| Fibroblast growth factor (FGF) signaling is involved in development, adult homeostasis and associated diseases. Variation in size, sulfation pattern and topography of heparan sulfate (HS) oligosaccharides impacts FGF signaling by regulating the interaction of FGF with pre-formed inactive dimer of FGFRs for formation of stable active FGF-HS-FGFR signaling complex. However, whether structurally specific motifs are at play for the assembly and activation of this complex are unclear. In this report, we utilize the high-quality stable recombinant GST-FGF as a bioaffinity reagent to separate and identify oligosaccharide motifs with differential affinities for FGF and examine their ability to support FGF signaling. We isolated a rare undersulfated octasaccharide mixture (7,8-S-OctaF7) with high affinity for GST-FGF7, which forms a binary complex with FGFR2IIIb competent to subsequently bind FGF7 and activate the FGF7-linked signaling pathways with high specific activity in mouse keratinocytes expressing FGFR2IIIb, relative to the more abundant and highly sulfated octasaccharides with lower affinity. Mass spectrometric, disaccharide composition and anticoagulant activity analysis indicated that 7,8-S-OctaF7 is comprised of 7 and 8 sulfated octasaccharides exhibiting a bisulfated disaccharide containing the rare N-unsubstituted glucosamine and at least one component in which groups are distributed sufficient to support antithrombin-mediated inhibition of Factor Xa activity. However, 7,8-S-OctaF7 fails to support FGF1 signaling in both mouse fibroblasts expressing FGFRlIIIc and keratinocytes. In contrast, octasaccharides with high sulfation but lower affinity for FGF7 failing to support FGF7 signaling supports FGF1 signaling. GST-FGF1 bioaffinity fractionation revealed that FGF1 binds more oligosaccharides with high affinity than FGF7. The high affinity octasaccharides are much more heterogeneous than 7,8-S-OctaF7, and exhibit 8 or more sulfates with the most abundant 11 sulfates, which is generally similar to lower affinity fractions eluting from both FGF1 and FGF7 at 0.30--0.60 M salt. Both fractions exhibit high specific activity for support of FGF1 signaling in fibroblasts and keratinocytes, but are deficient in FGF7-induced activity in keratinocytes. The results suggest that in a tissue setting where multiple FGF and FGFR isotypes are present, rare HS oligosaccharide motifs with specific monosaccharides and disposition of side groups beyond more abundant high charge density and longer length discriminate between FGFs for signaling of specific FGFRs. |
