Abstract
Heparin-like saccharides play an essential role in binding to both fibroblast growth factors (FGF) and their receptors at the cell surface. In this study we prepared a series of heparin oligosaccharides according to their size and sulfation level. We then investigated their affinity for FGF2 and their ability to support FGF2 mitogenesis of heparan sulfate-deficient cells expressing FGFR1c. Tetra- and hexasaccharides bound FGF2, but failed to dimerize the growth factor. Nevertheless, these saccharides promoted FGF2-mediated cell growth. Furthermore, whereas enzymatic removal of the non-reducing end 2-O-sulfate group had little effect on the 1:1 interaction with FGF2, it eliminated the mitogenic activity of these saccharides. This evidence supports the symmetric two-end model of ternary complex formation. In contrast, even at very low concentrations, octasaccharide and larger heparin fragments conferred a potent mitogenic activity that was independent of terminal 2-O-sulfation. This correlated with the ability to dimerize FGF2 in an apparently cooperative manner. This data suggests that potent mitogenic signaling results from heparin-mediated trans-dimerization of FGF2, consistent with the asymmetric model of ternary complex formation. We propose that, depending on saccharide structure, there are different architectures and modes of ternary complex assembly that differ in stability and/or efficiency of transmembrane signaling.
Highlights
FGF1 and FGF2 are the prototypic members of the fibroblast growth factor (FGF)3 family
At the biochemical level the potent FGF2 activators can be distinguished from the weak activator by their propensity to stably dimerize FGF2, and do so in a strongly cooperative manner. It has been appreciated for some time that FGF signaling requires the formation of a ternary complex between the FGF ligand, FGFR, and Heparan sulfate (HS) [35]; the stoichiometry and molecular arrangement of these components has remained controversial
The asymmetrical model [26] described a 2:2:1 FGF11⁄7FGFR2c1⁄7heparin complex, with the two FGF1⁄7FGFR units dimerized in a trans configuration upon a heparin decasaccharide (Fig. 1B)
Summary
Materials—The FGFR-transfected BaF3 cell lines, originally created by Dr D. Bio-Gel P-10 resin was purchased from BioRad. Invitrogen supplied cell culture media and horse serum, whereas the CytoliteTM cell proliferation assay kit was from PerkinElmer Life Science. Fractions (1 ml) were collected and each peak was pooled separately, freeze-dried several times to remove residual ammonium hydrogen carbonate. BaF3 cells were serum starved for 2 days, washed three times with phosphatebuffered saline at 37 °C to remove residual IL-3. (2 nmol) was added to heparin samples (1 nmol) suspended in Subfractions of the sized heparin oligosaccharide pools were column buffer (final volume of 250 l) and incubated at room prepared by resolution on an analytical ProPac PA1 Each pooled sample was reapplied to the ProPac PA1 column to confirm purity
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