Heparan Sulfate Domains Required for Fibroblast Growth Factor 1 and 2 Signaling through Fibroblast Growth Factor Receptor 1c

Dixy E Green,Fuming Zhang,Lingyun Li,Xinyue Liu,Yanlei Yu,Yongmei Xu,Jian Liu,Paul L Deangelis,Mathew Suflita,Xing Zhang,Victor Schultz,Robert J Linhardt

doi:10.1074/jbc.m116.761585

Abstract

A small library of well defined heparan sulfate (HS) polysaccharides was chemoenzymatically synthesized and used for a detailed structure-activity study of fibroblast growth factor (FGF) 1 and FGF2 signaling through FGF receptor (FGFR) 1c. The HS polysaccharide tested contained both undersulfated (NA) domains and highly sulfated (NS) domains as well as very well defined non-reducing termini. This study examines differences in the HS selectivity of the positive canyons of the FGF12-FGFR1c2 and FGF22-FGFR1c2 HS binding sites of the symmetric FGF2-FGFR2-HS2 signal transduction complex. The results suggest that FGF12-FGFR1c2 binding site prefers a longer NS domain at the non-reducing terminus than FGF22-FGFR1c2 In addition, FGF22-FGFR1c2 can tolerate an HS chain having an N-acetylglucosamine residue at its non-reducing end. These results clearly demonstrate the different specificity of FGF12-FGFR1c2 and FGF22-FGFR1c2 for well defined HS structures and suggest that it is now possible to chemoenzymatically synthesize precise HS polysaccharides that can selectively mediate growth factor signaling. These HS polysaccharides might be useful in both understanding and controlling the growth, proliferation, and differentiation of cells in stem cell therapies, wound healing, and the treatment of cancer.

Highlights

A small library of well defined heparan sulfate (HS) polysaccharides was chemoenzymatically synthesized and used for a detailed structure-activity study of fibroblast growth factor (FGF) 1 and FGF2 signaling through fibroblast growth factors (FGFs) receptor (FGFR) 1c
Because of the topological constraints of the two heparan sulfate chains being attached to the core protein through their reducing ends, the interacting NS domains must be located on the non-reducing end of each
The electrostatic and topological characteristics of the basic canyon are different for each protein signaling complex; i.e. the canyon of FGF12FGFR1c2 should be distinct from that of FGF22-FGFR1c2 [57]

Summary

Results

Design of Synthetic Heparan Sulfate Targets for Testing—The domains present within natural heparan sulfate consist of sequences of high sulfation (NS domains) and low sulfation (NA domains) [2,3,4]. Eight heparan sulfate chains were designed to begin to study the contribution of domain structures in HS for FGF-FGFR signaling (Fig. 1, 1-8) These chains consist of one or two long domains assembled at the non-reducing end of a GlcA-pNP acceptor. A second chain, 1, contained a long (ϳ15-repeat) NA domain assembled at the non-reducing end of the acceptor (Fig. 3), resembling a single domain heparan sulfate chain, serving as a negative control in signaling assays. Each TFA-containing block copolymer was dissolved in a mildly basic solution of Et3N, MeOH, and H2O and stirred overnight Under these conditions, the GlcNTFA residues were completely deprotected, exposing free amino groups, which were chemically N-sulfonated using NMe31⁄7SO3 to afford GlcNAc and GlcNS domains containing block copolymers, the NSulfo precursors to synthetic heparan sulfates 2-8. TriS, NS6S, NS2S, and NS disaccharides arise from the NS domains, and 2S6S, 6S, 2S, and 0S disaccharides arise from the NS domains. —, not detected

Disaccharide composition

Discussion

Experimental Procedures