Optimization of the conditions for the immobilization of glycopolypeptides on hydrophobic silica particulates and simple purification of lectin using glycopolypeptide-immobilized particulates.

Glycopolypeptide-immobilized particulates exhibit high binding selectivities and affinities for several analytes. However, to date, the conditions for the synthesis of glycopolypeptide-immobilized particulates have not been optimized and the application of these particulates as carriers for affinity chromatography has not been reported. Accordingly, herein, as a model compound for determining the optimal conditions for the immobilization of an artificial glycopolymer on hexyl-containing hybrid silica particulates (HSPs), the glycopolypeptide poly [GlcNAcß1,4GlcNAc-ß-NHCO-(CH2)5NH-/CH3(CH2)9NH-/γ-PGA] (3) containing multivalent chitobiose moieties and multivalent decyl groups with a γ-polyglutamic acid backbone was synthesized. Immobilization of 3 on HSPs under each condition was evaluated by a lectin-binding assay using wheat germ (Triticum vulgaris) agglutinin (WGA), which is an N-acetylglucosamine-binding lectin. As a result, the optimal immobilization conditions for HSPs at 25 mg/mL were obtained at dimethyl sulfoxide (DMSO) concentration of reaction solvent in the range of 1(DMSO):9(water) to 4(DMSO):6(water) and a compound 3 concentration in the range of 125 nM-1250 nM. Furthermore, the influence of the alkyl group structure introduced into glycopolypeptide for imparting hydrophobicity to it on the immobilization of glycopolypeptide on HSPs was investigated. As a result of comparing three types, poly [GlcNAcß1,4GlcNAc-ß-NHCO-(CH2)5NH-/γ-PGA] (1) with no alkyl group, poly [GlcNAcß1,4GlcNAc-ß-NHCO-(CH2)5NH-/CH3(CH2)4NH-/γ-PGA] (2) with a pentyl group, and 3 with a decyl group, 3 showed the best immobilization efficiency on HSPs. Finally, 1 mg 3-immobilized HSPs prepared under the optimum conditions adsorbed approximately 7.5 µg WGA in a structure-specific manner. We also achieved a simple WGA purification from raw wheat germ extract as a practical example using 3-immobilized HSPs. We believe that in the future, these glycopolypeptide-immobilized particulates will be used not only for the purification of plant lectins, but also as specific adsorbents for various lectins-like substances such as in vivo lectins, pathogenic viruses, and toxin proteins.

Anti-viral activity of culinary and medicinal mushroom extracts against dengue virus serotype 2: an in-vitro study.

BACKGROUND: Dengue is a mosquito-borne viral infection that has become a major public health concern worldwide. Presently, there is no specific vaccine or treatment available for dengue viral infection. METHODS: Lignosus rhinocerotis, Pleurotus giganteus, Hericium erinaceus, Schizophyllum commune and Ganoderma lucidium were selected for evaluation of their in-vitro anti-dengue virus serotype 2 (DENV-2) activities. Hot aqueous extracts (HAEs), ethanol extracts (EEs), hexane soluble extracts (HSEs), ethyl acetate soluble extracts (ESEs) and aqueous soluble extracts (ASEs) were prepared from the selected mushrooms. The cytotoxic effects of the extracts were evaluated by the MTT assay. The anti-DENV-2 activities of the extracts were evaluated in three different assays: simultaneous, attachment and penetration assays were perfomed using plaque reduction assays and RT-qPCR assays. The effect of the addition time on viral replication was assessed by the time of addition assay, and a virucidal assay was carried out to evaluate the direct effect of each mushroom extract on DENV-2. The chemical composition of glucans, and the protein and phenolic acid contents in the extracts were estimated. RESULTS: We found that the HAEs and ASEs of L. rhinocerotis, P. giganteus, H. erinaceus and S. commune were the least toxic to Vero cells and showed very prominent anti-DENV2 activity. The 50% inhibitory concentration (IC50) values of the ASEs ranged between 399.2-637.9 µg/ml, while for the HAEs the range was 312.9-680.6 µg/ml during simultaneous treatment. Significant anti-dengue activity was also detected in the penetration assay of ASEs (IC50: 226.3-315.4 µg/ml) and HAEs (IC50: 943.1-2080.2 µg/ml). Similarly, we observed a marked reduction in the expression levels of the ENV and NS5 genes in the simultaneous and penetration assays of the ASEs and HAEs. Time-of-addition experiments showed that the highest percent of anti-DENV2 activity was observed when the mushroom extracts were added immediately after virus adsorption. None of the extracts exhibited virucidal effect. Chemical composition analysis showed that the major components in the mushroom HAEs and ASEs were glucan (beta D-glucan) and proteins, however, there was no significant correlation between the anti-dengue activity and the concentration of glucans and proteins. CONCLUSION: These findings demonstrated the potential of mushroom extracts as anti-dengue therapeutic agents with less toxic effects.

