title: Heparan Sulfate Regulates the Structure and Function of Osteoprotegerin in Osteoclastogenesis.
abstract: Osteoprotegerin (OPG), a decoy receptor secreted by osteoblasts, is a major negative regulator of bone resorption. It functions by neutralizing the receptor activator of nuclear factor κB ligand (RANKL), which plays a central role in promoting osteoclastogenesis. OPG is known to be a high-affinity heparan sulfate (HS)-binding protein. Presumably, HS could regulate the function of OPG and affect how it inhibits RANKL. However, the molecular detail of HS-OPG interaction remains poorly understood, which hinders our understanding of how HS functions in osteoclastogenesis. Here we report mapping of the HS-binding site of OPG. The HS-binding site, identified by mutagenesis study, consists of eight basic residues that are located mostly at the junction of the second death domain and the C-terminal domain. We further show that heparin-derived dodecasaccharide is able to induce dimerization of OPG monomers with a stoichiometry of 1:1. Small-angle X-ray scattering analysis revealed that upon binding of HS, OPG undergoes a dramatic conformational change, resulting in a more compact and less flexible structure. Importantly, we present here three lines of evidence that HS, OPG, and RANKL form a stable ternary complex. Using a HS binding-deficient OPG mutant, we further show that in an osteoblast/bone marrow macrophage co-culture system, immobilization of OPG by HS at the osteoblast cell surface substantially lowers the inhibitory threshold of OPG toward RANKL. These discoveries strongly suggest that HS plays an active role in regulating OPG-RANKL interaction and osteoclastogenesis.
authors.affiliation: From the Department of Oral Biology, School of Dental Medicine, University at Buffalo, the State University of New York, Buffalo, New York 14214.
authors.forename: Miaomiao
authors.initials: M
authors.lastname: Li
authors.affiliation: From the Department of Oral Biology, School of Dental Medicine, University at Buffalo, the State University of New York, Buffalo, New York 14214.
authors.forename: Shuying
authors.initials: S
authors.lastname: Yang
authors.affiliation: From the Department of Oral Biology, School of Dental Medicine, University at Buffalo, the State University of New York, Buffalo, New York 14214 dingxu@buffalo.edu.
authors.forename: Ding
authors.identifier: http://orcid.org/0000-0001-9380-2712
authors.initials: D
authors.lastname: Xu
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D018920:Coculture Techniques; D006497:Heparan Sulfate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008264:Macrophages / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism*; D053244:Osteoprotegerin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053245:RANK Ligand / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D006497:Heparan Sulfate; D053244:Osteoprotegerin; D053245:RANK Ligand; C506031:Tnfrsf11b protein, mouse; C505689:Tnfsf11 protein, mouse
keywords: RANKL; conformational change; dimerization; heparan sulfate; osteoblast; osteoclastogenesis; osteoprotegerin; small-angle X-ray scattering (SAXS); ternary complex