‘GlycosaminoGlycome’ microarrays: Applications in studies of recognition by anti-human stem cell antibodies, growth factors and morphogens
File(s)
Author(s)
Wu, Nian
Type
Thesis
Abstract
This thesis is focused on construction of microarrays of the GlycosaminoGlycome including glycosaminoglycan (GAG) polysaccharides [chondroitin sulphates, heparan sulphates, heparin, keratan sulphate (KS) and hyaluronan] and the derived oligosaccharides as neoglycolipids (NGLs), and their application to characterization of a stem cell antigen R-10G and investigation of GAG-binding preferences of growth factors and morphogens to which human stem cells respond.
R-10G antigen is of special interest as it is expressed on human embryonic stem (ES) and induced pluripotent stem (iPS) cells, but not on human embryonal carcinoma cells. Screening analysis using the GAG polysaccharide microarray showed strong binding of antibody R-10G specifically to bovine cornea KS, but little or no binding to other KS preparations or GAG polysaccharides. ‘Designer’ arrays were then constructed from KS oligosaccharides obtained by partial depolymerisation using keratanases and mild acid treatment. The oligosaccharides were fractionated by gel-filtration and HPLC, with monitoring by mass spectrometry (MS) before conversion to NGLs for antigenic analysis. KS oligosaccharide fractions terminating with Gal (keratanase II digestion products) rather than GlcNAc at the non-reducing end were strongly bound by R-10G, and the minimum chain length was hexasaccharide. Among oligosaccharides separated from the hexasaccharide faction, Structure A (overleaf) assigned by NMR and Structure B assigned by MS/MS are antigen-positive, whereas Structure C is antigen-negative.
Galβ1-4GlcNAc(6S)β1-3Galβ1-4GlcNAc(6S)β1-3Galβ1-4GlcNAc(6S) (A)
Galβ1-4GlcNAc(6S)β1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc(6S) (B)
Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc(6S)β1-3Galβ1-4GlcNAc(6S) (C)
This suggests that the sulphate at the internal GlcNAc residue is not required and the sulphate at the non-reducing terminal GlcNAc residue is the key element.
A novel finding in screening of growth factors and morphogens using the GlycosaminoGlycome microarray is that KS is bound by fibroblast growth factor 2 and bone morphogenetic protein (BMP) 2, but not wingless-3a, sonic hedgehog and BMP-4. This opens the way into searching for a role for fully and undersulphated KS on stem cells.
R-10G antigen is of special interest as it is expressed on human embryonic stem (ES) and induced pluripotent stem (iPS) cells, but not on human embryonal carcinoma cells. Screening analysis using the GAG polysaccharide microarray showed strong binding of antibody R-10G specifically to bovine cornea KS, but little or no binding to other KS preparations or GAG polysaccharides. ‘Designer’ arrays were then constructed from KS oligosaccharides obtained by partial depolymerisation using keratanases and mild acid treatment. The oligosaccharides were fractionated by gel-filtration and HPLC, with monitoring by mass spectrometry (MS) before conversion to NGLs for antigenic analysis. KS oligosaccharide fractions terminating with Gal (keratanase II digestion products) rather than GlcNAc at the non-reducing end were strongly bound by R-10G, and the minimum chain length was hexasaccharide. Among oligosaccharides separated from the hexasaccharide faction, Structure A (overleaf) assigned by NMR and Structure B assigned by MS/MS are antigen-positive, whereas Structure C is antigen-negative.
Galβ1-4GlcNAc(6S)β1-3Galβ1-4GlcNAc(6S)β1-3Galβ1-4GlcNAc(6S) (A)
Galβ1-4GlcNAc(6S)β1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc(6S) (B)
Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc(6S)β1-3Galβ1-4GlcNAc(6S) (C)
This suggests that the sulphate at the internal GlcNAc residue is not required and the sulphate at the non-reducing terminal GlcNAc residue is the key element.
A novel finding in screening of growth factors and morphogens using the GlycosaminoGlycome microarray is that KS is bound by fibroblast growth factor 2 and bone morphogenetic protein (BMP) 2, but not wingless-3a, sonic hedgehog and BMP-4. This opens the way into searching for a role for fully and undersulphated KS on stem cells.
Version
Open Access
Date Issued
2017-10
Date Awarded
2018-01
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Feizi, Ten
Chai, Wengang
Pickering, Matthew
Sponsor
China Scholarship Council
Publisher Department
Department of Medicine
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)