Binding specificity of R-10G and TRA-1-60/81, and substrate specificity of keratanase II studied with chemically synthesized oligosaccharides.

Recently, we established a mouse monoclonal antibody specific to hiPS/ hES cells, R-10G, which recognizes a type of keratan sulfate. Keratan sulfates (KS) comprise a family of glycosaminoglycans consisting of the repeating unit of [Gal-GlcNAc(6S)]. However, there is a diversity in the degree of sulfation at Gal and GlcNAc residues, and also in the mode of linkage, Galß1 - 3GlcNAc (type 1) or Galß1 - 4GlcNAc (type 2). To gain more insight into the binding specificity of R-10G, we carried out an ELISA test on avidin-coated plates using polyethylene glycol (PEG)3-biotinylated derivatives of a series of N-acetyllactosamine tetrasaccharides (keratan sulfates (KSs)). The results suggested that the minimum epitope structure is Galß1 - 4GlcNAc(6S)ß1 - 3Galß1 - 4GlcNAc(6S)ß1 (type 2- type 2 keratan sulfate). Removal of sulfate from GlcNAc(6S) or addition of sulfate to Gal abolished the binding activity almost completely. We also examined the binding specificity of TRA-1-60/81 in the same assay system. The minimum epitope structure was shown to be Galß1 - 3GlcNAcß1 - 3Galß1 - 4GlcNAcß1 in agreement with the previous study involving glycan arrays (Natunen et al., Glycobiology, 21, 1125-1130 (2011)). Interestingly, however, TRA-1-60/81 was shown to bind to Galß1 - 3GlcNAc(6S)ß1 - 3Galß1 - 4GlcNAc(6S)ß1 (type 1- type 2 keratan sulfate) dose-dependently, being more than one-third the binding activity toward Galß1 - 3GlcNAcß1 - 3Galß1 - 4GlcNAcß1 than in the case of TRA-1-60. In addition, a substrate specificity study on keratanase II revealed that keratanase II degraded not only "type 2-type 2 keratan sulfate" but also "type 1-type 2 keratan sulfate", significantly.

Fluorous-Tag Assisted Syntheses of Sulfated Keratan Sulfate Oligosaccharide Fragments.

The first block iteration strategy for iterative solution-phase synthesis of protected keratan sulfate (KS)-like fragments is reported. Obstacles in a strategy using galactose-glucosamine (Gal-GlcNAc) modules led to the discovery of a differentially protected GlcNAc-Gal module that could be used to synthesize KS-like fragments using a fluorous tag that maintained solubility in organic solvents for purification of all intermediates via fluorous solid-phase extraction.