Hemolytic Lectin CEL-III Heptamerizes via a Large Structural Transition from α-Helices to a β-Barrel during the Transmembrane Pore Formation Process

CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-binding domains (domains 1 and 2) and one oligomerization domain (domain 3). After binding to the cell surface carbohydrate chains through domains 1 and 2, domain 3 self-associates to form transmembrane pores, leading to cell lysis or death, which resembles other pore-forming toxins of diverse organisms. To elucidate the pore formation mechanism of CEL-III, the crystal structure of the CEL-III oligomer was determined. The CEL-III oligomer has a heptameric structure with a long β-barrel as a transmembrane pore. This β-barrel is composed of 14 β-strands resulting from a large structural transition of α-helices accommodated in the interface between domains 1 and 2 and domain 3 in the monomeric structure, suggesting that the dissociation of these α-helices triggered their structural transition into a β-barrel. After heptamerization, domains 1 and 2 form a flat ring, in which all carbohydrate-binding sites remain bound to cell surface carbohydrate chains, stabilizing the transmembrane β-barrel in a position perpendicular to the plane of the lipid bilayer

Characterization of Domain 3 and Its α-Helical Region of the Hemolytic Lectin CEL-III Expressed as Glutathione S-Transferase-Fusion Proteins

CEL-III is a hemolytic lectin containing two carbohydrate-binding domains (domains 1 and 2) and a β-sheet-rich domain (domain 3). In domain 3, there is a hydrophobic region containing two α-helices (H8 and H9) and a loop between them, in which alternate hydrophobic residues, especially Val residues, are present. Synthetic peptides corresponding to the loop and second α-helix (H9) showed the strongest antibacterial activity. The recombinant glutathione S-transferase (GST)-fusion proteins containing domain 3 or the α-helical region peptide formed self-oligomers, whereas mutations in the alternate Val residues in the α-helical region lead to decreased oligomerization ability of the fusion proteins. These results suggest that the α-helical region, particularly its alternate Val residues are important for its oligomerization.Nagasaki Symposium on Nano-Dynamics 2008 (NSND2008) 平成20年1月29日(火)於長崎大学 Poster Presentatio

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

Expression and X-Ray Crystallographic Analysis of the Recombinant Hemolytic Lectin CEL-III

Recombinant hemolytic lectin CEL-III (rCEL-III) was expressed in Escherichia coli cells. Its hemolytic activity was much less than that of the native protein. X-ray crystallographic analysis of rCEL-III and native CEL-III (nCEL-III) revealed a slight difference in their tertiary structures, which may be caused by the amino acid replacements. It was inferred that these changes led to a decreased hemolytic activity of rCEL-III.Nagasaki Symposium on Nano-Dynamics 2008 (NSND2008) 平成20年1月29日(火)於長崎大学 Poster Presentatio

Effects of amino acid mutations in the pore-forming domain of the hemolytic lectin CEL-III

The hemolytic lectin CEL-III forms transmembrane pores in the membranes of target cells. A study on the effect of site-directed mutation at Lys405 in domain 3 of CEL-III indicated that replacements of this residue by relatively smaller residues lead to a marked increase in hemolytic activity, suggesting that moderately destabilizing domain 3 facilitates formation of transmembrane pores through conformational changes

Annual report of Perinatology Committee, Japan Society of Obstetrics and Gynecology, 2013: Development of Perinatal Emergency Care Systems and Suggestions

Placetal abruption is a disease occurring irrespective of the time and location and requiring maternal-fetal emergency care, so early delivery is indispensable, and the time from the occurrence of placental abruption to delivery should be shortened as much as possible

Annual report of Subcommittee for Examination of Causes of Maternal Death and their Prevention in Perinatology Committee, Japan Society of Obstetrics and Gynecology, 2013

As the fibrinogen level decreases early in atonic bleeding, early administration of FFP may be important as an initial approach to treat atonic bleeding.Amniotic fluid embolism is classified into 2 types, conventional type and uterus-type

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

Background CEL-I is a galactose/N-acetylgalactosamine-specific C-type lectin isolated from the sea cucumber Cucumaria echinata. Its carbohydrate-binding site contains a QPD (Gln-Pro-Asp) motif, which is generally recognized as the galactose specificity-determining motif in the C-type lectins. In our previous study, replacement of the QPD motif by an EPN (Glu-Pro-Asn) motif led to a weak binding affinity for mannose. Therefore, we examined the effects of an additional mutation in the carbohydrate-binding site on the specificity of the lectin. Methods Trp105 of EPN-CEL-I was replaced by a histidine residue using site-directed mutagenesis, and the binding affinity of the resulting mutant, EPNH-CEL-I, was examined by sugar-polyamidoamine dendrimer assay, isothermal titration calorimetry, and glycoconjugate microarray analysis. Tertiary structure of the EPNH-CEL-I/mannose complex was determined by X-ray crystallographic analysis. Results Sugar-polyamidoamine dendrimer assay and glycoconjugate microarray analysis revealed a drastic change in the specificity of EPNH-CEL-I from galactose/N-acetylgalactosamine to mannose. The association constant of EPNH-CEL-I for mannose was determined to be 3.17 × 103 M- 1 at 25 °C. Mannose specificity of EPNH-CEL-I was achieved by stabilization of the binding of mannose in a correct orientation, in which the EPN motif can form proper hydrogen bonds with 3- and 4-hydroxy groups of the bound mannose. Conclusions Specificity of CEL-I can be engineered by mutating a limited number of amino acid residues in addition to the QPD/EPN motifs. General significance Versatility of the C-type carbohydrate-recognition domain structure in the recognition of various carbohydrate chains could become a promising platform to develop novel molecular recognition proteins

cDNA cloning and characterization of a rhamnose-binding lectin SUL-I from the toxopneustid sea urchin Toxopneustes pileolus venom

The globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus contain several biologically active proteins. Among these, a galactose-binding lectin SUL-I isolated from the venom in the large globiferous pedicellariae shows several activities such as mitogenic, chemotactic, and cytotoxic activities through binding to the carbohydrate chains on the cells. We cloned cDNA encoding SUL-I by reverse transcription-PCR using the degenerate primers designed on the basis of the N-terminal amino acid sequence of the protein and expressed the recombinant SUL-I (rSUL-I) in Escherichia coli cells. The SUL-I gene contains an open reading frame of 927 nucleotides corresponding to 308 amino acid residues, including 24 residues of a putative signal sequence. The mature protein with 284 residues is composed of three homologous regions, each showing similarity with the carbohydrate-recognition domains of the rhamnose-binding lectins, which have been mostly found in fish eggs. While rSUL-I exhibited binding activity for several galactose-related sugars, the highest affinity was found for l-rhamnose among carbohydrates tested, confirming that SUL-I is a rhamnose-binding lectin. rSUL-I also showed hemagglutinating activity toward rabbit erythrocytes, indicating the existence of more than one carbohydrate-binding site to cross-link the carbohydrate chains on the cell surface, which may be closely related to its biological activities