Compositional Analysis of Heparin/Heparan Sulfate Interacting with Fibroblast Growth Factor 3

摘要

ABSTRACT: Heparan sulfate (HS) proteoglycans (PGs) interact with a number of extracellular signalingnproteins, thereby playing an essential role in the regulation of many physiological processes. One majornfunction of HS is to interact with fibroblast growth factors (FGFs) and their receptors (FGFRs) and formnFGF 3nHS 3nFGFR signaling complexes. Past studies primarily examined the selectivity of HS for FGF ornFGFR. In this report, we used a new strategy to study the structural specificity of HS binding to 10 differentnFGF 3nFGFR complexes. Oligosaccharide libraries prepared from heparin, 6-desufated heparin, and HS werenused for the interaction studies by solution competition surface plasmon resonance (SPR) and filter trappingnassays. Specific oligosaccharides binding to FGF 3nFGFR complexes were subjected to polyacrylamide gelnelectrophoresis (PAGE) analysis and disaccharide compositional analysis using liquid chromatography andnmass spectrometry. The competition SPR studies using sized oligosaccharidemixtures showed that binding ofneach of the tested FGFs or FGF 3nFGFR complexes to heparin immobilized to an SPR chip was size-ndependent. The 6-desulfated heparin oligosaccharides exhibited a reduced level of inhibition of FGF andnFGF 3nFGFR complex binding to heparin in the competition experiments. Heparin and the 6-desulfatednheparin exhibited higher levels of inhibition of the FGF 3nFGFR complex binding to heparin than of FGFnbinding to heparin. In the filter trapping experiments, PAGE analysis showed different affinities between thenFGF 3nFGFR complexes and oligosaccharides. Disaccharide analysis showed that HS disaccharides with andegree of polymerization of 10 (dp10) had high binding selectivity, while dp10 heparin and dp10 6-desulfatednheparin showed reduced or no selectivity for the different FGF 3nFGFR complexes tested.