Toxinological characterization of venom from Leptodeira annulata (Banded cat-eyed snake; Dipsadidae, Imantodini)

Highlights

• Leptodeira annulata venom contains mainly metalloproteinases and PLA₂.

• The extensive proteolytic activity includes fibrinogen/fibrin degradation.

• The proteolytic activity and hemorrhage are mediated by metalloproteinases.

• The venom increases vascular permeability through the action of PLA₂.

• These activities may contribute to local effects of envenomation by L. annulata.

Abstract

We investigated the histology of Duvernoy’s venom gland and the biochemical and biological activities of Leptodeira annulata snake venom. The venom gland had a lobular organization, with secretory tubules formed by serous epithelial cells surrounding each lobular duct. The latter drained into a common lobular duct and subsequently into a central cistern. In contrast, the supralabial gland was mucous in nature. SDS-PAGE revealed a profile of venom components that differed from pitviper (Bothrops spp.) venoms. RP-HPLC also revealed greater complexity of this venom compared to Bothrops venoms. The venom had no esterase, l-amino acid oxidase or thrombin-like activity, but was proteolytic towards elastin-Congo red, fibrin, fibrinogen, gelatin and hide powder azure. The venom showed strong α-fibrinogenase and fibrinolytic activities and reduced the rate and extent of plasma recalcification. The proteolytic activity was inhibited by EDTA and 1,10-phenanthroline (metalloproteinase inhibitors) but not by AEBSF and PMSF (serine proteinase inhibitors). The venom had phospholipase A₂ (PLA₂) activity that was inhibited by varespladib. The venom cross-reacted with antivenoms to lancehead (Bothrops spp.), coralsnake (Micrurus spp.) and rattlesnake (Crotalus durissus terrificus) venoms. The venom did not aggregate rat platelets or inhibit collagen-induced aggregation, but partially inhibited thrombin-induced aggregation. The venom was hemorrhagic (inhibited by EDTA) and increased the vascular permeability (inhibited by varespladib) in rat dorsal skin. In gastrocnemius muscle, the venom caused myonecrosis and increased serum creatine kinase concentrations. In conclusion, L. annulata venom has various enzymatic and biological activities, with the local effects being mediated primarily by metalloproteinases and PLA₂.

Introduction

Colubroidean snakes account for ∼52% of currently recognized snake species [1], although to-date only a small number of these have been reported to cause severe or fatal envenomation in humans [2,3]. In recent years, various studies have shown that these venoms contain many of the biological activities and major components typical of venoms of the Viperidae and Elapidae, including phospholipases A₂ (PLA₂), snake venom metalloproteinases (SVMPs), snake venom serine proteases (SVSPs) and three-finger toxins (3-FTx) [4,5], with some toxins in the latter group showing taxon-specific activity [[6], [7], [8]]. The usefulness of these venoms as a source of novel compounds with potential therapeutic applications has also been investigated [9].

The genus Leptodeira (generally referred to as ‘cat-eyed snakes’; family Dipsadidae and tribe Imantodini) consists of 12 species with a wide distribution from the southern tip of Texas in the USA, through Mexico and Central America (the main centers of diversity) and into South America as far south as Argentina [1,[10], [11], [12], [13]]. Most species are diurnal, <1 m in length and feed primarily on amphibians [11]. Despite the numerous Leptodeira species, few cases of human envenomation have been reported in the literature, possibly because of these snakes’ generally non-aggressive behavior and the incorrect attribution of envenomation to venomous pitvipers. Human envenomation by Leptodeira spp. has been reported for L. septentrionalis (northern cat-eyed snake) [14], L. frenata (rainforest cat-eyed snake) [15] and subspecies of L. annulata (banded cat-eyed snake) [2,16] and is generally characterized by transient local symptoms that include intense pain, edema, hemorrhagic blistering and erythema reminiscent of mild to moderate local envenoming caused by crotaline snakes of the genus Bothrops (lanceheads) and certain species of Crotalus (rattlesnakes). Arthralgia of ∼5 weeks duration was noted in envenomation by L. frenata [15]. In addition to the limited number of reports of human envenomation by Leptodeira spp., there is also very little information on the biochemistry and pharmacology of these venoms.

In common with other non-front-fanged colubroids [[17], [18], [19]], L. annulata venom is produced by paired venom (Duvernoy) glands located posterior to the eyes, although the structural organization of these glands has not yet been analyzed. Mebs [20,21] reported that L. annulata venom (geographic origin unstated) had low PLA₂ and phosphodiesterase activities, high proteolytic activity towards casein (comparable to various Bothrops spp.), but no l-amino acid oxidase, ATPase, cholinesterase, esterase, fibrinogen-coagulase (thrombin-like activity), kinin-releasing or 5′-nucleotidase activities. Lemoine et al. [22] also reported proteolytic activity towards gelatin, as well as neurotoxicity and hemorrhagic activity in vivo and in vitro, for L. annulata ashmeadii venom from Venezuela. More recently, Torres-Bonilla et al. [23] reported that the venom of Colombian L. annulata caused partial neuromuscular blockade in chick biventer cervicis neuromuscular preparations in vitro, along with morphological alterations presumably caused by the high proteolytic activity mediated by SVMPs.

In this study, we investigated the biochemical and biological activities of L. annulata venom in order to understand the previously reported effects of envenomation by this species in relation to the venom composition. We also examined the histological organization of the venom gland.