Application of Novel Sialoglyco Particulates Enhances the Detection Sensitivity of the Equine Influenza Virus by Real-Time Reverse Transcriptase Polymerase Chain Reaction.

Sialoglyco particulates carrying an N-glycolylneuraminyl-α-(2 → 3)-N-acetyllactosamine (Neu5Gcα2,3LacNAc) residue that displays a high level of affinity for the equine influenza virus (EIV) were generated using sialoglycopolypeptide and hexyl-containing hybrid silica particulates. The particulates were spherical with a diameter of approximately 950 nm and found to have good dispersibility in aqueous solution. Interaction between the sialoglyco particulates and the EIV was investigated by real-time reverse transcriptase polymerase chain reaction (rRT-PCR) of the EIV genome captured on the particulates. The number of EIV-specific genes detected by rRT-PCR on a nasal swab obtained from infected horses clearly increased when the sample was treated with sialoglyco particulates. Our results show these novel sialoglyco particulates can be used as a highly sensitive tool for detecting low levels of EIV that were previously undetectable in the early or late stage of infection.

Chemical synthesis of 4-azido-ß-galactosamine derivatives for inhibitors of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase.

Chondroitin sulfate E (CS-E) plays a crucial role in diverse processes ranging from viral infection to neuroregeneration. Its regiospecific sulfation pattern, generated by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST), is the main structural determinant of its biological activity. Inhibitors of GalNAc4S-6ST can serve as powerful tools for understanding physiological functions of CS-E and its potential therapeutic leads for human diseases. A family of new 4-acylamino-ß-GalNAc derivatives and 4-azido-ß-GalNAc derivatives were synthesized for their potential application as inhibitors of GalNAc4S-6ST. The target compounds were evaluated for their inhibitory activities against GalNAc4S-6ST. The results revealed that 4-pivaloylamino- and 4-azido-ß-GalNAc derivatives displayed evident activities against GalNAc4S-6ST with IC50 value ranging from 0.800 to 0.828 mM. They showed higher activities than benzyl D-GalNAc4S that was used as control.

Chemoenzymatic synthesis and characterization of N-glycolylneuraminic acid-carrying sialoglycopolypeptides as effective inhibitors against equine influenza virus hemagglutination.

A series of novel sialoglycopolypeptides carrying N-glycolylneuraminic acid (Neu5Gc)-containing trisaccharides having α(2 â†’ 3)- and α(2 â†’ 6)-linkages in the side chains of γ-polyglutamic acid (γ-PGA) were designed as competitive inhibitors against equine influenza viruses (EIV), which critically recognize the Neu5Gc residue for receptor binding. Using horse red blood cells (HRBC) we successfully evaluated the binding activity of the multivalent Neu5Gc ligands to both equine and canine influenza viruses in the hemagglutination inhibition (HI) assay. Our findings show the multivalent α2,3-linked Neu5Gc-ligands (3a-c and 7) selectively inhibit hemagglutination mediated by both influenza viruses and display a strong inhibitory activity. Our results indicate that the multivalent Neu5Gc-ligands can be used as novel probes to elucidate the mechanism of infection/adhesion of Neu5Gc-binding influenza viruses.

