Elsevier

Toxicon

Volume 47, Issue 4, 15 March 2006, Pages 465-472
Toxicon

Cloning of two novel P-III class metalloproteinases from Trimeresurus stejnegeri venom gland

https://doi.org/10.1016/j.toxicon.2006.01.003Get rights and content

Abstract

Hemorrhagic toxins are widely distributed in viperid and crotalid snake venoms. Envenomation of Trimeresurus stejnegeri, a member of Crotalidae family, caused potent systemic and local hemorrhage. Up to now, there is no report on hemorrhage toxins from this venom. In this work, we cloned two cDNAs of P-III metalloproteinase precursors, designated as stejnihagin-A and stejnihagin-B, respectively, from T. stejnegeri venom gland. Both cDNAs encode an opening reading frame of 600 amino acid residues, containing a signal sequence, a proprotein domain, a metalloproteinase domain, a disintegrin-like domain and a cystetine-rich domain. Sequence analysis suggested that these two sequences shared highest similarity to the hemorrhagic toxin HR1b from T. flavoviridis. Aligning the deduced mature protein sequences of stejnihagin-A and stejnihagin-B with other snake venom metalloproteinases (SVMPs), we observed that stejnihagin-A and stejnihagin-B, together with HR1b shared the common cysteinyl residue at the position 100 in the metalloproteinase domain. In combination with the phylogenetic analysis, we presumed that stejnihagin-A, stejnihagin-B and HR1b might constitute a novel subclass of P-III SVMPs, named P-IIIc.

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).

References (29)

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The sequence data of stejnihagin-A and stejnihagin-B reported in this paper have been submitted to the GenBank database under accession no. DQ195154 and DQ195153, respectively.

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