Microcystis viridis NIES-102 Cyanobacteria Lectin (MVL) Interacts with SARS-CoV-2 Spike Protein Receptor Binding Domains (RBDs) via Protein-Protein Interaction.

The emergence of coronavirus disease 2019 (COVID-19) posed a major challenge to healthcare systems worldwide, especially as mutations in the culprit Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) complicated the development of vaccines and antiviral drugs. Therefore, the search for natural products with broad anti-SARS-CoV-2 capabilities is an important option for the prevention and treatment of similar infectious diseases. Lectins, which are widely recognized as antiviral agents, could contribute to the development of anti-SARS-CoV-2 drugs. This study evaluated the binding affinity of six lectins (including the cyanobacterial lectin from Microcystis viridis NIES-102 (MVL), and Jacalin, a lectin from the breadfruit, Artocarpus altilis) to the receptor binding domain (RBD) of the spike protein on the original (wild) SARS-CoV-2 and three of its mutants: Alpha, Delta, and Omicron. MVL and Jacalin showed distinct binding affinity to the RBDs of the four SARS-CoV-2 strains. The remaining four lectins (DB1, ConA, PHA-M and CSL3) showed no such binding affinity. Although the glycan specificities of MVL and Jacalin were different, they showed the same affinity for the spike protein RBDs of the four SARS-CoV-2 strains, in the order of effectiveness Alpha > Delta > original > Omicron. The verification of glycan-specific inhibition revealed that both lectins bind to RBDs by glycan-specific recognition, but, in addition, MVL binds to RBDs through protein-protein interactions.

Specific Mutations near the Amyloid Precursor Protein Cleavage Site Increase γ-Secretase Sensitivity and Modulate Amyloid-ß Production.

Amyloid-ß peptides (Aßs) are produced via cleavage of the transmembrane region of the amyloid precursor protein (APP) by γ-secretase and are responsible for Alzheimer's disease. Familial Alzheimer's disease (FAD) is associated with APP mutations that disrupt the cleavage reaction and increase the production of neurotoxic Aßs, i.e., Aß42 and Aß43. Study of the mutations that activate and restore the cleavage of FAD mutants is necessary to understand the mechanism of Aß production. In this study, using a yeast reconstruction system, we revealed that one of the APP FAD mutations, T714I, severely reduced the cleavage, and identified secondary APP mutations that restored the cleavage of APP T714I. Some mutants were able to modulate Aß production by changing the proportions of Aß species when introduced into mammalian cells. Secondary mutations include proline and aspartate residues; proline mutations are thought to act through helical structural destabilization, while aspartate mutations are thought to promote interactions in the substrate binding pocket. Our results elucidate the APP cleavage mechanism and could facilitate drug discovery.

Specific Mutations in Aph1 Cause γ-Secretase Activation.

Amyloid beta peptides (Aßs) are generated from amyloid precursor protein (APP) through multiple cleavage steps mediated by γ-secretase, including endoproteolysis and carboxypeptidase-like trimming. The generation of neurotoxic Aß42/43 species is enhanced by familial Alzheimer's disease (FAD) mutations within the catalytic subunit of γ-secretase, presenilin 1 (PS1). FAD mutations of PS1 cause partial loss-of-function and decrease the cleavage activity. Activating mutations, which have the opposite effect of FAD mutations, are important for studying Aß production. Aph1 is a regulatory subunit of γ-secretase; it is presumed to function as a scaffold of the complex. In this study, we identified Aph1 mutations that are active in the absence of nicastrin (NCT) using a yeast γ-secretase assay. We analyzed these Aph1 mutations in the presence of NCT; we found that the L30F/T164A mutation is activating. When introduced in mouse embryonic fibroblasts, the mutation enhanced cleavage. The Aph1 mutants produced more short and long Aßs than did the wild-type Aph1, without an apparent modulatory function. The mutants did not change the amount of γ-secretase complex, suggesting that L30F/T164A enhances catalytic activity. Our results provide insights into the regulatory function of Aph1 in γ-secretase activity.

Biochemical properties of CumA multicopper oxidase from plant pathogen, Pseudomonas syringae.