Isolation and characterization of monoclonal antibodies specific for chondroitin sulfate E.

Chondroitin sulfate E (CSE) is a polysaccharide containing mainly disaccharide units of D-glucuronic acid (GlcA) and 4,6-O-disulfated N-acetyl-D-galactosamine (GalNAc) residues (E-unit) in the amount of ∼ 60%. CSE is involved in many biological and pathological processes. In this study, we established new monoclonal antibodies, termed E-12C and E-18H, by using CSE that contained more than 70% of E-units as an immunogen. These antibodies recognized CSE but not other CSs isomers or dermatan sulfate (DS). We evaluated the reactivities of the antibodies to 6-O-sulfated CSA (6S-CSA) and DS (6S-DS) that possessed ∼ 60% of GalNAc (4S, 6S) moieties in their structures. Neither of the antibodies reacted with 6S-DS. The antibodies strictly distinguished the structural difference of GlcA and L-iduronic acid in the polysaccharide. Binding affinities of the antibodies were determined by a surface plasmon resonance assay using CSE and 6S-CSA. The binding affinities were strongly associated with the molecular weight of CSE and the E-unit content of 6S-CSA. Moreover, we demonstrated that the antibodies are applicable to histochemical analysis. In conclusion, the new anti-CSE monoclonal antibodies specifically recognize the E-unit of CSE. The antibodies will become useful tools for the investigation of the biological and pathological significance of CSE.

Measurement of aberrant glycosylation of prostate specific antigen can improve specificity in early detection of prostate cancer.

INTRODUCTION: We previously identified prostate cancer (PCa)-associated aberrant glycosylation of PSA, where α2,3-linked sialylation is an additional terminal N-glycan on free PSA (S2,3PSA). We then developed a new assay system measuring S2,3PSA using a magnetic microbead-based immunoassay. We compared the diagnostic accuracy of conventional PSA and percent-free PSA (%fPSA) tests. METHODS: We used MagPlex beads to measure serum S2,3PSA levels using anti-human fPSA monoclonal antibody (8A6) for capture and anti-α2,3-linked sialic acid monoclonal antibody (HYB4) for detection. We determined the cutoff values in a training test and measured serum S2,3PSA levels in 314 patients who underwent biopsy, including 138 PCa and 176 non-PCa patients with PSA of <10.0 ng/ml. Serum S2,3PSA levels were presented as mean fluorescence intensity (MFI). Receiver operating characteristic curves were used to evaluate the diagnostic accuracy of total PSA, %fPSA, and S2,3PSA. RESULTS: We determined an MFI cutoff value of 1130 with a sensitivity of 95.0% and specificity of 72.0% for the diagnosis of PCa in the training test. In the validation study, the area under the curve for the detection of PCa with S2,3PSA was 0.84, which was significantly higher than that with PSA or %fPSA. CONCLUSIONS: Although the present study is small and preliminary, these results suggest that the measurement of serum S2,3PSA using a magnetic microbead-based immunoassay may improve the accuracy of early detection of PCa and reduce unnecessary prostate biopsy.

Computational design of a sulfoglucuronide derivative fitting into a hydrophobic pocket of dengue virus E protein.

We performed first-principles calculations based on the ab initio fragment molecular orbital method on dengue virus envelope protein with a hydrophobic ligand, octyl-ß-D-glucose to develop an entry inhibitor. As several polar amino acid residues are present at the edge of the pocket, the glucose moiety was chemically modified with hydrophilic groups. Introduction of both sulfated and carboxylated groups on glucose enhanced not only binding affinity to the protein but also inhibition of dengue virus entry. Octyl-2-O-sulfo ß-D-glucuronic acid may serve as a molecular probe to study the dengue virus entry process.

