Characterization of the interaction of hemolytic lectin CEL-III from the marine invertebrate, Cucumaria echinata, with artificial lipid membranes: involvement of neutral sphingoglycolipids in the pore-forming process.
The hemolytic lectin, CEL-III, is a Ca2+-dependent, galactose/N-acetylgalactosamine-specific lectin purified from the marine invertebrate, Cucumaria echinata (Holothuroidea). After binding to specific carbohydrates on the erythrocyte surface, CEL-III forms ion-permeable pores by oligomerizing in the membrane, which leads to colloid osmotic rupture of the cells. When incubated with liposomes composed of total lipids from the human erythrocyte membrane, CEL-III efficiently induced the leakage of carboxyfluorescein (CF) trapped in the vesicles, suggesting the presence of its receptor in the membrane lipids. The rate of CF-leakage increased with increasing temperature, although the hemolytic activity of CEL-III had been found to be much higher at lower temperatures (around 10 degrees C). Identification of the receptor for CEL-III was performed by examining the ability of individual lipids from human erythrocytes to induce CF-leakage from DOPC-liposomes. As a result, the most effective receptor was found to be lactosyl ceramide (LacCer), while globoside (Gb4Cer) also showed slight induction of CF-leakage. On the other hand, a binding assay involving CEL-III-horseradish peroxidase conjugate indicated that CEL-III exhibits similar affinity for LacCer and Gb4Cer, suggesting that the structure or length of the carbohydrate portion of sphingoglycolipids is also relevant as to their ability to induce CF-leakage in addition to their affinity. Electron micrographs of CEL-III-treated liposomes revealed that CEL-III induced considerable morphological changes in the vesicles, while a clearly distinguishable oligomeric structure of the protein was not observed.
Hatakeyama
T
T
Department of Applied Chemistry, Faculty of Engineering, Nagasaki University, Nagasaki, 852-8521, Japan. thata@net.nagasaki-u.ac.jp
Sato
T
T
Taira
E
E
Kuwahara
H
H
Niidome
T
T
Aoyagi
H
H
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006017:Glycolipids / Q000378:metabolism; D006028:Glycosphingolipids / Q000378:metabolism*; D006801:Humans; D037102:Lectins / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism / Q000648:ultrastructure; D008386:Marine Biology; D008567:Membranes, Artificial; D008854:Microscopy, Electron; D012616:Sea Cucumbers / Q000378:metabolism*