Cytotoxicity of a GalNAc-Specific C-Type Lectin CEL-I toward Various Cell Lines

• Published in: Journal of biochemistry (Tokyo) 2005, Vol.137 (1), p.41-50
• Main Authors: Kuramoto, Takuya, Uzuyama, Hitomi, Hatakeyama, Tomomitsu, Tamura, Tadashi, Nakashima, Takuji, Yamaguchi, Kenichi, Oda, Tatsuya
• Format: Article
• Language: English
• Subjects: 6-carboxyfluorescein diacetate; Animals; apoptosis; C-type lectin; C-type lectin proteins; carboxyfluorescein; Cell Line; cellular morphological changes; CFDA; CLPs; Con A; concanavalin A; Cricetinae; Cucumaria - chemistry; Cucumaria echinata; cytotoxicity; Dose-Response Relationship, Drug; F-CEL-I; FBS; fetal bovine serum; FITC; FITC-labeled CEL-I; fluorescein isothiocyanate; GalNAc; GlcNAc; Humans; Lectins, C-Type - isolation & purification; Lectins, C-Type - metabolism; MEM; Mice; minimal essential medium; N-acetylgalactosamine; N-acetylglucosamine; PBS; PHA-L; phosphate-buffered saline; phytohemagglutinin-L (Phaseolus vulgaris agglutinin); Rats; TBS; Tris-buffered saline; WGA; wheat germ agglutinin
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/jb/mvi005

We found that CEL-I was a potent cytotoxic lectin. MDCK, HeLa, and XC cells were highly sensitive to CEL-I cytotoxicity and killed in a dose-dependent manner, whereas CHO, L929, and RAW264.7 cells were relatively resistant to CEL-I, and no significant toxicity was observed up to 10 [micro]g/ml. Among these cell lines, MDCK cells showed the highest susceptibility to CEL-I cytotoxicity. A binding study using FITC-labeled CEL-I (F-CEL-I) revealed that the amounts of bound F-CEL-I on the sensitive cell lines were evidently greater than those on the resistant cell lines, suggesting that the different susceptibility of the cell lines to CEL-I cytotoxicity is partly explained by different efficiencies of binding of CEL-I to these cell lines. Interestingly, the cytotoxicity of CEL-I toward MDCK cells was more potent than those of other lectins such as WGA, PHA-L, and Con A, even though these lectins were capable of binding to MDCK cells at comparable levels to CEL-I. Since the cytotoxicity of CEL-I was strongly inhibited by GalNAc, the binding to cell surface specific carbohydrates is essential for the CEL-I cytotoxicity. The trypan blue dye exclusion test indicated that CEL-I caused a disorder of plasma membrane integrity as a relatively early event. CEL-I failed to induce the release of carboxyfluorescein (CF) from CF-loaded MDCK cells as seen for pore-forming hemolytic isolectin CEL-III, suggesting that the primary cellular target of CEL-I may be the plasma membrane, but its action mechanism differs from that of CEL-III. Although CEL-I induced dramatic cellular morphological changes in MDCK cells, neither typical apoptotic nuclear morphological changes nor DNA fragmentation was observed in CEL-I-treated MDCK cells even after such cellular changes. Our results demonstrated that CEL-I showed a potent cytotoxic effect, especially on MDCK cells, by causing plasma membrane disorder without induction of apoptosis.