Hemolytic Lectin Research Articles

Development of Antibacterial Peptides with Membrane Disruption and Folate Pathway Inhibitory Activities against Methicillin-Resistant Staphylococcus aureus.

Antimicrobial peptides (AMPs) offer an opportunity to overcome multidrug resistance. Here, novel peptides were designed based on AMP fragments derived from sea cucumber hemolytic lectin to enhance anti-methicillin-resistant Staphylococcus aureus (MRSA) activity with less side effects. Two designed peptides, CGS19 (LARVARRVIRFIRRAW-NH2) and CGS20 (RRRLARRLIFFIRRAW-NH2), exhibited strong antibacterial activities against clinically isolated MRSA with MICs of 3-6 μM, but no obvious cytotoxicity was observed. Consistently, CGS19 and CGS20 exerted rapid bactericidal activity and effectively induced 5.9 and 5.8 log reduction of MRSA counts in mouse subeschar, respectively. Further, CGS19 and CGS20 kill bacteria not only through disturbing membrane integrity but also by binding formate-tetrahydrofolate ligase, a key enzyme in the folate metabolism pathway, thereby inhibiting the folate pathway of MRSA. CGS19 and CGS20 are promising lead candidates for drug development against MRSA infection. The dual mechanisms on the identical peptide sequence or scaffold might be an underappreciated manner of treating life-threatening pathogens.

Functional expression and mutant analysis of thioredoxin-fused CEL-III, a hemolytic lectin from the marine invertebrate Cucumaria echinata.

CEL-III is a hemolytic lectin purified from the marine invertebrate Cucumaria echinata. New expression system of CEL-III was constructed, and the recombinant thioredoxin-fused CEL-III (Trx-CEL-III) showed strong hemolytic and carbohydrate-binding activity as same as authentic CEL-III. Mutation analysis of Trx-CEL-III suggested that carbohydrate binding to subdomain 1α and 2β of CEL-III might be important for the hemolytic activity.

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.

Structural analysis of Pore-Forming CEL-III

CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-binding domains (domains 1-2) and one oligomerization domain (domain 3). After binding to the cell surface carbohydrate chains through domains 1-2, 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 1-2 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 1-2 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.

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.

Purification and characterization of a novel C-type hemolytic lectin for clot lysis from the fresh water clam Villorita cyprinoides: A possible natural thrombolytic agent against myocardial infarction

Villorita cyprinoides (black clam) is a fresh water clam that belongs as a bivalve to the group of mollusc. The saline extracts from the muscle reveal high titers of agglutination potency on trypsin-treated rabbit erythrocytes. With the help of affinity chromatography a hemolytic protein with lectin activity which could all be inhibited by d-galactose were isolated. The lectins were separated on DEAE-cellulose and the main component was purified after an additional step of gel filtration on sephadex G-75. The main component is a non-glycosylated protein with a molecular weight of 96,560 Da determined by MALDI-ToF, consisting of a single protein chain and characterized by the lack of polymers and intermediate disulfide bonds. The pure main lectin with clot lytic feature shows two bands at molecular weights 36,360 and 26, 520 Da. Optimal inhibition of the pure lectin is achieved by d-galactose containing oligo- and polysaccharides. The lectin activity decreased above 40 °C and was lost at 62 °C, the stability over the pH range between 7.0 and 8.0 and requires divalent cations for their activity. The novel C-type hemolytic lectin for clot lysis from the clam Villorita cyprinoides was identified and evaluated, the purified hemolytic lectin (0.35 mg/ml and 0.175 mg/ml) enhanced clot lysis activity when compared to the different concentration (5 mg/ml and 1 mg/ml) of commercial streptokinase. In the present study identified hemolytic lectin was a rapid and effective clot lytic molecule and could be developed as new drug molecule in future.

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

BackgroundCEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata that shows Ca2+-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. MethodsSeveral amino acid residues in domain 3 were replaced by alanine, and hemolytic activity of the mutants was examined. ResultsK344A, 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. ConclusionsSeveral 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 significanceThe results provide important clues to the membrane pore-forming mechanism of CEL-III, which is also related to that of bacterial pore-forming toxins.

Effects of Detergents on the Oligomeric Structures of Hemolytic Lectin CEL-III as Determined by Small-Angle X-Ray Scattering

Hemolytic lectin CEL-III isolated from the sea cucumber Cucumaria echinata forms transmembrane pores by self-oligomerization in target cell membranes. It also formed soluble oligomers in aqueous solution upon binding with specific carbohydrates under conditions of high pH and a high salt concentration. The size of the soluble CEL-III oligomers decreased when treated with detergents such as Triton X-100 and SDS. Small-angle X-ray scattering measurements suggested that the dissociated unit of the oligomer was a tightly associated CEL-III heptamer. Without detergents in solution, these heptamers further assembled into larger 21mer oligomers, comprising three heptamers held together by relatively weak hydrophobic interactions.

