Taichi Usui

Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to human-type receptors.

H5N1 influenza A viruses have spread to numerous countries in Asia, Europe and Africa, infecting not only large numbers of poultry, but also an increasing number of humans, often with lethal effects. Human and avian influenza A viruses differ in their recognition of host cell receptors: the former preferentially recognize receptors with saccharides terminating in sialic acid-α2,6-galactose (SAα2,6Gal), whereas the latter prefer those ending in SAα2,3Gal (refs 3–6). A conversion from SAα2,3Gal to SAα2,6Gal recognition is thought to be one of the changes that must occur before avian influenza viruses can replicate efficiently in humans and acquire the potential to cause a pandemic. By identifying mutations in the receptor-binding haemagglutinin (HA) molecule that would enable avian H5N1 viruses to recognize human-type host cell receptors, it may be possible to predict (and thus to increase preparedness for) the emergence of pandemic viruses. Here we show that some H5N1 viruses isolated from humans can bind to both human and avian receptors, in contrast to those isolated from chickens and ducks, which recognize the avian receptors exclusively. Mutations at positions 182 and 192 independently convert the HAs of H5N1 viruses known to recognize the avian receptor to ones that recognize the human receptor. Analysis of the crystal structure of the HA from an H5N1 virus used in our genetic experiments shows that the locations of these amino acids in the HA molecule are compatible with an effect on receptor binding. The amino acid changes that we identify might serve as molecular markers for assessing the pandemic potential of H5N1 field isolates.

Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus.

The ‘Spanish’ influenza pandemic of 1918–19 was the most devastating outbreak of infectious disease in recorded history. At least 20 million people died from their illness, which was characterized by an unusually severe and rapid clinical course. The complete sequencing of several genes of the 1918 influenza virus has made it possible to study the functions of the proteins encoded by these genes in viruses generated by reverse genetics, a technique that permits the generation of infectious viruses entirely from cloned complementary DNA. Thus, to identify properties of the 1918 pandemic influenza A strain that might be related to its extraordinary virulence, viruses were produced containing the viral haemagglutinin (HA) and neuraminidase (NA) genes of the 1918 strain. The HA of this strain supports the pathogenicity of a mouse-adapted virus in this animal. Here we demonstrate that the HA of the 1918 virus confers enhanced pathogenicity in mice to recent human viruses that are otherwise non-pathogenic in this host. Moreover, these highly virulent recombinant viruses expressing the 1918 viral HA could infect the entire lung and induce high levels of macrophage-derived chemokines and cytokines, which resulted in infiltration of inflammatory cells and severe haemorrhage, hallmarks of the illness produced during the original pandemic.

A convenient synthesis of β-d-galactosyl disaccharide derivatives using the β-d-galactosidase from Bacillus circulans

Abstract β- d -Gal-(1 → 4)-β- d -GlcNAc-OC 6 H 4 NO 2 - p ( p -nitrophenyl N -acetyl-β-lactosaminide) and β- d -Gal-(1 → 6)-β- d -GlcNAc-OC 6 H 4 NO 2 - p ( p -nitrophenyl N -acetyl-β-isolactosaminide) were regioselectively synthesized from lactose and p -nitrophenyl 2-acetamido-2-deoxy-glucopyranoside, employing transglycosylation by the β- d -galactosidase from Bacillus circulans and by controlling the concentration of organic solvent in the reaction system. The (1 → 4)-linked disaccharide was formed exclusively when the concentration of organic solvent was high, whereas the (1 → 6)-linked isomer was produced with a low concentration. Further utilization of the transglycosylation by the enzyme led to the regioselective formation of β- d -Gal-(1 → 4)- d -GalNAc and β- d -Gal-(1 → 4)-β- d -GalNAc-OC 6 H 4 NO 2 - p . With the enzyme, β- d -galactosyl transfer occurred preferentially at the O-4 position of GlcNAc and GalNAc, regardless of the configuration of the hydroxyl group.

