title: Identification of the amino acid residues involved in the hemolytic activity of the Cucumaria echinata lectin CEL-III.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Laboratory of Biomolecular Chemistry, Nagasaki University, Nagasaki, Japan.
authors.forename: Keigo
authors.initials: K
authors.lastname: Hisamatsu
authors.forename: Tomonao
authors.initials: T
authors.lastname: Nagao
authors.forename: Hideaki
authors.initials: H
authors.lastname: Unno
authors.forename: Shuichiro
authors.initials: S
authors.lastname: Goda
authors.forename: Tomomitsu
authors.initials: T
authors.lastname: Hatakeyama
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D006461:Hemolysis / Q000187:drug effects*; D037102:Lectins / Q000737:chemistry / Q000494:pharmacology*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D055503:Protein Multimerization; D011817:Rabbits; D013329:Structure-Activity Relationship
chemical_list: C402644:CEL-III lectin, Cucumaria echinata; D037102:Lectins