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 intensity of the probe. The increase in the ANS fluorescence caused by CEL-III was Ca2+-dependent and strongly inhibited by 0.1 M lactose, indicating that Ca2+-dependent binding of CEL-III to specific carbohydrate receptors on the plasma membrane is responsible for this phenomenon. In contrast, no significant effect of CEL-III on the ANS fluorescence was observed in CHO cells, which are highly resistant to CEL-III cytotoxicity. In MDCK cells, energy transfer from tryptophan residues to bound ANS molecules was observed in the presence of CEL-III, but not in CHO cells. Furthermore, the amount of ANS bound to MDCK cells increased as the concentration of CEL-III increased. Therefore, a simple interpretation is that the CEL-III-induced increase in ANS fluorescence is attributable to an increase of the hydrophobic region in the plasma membrane where ANS could bind. Immunoblotting analysis of proteins from cells treated with CEL-III indicated that CEL-III oligomers were irreversibly 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 hydrophobicity of CEL-III molecules, which in turn provides new ANS-binding sites. The difference in susceptibility of MDCK and CHO cells to CEL-III cytotoxicity may be due to a difference in oligomerization of bound CEL-III.
Oda
T
T
Division of Biochemistry, Faculty of Fisheries, Faculty of Engineering, Nagasaki University, Bunkyo-machi, Nagasaki, 852-8521, Japan. t-oda@net.nagasaki-u.ac.jp
Shinmura
N
N
Nishioka
Y
Y
Komatsu
N
N
Hatakeyama
T
T
Muramatsu
T
T
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D006224:Cricetinae; D003603:Cytotoxins / Q000494:pharmacology; D004285:Dogs; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007785:Lactose / Q000494:pharmacology; D037102:Lectins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D008856:Microscopy, Fluorescence; D012616:Sea Cucumbers; D013050:Spectrometry, Fluorescence; D013696:Temperature
D000817:Anilino Naphthalenesulfonates; D003603:Cytotoxins; D005456:Fluorescent Dyes; D037102:Lectins; C027132:1-anilino-8-naphthalenesulfonate; D007785:Lactose; D002118:Calcium