Multicopper oxidases have a wide range of substrate specificity to be involved in various physiological reactions. Pseudomonas syringae, a plant pathogenic bacterium, has a multicopper oxidase, CumA. Multicopper oxidases have ability to degrade plant cell wall component, lignin. Once P. syringae enter apoplast and colonize, they start to disrupt plant immunity. Therefore, deeper understanding of multicopper oxidases from plant pathogens helps to invent measures to prevent invasion into plant cell, which brings agricultural benefits. Several biochemical studies have reported lower activity of CumA compared with other multicopper oxidase called CotA. However, the mechanisms underlying the difference in activity have not yet been revealed. In order to acquire insight into them, we conducted a biophysical characterization of PsCumA. Our results show that PsCumA has weak type I copper EPR signal, which is essential for oxidation activity. We propose that difference in the coordination of copper ions may decrease reaction frequency.

Usefulness of hemoglobin examination in gingival crevicular fluid during supportive periodontal therapy to diagnose the pre-symptomatic state in periodontal disease.

OBJECTIVES: The absence of bleeding on probing (BOP) is a good predictor of disease stability. This study investigated whether detection of hemoglobin (Hb) in gingival crevicular fluid (GCF) indicates minute signs of periodontal disease, even in BOP (-) cases. MATERIALS AND METHODS: GCF was collected from gingival sulci of 152 sound maxillary and mandibular teeth from 76 patients who had entered supportive periodontal therapy (SPT) using the split-mouth design. As clinical parameters, plaque index, GCF amount, gingival index, probing depth (PD), clinical attachment level, BOP, and alveolar bone resorption ratio were then recorded. As biochemical parameters, Hb amount, alkaline phosphatase (ALP) activity, and protein amount in GCF were measured. Periodontal conditions of diseased sites (PD ≥ 4 mm, BOP (+)) and healthy sites (PD ≤ 4 mm, BOP (-)) were further classified into two groups using the Hb cutoff value determined by PD and BOP and analyzed. RESULTS: Despite being healthy, ALP activity and protein amount in sulci of the group with Hb level greater than the cutoff value were significantly higher than those in the group with Hb level less than the cutoff value (P < 0.01). CONCLUSIONS: This study indicates that Hb examination is a promising candidate marker of pre-symptomatic periodontal disease because Hb presence in GCF suggests slight tissue damage, even in healthy sites defined as BOP (-). CLINICAL RELEVANCE: Hb examination of GCF is a powerful diagnostic tool for pre-symptomatic diagnosis of periodontal disease.

Diversified Biomineralization Roles of Pteria penguin Pearl Shell Lectins as Matrix Proteins.

Previously, we isolated jacalin-related lectins termed PPL2, PPL3 (PPL3A, 3B and 3C) and PPL4 from the mantle secretory fluid of Pteria penguin (Mabe) pearl shell. They showed the sequence homology with the plant lectin family, jacalin-related ß-prism fold lectins (JRLs). While PPL3s and PPL4 shared only 35%-50% homology to PPL2A, respectively, they exhibited unique carbohydrate binding properties based on the multiple glycan-binding profiling data sets from frontal affinity chromatography analysis. In this paper, we investigated biomineralization properties of these lectins and compared their biomineral functions. It was found that these lectins showed different effects on CaCO3 crystalization, respectively, although PPL3 and PPL2A showed similar carbohydrate binding specificities. PPL3 suppressed the crystal growth of CaCO3 calcite, while PPL2A increased the number of contact polycrystalline calcite composed of more than one crystal with various orientations. Furthermore, PPL4 alone showed no effect on CaCO3 crystalization; however, PPL4 regulated the size of crystals collaborated with N-acetyl-D-glucosamine and chitin oligomer, which are specific in recognizing carbohydrates for PPL4. These observations highlight the unique functions and molecular evolution of this lectin family involved in the mollusk shell formation.

Risk factors for tooth loss with a mean follow-up period of 13.9 years in supportive periodontal therapy patients.

