β-Xylosides have been used as an artificial initiator of glycosaminoglycan (GAG) biosynthesis to investigate its mechanism and to obtain these oligosaccharides. In GAG biosynthesis, phosphorylation on the xylose residue is a crucial step. However, little attention has been paid to phosphorylated oligosaccharides obtained from β-xylosides. In a previous study, we demonstrated that a novel β-xyloside, N-lauryl-O-β-xyloyranosyl-serinamide (Xyl-Ser-C12), had excellent GAG-type oligosaccharide priming ability, whereas phosphorylated oligosaccharides were not found in the primed oligosaccharides. This study examines the potential of Xyl-Ser-C12 and three of its derivatives for use as a probe to investigate the GAG biosynthesis mechanism. Glycosylated products were obtained by incubation of the β-xylosides in normal human dermal fibroblast cells and compared by liquid chromatography–electrospray ionization-mass spectrometry. By the optimized method to detect phosphorylated products, Xyl-Ser-C12 was demonstrated to prime not only GAG-type oligosaccharides but also a variety of xylose-phosphorylated products. Among the synthesized β-xylosides, those consisting of xylosyl-serine primed large amounts of phosphorylated and GAG-type oligosaccharides, whereas the others primed sialyloligosaccharides mainly. The majority of the phosphorylated products were considered to be GAG intermediates, which are less observed in nature. To our best knowledge, this is the first report showing that the amino acid residues around the Xyl attachment position strongly affect the phosphorylation efficiency and GAG chain-priming ability of β-xylosides. This study leads to the possibility of the use of β-xyloside as a probe to observe the Xyl phosphorylation process during GAG biosynthesis and investigate comparative glycosaminoglycomics between different cells.Topics: Carbohydrates; Cell and Molecular biology; Polysaccharides;

Glycosphingolipids are major components of the membrane raft, and several kinds of viruses and bacterial toxins are known to bind to glycosphingolipids in the membrane raft. Since the viral genes and pathogenic proteins that are taken into cells are directly delivered to their target organelles, caveolae/raft-mediated endocytosis represents a promising pathway for specific delivery. In the present study, we demonstrated the ability of an artificial pentadecapeptide, which binds to ganglioside GM3, to deliver protein into cells by caveolae/raft-mediated endocytosis. The cellular uptake of a biotinylated GM3-binding peptide (GM3BP)–avidin complex into HeLa cells was observed, and the cellular uptake of this complex was inhibited by an incubation with sialic acid or endocytic inhibitors such as methyl-ß-cyclodextrin, and also by an incubation at 4 °C. These results indicate that the GM3BP-avidin complex bind to GM3 in membrane raft, and are taken into cell through caveolae/raft-mediated endocytosis. The GM3BP-avidin complex was transported into cells and localized around the nucleus more slowly than a human immunodeficiency virus type 1 TAT peptide. Furthermore, the uptake of a green fluorescent protein (GFP) linked with GM3BP into HeLa cells was similar to that of the GM3BP–avidin complex, and the localization of the GM3BP-GFP fusion protein was markedly different with that of the TAT-GFP fusion protein. The uptake and trafficking of GM3BP were distinguished from conventional cell-penetrating peptides. GM3BP has potential as a novel peptide for the selective delivery of therapeutic proteins and materials into cells in addition to being a cell-penetrating peptide.

The initial attachment of influenza virus to cells is the binding of hemagglutinin (HA) to the sialyloligosaccharide receptor; therefore, the small molecules that inhibit the sugar–protein interaction are promising as HA inhibitors to prevent the infection. We herein demonstrate that sialic acid-mimic heptapeptides are identified through a selection from a primary library against influenza virus HA. In order to obtain lead peptides, an affinity selection from a phage-displayed random heptapeptide library was performed with the HAs of the H1 and H3 strains, and two kinds of the HA-binding peptides were identified. The binding of the peptides to HAs was inhibited in the presence of sialic acid, and plaque assays indicated that the corresponding N-stearoyl peptide strongly inhibited infections by the A/Aichi/2/68 (H3N2) strain of the virus. Alanine scanning of the peptides indicated that arginine and proline were responsible for binding. The affinities of several mutant peptides with single-amino-acid substitutions against H3 HA were determined, and corresponding docking studies were performed. A Spearman analysis revealed a correlation between the affinity of the peptides and the docking study. These results provide a practicable method to design of peptide-based HA inhibitors that are promising as anti-influenza drugs.

