Roles of N-glycosylation and lipidation in Wg secretion and signaling

Abstract

Wnt members act as morphogens essential for embryonic patterning and adult homeostasis. Currently, it is still unclear how Wnt secretion and its gradient formation are regulated. In this study, we examined the roles of N-glycosylation and lipidation/acylation in regulating the activities of Wingless (Wg), the main Drosophila Wnt member. We show that Wg mutant devoid of all the N-glycosylations exhibits no major defects in either secretion or signaling, indicating that N-glycosylation is dispensable for Wg activities. We demonstrate that lipid modification at Serine 239 (S239) rather than that at Cysteine 93 (C93) plays a more important role in regulating Wg signaling in multiple developmental contexts. Wg S239 mutant exhibits a reduced ability to bind its receptor, Drosophila Frizzled 2 (dFz2), suggesting that S239 is involved in the formation of a Wg/receptor complex. Importantly, while single Wg C93 or Wg S239 mutants can be secreted, removal of both acyl groups at C93 and S239 renders Wg incapable of reaching the plasma membrane for secretion. These data argue that lipid modifications at C93 and S239 play major roles in Wg secretion. Further experiments demonstrate that two acyl attachment sites in the Wg protein are required for the interaction of Wg with Wntless (Wls, also known as Evi or Srt), the key cargo receptor involved in Wg secretion. Together, our data demonstrate the in vivo roles of N-glycosylation and lipid modification in Wg secretion and signaling.

Figure 1. Signaling activities of wild-type and mutated Wg in cultured S2 cells and embryos

(A) Autocrine activation luciferase assay. Cells were co-transfected with plasmids encoding Wg (or Wg mutants), dFz2, Renilla luciferase and Firefly luciferase. Cell lysates were processed for topflash assay. (B) Paracrine activation luciferase assay. Donor cells were transfected with plasmids encoding Wg (or Wg mutants) and Renilla luciferase. Receiving cells were transfected with plasmids encoding dFz2 and Firefly luciferase. After two groups of cells were mixed and cocultured for a certain period, cell lysates were processed for topflash assay. In both A and B, relative luciferase activities are reflected by the ratio of Firefly luciferase activity versus Renilla luciferase activity of the same sample lysate. (C–J) Autocrine Wg signaling in the embryos. Ventral cuticles of embryos over-expressing wild-type Wg or Wg mutants under the control of da^Gal4. All embryos shown in this paper are oriented anterior to the left. (K–Q) Paracrine Wg signaling in the embryos. (K) Immuno-staining of GFP reporter indicates the expression of sim^Gal4 in the ventral midline of the embryo. (L) Denticles are fused and form a lawn in Wg null embryos. (M–Q) Cuticle preparations from Wg null embryos rescued by armadillo.S10 (M), wild-type Wg (N), WgC93A (O), WgS239A (P) or WgNN (P) under the control of sim^Gal4.

Figure 2. Signaling activities of wild-type and mutated Wg in the wing imaginal discs

(A) Schematic drawing of wing disc in the late third instar larvae showing A/P and D/V compartments. (B-B″) Wg-dependent Sens expression in the wild-type wing disc is in two narrow stripes abutting Wg-expressing cells. All confocal images in this paper are captured as single optical sections. (C–F″) Sens expression induced by wild-type Wg and Wg mutants. The expression of Wg variants (red) was driven by dpp^Gal4 along the anterior-posterior boundary. Sens activation indicates the short-range Wg signaling (green). All discs in this paper are oriented as shown in Figure 2A.

Figure 3. WgS239A is secreted by S2 cells and the wing disc cells

(A–D′) In somatic clones in the wing discs, wild-type and mutated Wg are expressed under the control of act^Gal4. Note that the punctated structures are missing in cells surrounding the clones in C. (E–G″) Extracellular distribution of wild-type Wg and lipidation mutants in wing discs. Wild-type and mutated Wg were over-expressed in the dorsal compartment of the wing disc under the control of ap^Gal4. The extracellular distribution of Wg was detected by mouse monoclonal antibody and a guinea pig polyclonal antibody was applied to detect the overall expression levels of Wg. (H) Secretion of wild-type Wg and lipidation mutants in S2 cells. S2 cells were transfected with wild-type Wg and two lipidation mutants respectively. Both conditioned medium and lysate were collected and processed for Western blots.

Figure 4. The weaker binding of WgS239A with dFz2 receptor

(A) Co-IP assay. S2 cells were transfected with plasmids encoding wild-type Wg (or WgS239A) and V5-tagged dFz2 (dFz2-V5). Cell lysates were immunoprecipitated and then analyzed by Western blotting with the antibodies indicated. IP, immunoprecipitation; IB, Immunoblot. (B) Quantification of the intensity of the co-precipitated dFz2 bands from co-IP assay in A. The relative intensity (ratio of mutants to wild-type) is shown as mean±s.d. (n= 3, *P<0.05, ** p<0.01 and *** p<0.001, t-test) (C–E″) Cell labeling assay. S2 cells transfected with a dFz2-V5 expression vector were equally split into three samples. Each sample was incubated with the conditioned medium containing similar amount of wild-type Wg (C-C″) or WgC93A (D-D″) or WgS239A (E-E″). Wg proteins trapped on the cell surface by dFz2 were determined by Wg staining. (F) Amount of Wg variants in the conditioned media in the cell labeling assay was determined by Western blotting. Similar protein levels were detected. (G) Quantification of the Wg accumulation in cell labeling assay. Fluorescence intensities of Wg were normalized to the expression levels of dFz2 in individual cells. The relative dFz2 binding (ratio of mutants to wild-type) is shown as mean±s.d. (n[ eld of view]=5–7, *P<0.05, ** p<0.01 and *** p<0.001, t-test)

Figure 5. Removal of double lipidation abolishes Wg signaling activity

The signaling activities of WgCS were examined by the luciferase reporter in S2 cells (A), cuticle patterning in embryos (B) and Sens induction in wing imaginal discs (C-C″). Same experiments were done as shown in Figure 1 and Figure 2.

Figure 6. WgCS is retained in the producing cells

(A–B″) Extracellular distribution of wild-type Wg (A-A″) and WgCS (B-B″) in transfected S2 cells. (C–D″) Extracellular distribution of wild-type Wg (C-C″) and WgCS (D-D″) in wing discs. Expression of Wg variants in the wing disc was driven by ap^Gal4. The membrane-associated WgCS was barely detectable in transfected cells and in wing disc cells. (E) Secretion of wild-type Wg and WgCS in S2 cells. Minimal amount of WgCS was present in the conditioned medium. A dsRed expression vector was co-transfected and blotted as a control for transfection efficiency.

Figure 7. Lipidation promotes the interaction of Wg with Wls

(A–D″) Subcellular localization of Wls-V5 co-expressed with wild-type Wg or lipidation mutants in wing disc cells. Regions far from endogenous Wg pool at the DV boundary were selected for images. (E) Co-IP of Wls with Wg variants. S2 cells were transfected with plasmids encoding V5-tagged Wls without (panel 1) or with one of the four Wg variants (panel 2–4). Cell lysates were immunoprecipitated and then analyzed by Western blotting with the antibodies indicated. (F) Quantification of the intensity of the co-precipitated Wls bands from co-IP assay in E. The relative intensity (ratio of mutants to wild-type) is shown as mean±s.d. (n= 3, ** p<0.01)