BACKGROUND: Clinical evidence indicates that there are various risk factors of tooth loss. However, the degree of this risk among other risk factors remains unclear. In this retrospective cohort study, the authors evaluated the hazard ratios of several risk factors for tooth loss. METHODS: Included patients had all been treated for dental disorders, were in the supportive phase of periodontal therapy by dental hygienists, and visited a Japanese dental office continually during a 10-year period. Periodontal parameters, tooth condition, and general status of all teeth (excluding third molars) at the initial visit and at least 10 years later were evaluated by using multiple classification analysis. RESULTS: The authors evaluated a total of 7584 teeth in 297 patients (average age: 45.3, mean follow-up time: 13.9 years) Non-vital pulp was the most significant predictor of tooth loss according to Cox hazards regression analysis (hazard ratio: 3.31). The 10-year survival rate was approximately 90% for teeth with non-vital pulp and 99% for teeth with vital pulp. Fracture was the most common reason for tooth loss. CONCLUSIONS: Non-vital pulp had the most significant association with tooth loss among the parameters. Therefore, it is very important to minimize dental pulp extirpation.

Clinical and microbiological effect of frequent subgingival air polishing on periodontal conditions: a split-mouth randomized controlled trial.

The objective of this study was to evaluate the clinical effects of repeated subgingival debridement by air polishing during supportive periodontal therapy. A double-blind, randomized controlled trial of 6 months in duration was conducted on 19 recall patients who were previously treated for chronic periodontitis. Three sites with probing pocket depths (PPD) of 4-9 mm in each of the patients were randomly assigned to the following treatments: Glycine powder/air polishing every 30 days (group 1), glycine powder/air polishing at baseline and on day 90 (group 2), or water irrigation every 30 days (group 3). Clinical parameters were recorded and microbiological sampling was performed at 0, 90, and 180 days post-treatment. Subgingival samples were analyzed using real-time PCR methods for Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. Between baseline and 90 days, group 1 showed significantly more PPD reduction compared to group 3 and no significant differences with group 2. Between baseline and 180 days, group 1 displayed a significant increase in clinical attachment level compared with group 3. No differences were observed among the groups in numbers of total bacteria or percentage of investigated bacteria at any time point. This study revealed that routine subgingival air polishing at 30-day intervals had significant clinical effects in moderately deep pockets in patients who underwent supportive periodontal therapy.

Protein encapsulation in the hollow space of hemocyanin crystals containing a covalently conjugated ligand.

Encapsulation of guest molecules into the vacant space of biomacromolecular crystals has been utilized for various purposes including functioning as a protein container to protect against physical stress and structural determination of the guest. Todarodes pacificus hemocyanin (TpHc) is a hollow cylindrical decameric protein complex with an inner space 110 Šin diameter and 160 Šin height. In the crystal, TpHc forms a straw-like bundle and contains one reactive Cys (Cys3246) in the inner domain of each protomer. Here, we conjugated biotin onto Cys3246 of TpHc followed by incubation with streptavidin. The streptavidin was immobilized into the inner space of TpHc due to its interaction with biotin. Moreover, the complex containing TpHc and streptavidin was crystallized under the same conditions used for unmodified TpHc. In order to expand this methodology for a variety of proteins, we conjugated the ligand nitrilotriacetic acid (NTA) chelated to a Ni2+ ion (Ni2+-NTA) to TpHc. We found that His-tagged green fluorescent protein (GFP) was encapsulated into the Ni2+-NTA-conjugated TpHc via the interaction between the His-tag and the Ni2+-NTA group. X-ray crystallography demonstrated that the crystal packing of the complex containing TpHc and GFP was identical to that of the unmodified TpHc. Our guest immobilization method is distinct from previous approaches that are dependent on diffusion of the guest into the host crystal. Thus, our findings may accelerate the development of proteinaceous crystal engineering.

Encapsulation of biomacromolecules by soaking and co-crystallization into porous protein crystals of hemocyanin.