Improvement of depression-like behavior and memory impairment with the ethanol extract of Pleurotus eryngii in ovariectomized rats.

Ethanol extract of Pleurotus eryngii (DC.) QUÉL has estrogen-like activities that protect against bone loss caused by estrogen deficiency. In the present study, we investigated the effect of P. eryngii on depression-like behavior and memory impairment in ovariectomized (OVX) rats. Immobility time during a forced swimming test was significantly longer for OVX rats than for sham-operated rats. The depression-like behavior in OVX rats was improved by long-term administration of the ethanol extract of P. eryngii (500 mg/kg body weight (b.w.)/d). Spatial memory impairment in OVX rats assessed by the Morris water maze test was also improved by P. eryngii extract without any effect on motility. These results suggested that P. eryngii extract has estrogen-like improvement activity against depression-like behavior and memory impairment in OVX rats. Additionally, increase in the amount of synaptosomal zinc after ovariectomy was inhibited by P. eryngii extract. Since zinc in synaptic vesicles is important for memory function and is linked to the pathophysiology of depression, normalization of zinc signaling would be involved in the beneficial effect of P. eryngii extract on neurological disorders after ovariectomy.

Influenza virus utilizes N-linked sialoglycans as receptors in A549 cells.

Influenza viruses (IFVs) recognize sialoglycans expressed on the host cell surface. To understand the mechanisms underlying tissue and host tropisms of IFV, it is essential to elucidate the molecular interaction of the virus with the host sialoglycan receptor. We established and applied a new monoclonal antibody, clone HYB4, which specifically recognizes the Neu5Acα2-3 determinant at the non-reducing terminal Gal residue of both glycoproteins and gangliosides to investigate the biochemical properties of IFV receptors in A549 cells. HYB4 significantly blocked virus binding to A549 cells in a dose-dependent manner. Virus overlay assay indicated that several glycoproteins with molecular masses of 80-120 kDa of A549 cells were commonly recognized by different subtypes of IFV, such as H1N1 and H3N2. H1N1 virus binding to the glycoproteins was diminished by pretreatment with either sialidase or PNGase F. On TLC-immunostaining experiments with HYB4, GM3 ganglioside was only detected in A549 cells. Interestingly, this antibody bound to GM3 gangliosides on TLC and plastic surfaces, but not on lipid bilayers. In comparison with the recognition of Maackia amurensis lectins, HYB4 exclusively recognized Neu5Acα2-3Galß1-4GlcNAc residues expressed on glycoproteins. These results strongly suggest that N-linked sialoglycans with the Neu5Acα2-3 determinant on several glycoproteins are receptors for influenza virus in A549 cells.

Carbohydrate-related inhibitors of dengue virus entry.

Dengue virus (DENV), which is transmitted by Aedes mosquitoes, causes fever and hemorrhagic disorders in humans. The virus entry process mediated through host receptor molecule(s) is crucial for virus propagation and the pathological progression of dengue disease. Therefore, elucidation of the molecular mechanisms underlying virus entry is essential for an understanding of dengue pathology and for the development of effective new anti-dengue agents. DENV binds to its receptor molecules mediated through a viral envelope (E) protein, followed by incorporation of the virus-receptor complex inside cells. The fusion between incorporated virus particles and host endosome membrane under acidic conditions is mediated through the function of DENV E protein. Carbohydrate molecules, such as sulfated glycosaminoglycans (GAG) and glycosphingolipids, and carbohydrate-recognition proteins, termed lectins, inhibit virus entry. This review focuses on carbohydrate-derived entry inhibitors, and also introduces functionally related compounds with similar inhibitory mechanisms against DENV entry.

Design and synthesis of a novel ganglioside ligand for influenza A viruses.

A novel ganglioside bearing Neua2-3Gal and Neua2-6Gal structures as distal sequences was designed as a ligand for influenza A viruses. The efficient synthesis of the designed ganglioside was accomplished by employing the cassette coupling approach as a key reaction, which was executed between the non-reducing end of the oligosaccharide and the cyclic glucosylceramide moiety. Examination of its binding activity to influenza A viruses revealed that the new ligand is recognized by Neua2-3 and 2-6 type viruses.

