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. 2009 Aug 21;284(34):23159-68.
doi: 10.1074/jbc.M109.025478. Epub 2009 Jun 24.

Characterization of wise protein and its molecular mechanism to interact with both Wnt and BMP signals

Katherine B Lintern  1 , Sonia Guidato, Alison Rowe, José W Saldanha, Nobue Itasaki
Affiliations

Characterization of wise protein and its molecular mechanism to interact with both Wnt and BMP signals

Katherine B Lintern et al. J Biol Chem. .

Abstract

Cross-talk of BMP and Wnt signaling pathways has been implicated in many aspects of biological events during embryogenesis and in adulthood. A secreted protein Wise and its orthologs (Sostdc1, USAG-1, and Ectodin) have been shown to modulate Wnt signaling and also inhibit BMP signals. Modulation of Wnt signaling activity by Wise is brought about by an interaction with the Wnt co-receptor LRP6, whereas BMP inhibition is by binding to BMP ligands VSports手机版. Here we have investigated the mode of action of Wise on Wnt and BMP signals. It was found that Wise binds LRP6 through one of three loops formed by the cystine knot. The Wise deletion construct lacking the LRP6-interacting loop domain nevertheless binds BMP4 and inhibits BMP signals. Moreover, BMP4 does not interfere with Wise-LRP6 binding, suggesting separate domains for the physical interaction. Functional assays also show that the ability of Wise to block Wnt1 activity through LRP6 is not impeded by BMP4. In contrast, the ability of Wise to inhibit BMP4 is prevented by additional LRP6, implying a preference of Wise in binding LRP6 over BMP4. In addition to the interaction of Wise with BMP4 and LRP6, the molecular characteristics of Wise, such as glycosylation and association with heparan sulfate proteoglycans on the cell surface, are suggested. This study helps to understand the multiple functions of Wise at the molecular level and suggests a possible role for Wise in balancing Wnt and BMP signals. .

