An international influenza outbreak represents one of the most serious public health crises of global concern. The dissemination of influenza A virus variants necessitates continuous monitoring owing to the high mutation rate of the virus. Therefore, this study aimed to engineer a nanocarbon-based biosensor that is capable of identifying the preferred host of the virus. The practical implementation of biosensors for constant surveillance requires their preservation for several years. This paper documents the preservability of sugar chain probes using pyrenylated sialylglycopeptide and neoglycoprotein and examines relative errors associated with fluctuations in binding activity. Atomic force microscopy revealed that substantial relative errors may be associated with partially degraded sugar chains, and that the cleaning process appears to be crucial in probe preparation.

Clade 2.2 H5N1 influenza viruses have caused an unusually high number of human infections, providing a unique opportunity to investigate early molecular steps associated with host adaptation. Although most work has focused on hemagglutinin (HA), the contribution of neuraminidase (NA) to these early adaptive events has remained unclear. By analyzing publicly available sequences from clade 2.2-infected patients, we identified 20 NA mutations and compared their phenotypes to 20 mutations acquired during diversification in primary human airway cells under drug-free conditions. Most patient-derived NA mutations resulted in modest reductions in sialidase activity, keeping activity within a functional range that supported improved replication in α2,6 sialylglycan (α2,6 Sia)-dominant environments, whereas excessive reduction impaired fitness. Notably, the phenotypes of culture-selected and patient-derived mutations were highly concordant, suggesting that these NA changes arose through natural selection rather than antiviral pressure. Re-analysis of patient sequences further revealed that many adaptive NA mutations co-occur with HA mutations that confer only weak, partial α2,6 Sia binding. Using reverse genetics, we found that such naturally occurring HA/NA mutation pairs acted cooperatively in a receptor-context-dependent manner to support α2,6-associated replication relative to HA-only mutants, placing these variants within a constrained "early-adaptation space" characterized by limited α2,6 engagement and moderately reduced NA activity. Together, these findings indicate that early human adaptation of clade 2.2 H5N1 involves not only HA and PB2, but also incremental, cooperative tuning of NA function. Monitoring coordinated HA-NA evolution may therefore improve risk assessment frameworks for zoonotic influenza viruses poised at early stages of human host adaptation.

IntroductionInflammatory bowel diseases (IBD), especially ulcerative colitis, are associated with a high risk of carcinogenesis. D-allose, a D-glucose epimer, exhibits antioxidant and antitumor activities. This study aimed to examine the effects of D-allose on colitis-associated carcinogenesis.MethodsA mouse model of colitis-associated carcinogenesis was established followed by treatment with D-allose. In vitro, ER stress and mitochondrial function in RAW 264.7 macrophages and the migration and proliferation of Caco-2 cells were analyzed to elucidate the underlying mechanisms. Colonic tissues obtained from IBD patients with were subjected to analyze ER stress in macrophages.ResultsD-allose administration significantly reduced the tumor number, hemorrhage, inflammation score, and macrophage infiltration in the AOM/DSS model. D-allose suppressed ER stress signal and mitochondrial dysfunction in LPS treated RAW 264.7 macrophages. D-allose suppressed ER stress marker Bip and CHOP expression in thapsigargin treated RAW 264.7. In IBD patient’s colon, ER stress marker Bip and CHOP positive macrophage infiltration was detected in both inflammatory and tumor areas. The level of fluorescence labeled M6~G1M9 oligosaccharides increased in the LPS-treated RAW 264.7 macrophages, while thapsigargin or D-allose had no effect. In Caco-2 cells, D-allose suppressed phosphorylated AMPK expression, reduced migratory activity. D-allose inhibited glycolysis, and decreased cell proliferation through TXNIP upregulation.ConclusionD-allose suppressed inflammation and tumor development in a colitis-associated carcinogenesis model. D-allose restoring macrophage ER stress and mitochondrial dysfunction, and inhibiting colon cancer cell migration and proliferation. Therefore, D-allose may represent as a promising therapeutic and preventive agent for IBD and inflammation-associated carcinogenesis.

Rare sugars are defined as monosaccharides and their derivatives that do not exist in nature at all or that exist in extremely limited amounts despite being theoretically possible. At present, no comprehensive dogma has been presented regarding how and why these rare sugars have deviated from the naturally selected monosaccharides. In this minireview, we adopt a hypothesis on the origin and evolution of elementary hexoses, previously presented by one of the authors (Hirabayashi, Q Rev Biol, 1996, 71:365-380). In this scenario, monosaccharides, which constitute various kinds of glycans in nature, are assumed to have been generated by formose reactions on the prebiotic Earth (chemical evolution era). Among them, the most stable hexoses, i.e., fructose, glucose, and mannose remained accumulated. After the birth of life, the "chemical origin" saccharides thus survived were transformed into a variety of "bricolage products", which include galactose and other recognition saccharides like fucose and sialic acid through the invention of diverse metabolic pathways (biological evolution era). The remaining monosaccharides that have deviated from this scenario are considered rare sugars. If we can produce rare sugars as we wish, it is expected that various more useful biomaterials will be created by using them as raw materials. Thanks to the pioneering research of the Izumori group in the 1990 ' s, and to a few other investigations by other groups, almost all monosaccharides including L-sugars can now be produced by combining both chemical and enzymatic approaches. After briefly giving an overview of the origin of elementary hexoses and the current state of the rare sugar production, we will look ahead to the next generation of monosaccharide research which also targets glycosides including disaccharides.

