Characterization and molecular cloning of neurotoxic phospholipases A2 from Taiwan viper (Vipera russelli formosensis).
Two phospholipases A2 (PLA2s), designated as RV-4 and RV-7 were purified from venom of the Taiwan Russell's viper (Vipera russelli formosensis) by gel-filtration and reverse-phase HPLC. Their primary structures were solved by both protein sequencing and cDNA cloning and sequencing. The cDNA synthesized was amplified by the polymerase-chain reaction using a pair of synthetic oligonucleotide primers corresponding to the N- and the C-terminal flanking regions of the enzymes. The deduced amino acid sequences of RV-4 and RV-7 were 92% identical to those of the vipoxin and vipoxin inhibitor, respectively, from the Bulgarian Vipera a. ammodytes. RV-4 itself was neurotoxic, whereas RV-7 had much lower enzymatic activity and was not toxic. The low enzymatic activity of RV-7 may be attributed to five acidic residues at positions 7, 17, 59, 114 and 119, which presumably impair its binding to aggregated lipid substrates. Based on the sequence comparison among all the known group II PLA2s, residues 6, 12, 76-81, and 119-125 were identified as important for the neurotoxicity. RV-4 and RV-7 exist in the crude venom as heterodimers, which were again formed by mixing together the HPLC-purified RV-4 and RV-7. Moreover, RV-7 inhibited the enzymatic activity of RV-4 in vitro but potentiated its lethal potency and neurotoxicity. It is suggested that RV-7 may facilitate the specific binding of RV-4 to its presynaptic binding sites, probably by preventing its non-specific adsorption.
Wang
Y M
YM
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, Republic of China.
Lu
P J
PJ
Ho
C L
CL
Tsai
I H
IH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D051379:Mice; D008969:Molecular Sequence Data; D009469:Neuromuscular Junction / Q000187:drug effects / Q000502:physiology; D009498:Neurotoxins / Q000633:toxicity; D009838:Oligodeoxyribonucleotides; D010741:Phospholipases A / Q000235:genetics* / Q000302:isolation & purification / Q000633:toxicity; D010974:Platelet Aggregation / Q000187:drug effects; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000633:toxicity; D017386:Sequence Homology, Amino Acid; D012911:Snakes; D014757:Viper Venoms / Q000235:genetics* / Q000633:toxicity
D009498:Neurotoxins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014757:Viper Venoms; D004247:DNA; D010741:Phospholipases A