C ‐mannosylation of R‐spondin3 regulates its secretion and activity of Wnt/β‐catenin signaling in cells

R‐spondin3 (Rspo3) is a secreted protein, which acts as an agonist of canonical Wnt/β‐catenin signaling that plays an important role in embryonic development and homeostasis. In this study, we focused on C ‐mannosylation, a unique type of glycosylation, of human Rspo3. Rspo3 has two putative C ‐mannosylation sites at Trp 153 and Trp 156 ; however, it had been unclear whether these sites are C ‐mannosylated or not. We demonstrated that Rspo3 was C ‐mannosylated at both Trp 153 and Trp 156 by mass spectrometry. Using C ‐mannosylation‐defective Rspo3 mutant‐overexpressing cell lines, we found that C ‐mannosylation of Rspo3 promotes its secretion and activates Wnt/β‐catenin signaling.