Abstract
Proteins that belong to the fibroblast growth factor (FGF) family regulate proliferation, migration, and differentiation of many cell types. Several FGFs, including the prototype factors FGF-1 and FGF-2, depend on interactions with heparan sulfate (HS) proteoglycans for activity. We have assessed tissue-derived HS fragments for binding to FGF-1 and FGF-2 to identify the authentic saccharide motifs required for interactions. Sequence information on a range of N-sulfated HS octasaccharides spanning from low to high affinity for FGF-1 was obtained. All octasaccharides with high affinity for FGF-1 (> or =0.5 m NaCl required for elution) contained an internal IdoUA(2-OSO(3))-GlcNSO(3)(6-OSO(3))-IdoUA(2-OSO(3))-trisaccharide motif. Octasaccharides with a higher overall degree of sulfation but lacking the specific trisaccharide motif showed lower affinity for FGF-1. FGF-2 was shown to bind to a mono-O-sulfated HS 6-mer carrying a single internal IdoUA(2-OSO(3))-unit. However, a di-O-sulfated -IdoUA(2-OSO(3))-GlcNSO(3)-IdoUA(2-OSO(3))-trisaccharide sequence within a HS 8-mer gave stronger binding. These findings show that not only the number but also the positions of individual sulfate groups determine affinity of HS for FGFs. Our findings support the notion that FGF-dependent processes can be modulated in vivo by regulated expression of distinct HS sequences.
Highlights
Proteins that belong to the fibroblast growth factor (FGF) family regulate proliferation, migration, and differentiation of many cell types
A di-O-sulfated -IdoUA(2-OSO3)-GlcNSO3-IdoUA(2OSO3)-trisaccharide sequence within a heparan sulfate (HS) 8-mer gave stronger binding. These findings show that the number and the positions of individual sulfate groups determine affinity of HS for FGFs
Sequence Analysis of FGF-1-Binding HS Octasaccharides— Affinity and Charge Fractionation of Octasaccharides - Oligosaccharides corresponding to NS domains were obtained from pig intestinal mucosa HS as described under “Experimental Procedures” and were reduced with NaB3H4 to introduce a terminal [1-3H]aManR label
Summary
Materials—HS from pig intestinal mucosa was a gift from G. van Dedem (Diosynth, Oss, The Netherlands). Pooled oligosaccharide fractions were desalted on PD-10 columns (Amersham Pharmacia Biotech) and further resolved by anion-exchange chromatography on a 4 ϫ 250-mm Propac PA-1 column (Dionex, Surrey, UK) eluted with a linear gradient (total volume, 100 ml) from 0 to 1.5 M NaCl in H2O adjusted to pH 3 with HCl. Saccharide fractions of similar charge density were pooled, desalted, and dried in a centrifugal evaporator. N-Sulfated, [1-3H]aManR end group-labeled oligosaccharides (mostly octasaccharides, occasionally hexasaccharides) were subjected to partial cleavage with nitrous acid (pHNO2) by incubation with 2– 4 mM NaNO2 in 20 – 40 mM HCl in a total volume of 20 l on ice. The precise reaction conditions were adjusted as required to yield appropriate (ideally equimolar) ratios of labeled di-, tetra-, and hexasaccharide degradation products (monitored by pilot runs on the Propac PA-1 column). Mass spectra were calibrated with cytochrome c (Sigma) and adrenocorticotropic hormone (Sigma)
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