Enzymic synthesis of useful chito-oligosaccharides utilizing transglycosylation by chitinolytic enzymes in a buffer containing ammonium sulfate

A chitinase purified from culture filtrates of Trichoderma resei KDR-11 efficiently catalyzed a transglycosylation reaction on tetra-N-acetylchitotetraoside in a buffer medium containing ammonium sulfate, converting the tetrasaccharide into hexa-N-acetylchitohexaose (39.6%) and di-N-acetylchitobiose (55.7%) as the major products. Sugar-chain elongation from di-N-acetylchitobiose as the initial substrate to hexa-N-acetyl-chitohexaose and hepta-N-acetylchitoheptaose was also efficiently induced through lysozyme catalysis in the presence of ammonium sulfate at high (30%) concentration. In this case, the addition of ammonium sulfate to the reaction system resulted in a remarkable increase of the hexamer and heptamer productions, which are desirable as biologically active oligosaccharides.

Enzymatic Syntheses of N-Acetyllactosamine and N-Acetylallolactosamine by the Use of β-D-Galactosidases

Abstract A β-D-galactosidase from Bacillus circulans induced β-D-galactopyranosyl transfer from lactose predominantly to a secondary (OH-4) rather than the primary hydroxyl group (OH-6) of 2-acetamido-2-deoxy-D-glucopyranose. 4-O-β-D-Galacto-pyranosyl-2-acetamido-2-deoxy-D-glucopyranose (N-acetyl-lactosamine) was thus readily synthesized on a gram scale and conveniently isolated by chromatography on a column of charcoal-Celite. On the other hand, the glycosyl transfer to the 6-position predominantly was efficiently induced to give 6-O-β-D-galactopyranosyl-2-acetamido-2-deoxy-D-glucopyranose (N-acetyl-allolactosamine) by consecutive use of β-D-galactosidases from Kluyveromyces lactis and B. circulans. These enzyme reactions were efficient enough to allow the one-pot preparation of the desired disaccharides.

Transglycosylation reaction of a chitinase purified from Nocardia orientalis

Chitinase from the culture filtrates of Nocardia orientalis IFO 12806 was purified to apparent homogeneity by precipitation with ammonium sulfate followed by successive chromatography on CM-Sephadex and Bio-Gel P-60, and finally by affinity chromatography on a phenyl-Sepharose CL-4B column. The enzyme, which is essentially a hydrolase, also catalyzed a transglycosylation reaction on tetra-N-acetyl-chitotetraose (GlcNAc)4 and penta-N-acetyl-chitopentaose (GlcNAc)5. The enzyme converted the tetrasaccharide into hexa-N-acetyl-chitohexaose (GlcNAc)6 (21%) and di-N-acetyl-chitobiose (GlcNAc)2 (63%) as the major products. The corresponding values for penta-N-acetyl-chitopentaose (GlcNAc)5 were hepta-N-acetyl-chitoheptaose (GlcNAc)7 23% and tri-N-acetyl-chitotriose (GlcNAc)3 59%. The rate of the transglycosylation depended on the temperature, the concentration of substrate and the pH.

A lectin from mycelia of the fungus Ganoderma lucidum

A lectin (GLL-M) was isolated from mycelia of Ganoderma lucidum using affinity chromatography on BSM-Toyopearl. GLL-M is a monomer in its native form with a M(r) of 18,000. Another lectin was also purified from fruiting bodies of the same fungus. The two lectins were partially compared with each other.