A lectin-like protein was discovered in Agaricus bisporus as part of the mushroom tyrosinase complex. The protein has a β-trefoil fold, which is typical of the ricin B-like-type lectin family. The structure of the recombinant protein has been elucidated, and its specific sugar-binding affinity towards mannose and mannitol has also been reported; therefore, the protein was named A. bisporus mannose-binding protein (Abmb). Although the sugar-binding site of Abmb is predicted to be close to the C-terminus, the sugar-binding site has not yet been determined. In this study, a variant of recombinant Abmb with a longer C-terminal region including a 6×His-tag was constructed and its structure was solved at 1.51 and 2.34 Å resolution in an orthorhombic and a monoclinic space group, respectively. The overall structure showed a β-trefoil fold as previously reported; however, several surface loop regions including the C-terminal region showed high flexibility. In addition, a glycan-search assay of this variant showed weak binding affinity towards β-D-galactose but no affinity towards α-D-mannose. The plasticity of the C-terminal tail could be related to the differences in the carbohydrate-binding affinity of Abmb.

Graphene biosensors have the potential to be excellent applications of two-dimensional materials, because graphene has high mobility and specific surface area. However, it is difficult to stably obtain consistent responses from graphene biosensors owing to external disturbances and the lack of understanding their sensing mechanism. We propose a graphene biosensor coated with a gas-permeable silicone elastomer, poly(dimethylsiloxane). The elastomer coating allows only the gas molecules to reach the graphene surface, suppressing the disturbances from other factors, and thereby eliciting stable responses to target ammonia molecules in solution. It allowed us to clarify the relationship between ammonia production via urease reactions and conductivity changes of the graphene biosensor. The biosensor responses were modeled using the combination of the dissociation equilibrium of ammonia, Langmuir's adsorption isotherm, and Michaelis-Menten equation. Findings of this study lay the foundation for practical applications of stable graphene biosensors based on our reasonable response model.

SARS-CoV-2 has evolved continuously and accumulated spike mutations with each variant having a different binding for the cellular ACE2 receptor. It is not known whether the interactions between such mutated spikes and ACE2 glycans are conserved among different variant lineages. Here, we focused on three ACE2 glycosylation sites (53, 90 and 322) that are geometrically close to spike binding sites and investigated the effect of their glycosylation pattern on spike affinity. These glycosylation deletions caused distinct site-specific changes in interactions with the spike and acted cooperatively. Of note, the particular interaction profiles were conserved between the SARS-CoV-2 parental virus and the variants of concern (VOCs) Delta and Omicron. Our study provides insights for a better understanding of the importance of ACE2 glycosylation on ACE2/SARS-CoV-2 spike interaction and guidance for further optimization of soluble ACE2 for therapeutic use.

The nodes of Ranvier are unmyelinated gaps in the axon, important for the efficient transmission of action potentials. Despite the identification of several glycoproteins involved in node formation and maintenance, glycans' structure and formation in the node remain unclear. Previously, we developed a recombinant lectin from the Clostridium botulinum neurotoxin complex, specific to the galactose and N-acetylgalactosamine terminal epitopes (Gg). Gg stained Neuro2a cells. Here, we show Gg punctuate staining in mouse brain cryosections. Thus, we hypothesized that Gg could help study glycans in the node of Ranvier. Lectin histochemistry on mouse brain cryosections confirmed that Gg binds specifically to the node of Ranvier in the central nervous system (CNS). Using a combination of lectin blotting, glycosidase treatment on tissue sections, and lectin histochemistry, Gg ligands were identified as α-galactose terminal glycoproteins in the perinodal extracellular matrix. Furthermore, we detected the spatiotemporal distribution of galactosylated glycans in the CNS node of Ranvier in mouse brain tissues at different postnatal times. Finally, we observed impaired clustering of galactosylated glycans in the nodes during demyelination and remyelination in cuprizone-induced demyelination and remyelination mouse model. In conclusion, Gg can serve as a novel brain imaging tool in glycobiology and report glycoprotein formation and alterations in the CNS node of Ranvier. Our findings might serve as a first step to establish the role of glycans in the node of Ranvier.