3-O-sulfated glucuronide derivative as a potential anti-dengue virus agent.

A series of 12 carbohydrate compounds were synthesized by introduction of a sulfated group at specific positions and evaluated for their activities against dengue virus (DENV) infection as well as binding to BHK-21 cells. 3-O-sulfated GlcA was active against DENV infection, whereas 2-O-sulfated GlcA and 3,6-di-O-sulfated Glc showed negligible activity. Persulfated compounds did not inhibit DENV infection. These results provided a rationale for designing sulfated carbohydrate compounds with low molecular mass as anti-DENV agents targeting E protein functions. 3-O-Sulfated GlcA showed no significant cytotoxicity at 1mM. The EC(50) value (120 µM) was lower than that of sucrose octasulfate (SOS), a small molecular weight inhibitor of DENV infection. Two negatively charged groups, 3-O-sulfate and 6-C-carboxylic acid, appear to be essential for anti-DENV activity. We performed docking study to investigate the binding potential of 3-O-sulfated GlcA with respect to DENV E protein. The docking study showed that distance and conformation of these negative charges on the carbohydrate may be suitable for association with three amino acid residues of E protein critically involved in virus adsorption (Lys295, Ser145, and Gly159). This interaction may competitively prevent functional DENV binding to receptor(s) on host cells. In conclusion, 3-O-sulfated GlcA is a chemical probe that may facilitate exploration of the molecular mechanisms underlying manifestations of dengue diseases.

No evidence of horizontal infection in horses kept in close contact with dogs experimentally infected with canine influenza A virus (H3N8).

BACKGROUND: Since equine influenza A virus (H3N8) was transmitted to dogs in the United States in 2004, the causative virus, which is called canine influenza A virus (CIV), has become widespread in dogs. To date, it has remained unclear whether or not CIV-infected dogs could transmit CIV to horses. To address this, we tested whether or not close contact between horses and dogs experimentally infected with CIV would result in its interspecies transmission. METHODS: Three pairs of animals consisting of a dog inoculated with CIV (10(8.3) egg infectious dose 50/dog) and a healthy horse were kept together in individual stalls for 15 consecutive days. During the study, all the dogs and horses were clinically observed. Virus titres in nasal swab extracts and serological responses were also evaluated. In addition, all the animals were subjected to a gross pathological examination after euthanasia. RESULTS: All three dogs inoculated with CIV exhibited clinical signs including, pyrexia, cough, nasal discharge, virus shedding and seroconversion. Gross pathology revealed lung consolidations in all the dogs, and Streptococcus equi subsp. zooepidemicus was isolated from the lesions. Meanwhile, none of the paired horses showed any clinical signs, virus shedding or seroconversion. Moreover, gross pathology revealed no lesions in the respiratory tracts including the lungs of the horses. CONCLUSIONS: These findings may indicate that a single dog infected with CIV is not sufficient to constitute a source of CIV infection in horses.

Antiviral agents targeting glycans on dengue virus E-glycoprotein.

The molecular recognition of dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) with arthropod-derived N-glycans on E-glycoprotein is essential for dengue virus (DENV) infection in humans. Therefore, the specific interaction of DC-SIGN with N-glycans on E-glycoprotein is a promising target for development of virus replication inhibitors. This article discusses the findings of a recent report describing antiviral activities of carbohydrate-binding agents, such as lectins and a small molecular weight compound against DENV infection of monocyte-derived dendritic cells. Several plant lectins and the small molecular weight compound Pladimicin-S showed potent inhibition of cellular infection by four DENV serotypes.

Unique heparan sulfate from shrimp heads exhibits a strong inhibitory effect on infections by dengue virus and Japanese encephalitis virus.

