Glycan Markers of Human Stem Cells Assigned with Beam Search Arrays

Glycan antigens recognized by monoclonal antibodies have served as stem cell markers. To understand regulation of their biosynthesis and their roles in stem cell behavior precise assignments are required. We have applied state-of-the-art glycan array technologies to compare the glycans bound by five antibodies that recognize carbohydrates on human stem cells. These are: FC10.2, TRA-1-60, TRA-1-81, anti-i and R-10G. Microarray analyses with a panel of sequence-defined glycans corroborate that FC10.2, TRA-1-60, TRA-1-81 recognize the type 1-(Gal beta-3GlcNAc)-terminating backbone sequence, Gal beta-3GlcNAc beta-3Gal beta-4GlcNAc beta-3Gal beta-4Glc-NAc, and anti-i, the type 2-(Gal beta-4GlcNAc) analog, Gal beta-4GlcNAc beta-3Gal beta-4GlcNAc beta-3Gal beta-4GlcNAc, and we determine substituents they can accommodate. They differ from R-10G, which requires sulfate. By Beam Search approach, starting with an antigen-positive keratan sulfate polysaccharide, followed by targeted iterative microarray analyses of glycan populations released with keratanases and mass spectrometric monitoring, R-10G is assigned as a monosulfated type 2 chain with 6-sulfation at the penultimate N-acetylglucosamine, Gal beta-4GlcNAc(6S)beta-3Gal beta-4GlcNAc beta-3Gal beta-4GlcNAc. Microarray analyses using newly synthesized glycans corroborate the assignment of this unique determinant raising questions regarding involvement as a ligand in the stem cell niche.