Crystal Structure of the Galectin-9 N-terminal Carbohydrate Recognition Domain from Mus musculus Reveals the Basic Mechanism of Carbohydrate Recognition

The galectins are a family of β-galactoside-binding animal lectins with a conserved carbohydrate recognition domain (CRD). They have a high affinity for small β-galactosides, but binding specificity for complex glycoconjugates varies considerably within the family. The ligand recognition is essential for their proper function, and the structures of several galectins have suggested their mechanism of carbohydrate binding. Galectin-9 has two tandem CRDs with a short linker, and we report the crystal structures of mouse galectin-9 N-terminal CRD (NCRD) in the absence and the presence of four ligand complexes. All structures form the same dimer, which is quite different from the canonical 2-fold symmetric dimer seen for galectin-1 and -2. The β-galactoside recognition mechanism in the galectin-9 NCRD is highly conserved among other galectins. In the apo form structure, water molecules mimic the ligand hydrogen-bond network. The galectin-9 NCRD can bind both N-acetyllactosamine (Galβ1–4GlcNAc) and T-antigen (Galβ1–3GalNAc) with the proper location of Arg-64. Moreover, the structure of the N-acetyllactosamine dimer (Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAc) complex shows a unique binding mode of galectin-9. Finally, surface plasmon resonance assay showed that the galectin-9 NCRD forms a homophilic dimer not only in the crystal but also in solution.

ANALYSIS OF SPECIFIC INTERACTIONS OF SYNTHETIC GLYCOPOLYPEPTIDES CARRYING N-ACETYLLACTOSAMINE AND RELATED COMPOUNDS WITH LECTINS

Analysis of interactions of synthetic glycopolypeptides with lectins was performed with a biosensor based on surface plasmon resonance (SPR). A series of synthetic oligosaccharide-substituted poly(L-glutamic acid)s were immobilized on sensor surfaces via the gamma-carboxyl groups of their peptide moieties by the surface thiol coupling method. Artificial glycopolypeptides: an N-acetyllactosamine-substituted polymer (1), an N-acetylisolactosamine-substituted polymer (2), a (GlcNAc)3-substituted polymer (3), a (GlcNAc)2-substituted polymer (4), and a p-aminophenyl N-acetyl-beta-lactosaminide-substituted polymer (5), were used as the ligands. On analysis by SPR, surface-bound polymers 1 and 5 reacted with Erythrina cristagalli agglutinin (ECA), Lycopersicon esculentum agglutinin (LEA), Ricinus communis agglutinin-120 (RCA120), and wheat germ (Triticum vulgaris) agglutinin (WGA). Polymer 2 reacted with WGA and RCA120, but did not with ECA and LEA. The results indicate that beta-(1-->4)-linked galactosyl residues are needed for binding to ECA and LEA. Polymer 3 reacted strongly with LEA and WGA, but polymer 4 reacted strongly only with WGA. Affinity constants (KA) for surface-bound polymer 5-lectin interactions were also about 4-61 times as strong as those for surface-bound polymer 1-lectin interactions. These artificial glycopolypeptides were shown to be useful as tools and probes of carbohydrate recognition and modeling in the analysis of glycoprotein-lectin interactions.

Immunomodulation of monocyte-derived dendritic cells through ligation of tumor-produced mucins to Siglec-9

Dendritic cells (DCs) play an essential role in the induction and maintenance of an effective immune response and express multiple siglecs. In the present study, we investigated whether or not the ligation of tumor-produced mucins with Siglec-9 expressed on immature DCs is related to escape from immunosurveillance in the tumor-bearing state. Expression of Siglec-9 was up-regulated on the development of monocytes into immature DCs and was decreased in mature DCs. Binding of various mucins and artificial glycopolymers carrying poly (NeuAc α2,6 LacNAc) or poly (NeuAc α2,3 LacNAc) to Siglec-9 was demonstrated by means of a plate assay. These mucins also bound to the surface of immature DCs. When immature DCs were treated with LPS in the presence of these mucins or artificial glycopolymers, the production of IL-12 was significantly reduced, but that of IL-10 was not. Furthermore, IL-12 production was decreased to a similar level on treatment with anti-Siglec-9 mAb. Mucins prepared from serum of cancer patients actually could bind to Siglec-9. These results suggest that Siglec-9 expressed on DCs is involved in immunoregulation through ligation with mucins in an epithelial cancer patient.