Encapsulation of guest molecules into the hollow spaces of crystals has been applied for a variety of purposes such as structure determination, separation, and catalysis of the guest. Although host-guest studies have been developed mainly in crystals of small molecules, those of biomacromolecules have recently been applied. In those reports, a huge hollow space in the protein crystal is commonly used for encapsulation of the guest. Our previous study revealed that cylindrical hemocyanins stack inside the crystal as a linear hollow structure. The diameter of the linear hollow is approximately 110 Å, which is large enough for most proteins to pass through. In the present study, we evaluated the potential of hemocyanin crystals as a host to encapsulate biomacromolecules. Confocal microscopy revealed that hemocyanin crystals encapsulate proteins of molecular mass up to 250 kDa, i.e., 27 kDa green fluorescence protein, 105 kDa allophycocyanin, 220 kDa C-phycocyanin, and 250 kDa phycoerythrin, and DNAs up to 200-bp long, whereas 440 kDa ferritin not. Further analysis revealed that hemocyanin crystals prefer a negatively charged guest rather than a positive charge to encapsulate. Moreover, a photobleaching experiment showed that the guest does not move once entrapped. This knowledge of the host-guest study using the hollow hemocyanin crystal should be of significance for further application of hollow proteinaceous crystals as a host.

CHAC1 overexpression in human gastric parietal cells with Helicobacter pylori infection in the secretory canaliculi.

BACKGROUND: Cation transport regulator 1 (CHAC1), a newly discovered enzyme that degrades glutathione, is induced in Helicobacter pylori (H. pylori)-infected gastric epithelial cells in culture. The CHAC1-induced decrease in glutathione leads to an accumulation of reactive oxygen species and somatic mutations in TP53. We evaluated the possible correlation between H. pylori infection and CHAC1 expression in human gastric mucosa. MATERIALS AND METHODS: Both fresh-frozen and formalin-fixed paraffin-embedded tissue samples of gastric mucosa with or without H. pylori infection were obtained from 41 esophageal cancer patients that underwent esophago-gastrectomy. Fresh samples were used for real-time polymerase chain reaction for H. pylori DNA and CHAC1 mRNA, and formalin-fixed samples were used for immunohistochemistry with anti-CHAC1 and anti-H. pylori monoclonal antibodies. Double-enzyme or fluorescence immunohistochemistry and immuno-electron microscopy were used for further analysis. RESULTS: Significant CHAC1 overexpression was detected in H. pylori-infected parietal cells that expressed the human proton pump/H,K-ATPase α subunit, whereas a constitutively low level of CHAC1 mRNA expression was observed in the other samples regardless of the H. pylori infection status, reflecting the weak CHAC1 expression detected by immunohistochemistry in the fundic-gland areas. Immuno-electron microscopy revealed intact H. pylori cells in the secretory canaliculi of infected parietal cells. Some parietal cells exhibited positive nuclear signals for Ki67 in the neck zone of the gastric fundic-gland mucosa with H. pylori infection. CONCLUSION: Cation transport regulator 1 overexpression in H. pylori-infected parietal cells may cause the H. pylori-induced somatic mutations that contribute to the development of gastric cancer.

Glycan Binding Profiling of Jacalin-Related Lectins from the Pteria Penguin Pearl Shell.

We determined the primary structures of jacalin-related lectins termed PPL3s (PPL3A, 3B, and 3C, which are dimers consisting of sequence variants α + α, α + ß, ß + ß, respectively) and PPL4, which is heterodimer consisting of α + ß subunits, isolated from mantle secretory fluid of Pteria penguin (Mabe) pearl shell. Their carbohydrate-binding properties were analyzed, in addition to that of PPL2A, which was previously reported as a matrix protein. PPL3s and PPL4 shared only 35-50% homology to PPL2A, respectively; they exhibited significantly different carbohydrate-binding specificities based on the multiple glycan binding profiling data sets from frontal affinity chromatography analysis. The carbohydrate-binding specificity of PPL3s was similar to that of PPL2A, except only for Man3Fuc1Xyl1GlcNAc2 oligosaccharide, while PPL4 showed different carbohydrate-binding specificity compared with PPL2A and PPL3s. PPL2A and PPL3s mainly recognize agalactosylated- and galactosylated-type glycans. On the other hand, PPL4 binds to high-mannose-and hybrid-type N-linked glycans but not agalactosylated- and galactosylated-type glycans.

Structures of jacalin-related lectin PPL3 regulating pearl shell biomineralization.

