Mutations on the N-terminal region abolish differentially the enzymatic activity, membrane-damaging activity and cytotoxicity of Taiwan cobra phospholipase A2.
To examine the functional contribution of the N-terminal region to the activities of Naja naja atra phospholipase A(2) (PLA(2)), studies on three N-terminally mutated PLA(2) were carried out in the present work. Removal of N-terminal heptapeptide caused a complete loss of membrane-damaging activity, whilst the mutants with an extra Met before Asn-1 or substituting Asn-1 with Met still retained approximately 40.9% and 82.9% membrane-damaging activity of the native enzyme, respectively. Mutations on the N-terminal region did not greatly affect the Ca(2+)-binding ability but caused a precipitous drop in PLA(2) activity. Moreover, the gross conformation of the mutants was different from that of the native enzyme as revealed by CD spectra. Nevertheless, the mutants as well as native PLA(2) induced apoptotic death of U937 cells, and the cytotoxicity of mutants was similar to or even greater than that of the native PLA(2). These results indicate that mutations on the N-terminus abolish the enzymatic activity, membrane-damaging activity and cytotoxicity of N. naja atra PLA(2) in different ways, and suggest a feasible approach to selective elimination of the multiple activities of PLA(2) enzymes.
Chiou
Yi-Ling
YL
Institute of Biomedical Sciences, National Sun Yat-Sen University-Kaohsiung Medical University Joint Research Center, National Sun Yat-Sen University, Kaohsiung 804, Taiwan.
Cheng
Yun-Ching
YC
Kao
Pei-Hsiu
PH
Wang
Jeh-Jeng
JJ
Chang
Long-Sen
LS
D000818:Animals; D018076:DNA, Complementary / Q000737:chemistry; D004546:Elapid Venoms / Q000737:chemistry* / Q000201:enzymology; D017815:Elapidae; D009154:Mutation; D054467:Phospholipases A2 / Q000737:chemistry*; D013624:Taiwan
D018076:DNA, Complementary; D004546:Elapid Venoms; D054467:Phospholipases A2