Abstract:
:We prepared a streptavidin-based neoglycoprotein which carries more than 140 GT1b oligosaccharides. GT1b oligosaccharides were covalently coupled to streptavidin by reductive amination, yielding a monomer form of streptavidin carrying 13 oligosaccharides. The monomer form of glycosylated streptavidin was polymerized with biotinylated-bovine serum albumin, which yielded a polymer carrying more than 140 oligosaccharides. Both the monomer and the polymer bound to Chinese hamster ovary cells expressing murine sialoadhesin. The relative binding potencies determined with the polymer, monomer, and free GT1b oligosaccharides were 3,500, 83, and 1, respectively, indicating that an increase in the number of oligosaccharide ligands is critical for high avidity. The high avidity of the polymer enabled us to develop a sensitive and quantitative binding assay, and the assay was applied to characterization of the binding specificity of sialoadhesin. The polymer binding was inhibited by various gangliosides, the order of the inhibitory potencies being GM3 (IC50 = 40 microM) > GD1a (100 microM) > sialylparagloboside (120 microM) > GT1b (310 microM) > GM2 (640 microM) > GM4 (2,100 microM) > GD1b>LacCer = GM1 = paragloboside (no inhibition). These results indicate that the binding specificity is comparable to that reported, i.e. the determinant structure is NeuAcalpha2-3Galbeta1-linked to either 3GalNAc, 3(4)GlcNAc, or 4Glc, and that the oligosaccharide structure on the polymer is properly presented to sialoadhesin on the cell surface. To determine the precise requirement of the NeuAc structure for binding, NeuAc of GM3 was converted into various derivatives, the inhibitory potencies of which were examined; i.e. GM3 containing NeuAc, IC50 = 40 microM; C7- or C8-aldehyde, 500 microM; C7- or C8-alcohol, 700 microM; C1-alcohol, 2,000 microM; C1-amide, 2,200 microM; and NeuGc,>3,000 microM. These results confirmed the requirement of the hydroxyl group at C9 and/or C8, the carboxyl group at C1, and the methyl group of the N-acetyl residue of NeuAc in a quantitative manner. Thus, this streptavidin-based neoglycoprotein is a useful multivalent glycoprobe, which exhibits high affinity and specificity to murine sialoadhesin on the cell surface.
journal_name
J Biochemjournal_title
Journal of biochemistryauthors
Hashimoto Y,Suzuki M,Crocker PR,Suzuki Adoi
10.1093/oxfordjournals.jbchem.a021960subject
Has Abstractpub_date
1998-03-01 00:00:00pages
468-78issue
3eissn
0021-924Xissn
1756-2651journal_volume
123pub_type
杂志文章abstract::We recently described the cloning of putative human CMP-sialic acid transporter (hCST) cDNA [Ishida, N. et al. (1996) J. Biochem. 120, 1074-1078]. The hCST cDNA coded for a hydrophobic protein with an amino acid sequence showing a high degree of similarity (92% identity) to that of murine CMP-sialic acid transporter. ...
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abstract::Three glucosamine-containing sphingoglycolipids were isolated from human lung tissue of a blood group-A subject. They were hexaglycosyl, pentaglycosyl, and tetraglycosyl ceramides. The hexaglycosyl ceramide exhibited blood group-A antigenicity, and the following chemical structure was proposed on the basis of sequenti...
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abstract::Identification of single-stranded regions in Torulopsis utilis 5S RNA was attempted by the use of Nuclease S1, a single-strand specific endonuclease. When T. utilis 5S RNA was subjected to prolonged incubation with Nuclease S1, about 50% of the substrate 5S RNA remained as large oligonucleotide "cores." Such Nuclease...
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journal_title:Journal of biochemistry
pub_type: 杂志文章
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