To enable the study of HS biological specificity, complex saccharide fraction libraries from tissue HS have been produced and their ability to bind and activate FGF growth factors investigated. Heparitinase-derived saccharides from porcine mucosal HS were purified by Superdex 30 size exclusion, and SAX and reverse phase HPLC. A library of fractions (~100, 6-10mers) was screened for binding to FGF1 and FGF2 using a competition ELISA, and for corresponding bioactivity (FGF1 or 2 signalling in BaF3 cell assays with defined FGF receptors). We identified fractions that bound and activated these ligands, and also ones that did not. Selected structures were sequenced using a novel approach employing a mass tag and electro-spray mass spectrometry (ES-MS) analysis. We have also developed array-format bioassays that allow live cell responses to immobilized glycans to be measured. Our data demonstrate that:
• structures of identical size and sulfation content, but distinct sulfation sequences, differed widely in their bioactivity
• binding data alone does not readily correlate with activity
• bioassay data is the ultimate arbiter of biological specificity
We conclude that chemical information that conveys functional specificity is encoded in the complex sulfation sequences of HS chains. Further application of this strategy will permit glycomics approaches to decode the molecular basis of HS function.