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Epitope Mapping by a Wnt-blocking Antibody: Evidence of the Wnt Binding Domain in Heparan Sulfate

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Epitope Mapping by a Wnt-blocking Antibody: Evidence of the Wnt Binding Domain in Heparan Sulfate

Wei Gao et al. Sci Rep.

Abstract

Heparan sulfate (HS) is a polysaccharide known to modulate many important biological processes, including Wnt signaling. However, the biochemical interaction between HS and Wnt molecules is not well characterized largely due to the lack of suitable methods. To determine the Wnt binding domain in HS, we used a Wnt signaling-inhibitory antibody (HS20) and a panel of synthetic HS oligosaccharides with distinct lengths and sulfation modifications. We found that the binding of HS20 to heparan sulfate required sulfation at both the C2 position (2-O-sulfation) and C6 position (6-O-sulfation). The oligosaccharides with the greatest competitive effect for HS20 binding were between six and eight saccharide residues in length. Additionally, a four residue-long oligosaccharide could also be recognized by HS20 if an additional 3-O-sulfation modification was present. Furthermore, similar oligosaccharides with 2-O, 6-O and 3-O-sulfations showed inhibition for Wnt activation. These results have revealed that HS20 and Wnt recognize a HS structure containing IdoA2S and GlcNS6S, and that the 3-O-sulfation in GlcNS6S3S significantly enhances the binding of both HS20 and Wnt. This study provides the evidence for identifying the Wnt binding domain in HS and suggests a therapeutic approach to target the interaction of Wnt and HS in cancer and other diseases.

Figures

Figure 1
Figure 1. The binding properties of HS20 monoclonal antibody.
(a) The schematic structure of GPC3, the human monoclonal antibody HS20 recognizing the heparan sulfate chains on GPC3 and the mouse monoclonal antibody YP7 recognizing a C-terminal region of the GPC3 core protein. (b) HS20 did not bind the mutant GPC3 without heparan sulfate chains (GPC3ΔHS). ELISA results showed the binding properties of HS20 and YP7 on wild type GPC3 and GPC3ΔHS. BSA was used as the negative antigen control. Values represent mean ± SD, t-test. (c) The binding property of HS20 to various glypicans. ELISA results show the binding affinities of HS20 for GPC1, GPC3, GPC5 and GPC6. The X-axis represents the concentration of HS20, while the Y axis indicates protein binding as determined by the OD450 nm. (d) HS20 lost binding capacity on the cells without heparan sulfate. Flow results showed the binding of HS20 for CHO-K1 cells and pgsA-745 cells, which lack the ability to produce HS and CS. (e) HS but not CS inhibited HS20 binding for GPC3. A competitive ELISA where HS20 (5 μg/ml) was pre-incubated with increasing concentrations of HS or bikunin (CS) for 30 minutes, and then added to an ELISA plate to detect the binding to GPC3.
Figure 2
Figure 2. HPLC chromatograms of synthesized oligosaccharides.
Both DEAE-HPLC and PAMN-HPLC methods were used to analyze the purity of each individual oligosaccharide. Overall, the purity for each compound was determined to be >90%.
Figure 3
Figure 3. The binding of HS20 required both 2-O-sulfation and 6-O-sulfation.
(a) Structures of the synthetic oligosaccharides (12mers) with different sulfations. (b) Both 2-O-sulfation and 6-O-sulfation were required for HS20 binding in a competitive ELISA. Indicated oligosaccharides were pre-incubated with the HS20 antibody for 30 minutes, and then the mixture was added into an ELISA plate to detect the binding of GPC3 or GPC1. HS was used as a positive control inhibitor.
Figure 4
Figure 4. The minimal HS binding motif of HS20 was a 6mer with both 2-O-sulfation and 6-O-sulfation.
(a) Structures of the synthetic oligosaccharides (4mer to 12mer) with both 2-O and 6-O-sulfation. (b) The 2-O and 6-O sulfated oligosaccharides with lengths longer than 4mer inhibited HS20 binding. Competitive ELISA was performed by pre-incubating oligosaccharides with HS20 for 30 minutes, and then adding the mixture into an ELISA plate to detect the binding to GPC3.
Figure 5
Figure 5. The addition of a 3-O-sulfation to oligosaccharides further inhibits HS20 binding.
(a) Structures of the synthetic oligosaccharides (4mer to 12mer) with both 2-O and 6-O-sulfations (mer-5), with an extra 3-O-sulfation (mer-6), and with two extra 3-O-sulfations (mer-7). (b) The oligosaccharides with an extra 3-O-sulfation showed more inhibition of HS20 binding. Competitive ELISAs were performed on various 4mer, 6mer and 12mer oligosaccharides. Dashed lines indicate the value of IC50. (c) A competitive ELISA with various 12mers that have differences in the sulfation position and number.
Figure 6
Figure 6. The effect of oligosaccharide variation on Wnt binding.
(a) Wnt binding HS required 6-O-sulfation. Topflash assay: 50% Wnt3aCM was pre-incubated with HS or indicated oligosaccharides for 30 minutes and then added to HEK293Topflash cells. Luciferase activity was measured 6 hours later and normalized by total protein. HS was set up as a positive inhibitor. (b) 8mer, 10mer and 12mer polysaccharides with 2-O and 6-O sulfations inhibited Wnt activation. A Topflash assay was conducted as with 6mer, 8mer, 10mer, and 12mer oligosaccharides that contained 2-O and 6-O sulfations. The experiment followed the same procedure that was previously mentioned. (c) A 3-O sulfation on 2-O and 6-O sulfated oligosaccharides had enhanced Wnt inhibition. A Topflash assay was used to determine the effect of adding a 3-O sulfation on 2-O and 6-O sulfated oligosaccharides had enhanced Wnt inhibition to the various length oligosaccharides on Wnt signaling. The experiment followed the same procedure that was previously mentioned. (d) Two extra 3-O sulfations on 2-O and 6-O sulfated oligosaccharides showed further inhibition on Wnt activation. A Topflash assay was used to determine the effect of adding a second 3-O sulfation to a 12mer oligosaccharide. For all figures, values represent mean ± s.d.
Figure 7
Figure 7. A comparison of Wnt and HS20 binding motifs on oligosaccharides.
(a) Hypothetical schematic structures for HS20-binding HS. HS20-binding oligosaccharides require both 2-O and 6-O sulfations (bold). The site of 3-O-sulfation (bold) is shown by a green box. The minimal oligosaccharide recognized by HS20 is shown as a 6mer (light blue region) or as a 4mer with a 3-O-sulfation (purple region). (b) Hypothetical schematic structures for Wnt-binding HS. Wnt-recognized oligosaccharides require 6-O sulfation (bold). The sites of 3-O-sulfation are shown by green boxes. The minimal oligosaccharide recognized by Wnt is an 8mer (light pink region) or a 6mer with a 3-O-sulfation (orange region).

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