Edited by Maurice MontalKeywords: Clostridium botulinum Hemagglutinin Sialylated oligosaccharide X-ray structure a b s t r a c t Clostridium botulinum produces the botulinum neurotoxin, forming a large complex as progenitor toxins in association with non-toxic non-hemagglutinin and/or several different hemagglutinin (HA) subcomponents, HA33, HA17 and HA70, which bind to carbohydrate of glycoproteins from epithelial cells in the infection process. To elucidate the carbohydrate recognition mechanism of HA70, X-ray structures of HA70 from type C toxin (HA70/C) in complexes with sialylated oligosaccharides were determined, and a binding assay by the glycoconjugate microarray was performed. These results suggested that HA70/C can recognize both a2-3-and a2-6-sialylated oligosaccharides, and that it has a higher affinity for a2-3-sialylated oligosaccharides.
There is increasing interest in biologics, i.e. human-originated biological pharmaceutics. Most of the protein drugs developed so far, such as immunoglobulins and erythropoietin, are secreted glycoproteins; as a result, any non-human-type glycans, such as αGal and NeuGc, derived from animal cells and sera must be removed to circumvent undesirable immunogenic reactions. In this study, we made an extensive search for potential xenoantigenic glycans among a panel of mammalian sera. As a result, sera belonging to the order Artiodactyla, i.e. bovine, lamb and goat sera, were found to contain substantial amounts of hypersialylated biantennary glycans closely associated with a type-I lactosamine structure containing a unique tetrasaccharide, Siaα2-3Galβ1-3(Siaα2-6)GlcNAc. In all three Artiodactyla sera, the most abundant structure was Siaα2-3Galβ1-3(Siaα2-6)GlcNAcβ1-2Manα1-3[Siaα2-6Galβ1-4GlcNAcβ1-2Manα1-6]Manβ1-4GlcNAcβ1-4GlcNAc. A dually hypersialylated biantennary structure, Siaα2-3Galβ1-3(Siaα2-6)GlcNAcβ1-2Manα1-3[Siaα2-3Galβ1-3(Siaα2-6)GlcNAcβ1-2Manα1-6]Manβ1-4GlcNAcβ1-4GlcNAc, was also abundant (10%) in bovine serum. The amount of hypersialylated glycans among total sialylated glycans was 46, 26 and 23% in bovine, lamb and goat sera, respectively. On the other hand, such structures could not be detected in the sera of other animals including human. The biological functions and the immunogenicity of the hypersialylated glycans in these animals remain to be elucidated; however, it is worth noting that glycoproteins biosynthesized from Artiodactyla cells and those contaminated with bovine serum might enhance undesirable antigenicity in human patients.
We previously showed that galectin-9 suppresses degranulation of mast cells through protein-glycan interaction with IgE. To elucidate the mechanism of the interaction in detail, we focused on identification and structural analysis of IgE glycans responsible for the galectin-9-induced suppression using mouse monoclonal IgE (TIB-141). TIB-141 in combination with the antigen induced degranulation of RBL-2H3 cells, which was almost completely inhibited by human and mouse galectin-9. Sequential digestion of TIB-141 with lysyl endopeptidase and trypsin resulted in the identification of a glycopeptide (H-Lys13-Try3; 48 amino acid residues) with a single N-linked oligosaccharide near the N terminus capable of neutralizing the effect of galectin-9 and another glycopeptide with two N-linked oligosaccharides (H-Lys13-Try1; 16 amino acid residues) having lower activity. Enzymatic elimination of the oligosaccharide chain from H-Lys13-Try3 and H-Lys13-Try1 completely abolished the activity. Removal of the C-terminal 38 amino acid residues of H-Lys13-Try3 with glutamyl endopeptidase, however, also resulted in loss of the activity. We determined the structures of N-linked oligosaccharides of H-Lys13-Try1. The galectin-9-binding fraction of pyridylaminated oligosaccharides contained asialo-and monosialylated bi/tri-antennary complex type oligosaccharides with a core fucose residue. The structures of the oligosaccharides were consistent with the sugar-binding specificity of galectin-9, whereas the nonbinding fraction contained monosialylated and disialylated biantennary complex type oligosaccharides with a core fucose residue. Although the oligosaccharides linked to H-Lys13-Try3 could not be fully characterized, these results indicate the possibility that cooperative binding of oligosaccharide and neighboring polypeptide structures of TIB-141 to galectin-9 affects the overall affinity and specificity of the IgElectin interaction.
We provide a novel dimension-free uniform concentration bound for the empirical risk function of constrained logistic regression. Our bound yields a milder sufficient condition for a uniform law of large numbers than conditions derived by the Rademacher complexity argument and McDiarmid's inequality. The derivation is based on the PAC-Bayes approach with second-order expansion and Rademacher-complexity-based bounds for the residual term of the expansion.
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