Molecular Cloning, Functional Expression, and Characterization of Isolectin Genes of Hemolytic Lectin CEL-III from the Marine InvertebrateCucumaria echinata

CEL-III is a hemolytic lectin purified from the marine invertebrate Cucumaria echinata. Previous research has indictated that CEL-III is composed of several isoforms. Here we identified five CEL-III isolectin genes, designated CEL-III-L1, CEL-III-L2, CEL-III-S1, CEL-III-S2, and CEL-III-LS1, by cDNA cloning. The deduced amino acid sequences suggested they shared 94.0-99.8% identical residues. Among the amino acid residues involved in carbohydrate binding, the His residue, which contributes to stacking with sugar, in subdomain 1α was replaced by Tyr in CEL-III-L2. The recombinant proteins were expressed in Escherichia coli or insect cells. rCEL-III-L2 showed higher hemolytic activity than those of the other isolectins. Furthermore, an apparent oligomer band of rCEL-III-L2 was detected on erythrocyte membranes, although the other isolectins showed smear bands. These results suggest that Tyr36 of CEL-III-L2 is important for the expression of hemolytic activity and oligomerization.

Cytotoxic proteins of Amanita virosa Secr. mushroom: Purification, characteristics and action towards mammalian cells

A new hemolytic lectin was purified from the fruit bodies of Amanita virosa Secr. mushroom by the affinity chromatography on the cross-linked ovomucin. This lectin destroyed erythrocytes of human and animals of various species, and its hemolytic activity decreased in the row: rabbit > rat > human > dog. The erythrocytes of sheep, cow and carp were resistant to such hemolytic action of the lectin (1 mg/mL). The lectin-mediated hemolysis was blocked by the polyethylene glycol with molecular mass over 1350. A. virosa lectin, unlike Amanita phalloides lectin, did not interact with tested monosaccharides. However, the 4-nitrophenyl derivates of the monosaccharides inhibited the action of A. virosa lectin which did not prefer targeting O-type glycoproteins over the N-type glycoproteins. Murine leukemia cells of L1210 line and human leukemia T-cells of CEM T4 and Jurkat lines were shown to be sensitive to toxic effect of the lectin and another protein toxovirin isolated from A. virosa fruit bodies It was found that toxovirin possessed an enzymatic activity of l-amino acid oxidase. Since both toxic proteins – the lectin and toxovirin – are sensitive to an elevated temperature, it is suggested that they play a significant role in human poisoning only when the unbaked mushroom is eaten.

1P014 ストップドフローX線小角散乱測定による溶血性レクチンの構造変化の解析(蛋白質-構造,第48回日本生物物理学会年会)

1P014 ストップドフローX線小角散乱測定による溶血性レクチンの構造変化の解析(蛋白質-構造,第48回日本生物物理学会年会)

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 Ca(2+). This suggests that Ca(2+) supported correct folding of the carbohydrate-binding domains of rCEL-III, leading to effective binding to the cell surface and subsequent self-oligomerization.

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.

Carbohydrate-Recognition and Hemolytic Mechanism of the Hemolytic Lectin from Marine Invertebrate

CEL-III is a Ca2+-dependent hemolytic lectin isolated from the sea cucumber Cucumaria echinata. X-ray crystallographic analysis revealed that CEL-III is composed of two ricin-like carbohydrate-binding domains (domain 1/2) and an oligomerization domain (domain 3). After binding to the specific carbohydrate chains on erythrocyte membrane, CEL-III undergoes conformational change, which leads to formation of membrane pores composed of its oligomers. Carbohydrate-recognition mechanism of CEL-III and its relationship with self-oligomerization mediated by domain 3 are discussed.

