Isoglobotrihexosylceramide (iGb3), a well-characterized natural killer T cell ligand found in mammalian tissues, is also known as a glycosphingolipid that contains the human IgE epitope α-Gal (Galα1,3Gal) structure. Here, we analyzed the reactivity of several mice and human serum immunoglobulins against iGb3. Additionally, we isolated and characterized the variable region sequences of a monoclonal antibody that specifically recognizes iGb3. No IgE reactive with iGb3 was detected in sera from MRL/lpr mice, which are known to produce autoreactive antibodies, or in sera from healthy human donors. Furthermore, no induction of IgE and IgG was observed in the sera of mice immunized with iGb3; only IgM reactivity to iGb3 was detected. Further analysis of an anti-iGb3 monoclonal antibody generated from the splenocytes of an iGb3-immunized mouse revealed that the nucleotide sequences of the variable regions exhibited high homology to those of antibodies recognizing glycoconjugates containing Galα1,3 or Galα1,4 structures. These results indicate that the mouse genome harbors genes capable of encoding antibodies that recognize α-linked galactose-containing glycans, including iGb3, but that iGb3 is not sufficiently immunogenic to induce IgE in mammalian lymphocytes.
Hiono T, Matsuda A, Wagatsuma T, Okamatsu M, Sakoda Y, et al.
Virology •
Glycan structures on hemagglutinin (HA) of influenza A viruses have been analyzed previously to understand their significance. However, the formerly established methods using mass spectrometry present disadvantages such as procedure complexity, sensitivity, and throughput. Our study has established a novel method for analyzing glycan profiles of HA using lectin microarray techniques. We successfully obtained glycan profiles of HA starting from 1 ml of the 10 TCID samples through simple antigen enrichment using optimized immunoprecipitation. The profiles were reasonably consistent with known glycan structures of HA. Next, we compared glycan profiles of the HAs prepared from chicken embryos, MDCK, Vero, and A549 cells, and demonstrated the host cell-specific HA glycan profiles. Notably, the HA from MDCK cells was α1-3 galactosylated. Our method provides a highly sensitive and simple procedure for glycan profiling of the viral glycoproteins, thereby paving way for direct glycan analyses of human- and animal-derived virions.
Suzuki H, Kameyama A, Tachibana K, Narimatsu H, Fukui K
Analytical chemistry •
Mechanisms of fragmentation of glycosyl bond linkages in various saccharides were investigated by using computational calculations to find general rules of fragmentation of sodiated oligosaccharides in mass spectrometry. The calculations revealed that alpha-Glc, alpha-Gal, beta-Man, alpha-Fuc, beta-GlcNAc, and beta-GalNAc linkages were cleaved more easily than beta-Glc, beta-Gal, and alpha-Man linkages because the transition states of the former were stabilized by the anomeric effect. The 1-6 linkage was more stable than the others, since saccharides with flexible 1-6 linkages were more stabilized in energy than the other linkages by the sodium cation. The sialyl linkage was the most labile of all the linkages investigated. Comparison of activation energies and binding affinities to the sodium cation revealed an increase in activation energy in proportion to the increment in binding affinity. The calculated stabilities of glycosyl bonds were: alpha-Man (Manalpha1-3Man, Manalpha1-4Man, Manalpha1-6Man) > beta-Gal (Galbeta1-4Gal) > alpha-GalNAc (GalNAcalpha1-4GalNAc) > beta-Man (Manbeta1-4GlcNAc) > alpha-Gal (Galalpha1-3Gal, Galalpha1-4Gal, Galalpha1-6Gal) > beta-Man (Manbeta1-4Man) > beta-GalNAc (GalNAcbeta1-4GalNAc) > alpha-Fuc (Fucalpha1-6GlcNAc) > alpha-Fuc (Fucalpha1-4GlcNAc) > beta-GlcNAc (GlcNAcbeta1-4GlcNAc) > alpha-Fuc (Fucalpha1-3GlcNAc) > alpha-NeuNAc (NeuNAcalpha2-3Gal, NeuNAcalpha2-6Gal); this result was close to the experimentally deduced trend. These theoretically and experimentally derived general rules for fragmentation should be useful for analyzing the experimentally obtained mass spectra of oligosaccharides.
Uzawa H, Nishida Y, Sasaki K, Minoura N, Kobayashi K
Chembiochem : a European journal of chemical biology •
The substrate specificities of three molluscan sulfatases (E.C. 3.1.6.1; snail, abalone, and limpet origins) were investigated with assorted p-nitrophenyl (pNP) di-O-sulfonated beta-D-galactopyranosides and beta-lactosides [3,6-SO(3) Gal (1), 3',6'-SO(3) Lac (2), 4, 6SO(3) Gal (3), 2,6-SO(3) Gal (4), 3,4-SO(3) Gal (5), and 3,6-SO(3) GalNAc (6); Ac, acetyl; Gal, galactose; Lac, lactose] together with mono-O-sulfonated beta-D-galactopyranoside [pNP 3SO(3)-Gal (7)] and tri-O-sulfonated alpha-D-galactopyranoside [2,3,6-SO(3)-alpha-Gal (11)]. Some notable differences between the substrate specificity of the three sulfatases were disclosed; snail sulfatase hydrolyzed the 3O- and 2O-sulfo groups of 1 and 4, respectively, to afford 6SO(3) Gal (9) in high yields, while the abalone enzyme did not act on 4. Only the limpet enzyme could cleave the 3O-sulfo groups of 7 to give pNP beta-galactoside. In contrast, every enzyme could utilize 11 as a good substrate to afford a mixture of 6SO(3)-alpha-Gal (13) and 2,6-SO(3) alpha-Gal (12). None of the enzymes could cleave the O-sulfo groups of 5 and 6, which indicates that a primary 6O-sulfo group tends to promote the enzymatic hydrolysis of O-sulfo groups at the secondary positions.
