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. 2014 Apr 4;289(14):9754-65.
doi: 10.1074/jbc.M113.546937. Epub 2014 Feb 22.

Fibroblast growth factor-based signaling through synthetic heparan sulfate blocks copolymers studied using high cell density three-dimensional cell printing

Affiliations

Fibroblast growth factor-based signaling through synthetic heparan sulfate blocks copolymers studied using high cell density three-dimensional cell printing

Eric Sterner et al. J Biol Chem. .

Abstract

Four well-defined heparan sulfate (HS) block copolymers containing S-domains (high sulfo group content) placed adjacent to N-domains (low sulfo group content) were chemoenzymatically synthesized and characterized. The domain lengths in these HS block co-polymers were ~40 saccharide units. Microtiter 96-well and three-dimensional cell-based microarray assays utilizing murine immortalized bone marrow (BaF3) cells were developed to evaluate the activity of these HS block co-polymers. Each recombinant BaF3 cell line expresses only a single type of fibroblast growth factor receptor (FGFR) but produces neither HS nor fibroblast growth factors (FGFs). In the presence of different FGFs, BaF3 cell proliferation showed clear differences for the four HS block co-polymers examined. These data were used to examine the two proposed signaling models, the symmetric FGF2-HS2-FGFR2 ternary complex model and the asymmetric FGF2-HS1-FGFR2 ternary complex model. In the symmetric FGF2-HS2-FGFR2 model, two acidic HS chains bind in a basic canyon located on the top face of the FGF2-FGFR2 protein complex. In this model the S-domains at the non-reducing ends of the two HS proteoglycan chains are proposed to interact with the FGF2-FGFR2 protein complex. In contrast, in the asymmetric FGF2-HS1-FGFR2 model, a single HS chain interacts with the FGF2-FGFR2 protein complex through a single S-domain that can be located at any position within an HS chain. Our data comparing a series of synthetically prepared HS block copolymers support a preference for the symmetric FGF2-HS2-FGFR2 ternary complex model.

Keywords: Block Copolymers; Fibroblast Growth Factor (FGF); Fibroblast Growth Factor Receptor (FGFR); Fibroblast Growth Factors; Glycosaminoglycan; Heparan Sulfate; Polysaccharide; Signaling Complex; Three-dimensional Cellular Printing.

