Dataset Information

Identification, Characterization, and X-ray Crystallographic Analysis of a Novel Type of Lectin AJLec from the Sea Anemone Anthopleura japonica.

ABSTRACT: A novel galactose-specific lectin, AJLec (18.5?kDa), was isolated from the sea anemone, Anthopleura japonica. AJLec was characterized using the hemagglutination assay, isothermal titration calorimetry (ITC), and glycoconjugate microarray analysis and we found that AJLec has a specificity for galactose monomers and ?-linked terminal galactose residues in complex carbohydrates, but not for N-acetylgalactosamine (GalNAc), which is commonly recognized by galactose-binding lectins. The primary structure of AJLec did not show homology with known lectins, and a crystal structural analysis also revealed a unique homodimeric structure. The crystal structure of AJLec complexed with lactose was solved by measuring the sulfur single-wavelength anomalous diffraction (S-SAD) phasing with an in-house Cu K? source method. This analysis revealed that the galactose residue in lactose was recognized via its O2, O3, and O4 hydroxyl groups and ring oxygen by calcium coordination and two hydrogen bonds with residues in the carbohydrate-binding site, which demonstrated strict specificity for the ?-linked terminal galactose in this lectin.

SUBMITTER: Unno H

PROVIDER: S-EPMC6070535 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Identification, Characterization, and X-ray Crystallographic Analysis of a Novel Type of Lectin AJLec from the Sea Anemone Anthopleura japonica.

Unno Hideaki H Nakamura Azusa A Mori Shingo S Goda Shuichiro S Yamaguchi Kenichi K Hiemori Keiko K Tateno Hiroaki H Hatakeyama Tomomitsu T

Scientific reports 20180801 1

A novel galactose-specific lectin, AJLec (18.5 kDa), was isolated from the sea anemone, Anthopleura japonica. AJLec was characterized using the hemagglutination assay, isothermal titration calorimetry (ITC), and glycoconjugate microarray analysis and we found that AJLec has a specificity for galactose monomers and β-linked terminal galactose residues in complex carbohydrates, but not for N-acetylgalactosamine (GalNAc), which is commonly recognized by galactose-binding lectins. The primary struct ...[more]

PMID: 30068923

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Project description:BackgroundPore-forming proteins (PFP) are a class of toxins abundant in the venom of sea anemones. Owing to their ability to recognize and permeabilize cell membranes, pore-forming proteins have medical potential in cancer therapy or as biosensors. In the present study, we showed the partial purification and sequencing of a pore-forming protein from Anthopleura dowii Verrill (1869). 17.MethodsCytolytic activity of A. dowii Verrill (1869) venom was determined via hemolysis assay in the erythrocytes of four mammals (sheep, goat, human and rabbit). The cytotoxic activity was analyzed in the human adherent lung carcinoma epithelial cells (A549) by the cytosolic lactate dehydrogenase (LDH) assay, and trypan blue staining. The venom was fractionated via ammonium sulfate precipitation gradient, dialysis, and ion exchange chromatography. The presence of a pore-forming protein in purified fractions was evaluated through hemolytic and cytotoxic assays, and the activity fraction was analyzed using the percent of osmotic protections after polyethylene glycol (PEG) treatment and mass spectrometry. 18.ResultsThe amount of protein at which the venom produced 50% hemolysis (HU50) was determined in hemolysis assays using erythrocytes from sheep (HU50 = 10.7 ± 0.2 μg), goat (HU50 = 13.2 ± 0.3 μg), rabbit (HU50 = 34.7 ± 0.5 μg), and human (HU50 = 25.6 ± 0.6 μg). The venom presented a cytotoxic effect in A549 cells and the protein amount present in the venom responsible for producing 50% death (IC50) was determined using a trypan blue cytotoxicity assay (1.84 ± 0.40 μg/mL). The loss of membrane integrity in the A549 cells caused by the venom was detected by the release of LDH in proportion to the amount of protein. The venom was fractionated; and the fraction with hemolytic and cytotoxic activities was analyzed by mass spectrometry. A pore-forming protein was identified. The cytotoxicity in the A549 cells produced by the fraction containing the pore-forming protein was osmotically protected by PEG-3350 Da molecular mass, which corroborated that the loss of integrity in the plasma membrane was produced via pore formation. 19. Conclusion: A. dowii Verrill (1869) venom contains a pore-forming protein suitable for designing new drugs for cancer therapy.

Project description:BackgroundAlthough much is known about how circadian systems control daily cycles in the physiology and behavior of Drosophila and several vertebrate models, marine invertebrates have often been overlooked in circadian rhythms research. This study focuses on the starlet sea anemone, Nematostella vectensis, a species that has received increasing attention within the scientific community for its potential as a model research organism. The recently sequenced genome of N. vectensis makes it an especially attractive model for exploring the molecular evolution of circadian behavior. Critical behavioral data needed to correlate gene expression patterns to specific behaviors are currently lacking in N. vectensis.Methodology/principal findingsTo detect the presence of behavioral oscillations in N. vectensis, locomotor activity was evaluated using an automated system in an environmentally controlled chamber. Animals exposed to a 24 hr photoperiod (12 hr light: 12 hr dark) exhibited locomotor behavior that was both rhythmic and predominantly nocturnal. The activity peak occurred in the early half of the night with a 2-fold increase in locomotion. Upon transfer to constant lighting conditions (constant light or constant dark), an approximately 24 hr rhythm persisted in most animals, suggesting that the rhythm is controlled by an endogenous circadian mechanism. Fourier analysis revealed the presence of multiple peaks in some animals suggesting additional rhythmic components could be present. In particular, an approximately 12 hr oscillation was often observed. The nocturnal increase in generalized locomotion corresponded to a 24 hr oscillation in animal elongation.Conclusions/significanceThese data confirm the presence of a light-entrainable circadian clock in Nematostella vectensis. Additional components observed in some individuals indicate that an endogenous clock of approximately 12 hr frequency may also be present. By describing rhythmic locomotor behavior in N. vectensis, we have made important progress in developing the sea anemone as a model organism for circadian rhythm research.

Dataset Information

Identification, Characterization, and X-ray Crystallographic Analysis of a Novel Type of Lectin AJLec from the Sea Anemone Anthopleura japonica.

Publications

Identification, Characterization, and X-ray Crystallographic Analysis of a Novel Type of Lectin AJLec from the Sea Anemone Anthopleura japonica.

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