Abstract
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 IgE-lectin interaction.
Highlights
Subsequent studies in mice (Gray et of., 1963 ; Gray and Rauh, 1967) indicated that this antagonist action of reserpine was due to the depletion of norepinephrine in brain, i.e., norepinephnine is required in brain in order for inhibitors of carbonic anhydrase to have an anticonvulsant effect
Reserpine vehicle alone methazolamide and the of amines in brain was Because treatment with or in combination with vehicles for a-methyltyrosine or p-chlorophenylalanjne the concentration of amines, had no influence on the means associated with these treatments were pooled to give a general estimate of the mean concentration of each amine in brain
Percent depletions were calculated with reference to these estimates from the mean concentrations 24, 29.5 and 77 hours after treatment of amines 5, with 4 mg/kg of reserpine alone or in combination with methazolamide and the vehicles for cx-methyltyrosine or p-chlorophenylalanine
Summary
The effect of p-chtorophenytatanine (PCPA) atone and in reserpine-pretreated the anticonvulsant potency of methazolamide rats on brain amines and on see table 2 for relevant details. The effect of reserpine on brain aniines and on the anticonvulsant potency of methazolamide in rats pietreated with p-chlorophenylalanine (PCPA)
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