Endo-β-N-acetylglucosaminidases (ENGases) are enzymes that hydrolyze the N-linked oligosaccharides. Many ENGases have already been identified and characterized. However, there are still a few enzymes that have hydrolytic activity toward multibranched complex-type N-glycans on glycoproteins. In this study, one novel ENGase from Bacteroides nordii (Endo-BN) species was identified and characterized. The recombinant protein was prepared and expressed in Escherichia coli cells. This Endo-BN exhibited optimum hydrolytic activity at pH 4.0. High performance liquid chromatography (HPLC) analysis showed that Endo-BN preferred core-fucosylated complex-type N-glycans, with galactose or α2,6-linked sialic acid residues at their non-reducing ends. The hydrolytic activities of Endo-BN were also tested on different glycoproteins from high-mannose type to complex-type oligosaccharides. The reaction with human transferrin, fetuin, and α1-acid glycoprotein subsequently showed that Endo-BN is capable of releasing multi-branched complex-type N-glycans from these glycoproteins.

In general, viruses recognize host cell surface glycans, but the measurement of virus-host cell glycan interaction is not widely operated. This is not only because commercially available, structure-defined glycans are limited, but also because such interactions, if any, between viruses and isolated glycans are relatively weak, and thus, difficult to detect by conventional methods, e.g., enzyme-linked immune-sorbent assay. We describe a practical method to detect virus binding to glycans; for this, preparation of glycan arrays using glycopeptides derived from biomaterials is necessary. In this context, neoglycoprotein is produced using bovine serum albumin (BSA) and commercially available glycopeptides, with which influenza viruses are detected using an evanescent-field-activated fluorescence scanner. It is clearly shown that H1N1 strains of influenza virus recognize BSA, to which DiNeuα2-6bianntena-peptide (SGP) is covalently linked, while on the other hand H5N1 strains recognize BSA linked to DiNeuα2-3bianntena-peptide (α2,3SGP).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been transmitted across all over the world, in contrast to the limited epidemic of genetically- and virologically-related SARS-CoV. However, the molecular basis explaining the difference in the virological characteristics among SARS-CoV-2 and SARS-CoV has been poorly defined. Here we identified that host sialoglycans play a significant role in the efficient spread of SARS-CoV-2 infection, while this was not the case with SARS-CoV. SARS-CoV-2 infection was significantly inhibited by α2-6-linked sialic acid-containing compounds, but not by α2–3 analog, in VeroE6/TMPRSS2 cells. The α2-6-linked compound bound to SARS-CoV-2 spike S1 subunit to competitively inhibit SARS-CoV-2 attachment to cells. Enzymatic removal of cell surface sialic acids impaired the interaction between SARS-CoV-2 spike and angiotensin-converting enzyme 2 (ACE2), and suppressed the efficient spread of SARS-CoV-2 infection over time, in contrast to its least effect on SARS-CoV spread. Our study provides a novel molecular basis of SARS-CoV-2 infection which illustrates the distinctive characteristics from SARS-CoV.

In the fission yeast Schizosaccharomyces pombe, alpha 1,2- and alpha 1,3-linked D-galactose (Gal) residues are transferred to N- and O-linked oligosaccharides of glycoproteins by galactosyltransferases. Although the galactomannans are important for cell-cell communication in S. pombe (e.g., in nonsexual aggregation), the mechanisms underlying galactosylation in cells remain unclear. Schizosaccharomyces pombe has 10 galactosyltransferase-related genes: seven belonging to glycosyltransferase (GT) family 34 and three belonging GT family 8. Disruption of all 10 alpha-galactosyltransferases (strain Delta 10GalT) has been shown to result in a complete lack of alpha-Gal residues. Here, we have investigated the function and substrate specificities of galactosyltransferases in S pombe by using strains expressing single alpha-galactosyltransferases in the Delta 10GalT background. High-performance liquid chromatography (HPLC) analysis of pyridylaminated O-linked oligosaccharides showed that two GT family 34 alpha 1,2-galactosyltransferases (Gma12p and Gmh6p) and two GT family 8 alpha 1,3-galactosyltransferases (Otg2p and Otg3p) are involved in galactosylation of O-linked oligosaccharide. Moreover, H-1-NMR of N-glycans revealed that three GT family 34 alpha 1,2-galactosyltransferases (Gmh1p, Gmh2p and Gmh3p) are required for the galactosylation of N-linked oligosaccharides. Furthermore, HPLC and lectin-blot analysis revealed that Otg1p showed alpha 1,3-galactosyltransferase activity under conditions of co-expression with Gmh6p, indicating that alpha-1,2-linked galactose is required for the galactosylation activity of Otg1p in S. pombe. In conclusion, eight galactosyltransferases have been shown to have activity in S. pombe with different substrate specificities. These findings will be useful for genetically tailoring the galactosylation of both N- and O-glycans in fission yeast.