The nacreous layer of pearl oysters is one of the major biominerals of commercial and industrial interest. Jacalin-related lectins, including PPL3 isoforms, are known to regulate biomineralization of the Pteria penguin pearl shell, although the molecular mechanisms are largely unknown. The PPL3 crystal structures were determined partly by utilizing microgravity environments for 3 isoforms, namely, PPL3A, PPL3B, and PPL3C. The structures revealed a tail-to-tail dimer structure established by forming a unique inter-subunit disulfide bond at C-termini. The N-terminal residues were found in pyroglutamate form, and this was partly explained by the post-translational modification of PPL3 isoforms implied from the discrepancy between amino acid and gene sequences. The complex structures with trehalose and isomaltose indicated that the novel specificity originated from the unique α-helix of PPL3 isoforms. Docking simulations of PPL3B to various calcite crystal faces suggested the edge of a ß-sheet and the carbohydrate-binding site rich in charged residues were the interface to the biomineral, and implied that the isoforms differed in calcite interactions.

Optical Fiber-Type Sugar Chip Using Localized Surface Plasmon Resonance.

Optical fiber-type Sugar Chips were developed using localized surface plasmon resonance (LSPR) of gold (Au) nanoparticles. The endface of an optical fiber was first aminosilylated and then condensed with α-lipoic acid containing a dithiol group. Second, gold nanoparticles were immobilized onto the endface via an Au-S covalent bond. Finally, sugar moieties were attached to the gold nanoparticle using our original sugar chain-ligand conjugates to obtain fiber-type Sugar Chips, by which the sugar moiety-protein interaction was analyzed. The specificity, sensitivity, and quantitative binding potency against carbohydrate-binding protein were found to be identical to that of a conventional SPR sensor. In this analysis, only a small sample volume (approximately 10 µL) was required compared with 100 µL for the conventional SPR sensor, suggesting that the fiber-type Sugar Chip and LSPR are applicable for nonpure small masses of proteins.

Regulation of axon arborization pattern in the developing chick ciliary ganglion: Possible involvement of caspase 3.

During a certain critical period in the development of the central and peripheral nervous systems, axonal branches and synapses are massively reorganized to form mature connections. In this process, neurons search their appropriate targets, expanding and/or retracting their axons. Recent work suggested that the caspase superfamily regulates the axon morphology. Here, we tested the hypothesis that caspase 3, which is one of the major executioners in apoptotic cell death, is involved in regulating the axon arborization. The embryonic chicken ciliary ganglion was used as a model system of synapse reorganization. A dominant negative mutant of caspase-3 precursor (C3DN) was made and overexpressed in presynaptic neurons in the midbrain to interfere with the intrinsic caspase-3 activity using an in ovo electroporation method. The axon arborization pattern was 3-dimensionally and quantitatively analyzed in the ciliary ganglion. The overexpression of C3DN significantly reduced the number of branching points, the branch order and the complexity index, whereas it significantly elongated the terminal branches at E6. It also increased the internodal distance significantly at E8. But, these effects were negligible at E10 or later. During E6-8, there appeared to be a dynamic balance in the axon arborization pattern between the "targeting" mode, which is accompanied by elongation of terminal branches and the pruning of collateral branches, and the "pathfinding" mode, which is accompanied by the retraction of terminal branches and the sprouting of new collateral branches. The local and transient activation of caspase 3 could direct the balance towards the pathfinding mode.

Isolation of Rice Bran Lectins and Characterization of Their Unique Behavior in Caco-2 Cells.

Rice bran lectins, named as RBA1 and RBA2, were isolated from Oryza sativa in two chromatography steps: affinity chromatography and cation-exchange chromatography. RBA1 was found to be composed of a covalently linked heterodimer of 20- and 12-kDa subunits, and RBA2 was a noncovalently linked dimer of 12-kDa subunits. Both RBA1 and RBA2 bound to desialylated complex glycoproteins such as fetuin, α1-acid glycoprotein, and transferrin, and agalactosylated complex glycoproteins such as agalacto fetuin, agalacto-α1-acid glycoprotein, and agalacto-transferrin, in addition to chitooligosacchrides. RBAs were heat stable up to 80 °C and stable at pH 4-10. RBA1 increased the transport of the fluorescent marker, rhodamine 123, which is known to be transported via the P-glycoprotein-mediated efflux pathway across human intestinal Caco-2 cell monolayers. Furthermore, RBA1 itself was transported to the basolateral side of the monolayers via an endocytotic pathway.

Effect of chum salmon egg lectin on tight junctions in Caco-2 cell monolayers.

