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Mol Cell Proteomics. 2019 Oct;18(10):1981-2002. doi: 10.1074/mcp.RA119.001309. Epub 2019 Jul 15.

Glycan Markers of Human Stem Cells Assigned with Beam Search Arrays.

Author information

1
Glycosciences Laboratory, Department of Medicine, Imperial College London, London W12 0NN, United Kingdom.
2
Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215.
3
Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180.
4
Research Center for Glycobiotechnology, Ritsumeikan University, Noji-Higashi, 1-1-1, Kusatsu Shiga 525-8577, Japan.
5
Glycosciences Laboratory, Department of Medicine, Imperial College London, London W12 0NN, United Kingdom w.chai@imperial.ac.uk.
6
Glycosciences Laboratory, Department of Medicine, Imperial College London, London W12 0NN, United Kingdom t.feizi@imperial.ac.uk.

Abstract

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-(Galβ-3GlcNAc)-terminating backbone sequence, Galβ-3GlcNAcβ-3Galβ-4GlcNAcβ-3Galβ-4GlcNAc, and anti-i, the type 2-(Galβ-4GlcNAc) analog, Galβ-4GlcNAcβ-3Galβ-4GlcNAcβ-3Galβ-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 mono-sulfated type 2 chain with 6-sulfation at the penultimate N-acetylglucosamine, Galβ-4GlcNAc(6S)β-3Galβ-4GlcNAcβ-3Galβ-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.

KEYWORDS:

NMR; Stem cells; antibodies; glycan arrays; glycan markers; glycan roles; glycomics; growth factors; keratan sulfate; mass spectrometry; micro arrays

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