2P-014 海鼠(グミ)由来溶血性レクチンCEL-IIIの多量化機構のX線小角散乱による解析(蛋白質・構造(2),第46回日本生物物理学会年会)

2P-014 海鼠(グミ)由来溶血性レクチンCEL-IIIの多量化機構のX線小角散乱による解析(蛋白質・構造(2),第46回日本生物物理学会年会)

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

CEL-III is a Ca(2+)-dependent hemolytic lectin, isolated from the marine invertebrate Cucumaria echinata. The three-dimensional structure of CEL-III/GalNAc and CEL-III/methyl alpha-galactoside complexes was solved by x-ray crystallographic analysis. In these complexes, five carbohydrate molecules were found to be bound to two carbohydrate-binding domains (domains 1 and 2) located in the N-terminal 2/3 portion of the polypeptide and that contained beta-trefoil folds similar to ricin B-chain. The 3-OH and 4-OH of bound carbohydrate molecules were coordinated with Ca(2+) located at the subdomains 1alpha, 1gamma, 2alpha, 2beta, and 2gamma, simultaneously forming hydrogen bond networks with nearby amino acid side chains, which is similar to carbohydrate binding in C-type lectins. The binding of carbohydrates was further stabilized by aromatic amino acid residues, such as tyrosine and tryptophan, through a stacking interaction with the hydrophobic face of carbohydrates. The importance of amino acid residues in the carbohydrate-binding sites was confirmed by the mutational analyses. The orientation of bound GalNAc and methyl alpha-galactoside was similar to the galactose moiety of lactose bound to the carbohydrate-binding site of the ricin B-chain, although the ricin B-chain does not require Ca(2+) ions for carbohydrate binding. The binding of the carbohydrates induced local structural changes in carbohydrate-binding sites in subdomains 2alpha and 2beta. Binding of GalNAc also induced a slight change in the main chain structure of domain 3, which could be related to the conformational change upon binding of specific carbohydrates to induce oligomerization of the protein.

1P024 海鼠(グミ)由来溶血性レクチンCEL-IIIの多量化機構のX線小角散乱による解析(蛋白質(構造・構造機能相関),口頭発表,第45回日本生物物理学会年会)

1P024 海鼠(グミ)由来溶血性レクチンCEL-IIIの多量化機構のX線小角散乱による解析(蛋白質(構造・構造機能相関),口頭発表,第45回日本生物物理学会年会)

Вивчення вуглеводної специфічності гемолітичного лектину блідої поганки (Amanita phalloides (Vaill. Fr.) Secr)

Вивчення вуглеводної специфічності гемолітичного лектину блідої поганки (Amanita phalloides (Vaill. Fr.) Secr)

Antibacterial activity of peptides derived from the C-terminal region of a hemolytic lectin, CEL-III, from the marine invertebrate Cucumaria echinata.

Several synthetic peptides derived from the C-terminal domain sequence of a hemolytic lectin, CEL-III, were examined as to their action on bacteria and artificial lipid membranes. Peptide P332 (KGVIFAKASVSVKVTASLSK-NH(2)), corresponding to the sequence from residue 332, exhibited strong antibacterial activity toward Gram-positive bacteria. Replacement of each Lys in P332 by Ala markedly decreased the activity. However, when all Lys were replaced by Arg, the antibacterial activity increased, indicating the importance of positively charged residues at these positions. Replacement of Val by Leu also led to higher antibacterial activity, especially toward Gram-negative bacteria. The antibacterial activity of these peptides was correlated with their membrane-permeabilizing activity toward the bacterial inner membrane and artificial lipid vesicles, indicating that the antibacterial action is due to perturbation of bacterial cell membranes, leading to enhancement of their permeability. These results also suggest that the hydrophobic region of CEL-III, from which P332 and its analogs were derived, may play some role in the interaction with target cell membranes to trigger hemolysis.

Molecular Cloning, Expression, and Characterization of Novel Hemolytic Lectins from the Mushroom Laetiporus sulphureus, Which Show Homology to Bacterial Toxins

We describe herein the cDNA cloning, expression, and characterization of a hemolytic lectin and its related species from the parasitic mushroom Laetiporus sulphureus. The lectin designated LSL (L. sulphureus lectin), is a tetramer composed of subunits of approximately 35 kDa associated by non-covalent bonds. From a cDNA library, three similar full-length cDNAs, termed LSLa, LSLb, and LSLc, were generated, each of which had an open reading frame of 945 bp encoding 315 amino acid residues. These proteins share 80-90% sequence identity and showed structural similarity to bacterial toxins: mosquitocidal toxin (MTX2) from Bacillus sphaericus and alpha toxin from Clostridium septicum. Native and recombinant forms of LSL showed hemagglutination and hemolytic activity and both activities were inhibited by N-acetyllactosamine, whereas a C-terminal deletion mutant of LSLa (LSLa-D1) retained hemagglutination, but not hemolytic activity, indicating the N-terminal domain is a carbohydrate recognition domain and the C-terminal domain functions as an oligomerization domain. The LSL-mediated hemolysis was protected osmotically by polyethylene glycol 4000 and maltohexaose. Inhibition studies showed that lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc) was the best inhibitor for LSL. These results indicate that LSL is a novel pore-forming lectin homologous to bacterial toxins.