Crystal Structure of the Hemolytic Lectin CEL-III Isolated from the Marine Invertebrate Cucumaria echinata

Abstract

CEL-III is a Ca2؉ -dependent and galactose-specific lectin purified from the sea cucumber, Cucumaria echinata, which exhibits hemolytic and hemagglutinating activities. Six molecules of CEL-III are assumed to oligomerize to form an ion-permeable pore in the cell membrane. We have determined the crystal structure of CEL-III by using single isomorphous replacement aided by anomalous scattering in lead at 1.7 Å resolution. CEL-III consists of three distinct domains as follows: the N-terminal two carbohydrate-binding domains (1 and 2), which adopt ␤-trefoil folds such as the B-chain of ricin and are members of the (QXW) 3 motif family; and domain 3, which is a novel fold composed of two ␣-helices and one ␤-sandwich. CEL-III is the first Ca 2؉ -dependent lectin structure with two ␤-trefoil folds. Despite sharing the structure of the B-chain of ricin, CEL-III binds five Ca 2؉ ions at five of the six subdomains in both domains 1 and 2. Considering the relatively high similarity among the five subdomains, they are putative binding sites for galactose-related carbohydrates, although it remains to be elucidated whether bound Ca 2؉ is directly involved in interaction with carbohydrates. The paucity of hydrophobic interactions in the interfaces between the domains and biochemical data suggest that these domains rearrange upon carbohydrate binding in the erythrocyte membrane. This conformational change may be responsible for oligomerization of CEL-III molecules and hemolysis in the erythrocyte membranes.

C-type Lectin-like Carbohydrate Recognition of the Hemolytic Lectin CEL-III Containing Ricin-type β-Trefoil Folds

“…This is consistent with an increase in ␤-sheet structure of the entire CEL-III oligomer (4 fore, it seems reasonable to conclude that oligomerization of CEL-III is mainly mediated by interactions between its domain 3 after binding to cell surface carbohydrates. We have solved the crystal structure of CEL-III (21), which confirmed the domain structure as suggested from the amino acid sequence; domains 1 and 2 adopt a ␤-trefoil structure, each consisting of three subdomains or motifs. They showed apparent similarity with the B-chains of ricin and abrin despite their relatively low sequence identity.…”

“…CEL-III was finally purified by gel filtration through Sephadex G-75 in TBS. Crystallization of CEL-III/carbohydrate complexes was done under similar conditions as those for native CEL-III crystals (21) in the presence of 10 mM carbohydrates. Briefly, the protein solution (7 mg/ml, 2-4 l) in TBS containing 10 mM CaCl 2 was mixed with the same amount of reservoir solution (12% (w/v) polyethylene glycol 8000, 100 mM BisTris, NaOH, pH 6.5, and 200 mM magnesium acetate) and subjected to sitting drop vapor diffusion at 20°C.…”

“…The structures of these CEL-III/carbohydrate complexes were solved by a molecular replacement method using native CEL-III as a search model. Five Ca 2ϩ and two Mg 2ϩ ions were assigned to the same positions as those for native protein, considering their electron density, coordination numbers, bond distances, and ligand atoms (21). As shown in Fig.…”

“…Complexes-Crystals were prepared under similar conditions as for native CEL-III (21). We have tried to crystallize CEL-III complexed with several carbohydrates, such as GalNAc, lactose, lactulose, Me-␤-Gal, Me-␣-Gal, and galactose (3).…”

Galactose Recognition by a Tetrameric C-type Lectin, CEL-IV, Containing the EPN Carbohydrate Recognition Motif

“…These observations are consistent with the results obtained from hemagglutination assays (9) in which GalNAc showed the highest hemagglutination inhibition and also revealed a preference for ␤-GalNAc. CEL-III is a unique R-type lectin with a ␤-trefoil fold (31,32) containing Ca 2ϩ ions in the carbohydrate binding domains. This lectin showed the highest affinity for oligosaccharide number 729 and moderately high affinity for several N-linked glycans.…”