrhagic potencies remain elusive [2].