Fusion of sphingomyelin vesicles induced by proteins from Taiwan cobra (Naja naja atra) venom. Interactions of zwitterionic phospholipids with cardiotoxin analogues.
Egg sphingomyelin vesicles were used to assay aggregation/fusion activities of proteins from Taiwan (Naja naja atra) venom to avoid the problem of phospholipase A2 contamination during protein purification. It led to the identification of a new cardiotoxin (CTX) analogue protein (CTX V) with major aggregation/fusion, but few hemolysis, activities. On the contrary, cardiotoxin (CTX III) induced significant hemolysis of human red blood cells but exhibited few aggregation/fusion activities. To study the structure/activity relationship of these CTX-induced processes, the amino acid sequence of CTX V was determined and its aggregation/fusion activity was compared with that of CTX III by transmission electron microscopy, quasielastic laser light scattering, differential scanning calorimetry, and fluorescence spectroscopy. The results show that the CTX-induced fusion process at temperatures slightly above that of the gel to liquid-crystalline phase transition of sphingomyelin vesicles can ultimately convert small sonicated vesicles into large fused vesicles with sizes of 1-2 microns. The abilities of CTX V to induce the leakage of sphingomyelin vesicles content and to cause the fusion of vesicles are approximately 10-fold higher than those of CTX III. Based on the CTX structures determined in the present and other studies, it is suggested that the amino acid residue X within the well conserved sequence of -Cys-Pro-X-Gly-Lys-Gln-Leu-Cys- plays a role in the interaction of CTX with lipid molecules. The lipid phase transition could further enhance the protein-lipid interaction in the process leading to the fusion of vesicles.
Chien
K Y
KY
Institute of Life Sciences, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
Huang
W N
WN
Jean
J H
JH
Wu
W G
WG
D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D004179:Cobra Cardiotoxin Proteins / Q000737:chemistry* / Q000633:toxicity; D004546:Elapid Venoms / Q000737:chemistry*; D006168:Guinea Pigs; D006461:Hemolysis / Q000187:drug effects; D008563:Membrane Lipids / Q000737:chemistry; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013109:Sphingomyelins / Q000737:chemistry*
D004179:Cobra Cardiotoxin Proteins; D004546:Elapid Venoms; D008563:Membrane Lipids; D010743:Phospholipids; D013109:Sphingomyelins