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.
Otsuka I, Blanchard B, Borsali R, Imberty A, Kakuchi T
Chembiochem : a European journal of chemical biology •
A series of poly(phenylacetylene)s bearing diverse saccharide pendants--N-acetyl-D-glucosamine, D-lactose, and N-acetyl-D-neuraminic acid--were synthesized by rhodium-mediated polymerizations of the corresponding acetyl-protected glycosylated phenylacetylenes followed by deprotection. The circular dichroism spectra of these glycosylated poly(phenylacetylene)s each displayed split-type Cotton effects in the long absorption region of the conjugated polymer backbone (260-500 nm), thus indicating predominantly one-handed helical conformations in their backbones. The binding affinities of these glycosylated poly(phenylacetylene)s, and those of previously reported phenylacetylenes bearing D-galactose, towards plant and bacterial lectins were investigated by hemagglutination inhibition assay and isothermal titration calorimetry (ITC). The stoichiometries of binding vary strongly, depending on the lectin binding sites and the accessibilities of the carbohydrate residues in the helices. The measured affinities also vary, with the maximum value observed for the interaction between poly-PA-α-Gal and lectin I from Pseudomonas aeruginosa, with a K(d) value of 4 μM per monosaccharide representing a 200-fold increase relative to the corresponding monomer.
Uematsu R, Shinohara Y, Nakagawa H, Kurogochi M, Furukawa J, et al.
Molecular & cellular proteomics : MCP •
Glycosylation of proteins greatly affects their structure and function, but traditional genomics and transcriptomics are not able to precisely capture tissue- or species-specific glycosylation patterns. We describe here a novel approach to link different "omics" data based on exhaustive quantitative glycomics of murine dermis and epidermis. We first examined the dermal and epidermal N-glycome of mouse by a recently established glycoblotting technique. We found that the Galalpha1-3Gal epitope was solely expressed in epidermis tissue and was preferentially attached to adhesion molecules in a glycosylation site-specific manner. Clarified glycomic and protemic information combined with publicly available microarray data sets allowed us to identify galectin-3 as a receptor of Galalpha1-3Gal epitope. These findings provide mechanistic insight into the causal connection between the genotype and the phenotype seen in alpha3GalT-1-deficient mice and transgenic mice expressing endo-beta-galactosidase C. Because humans do not possess the Galalpha1-3Gal structure on their tissues, we further examined the human dermal and epidermal N-glycome. Comparative glycomics revealed that the GalNAcbeta1-4GlcNAc (N,N'-diacetyllactosediamine) epitope, instead of the Galalpha1-3Gal epitope, was highly expressed in human epidermis.
