Purification and Characterization of Four Ca2+-Dependent Lectins from the Marine Invertebrate, Cucumaria echinata

Abstract

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.

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

“…This lectin strongly bound to oligosaccharide numbers 702, 708, 716, and 717, which contain GalNAc␤1-3/4 structures, and weakly bound to oligosaccharide numbers 719, 720, and 721, which contain GalNAc␣1-3 linkages. 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.…”

“…Purification of Native CEL-IV-CEL-IV was purified from C. echinata by affinity chromatography using carbohydrateimmobilized columns essentially as reported previously (9). However, in this study, we used a column with raffinose (Gal␣1-6Glc␣1-2␤Fru) as an immobilized carbohydrate for the initial separation of CEL-IV because this lectin shows relatively a high binding specificity for ␣-galactoside-containing carbohydrates.…”

“…This lectin shows Gal/GalNAc-specificity, especially for ␣-galactosides, whereas CEL-I and CEL-III show higher specificity for ␤-galactosides (9). In general, C-type CRDs contain carbohydrate recognition motifs composed of three amino acid residues, Gln-Pro-Asp (QPD) or Glu-Pro-Asn (EPN), which are considered to determine their galactose or mannose specificities, respectively (10).…”

Mannose-recognition mutant of the galactose/N-acetylgalactosamine-specific C-type lectin CEL-I engineered by site-directed mutagenesis

“…These glycoconjugates contain galactose and/or N-acetylgalactosamine ( Table 2). As we reported previously, CEL-I is highly specific to N-acetylgalactosamine, particularly disaccharides containing Nacetylgalactosamine at the nonreducing end [10,12,33]. Thus, the results of glycoconjugate array analysis confirmed such previous observations.…”

Antibacterial and hemolytic effects of aqueous and organic extracts from different tissues of sea urchin Echinometra mathaei on pathogenic streptococci

“…Several hemolytic factors have been isolated from echinoderms, including proteins (Canicatti and D'Ancona 1990), lectins (Hatakeyama et al 1994), complement-like factors (Bertheussen 1983), and a variety of saponins (Iorizzi et al 2001). From a pharmaceutical point of view, it is an advantage when antibacterial drugs have no side effects such as hemolytic activity (Haug et al 2002).…”