Abstract
Secretory phospholipase A2 (sPLA2) is known as a major component of snake venoms and displays higher-order catalytic hydrolysis functions as well as a wide range of pathological effects. Atheris is not a notoriously dangerous genus of snakes although there are some reports of fatal cases after envenomation due to the effects of coagulation disturbances and hemorrhaging. Molecular characterization of Atheris venom enzymes is incomplete and there are only a few reports in the literature. Here, we report, for the first time, the cloning and characterization of three novel cDNAs encoding phospholipase A2 precursors (one each) from the venoms of the Western bush viper (Atheris chlorechis), the Great Lakes bush viper (Atheris nitschei) and the Variable bush viper (Atheris squamigera), using a “shotgun cloning” strategy. Open-reading frames of respective cloned cDNAs contained putative 16 residue signal peptides and mature proteins composed of 121 to 123 amino acid residues. Alignment of mature protein sequences revealed high degrees of structural conservation and identity with Group II venom PLA2 proteins from other taxa within the Viperidae. Reverse-phase High Performance Liquid Chromatography (HPLC) profiles of these three snake venoms were obtained separately and chromatographic fractions were assessed for phospholipase activity using an egg yolk suspension assay. The molecular masses of mature proteins were all identified as approximately 14 kDa. Mass spectrometric analyses of the fractionated oligopeptides arising from tryptic digestion of intact venom proteins, was performed for further structural characterization.
Highlights
As major components of snake venoms, proteins of the secretory phospholipase A2 family have been structurally-defined, characterized and their catalytic mechanisms have been elucidated [1].Traditionally, these snake venom enzymes have been divided into two main groups, Group I (GI)and Group II (GII), used to distinguish between molecules based on their amino acid sequences, disulfide-bridging patterns, unique functional loops and extension amino acids [2]
Novel disintegrins had been isolated and identified in a previous study [20], and in this work, cloning of precursor-encoding cDNAs and characterization of the mature phospholipase A2 proteins from Atheris chlorechis (A. chlorechis), Atheris nitschei (A. nitschei) and Atheris squamigera (A. squamigera) venoms, were carried out in an attempt to explain the toxic effects of the venom of Atheris snakes in more depth
Except for phospholipase A2 (PLA2)‐A.N., with two additional cysteine residues, the positions of the 12 cysteines are homologous in the three precursors, meaning that this uniform character of disulfide bridging pattern should lead to similar crystal structures of those three PLA2s
Summary
As major components of snake venoms, proteins of the secretory phospholipase A2 (sPLA2 ) family have been structurally-defined, characterized and their catalytic mechanisms have been elucidated [1]. These snake venom enzymes have been divided into two main groups, Group I (GI). Of the 400 species of snakes in Africa, bush vipers (Atheris, Viperidae) are not considered to be highly-dangerous in terms of the numbers of documented bites and deaths. This may in part be due to their arboreal behavior and their distribution in often inhospitable habitats [13]. Novel disintegrins had been isolated and identified in a previous study [20], and in this work, cloning of precursor-encoding cDNAs and characterization of the mature phospholipase A2 proteins from Atheris chlorechis (A. chlorechis), Atheris nitschei (A. nitschei) and Atheris squamigera (A. squamigera) venoms, were carried out in an attempt to explain the toxic effects of the venom of Atheris snakes in more depth
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