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.