Guanidination of notexin promotes its phospholipase A(2) activity-independent fusogenicity on vesicles with lipid-supplied negative curvature.
To address the requirement of phospholipase A(2) (PLA(2)) activity in membrane fusion events and membrane perturbation activity of notexin and guanidinated notexin (Gu-notexin), the present study was conducted. Notexin and Gu-notexin did not show PLA(2) activity after the removal of Ca(2+) with EDTA. Metal-free notexin and Gu-notexin were found to induce membrane leakage and fusion of phospholipid vesicles. Fusogenic activity of native and modified notexin correlated positively with their membrane-damaging activity underlying the deprivation of PLA(2) activity. Compared with Ca(2+)-bound Gu-notexin, fusogenicity of metal-free Gu-notexin was notably increased by incorporation of cholesterol, cholesterol sulfate, phosphatidylethanolamine, α-tocopherol and phosphatidic acid that supplied negative curvature into phospholipid bilayer. The ability of Gu-notexin to induce membrane fusion of vesicles with lipid-supplied negative curvature was higher than that of notexin regardless of the absence or presence of Ca(2+). Consistently, metal-free Gu-notexin markedly induced membrane fusion of red blood cells (RBCs) compared with metal-free notexin, and fusion activity of metal-free Gu-notexin on cholesterol-depleted RBCs notably reduced. Compared with notexin, Gu-notexin highly induced uptake of calcein-loaded phosphatidylcholine (PC)/cholesterol and PC/cholesterol sulfate vesicles by K562 cells in the presence of EDTA. Taken together, our data suggest that notexin and Gu-notexin could induce vesicle leakage and fusion via a PLA(2) activity-independent mechanism, and guanidination promotes PLA(2) activity-independent fusogenicity of notexin on vesicles with lipid-supplied negative curvature.
Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan.
Pei-Hsiu
PH
Kao
Yi-Ling
YL
Chiou
Ying-Jung
YJ
Chen
Shinne-Ren
SR
Lin
Long-Sen
LS
Chang
D002118:Calcium / Q000737:chemistry; D002463:Cell Membrane Permeability / Q000187:drug effects; D004546:Elapid Venoms / Q000737:chemistry / Q000302:isolation & purification / Q000633:toxicity*; D004912:Erythrocytes / Q000187:drug effects / Q000648:ultrastructure; D005452:Fluoresceins / Q000032:analysis / Q000378:metabolism; D019791:Guanidine / Q000737:chemistry*; D006801:Humans; D020014:K562 Cells; D050356:Lipid Metabolism / Q000187:drug effects; D008081:Liposomes / Q000648:ultrastructure; D008560:Membrane Fluidity / Q000187:drug effects; D008561:Membrane Fusion / Q000187:drug effects* / Q000502:physiology; D054467:Phospholipases A2 / Q000737:chemistry / Q000502:physiology*
D004546:Elapid Venoms; D005452:Fluoresceins; D008081:Liposomes; C072139:Notechis 11'2; C005082:notexin; D054467:Phospholipases A2; D019791:Guanidine; D002118:Calcium; C007740:fluorexon