Venom phospholipases A2 of bamboo viper (Trimeresurus stejnegeri): molecular characterization, geographic variations and evidence of multiple ancestries

Abstract

Phospholipases A2 (PLA2s) were purified from the Trimeresurus stejnegeri venom obtained from various localities in Taiwan and three provinces in China, by gel filtration followed by reversed-phase HPLC. The precise molecular mass and N-terminal sequence of each PLA2 were determined. In addition to the six previously documented PLA2 isoforms of this species, we identified ten novel isoforms. The venom gland cDNAs of individual specimens of the viper from four localities were used for PCR and subsequent cloning of the PLA2s. The molecular masses and partial sequences of most of the purified PLA2s matched with those deduced from a total of 13 distinct cDNA sequences of these clones. Besides the commonly known Asp49 or Lys-49 PLA2s of crotalid venoms, a novel type of PLA2 with Asn-49 substitution at the Ca2+-binding site was discovered. This type of PLA2 is non-catalytic, but may cause local oedema and appears to be a venom marker of many tree vipers. In particular, we showed that T. stejnegeri displayed high geographic variations of the PLA2s within and between their Taiwanese and Chinese populations, which can be explained by geological isolation and prey ecology. A phylogenetic tree of the acidic venom PLA2s of this species and other related Asian vipers reveals that T. stejnegeri contains venom genes related to those from several sympatric pit vipers, including the genera Tropedolaemus and Gloydius besides the Trimeresurus itself. Taken together, these findings may explain the exceptionally high variations in the venom as well as the evolutionary advantage of this species.

Revised Systematics of Taiwanese Viperid Snakes and the Correlation to Venom Diversity and Evolution

“…Notably, disintegrins of T. stejnegeri showed high diversification and more than four isoforms could be identified. This is in accord with the unusually high diversity of its venom PLAs (Tsai et al, 2004). The disintegrins of T. stejnegeri and Gloydius halys are at the basal positions relative to those from other crotalid genera, and trigramin β is identical to albolabrin of T. albolabris.…”

“…However, the genetic relationships may also be complicated by intra-species variations, possibly resulting from prey-ecology (Daltry et al, 1997), geographic separation, ontology (Warrell, 1997), or other factors. Indeed, we found that the three Taiwanese viperid species, D. r. siamensis (Tsai et al, 1996), P. mucrosquamatus venoms (Tsai et al, 2001), and T. stejnegeri (Tsai et al, 2004), show considerable intra-species variations in their venom PLA isoforms, mainly due to differential expression of each isoforms and geographic separation of the snakes. Usually, more PLA sequences can be deduced from cloning the venom gland cDNAs than are predicted from venom purification because not all of the mRNA are translated into venom proteins (Chijiwa et al, 2000).…”

“…One of the most common components found in the pit viper venoms are the acidic Glu6-PLAs (Wang et al, 1999;Tsai et al, 2004), which may inhibit the aggregation of platelets or play other roles (Tsai et al, 2003). The phylogeny tree of venom acidic E6-PLA from Asiatic pit vipers is shown in Fig.…”

“…AzE6a (the out-group) is an unpublished full sequence for the acidic PLA from Azemiops feae venom. Other E6-PLA sequences used are published amino acid sequences (Tsai et al, 2004;Wang et al, 1996b). Three partial sequences (with N -terminal 32 residues) were denoted with asterisks; Omm-E6-PLA is an E6-PLA from O. m. monticola venom.…”

“…We have previously purified, cloned, and characterized PLAs from the venom of Taiwan vipers, including D. r. siamensis (Tsai et al, 1996;Wang et al, 1992), P. mucrosquamatus (Liu et al, 1991;Tsai et al, 1995;Tsai et al, 2001;Wang and Tsai, 1994), D. acutus (Wang et al, 1996a;Wang et al, 1996b), and T. stejnegeri (Tsai et al, 2004). One of the most common components found in the pit viper venoms are the acidic Glu6-PLAs (Wang et al, 1999;Tsai et al, 2004), which may inhibit the aggregation of platelets or play other roles (Tsai et al, 2003).…”