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.

Keywords: RANKL; conformational change; dimerization; heparan sulfate; osteoblast; osteoclastogenesis; osteoprotegerin; small-angle X-ray scattering (SAXS); ternary complex.

FIGURE 1.

Schematic view of the OPG domain structure. The N-terminal cysteine-rich domains (CRD1–4) are responsible for RANKL binding. The structure and exact function of the two death domains (D1 and D2) and the last C-terminal domain (Tail) are largely unknown. Amino acid numbering is based on mouse OPG.

FIGURE 2.

Binding of OPG to osteoblasts is HS dependent. A, binding of recombinant mouse OPG (mOPG, 20 ng to 1 μg/ml) to MC3T3 cells was determined by a FACS-based binding assay. The bound mOPG were detected by staining with a goat anti-mOPG antibody, followed by anti-goat IgG-Alexa 647. The shaded histogram is from cells stained with only with antibodies. B, binding of mOPG (100 ng/ml) to untreated MC3T3 cells or cells pretreated with heparin lyase III (HL-III). C, the inhibitory effects of heparin-derived octasaccharide (H8), decasaccharide (H10), and dodecasaccharide (H12) on mOPG binding to MC3T3 cells was determined by FACS assay. Oligosaccharides were used at 10 nm to 40 μm. Data are representative of at least three separate assays. D, comparison of the inhibitory effects of H12 and H14. Oligosaccharides were used at 30 nm (narrow line) and 300 nm (thick line).

FIGURE 3.

2-O-Sulfation plays a critical role in mediating HS-OPG interactions. A, binding of mOPG (100 ng/ml) to CHO-K1 and pgsF (2-O-sulfotransferase deficient CHO) cells are determined by FACS. B, binding of mOPG (100 ng/ml) to pgsD CHO cells. pgsD cells express no HS but abundant chondroitin sulfate is at the cell surface. Data are representative of at least three separate assays.

FIGURE 4.

HS induces OPG dimerization. A, HS forms a stable complex with OPG dimer. 40 μg (0.8 nmol) of mOPG dimer was incubated with 3 μg (0.8 nmol) of heparin-derived dodecasaccharide (H12) at room temperature for 2 h and resolved on a SEC column (Enrich650, Bio-Rad). B, HS induces dimerization of the OPG monomer. 20 μg (0.4 nmol) of mOPG monomer was incubated with 0.35 (0.1 nmol), 0.7 (0.2 nmol), or 1.4 μg (0.4 nmol) of H12 at room temperature for 2 h and resolved on a SEC column.

FIGURE 5.

Mechanism of OPG dimerization. A, naturally occurring OPG dimer requires formation of intermolecular disulfide bond. Purification of WT mOPG or C400A and C400S mutants on heparin-Sepharose is shown. The dotted line represents the salt gradient (in conductivity mS/cm). The WT mOPG was eluted as two peaks (low-salt peak is monomer, high-salt peak is dimer), whereas both cysteine 400 mutants were eluted as a single low-salt peak. B, HS-induced OPG dimer does not require intermolecular disulfide bond. 30 μg of C400A monomer was incubated with 2 μg of H12 at room temperature for 2 h and resolved on a SEC column.

FIGURE 6.

HS binding induces conformational change of OPG. A, raw SAXS scattering curves of SEC purified mOPG dimer or dimer-H12 complex collected at 4 mg/ml. B, P(r) function plots of mOPG dimer or dimer-H12 complex. C, Kratky plots of mOPG dimer or dimer-H12 complex. D, raw SAXS scattering curves of SEC purified mOPG dimer-H12 and monomer-H12 complexes collected at 4 mg/ml.

FIGURE 7.

HS, OPG, and RANKL form a ternary complex. A, binding of purified RANKL (60 μg) to heparin-Sepharose. Dotted line represents the salt gradient. B, OPG dimer or purified OPG dimer-RANKL complex were bound to a heparin-Sepharose column and eluted with a salt gradient (dotted line). Fractions (16 to 19 ml, 0.5 ml/fraction) of the OPG-RANKL complex run were resolved by SDS-PAGE and visualized by silver staining. C, OPG monomer (20 μg), RANKL (20 μg), and H12 (2 μg) were incubated in different combinations as indicated for 4 h at 25 °C. The complexes were resolved on an SEC column. The elution positions of the M_r standards BSA, IgY, and ferritin are marked with black triangles (13.5, 12.05, and 11.05 ml, respectively). Two peak fractions of the ternary complex (11.8 ml peak) were visualized by silver stain. D, binding of RANKL or preformed RANKL-OPG complex (100 ng/ml) to MC3T3 cells determined by FACS. RANKL binding was detected by a phycoerythrin (PE)-conjugated anti-mouse RANKL mAb. E, binding of the RANKL-OPG complex to MC3T3 cells shown in double staining dot plot using anti-RANKL-PE and goat anti-mOPG followed by anti-goat IgG-Alexa 647.

FIGURE 8.

The HS-binding site consists of residues from D2 and Tail domains. A, binding of purified WT or mutants (all dimers) to heparin was analyzed by a 1-ml HiTrap heparin-Sepharose column at pH 7.1. The salt concentrations required for elution are plotted. Mutants that show >100 mm reduction in elution salt concentration are marked in bold. B, binding of OPG mutants (R370A/R379A or K359A/R370A/R379A, all dimers, 33 ng/ml) and wild-type mOPG (dimer and monomer, 33 ng/ml) to cell surface HS was determined by FACS.

FIGURE 9.

HS-OPG interactions lower the inhibitory threshold of OPG against RANKL. A, inhibition of osteoclastogenesis in osteoblasts/bone marrow macrophage co-culture by WT OPG dimer or triple mutant K359A/R370A/R379A at 1, 3, 10, 30, and 100 ng/ml. The extent of osteoclastogenesis is assessed by an enzymatic assay of TRAP activity in whole cell lysate. n = 3. Error bars represent S.D. * represents p < 0.01 by Student's t test. Data are representative of at least three separate assays. B–E, representative images of differently treated co-culture. Mature osteoclasts are visualized by TRAP staining. The pink multinucleated cells are osteoclasts and the round clear cells are undifferentiated bone marrow macrophages. F, inhibition of osteoclastogenesis in bone marrow macrophage monoculture by WT OPG dimer or triple mutant OPG at 50, 100, 200, and 500 ng/ml. Osteoclastogenesis is induced by recombinant soluble RANKL (50 ng/ml) and M-CSF (20 ng/ml).

FIGURE 10.

The proposed model by which HS regulates OPG-RANKL interactions. Secreted WT OPG is immobilized on cell surface HSPG through binding to the HS chain, which brings OPG to close proximity to the membrane-bound RANKL and increases the probability of successful engagement between OPG and RANKL. Binding to HS also induces a significant conformation change of OPG, which might further facilitate OPG-RANKL interactions. In contrast, HS binding-deficient mutant OPG can only access membrane-bound RANKL through diffusion, which leads to less efficient inhibition of RANKL.