Molecular Characterization of Three Novel Phospholipase A₂ Proteins from the Venom of Atheris chlorechis, Atheris nitschei and Atheris squamigera.
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.
School of Integrative Medicine, Fujian University of Traditional Chinese Medicine, No.1 Qiu Yang Road, Shangjie Town, Fuzhou 350122, Fujian, China. hwang11@qub.ac.uk.
He
H
Wang
School of Pharmacy, Fujian Medical University, No.1 Xueyuan Road, Shangjie Town, Fuzhou 350004, Fujian, China. wh4401@hotmail.com.
Xiaole
X
Chen
Natural Drug Discovery Group, School of Pharmacy, Queen's University Belfast, University Road, Belfast BT7 1NN, UK. m.zhou@qub.ac.uk.
Mei
M
Zhou
Natural Drug Discovery Group, School of Pharmacy, Queen's University Belfast, University Road, Belfast BT7 1NN, UK. l.wang@qub.ac.uk.
Lei
L
Wang
Natural Drug Discovery Group, School of Pharmacy, Queen's University Belfast, University Road, Belfast BT7 1NN, UK. t.chen@qub.ac.uk.
Tianbao
T
Chen
Natural Drug Discovery Group, School of Pharmacy, Queen's University Belfast, University Road, Belfast BT7 1NN, UK. chris.shaw@qub.ac.uk.
Chris
C
Shaw
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D004530:Egg Yolk / Q000378:metabolism; D054467:Phospholipases A2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D030162:Reptilian Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014757:Viper Venoms / Q000201:enzymology*; D017819:Viperidae
D018076:DNA, Complementary; D030162:Reptilian Proteins; D014757:Viper Venoms; D054467:Phospholipases A2
Atheris; mass spectrometry; molecular cloning; phospholipase A2; snake venom