Effect of the hemolytic lectin CEL-III from Holothuroidea Cucumaria echinata on the ANS fluorescence responses in sensitive MDCK and resistant CHO cells

The addition of CEL-III to sensitive MDCK cells preincubated with 8-anilino -1-naphthalenesulfonate (ANS) caused an increase in the fluorescence intens ity of the probe. The increase in the ANS fluorescence caused by CEL-III wa s Ca2+-dependent and strongly inhibited by 0.1M lactose, indicating that Ca 2+-dependent binding of CEL-III to specific carbohydrate receptors on the p lasma membrane is responsible for this phenomenon. In contrast, no signific ant effect of CEL-III on the ANS fluorescence was observed in CHO cells, wh ich are highly resistant to CEL-III cytotoxicity, In MDCK cells, energy tra nsfer from tryptophan residues to bound ANS molecules was observed in the p resence of CEL-III, but not in CHO cells. Furthermore, the amount of ANS bo und to MDCK cells increased as the concentration of CEL-III increased. Ther efore, a simple interpretation is that the CEL-III-induced increase in ANS fluorescence is attributable to an increase of the hydrophobic region in th e plasma membrane where ANS could bind. Immunoblotting analysis of proteins from cells treated with CEL-III indicated that CEL-III oligomers were irre versibly bound to the cells, and the amount of oligomer bound to MDCK cells was much greater than that bound to CHO cells under any conditions tested. The oligomerization may be accompanied by an enhancement of the hydrophobi city of CEL-III molecules, which in turn provides new ANS-binding sites. Th e difference in susceptibility of MDCK and CHO cells to CEL-III cytotoxicit y may be due to a difference in oligomerization of bound CEL-III.