The structure and biological activities of a highly sulfated heparan sulfate (HS) extracted from shrimp (Penaeus brasiliensis) heads were characterized. Structurally the shrimp HS was more heterogenous than heparin, although it is still highly sulfated. The molecular mass of the shrimp HS preparation was determined to be 32.3 kDa by gel filtration HPLC. Analysis by surface plasmon resonance demonstrated that various growth/differentiation factors specifically bound to the shrimp HS with comparable affinity. Notably, the shrimp HS had a greater inhibitory effect against infections by dengue virus type 2 as well as Japanese encephalitis virus than heparin. Experiments on anticoagulant activity indicated that the shrimp HS exhibited significant anti-thrombin activity, but less than the commercial heparin. Hence, the HS preparation from shrimp heads, an industrial waste, is a prospective agent for a variety of clinical applications.

Dengue virus type 2 recognizes the carbohydrate moiety of neutral glycosphingolipids in mammalian and mosquito cells.

Dengue viruses infect cells by attaching to a surface receptor which remains unknown. The putative receptor molecules of dengue virus type 2 on the surface of mosquito (AP-61) and mammalian (LLC-MK2) cell lines were investigated. The immunochemical detection and structural analysis of carbohydrates demonstrated that the neutral glycosphingolipids, L-3 (GlcNAcß1-3Manß1-4Glcß1-1'Cer) in AP-61 cells, and nLc(4) Cer (Galß1-4GlcNAcß1-3Galß1-4Glcß1-1'Cer) in LLC-MK2 cells were recognized by the virus. These findings strongly suggest that neutral glycosphingolipids share the key determinant for virus binding and that the ß-GlcNAc residue may play an important role in dengue virus binding to the host cell surface.

Distribution of influenza virus sialoreceptors on upper and lower respiratory tract in horses and dogs.

It is strongly suspected that equine influenza virus (EIV) is the origin of canine influenza virus (CIV, H3N8), which was first isolated in U.S.A. in 2004, on the basis of phylogenetic analyses. Although the distribution of influenza virus sialoreceptors seems to be associated with this interspecies transmission, there have been scant data of comparison about distributions of sialoreceptors on the whole respiratory tract between horses and dogs. We examined the histological distribution of influenza virus sialoreceptors on the upper and lower respiratory tract in detail in both animals using double lectin staining with Maackia amurensis (specific for SAα2,3Gal) and Sambucus sieboldiana (specific for SAα2,6Gal). SAα2,3Gal was observed on the surface of ciliated epithelial cells in the nasal mucosa, trachea and bronchus in both animals. The results may indicate that dogs are susceptible to EIV without alteration of receptor binding specificity.

Dengue virus receptor.

Dengue virus is an arthropod-borne virus transmitted by Aedes mosquitoes. Dengue virus causes fever and hemorrhagic disorders in humans and non-human primates. Direct interaction of the virus introduced by a mosquito bite with host receptor molecule(s) is crucial for virus propagation and the pathological progression of dengue diseases. Therefore, elucidation of the molecular mechanisms underlying the interaction between dengue virus and its receptor(s) in both humans and mosquitoes is essential for an understanding of dengue pathology. In addition, understanding the molecular mechanism(s) of virus entry is crucial for the development of effective new therapies to treat dengue patients. Binding of dengue virus to its receptor molecules is mediated through a viral envelope glycoprotein, termed E protein. We present a summary and describe the structures, binding properties, and pathological relevance of dengue virus receptor molecules proposed to date. In mammalian cells, there are many candidate molecules that may act as receptors, such as sulfated glycosaminoglycans (GAGs), lectins that recognize carbohydrates, glycosphingolipid (GSL), proteins with chaperone activity, laminin-binding proteins, and other uncharacterized proteins. There are also several lines of evidence for receptor molecules such as GSLs, proteins with chaperone activity, laminin-binding proteins, and other uncharacterized proteins in mosquito cells and organs. This review focuses on several molecules involved in carbohydrate-dependent binding of the virus.