The progression of influenza varies according to age and the presence of an underlying disease; appropriate treatment is therefore required to prevent severe disease. Anti-influenza therapy, such as with neuraminidase inhibitors, is effective, but diagnosis at an early phase of infection before viral propagation is critical. Here, we show that several dozen plaque-forming units (pfu) of influenza virus (IFV) can be detected using a boron-doped diamond (BDD) electrode terminated with a sialic acid-mimic peptide. The peptide was used instead of the sialyloligosaccharide receptor, which is the common receptor of influenza A and B viruses required during the early phase of infection, to capture IFV particles. The peptide, which was previously identified by phage-display technology, was immobilized by click chemistry on the BDD electrode, which has excellent electrochemical characteristics such as low background current and weak adsorption of biomolecules. Electrochemical impedance spectroscopy revealed that H1N1 and H3N2 IFVs were detectable in the range of 20–500 pfu by using the peptide-terminated BDD electrode. Our results demonstrate that the BDD device integrated with the receptor-mimic peptide has high sensitivity for detection of a low number of virus particles in the early phase of infection.

A series of carbosilane dendrimers uniformly functionalized with hemagglutinin (HA) binding peptide (sialic acid-mimic peptide, Ala-Arg-Leu-Pro-Arg) was systematically synthesized, and their anti-influenza virus activity was evaluated. The carbosilane-based peptide dendrimers, unlike sialylated dendrimers, cannot be digested by virus neuraminidases. The peptide dendrimers exhibited intriguing biological activities depending on the form of their core frame, with a dumbbell-type peptide dendrimer showing particularly strong inhibitory activities against two human influenza viruses, A/PR/8/34 (H1N1) and A/Aichi/2/68 (H3N2). The IC50 values of the dumbbell-type peptide dendrimer for both strains were 0.60 μM, the highest activity among the HA-binding peptide derivatives. The results suggest that a dumbbell-shaped carbosilane dendrimer is the most suitable core scaffold for HA-binding peptide dendrimers.

O-GalNAcα-modified proteins are the precursor of mucin-type O-glycosylated proteins. Homogeneously O-glycosylated proteins are required to investigate the biological functions of glycoproteins and to develop biopharmaceuticals. Here we show that the incorporation of GalNAcα-Thr into proteins successfully proceeded by the use of a chemically aminoacylated tRNA. GalNAcα-Thr was chemoenzymatically attached to amber suppressor tRNA and the product was subjected to in vitro translation together with streptavidin mRNA containing the UAG codon. Gel electrophoresis and mass analysis showed that GalNAcα-Thr was successfully incorporated into the N-terminus, although it was not incorporated at the interior. This method will facilitate the preparation of homogeneous GalNAcα-proteins.

To know the involvement of glycosaminoglycans (GAGs) in the metastasis of mouse FBJ osteosarcoma cells, Nα-lauroyl-O-(β-d-xylopyranosyl)-l-serinamide (Xyl-Ser-C12), which initiates elongation of GAG chains using the glycan biosynthesis system in cells, was administered to FBJ cells with different metastatic capacities. Production of glycosylated products derived from Xyl-Ser-C12, especially heparan sulfate (HS) GAG-type oligosaccharides such as GalNAc-GlcA-GlcNAc-GlcA-Gal-Gal-Xyl-Ser-C12, was indicated in poorly metastatic FBJ-S1 cells more than in highly metastatic FBJ-LL cells by LC–MS. The results of RT-PCR revealed that HS synthases, Ext1 and Ext2, were expressed in FBJ-S1 cells more than in FBJ-LL cells. Furthermore, siRNA against Ext1 suppressed the expression of HS and enhanced the motility of FBJ-S1 cells. In addition, the expression of heparanase (HPSE) was enhanced in Ext-1-knockdown FBJ-S1 cells, and responsible for the increase in cell motility caused by the down-regulation of Ext1 expression. Our data provide the first evidence that Ext1 regulates the expression of HPSE and also indicated that levels of Ext1 and HPSE influenced the motility of FBJ cells.

The deposition of amyloid β-protein (Aβ) is a pathological hallmark of Alzheimer’s disease (AD). We previously found that the ganglioside-enriched microdomains (ganglioside clusters) in presynaptic neuronal membranes play a key role in the initiation of the Aβ assembly process. However, not all ganglioside clusters accelerate Aβ assembly. In the present study, we directly observed a spherical Aβ in an atomic force microscopic study on the morphology of a reconstituted lipid bilayer composed of lipids that were extracted from a detergent-resistant membrane microdomain (DRM) fraction of synaptosomes prepared from aged mouse brain. The Aβ assembly was generated on a distinctive GM1 domain, which was characterized as the Aβ-sensitive ganglioside nanocluster (ASIGN). By using an artificial GM1 cluster-binding peptide, ASIGN was found to have a high density of GM1; therefore, there would be a critical density of GM1 in nanoclusters to induce Aβ binding and assembly. These results suggest that ganglioside-bound Aβ (GAβ), which acts as an endogenous seed for Aβ fibril formation in AD brains, is generated on ASIGN on synaptosomal membranes.

