CEL-III is one of four Ca(2+)-dependent galactose/N-acetylgalactosamine (GalNAc)-binding lectins from the marine invertebrate Cucumaria echinata which exhibits hemolytic activity, especially toward rabbit and human erythrocytes. The hemolytic activity of CEL-III was also Ca(2+)-dependent and was found to be inhibited by galactose or GalNAc-containing carbohydrates, suggesting that the hemolysis was caused by CEL-III binding to specific carbohydrates on the erythrocyte membrane by Ca(2+)-dependent lectin activity, followed by partial destruction of the membrane. The activity of CEL-III was highest at 10 degrees C and decreased markedly with increasing temperature, unlike usual enzymatic reactions. The hemolytic activity of CEL-III increased with increasing pH from neutral to 10, but almost no hemolysis was observed below pH 6.5. Immunoblotting analysis of proteins from the erythrocyte membrane after treatment with CEL-III indicated that CEL-III aggregates were irreversibly bound to the membrane. When erythrocytes were incubated with CEL-III in the presence of dextran with molecular masses greater than 4 kDa, lysis was impeded considerably, while a concomitant release of ATP was detected from these osmotically protected cells. It was found that CEL-III released carboxyfluorescein from artificial globoside-containing lipid vesicles, and it is suggested that CEL-III is a novel pore-forming protein with the characteristics of a Ca(2+)-dependent lectin, which may act as a toxic protein to foreign microorganisms.
Four Ca(2+)-dependent, N-acetylgalactosamine/galactose-specific lectins were purified from the marine invertebrate, Cucumaria echinata (Holothuroidea), by column chromatography on lactosyl-Sepharose 4B, Sephacryl S-200, and Q-Sepharose. The molecular masses of these lectins were estimated to be 27 kDa (CEL-I), 35 kDa (CEL-II), 45 kDa (CEL-III), and 68 kDa (CEL-IV) on SDS-PAGE under nonreducing conditions. Among these lectins, CEL-I and CEL-IV strongly agglutinated rabbit and human erythrocytes, and were found to recognize N-acetylgalactosamine and galactose-containing carbohydrates from the results of a hemagglutination inhibition assay. In contrast, CEL-II failed to agglutinate any erythrocytes tested, although its carbohydrate-binding ability was confirmed by a carbohydrate-binding assay involving asialofetuin-horseradish peroxidase. Interestingly, CEL-III caused hemolysis of rabbit and human erythrocytes, while it showed only hemagglutination of chicken and horse erythrocytes at relatively high concentrations. The hemolytic activity of CEL-III was also dependent on the Ca(2+)-concentration, and inhibited by N-acetylgalactosamine and galactose-containing carbohydrates, suggesting that the hemolysis was caused by Ca(2+)-dependent binding of CEL-III to specific carbohydrate chains on the erythrocyte surface and the following partial destruction of the membrane.
The hemolytic lectin CEL-III is a Ca2+-dependent, galactose/GalNAc-specific lectin purified from the marine invertebrate Cucumaria echinata (Holothuroidea). We found that this lectin forms ion-permeable pores in erythrocyte and artificial lipid membranes that have specific carbohydrate ligands on the surface. The hemolytic activity of CEL-III exhibited characteristic pH dependence; activity increased remarkably with pH in the alkaline region, especially above pH 9. When rabbit erythrocyte membrane was examined by immunoblotting using anti-CEL-III antiserum after treatment with CEL-III, the irreversible binding of the CEL-III oligomer increased with pH, indicating that the increase in hemolytic activity at higher pH is associated closely with the amount of oligomer irreversibly bound to the membrane. Surface hydrophobicity of CEL-III, as measured by the fluorescent probe 8-anilino-1-naphthalenesulfonate, increased markedly with the binding of specific ligands such as lactose, lactulose, and N-acetyllactosamine at pH 9-10 in the presence of 1 M NaCl. The enhancement of surface hydrophobicity induced by the binding of carbohydrates was also accompanied by the formation of a CEL-III oligomer, which was found to be the same size on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as the oligomer that formed in CEL-III-treated erythrocyte membranes. Far-UV circular dichroism spectra of CEL-III and the oligomer revealed a definite difference in secondary structure. These data suggest that the binding of CEL-III to specific carbohydrate ligands on the erythrocyte surface induces a conformational change in the protein, leading to the exposure of a hydrophobic region which triggers oligomerization and the irreversible binding of the protein to the membrane.
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.
The carbohydrate-binding properties of the hemolytic lectin CEL-III from the Holothuroidea Cucumaria echinata were studied using the microplate assay system which we have recently developed [Hatakeyama et al. (1996) Anal. Biochem. 237, 188-192]. When the binding of CEL-III to lactose covalently immobilized on a microplate was examined using colloidal gold solution, the binding was detected with as little as 1 microgram/ml protein. Affinity of several carbohydrates to CEL-III was assessed by means of an inhibition experiment using the lactose-coated plate and it was found that N-acetylgalactosamine has the highest affinity for CEL-III, followed by lactose and lactulose. Examination of the binding of CEL-III to the lactose-coated plate at various pH values and temperatures revealed that the affinity is higher in the acidic pH region and at lower temperatures. From the Ca(2+)-dependence profile for the binding of CEL-III to the lactose-coated plate, the apparent dissociation constant for Ca2+ was estimated to be 2.3 mM. These results suggested that the carbohydrate-binding properties of CEL-III are closely related to its hemolytic activity, although an additional interaction between the protein and the lipid bilayer, which is enhanced in the alkaline pH region, also seems to be necessary for its hemolytic action.
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