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, GEL-III, is a Ca2+-dependent, galactose/N-acetylgalac tosamine-specific lectin purified from the marine invertebrate, Cucumaria e chinata (Holothuroidea). After binding to specific carbohydrates on the ery throcyte surface, GEL-III forms ion-permeable pores by oligomerizing in the membrane, which leads to colloid osmotic rupture of the cells, When incuba ted with liposomes composed of total lipids from the human erythrocyte memb rane, GEL-III efficiently induced the leakage of carboxyfluorescein (CF) tr apped in the vesicles, suggesting the presence of its receptor in the membr ane lipids. The rate of CF-leakage increased with increasing temperature, a lthough the hemolytic activity of GEL-III had been found to be much higher at lower temperatures (around 10 degrees C). Identification of the receptor for GEL-III was performed by examining the ability of individual lipids fr om human erythrocytes to induce CF-leakage from DOPC-liposomes. As a result , the most effective receptor was found to be lactosyl ceramide (LacCer), w hile globoside (Gb(4)Cer) also showed slight induction of CF-leakage, On th e other hand, a binding assay involving CEL-III-horseradish peroxidase conj ugate indicated that CEL-III exhibits similar affinity for LacCer and Gb(4) Cer, suggesting that the structure or length of the carbohydrate portion of sphingoglycolipids is also relevant as to their ability to induce CF-leaka ge in addition to their affinity. Electron micrographs of CEL-III-treated l iposomes revealed that GEL-III induced considerable morphological changes i n the vesicles, while a clearly distinguishable oligomeric structure of the protein was not observed.