Sialyloligosaccharides of glycoproteins and glycosphingolipids play important roles in biological events on cell membranes. GT1b is a ganglioside having a trisialyloligosaccharide and is a receptor for tetanus toxin. In the present study, pentadecapeptide ligands for GT1b were obtained by phage display selection from a random peptide library with the use of a GT1b monolayer. The artificial pentadecapeptides had high affinity for GT1b which tended to increase depending on the number of sialic acids in sialyloligosaccharides. Arg, Ser, and hydrophobic amino acids were found in a consensus motif and may contribute to carbohydrate recognition. The consensus motif of the GT1b-binding peptides was different from that of GM1-, GM2-, GM3-, or GD1a-binding peptides. Peptide ligands for GT1b should be investigated for trisialyloligosaccharide functions and the development of therapeutic agents against trisialyloligosaccharide-related diseases.

Nα-Lauryl-O-(β-d-xylopyranosyl)-l-serinamide (Xyl-Ser-C12) was synthesized as a saccharide primer to obtain oligosaccharides of glycosaminoglycan using the glycan biosynthetic potential of mouse osteosarcoma FBJ-S1 cells and Chinese hamster ovary (CHO) cells. The glycosylated products secreted into the culture medium were collected and analyzed by liquid chromatography–mass spectrometry and glycosidase digestion. The structure of the Xyl-Ser-C12 derivatives was investigated. Several glycosaminoglycan-type oligosaccharides, such as GalNAc-(GlcA-GlcNAc)n-GlcA-Gal-Gal-Xyl-Ser-C12, were detected, and identified as intermediates of the biosynthesis of heparan sulfate glycosaminoglycans. Xyl-Ser-C12 exhibited greater acceptor activity for the glycosylation of glycosaminoglycan-type oligosaccharides than p-nitrophenyl-β-d-xylopyranoside.Graphical abstractThe structure of Nα-lauryl-O-(β-d-xylopyranosyl)-l-serinamide (Xyl-Ser-C12).Highlights► Xyl-Ser-C12 saccharide primer was synthesized to obtain glycosaminoglycan oligosaccharides. ► Glycosylated products of Xyl-Ser-C12 from cells were analyzed by LC–MS/MS and glycosidase digestion. ► Xyl-Ser-C12 exhibited good acceptor activity for the glycosylation of glycosaminoglycan-type oligosaccharides.

Influenza is an infectious disease caused by the influenza virus, and each year many people suffer from this disease. Hemagglutinin (HA) in the membrane of type A influenza viruses recognizes sialylglycoconjugate receptors on the host cell surface at an initial step in the infection process; consequently, HA inhibitors are considered potential candidates for antiviral drugs. We identified peptides that bind to receptor-binding sites through a multiple serial selection from phage-displayed random peptide libraries. Using the HA of the H1 and H3 strains as target proteins, we obtained peptides that bind to both HAs. The binding affinities of peptides for these HAs were improved by secondary and tertiary selections from the corresponding sublibraries. A docking simulation suggested that, similar to sialic acid, the peptides are recognized by the receptor-binding site in HA, which indicates that these peptides mimic the sialic acid structure. N-stearoyl peptides inhibited infections by the A/Puerto Rico/8/34 (H1N1) and A/Aichi/2/68 (H3N2) strains of influenza virus. Such HA-inhibitors are promising candidates for novel antiviral drugs.

Glycosphingolipids (GSLs) in membrane microdomains participate in important biological functions. In the present paper, we propose a novel model of the distribution of GSLs in membrane microdomains composed of sphingomyelin (SM) and cholesterol. We investigated the distribution of the ganglioside GM3 in a lipid membrane reconstituted with lipid extract from mouse B16 melanoma cells using an atomic force microscope (AFM). The surface topography of the reconstituted lipid bilayer showed three areas of different heights. The highest area was confirmed to be a GM3 domain by labeling with wheat germ agglutinin. To identify the lipids which are contributed to make the topography, the topographies of the artificial lipid bilayers composed of GM3, SM, 1-palmitoyl-2-oleoyl-phosphatidylcholine, and cholesterol were investigated. AFM images of the artificial lipid bilayers showed that the GM3 domain surrounded by a SM-containing phase only occurred, and its formation was found to depend on the cholesterol content.The segregation of a GM3 domain in another phase was indicated by the AFM-based observation of reconstituted membrane of lipid extracts from B16 membrane cells and artificial membrane.

