title: Epitope mapping by a Wnt-blocking antibody: evidence of the Wnt binding domain in heparan sulfate.
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.
authors.affiliation: Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.|Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Cell Biology, School of Basic Medical Science, Nanjing Medical University, Nanjing, Jiangsu, 210029, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Gao
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA.
authors.forename: Yongmei
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA.
authors.forename: Jian
authors.initials: J
authors.lastname: Liu
authors.affiliation: Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Mitchell
authors.initials: M
authors.lastname: Ho
mesh_terms: D000818:Animals; D019138:Antibodies, Blocking / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000918:Antibody Specificity; D001665:Binding Sites; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002460:Cell Line; D003412:Cricetulus; D018604:Epitope Mapping / Q000379:methods*; D053673:Glypicans / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D006497:Heparan Sulfate / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007739:L Cells; D051379:Mice; D009844:Oligosaccharides / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains; D015398:Signal Transduction; D051153:Wnt Proteins / Q000037:antagonists & inhibitors* / Q000276:immunology* / Q000378:metabolism
chemical_list: D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; D053673:Glypicans; D006497:Heparan Sulfate; D009844:Oligosaccharides; D051153:Wnt Proteins; C105937:GPC3 protein, human