Apicobasal secretion of Wnt11 and Wnt3a in polarized epithelial cells is regulated by distinct mechanisms

Abstract

SummaryWnts are glycan-and lipid-modified morphogens that are important for cellular responses, but how Wnts are secreted in polarized epithelial cells remains unclear. Although Wntless (Wls) has been shown to interact with Wnts and support their secretion, the role of Wls in the sorting of Wnts to the final destination in polarized epithelial cells have not been clarified. Glycosylation was shown to be important for the sorting of some transmembrane and secreted proteins, but glycan profiles and their roles in the polarized secretion of Wnts has not yet been demonstrated. Here we show the apical and basolateral secretion of Wnts is regulated by different mechanisms. Wnt11 and Wnt3a were secreted apically and basolaterally, respectively, in polarized epithelial cells. Wls was localized to the basolateral membrane. Mass-spectrometric analyses revealed that Wnt11 is modified with complex/hybrid(Asn40)-, highmannose(Asn90)-and high-mannose/hybrid(Asn300)-type glycans and that Wnt3a is modified with two high-mannose-type glycans (Asn87 and Asn298). Glycosylation processing at Asn40 and galectin-3 were required for the apical secretion of Wnt11, whereas clathrin and adaptor protein-1 were required for the basolateral secretion of Wnt3a. By the fusion of the Asn40 glycosylation site of Wnt11, Wnt3a was secreted apically. The recycling of Wls by AP-2 was necessary for the basolateral secretion of Wnt3a but not for the apical secretion of Wnt11. These results suggest that Wls has different roles in the polarized secretion of Wnt11 and Wnt3a and that glycosylation processing of Wnts decides their secretory routes.

Basolateral secretion of Wnt5a in polarized epithelial cells is required for apical lumen formation

“…Wls is essential for the secretion of Wnts, and is trafficked to the basolateral membranes by AP-1 and clathrin from the trans-Golgi network (Port and Basler, 2010;Yamamoto et al, 2013). Consistent with these observations, knockdown of Wls completely suppressed Wnt5a secretion in polarized MDCK II/ Wnt5a cells, although the polarized distribution of podocalyxin and TfR was not impaired ( Fig.…”

“…Our previous study also revealed that the majority of Wnt3a and Wnt11 are secreted basolaterally and apically, respectively, in polarized Madin-Darby canine kidney (MDCK) epithelial cells (Yamamoto et al, 2013). The basolateral traffic of Wnt3a requires adaptor protein 1 (AP-1) and clathrin, which are known to be important for the sorting of basolateral membrane proteins (Boehm and Bonifacino, 2001;Deborde et al, 2008;Fölsch, 2005).…”

“…It is generally thought that glycosylation of secretary proteins is necessary for their folding in the endoplasmic reticulum (ER) or exit from the ER (Spiro, 2002). Wnt3a and Wnt11 were glycosylated at multiple Asn residues, and their glycosylation profiles differed (Yamamoto et al, 2013). The complex-type glycan attached to Wnt11 at the N-terminal region is unique, and glycosylation is necessary for the apical secretion of Wnt11.…”

“…In addition, Wnt3a and Wnt11 are modified with palmitoleic acid at a conserved Ser residue (Takada et al, 2006;Yamamoto et al, 2013). The lipidation of Wnt3a and Wnt11 occurs in the ER, is catalyzed by porcupine and is essential for their exit from the ER.…”

“…Wntless (Wls), which plays a crucial role in the trafficking of Wnt, recognizes palmitoleic-acid-modified Wnts in the ER and cycles between the plasma membranes and the ER through the Golgi (Port and Basler, 2010;Yu et al, 2014). Wls itself is trafficked to the basolateral membranes and its recycling is not required for the apical sorting of Wnt11 from the transGolgi (Yamamoto et al, 2013). Crystal structural analyses have revealed that the lipid modification of Xenopus Wnt8 inserts into the groove of the extracellular domain of mouse Fz8, suggesting the necessity of lipid modification of Wnts for the recognition and activation of receptors (Janda et al, 2012).…”