Capillary electrophoresis: a tool for studying interactions of glycans/proteoglycans with growth factors.
Heparan sulfate (HS) and heparin bind to various growth factors and modulate their activities. Interactions of heparin and HS with members of the fibroblast growth factor (FGF) family are prerequisites for binding of FGFs to their high affinity cell receptors. The sulfation patterns of distinct oligosaccharide domains within heparin and HS chains determine their high affinity binding with basic FGF (bFGF). In order to study the structural basis of interactions of HS with bFGF, we developed a capillary electrophoresis (CE) method in order to monitor the ability of HS-derived oligosaccharides to bind this growth factor. HS was degraded to Delta-di- and Delta-oligosaccharides with digestion with heparitinase and the obtained Delta-saccharides were analyzed by capillary zone electrophoresis (CZE), using 50 mM phosphate, pH 3.5, as operating buffer, reversed polarity (30 kV) and detection at 232 nm. Under these conditions all differently sulfated HS Delta-disaccharides and the various Delta-oligosaccharide groups were resolved. Following incubation of the digest with bFGF and re-electrophoresis of the mixture, the bFGF interacting oligosaccharide groups were easily detected and identified. In view of the obtained results, CE is a multipotent analytical tool for determining disaccharide composition in HS, separating the various oligosaccharide groups produced by the action of heparitinase and identifying those interacting with bFGF.
Militsopoulou
Maria
M
Section of Organic Chemistry, Biochemistry and Natural Products, Laboratory of Biochemistry, Department of Chemistry, University of Patras, 26500 Patras, Greece.
Lamari
Fotini
F
Karamanos
Nikos K
NK
D000818:Animals; D002417:Cattle; D019075:Electrophoresis, Capillary / Q000379:methods; D006133:Growth Substances / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D011134:Polysaccharides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D011509:Proteoglycans / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D013678:Technology, Pharmaceutical / Q000379:methods*
D006133:Growth Substances; D011134:Polysaccharides; D011509:Proteoglycans