The effect of a chum salmon egg lectin (CSL3) on tight junction (TJ) of Caco-2 cell monolayers was investigated. The lectin opened TJ as indicated by the decrease of the transepithelial electrical resistance (TER) value and the increase of the permeation of lucifer yellow, which is transported via the TJ-mediated paracellular pathway. The effects of CSL3 were inhibited by the addition of 10 mM L-rhamnose or D-galactose which were specific sugars for CSL3. The lectin increased the intracellular Ca2+ of Caco-2 cell monolayers, that could be inhibited by the addition of L-rhamnose. The fluorescence immunostaining of ß-actin in Caco-2 cell monolayers revealed that the cytoskeleton was changed by the CSL3 treatment, suggesting that CSL3 depolymerized ß-actin to cause reversible TJ structural and functional disruption. Although Japanese jack bean lectin and wheat germ lectin showed similar effects in the decrease of the TER values and the increase of the intracellular Ca2+, they could not be inhibited by the same concentrations of simple sugars, such as D-glucose and N-acetyl-D-glucosamine.

Isolation and biochemical characterization of Apios tuber lectin.

Apios tuber lectin, named ATL, was isolated from Apios americana Medikus by two chromatography steps, hydrophobic chromatography and anion-exchange chromatography. The minimum concentration required for the hemagglutination activity toward rabbit erythrocytes of ATL was 4 µg/mL. ATL was composed of a homodimer of 28.4 kDa subunits. The amino acid sequence of ATL was similar to those of other legume lectins. The lectin showed moderate stability toward heating and acidic pH, and the binding affinity against several monosaccharides, such as D-glucosamine and D-galactosamine. ATL also bound to desialylated or agalactosylated glycoproteins such as asialo and agalacto transferrin. ATL decreased the transepithelial electrical resistance across human intestinal Caco-2 cell monolayers, suggesting the effect on the tight junction-mediated paracellular transport.

Biochemical characterization of Acacia schweinfurthii serine proteinase inhibitor.

One of the many control mechanisms of serine proteinases is their specific inhibition by protein proteinase inhibitors. An extract of Acacia schweinfurthii was screened for potential serine proteinase inhibition. It was successfully purified to homogeneity by precipitating with 80% (v/v) acetone and sequential chromatographic steps, including ion-exchange, affinity purification and reversed-phase high performance liquid chromatography. Reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis conditions revealed an inhibitor (ASTI) consisting of two polypeptide chains A and B of approximate molecular weights of 16 and 10 kDa, respectively, and under non-reducing conditions, 26 kDa was observed. The inhibitor was shown to inhibit bovine trypsin (Ki of 3.45 nM) at an approximate molar ratio of inhibitor:trypsin (1:1). The A- and B-chains revealed complete sequences of 140 and 40 amino acid residues, respectively. Sequence similarity (70%) was reported between ASTI A-chain and ACTI A-chain (Acacia confusa) using ClustalW. The B-chain produced a 76% sequence similarity between ASTI and Leucaena leucocephala trypsin inhibitor.

Establishment of appropriate conditions for the efficient determination of multiple mycotoxins in tea samples and assessment of their drinking risks.

Conditions were determined for rapid, convenient, and efficient determination of 16 common mycotoxins in tea samples. Mycotoxins in tea leaves and tea infusion samples were extracted using solid-liquid extraction/liquid-liquid extraction combined with ultrasonic-assisted extraction. The extraction solvent was 2-butanone/ethyl acetate (9/1 v/v) with 0.1 % formic acid. The established conditions enabled the analysis of these mycotoxins by ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) in 5.5 min. In addition, HPLC with a temperature-controlled fluorescence detector was able to simultaneously determine 8 mycotoxins with fluorescent properties in 10 min without derivatization. Aflatoxin M1 (2.15 and 3.01 µg/kg), fumonisin B2 (198.89 µg/kg), and zearalenone (87.54 µg/kg) could be detected in commercially available pu-erh tea, green tea, and black tea products, and their total transfer rates from the products to brewed tea infusions were 64.08-65.13 %, 90.59 %, and 25.99 %, respectively. The risks of drinking mycotoxins from these tea infusions mostly showed low levels of concern.