Abstract
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.
Highlights
We purified four Ca2ϩ-dependent, galactose/N-acetylgalactosamine (GalNAc)-specific lectins from the Holothuroidea Cucumaria echinata (1)
The pH Dependence of the Irreversible Binding of CEL-III to the Erythrocyte Membrane—Our previous results indicated that hemolysis by CEL-III depends largely on pH; little hemolytic activity was observed in the acidic region, hemolysis by CEL-III increased remarkably with pH, especially above pH 9 (2)
The immunoblot gave stronger bands of CEL-III oligomer with a molecular mass of about 270 kDa at higher pH, whereas only very weak bands were seen in the acidic region. This result indicates that the strong hemolytic activity at higher pH is closely related to the effective irreversible binding of CEL-III to the erythrocyte membrane and its oligomerization in this pH region, which probably damages the membrane with the formation of ion-permeable pores
Summary
We purified four Ca2ϩ-dependent, galactose/N-acetylgalactosamine (GalNAc)-specific lectins from the Holothuroidea Cucumaria echinata (1). We found that the binding of specific carbohydrates induced an enhancement of surface hydrophobicity as well as oligomerization of CEL-III in aqueous solution, which suggests that binding to carbohydrate receptors on the cell surface may lead to a conformational
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