Temperature- and pH-dependent cytotoxic effect of the hemolytic lectin CEL-III from the marine invertebrate Cucumaria echinata on various cell lines.
We investigated the cytotoxicity of CEL-III, one of four Ca2+-dependent galactose/N-acetylgalactosamine (GalNAc)-binding lectins from the marine invertebrate Cucumaria echinata. Among six cell lines tested, MDCK cells showed the highest susceptibility to CEL-III cytotoxicity and its LD50 was estimated to be 53 ng/ml, while no significant cytotoxicity of CEL-III was observed in CHO cells up to 10,000 ng/ml. In the presence of 0.1 M lactose, the cytotoxicity of CEL-III was strongly inhibited. The binding studies using FITC-labeled CEL-III revealed that the amount of CEL-III bound to MDCK cells was about 2-fold greater than that in the case of CHO cells. The cytotoxicity of CEL-III increased with decreasing temperature. The surviving fractions of Vero cells exposed to CEL-III at 4 degrees C were immediately decreased, and more than 90% of exposed cells were killed within 20 min, whereas at 37 degrees C much longer exposure period (more than 10 h) was required to kill 50% of the cells. CEL-III induced the release of carboxyfluorescein (CF) from CF-loaded MDCK cells and this activity was markedly increased at alkaline pH (pH 10) and at lower temperature (4 degrees C). Even in CHO cells, considerable CF release was induced by CEL-III at 4 degrees C and at pH 10 but not at pH 7.5 at both temperatures. In agreement with these results, CHO cells exposed to CEL-III at 4 degrees C and at pH 10 were killed in a dose-dependent manner. These results suggest that CEL-III exhibits cytotoxicity through damaging the plasma membrane by pore-formation in a temperature- and pH-dependent manner. Different susceptibility of each cell line to CEL-III cytotoxicity may be due to differences in the processes leading to pore-formation after binding to cell-surface carbohydrates.
Oda
T
T
Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Bunkyo-machi. t-oda@net.nagasaki-u.ac.jp
Tsuru
M
M
Hatakeyama
T
T
Nagatomo
H
H
Muramatsu
T
T
Yamasaki
N
N
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D037102:Lectins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D008387:Marine Toxins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D013696:Temperature
D037102:Lectins; D008387:Marine Toxins