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Figures

FIGURE 1.
FIGURE 1.
Structure of chick Wise protein. A, stereo ribbon representation of the chick Wise three-dimensional structural model (residues 68–186). Purple, β-strands; green, loop regions. Yellow, disulfide bonds in the cystine knot plus a further disulfide (cysteines 89 and 147) linking two fingers of the structure. N- and C-terminal ends are indicated. B, schematic drawing of the full-length chick Wise structure. Arrowhead, the predicted signal sequence cleavage site for secretion; black dot, asparagine at position 47 (N47), the glycosylated site revealed in this study. Six cysteine residues forming the “cystine knot” are shown in circles, and disulfide bonds for the knot formation are shown by dotted lines. Three loops (Finger 1, Heel, and Finger 2) are indicated. The scheme also shows the deleted parts of Wise constructs ΔN, Δheel, and ΔC.
FIGURE 2.
FIGURE 2.
Glycosylation of Wise. A–D, conditioned media from cells transfected with Wise (A), Wise(ΔC) (B), Wise(ΔN) (C), or Wise(N47A) (D), treated with O-glycosidase or PNGase F. Wise and Wise(ΔC) are sensitive to PNGase F and not to O-glycosidase. In B, the cell extract was also loaded, showing that the majority of Wise protein in the cell extract is a non-glycosylated form, and only a small amount of the glycosylated form exists. Wise(ΔN) and Wise(N47A) are not sensitive to PNGase F. E, conditioned medium of Wise treated with endo-β-N-acetylglucosaminidase D (Endo D) or endo-β-N-acetylglucosaminidase H (Endo H). Wise is sensitive to endo-β-N-acetylglucosaminidase D and resistant to endo-β-N-acetylglucosaminidase H.
FIGURE 3.
FIGURE 3.
Secretion and proteoglycan binding of Wise. A, HEK293 cells were transfected with DNA constructs, as indicated above the blots, together with green fluorescent protein DNA, and the cell extracts and conditioned media were analyzed on Western blots. β-Tubulin is a loading control for the cell extract, and green fluorescent protein serves as a control for transfection efficiency. Secretion efficiency of Wise(ΔN) is significantly low, whereas that of Wise(Δheel) is high. Wise(ΔC) in the cell extract is below the detectable level. B and C, HEK293 cells were transfected with Myc-LRP6 (green) and treated with either Wise (B, red) or Wise(Δheel) (C, red) conditioned medium. Wise is bound strongly to the LRP6-transfected cells in addition to non-transfected cells. Wise(Δheel) does not bind to LRP6-transfected cells or to non-transfected cells. D, release of Wise protein into the conditioned medium after treatment with sodium chlorate. Wise-transfected HEK293 cells treated with sodium chlorate show an increased amount of Wise protein in the conditioned medium and a lesser amount in the cell extract. E, HEK293 cells were transfected with Myc-LRP6 (green) and treated with Wise (red) medium along with heparan sulfate. In contrast to B, Wise is bound specifically to LRP6-transfected cells.
FIGURE 4.
FIGURE 4.
Effect of Wise deletion constructs on LRP6. A, immunoprecipitation assay. HEK293 cells were transfected with control or FLAG-tagged Wise constructs, as indicated, together with a construct encoding the LRP6 extracellular domain with a Myc tag (LRP6ECD). The amount of DNA used for Wise(ΔN) and Wise(Δheel) was either increased or decreased, respectively, due to different secretion efficacies seen in Fig. 3A. The conditioned media were collected and used for immunoprecipitation (IP) with anti-FLAG antibody. A small volume of the media from each sample was loaded separately to examine the expression of proteins (top two panels). Immunoprecipitation of Wise and LRP6ECD is shown in the bottom panels. Despite the large amount of Wise(Δheel) in the conditioned medium, it fails to precipitate LRP6ECD. B, TOPflash reporter assay. HEK293 cells were transfected with TOPflash and control Renilla reporters and Wnt1 construct and then treated with conditioned media of various Wise mutants. The conditioned media were first concentrated five times and checked on Western blots. The required samples were then diluted so that each of the media contained a comparable amount of Wise proteins. After the adjustment, the media were applied to the transfected cells and also reexamined on Western blots (shown in supplemental Fig. 4). The graph shows the relative luciferase units, normalized to the control sample (no Wnt1). Wise(Δheel) shows a decreased ability to block Wnt1-dependent reporter activity.
FIGURE 5.
FIGURE 5.
Effect of Wise deletion constructs on BMP4. A, immunoprecipitation (IP) assay. HEK293 cells were transfected with control or FLAG-tagged Wise constructs, as indicated above, together with a construct encoding Myc-tagged BMP4. The amounts of DNA used for Wise(ΔN) and Wise(Δheel) were adjusted in a manner similar to that in Fig. 4A. The conditioned media were collected from each and used for immunoprecipitation with anti-FLAG antibody. Input samples are shown in the top two panels. Immunoprecipitation of Wise and BMP4 is shown in the bottom two panels. There was no significant difference in the precipitation efficiency except that Wise(ΔN) showed less precipitation of BMP4 relative to the abundant amount of Wise(ΔN) inputs. This trend was not consistently seen in other experiments. B, phospho-Smad1/5/8 assay. HEK293 cells were treated with a mixture of recombinant BMP4 protein and Wise conditioned medium (Wise, N47A, or Δheel), as indicated, and the cells were collected to detect phosphorylated Smad (p-Smad)1/5/8 (red) on a Western blot with a fluorescent secondary antibody. β-Tubulin (green) is a loading control. Each channel is also shown in black and white. The bottom panel shows a Western blot of input Wise media. *, a nonspecific band. C, BRE reporter assay. HEK293 cells were transfected with BMP4 and Wise constructs, as indicated, together with BRE and control Renilla reporters. The graph shows the relative luciferase units, normalized to the control sample (no BMP4). Wise, N47A, and Δheel suppress BMP4 activity.
FIGURE 6.
FIGURE 6.
Function of Wise on LRP6 and BMP4. A, immunoprecipitation (IP) assay. Conditioned media of HEK293 cells separately transfected with Wise or LRP6IgG were mixed together with bovine serum albumin or recombinant BMP4, as indicated (+, 100 ng; ++, 200 ng/700 μl) and immunoprecipitated with LRP6IgG, and the precipitated samples were analyzed on Western blots using anti-FLAG (Wise), anti-BMP4, or anti-Fc antibodies. Inputs of BMP4 and LRP6 indicate the presence of these proteins in the mix of relevant conditioned media and proteins. Input Wise is not shown because it was undetectable after diluting with other conditioned media. Concentrated Wise medium was checked on a separate blot prior to use, and an equal volume of the medium from the same batch was used for each of the immunoprecipitation samples. Immunoprecipitation of Wise (upper panel) is not compromised by the presence of BMP4. *, nonspecific bands. B, immunostaining of HEK293 cells stably expressing Myc-tagged LRP6 (green), treated with conditioned medium of FLAG-tagged Wise (red), without or with recombinant BMP4 (100 ng/ml). Additional BMP4 does not interfere with the binding of Wise to LRP6. C, TOPflash reporter assay. HEK293 cells were transfected with Wnt1 (0.1 μg), Wise (0.1 μg), and/or BMP4 (+, 0.1 μg; ++, 0.2 μg) constructs, as indicated, together with TOPflash and control Renilla reporters. Activation of TOPflash reporter by Wnt1 is suppressed by Wise. The addition of BMP4 does not affect the function of Wise in Wnt1 inhibition. D, BRE reporter assay. HEK293 cells were transfected with BMP4 (0.05 μg), Wise (0.3 μg), and/or LRP6 (+, 0.005 μg; ++, 0.01 μg) constructs, as indicated, together with BRE and control Renilla reporters. Suppression of BMP4 activity by Wise is largely prevented by additional LRP6. BSA, bovine serum albumin.
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