Keigo Hisamatsu

Roles of the Valine Clusters in Domain 3 of the Hemolytic Lectin CEL-III in Its Oligomerization and Hemolytic Abilities

The hemolytic lectin CEL-III and its site-directed mutants were expressed in Escherichia coli cells. Replacement of the valine clusters in domain 3 with alanine residues led to increased self-oligomerization in solution and higher hemolytic activity. The results suggest the involvement of these valine clusters in CEL-III oligomerization and hemolytic activity.

Identification of the amino acid residues involved in the hemolytic activity of the Cucumaria echinata lectin CEL-III.

BACKGROUND CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata that shows Ca(2+)-dependent Gal/GalNAc-binding specificity. This lectin is composed of two carbohydrate-recognition domains (domains 1 and 2) and an oligomerization domain (domain 3) that facilitates CEL-III assembly in the target cell membrane to form ion-permeable pores. METHODS Several amino acid residues in domain 3 were replaced by alanine, and hemolytic activity of the mutants was examined. RESULTS K344A, K351A, K405A, K420A and K425A showed marked increases in activity. In particular, K405A had activity that was 360-fold higher than the wild-type recombinant CEL-III and 3.6-fold higher than the native protein purified from sea cucumber. Since these residues appear to play roles in the stabilization of domain 3 through ionic and hydrogen bonding interactions with other residues, the mutations of these residues presumably lead to destabilization of domain 3, which consequently induces the oligomerization of the protein through association of domain 3 in the membrane. In contrast, K338A, R378A and R408A mutants exhibited a marked decrease in hemolytic activity. Since these residues are located on the surface of domain 3 without significant interactions with other residue, they may be involved in the interaction with components of the target cell membrane. CONCLUSIONS Several amino acid residues, especially basic residues, are found to be involved in the hemolytic activity as well as the oligomerization ability of CEL-III. GENERAL SIGNIFICANCE The results provide important clues to the membrane pore-forming mechanism of CEL-III, which is also related to that of bacterial pore-forming toxins.

Crystallization and preliminary crystallographic study of oligomers of the haemolytic lectin CEL-III from the sea cucumber Cucumaria echinata

CEL-III is a Ca(2+)-dependent haemolytic lectin isolated from the marine invertebrate Cucumaria echinata. This lectin binds to Gal/GalNAc-containing carbohydrate chains on the cell surface and, after conformational changes, oligomerizes to form ion-permeable pores in cell membranes. CEL-III also forms soluble oligomers similar to those formed in cell membranes upon binding of specific carbohydrates in high-pH and high-salt solutions. These soluble and membrane CEL-III oligomers were crystallized and X-ray diffraction data were collected. Crystals of soluble oligomers and membrane oligomers diffracted X-rays to 3.3 and 4.2 Å resolution, respectively, using synchrotron radiation and the former was found to belong to space group C2. Self-rotation functional analysis of the soluble oligomer crystal suggested that it might be composed of heptameric CEL-III.

Effects of Ca2+ on Refolding of the Recombinant Hemolytic Lectin CEL-III

CEL-III is a hemolytic lectin isolated from Cucumaria echinata. Although recombinant CEL-III (rCEL-III) expressed in Escherichia coli showed very weak hemolytic activity compared with native protein, it was considerably enhanced by refolding in the presence of Ca2+. This suggests that Ca2+ supported correct folding of the carbohydrate-binding domains of rCEL-III, leading to effective binding to the cell surface and subsequent self-oligomerization.