RWD1 inhibits Wnt signaling and Ryk function in neurons.

<p>(A) Western blot analysis of lysates from SN4741 cells treated with Wnt5a (300 ng/mL) or vehicle (PBS; C) and with human IgG (50 µg/mL) or RWD1 (50 µg/mL). P-Dvl2, phosphorylated Dvl2. Molecular mass standards are shown at left in kDa. The three lanes were non-consecutive but on the one membrane. The normalized ratio P-Dvl2/total Dvl2 (corrected for β-actin) is shown beneath each lane. (B) Quantification of neurite growth from E15.5 mouse cortical neurons treated with human IgG (50 µg/mL), RWD1 (50 µg/mL), Wnt5a (300 ng/ml) and/or vehicle (PBS; C). Results represent the mean±SEM of four independent experiments. *P&lt;0.05; **P&lt;0.01; ***P&lt;0.001.</p

Proteolytic processing of the mouse Ryk extracellular region.

<p>(A) Proteolysis of Ryk in mammalian cell lines. Cells were transiently transfected with pcDNA3.mM2RFCT and lysed 48 h later. Anti-FLAG immunoprecipitates (IP) were immunoblotted (IB) with rabbit anti-Ryk^IC polyclonal antibody. Molecular mass standards are shown at left in kDa. MEFs, mouse embryonic fibroblasts; RDTI.2, Ryk-deficient large T antigen-immortalized fibroblasts derived from a <i>Ryk</i>^−/− embryo. (B) Consensus cleavage sites in the mouse Ryk extracellular region for PC1, PC2, furin, PC4, PC5, PACE4 and PC7 (single-letter amino acid code; basic residues conforming to the consensus in blue; residues numbered according to NCBI Reference Sequence NP_038677.3; (K/R)X_n(K/R)↓, where X is any residue, n = 0, 2, 4 or 6 and the downward arrow represents cleavage <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075447#pone.0075447-Seidah1" target="_blank">[53]</a>). The furin cleavage site in the mouse c-Met extracellular domain is shown for comparison. (C) COS-7 cells were transiently transfected with plasmids encoding mM2RFCT or the derivatives V594A; K186Q (monobasic, MB); KK181→QQ181 (dibasic, DB); KRRK176→QQQQ176 (tetrabasic, TB); and QQQQ176;QQ181;Q186 (compound mutant; CM). The α₁-PDX.FLAG protein (p54, p57 and p64 isoforms indicated) was expressed to inhibit endogenous furin. Mouse c-Met.FLAG was expressed as a positive control for inhibition of furin. The location and identity of Ryk-CTF was confirmed using anti-Ryk^IC polyclonal antibody (bottom panel). Molecular mass standards are shown at left in kDa. (D) Transiently transfected COS-7 cells were treated with potential activators of receptor shedding for 30 min. Anti-Myc IPs were prepared from the conditioned medium and immunoblotted with an anti-Myc antibody. Molecular mass standards are shown at left in kDa. V, empty vector (pcDNA3)-transfected cells; PBSS, phosphate-buffered saline with calcium and magnesium used as diluent for pervanadate. (E) Transiently transfected COS-7 cells were pre-treated with protease inhibitors, then shedding was activated with TFP (100 µM, 30 min). Anti-Myc IPs were analyzed as in (C). Molecular mass standards are shown at left in kDa. (F) Model for sequential proteolysis of Ryk. The metalloprotease-mediated cleavage in step (ii) has constitutive and inducible components. Green, WIF domain; gold, PTK domain; red, transmembrane helix; Ryk-FL, full-length uncleaved Ryk; Ryk-CTF, Ryk carboxyl-terminal fragment; Ryk-ICD, Ryk intracellular domain fragment; Rβ, predicted membrane-associated peptide analogous to amyloid-β peptide.</p

Generation of mouse MAbs to the RYK extracellular region and epitope mapping.

<p>(A) Flow cytometry using purified mouse anti-RYK MAbs 1B4, 1G8, 5E3 and 6G1 on 293-EBNA cells stably expressing pVITRO3-mcs (empty vector control; V) or hRYKFCT (RYK). All antibodies detected RYK in hRYKFCT-transfected but not vector-transfected cells. (B) Schematic of the mouse Ryk fusion proteins used in this study. EC, extracellular region; WD, WIF domain. (C) Western blot analysis of purified mouse Ryk fusion proteins using mouse anti-RYK MAbs 1B4 and 6G1. The pattern of binding was the same for both antibodies. The presence of all the fusion proteins was confirmed by stripping the membrane and reprobing with rabbit anti-Ryk^EC polyclonal antibody. Molecular mass standards are shown at left in kDa. IB, immunoblot. (D) ELISA results using mouse anti-RYK MAbs 1B4 and 6G1 on an immobilized peptide library of the entire human RYK extracellular region. Peptides 3 to 37: RYK WIF domain; peptide 47: FLAG epitope (incubated with mouse anti-FLAG M2 MAb; positive control); well 48: empty (negative control). The MAbs were used at 2 µg/mL. All antibodies bound to the same epitope, in peptides 40−42. The location of the epitope is shown schematically (bottom). Epitopes for the 1G8 and 5E3 antibodies were identical (data not shown). OD, optical density.</p

Characterization of the human inhibitory anti-RYK IgG_1κ antibody (RWD1).

<p>(A) RWD1 IPs from lysates of HEK293T cells transiently transfected with vectors encoding human RYK domain swap derivatives (lanes 1–5) were immunoblotted (IB) with anti-FLAG antibody. Lysate input (lanes 6–9) is shown at right. Molecular mass standards are shown at left in kDa. WIF1-WD, WIF domain from human WIF1; ROR2-CRD, CRD from human ROR2; H, heavy chain; L, light chain. (B) Anti-FLAG IPs were immunoblotted (IB) with an anti-Myc antibody to detect Wnt binding to hRYK.Fc. Molecular mass standards are shown at left in kDa. (C) ELISA analysis of immobilized RWD1 probed with hRYK.Fc. Increased hRYK.Fc binding to the antibody was observed with higher concentrations of either immobilized RWD1 (left panel) or soluble hRYK.Fc (right panel). Results represent the mean±standard deviation of two or three independent experiments. IgG, human IgG; OD, optical density. (D) HEK293T cells transiently transfected with the plasmids indicated (at right) were fixed as shown (above), paraffin-embedded and subjected to IHC using RWD1 biotinylated on <i>N</i>-glycan chains. Bar in panel (iv) represents 50 µm. NBF, neutral-buffered formalin; PFA, paraformaldehyde. (E) Example of IHC on a tumor from a formalin-fixed and paraffin-embedded human breast cancer tissue microarray stained with bRWD1 (upper panel) or human MAb isotype control (bIgG_1κ; lower panel). Open arrow, RYK on a cancer cell; filled arrow, RYK on tumor stroma; arrowhead, RYK-positive cancer cell nucleus; b, biotinylated on <i>N</i>-glycan chains. Bar represents 50 µm.</p