Three ether-linked alkyl 5a-carba-glycopyranosides 1b,d, and 5b, and 5a′-carba-lactoside 7b were examined as potent primers in mouse B16 melanoma cells for their feasibility as building blocks for oligosaccharide biosynthesis. Uptake by B16 cells was first observed for all carba-glycoside primers, and, especially, the 5a-carba-sugar analogues of N-acetyl-β-d-glucosaminide 1b and β-d-glucoside 1d were shown to produce two-to-four-fold larger amounts of glycosylated products than the corresponding true sugar primers 1a and 1c. The carba glycoside uptake by cells resulted in β-galactosylation and subsequent sialylation of the incorporated galactose residues, giving rise to glycosylated products 3b and 3d having similar glycan structures as the ganglioside GM3. According to efficient uptake in cells, in addition to stability of the ether-linked pseudo-reducing ends of the oligosaccharides that formed, the carba glycoside primers have been demonstrated to be versatile building blocks for these biocombinatorial syntheses of glycolipid oligosaccharide mimetics. On the other hand, uptake for 5a-carba-galactopyranoside residue was found to be decreased by one-third for dodecyl 5a-carba-β-d-galactopyranoside 5b. Observation of similar levels for 5a′-carba-β-lactoside 7b under both cellular and cell-free conditions suggested that enzymes are likely to recognize the pyranose oxygen atom.

We obtained a novel carbohydrate-binding peptide having a helix−loop−helix scaffold from a random peptide library. The helix−loop−helix peptide library randomized at five amino acid residues was displayed on the major coat protein of a filamentous phage. Affinity selection with a ganglioside, Galβ1−3GalNAcβ1−4(Neu5Acα2−3)Galβ1−4Glcβ1−1′Cer (GM1), gave positive phage clones. Surface plasmon resonance spectroscopy showed that a corresponding 35-mer synthetic peptide had high affinity for GM1 with a dissociation constant of 0.24 µM. This peptide preferentially binds to GM1 rather than asialo GM1 and GM2, suggesting that a terminal galactose and sialic acid are required for the binding as for cholera toxin. Circular dichroism spectroscopic studies indicated that a helical structure is important for the affinity and specificity. Furthermore, alanine scanning at randomized positions showed that arginine and phenylalanine play an especially important role in the recognition of carbohydrates. Such a de novo helix−loop−helix peptide would be available for the design of carbohydrate-binding proteins.

Syntheses of oligosaccharides expressed on cells are indispensable for the improvement of the functional analyses of the oligosaccharides and their applications. We are developing saccharide primers for synthesizing oligosaccharides using living cells. In this study, dodecyl 2-acetamido-2-deoxy-β-d-glucopyranoside (GlcNAc-C12) and dodecyl β-d-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-d-glucopyranoside (LacNAc-C12) were examined for their abilities to prime the syntheses of neolacto-series oligosaccharides in HL60 cells. When GlcNAc-C12 was incubated with HL60 cells in serum-free medium for 2 days, 14 kinds of glycosylated products were collected from the culture medium. They were separated by high-performance liquid chromatography. The sequences of the products were determined to be neolacto-series oligosaccharides including LewisX, sialyl LewisX, polylactosamine, and sialylpolylactosamine by mass spectrometry. GlcNAc-C12 was also glycosylated by B16 cells and gave sialyllactosamine. Furthermore, LacNAc-C12 gave similar glycosylated products to GlcNAc-C12.

Influenza virus hemagglutinin recognizes sialyloligosaccharides of glycoproteins and glycolipids as cell surface receptors in the initial stage of the infection process. We demonstrate that pentadecapeptides that bind to a sialylgalactose structure (Neu5Ac−Gal) inhibited the infection of cells by influenza virus. The pentadecapeptides were identified through affinity selection from a phage-displayed random peptide library using a monolayer of the ganglioside Neu5Acα2−3Galβ1−4Glcβ1−1′Cer (GM3). The peptides were found to have affinity for GM3, and alanine scanning showed seven amino acid residues that contribute to carbohydrate recognition. The binding of peptides to the cell surface was significantly inhibited in the presence of sialic acid or by the digestion of cell surface sialyl residues by neuraminidase. Plaque assays indicated that a molecular assembly of alkylated peptides inhibited the infection of Madin−Darby canine kidney cells by influenza virus. Carbohydrate-binding peptides that inhibit carbohydrate−virus interaction showed inhibitory activity. These results may lead to a new approach to the design of antiviral drugs.