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Title: 'GlycosaminoGlycome' microarrays : applications in studies of recognition by anti-human stem cell antibodies, growth factors and morphogens
Author: Wu, Nian
ISNI:       0000 0004 7969 8260
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2018
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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.
Supervisor: Feizi, Ten ; Chai, Wengang ; Pickering, Matthew Sponsor: China Scholarship Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral