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 modifications 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 protein involved in Wg secretion. Together, our data demonstrate the in vivo roles of N-glycosylation and lipid modification in Wg secretion and signaling.

A dynamic population of stromal cells contributes to the follicle stem cell niche in theDrosophilaovary

“…Many of these foci co-stained with an antibody against Wntless, which is known to be co-trafficked with Wg in vesicles ( Fig. 3A) (Tang et al, 2012). This indicates that Wg protein is indeed present in escort cells.…”

N-glycosylation requirements in neuromuscular synaptogenesis

“…In testing three well-characterized signaling pathways, we found that WG accumulates, whereas both GBB and JEB are reduced in the Mgat1 null synaptomatrix. WG has two N-glycosylation sites, but these do not regulate ligand expression (Tang et al, 2012), suggesting WG build-up occurs owing to lost synaptomatrix Nglycosylation. Importantly, WG overexpression increases NMJ bouton formation similarly to the phenotype of Mgat1 nulls (Ataman et al, 2008;Korkut et al, 2009), suggesting a possible causal mechanism.…”

Making, Exporting, and Modulating Wnts

“…All Wnts bar one (Drosophila WntD, see below) undergo post-translational glycosylation and acylation. The role of glycosylation is unclear, with some reports suggesting that it is largely dispensable (Mason et al, 1992, Tang et al, 2012, whilst others propose that glycosylation is important for efficient secretion (Komekado et al, 2007, Kurayoshi et al, 2007 but not for signalling activity (Kurayoshi et al, 2007). In contrast, acylation is essential for the secretion of Wnts as well as their signalling activity (Willert et al, 2003).…”

“…No other secreted protein is known to undergo such modification (Hedgehog family members carry distinct lipids, namely palmitate and cholesterol). Several studies have investigated the role of Wnts' palmitoleate (Komekado et al, 2007, Tang et al, 2012. It is now widely accepted that lipidation occurs at a single serine residue and is essential for Wnt function (Janda et al, 2012, Takada et al, 2006.…”