Lectins are proteins with the ability to recognize and bind to specific glycan structures. These molecules play important roles in many biological systems and are actively being studied because of their ability to detect glycan biomarkers for many diseases. Hemagglutinin (HA) proteins from Clostridium botulinum type C neurotoxin complex; HA1, HA2, and HA3 are lectins that aid in the internalization of the toxin complex by binding to glycoproteins on the cell surface. HA1 mutants have been previously reported, namely HA1 W176A/D271F and HA1 N278A/Q279A which are specific to galactose (Gal)/N-acetylgalactosamine (GalNAc) and N-acetylneuraminic acid (Neu5Ac) sugars, respectively. In this study, we utilized HA1 mutants and expressed them in complex with HA2 WT and HA3 WT to produce glycan detecting tools with high binding affinity. Particularly, two types were made: Gg and Rn. Gg is an Alexa 488 conjugated lectin complex specific to Gal and GalNAc, while Rn is an Alexa 594 conjugated lectin complex specific to Neu5Ac. The specificities of these lectins were identified using a glycan microarray followed by competitive sugar inhibition experiments on cells. In addition, we confirmed that Gg and Rn staining is clearly different depending on cell type, and the staining pattern of these lectins reflects the glycans present on the cell surface as shown in enzyme treatment experiments. The availability of Gg and Rn provide us with new promising tools to study Gal, GalNAc, and Neu5Ac terminal epitopes which can aid in understanding the functional role of glycans in physiological and pathological events.

In the fission yeast Schizosaccharomyces pombe, α1,2- and α1,3-linked D-galactose (Gal) residues are transferred to N- and O-linked oligosaccharides of glycoproteins by galactosyltransferases. Although the galactomannans are important for cell-cell communication in S. pombe (e.g., in non-sexual aggregation), the mechanisms underlying galactosylation in cells remain unclear. S. pombe has 10 galactosyltransferase-related genes: seven belonging to glycosyltransferase (GT) family 34 and three belonging GT family 8. Disruption of all 10 α-galactosyltransferases (strain Δ10GalT) has been shown to result in a complete lack of α-Gal residues. Here, we have investigated the function and substrate specificities of galactosyltransferases in S pombe by using strains expressing single α-galactosyltransferases in the Δ10GalT background. High-performance liquid chromatography (HPLC) analysis of pyridylaminated O-linked oligosaccharides showed that two GT family 34 α1,2-galactosyltransferases (Gma12p and Gmh6p) and two GT family 8 α1,3-galactosyltransferases (Otg2p and Otg3p) are involved in galactosylation of O-linked oligosaccharide. Moreover, 1H-NMR of N-glycans revealed that three GT family 34 α1,2-galactosyltransferases (Gmh1p, Gmh2p, and Gmh3p) are required for galactosylation of N-linked oligosaccharides. Furthermore, HPLC and lectin-blot analysis revealed that Otg1p showed α1,3-galactosyltransferase activity under conditions of co-expression with Gmh6p, indicating that α-1,2-linked galactose is required for the galactosylation activity of Otg1p in S. pombe. In conclusion, eight galactosyltransferases have been shown to have activity in S. pombe with different substrate specificities. These findings will be useful for genetically tailoring the galactosylation of both N- and O- glycans in fission yeast.

Small solid-state devices are candidates for accelerating biomedical assays/drug discovery, however their potential remains unfulfilled. Here, we demonstrate that graphene-field effect transistors (FET) can be used to successfully detect the key molecular events underlying viral infections and the effect of antiviral drugs. Our device success is achieved by bio-mimicking the host-cell surface during an influenza infection at the graphene channel. In-situ AFM confirms the biological interactions at the sialic acid-functionalized graphene: viral hemagglutinin (HA) binds to sialic acid, and neuraminidase (NA) reacts with the sialic acid-HA complex. The graphene-FET detects HA binding to sialic acid, and NA cleavage of sialic acid. The inhibitory effect of the drug “zanamivir” on NA-sialic acid interactions is monitored in real-time; the reaction rate constant of NA-sialic acid reaction was successfully determined. We demonstrate that graphene-FETs are powerful platforms for measurement of biomolecular interactions and contribute to future deployment of solid-state devices in drug discovery/biosensing.

We describe a method to detect influenza virus using an evanescent-field-activated fluorescence scanner type glycan array and ELISA system. Neoglycoprotein was prepared by combination of organic chemistry and biomaterial preparation. These ligands were spotted on a glass plate or plastic well to make a glycan array and ELISA plate. We detected cultured influenza virus using glycan array and ELISA. Then, we showed that the neoglycoprotein binds to Cy3-labeled hemagglutinins (H1 and H5), a NeuAc alpha 2,6LacNAc or NeuAc alpha 2,3LacNAc recognized protein, as detected.