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Figures

FIGURE 1.
FIGURE 1.
Synthesis of di-block and tri-block HS copolymer using a chemoenzymatic approach. A, a recombinant catalyst composed of parts of both PmHS1 and -2 heparosan synthases, Chimera G, drives the elongation of a short acceptor into the first ∼8-kDa molecular mass section of the block copolymer with either protection (R = TFA) or acetylation (R = CH3CO) at the amine position of the glucosamine residue. B, after this first series of elongations, a second elongation, of the initial ∼8 kDa polymer to a total molecular mass of ∼16 kDa, is done to form a di-block copolymer. In the second series, the R′ modification of the amine residue is the reverse of the R-group in the first series. C, for tri-block copolymers, a final elongation was performed to a total molecular mass of ∼24 kDa, with R-groups identical to the first series. After the synthesis of these block copolymers, the substrates were de-NTFA-protected and N-sulfonated. Finally, the di-block and tri-block copolymers underwent treatment with C5-epimerase/2-O sulfotransferase (C5-Epi, 2-OST) and 6-O sulfotransferase isoform 1 and 3 (6-OST-1,3) in sequential steps. The sulfonated di-block and tri-block copolymers were ∼20 and 30 kDa in molecular mass, respectively (see Fig. 3B for their structures).
FIGURE 2.
FIGURE 2.
The formation of the FGF, HS, and FGFR ternary complex has previously been described by two unique mechanisms. A, the FGF2-HS2-FGFR2 model was first described by Schlessinger et al. (50) and describes a model in which the non-reducing end of two HSPGs interacts with dimeric complex of FGF2-FGFR2 to complete the ternary complex and initiate cell signaling. B, in the Pellegrini model (51), only a single molecule of HS is required for interaction with the FGF2-FGFR2 dimeric complex. In this model, the domain specificity of the sulfation pattern was less significant, as the whole HS chain was considered in the ternary complex. C, a goal of the current work is to test the domain sulfation pattern (red = high sulfation; green = low sulfation) of HS against cellular proliferation promoted by the formation of the FGF-HS-FGFR ternary complex. Each HS block copolymer is unique, in that is contains only reducing end sulfation (NS), only non-reducing end sulfation (SN), sulfation of both ends (SNS), or sulfation of neither (NSN). The arrows at the end of these substrates indicate the reducing end of the substrate. The + and − shown in the parentheses indicate the relative strength of signaling shown by each HS block copolymer.
FIGURE 3.
FIGURE 3.
Polyacrylamide gel electrophoresis of the final block copolymer products and their structures. A, PAGE analysis shows six lanes, a heparin decasaccharide standard, a mixture of heparin-derived oligosaccharide standards, and the SN, NS, NSN and SNS block copolymers. B, the chemical structures of the HS block copolymer final products are shown with their sulfo groups highlighted in yellow and the carboxyl groups of their iduronic acid residues highlighted in pink. The letters a, b, and c correspond to the repeat number of disaccharides in each block.
FIGURE 4.
FIGURE 4.
1H NMR spectra of block copolymer intermediates afforded after N-de-trifluoroacetylation and N-sulfonation. A, SN; B, NS; C, NSN; and D, SNS.
FIGURE 5.
FIGURE 5.
LC-MS analysis of AMAC-labeled disaccharides prepared from block copolymer intermediates (before O-sulfotransferases and C5-epimerase treatment) and final products (after O-sulfotransferases and C5-epimerase treatment) using heparin lyase digestion. Total ion chromatogram A, standard AMAC-labeled disaccharides. B, block copolymer intermediate SN. C, block copolymer intermediate NS. D, block copolymer intermediate NSN. E, block copolymer intermediate SNS. F, block copolymer final product SN. G, block copolymer final product NS. H, block copolymer final product NSN. I, block copolymer final product SNS. Different AMAC-labeled disaccharides show different response factors, thus a standard curve was constructed using each to calculate the molar ratio, presented in Table 1.
FIGURE 6.
FIGURE 6.
A 96-well microtiter plate based assay probing the affect of block copolymers on cellular proliferation via FGFR3c expressing BaF3 cells. In the case of the 30-kDa block copolymers (SNS and NSN), interactions with FGF1 (A) and FGF2 (B) indicated that the block copolymers with high levels of non-reducing end sulfation, SNS, appeared to be a better promoter of cellular proliferation through the FGF-HS-FGFR ternary complex than its complement, NSN. In the case of FGF7 (C) there was no clear difference between the levels of cellular proliferation promoted by SNS against NSN. These near-zero levels of proliferation were expected from FGF7 and are consistent with previously published literature (48). When comparing the 20-kDa block copolymers (SN and NS), better levels of proliferation are seen from the block copolymer with high levels of non-reducing end sulfation (SN) versus the block copolymer with high reducing end sulfation (NS). In the cases of FGF1 (D) and FGF2 (E), there are obvious differences in the levels of proliferation, similar to those seen in panel A and B. There were low-to-zero background levels of proliferation for F, the interaction with FGF7. Each of these FGF-block copolymer-FGFR combinations was tested in 8 replicates in a 96-well plate. These data were normalized against a positive control of FGF1-heparin-FGFR3c growth and a negative control of zero GAG added.
FIGURE 7.
FIGURE 7.
High cell density microarray-based printing allows for probing the HS block copolymer-mediated FGF-FGFR signaling allowing the direct comparison of large numbers of replicates on a single slide. On the microarray chip, 48 replicates can be assayed using the same amount of material required for 1 replicate in a 96-well microtiter plate assay. This improvement allows for increased statistical significance in experimentation while also reducing the amount of overall material needed. Shown are 16-spot snapshots of SNS-induced growth (A), NSN-induced growth (B), SN-induced growth (C), and NS-induced growth (D) with FGF2 and FGFR3c expressing cells. Within each snapshot, these fluorescent intensity images allow for the qualitative assessment of each slide before a more thorough fluorescent intensity quantification using computational software. Block copolymers probed against FGF2 and FGFR1c (E), FGFR2c (F), or FGFR3c (G) indicate that non-reducing end sulfonation is highly important to completing the FGF-HS-FGFR ternary complex. In all cases there were statistical (* = p < 0.05; ** = p < 0.01; *** = p < 0.001) differences in cellular proliferation when comparing SNS and NSN or SN and NS. The relative proliferation percentage (plotted on the y axis) was normalized against a positive control of FGF-heparin-FGFR proliferation and a negative control of no GAG added, non-growth.
FIGURE 8.
FIGURE 8.
Using high cell density microarray printing, a basic dose-response curve was constructed using cells that express FGFR1c, FGF2, and the tri-block copolymers, SNS (●) and NSN (■). Based on these results, we demonstrate that SNS, the tri-block copolymer with a highly sulfated domains at both its reducing and non-reducing ends, promoted cellular proliferation at lower levels of FGF2 concentration than the NSN tri-block copolymer having under sulfated domains at both its reducing and non-reducing ends. These results suggest that a 2:2:2 model of FGF-HSPG-FGFR interaction is preferred over the formation of a 2:1:2 model.

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References

    1. Ly M., Laremore T. N., Linhardt R. J. (2010) Proteoglycomics. Recent progress and future challenges. OMICS 14, 389–399 - PMC - PubMed
    1. Caterson B., Flannery C. R., Hughes C. E., Little C. B. (2000) Mechanisms involved in cartilage proteoglycan catabolism. Matrix Biol. 19, 333–344 - PubMed
    1. Kresse H., Schönherr E. (2001) Proteoglycans of the extracellular matrix and growth control. J. Cell. Physiol. 189, 266–274 - PubMed
    1. Sasisekharan R., Venkataraman G. (2000) Heparin and heparan sulfate. Biosynthesis, structure, and function. Curr. Opin. Chem. Biol. 4, 626–631 - PubMed
    1. Jin L., Abrahams J. P., Skinner R., Petitou M., Pike R. N., Carrell R. W. (1997) The anticoagulant activation of antithrombin by heparin. Proc. Natl. Acad. Sci. U.S.A. 94, 14683–14688 - PMC - PubMed

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