Highly sulfated nonreducing end-derived heparan sulfate domains bind fibroblast growth factor-2 with high affinity and are enriched in biologically active fractions

Abstract

Human fibroblast growth factor-2 (FGF2) regulates cellular processes including proliferation, adhesion, motility, and angiogenesis. FGF2 exerts its biological function by binding and dimerizing its receptor (FGFR), which activates signal transduction cascades. Effective binding of FGF2 to its receptor requires the presence of heparan sulfate (HS), a linear polysaccharide with N-sulfated domains (NS) localized at the cell surface and extracellular matrix. HS acts as a platform facilitating the formation of a functional FGF-FGFR-HS ternary complex. Crystal structures of the signaling ternary complex revealed two conflicting architectures. In the asymmetrical model, two FGFs and two FGFRs bind a single HS chain. In contrast, the symmetrical model postulates that one FGF and one FGFR bind to the free end of the HS chain and dimerization require these ends to join, bringing the two half-complexes together. In this study, we screened a hexasaccharide HS library for compositions that are able to bind FGF2. The library was composed primarily of NS domains internal to the HS chain with minor presence of non-reducing end (NRE) NS. The binders were categorized into low versus high affinity binders. The low affinity fraction contained primarily hexasaccharides with low degree of sulfation that were internal to the HS chains. In contrast, the high affinity bound fraction was enriched in NRE oligosaccharides that were considerably more sulfated and had the ability to promote FGFR-mediated cell proliferation. The results suggest a role of the NRE of HS in FGF2 signaling and favor the formation of the symmetrical architecture on short NS domains.

FIGURE 1.

Binding stoichiometry of FGF2 to dp6 estimated using SEC. The stoichiometry of FGF2:dp6 was assessed by running the complex on a Superdex 75 SEC column calibrated with protein standards. The complex elutes at an earlier time in comparison to the free FGF2. By extrapolation from the calibration curve, the FGF2-dp6 complex has a molecular mass of 18.3 kDa. This is suggestive that FGF2 binds a hexasaccharide with a ratio FGF2:dp6 of 1:1.

FIGURE 2.

MS analysis of FGF in complex with HS. A, recombinant FGF2 used in the experiment is provided as a mixture of 4 isoforms (A, B, C, and D) that differ from each other by one or more amino acid losses. A is the most abundant isoform in the mixture. B, SEC collected FGF2-dp6 complex at 100 mm ammonium acetate was studied by direct infusion to confirm the stoichiometry of FGF2 to dp6. The dp6 compositions are given by the following numerical code [ΔHexA,HexA,GlcN,SO₃,Acetate]. The most abundant complexes present contain protein A and the oligosaccharides [1,2,3,4,1] [1,2,3,51], and [1,2,3,6,1], respectively, in a ratio FGF:dp6 of 1:1. NRE heptasaccharides are also present at low abundance and bind with the same stoichiometry as the hexasaccharides. C, FGF2-dp6 complex was collected after separation on a size exclusion column running at 200 mm instead of 100 mm ammonium acetate. These conditions were used in order to keep bound oligosaccharides of highest affinity. The most abundant complexes formed are A+[0,3,4,6,1], A+[0,3,4,7,1], A+[0,3,4,8,1], which are complexes containing 1 NRE heptasaccharide and 1 FGF2 subunit.

FIGURE 3.

LC/MS analysis of low stringency wash resistant-bound fraction. A, collected FGF-dp6 complex eluting from the SEC column was trapped onto a C-18 column, washed with 100 mm ammonium acetate and eluted by high salt. The bound oligosaccharides were analyzed for their sulfate and acetate content and quantified HILIC LC/MS. The upper panel shows the LC/MS spectrum of the HS library while the lower one shows that of the 100 mm wash resistant-bound fraction. In the bound fraction, low sulfation NS domains such as [1,2,3,2,1] and [1,2,3,3,1] disappear in favor of the higher sulfation NS domains. NRE originating NS domains such as [0,3,3,6,1] are also enriched to 10% of total binders. B, quantification of the abundances of the compositions shown in A.

FIGURE 4.

LC/MS analysis of high stringency wash resistant bound fraction. A, SEC collected FGF-dp6 complex was bound to a C-18 column and washed with a higher stringency salt (200 mm) to remove low affinity binders. The LC/MS spectra of the HS library (upper panel) and 200 mm resistant-bound fraction (lower panel) are shown. The high affinity bound fraction shows a pronounced enrichment of NRE compositions occurring at 73% of total binders while present at 1% in the initial library. Asterisks correspond to singly charged background ions. B, quantification of the abundances of the compositions shown in A.

FIGURE 5.

Proliferative activity of the FGF2-bound fractions. The unfractionated dp6 library, the 100 mm resistant-bound fraction and 200 mm resistant-bound fraction were tested for their ability to induce BaF-32 cell proliferation. The unfractionated dp6 library shows only basal proliferation levels comparable to those achieved in the absence of HS. However, the purified fractions show higher activity in promoting cell proliferation compared with the dp6 library. As expected, the fractions reached activation levels below those induced by full-length HS. The biological activity correlates with the degree of enrichment of the high affinity NRE structures in the tested fractions. One proliferative activity unit represents a 2-fold increase in BaF-32 cell number compared with FGF2-treated control.